ISEV2019 Abstract Book

Journal of Extracellular Vesicles

ISSN: (Print) 2001-3078 (Online) Journal homepage: https://www.tandfonline.com/loi/zjev20

To cite this article: (2019) ISEV2019 Abstract Book, Journal of Extracellular Vesicles, 8:sup1, 1593587, DOI: 10.1080/20013078.2019.1593587

To link to this article: https://doi.org/10.1080/20013078.2019.1593587

Published online: 23 Apr 2019.

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Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=zjev20 JOURNAL OF EXTRACELLULAR VESICLES 2019, VOL. 8, 1593587 https://doi.org/10.1080/20013078.2019.1593587

ISEV2019 Abstract Book

About ISEV The International Society for Extracellular Vesicles is the leading professional society for researchers and scientists involved in the study of microvesicles and exosomes. With nearly 1000 members, ISEV continues to be the leader in advancing the study of extracellular vesicles. Founded in 2012 in Sweden, ISEV has since moved its headquarters to the USA. Through its programmes and services, ISEV provides essential training and research opportunities for those involved in exosome and microvesicle research.

Mission statement Advancing extracellular vesicle research globally.

Vision Our vision is to be the leading advocate and guide of extracellular vesicle research and to advance the understanding of extracellular vesicle biology.

ISEV2019 Annual Meeting The International Society for Extracellular Vesicles is the premier international conference of extracellular vesicle research, covering the latest in exosomes, microvesicles and more. With an anticipated 1000 attendees, ISEV2019 will feature presentations from the top researchers in the field, as well as providing opportunities for talks from students and early career researchers.

ISEV2019 International Organizing Committee IOC Chair: Hidetoshi Tahara, Ph.D. (Japan); Andrew Hill, Ph.D. (Australia), Carolina Soekmadji, Ph.D. (Australia), Cherie Blenkiron, Ph.D. (New Zealand), Edit Buzas, Ph.D. (Hungary), Hang Hubert Yin, Ph.D. (China), Juan Falcon Perez, Ph.D. (Spain), Kazunari Akiyoshi, Ph.D. (Japan), Kenneth W. Witwer, Ph.D. (USA), Kyoko Hida, Ph.D. (Japan), Lei Zheng (China), Marca Wauben, Ph.D. (The Netherlands), Mariko Ikuo, Ph.D. (Japan), Masahiko Kuroda, M.D., Ph.D. (Japan), Nobuyoshi Kosaka, Ph.D. (Japan), Ryou-u Takahashi, Ph.D. (Japan), Sai-Kiang Lim, Ph.D. (Singapore), Susmita Sahoo, Ph.D. (USA), Takahiro Ochiya, Ph.D. (Japan), Tang- Long Shen, Ph.D. (Taiwan), Yong Song Gho, Ph.D. (Korea) and Yoshinobu Takakura, Ph.D. (Japan)

Journal of extracellular vesicles: Editors-in-Chief Clotilde Thery, Ph.D. (France)

© 2019 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group on behalf of The International Society for Extracellular Vesicles. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 2 ISEV2019 ABSTRACT BOOK JOURNAL OF EXTRACELLULAR VESICLES 3

Plenary Session 1: Standardizations Chairs: Andrew Hill; Hidetoshi Tahara Location: Level 3, Hall B

NanoCosmos: extracellular vesicles as nanosized extracellular extracellular vesicles derived from host, bacteria, diet organelles delivering the complex messages between cells and and environments including their physical, biochem- organisms Yong Song Gho ical and biological complex properties (http://evpedia.

Department of Life Sciences, POSTECH, Pohang, Republic of Korea info). Then, this presentation focuses on our recent progress in novel extracellular vesicle-mimetic technol- The secretion of nanosized lipid bilayered extracellular ogies for targeted drug delivery, theranostics and epi- vesicles is a universal cellular process occurring from genetic reprogramming as well as for adjuvant-free, simple organisms to complex multicellular organisms. non-toxic vaccine delivery system against bacterial Recent progress in this area has revealed that extracel- infection. Furthermore, bacterial extracellular vesicle- lular vesicles play multifaceted pathophysiological based cancer immunotherapy will be introduced. Based functions by delivering the complex messages between on the concept of emergent properties of heteroge- cells and organisms, suggesting that extracellular vesi- neous extracellular vesicles, future research directions cles are NanoCosmos, i.e., extracellular organelles that to decode the complexity of extracellular vesicle- play diverse roles in intercellular and interkingdom mediated intercellular communication network, either communication. This presentation briefly introduces at the single vesicle level or at a systems level as a our last 20 year’s comprehensive research on whole, and the secret of life will be briefly introduced. 4 ISEV2019 ABSTRACT BOOK

Symposium Session 1: Cardiovascular Disease Thursday 25 April 2019 Chairs: J. Brian Byrd; Pia Siljander Location: Level B1, Hall B 11:00–12:30

OT01.01 otherwise wild-type, naïve mice mobilize splenic neutrophils to peripheral blood (P <0.001). Summary/Conclusion: Neutrophils appear at sites of Extracellular vesicles mediate neutrophil cell deployment from the injury in the immediate hours after ischemic injury. spleen following acute myocardial infarction Neutrophil interactions with EC-EV may mediate their Naveed Akbar and Robin Choudhury splenic liberation and transcriptional programming fol- University of Oxford, Oxford, UK lowing AMI, en route to the injured myocardium. The Introduction: Acute myocardial infarction (AMI) mobi- splenic neutrophil reserve may be a novel therapeutic lizes monocytes from the splenic reserve and induces target in AMI. transcriptional activation en route to the injured myo- Funding: British Heart Foundation. cardium, possibly through interactions involving plasma liberated extracellular vesicles (EVs). Neutrophils also OT01.02 reside in the spleen and are the first cells to arrive at sites of injury and mediate further damage. Here, we describe neutrophil deployment from the spleen in AMI In vivo characterization of endogenous cardiovascular extracellular vesicles and their response to ischaemic injury and by endothelial cell (EC)-derived EVs. Aaron Scotta, Costanza Emanuelib and Rebecca Richardsonc Methods: Patients provided informed consent as part of aUniversity of Bristol, Uffculme, UK; bImperial College London, London, UK; the Oxford Acute Myocardial Infarction Study. EV were cUniversity of Bristol, Bristol, UK isolated using ultra centrifugation (120,000g 2h)and characterized for size and concentration by Nanoparticle Introduction: Cardiomyocytes and endothelial cells are Tracking Analysis, EV markers (TSG101, ALIX, CD63/ counted among the cell types that secrete extracellular CD69) by western blot, and microRNAs (miRNAs) by vesicles (EVs). EVs mediate the targeted transfer of RT-qPCR. Mouse and human EC were used in vitro to lipids, proteins and nucleic acids by traversing the derive EC-EV. extracellular milieu. Recent studies suggest that EVs Results:PatientspresentingwithAMI(n =15)have2.2- play a functional role in cardiovascular disease and fold more plasma EV at time of injury vs. a 6-month cardiac repair. For example, a population of exosomes follow-up measurement (P =0.008).PlasmaEVsatthe carrying proangiogenic miRNAs was found in the peri- time of presentation correlate significantly with the extent cardial fluid of patients undergoing heart surgery. of ischemic injury (R =0.046,P =0.006)andplasma Further investigation will be required to determine neutrophils (R =0.37,P =0.017).ExperimentalAMIin which cardiac cells are producing these EVs, the cell wild type, naïve (C57B6/J) mice induces splenic-neutro- type receiving them and the functional relevance of phil deployment (P =0.004).HumanplasmaEV- this. miRNAs are significantly altered post-AMI. AMI plasma Methods: A complete understanding of this process EV-miRNA-mRNA targets (IPA, Qiagen) are signifi- requires a comprehensive in vivo model. The zebrafish cantly over represented when compared to neutrophil is an amenable vertebrate model with genetic tractabil- Gene Ontology terms for degranulation (P <0.001),acti- ity and optical transparency allowing for subcellular vation (P <0.001),chemotaxis(P =0.008)andmigration observation in a living organism. The use of stable (P =0.008).HumanECreleasesmoreEVafterinflam- transgenic lines with cell-type-specific promoters driv- matory stimulation (control 2.4 ×108 ± 4.9 x 107 EVs/ ing the expression of membrane tethered fluorophores mL vs. tumour necrosis factor-alpha stimulated, allows labelling of the cell membrane and the EVs 1.4 ×109 ± 3.0 ×108 EVs/mL, P = 0.003) and contains produced by individual cell types. Light sheet micro- many of the miRNAs enriched in human plasma-EV scopy permits cardiovascular-specific EVs to be tracked following AMI. Mouse EC-EV tail vein injected into in vivo and an established ischaemic injury model JOURNAL OF EXTRACELLULAR VESICLES 5 allows EV profiles from uninjured, injured and repair- assessed by flow cytometry (Reddel et al. Thromb ing/regenerating cardiac tissue to be determined and Haemost. 2018; 118(4): 723–733) using fluorescent sur- compared. face markers for phosphatidylserine and cell origin Results: Live imaging of transgenic zebrafish with including platelets (CD41a), leukocytes (CD45) and endothelial cell-derived EVs labelled with mCherry MAC-1 (CD11b). Positive events were defined with reveals large numbers of EVs in the peripheral circula- supernatant of ultracentrifuged pooled normal plasma tion, interactions with downstream endothelial cells as negative control. Changes pre–post RIPC were and release in to the blood flow from filopodia-like assessed by paired t-test. The study was approved by protrusions. Cardiomyocyte-derived EVs are observed the local ethics committee. in the pericardial fluid surrounding the heart and are Results: In the whole population, there was no effect of often seen interacting with cells of the pericardial wall. RIPC on fibrinolytic factors but a decrease in platelet- Additionally, a modified FACS protocol reveals how derived EV. However, in non-diabetic patients and not cardiomyocyte-derived EV numbers fluctuate in in diabetic patients, RIPC increased overall fibrinolytic response to cardiac injury. potential and CD45+ and CD11b+ EV. These effects Summary/Conclusion: This data present exciting were not seen after sham treatment. opportunities to further dissect the cargo being carried Summary/Conclusion: There is a global increase in by these EVs in a vertebrate model of human disease. fibrinolytic potential after RIPC treatment in CAD Funding: British Heart Foundation. patients without diabetes mellitus, which may be contributed to by increased leukocyte-derived EV and/or decreased platelet-derived EV. Ongoing work aims to OT01.03 directly identify this contribution in patients who undergo RIPC.

Enhanced fibrinolysis and altered extracellular vesicles after remote ischaemic preconditioning in non-diabetic coronary artery disease patients Caroline J. Reddela, Jerrett Laub, Gabrielle Penningc, Vivien Chend and OTO1.04 Leonard Kritharidese aANZAC Research Institute, University of Sydney, Concord Repatriation b General Hospital, Concord, Australia; Department of Cardiology, Concord Urinary extracellular vesicle concentration, microRNA-155 expression c Repatriation General Hospital, Concord, Australia; ANZAC Research and inflammatory surface marker expression are altered in patients Institute, University of Sydney, Concord Repatriation General Hospital, with symptomatic coronary artery disease d Concord, Australia; ANZAC Research Institute and Department of Stephen Fitzsimonsa,SilviaOggerob,NiallMahonc,NicolaRyanc,Mauro Haematology, Concord Repatriation General Hospital, Concord, Australia; Perrettid and Orina Beltona eANZAC Research Institute and Department of Cardiology, Concord a b Repatriation General Hospital, Concord, Australia University College Dublin, Dublin, Ireland; Queen Mary University of London, London, UK; cThe Mater Misericordiae University Hospital, Dublin, Ireland; dWilliam Harvey Research institute, Queen Mary Introduction: Brief non-harmful ischaemia, remote University of London, London, UK ischaemic preconditioning (RIPC) has been shown to confer benefit to patients with coronary artery disease Introduction: Urinary extracellular vesicles (uEVs) (CAD). Some studies indicate lesser benefit in patients (exosomes, microvesicles and apoptotic bodies) have with diabetes. RIPC may enhance fibrinolysis. potential as diagnostic and prognostic biomarkers. In Hypothesis: RIPC causes an increase in fibrinolytic atherosclerosis, the underlying cause of heart attack potential through release of fibrinolytic factors from and stroke, EV release can be dysregulated and their the endothelium or fibrinolysis-supporting extracellu- contents can mediate pro-inflammatory effects. Several lar vesicles (EVs) and this effect is less evident in markers have been previously identified on uEV patients with diabetes. including exosome markers CD63 and CD9, CD45 Methods: Patients at Concord Hospital with suspected (leukocyte marker), CD61 (platelet marker), CD14 CAD gave written informed consent and were admi- (monocyte/macrophage marker) and α/β integrins. nistered RIPC (sphygmomanometer on the arm, 3 ×5 The selectively packaged cargo of these membrane min cycles, n = 31) or sham (n = 29) before angiogra- bound carriers include microRNAs (miRs). miR-21 phy, with recruitment ongoing. Blood was collected and miR-155 are key regulatory miRs that are upregu- pre- and immediately post-RIPC/sham and platelet- lated in immune cells and are released in EVs following free plasma generated. Global coagulation/fibrinolytic exposure to pro-inflammatory stimuli. miR-155 has potential was measured by overall haemostatic poten- been reported to have pro-atherogenic effects and tial assay (Reddel et al. Thromb Res. 2013; 131(5): 457– miR-155 deficiency in murine models results in 462) and various fibrinolytic factors by ELISA. EV were reduced atherosclerotic lesion burden. 6 ISEV2019 ABSTRACT BOOK

Methods: Urine was collected from patients diagnosed development of an integrative CV risk prediction with coronary artery disease (CAD), classified as symp- model for diabetic ESRD patients. The foundation of tomatic (non-ST-elevation myocardial infarction, ST- this study is the Predictors of Arrhythmic and elevation myocardial infarction or unstable angina) or Cardiovascular Risk in End Stage Renal Disease asymptomatic (stable angina). uEVs from symptomatic (PACE) cohort (n = 571); a cohort of ESRD patients and asymptomatic patients were isolated via benchtop followed over four years, with a repository of clinical centrifugation. The concentration and size of uEVs data with matched biological samples. ExoQuick was were analysed via the NanoSight NS300 (n = 15 per used for the isolation of EVs from plasma, and their group). The expression of miR-155 and miR-21 was properties characterized through NanoSight analysis, investigated by RT-qPCR (n = 10 per group). uEV western blotting and electron microscopy. A high- surface marker expression was analysed by throughput microfluidics RT-qPCR platform ImageStreamX MK2 Imaging Flow Cytometer (12 per (n = 420) was used to examine potential associations group). between miRNA and clinical outcomes. We then vali- Results: uEV concentration in symptomatic patients dated functionality on human coronary endothelial (median; 6.46E+9 particles/mL) was significantly cells (n = 12, coronary artery disease vs. control) decreased (p < 0.05) compared to asymptomatic using mRNA-Seq. patients (median; 1.25E+10 particles/mL). CD11B+ Results: EV analysis revealed diabetic ESRD patients uEVs were increased and CD16+ uEVs were decreased have increased circulating vesicle concentrations; in in the symptomatic patients (p < 0.01). In addition, the addition to having vesicles of a larger mean size. Data concentration of CD45+ EVs were increased in symp- from our clinically translatable microfluidics-based RT- tomatic patients (p < 0.001). Although uEV miR-21 qPCR methodology identified numerous potential was unchanged, miR-155 expression was significantly microRNA biomarkers for CV complications of increased in the symptomatic group (p < 0.05). ESRD. Following miRNA identification, we utilized Summary/Conclusion: uEV concentration, miR-155 penalized regression models to generate a panel of expression and surface marker expression have diag- miRNAs which may serve as CV-risk predictors for nostic and prognostic potential. As CAD severity diabetic ESRD patients. In particular, miR-23b-3p increases, uEV concentration is reduced, surface mar- appears to be significantly associated with coronary ker expression is altered and uEV miR-155 expression artery disease severity. is increased. Summary/Conclusion: This data will be weighted with Funding: The Irish Research Council. the novel biomarker data and fully integrated to build a clinical risk prediction model for the development of CV complications in ESRD and reassessed in a new OT01.05 cohort (D4 Cohort) replicate the findings and validate the risk prediction model. Circulating extracellular vesicle-associated microRNAs as predictive Funding: This work was funded by AstraZeneca and biomarkers of cardiovascular complications in end-stage renal the Canadian Vascular Network. disease Dakota D. Gustafsona, Jessica Fitzpatrickb, Jason Fishc and Rulan Parekhb aDepartment of Laboratory Medicine and Pathobiology, University of OT01.06 Toronto, Toronto, ON, Canada; bChild Health Evaluative Sciences, Research Institute, The Hospital for Sick Children, Toronto, ON, Canada; cToronto General Hospital Research Institute, University Health Network, Toronto, ON, Canada Live tracking system for endogenous exosomes Weijia Luoa, Yuan Daia, Kelsey Andradea, Megha Chandnab, Pamela Ulloa- Francoc and Jiang Changa Introduction: Chronic kidney disease affects roughly aTexas A&M University College of Medicine, Houston, USA; bUniversity of three million Canadians and is accompanied by con- Texas-Austin, Houston, USA; cTrinity College, Hartford, USA siderable human suffering, premature death and costly medical care. The incidence of ESRD and its associated Introduction: Exosomes are emerging new category of cardiovascular (CV) complications continues to rise messengers that communicating among cells, tissues despite improved treatments for the primary etiologies and organs. Understanding the kinetic of exosome of diabetes and hypertension. communication in vivo is a critical foundation for Methods: We are using multiple biological approaches studying exosome functions and developing exosome- to better understand the organ-level effects of extracel- based drug-delivery models. Current studies of exo- lular vesicle (EV)/miRNA dysregulation and gather the some in vivo trafficking largely rely on the administra- detailed mechanistic insight necessary for the tion of exogenous exosomes labelled by fluorescent JOURNAL OF EXTRACELLULAR VESICLES 7 dyes or proteins. These methods may not fully repre- an inducible system. The exosome labelling and distribu- sent endogenous exosome kinetics due to ex vivo exo- tion were assessed by luciferase assay and noninvasive some manipulation. Here, we established the first bioluminescent live imaging. inducible endogenous exosome tracking mouse model Results: CD63NanoLuc expression was tightly con- that tracks endogenous exosome released by cardio- trolled and only detected in cardiomyocytes upon myocytes in vivo. induction. The endogenous exosomes released from Methods:Wegeneratedatransgenicmouseexpressing cardiomyocytes were labelled and detected in vitro in the bioluminescent reporter Nano-luciferase (NanoLuc)- cell culture supernatant, and in vivo in animal plasma. fusion protein. The ultrasensitive and stable NanoLuc A signature distribution profile of the endogenous car- reporter has a 150-fold stronger signal compared to the diomyocyte-releasing exosomes was achieved. traditional Firefly and Renilla luciferases and the longest Summary/Conclusion: This exosome tracking model luminescent half-life amongst all known luciferases. We enables elucidating the endogenous exosome traffick- fused NanoLuc reporter with exosome surface marker ing pattern, and allows the study of exosome behaviour CD63 for specific labelling of exosomes. Then, the cardi- under different conditions. It will provide a powerful omyocyte-specific αMHC promoter followed by a loxP- tool for the exploration of the biological functions, STOP-loxP cassette was engineered for precise spatial mechanisms and clinical applications of exosomes in labelling of exosomes originating from cardiomyocytes. a broad spectrum of research. We then crossed the cardiomyocyte-specific mouse with a Funding: AHA Innovative Project Award: 18IPA341 tamoxifen-inducible Cre mouse (R26CreERT2) to achieve 80012. 8 ISEV2019 ABSTRACT BOOK

Symposium Session 2: Nucleic Acid Biomarkers in Human Disease Chairs: Robert Kitchen; Louise Laurent Location: Level B1, Lecture Room 11:00–12:30

OT02.01 brain and blood of AD, PD, ALS and CJD patients. The miRNA candidates can be used to develop a blood- based diagnostic test highly relevant to a brain disease, miRNA exosomal biomarkers in brain derived and serum exosomes equivalent to noninvasive brain biopsy. associated with neurodegenerative diseases Lesley Chenga, Xia Lib, James Doeckec, Laura Vellad, Natasha Vassileffe, Funding: National Health and Medical Research Mitch C. Shambrooka, Robyn Sharplesf, Saima Zafarg, Inga Zerrh, Catriona Council, Australia MND, Australia Creutzfeldt-Jakob i d d d McLean , Malcolm Horne , Colin Masters , Kevin Barnham and Andrew disease Support Group Network, Australia Dementia Hillj Centre for Research Collaboration, Australia aDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, VIC, Australia, Melbourne, Australia; bDepartment of Mathematics and Statistics, La Trobe Institute for Molecular Science, La Trobe University, VIC, Australia, Melbourne, OT02.02 Australia; cCSIRO Digital Productivity Flagship/The Australian E-Health Research Centre, Herston, Queensland, Australia, Brisbane, Australia; dThe Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Parkville, Victoria, Australia; eThe Department of Biochemistry and Brain-derived extracellular vesicle microRNA signatures associated Genetics, La Trobe Institute for Molecular Science, La Trobe University, with in utero and postnatal oxycodone exposure: Implications for Bundoora, Victoria, Australia; fThe Department of Biochemistry and Genetics, altered synaptogenesis La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria Schaala, Dalia Mooreb, Peng Xiaoa, Sowmya V. Yelamanchilib, Victoria, Australia; gDepartment of Neurology, Clinical Dementia Centre, Gurudutt Pendyalaa University Hospital Göttingen, Göttingen, Germany; hThe Florey Institute of aUniversity of Nebraska Medical Center, Omaha, USA; bDepartment of Neuroscience and Mental Health, The University of Melbourne, Parkville, Pharmacology and Experimental Neuroscience, University of Nebraska Victoria, Australia., Melbourne, Australia; iThe Department of Biochemistry Medical Center, Omaha, USA and Genetics, La Trobe Institute for Molecular Science, La Trobe University, Bundoora, Victoria, Australia Introduction: Oxycodone (oxy) is a semi-synthetic Introduction: Several blood-based tests have been opioid commonly used as a pain medication which explored to detect Alzheimer’s disease (AD) and other also is a widely abused prescription drug. While very neurogenerative diseases; however, evidence is required limited studies have examined the effect of in utero oxy to determine whether blood sampling is an appropriate (IUO) exposure on neurodevelopment, a significant specimen to diagnose brain diseases. Exosomes are gap in knowledge is the effect of IUO compared with small extracellular membrane vesicles packaged with postnatal oxy (PNO) exposure on synaptogenesis – a RNA and protein cargo. Previously we isolated serum key process in the formation of synapses during brain exosomes from AD patients which displayed an abnor- development in the exposed offspring. In the present mal composition of 16 specific microRNA (miRNA) study, we isolated and characterized brain-derived biomarkers compared to controls. extracellular vesicle (BDE)-associated microRNA Methods: To provide evidence that our serum exoso- cargo from the brains of IUO and PNO offspring mal miRNA biomarkers are suitable for the detection using RNA seq. Several key miRNAs unique to both of a brain condition, we also profiled exosomes isolated the IUO and PNO groups were identified and validated from post-mortem human AD (n = 8), PD (n = 8), ALS using RT-PCR. To further gain mechanistic insights, (n = 7) and control (n =5–8 per group) brain tissues we characterized the miRNA cargo effects on changes using next-generation sequencing. in synaptic architecture using in vitro primary neurons Results: Brain-derived exosomes (BDEs) were found to during a key stage of brain development. contain a unique profile of small RNA, including Methods: Density gradient EV isolations from brain miRNA, compared to whole tissue. Furthermore, all tissue, transmission electron microscopy, RT-PCR, in 16 AD serum biomarkers, identified in our previous vitro primary neuronal cultures and spine density study, were detected in BDEs, together with differen- analysis. tiators for PD, ALS and CJD diagnosis in serum and in Results: Transmission electron microscopy revealed an some cases neural-derived exosomes. increase in BDE sizes in both the PNO and IUO groups Summary/Conclusion: This work has identified highly suggesting that oxy exposure can affect BDE size thus specific panels of miRNA that is both present in the indicating differential expression of molecular cargo. JOURNAL OF EXTRACELLULAR VESICLES 9

Next, RNA-Seq identified novel and distinct BDE Results: We identified a 16-mRNA signature by miRNAs unique to IUO and PNO which were further mining over 25,000 public and proprietary RNA-seq validated by RT-PCR. Bioinformatics analysis on these datasets, using a machine learning approach to rank differentially expressed BDEs, revealed key Gene genes based on dysregulation in bladder cancer, pre- Ontology terms involved in neurodevelopment such sence in urine exosomes and stability to haematuria. as neuron projection development, neuronal morpho- Using this signature, we trained a classifier to differ- genesis, pallium/cerebellum development in the IUO entiate samples based on presence/absence of bladder offspring. To determine, if BDEs impacted the synap- cancer, optimized for negative predictive value (NPV). todendritic architecture, we treated 14 days in vitro rat The model performs well in both newly diagnosed and cortical primary neurons with equal amounts of P14 recurrent cases, even in low-grade disease, with an BDEs from the three groups. Confocal imaging of overall performance of 100% NPV at 46% specificity. dendritic spines showed a significant reduction on As the model is based solely on exosomal mRNA treatment with PNO BDEs and which was further abundance, the score provides entirely new informa- exacerbated on treatment with the IUO BDEs. tion that would enable a clinician to further improve Summary/Conclusion: We conclude that BDEs from specificity by considering standard of care parameters. PNO and IUO offspring carry potentially distinct BDE Summary/Conclusion: Exosomal mRNAs have been miRNA cargo that subsequently damage the synapto- used to diagnose other malignancies but this represents dendritic architecture and could further lead to neuro- the first application of this form of liquid biopsy to nal dysfunction at a key stage of neurodevelopment. bladder cancer. While performance must be validated Funding: Start-up funds and NIH/NIDA. in a larger clinical trial, this signature could prevent ~50% of unnecessary biopsies, provide a noninvasive means of monitoring relapse and reduce the financial OT02.03 burden of early stage bladder cancer care.

OT02.04 Development of a high-performance urine exosomal-mRNA signature for identification of bladder cancer Sudipto Chakraborttya, Robert Kitchena, James Hurleya, Georg Stollb, Xuan Zhangc, Mikkel Noerholmd, Seth Yua and Johan Skoge Genome-wide methylation profiling of extracellular vesicle DNA a b allows brain tumour classification Exosome Diagnostics, Inc, Waltham, USA; Exosome Diagnostics, GmbH, a b b b c Franz Lennard. Ricklefs , Cecile Maire , Katharina Kolbe , Mareike Holz , Martinsried, Germany; Neuology and Radiology Services and program in Manfred Westphalb, Ullrich Schüllerb and Katrin Lamszusb Neuroscience, Harvard Medical School, Massachusetts General Hospital, Boston, USA; dExosome Diagnostics, GmbH, Martinsried, Germany; aUniversity medical center Hamburg-Eppendorf, Hamburg, Germany; eExosome Diagnostics, Inc., Waltham, Massachusetts, USA bUniversity Medical Center Hamburg-Eppendorf, Hamburg, Germany

Introduction: Blood in the urine is a common symp- Introduction: Genome-wide methylation profiling has tom of bladder cancer but of individuals who present recently been developed into a tool that allows subtype with haematuria on average only 8% will have cancer. tumour classification in central nervous system (CNS) Moreover, up to 70% of patients with a prior bladder tumours. Extracellular vesicles (EVs) are released by tumour will experience a relapse. The majority of these CNS tumour cells protecting their cargo, including individuals will therefore undergo invasive and expen- DNA, from degradation rendering EVs as optimal bio- sive testing (cystoscopy & CT scan) to confirm the markers to define subgroups, stratify patients and presence of a tumour, either for first diagnosis or active monitor therapy by liquid biopsy. It is unclear, how- surveillance of recurrence. A low-cost, noninvasive ever, if DNA derived from glioma EVs reflects gen- urine test capable of preventing unnecessary biopsies ome-wide methylation profiles and mutational statuses is a challenging but attractive proposition. that would allow tumour classification. Methods: Here, we present results from a clinical study Methods: DNA was isolated from glioma cell cultures in which exosomal mRNAs were profiled from voided (GSC) EVs, GSCs and matched tumour samples urine, collected prior to diagnosis, from individuals (n = 3). EVs were isolated through differential ultra- suspected of having either newly diagnosed or relapsed centrifugation and classified by nanoparticle tracking bladder cancer. We selected 81 individuals for the analysis (NTA), immunoblotting, imaging flow cyto- clinical study, 44 of whom were diagnosed with mostly metry (IFCM), multiplex EV assay and electron micro- early stage bladder cancer. The remaining individuals scopy. Genome-wide DNA methylation profiling was were healthy or diagnosed with a non-cancerous urin- performed using a 850-k Illumina EPIC array and ary disease. classified by the DKFZ brain tumour classifier. 10 ISEV2019 ABSTRACT BOOK

Results: GSCs secrete diverse EVs as measures by IFCM TaqMan® Array Human MicroRNA A + B Cards Set and multiplex EV assay that are high for common EV v3.0 (ThermoFisher). MiRNA expression was compared markers (a.e. CD9, CD63 and CD81). The range of EVs between MA-ACT and CTL using two-sample t-tests for was 120–150 nm measured by NTA. Genome-wide miRNA expressed in at least 50% of samples in at least methylation profiles of GSC EVs in addition to copy one of the two groups. Tobacco use was controlled for. number alterations and mutations matched their paren- Results:ThedatashowthatinMA-ACT(n =5)vs.CTL tal GSC and original tumour sample, being (n =5),fourofthefiveMA-ACThaveanincreasein Glioblastoma, IDH wildtype or mutant, with additional total plasma EVs relative to all five CTL. The average EV subclass analyses. Specifically, MGMT methylation sta- concentration in MA users (2 ×108 ± 4.5× 107 EV/μL) tuses could be obtained through EV DNA. is trending to increased levels, relative to CTL Summary/Conclusion: Here we report, that EV DNA (7.5 ×107 ± 0.9 ×107 EV/μL). EV counts relative to reflects the tumour methylation class as well as most size show a range of EVs with a mode of ~110 nm in copy number variations and mutations present in the both MA-ACT and CTL plasma, and equivalent median parental cells and the original tumour. DNA EV EV size. Of 226 miRNA in the EVs, there are 30 miRNAs methylation profiles may therefore be used to detect that meet have area under the curve (AUC) >0.65 and and classify CNS tumours. median difference >1, and 47 miRNAs with AUC >0.65 Funding: FLR received a scholarship of the German and mean difference >1. Twenty-three of these miRNAs Academic Foundation. overlap and are the current focus of target prediction. Summary/Conclusion: EV miRNA expression in subjects with MA use disorder was significantly different OT02.05 than in control participants, suggesting that MA may affect EV communication among cells. The differential miRNA expression also implicates a role for EVs in Methamphetamine use disorder alters plasma extracellular vesicle characteristics and microRNA expression behavioural and physiological effects specific to MA Ursula Sandaua, John Nolanb, Xiao Shic, Tracy Swansonc, Marilyn Huckansd, and suggests that there may be changes in expression d e f d William Schutzer , Kylie Sage , Jodi Lapidus , Jennifer Loftis , Aaron of miRNAs that are relevant to specific drugs of addic- Janowskyg and Julie A. Saugstada tion, as well as to a spectrum of drug-mediated addic- aDepartment of Anesthesiology & Perioperative Medicine, Oregon Health & Science University, Portland, USA; bScintillon Institute, San Diego, USA; cVA tion disorders. Portland Health Care System, and Department of Psychiatry, Oregon Health & Science University, Portland, USA; dVA Portland Health Care System, Department of Psychiatry, and Methamphetamine Abuse Research Center, Oregon Health & Science University, Portland, USA; eBiostatistics & Design OT02.06 Program, Oregon Health and Science University, Portland, USA; fBiostatistics & Design Program, Oregon Health and Science University, Oregon Health & Science University – Portland State University School of Public Health, Portland, USA; gVA Portland Health Care System, Departments of Use of extracellular vesicles purified from lymphatic exudative Psychiatry and Behavioral Neuroscience, and Methamphetamine Abuse seroma as surrogate markers of melanoma residual disease Research Center, Oregon Health & Science University, Portland, USA Susana Garcia-Silvaa, Alberto Benito-Martínb, Sara Sanchez-Redondoc, Alberto hernandez-Barrancoa, Pilar Ximénez-Embúnd, Laura Noguésa, Ana Isabel Amora, Kay Brinkmanne, Marina S Mazariegosc, Jasminka Boskovichf, ’ g h e i Introduction: Methamphetamine s(MA)rewarding Mercedes Robledo , Johan Skog , Mikkel Noerholm , Javier Muñoz , Pablo L. properties and addictive potential are correlated with Ortiz-Romeroj, José Luis Rodríguez-Peraltok, Piotr Rutkowskil and Héctor a increased synaptic dopamine availability following Peinado alterations in dopamine and vesicular monoamine trans- aMicroenvironment and Metastasis Laboratory, Molecular Oncology Programme. Spanish National Cancer Research Center (CNIO), 28029 porter function. We examined plasma extracellular vesi- Madrid, Spain, Madrid, USA; bChildren‘s Cancer and Blood Foundation cle (EV) number, size, protein markers and miRNA Laboratories, Departments of Pediatrics, Weill Cornell Medicine, New York, NY 10021, USA, New york, USA; cMicroenvironment and Metastasis content in human subjects who are actively using MA. Laboratory, Molecular Oncology Programme. Spanish National Cancer Methods:Plasmasamplesfrom10adultswithactiveMA Research Center (CNIO), 28029 Madrid, Spain, MAdrid, USA; dProteomics dependence (MA-ACT), and 10 non-dependent controls Unit – ProteoRed-ISCIII, Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain, Madrid, USA; eExosome Diagnostics, GmbH, (CTL) were obtained from the Methamphetamine Abuse Martinsried, Germany, Martinsried, USA; fElectron Microscopy Unit, Research Center Biorepository at Oregon Health & Spanish National Cancer Research Centre (CNIO), Madrid 28029, Spain, MAdrid, USA; gHereditary Endocrine Cancer Group, Spanish National Science University and the VA Portland Health Care Cancer Research Centre (CNIO), Madrid 28029, Spain, Madrid, USA; System. We used single Vesicle Flow Cytometry to hExosome Diagnostics, Inc., Waltham, Massachusetts, USA, Waltham, USA; iSpanish National Cancer Research Centre, Madrid, South Georgia & South directly measure plasma EV concentration and size. We Sandwich Islands; jDepartment of Dermatology, Medical School, Universidad used size-exclusion chromatography (iZON Science) to Complutense, Instituto i + 12, Hospital Universitario 12 de Octubre, Madrid ™ 28041, Spain, Madrid, USA; kDepartment of Pathology, Medical School, isolate plasma EVs. EV total RNA isolated by mirVana Universidad Complutense, Instituto i + 12, Hospital Universitario 12 de PARIS™ RNA Kit (ThermoFisher) was analysed on Octubre, Madrid 28041, Spain, Madrid, USA; lMaria Sklodowska-Curie JOURNAL OF EXTRACELLULAR VESICLES 11

Institute – Oncology Center, Department of Soft Tissue/Bone Sarcoma and demonstrated that they are enriched in melanoma Melanoma, Warsaw, Poland, Varsaw, USA oncogenic pathways together with immune-related Introduction: Liquid biopsies in melanoma patients pathways; however, proteomic analysis did not allow have the potential to improve prognosis. Exudative identify biomarkers of relapse or progression. seroma obtained in the drainage implanted post-lym- Importantly, detection of BRAFV600E mutation in ser- – phadenectomy has never been explored as a source of oma-derived EVs obtained 24 48 hours post-lympha- biomarkers. The use of circulating extracellular vesicles denectomy identified patients at risk of relapse (EVs) as surrogate markers of residual disease could be significantly (Log rank p = .0067) in a cohort of 17 a novel and powerful non-invasive tool. stage III melanoma patients followed up for 700 days. Methods: Exosomes were purified by ultracentrifuga- Summary/conclusion: Our data show for the first time tion from exudative seroma obtained after lymphade- that exudative seroma obtained post-lymphadenectomy nectomy from stage III melanoma patients, were is a novel biofluid enriched on EVs and DNA that can analysed by nanosight analysis and electron microbe interrogated for melanoma markers and BRAF scopy, and compared to plasma in a total of 92 sam- mutation. Analysis of BRAFV600 mutation identified ples. We profiled the proteomic profiles of exudative patients at risk of relapse with high significance. These seroma- and plasma-derived exosomes by mass spec- data support that our approach could be a novel factor trometry. Extracellular vesicle-associated nucleic acids to detect residual disease right after surgery in stage III (EV-NAs) and analysed BRAFV600E by an allele-spe- melanoma patients. Our analysis could be a novel cific PCR in exudative seroma samples as a novel approach to aid oncologists to identify high-risk groups parameter to detect residual disease of patients post-lymphadenectomy and improve Results: We found that exudative seroma is a novel patient outcome. biofluid highly enriched in EVs, higher size and Funding: MINECO, NIH, Starr Foundation, Ramon y increased DNA cargo in comparison to plasma. Cajal Programme, AECC, FERO foundation, and Proteomic analysis of seroma-derived exosomes MINECO-REDiEX. 12 ISEV2019 ABSTRACT BOOK

Symposium Session 3: EVs in Cancer Metastasis and Angiogenesis Chairs: Kyoko Hida; Alissa Weaver Location: Level 3, Hall B 11:00–12:30

OT03.01 Summary/Conclusion: Our results indicated that HLSC-EVs and TKIs have a synergistic anti-tumour effect on renal CSCs inducing an enhancement of Stem cell-derived extracellular vesicles increase cancer stem cell apoptosis by a combined effect on intracellular path- sensitivity to tyrosine kinase inhibitors through Akt/mTOR/PTEN- combined modulation ways pivotal in the induction of tumour growth and Benedetta Bussolatia, Valentina Fonsatob, Michela De Lenac, Stefania Trittad, survival. This effect appear to be due to an EV-depen- b e f g Alessia Brossa , Ruggero Calvetti , Ciro Tetta and Giovanni Camussi dent enhancement of TKI induced mechanisms and a Department of Molecular Biotechnology and Health Sciences, University of not to epigenetic changes induced by EV leading to Turin, Turin, Italy; bMolecular Biotechnology Center, University of Torino, Torino, Italy; cMolecular Biotechnology Center, University of Torino, Torino, increased TKI sensitivity. This study provides a rational Italy; dUniversità di Torino, Nichelino, Italy; eDepartment of Molecular for a combined use in tumour treatment. Biotechnology and Health Sciences, Torino, Italy; fUnicyte srl, Turin, Italy; gDepartment of Medical Sciences, University of Turin, Turin, Italy Funding: This study was supported by Associazione Italiana per la Ricerca sul Cancro (A.I.R.C.), project Introduction: Cancer Stem Cells (CSCs) are a small IG2012 and by grant no. 071215 from Unicyte. cell population able to sustain the maintenance and recurrence of tumours. In consideration of the high OT03.02 drug resistance and tumour initiating capability, targeting CSCs represents an important approach to eradicate tumours. We previously showed a pro-apoptotic Exosomal nidogen 1 drives liver cancer metastasis by inducing secretion of tumour necrosis factor receptor 1 from activated lung effect of extracellular vesicles (EVs) derived from fibroblasts human liver stem cells. In this study, we evaluated Xiaowen Mao, Sze Keong Tey and Judy Yam whether HLSC-EVs could act in synergy with tyrosine The University of Hong Kong, Hong Kong, Hong Kong kinase inhibitor drugs (TKIs) on apoptosis of CSCs isolated from renal carcinomas. Introduction: Hepatocellular carcinoma (HCC) is an Methods: We administered HLSC-EVs and TKIs to aggressive tumour with metastasis as a signature in the renal CSCs, as co-incubation or sequential administra- advanced stage. Acquisition of migratory and invasive tion. TKIs were also loaded in EVs. Intracellular phos- behaviours is fundamental to cancer cells to metasta- phoproteins were evaluated in the CSC lysates by the size. A supportive microenvironment for the coloniza- magnetic bead-based immunoassays Bio-Plex Pro cell- tion of incoming disseminated cancer cells during signalling assay and confirmed by Western Blot metastasis is also indispensable. Exosome shedding analysis. has emerged as an important channel for intercellular Results: We found that HLSC-EVs in combination communication in tumour microenvironment during with Sunitinb or Sorafenib significantly increased metastasis. renal CSCs apoptosis induced by low TKI dose. At Methods: Exosomes derived from HCC cell lines were variance, no synergistic effect was observed when functionally characterized by in vitro and in vivo bone marrow mesenchymal stem cell-derived EVs assays. Proteomic profiling and expression level of were used. CSC apoptosis was also enhanced when exosomal proteins were analysed by mass spectrometry TKIs were loaded in HLSC-EVs. In particular, renal and enzyme-linked immunosorbent assay (ELISA), CSCs chemosensitivity to TKIs was enhanced when respectively. The study of interplay between exosomes, HLSC-EVs were either co-administered with TKIs or HCC cells and lung fibroblasts were carried out using added after, but not before. By a mechanistic point of functional assays, immunofluorescent staining and view, Akt/mTOR and Erk and Creb intracellular path- ELISA. ways, known to be pivotal in the induction of tumour Results: Exosomes derived from metastatic HCC cells growth and survival, appeared modulated as conse- augmented cell migration and invasiveness. In animal quence of TKIs/HLSC-EVs co-administration as well model, metastatic-exosomes promoted liver tumour as by EV post-administration. formation, increased incidence of distant metastasis to JOURNAL OF EXTRACELLULAR VESICLES 13 lungs as well as facilitated colonization of hepatoma created an appropriate microenvironment for the cells in lungs and enhanced the permeability of pul- metastasis. monary vasculature. Proteomic profiling of exosomes Results: Our whole-transcriptome analysis of fibro- identified nidogen 1 (NID1) as a functional component blasts revealed that high metastatic DGC cell-derived responsible for the promoting effect of metastatic-exo- EVs strongly induced the expression of inflammatory somes. Our data showed that suppression of exosomal chemokines such as CXCL1 and CXCL8. However, it is NID1 (exo-NID1) significantly diminished the biologi- not observed in the fibroblast treated with EVs from cal activities of metastatic-exosomes. Apart from HCC low-metastatic DGC. Interestingly, both cancer-derived cells, exo-NID1 enhanced the growth and induced EVs did not affect the expression of alpha-smooth activation of lung fibroblasts. Tumour necrosis factor muscle actin (α-SMA), a typical marker of myofibro- receptor 1 (TNFR1), found to be released by lung blast phenotype. When fibroblasts were treated with fibroblast pretreated with metastatic-exosomes, showed TGFβ, α-SMA was clearly induced but suppressed potent effect in promoting HCC cell motility. Notably, inflammatory chemokine expression in the fibroblasts. the level of exo-NID1 was well correlated with the Immunocytochemical analysis of CXCL8 and α-SMA metastatic potential of parental HCC cells. showed distinct populations of activated fibroblasts in Encouragingly, the level of NID1 in circulating exo- the co-culture with high-metastatic DGC, suggesting somes of HCC late stage patients was higher than those that functional heterogeneity was generated by EVs. at early stage. We also identified that various miRNAs including Summary/Conclusion: Our study reveals the novel miR-193b were enriched in high metastatic DGC cell- role of NID1, in the form of exosomes, in HCC metas- derived EVs to induce chemokine expression in tasis and illuminates the expression profile of exosomal fibroblasts. NID1 with clinical significance. Our study implicates Summary/Conclusion: Our findings suggest that the that targeting signalling pathway mediated by exo- intercellular crosstalk of high-metastatic DGC and fibro- somes of metastatic HCC as a therapeutic strategy blasts via EVs contributes to forming the appropriate for HCC. tumour microenvironment toward the metastasis. Funding: RGC GRF (17,113,116) and SKLLR IRF 2017.

OT03.03 OT03.04

Cancer extracellular vesicles create functional heterogeneity of Extracellular vesicles from obese human adipose tissue alter the invasive and proliferative properties of prostate cancer cells cancer-associated fibroblasts in gastric cancer a Yutaka Naitoa, Yusuke Yamamotob, Akiko Kogurec, Iwao Shimomurad, Anca D. Dobrian , Ryan Huyck, Bronson Haynes, Vanessa Correll, Avennette Minami Kumazakic and Takahiro Ochiyad Pinto, James Reed, Matthew Freedman, William McPheat, Julius O. Nyalwidheb, O. John Semmesb a Division of Molecular and Cellular Medicine, National Cancer Center a b Research Institute, London, UK; bDivision of Molecular and Cellular Eastern Virginia Medical School, Norfolk, USA; Leroy T. Canoles Jr. Cancer Medicine, National Cancer Center Research Institute, Tokyo, USA; Research Center, Eastern Virginia Medical School, Norfolk, USA cDivision of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan; dDepartment of Molecular and Cellular Introduction: Obesity increases the risk and aggres- Medicine, Institute of Medical Science, Tokyo Medical University, Shinjyuku-ku, Japan siveness of multiple cancers including prostate cancer. Adipose tissue (AT) is a rich source of extracellular Introduction: Cancer-associated fibroblasts (CAFs) are vesicles (EVs) that were shown to contribute to vascu- the major stromal components in the various types of lar and metabolic pathologies. Here we characterized malignancies. It has been recognized that the func- the miRNA and proteome of EV isolated from human tional heterogeneity of CAFs provide an appropriate visceral (V) and subcutaneous (S) fat of bariatric sub- microenvironment for tumour progression. However, jects and explored their mechanistic effects on mole- it is still largely unknown how functional heterogeneity cular and functional phenotypes of metastatic prostate of CAF is governed by tumour cells. In this study, we cancer cells. investigated the role of extracellular vesicles (EVs) on Methods: Paired S and V AT collected intraoperatively the formation of CAF functional heterogeneity. were used to isolate EVs by ultracentrifugation Methods: We treated EVs derived from high-meta- (n = 27). DIO-labelled EV-S or EV-V was incubated static diffuse-type gastric cancer (DGC) cells or low- overnight with PC3-ML metastatic prostate cancer metastatic DGC cells to the fibroblasts. By comparing cells. EV uptake, proliferation, migration and invasion transcriptome profiles of fibroblasts with the EVs, we were quantified by fluorescence microscopy, BrdU sought to understand how high-metastatic DGC cells incorporation, wound healing and invasion assays, 14 ISEV2019 ABSTRACT BOOK respectively. The miRNA and proteome cargo of EVs used to evaluate in vivo effects of omental-exosomes on were measured using the Nanostring platform and LC/ gastric cancer tumour growth. MS/MS. Changes in gene expression in recipient PC3- Results: Initially, we demonstrate a robust uptake of ML cells were determined using Nanostring. omental fat exosomes by gastric cancer cells. We show Results: EV-S and EV-V produced similar effects on that these exosomes enhance gastric cancer cell prolif- recipient PC3-ML cells. EVs increased cell proliferation eration, migration and invasion. We also revealed that by ~1.8-fold (p < 0.05); had no effect on cell migration the number of exosomes is directly related to their but dramatically decreased cell invasion by 2.5-fold effect on gastric cancer cells. We further show that (p < 0.01) compared to untreated controls. Gene omental fat exosomes induce gastric cancer cellular expression in recipient PC3-ML cells showed signifi- chemoresistance to platinum-based therapy, and that cant two to three fold decrease in expression of 8 omental exosomes augment gastric cancer xenograft MMPs without changes in TIMP expression. tumour growth in-vivo. Using a cytokine array, we Mesenchymal markers Snail and Zeb were also signifi- characterized the proteome of omental fat exosomes cantly decreased and seven glycolytic and PPP enzymes compared to SC exosomes. miRNA profiling identified were 1.5- to 2.5-fold increased. Consistent with these several established oncomiRs. These vesicles carry changes, the miRNA cargo of EVs was shown to target numerous proteins and miRNAs implicated in cellular all the above pathways and the top pathways detected adhesion and chemotaxis, tumour growth and motility in the EV proteome were metabolism and energy as well as chemoresistance; some of these molecules production. have been reported as pro-tumourigenic factors in gas- Summary/Conclusion: AT EVs appear to induce a tric cancer. Finally, we demonstrate that omental fat- mesenchymal to epithelial transition in prostate cancer exosomes increase the expression of transcription fac- cells. This study reveals a novel role of EVs from tors, mRNA of extracellular matrix proteins and adhe- human AT on metastasis and suggests a new mechan- sion molecules within gastric cancer cells. istic link between obesity and prostate cancer. Summary/Conclusion: These observations demon- Funding: Commonwealth of Virginia Health Research strate for the first time the uptake of omental fat exo- Board. somes by cancer cells; these vesicles carry different molecules which promote gastric cancer cellular aggressiveness in vitro and in vivo. Taken together, OT03.05 our data imply that omental fat exosomes might play a role in gastric cancer peritoneal spread.

Novel vesicular mediators of peritoneal metastases Shelly Loewensteina, Fabian Gerstenhaberb, Nir Lubezkyb, Eran Nizrib, Joseph OT03.06 Klausnerb, Noam Shomronc, Guy Lahatb aTel Aviv Sourasky Medical Center, Tel Aviv, Israel; bSurgery Division, Tel Aviv Sourasky Medical Center, Tel-Aviv, Israel; cTel Aviv university, Tel Aviv, Israel Non-SUMOylated Cx43 changes the recruitment of cellular components into exosomes switching the role of these vesicles in metastatic melanoma Introduction: Malignant progression results from a Adrián Varela-Vázqueaa, María D. Mayán Santosb, Amanda Guitián- c d e dynamic crosstalk between stromal and cancer cells. Caamaño , Alejandro Castro-Iglesias , Tamara Camino Martínez , Susana B. Bravo-Lópezf, María Pardog, Teresa Calleja-Chucláh and Eduardo Fonsecac Recent data suggest that this crosstalk is mediated by aCellCOM research group. Instituto de Investigación Biomédica de A Coruña exosomes, nanovesicles secreted by various cell types (INIBIC). Servizo Galego de Saúde (SERGAS), A Coruña, Spain; bTranslational which allow the transfer of proteins, and nucleic acids Research in Cell Communication and Signalling (CellCOM). Instituto de Investigación Biomédica de A Coruña (INIBIC). Xubias de Arriba, 84 15006 between cells. We investigated the potential role of A Coruña, Spain., A Coruña, Spain; cCellCOM research group. Instituto de omental fat exosomes in gastric cancer peritoneal Investigación Biomédica de A Coruña (INIBIC). Servizo Galego de Saúde d metastasis. (SERGAS), A Coruña, Spain; Translational Research in Cell Communication and Signalling (CellCOM). Instituto de Investigación Biomédica de A Coruña Methods: Omental fat exosomes were produced from (INIBIC), A Coruña, Spain; eGrupo Obesidómica.Instituto de Investigación fresh human omental fat specimens. Proliferation, Sanitaria de Santiago de Compostela (IDIS). Complejo Hospitalario Universitario de Santiago. Travesía da Choupana s/n. 15706, Santiago de migration, invasion and chemoresistance were used to Compostela, Spain, Santiago de Compostela, Spain; fProteomics laboratory. evaluate the phenotypic behaviour of omental-exo- Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS), Complexo Hospitalario Universitario de Santiago de Compostela (CHUS), A somes treated gastric cancer cells. Using a comprehen- Coruña, Spain; gGrupo Obesidómica. Instituto de Investigación Sanitaria de sive cytokine array, we identified the proteome of Santiago de Compostela (IDIS). Complejo Hospitalario Universitario de Santiago. Travesía da Choupana s/n. 15706, Santiago de Compostela, Spain, omental-exosomes. Exosomal miRNAs were profiled Santiago de Compostela, Spain; hServicio de Farmacia Hospitalaria. CH- using NanoString technology. A xenograft model was Universitario A Coruña (XXIAC). Servizo Galego de Saúde (SERGAS), JOURNAL OF EXTRACELLULAR VESICLES 15

Universidade da Coruña. Xubias de Arriba, 84 15006 A Coruña, Spain., A vectors restored Cx43 membrane localization, raised Coruña, Spain GJIC and increased Cx43 in the EVs. EVs isolated Introduction: Connexin43 (Cx43), a transmembrane from BRAF-mutant melanoma cells overexpressing protein involved in cell communication and signalling, Cx43 only contains the non-SUMOylated Cx43. has been described as a tumour suppressor factor in When different melanoma cell lines were exposed to melanoma, however its role in disease progression exosomes containing Cx43, these EVs significantly remains under debate. Extracellular vesicles (EVs) decreased cell proliferation and blocked colonies released by melanoma cells provide signals and “edu- growth. The effect of exosomal Cx43 was compared cate” distant cells. The presence of Cx43 in EVs pro- to the overexpression of the protein. The presence of vides these particles with an additional capacity to Cx43 in EVs significantly increased the sensitivity of exchange small molecules such as RNAs, metabolites BRAF-mutant metastatic melanoma to drugs such as or ions with target cells via gap junction channels BRAF/MEK inhibitors. The RNA and proteomic com- (GJs).In this study, we have investigated the role of ponent identified by RNA-Seq and MS revealed that exosomal Cx43 in metastatic melanoma. exosomal Cx43 through its scaffolding function could Methods: Protein levels and activity were studied by be involved in the recruitment of proteins and small western-blot, immunofluorescence, colony formation RNAs to the EVs switching the messages and therefore and proliferation and migration assays. GJIC by the role of these EVs in melanoma. Scrape loading. EVs were isolated by ultracentrifuga- Summary/Conclusion: Our results indicate that exo- tion and analysed using the NanoSight and electron somal particles containing Cx43 are potent vehicles to microscopy. Their content was analysed by mass spec- combat metastatic melanoma. Further understanding trometry (MS) and by RNA-seq. of the role of Cx43 in EVs will have implications for Results: Low levels and SUMOylated Cx43 in BRAF- the development of new therapeutic strategies. For mutant human melanoma cells was associated with instance, we demonstrated their ability as drug carriers cytoplasmic distribution and low incidence of dye cou- to combat metastasic melanoma when these vesicles pling (GJIC). Ectopic Cx43 gene expression using contain Cx43. 16 ISEV2019 ABSTRACT BOOK

Symposium Session 4: EV Biogenesis I Chairs: Nobuyoshi Kosaka; Clotilde Théry Location: Level B1, Hall A 11:00–12:30

OT04.01 observed increased secretion of CD63+ but not CD9 + EVs. Summary/Conclusion: Our results demonstrate that small EVs can form both at the PM and inside multi- Linking the trafficking of CD63 and CD9 to their secretion mechanisms into extracellular vesicles vesicular endosomes. Our tools can be used to deter- a b a Mathilde Mathieu , José Ignacio Valenzuela , Mathieu Maurin , Mabel mine the respective effects of drugs and gene silencing Jouvea, Nathalie Nevoa, Gaëlle Boncompaina, Franck Perezb and Clotilde Theryc on secretion of each of these EVs aInstitut Curie, INSERM U932, Paris, France; bInstitut Curie, umr144, Paris, 3 France; Institue Curie, Paris, France OT04.02 Introduction: A major challenge in the study of extracellular vesicles is to characterize and separate the Interdependency of the multiple endosomal sorting mechanisms different extracellular vesicle (EV) subtypes of a differ- influencing exosome biogenesis Roberta Palmullia, Guillaume van Nielb, Frederik Verweijb, Xavier ent origin. Indeed, small EVs from the plasma mem- Heilingensteina, Eric Rubinsteinc and Graça Raposoa brane or from endosomes cannot be separated with the a Institut Curie, PSL Research University, CNRS UMR144, Paris, France; classical EV isolation methods. Moreover, even if some bCPN, Centre for Psychiatry and Neuroscience, Hôpital Saint-Anne, c of their molecular mechanisms of secretion are known, Universite´ Descartes, INSERM U894, Paris, France; Inserm U935 (ex. U1004) – Paul Brousse Hospital – André Lwoff Institute, Villejuif, France it is challenging to find specific mechanisms for one particular subtype (see perspective article: Mathieu et Introduction: Exosomes are generated as intraluminal al. Nat cell Biol 2019, in press). Understanding how vesicles (ILVs) in the multivesicular endosome (MVE). markers of subtypes of EVs are directed to similar or In the endosomal system, protein cargoes either are different EVs could help to differentiate them, even- sequestered to ILVs by inward budding or exit the tually to describe their specific functions. At least two system by outward budding. Sorting to ILVs is different populations of small EVs were previously mediated by various machineries, whose interdepen- described, one carrying the three tetraspanins CD63, dency is poorly understood, and is likely counterba- CD9 and CD81, and one with CD9 only (Kowal et al. lanced by recycling mechanisms that retrieve protein PNAS 2016). from MVEs. We have taken profit of the particular role Methods: We chose to study in HeLa cells the traffick- of CD63 in the balance between ESCRT-dependent and ing of CD63 and CD9 and its link with their secretion -independent biogenesis of ILVs and in the sorting of in EVs, using the RUSH system to synchronize and ApoE in melanoma cells to elucidate the interdepen- follow their post-Golgi trafficking (Boncompain et al. dency of different sorting mechanisms influencing exo- Nat Methods 2012). We used the RUSH system to some composition. perform live-cell imaging, electron microscopy, immu- Methods: After siRNA depletion of reported key actors nofluorescence and flow cytometry analyses at different of exosome production, EVs released by melanoma cells steps of trafficking, and to analyse EVs secreted after a were isolated by differential ultracentrifugation and floa- specific time of trafficking. tation on density gradient and characterized using bio- Results: Despite their presence in the same EVs, CD63 chemistry and electron microscopy. ILV biogenesis and and CD9 do not traffic to the same final compartments. sorting of specific cargoes throughout the endosomal While CD63 is endosomal, CD9 is located on the system was assessed by immunofluorescence or electron plasma membrane. We showed that CD9 could be microscopy after high-pressure freezing. found transiently with CD63 in intracellular compart- Results: Our data show that melanoma cells secrete ments before reaching the plasma membrane (PM), subpopulations of exosomes with different density while CD63 goes to the PM before being internalized. and composition. Investigation of known key regula- By forcing stable expression of CD63 at the PM, or tors of in- or outward budding in MVEs differently impairing post-Golgi and endosomal trafficking, we affected exosome subpopulations. In particular, CD63 JOURNAL OF EXTRACELLULAR VESICLES 17 modulates ApoE secretion on exosomes and its cellular bodies (MVBs) in cells allowing visualization of traf- localization, suggesting that CD63 is a master regulator ficking to the leading edge of migrating cells and of cargo trafficking in the endosomal system. uptake of external exosome deposits. Summary/Conclusion: Our data highlight that exo- Summary/Conclusion: Using pHLuorin_M153R- somes biogenesis is not only dependent on ILV bud- CD63 construct, we demonstrate superior visualization ding but also on a global regulation of endosomal of exosome secretion in multiple contexts and identify homeostasis. Our study provides a better perception a role for exosomes in promoting leader-follower beha- of the interconnections existing between sorting of viour in collective migration. By incorporating a cargoes to ILVs and their retrieval from the endosomal further non-pH-sensitive red fluorescent tag, this system. This broader view is crucial to understand the reporter allows visualization of the entire exosome life- precise roles of reported regulators of exosomes bio- cycle, including MVB trafficking, exosome secretion, genesis that are broadly used by the community. exosome uptake and endosome acidification. This new reporter will be a useful tool for understanding OT04.03 both autocrine and paracrine roles of exosomes. OT04.04 A bright, versatile live cell reporter of exosome secretion and uptake Bong Hwan Sunga and Alissa Weaverb aVanderbilt University, Nashville, USA; bDepartment of Cell and An explanation for “PS-negative” extracellular vesicles: endogenous Developmental Biology, Vanderbilt University School of Medicine, annexin-a5 from the cytosol cover externalized phosphatidylserines Nashville, USA on plasma membranes Anis Khiat, Dominique Charue, Sihem Sadoudi, Sylvain Le Jeune, Marie Lê- Introduction: Small extracellular vesicles (EVs) called Hoang, Chantal Boulanger, Olivier P. Blanc-brude exosomes affect a variety of autocrine and paracrine INSERM ‘ParCC’ Paris-Cariovascular Research Center, Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, and Université cellular phenotypes. Understanding the function of Sorbonne, Paris, France exosomes in these processes requires a variety of tools. We previously constructed a live-cell reporter, Introduction: Stressed cells shed extracellular vesicles pHLuorin-CD63 that allowed dynamic monitoring of (EVs) thought to bear externalized phosphatidylserine exosome secretion in migrating and spreading cells. (PS) at their surface and promote inflammation, coa- However, there were some caveats to its use, including gulation and tissue injury. Conversely, endogenous relatively low fluorescent expression in cells and the cytosolic annexins, such as annexin-A5, orchestrate inability to make cell lines that stably express the vesicle trafficking and membrane repair within multi- protein. ple cell types, via Ca2+-dependent binding to intracel- Methods: By incorporating a stabilizing mutation in lular PS. We hypothesized that endogenous annexin- the pHLuorin moiety, M153R, pHLuorin-CD63 now A5 binds to PS during vesiculation and gets externa- exhibits higher and stable expression in cells and super- lized with PS at the surface of EVs. ior monitoring of exosome secretion. Cancer cells sta- Methods: We purified healthy plasma and red blood bly expressing pHLuorin_M153R-CD63 were imaged cells and induced Ca2+-mediated vesiculation. We using a variety of microscopy techniques including a assessed annexin-A5 and EV distribution in superna- confocal and wide-field microscopy and a correlative tants by Western blots, FACS, ELISA, cryo-TEM. light-electron microscopy. Results: (1) About 20% cytosolic annexin-A5 leaked Results: pHLuorin_M153R-CD63 was exclusively out during vesiculation, but cytoskeletal proteins were detected in exosome-enriched small EV preparations. not released. (2) We separated supernatant EVs from Live-cell imaging revealed pHLuorin_M153R-CD63- “free” proteins by size-exclusion chromatography and positive puncta left behind migrating cells suggesting quantified EV-bound vs. “free” annexin-A5. All the deposition consists of exosomes. Those puncta and annexin-A5 remained bound to EVs. Other cytosolic trails were not only positive for other exosome markers proteins (haemoglobin) bound to EVs only partly. such as Alix and TSG101 but also correspond to small FACS with anti-annexin-A5 antibodies revealed the EVs observed by a scanning electron microscope. In presence of annexin-A5 at the EV surface. (3) We addition, follower cells exhibited pathfinding behaviour measured EV-bound and “free” annexin-A5 in plasma, over pHLuorin_M153R-CD63 deposits. Incorporation vs PS-, PS+, CD235a+ and annexin-A5+ EVs, and of mScarlet, a non-pH-sensitive red fluorescent tag, to made similar observations. Our study suggests that pHLuorin_M153R-CD63 further improves the ability endogenous annexin-A5 can cover externalized PSs to track trafficking and secretion of multivesicular on EVs in the presence of Ca2+. 18 ISEV2019 ABSTRACT BOOK

Summary/Conclusion: This new mechanism of PS- Results: miRNAs which suppressed the secretion of neutralization may explain previous reports of appar- EVs from melanoma cells were identified after the ently “PS-negative” EVs. Conventional detection of screening of nearly 2000 miRNAs. To understand the EVs with EXOgenous fluorescent annexin-A5 (FACS) molecular mechanisms mediated by these miRNAs, the may thus depend on PS not being engaged by endo- target genes of these miRNAs were identified and eval- genous annexin-A5 prior to detection. The physio- uated for their contribution to EV production in cancer pathological relevance of endogenous PS cells. Indeed, attenuation of these target genes declined neutralization may complement enzyme- and ATP- the secretion of EVs from melanoma cells, suggesting mediated internalization of PS in healthy cells. PS the contribution of these genes in EV production/ neutralization may become critical when internaliza- secretion. Furthermore, the expression of these genes tion mechanisms are overwhelmed, and serve to was higher in melanoma tumour tissues compared with restrain PS-mediated reactions and enforce anti- that in normal tissues. inflammatory and anti-thrombotic control when the Summary/Conclusion: These findings suggest that the integrity of a few cells only is compromised. On the miRNAs and their target genes were involved in EV other hand, dysfunctional annexin-A5 or calcium production/secretion, resulting in the promotion of metabolism may contribute to the release of pro- cancer progression. inflammatory and pro-thrombotic PS+ EVs. Funding: Funded by Fondation pour la Recherche Médicale and Sorbonne University. OT04.06

Distinct mechanisms of microRNA sorting into cancer cell-derived OT04.05 extracellular vesicle subtypes Morayma Temoche-Diaza, Matthew Shurtleffa, Ryan Nottinghamb, Jun Yaob, Alan Lambowitzb, Randy Schekmana

aUniversity of California, Berkeley, Berkeley, USA; bUniversity of Texas, Identification of EV secretion-associated gene involved in melanoma Austin, Austin, USA progression by microRNA-based screening Nobuyoshi Kosakaa, Fumihiko Urabeb, Tomofumi Yamamotoc, Yurika Sawad and Takahiro Ochiyaa Introduction: Extracellular vesicles (EVs) encompass a aDepartment of Molecular and Cellular Medicine, Institute of Medical variety of vesicles secreted to the extracellular space. b Science, Tokyo Medical University, Shinjyuku-ku, Japan; Division of EVs have been implicated in promoting tumour metas- Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan; cDepartment of Molecular and Cellular Medicine, tasis but the molecular compositions of tumour- Institute of Medical Science, Tokyo Medical University, Tokyo, Japan; derived EV sub-types and the mechanisms by which dDepartment of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan molecules are sorted into EVs remain mostly unknown. As such dissecting different EV sub-populations and Introduction:It has been shown that extracellular vesi- analysing the molecular mechanisms behind active cles (EVs) derived from cancer cells dictate their sur- cargo sorting is needed. rounding microenvironmental cells or distant cells in Methods:Thehighlymetastaticbreastcancercellline, the future metastatic organs for the benefit of cancer MDA-MB-231, was used as the model cell line for this cells. Thus, revealing the molecular mechanisms under- study. Iodixanol linear gradient allowed for the separation lying the production of EVs would prove to be a valu- of EV sub-populations. miRNA profiling and TGIRT- able contribution for establishing EV-targeted therapy sequencing was used to study the miRNA content of against cancer. However, the precise mechanism of EV the distinct EV sub-populations. Cell fractionation and production, especially in cancer cells, remains unclear. cell-free miRNA packaging reconstitutions, coupled with Here, we established a microRNA-based screening sys- in vivo confirmation, in cultured cells, were used to study tem to identify the molecules involved in EV produc- the molecular mechanisms of miRNA sorting. tion from melanoma cells. Results:WefoundthatatleasttwodistinctEVsub- Methods: Melanoma cell lines, A375 cells, were used in populations are released by MDA-MB-231 cells. Their this study. Combined with the ultra-sensitive EV detec- differential biochemical properties suggest different sub- tion method (Yoshioka), ExoScreen, we have screened cellular origins (endosomes vs. direct budding from the nearly 2000 miRNAs in melanoma cells. To confirm plasma membrane). Moreover, they are governed by dis- the results of ExoScreen, we employed the nanoparticle tinct mechanisms of miRNA sorting (active vs. passive). tracking analysis. Target genes of miRNAs were iden- By using biochemical and genetic tools, we found that the tified by the combination of gene expression analysis Lupus La protein is responsible for mir122 sorting into and target prediction bioinformatics. EVs in vitro and in vivo.Moreover,in vitro studies JOURNAL OF EXTRACELLULAR VESICLES 19 showed that the Lupus La protein interacts with mir122 231 cells. Their differential sub-cellular origin is with very high affinity. Finally, we uncovered the mir122 coupled with two distinct mechanisms of miRNA sort- motifs required for mir122-La high affinity interaction, ing. The Lupus La protein is responsible for the active and therefore mir122 sorting into EVs. sorting of mir122 into EVs in vitro and in vivo. Summary/Conclusion: Two EV sub-populations with Funding: Howard Hughes Medical Institute (HHMI). distinct sub-cellular origins, are released by MDA-MB- 20 ISEV2019 ABSTRACT BOOK

Oral with Poster Session 1 Chairs: Uta Erdbrügger; Kenneth Witwer Location: Level B1, Hall B 13:30–15:00

cancer cell lines, and induces the shedding of LO while OWP1.01=PS10.10 inhibiting the shedding of exosomes. Furthermore, miR-1227 induces the migration of poorly migratory miR-1227 alters extracellular vesicle shedding cancer cells and increases the expression of tumour Andrew R. China, Minyung Kima, Valentina R. Minciacchib, Tatyana supportive cytokines. Vagnera, Javier Mariscala, Cristiana Spinellia, Mandana Zandiana, Paolo Gandellinic, Nadia Zaffaronic, Shivani Sharmad, Sungyong Youa and Summary/Conclusion: Together these data hint that Dolores Di Vizioa miR-1227 may promote prostate cancer progression aCedars Sinai Medical Center, West Hollywood, USA; bCedars Sinai Medical through several mechanisms including alteration of Center, Frankfurt, Germany; cFondazione IRCCS Istituto Nazionale Tumori, EV shedding. Milan, USA; dUniversity of California, Los Angeles, Los Angeles, USA Funding: 2017–2022 R01CA218526. Introduction: Extracellular vesicles (EVs) play a key 2018–2020 Chesapeake Urology Associates Sanford role in cancer development and metastasis by influen- J. Siegel, MD Prostate Cancer Research Scholarship cing the behaviour of the primary tumour and by 2018–2020 Luke Wu-Jei Chang Discovery Fund aiding the establishment of a pre-metastatic niche in 2016–2019 PI DoD PCRP Award PC150836 distant organs. This process is due to the EV-mediated functional transfer of biologically active molecules including microRNA (miRNA). miR-1227 is a poorly OWP1.02=PF11.14 characterized miRNA that is enriched in EV secreted by prostate cancer (PC) cells in comparison to non- MSC exosome works through a multi-faceted mechanism of action in tumorigenic prostate epithelial cells. However, the role joint repair of miR-1227 in cancer is poorly understood. Our Shipin Zhanga, Yedan Wangb, Francis Keng Lin Wongc, Ming Wangb, Ruenn Chai Laid, James Hoi Po Huib, Sai Kiang Limd and Wei Seong Toha objective is to determine the role of miR-1227 in PC. a Methods: RNA sequencing from miR-1227 stably Faculty of Dentistry, National University of Singapore, Singapore, Singapore; bDepartment of Orthopaedic Surgery, Yong Loo Lin School of Medicine, expressing PC cells, RISCTRAP Immunoprecipitation National University of Singapore, Singapore, Singapore; cDepartment of of miR-1227 bound mRNA and five different in silico Orthopaedic Surgery, Sengkang General Hospital, Singhealth, Singapore, Singapore; dInstitute of Medical Biology, Agency for Science, Technology miRNA target prediction methods were used to iden- and Research, Singapore, Singapore tify putative miR-1227 targets. Exosomes and large oncosomes (LO) were isolated by differential ultracen- Introduction: MSC exosome is increasingly accepted trifugation followed by density gradient purification. as the principal agent that underpins the therapeutic Atomic force microscopy and TRPS were used to efficacy of mesenchymal stem cell (MSC) in tissue quantify exosomes and LO secreted by PC cells stably repair. Here, we aim to elucidate the mechanism of expressing miR-1227 or vector control. action (MoA) of MSC exosome in immunocompetent Results: A comparative analysis between different EV rat models of osteochondral defect and osteoarthri- subtypes indicates that miR-1227 is enriched in LO, a tis (OA). class of EV that are secreted by highly invasive and Methods: Exosomes were purified from conditioned metastatic amoeboid-migrating cells. LO carry more medium of human MSCs by size fractionation. RNA than the more widely studied exosomes indicat- Osteochondral defect creation or anterior cruciate liga- ing that LO may be a more robust source of EV- ment transection to induce OA were performed in 72 encapsulated miRNA. Gene ontology analysis from adult rats. Thereafter, weekly 100-µl intra-articular miR-1227 targets identified by RNA sequencing from injections of 100-µg exosome or PBS vehicle were miR-1227 stably expressing PC cells, RISCTRAP given. Analysis included weight distribution, histology, Immunoprecipitation of miR-1227 bound mRNA, and immunohistochemistry and cytokine assay. Cellular in silico miRNA target prediction highlighted several assays using chondrocytes were performed to deter- genes related to EV secretion. miR-1227 alters the mine the exosome-activated cellular processes and sig- localization of exosome and LO markers in multiple nalling pathways. JOURNAL OF EXTRACELLULAR VESICLES 21

Results: We observed that exosome-mediated repair of plasma concentrations of EVs labelled with antibodies osteochondral defects was characterized by increased for activated platelets (CD61, CD62p; PEVs), endothe- cellular infiltration and proliferation, enhanced matrix lial cells (CD146; EEVs) and red blood cells (CD235a; synthesis, together with a regenerative M2 macrophage RBC-EVs). Processing of 1,224 flow cytometry data phenotype and a reduction in pro-inflammatory cyto- files was performed using in-house developed, auto- kines IL-1β and TNF-α. In OA joints, MSC exosome mated software (MATLAB R2018a), enabling flow mediated an early suppression of pain and degenera- rate stabilization, diameter and refractive index deter- tion with reduced inflammation, followed by sustained mination, MESF calibration, fluorescent gate determi- proliferation and matrix restoration that led to cartilage nation and statistics reporting. and subchondral bone regeneration. Using chondro- Results: Between AMI patients and controls, PEV con- cyte cultures, we could attribute some of these cellular centrations in plasma were comparable (p = ns), EEV activities during exosome-mediated joint repair to exo- concentrations increased (p < 0.0001) and RBC-EV somal CD73-mediated adenosine activation of AKT concentrations decreased (p < 0.0001). Antiplatelet and ERK signalling. These effects were partially abro- drug ticagrelor decreased concentrations of PEVs gated by wortmannin or U0126, which inhibited AKT (p = 0.03), compared to less potent clopidogrel but and ERK phosphorylation, respectively. The role of did not affect EEVs and RBC-EVs. In turn, concentra- exosomal CD73 was confirmed using CD73 inhibitor tions of EEVs, but not PEVs and RBC-EVs, positively and theophylline that showed inhibition of exosome- correlated with the dose of atorvastatin (p < 0.001). The induced AKT and ERK phosphorylation. antioxidative β-blocker carvedilol increased concentra- Summary/Conclusion: Our observations suggest that tions of RBC-EVs, compared to nebivolol (p = 0.05) MSC exosome works through a multi-faceted MoA but did not affect PEVs and EEVs. that involved multiple cellular processes to restore Summary/Conclusion: Flow cytometry and automated joint homeostasis and promote regeneration. data processing were used to find biomarkers for AMI Funding:NationalMedicalResearchCouncilSingapore based on EVs in plasma. During treatment, ticagrelor (NMRC/CNIG/1168/2017 and NMRC/CIRG/1480/2017). decreased PEV concentrations, atorvastatin increased EEV concentrations and carvedilol increased RBC-EV OWP1.03=PS03.11 concentrations, suggesting that EVs might be used to monitor AMI treatment. AMI patients differed from controls regarding EEV and RBC-EV concentrations, Identification of extracellular vesicles as biomarkers for myocardial but not PEVs, likely because blood was collected 24 h infraction by flow cytometry and automated data processing Aleksandra Gaseckaa, Edwin van der Polb, Frank Coumansc, Kinga Plutad, after the start of antiplatelet therapy. In follow-up Grzegorz Opolskid, Krzysztof J. Filipiake, Rienk Nieuwlandc studies, it is crucial to collect blood prior to treatment. a1st Chair and Department of Cardiology, Medical University of Warsaw, Warsaw, Poland; bAmsterdam UMC, University of Amsterdam, Department of Biomedical Engineering and Physics, Amsterdam, Netherlands; OWP1.04=PF11.15 cAmsterdam UMC, University of Amsterdam, Laboratory of Experimental Clinical Chemistry, Amsterdam, Netherlands; d1st Chair and Department of Cardiology, Medical University of Warsaw, Warsaw, Poland; e1st Chair and Exosome mediated enhancement of cellular therapy in acute Department of Cardiology, Medical University of Warsaw, Warsaw, Poland myelogenous leukemia (AML) Theo Borgovana, Peter Quesenberryb, Mike Deltatto; Sicheng Wenc, Mark Introduction: Acute myocardial infarction (AMI) is a Doonerb major cause of death. To diagnose AMI, measuring aBrown University Department of Hematology Oncology; Rhode Island troponin concentration is the gold standard. Since tro- Hospital, Pawtucket, USA; bBrown University Department of Hematology Oncology; Rhode Island Hospital, providence, USA; cBrown University/ ponin is unspecific for AMI, novel biomarkers for AMI Rhode Island Hospital, Providence, USA are urgently needed. After the onset of AMI, platelets, endothelial cells and blood cells release specific extra- Introduction: Of the AML patients able to tolerate cellular vesicles (EVs). Our aim is to identify these EVs curative therapy with chemotherapy and stem cell as biomarkers for AMI diagnosis and treatment transplant many are challenged by treatment related monitoring. toxicities as well as graft vs. host disease. There is Methods: The study was approved by the medical novel work exploring the utility of haploidentical cel- ethics committee. Venous blood was collected 24 h, lular therapy infusion in order to incite purposeful 72 h and 6 months after AMI from fasting patients recipient immune response and subsequent cytokine (n = 60, 64.5 ± 10.8 years, 68% male) and healthy storm to treat refractory AML. Our group has demon- controls (n = 30, 57.7 ± 6.6 years, 62% male). Flow strated the healing potential of bone marrow derived cytometry (Apogee A60 Micro) was used to determine mesenchymal stem cell extracellular vesicles (MSC- 22 ISEV2019 ABSTRACT BOOK

EVs) across multiple disease states, most recently lung development via the administration of extracellular demonstrating the pro-apoptoic signalling imparted vesicles (EVs) derived from amniotic fluid stem cells by these nanoparticles on nascent leukemic cells in (AFSCs) in rat models of PH. Moreover, we report the vivo; as well as the potentiating effects of MSC-EVs microRNAs present in AFSC-EVs that are responsible for when used as an adjunct to standard cytarabine che- these beneficial effects. motherapy. We have also shown the protective role of Methods: AFSC-EVs were isolated by ultracentrifuga- hMSC EV on radiated BM and stem cell recovery. tion from conditioned medium (CM) of c-Kit+ rat Methods: Kasumi AML cells lines were seeded with AFSC that were grown in exosome-depleted FBS for MSC-derived EVs. Vesicles were isolated using an 18h. AFSC-EVs were assessed for size (nanoparticle established differential centrifugation technique, and tracking analysis), morphology (TEM), and expression were co-cultured with Kasumi cells for various time of CD63, Hsp70, Flo-1 and TSG101 (Western). points. To study cellular viability, we used a fluores- Ex vivo: Pregnant dams were gavaged nitrofen at E9.5 cence-based method for quantifying viable cells. to induce foetal PH. At E14.5, foetal lungs were har- We also explored various modes of death EVs may vested, and incubated with culture medium alone, illicit via a tri-dye Abcam assay designed to simulta- AFSC-CM, or AFSC-EVs. Foetal lungs from untreated neously monitor apoptotic, necrotic and healthy cells. dams served as control. Lungs were compared for Both assays were used to measure viability and apop- terminal bud density and surface area at 72 h, by two tosis in similar experiments employing cytarabine independent investigators. Results: AML cell Proliferation Decreased after 1– In vitro: Foetal rat lung organoids were generated with 6 days of co-culture with hMSC-derived EVs. epithelial cells from normal and hypoplastic lungs. Apoptosis is the primary mode of death induced. Organoids were cultured for 10 days in either medium AML cell proliferation decreased synergistic after 1– alone or medium supplemented with AFSC-EVs. Lung 6 days of co-culture with hMSC-derived EVs ± organoids from untreated normal pups served as con- Cytarabine. trol. Organoids were assessed for proliferation (Ki67) Summary/Conclusion: MSCs inhibits the proliferation and markers of epithelial cell differentiation via of the AML cell line in vitro and work synergistically immunofluorescence. with cytarabine chemotherapy to promote apoptotic RNA-sequencing: RNA was isolated using SeraMir, death in AML cell lines. Our prior work has shown constructed into libraries (CleanTag Small RNA) and that MSC-EVs can abate the effects of toxic chemo/ sequenced on NextSeq High Output single-end sequencing radiation and serve to protect stem cell allowing for run. quicker recover in cell blood counts. Results: Administration of AFSC-EVs increased term- Based on the innate ability of MSC-EV to directly inal bud density and surface area of lung explants back alter the cellular machinery of abnormal leukemic cell to control levels and promoted lung epithelial cell and of nascent immune cells our corollary hypothesis is differentiation in lung organoids (increased SPC and that BM-derived MSC-EVs may serve as suitable alter- CC10 expression). AFSC-EVs contain 901 microRNAs, native to conditioning chemo/radiation in the AML some of which are crucial for foetal lung development, setting and will enhance the effects seen by cellular such as miR17 ~ 92 cluster. therapy infusion. Summary/Conclusion: Administration of AFSC-EVs Funding: t32. rescues impaired foetal lung development in experimental models of PH. AFSC-EV regenerative ability is OWP1.05=PF12.09 exerted via the release of miRNAs some of which regulate genes involved in foetal lung development. AFSC-EVs represent a promising therapeutic strategy Extracellular vesicles derived from amniotic fluid stem cells rescue for PH in foetuses. impaired foetal lung development via the release of microRNAs Funding: CIHR-SickKids Foundation. Lina Antounians, Vincenzo Catania, Benjamin Liu, Areti Tzanetakis, Louise Montalva and Augusto Zani

The Hospital for Sick Children, Toronto, Canada OWP1.06=PS01.11 Introduction:Incompletelungdevelopment,alsoknown as pulmonary hypoplasia (PH), is a recognized cause of neonatal death. To date, there is no effective treatment that Extracellular vesicles from Fat-laden hypoxic hepatocytes activates pro-fibrogenic signals in Hepatic Stellate Cells promotes foetal lung growth and maturation. Herein, we Alejandra Hernandeza, Yana Gengb, Daniel Cabrerac, Nancy Solisd, Han describe a stem cell-based approach that enhances foetal Moshagee and Marco Arresed JOURNAL OF EXTRACELLULAR VESICLES 23 aPontificia Universidad Católica de Chile; University Medical Center of Groningen, Groningen, Netherlands; bUMCG, Groningen, Netherlands; OWP1.07=PS08.07 cPontificia Universidad Católica de Chile/Universidad Bernardo O´Higgins, SANTIAGO, Chile; dPontificia Universidad Católica de Chile, Santiago, Chile; eUniversity Medical Center Groningen, Groningen, Netherlands Exploration of the surface modification of outer membrane vesicles Maximilian Richtera, Eleonora Diamantib, Anna Hirschb, Gregor Fuhrmannc Introduction/Background: Transition from isolated aHelmholtz-Institute for Pharmaceutical Research Saarland, Biogenic steatosis to non-alcoholic steatohepatitis is a key issue Nanotherapeutics, Saarbruecken, Germany; bHelmholtz-Institute for in non-alcoholic fatty liver disease (NAFLD). Recent Pharmaceutical Research Saarland, Drug Design and Optimization, Saarbruecken, Germany; 3Helmholtz-Institute for Pharmaceutical Research observations in patients with obstructive sleep apnoea Saarland, BION, Saarbruecken, Germany syndrome (OSAS), suggest that hypoxia may contribute to disease progression mainly through activation Introduction:Introducingbacteria-bindingsmallmole- of hypoxia inducible factor 1α (HIF-1α)-related path- cules to the surface of outer membrane vesicles (OMVs) ways. Release of extracellular vesicles (EV) by injured could greatly improve their potential for antimicrobial hepatocytes may be involved in NAFLD progression. drug delivery too difficult to treat bacteria. Among the Aim: to explore whether hypoxia modulates the release small number of studies on surface modification of of EV from free fatty acid (FFA)-exposed hepatocytes OMVs, very few deal with small molecules. The aim of and assess cellular crosstalk between hepatocytes and the present study is to evaluate different methods of intro- LX-2 cells (human hepatic stellate cell line). ducing bacteria specific targeting moieties to OMVs. We Methods: HepG2 cells were treated with FFAs (250 μM assessed the modification of surface proteins using N- palmitic acid + 500 μM oleic acid) and chemical hydroxysuccinimide (NHS) esters, well established for hypoxia (CH) was induced with Cobalt (II) Chloride, mammalian extracellular vesicles (EVs), cholesterol inser- which is an inducer of HIF-1α. Induction of CH was tion, mainly applied for liposomes, and the novel applica- confirmed by Western blot (WB) of HIF-1α. EV isolation of diazo-transfer followed by click-chemistry. tion and quantification was performed by ultracentri- Methods:OMVswereobtainedfrommodelmyxo- fugation and nanoparticle tracking analysis bacteria by differential ultracentrifugation (UC) fol- respectively. EV characterization was performed by lowed by size-exclusion chromatography (SEC). For electron microscopy and WB of CD-81 marker. LX-2 cholesterol insertion and NHS ester-modification, cells were treated with 15 μg/ml of EV from hepato- purified OMVs were incubated with either cholesteryl cytes obtained from different groups and markers of PEG 2,000 FITC or sulfo cyanine7 NHS ester. For pro-fibrogenic signalling were determined by quantita- diazo transfer the pellet after UC was incubated with tive PCR (qPCR), WB and immunofluorescence (IF). adiazotransferagentandtheOMVssubsequently Results: FFA and CH-treatment of HepG2 cells conjugated with DBCO-AF594. Unincorporated dye increased gene expression of IL-1β and TGF-β1in was removed by SEC. Liposomes were composed of HepG2 cells and increased the release of EV compared DMPC and DPPC in 2:3 molar ratio. Results repre- to non-treated HepG2 cells. Treatment of LX-2 cells sent correlated fluorescence intensity and particle with EV from FFA-treated hypoxic HepG2 cells number. increased gene expression of TGF-β1, CTGF, α-SMA Results: Treatment with sulfo cyanine7 NHS ester led and Collagen1A1 compared to LX-2 cells treated with to the modification with 547 ± 163 molecules per EV from non-treated hepatocytes or LX-2 cells exposed OMVs, compared to 18 ± 1 for the control using to EV-free supernatant from FFA-treated hypoxic sulfo cyanine7 acid. Cholesterol insertion introduced HepG2 cells. Moreover, EV from FFA-treated hypoxic 4 ± 1 molecules per OMV, compared to 101 ± 23 for HepG2 cells increased Collagen1A1 and α-SMA pro- liposomes. First results for the diazo-transfer showed tein levels. 71 dye-molecules per OMV, with 32 for the control. Summary/Conclusion: CH promotes EV release from Summary/Conclusion: Of the three methods, NHS HepG2 cells. EV from hypoxic FFA-treated HepG2 cells ester-modification displayed the highest efficiency, evoke pro-fibrotic responses in LX-2 cells. Further geno- similar to published results for mammalian EVs. In mic and proteomic characterization of EV released by comparison, diazo transfer only yielded ~13% of the steatotic cells under hypoxia are necessary to further dye-molecules per particle. However, there are still delineate their role in the crosstalk between hepatocytes many parameters to be optimized for this method, and stellate cells in the setting of NAFLD and OSAS. including OMV concentration and incubation period. Funding: FONDECYT 1150327–1150311. Cholesterol insertion was unsuccessful for OMVs, 24 ISEV2019 ABSTRACT BOOK probably owing to their membrane structure. In this OWP1.09=LBT01.01 study, we aim to get important insights into the modification of OMVs for bacterial targeting and EV-surface engineering in general. Coagulation influences properties of extracellular vesicles isolated Funding: This project was funded by Studienstiftung from autologous blood derived products Andrea De Lunaa, Alexander Otahala, Olga Kutenb, Zsombor Laczac and des Deutschen Volkes and Bundesministerium fuer Stefan Nehrera Bildung und Forschung. aDanube University Krems, Krems, Austria; bOrthoSera GmbH, Krems, Austria; cOrthosera GmbH, Krems, Austria OWP1.08=LBT02.03 Introduction: Platelet rich plasma (PRP) is the most commonly used blood derivative in clinics due to its Isolation of neuron-specific extracellular vesicles high concentration of platelets and perceived high a b c b Dmitry Ter-Ovanesyan , Maia Kipman , Emma Kowal , Ju Hyun Lee , growth factor levels. Drawbacks of using PRP are dis- Wendy Trieub, Aviv Regevd, David Waltb and George Churchb crepancies among preparation protocols and the pre- aHarvard, Cambridge, USA; bWyss Institute, Boston, USA; cMIT, Cambridge, USA; dBroad Institute, Cambridge, USA sence of cells (platelets, leucocytes) which can evoke cellular processes (e.g. inflammation) when injected Introduction: Human biological fluids contain extra- into the host. One possibility is to isolate only the cellular vesicles (EVs) from different cell types. It active components of blood derivatives which may would be incredibly useful to be able to isolate EVs overcome this problem. In the current study, we that originated from specific cell types for diagnostic focused on extracellular vesicles (EVs) isolated from purposes as a way to gain molecular information two autologous blood derivatives, PRP and hyperacute (RNA, protein) from inaccessible cell types non- serum and investigated whether the clotting cascade invasively. influences EV properties. Methods: We have developed a general framework for Methods: EVs were isolated from citrate-anticoagu- identifying EV surface markers that can be used for lated PRP (CPRP) and hyperacute serum using differ- immuno-isolation of cell type specific EVs. As a proof ential ultracentrifugation followed by a size exclusion of principle, we have applied this framework to the chromatography. Particle concentration and size were isolation of neuron-derived EVs from human cere- determined by nanoparticle tracking analysis (NTA). brospinal fluid or plasma. In addition to the computa- Cryo-electronmicrosopy was performed to visualize tional analysis, we have developed an in-vitro system of isolated EVs. Expression of miRNAs transported human neurons differentiated from human induced within EVs as well as in their respective input material pluripotent (iPS) cells. We performed mass spectro- was analysed by qPCR. metry on EVs isolated from these neurons to identify Results: NTA revealed higher particle concentrations neuron-specific proteins. We also used this system to and bigger sized EVs within CPRP compared to hyper- develop a robust immune-isolation method for neuron acute serum. These findings were confirmed by cryo- EV markers. electronmicroscopy. Profound differences were Results: We have characterized the proteins present in detected regarding miRNA expression between the neuron exosomes by mass spectrometry and then used two blood derivatives. In total, 126 miRNAs were computational analysis of published gene expression identified which were expressed both in input material and proteomics data to come up with a list of candidate as well as in the corresponding EVs. The correlation neuron-specific EV markers. After developing methods between miRNAs in EVs and input material was higher for immuno-isolation of neuron EVs with these mar- in CPRP compared to hyperacute serum meaning that kers, we applied our methods to human cerebrospinal in hyperacute serum miRNAs were identified which fluid and plasma. were higher expressed in EVs than in the correspond- Summary/conclusion: We have developed a frame- ing input material. work for the isolation of cell type specific EVs through Summary/conclusion: EVs from autologous blood the combination of an experimental in vitro system and products represent a novel and cell-free regeneration computational analysis of gene expression and proteo- approach. We observed that the clotting cascade mics data. We have applied this framework to the (plasma versus serum) has an influence on concentra- isolation of neuron-specific EVs in human biological tion, size and miRNA expression patterns of EVs. fluids. We envision these methods being broadly These differences might have an impact on the biolo- applicable to the development of novel diagnostic bio- gical mode of action of blood-derived products used in markers for a variety of diseases. clinics. JOURNAL OF EXTRACELLULAR VESICLES 25

Funding: Financial support was received from the (but not in ectosomes) only upon LPS stimulus, and European Fund for Regional Development (EFRE) not in the EVs-free medium, suggesting that TG2 is a and the Science Fund of Lower Austria. miRNA cargo of exosomes during neuroinflammation. expression analysis was performed by TAmiRNA Summary/conclusion: TG2 is externalised through GmbH. Cryo-electronmicroscopy was conducted at astrocyte-derived exosomes upon neuroinflammatory the Core Facility of the Vienna Bio-Center. stimuli. Extracellular TG2 mediates the opening of L- type VOCCs in neurons and sets basal [Ca2+]i at higher levels, which could have a significant impact OWP1.10=LBF02.01 on neuronal activity in neuroinflammation. Funding: John Turland PhD bursary (NTU) and IBRO

Type-2 transglutaminase affects calcium homeostasis in neurons and travel fund. is released in association with astrocytes-derived exosomes Elisa Tonolia, Ilaria Pradab, Claudia Verderioc and Elisabetta Verderioa aNottingham Trent University, Nottingham, United Kingdom; bCNR OWP1.11=LBT01.02 Institute of Neuroscience, Milan, Italy., Milan, Italy; cCNR Institute of Neuroscience, Milan, Italy, Milan, Italy

Ev-avogadro project: towards a liposomal concentration standard for Introduction: Type-2 transglutaminase (TG2) has been extracellular vesicle research linked to calcium (Ca2+) dysregulation in conditions Gergo Bartaa, Diana Kitkaa, Andras Wachaa, Judith Mihalya, Attila Botaa, a a b c such as neurodegeneration. Recent evidences suggest Krisztina Nemeth , Pal Szabo , Jean-Luc Fraikin and Zoltan Varga that extracellular vesicles (EVs) contribute to the onset aResearch Centre for Natural Sciences HAS, Budapest, Hungary; bSpectradyne LLC, Torrance, USA; cResearch Centre for Natural Sciences, and progression of neurological diseases, and we have Hungarian Academy of Sciences, Budapest, Hungary recently shown that TG2 is a cargo of EVs in biological fluids (Furini et al., 2018). Here, we hypothesise that Introduction: There is an unmet need for standardiza- TG2 could be released by EVs, interact with neurons tion of concentration measurements in the field of and affect neuronal Ca2+ homeostasis. extracellular vesicles (EVs). Liposomes may serve an Methods: Primary hippocampal neurons were estab- ideal reference system for EVs, but the determination lished from E18 rat embryos. Extracellular TG2 was of the number concentration of liposomes from first modulated in neurons either by lipofectamine transfec- principles was not attempted so far. Inspired by the tion of a TG2-EGFP construct or by addition of pur- International Avogadro project, we aimed to determine ified TG2. Intracellular Ca2+ concentration ([Ca2+]i) the concentration of liposomes with well-defined size was assessed by live imaging in fura-2/AM-loaded neu- and composition via counting the number of phospho- rons. EVs were isolated from primary astrocytes (60 lipid molecules in these “nanospheres”. DIV) by serial centrifugation, characterised by western Methods: Liposomes composed of phosphocholine and blotting (flotillin-2 and alix) and nanoparticle tracking phosphoglycerol were prepared by the extrusion analysis (ZetaView). Experiments to assess TG2 influ- method. Wide-angle X-ray scattering (WAXS) was ence on exosomes-to-neural cells interactions, using a used to determine the area-per-lipid value. The size Renilla sensor based on miR-146a-5p-transfer, are still distribution of the liposomes was determined by ongoing. microfluidic resistive pulse sensing (MRPS) and Results: Increase of extracellular TG2 levels in neurons freeze-fracture combined TEM. Small-angle X-ray scat- induced an influx of extracellular Ca2+ ions, leading to tering (SAXS), differential scanning calorimetry (DSC) a significant raise in basal [Ca2+]i both in normal and infrared spectroscopy (IR) were used to prove the conditions (ΔF340/380 = 0.126 ± 0.014; N = 23; unilamellarity, the ideal miscibility of the lipids and the p <10–5) and with inhibited synaptic transmission ordered packing of the hydrocarbon chains of the (tetrodotoxin) (ΔF340/380 = 0.058 ± 0.005; N = 33; lipids, respectively. Concentration of the lipids was p <10–5). Nifedipine, a blocker of L-type voltage- determined by liquid chromatography–mass spectro- operated Ca2+ channels (VOCCs), partially prevented metry (LC-MS). TG2-dependent Ca2+ response (average inhibition Results: The prepared liposomes proved to be unila- 36%; N = 21; p <10–5), suggesting that Ca2+ influx mellar with narrow size distribution (83 nm avg.), as may occur through L-type VOCCs. To identify the obtained by MRPS and TEM. DSC and IR measure- source of extracellular TG2, we analysed EVs isolated ments confirmed that the phospholipid bilayer of these from rat primary astrocytes, previously reported to liposomes is in the liquid-ordered phase, hence the release TG2 into the matrix especially in inflammatory area-per-lipid of 0.41 nm2 was determined from conditions. TG2 was detected in astrocytic exosomes WAXS measurements. Using the concentration of 26 ISEV2019 ABSTRACT BOOK phospholipids from LC-MS measurements, the number Exosomes were incorporated into VSMCs, while the concentration of liposomes was determined (8E+13 internalization of SHR exosomes was significantly 1/mL). lower than WKY exosomes. Both WKY and SHR exo- Summary/conclusion: Liposomes containing saturated somes similarly stimulated proliferation, migration and phospholipids are in the liquid-ordered phase, which cytoskeletal changes such as formation of filopodia and can be utilized to determine the area-per-lipid using lamellipodia in VSMCs. Heparin, an inhibitor of exo- WAXS. This value, together with the independently some internalization, completely blocked the migration determined size, and lipid concentration can be used and proliferation. Protein expression of CD9 and to calculate the number concentration of liposomes. As CD63, an exosomal marker, was significantly higher the light scattering properties of liposomes matches in exosomes from WKY than SHR. The expression of that of EVs, liposome-based standards for optical mea- several microRNAs in SHR exosomes changed com- surements of EVs can be obtained with the presented pared with WKY exosomes. techniques. Summary/conclusion: These results suggest that Funding: This work was supported under grant num- plasma exosomes play physiological, but not patholo- bers PD 121326 and NVKP_16-1-2016-0007 by NKFIH gical, role on VSMCs irrespective of their origin (nor- (Hungary). ZV was supported by the János Bolyai motensive or hypertensive rats). Further research is Research Fellowship. required for determining whether the changes in molecular profiles of circulating exosomes mediate the development of high blood pressure in SHR. OWP1.12=LBF02.02 OWP1.13=LBF01.02 Plasma exosomes regulate proliferation and migration of vascular smooth muscle cells a b c b Kosuke Otani , Mai Yokoya , Muneyoshi Okada and Hideyuki Yamawaki Colorectal cancer cell-derived exosome enhances microenvironmental a angiogenesis through modulation of intracellular metabolism Laboratory of Veterinary Pharmacology, School of Veterinary Medicine, a b c Kitasato University, Towada, Japan; bKitasato University, Towada, Japan; Atsushi Ikeda , Satoshi Nagayama and Koji Ueda c Kitasato University, School of Veterinary Medicine, Laboratory of aCancer proteomics group, Cancer Precision Medicine Center, Japanese Veterinary Pharmacology, Towada, Japan Foundation for Cancer Research, Tokyo, Japan; bDepartment of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation Introduction: We previously reported that systolic for Cancer Research, Tokyo, Japan; cCancer Proteomics Group, Cancer Precision Medicine Center, Japanese Foundation for Cancer Research, blood pressure in spontaneously hypertensive rats, an Tokyo, Japan animal model of essential hypertension, was partly modulated by circulating exosomes (BBRC 2018). Introduction: For improvement of prognosis of color- Vascular wall remodelling regulated by proliferation ectal cancer (CRC), detection at an earlier stage of CRC and migration of vascular smooth muscle cells is essential. Exosomes are nanovesicles secreted from (VSMCs) mediates development of hypertension. We plasma membrane, and have potential to be served as aimed to clarify the effects of plasma exosomes derived biomarker carriers. In this study, we performed pro- from SHR and control Wistar Kyoto Rats (WKY) on teomic profiling of exosomes secreted from viable CRC proliferation and migration of VSMCs. tissues. Methods: Exosomes were isolated from rat plasma by Methods: To identify early detection biomarkers for an ultracentrifuge method, and identified through CRC, we performed comprehensive proteome analysis measurement of particle size distribution by a tunable of tissue-exudative extracellular vesicles (Te-EVs), resistance pulse sensing. For exploring exosome inter- which were obtained from culture media of freshly nalization in VSMCs, the isolated exosomes were resected viable CRC tissue or adjacent normal mucosa labelled with PKH67 dye and observed by a fluores- (n = 17). Among the identified Te-EV proteins, we cence microscopy. Proliferation and migration of narrowed down the biomarker candidate by selecting SMCs were determined by a bromodeoxyuridine incor- proteins which are statistically upregulated (p < .05, poration and Boyden chamber assay, respectively. fold change > 5.0) in Te-EVs from CRC tissues than Actin cytoskeleton was visualized by a rhodamine- those from adjacent normal tissues. Then we per- phalloidin staining. Expression of protein and formed functional analysis of the biomarker candidate microRNA in exosomes was determined by Western specifically. blotting and microarray, respectively. Results: Comprehensive LC/MS analysis identified Results: There was no difference in size and concen- 6,149 Te-EV proteins, in which 641 proteins showed tration of plasma exosomes between WKY and SHR. significant upregulation in Te-EVs from CRC tissues JOURNAL OF EXTRACELLULAR VESICLES 27

(p < .05, fold change > 5. 0) compared to those from gamma/LPS-activated macrophages. Incubation with adjacent normal mucosa. We focused especially on An5-MSC-EVs resulted in a significant induction in GAM (p = 7.0 ×10–5, fold change = 7.4) as a novel the expression of both pro- and anti-inflammatory biomarker candidate. GAM protein was significantly cytokines, including TNF-alfa, IL-1Beta, IL-6, IL-10 overexpressed in CRC tissues compared with adjacent and TGFbeta1. Incubation with free An5 induced normal mucosa. In EV-sandwich ELISA assay, the only pro-inflammatory cytokines without affecting IL- expression level of GAM on plasma EVs from CRC 10 and TGFbeta1 expression. The iNOS2/Arg1 ratio patients was significantly higher than that from healthy was reduced in both EV-treated groups, indicating a donors in EV-sandwich ELISA assay (n = 133, shift from M1 to M2 polarization. p = 4.0 ×10–7). In addition, the uptake of GAM-over- Summary/conclusion: In conclusion, both MSC-EVs expressing EVs enhanced vascular endothelial cell and An5-MSC-EVs shift the macrophage phenotype growth and angiogenesis through modulation of nitric from M1 to M2. The combined induction of oxide metabolism. TGFbeta1 and IL-10, observed only in An5-MSC-EV- Summary/conclusion: EV-GAM might have great stimulated macrophages, might be related to the potential as a target for both CRC diagnosis and ther- immune-modulating characteristics of these modified apy. Our strategy for identification of exosomal bio- EVs that contribute to the therapeutic effects observed marker by proteomic profiling of Te-EV proteins can in vivo. be applied to other cancers. Funding: The BROAD MEDICAL RESEARCH PROGRAM AT CCFA supported this work OWP1.14=LBS02.01 OWP1.15=LBS03.01

Annexin V binding modulates the response of macrophages to mesenchymal stromal cell-derived extracellular vesicles Membrane-radiolabelled exosomes for comparative biodistribution Michele Grassia, Michela Pozzobonb, Melania Scarpac, Damiana Incendid, analysis in immunocompetent and immunodeficient mice – A novel Alessia Giarraputoe, Anna Maria Tolomeoa, Marcin Jurgaf, Andrea g h and universal approach Porzionato and Maurizio Muraca a a b a Farid N. Faruqu , Julie Wang , Lizhou Xu , Luke McNickle , Ming-Yiu a c c d a aDepartment of Women’s and Children’s Health – University of Padova, Chong , Mark Gurney , Aled Clayton , Lesley A. Smyth , Robert Hider , e a Padova, Italy; bUniversity of Padova, Padova, Italy; cIstituto Oncologico Jane Sosabowski and Khuloud Al-Jamal d Veneto IOV-IRCCS, Padova, Italy; University di Padova Department DNS, a b King‘s College London, London, United Kingdom; School of Cancer and Padova, Italy; eUniversity of Padova, Padova, Italy; fThe Cell Factory, Niel, Pharmaceutical Sciences, King‘s College London, London, United Kingdom; Belgium; gDepartment of Neuroscience, University of Padua, Padova, Italy; cCardiff University, Cardiff, United Kingdom; dUniversity of East London, hUniversity of Padova, Padova, Italy London, United Kingdom; eQueen Mary University of London, London, United Kingdom Introduction: We have previously shown that Annexin a5 (An5) binding to mesenchymal stromal cell-derived Introduction: Exosomes have gained interest as novel extracellular vesicles (MSC-EVs) enhances the anti- drug nanocarriers due to their biological origin and inflammatory properties of these nanoparticles in an role in intercellular biomolecule delivery. In-depth animal model of colitis. However, the mechanisms knowledge of their in vivo biodistribution is therefore underlying these effects are unknown. Here, we inves- essential. This work aimed to develop a reliable and tigated the immunoregulatory effect of MSC-EVs with universal method to radiolabel exosomes to study in and without An5 binding on activated macrophages in vivo biodistribution in mice. vitro. Methods: Melanoma (B16F10 cells)-derived exosomes Methods: Macrophages were isolated from mouse bone (ExoB16) were isolated and characterised for size, yield, marrow and activated by INFgamma and LPS. Clinical purity, exosomal markers and morphology using grade Wharton Jelly-derived MSC-EVs were obtained Nanoparticle Tracking Analysis (NTA), protein mea- from The Cell Factory (Esperite NV, Niel, Belgium) surements, flow cytometry and electron microscopy. and quantified by Resistive Pulse Sensing analysis. 5,0E Two radiolabelling approaches were explored – intra- +05 macrophages were incubated with PBS (vehicle luminal labelling (111Indium entrapment via tropolone only, control, group 1) 5,0E+08 MSC-EVs (group 2), shuttling); and membrane labelling (111Indium chela- 5,0E+08 MSC-EVs added with 2 ug An5 (group 3) or tion by covalently attached bifunctional chelator). with 2 ug free An5 (group 4). After 24 h, the cells were Labelling efficiency and stability was assessed by gel analysed by flow cytometry and RNA was extracted for filtration and thin layer chromatography. Melanoma- RT-PCR analysis. bearing immunocompetent (C57BL/6) and immunode- Results: Incubation with MSC-EVs significantly ficient (NSG) mice were injected intravenously with increased only the expression of IL-10 in IFN- radiolabelled ExoB16 (1x1011 particles) followed by 28 ISEV2019 ABSTRACT BOOK metabolic cages study, whole body SPECT-CT imaging labelling approach rendered its result more reliable and and ex vivo gamma counting at 1, 4 and 24 h post- was used to compare ExoB16 biodistribution in mela- injection. noma-bearing immunocompromised (NSG) mice. Results: Membrane-labelled ExoB16 (ML-ExoB16) Similar biodistribution profile was observed in both showed superior radiolabelling efficiency and radioche- C57BL/6 and NSG mice, where prominent accumula- mical stability compared to intraluminal-labelled tion was seen in liver and spleen, apart from the lower ExoB16 (IL-ExoB16). Both IL- and ML-ExoB16 tumour accumulation observed in the NSG mice. showed prominent accumulation in liver and spleen. Summary/conclusion: Membrane radiolabelling of IL-ExoB16 showed higher tumour accumulation than exosomes is a reliable approach that allows for both ML- ExoB16 (6.7% and 0.6% ID/g tissue, respectively), live imaging and quantitative biodistribution studies to with the former showing similar value as its free tracer be performed on potentially all exosome types without ([111]Trop). The superior stability of the membrane- engineering parent cells. JOURNAL OF EXTRACELLULAR VESICLES 29

Oral with Poster Session 2 Chairs: Kazunari Akiyoshi; Muller Fabbri Location: Level B1, Lecture Room 13:30–15:00

OWP2.01=PS08.08 or ADAM10 on EVs. We hypothesize that this is due to a low abundance of these markers in PPP from healthy individuals. Identification of common EV markers in plasma using high-resolution Summary/Conclusion flow cytometry : Our findings demonstrate that Anders Askelanda, Jaco Bothab, Rikke Wehner Rasmussenb and Aase hFCM can be used for the characterization of smaller b Handberg EVs in PPP. Furthermore, we find that CD9+EVs do aAalborg University Hospital, Aalborg, Denmark; bDepartment of Clinical not universally express PS. From this point on, we plan Biochemistry, Aalborg University Hospital, Aalborg, Denmark to study enrichment of these EV phenotypes following a number of EV purification protocols, and determine Introduction: Recent advancements in flow cytometry whether EV isolation enable a more extensive charac- (FCM) have led to the development of high-resolution terization of smaller EVs. FCMs dedicated to the analysis of small particles (hFCM). hFCM studies have predominantly focused on the analysis of EVs expressing phosphatidylserine OWP2.02=PS08.09 (PS). PS is enriched in microvesicles (MVs), wherein it is involved in lipid rearrangements responsible for MV Software to automate calibration and processing of flow cytometry budding. While PS also is expressed on exosomes, it is data in clinical studies unknown whether it can be used as a universal marker Edwin van der Pols, Frank Coumansb, Leonie de Rondc, Aleksandra Gaseckad, Najat Hajjie, Rienk Nieuwlandb and Ton van Leeuwenf for smaller EVs. In this study, we attempted to char- aAmsterdam UMC, University of Amsterdam, Department of Biomedical acterize proteins enriched in smaller EVs (CD9, CD63, Engineering and Physics, Amsterdam, Netherlands; bAmsterdam UMC, CD81 and ADAM 10) and the relative co-expression of University of Amsterdam, Laboratory of Experimental Clinical Chemistry, c PS with each of these markers. Amsterdam, Netherlands; Amsterdam University Medical Centers, Amsterdam, USA; d1st Chair and Department of Cardiology, Medical Methods: FCM analysis was performed on an Apogee University of Warsaw, Warsaw, Poland; eAmsterdam University Medical A60 Micro-PLUS. In brief, platelet-poor plasma (PPP) Centers, Amsterdam, Netherlands; fdAmsterdam UMC, University of Amsterdam, Department of Biomedical Engineering and Physics, from healthy individuals was stained with lactadherin- Amsterdam, Netherlands FITC (PS+) and one of several EV surface markers enriched in smaller EVs. To evaluate the precise differ- Introduction: In search of new biomarkers, flow cyt- ences in PS and specific EV marker expression, the ometers are used in clinical studies to measure the analysis was performed twice, (1) triggering on lactad- concentration of specific extracellular vesicles (EVs). herin and (2) each EV marker (CD9-PE, CD81-PE, Flow cytometers measure light scattering and fluores- CD63-PE, ADAM10-PE), separately. All antibodies cence of single EVs in a fluid stream. However, to were matched with appropriate isotope controls and realize data interpretation and comparison, light scat- centrifuged at 17,000g for 10 min prior to antibody tering and fluorescence signals and the flow rate labelling. EVs were defined as lactadherin or EV sur- require calibration. Moreover, flow cytometers gener- face marker positive events ≤1000 nm. ate large datasets. For example, a clinical study invol- Results: Initial results indicate that CD9 is highly ving 60 patients, 30 controls, and 8 antibody labels expressed on EVs and is not universally associated to covers 1224 data files, >33 gigabytes of data and >0.3 PS. Triggering on PS revealed that 34.7% of all events billion events. To manually calibrate and analyse such a were CD9 positive (CD9+|PS+). Conversely, triggering dataset would take days if not weeks and is prone to on CD9 resulted in a 2.1-fold increase in total events, human mistakes. Therefore, an urgent need exists for where 17.0% of events were PS+ (CD9+|PS+). Inferring software to automate calibration and processing of flow size from silica nanospheres, it appeared that popula- cytometry data. tions containing CD9 (CD9+|PS+ and CD9+|PS-) were Methods: We have developed software (MATLAB smaller (94.4–99.7% <180 nm) compared to popula- R2018a) to automatically process multiple .fcs files tions that did not (PS+|CD9-; 85.6% <180 nm & 95.2% and (1) relate two scatter signals to the diameter in <300 nm). Interestingly, we did not detect CD81, CD63 nm and refractive index (RI) of EVs, (2) express 30 ISEV2019 ABSTRACT BOOK fluorescence signals in terms of molecules of equivalent platforms. (3) Finally, we propose controls, solutions soluble fluorochrome, (3) export calibrated channels to or workarounds for understanding and limiting the new .fcs files, (4) recognize unstable flow rates, (5) influence of each of these pitfalls. determine fluorescence thresholds, (6) apply gates, (7) Results: (1) High-resolution FCM and imaging FCM create PDFs with scatter plots and (8) report statistics. displayed greater sensitivity and resolution compared We are using clinical studies to validate and apply the to conventional FCM when measuring a mixture of software. nanospheres. Equally, both methods could detect larger Results: Compared to manual thresholding, automatic concentrations of specific EV phenotypes than conven- thresholding results in a systematic decrease in counts tional FCM, where imaging FCM outperformed high- of 10% and a maximum difference of 14% (n = 5). resolution FCM. Within day variability (n = 20 ali- Using a high-end laptop, data processing takes typically quots) was similar for conventional and high-resolu- a minute or several seconds per .fcs file with or without tion FCM, while imaging FCM had a markedly larger PDF reporting, respectively. Flow rate monitoring is variability. Between day variability (n =5×5 aliquots) useful for 61% of the data. The platelet marker CD61 was similar for all three platforms. (2) The three most stains 7% of the events with an RI >1.42, which are substantial pitfalls variably influencing interpretation lipoproteins, and the concentration of these lipopro- of results on the three platforms are non-specific bind- teins differed 4000-fold between individuals. ing of labels, antibody aggregates, and entities in the Summary/Conclusion: We have developed software to sample (i.e. lipoproteins) binding EV-defining dyes. (3) automate calibration and processing of flow cytometry The most important strategies for circumventing these data in clinical studies, thereby reducing analyses time, pitfalls are stringent matching, gating and comparison preventing human mistakes and providing new of antibodies and isotype controls, high-speed centri- insights. For example, non-specific labelling of antibo- fugation of antibodies and labels prior to staining, and dies to lipoproteins together with variations in lipopro- the use and interpretation of stained buffer controls tein concentrations emphasize the relevance of fasting and detergent treated samples. before venipuncture. Our next step is to extend the Summary/Conclusion: High-resolution and imaging software with machine learning. FCM hold great potential for EV characterization. Funding: NWO-TTW VENI 15924. However, increased sensitivity also leads to new artefacts and pitfalls. The solutions proposed in this pre- OWP2.03=PS08.10 sentation provide useful strategies for circumventing these.

Conventional, high-resolution and imaging flow cytometry: potentials, pitfalls and solutions for EV characterization Jaco Botha, Rikke Wehner Rasmussen, Mathilde Sanden and Aase Handberg OWP2.04=PS08.11

Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark Convolutional neural networks for classification of tumour derived extracellular vesicles Introduction: Flow cytometry (FCM) has long been a Wooje Leea, Aufried Lenferinka, Cees Ottob and Herman Offerhausa preferred method for characterizing EVs, however their aUniversity of Twente, Enschede, Netherlands; bMedical Cell Biophysics, small size have limited the applicability of conventional University of Twente, Enschede, Netherlands FCM to some extent. Thus, high-resolution and imaging FCMs have been developed but not yet system- Introduction: Raman spectroscopy probes molecular atically evaluated. The aim of this presentation is to vibration and thus reveals chemical information of a describe the applicability of high-resolution and ima- sample without labelling. This optical technique can be ging FCM in the context of EV characterization and used to study the chemical composition of diverse the most significant pitfalls potentially influencing data extracellular vesicles (EVs) subtypes. EVs have a com- interpretation. plex chemical structure and heterogeneous nature so Methods: (1) First, we present a side-by-side compar- that we need a smart way to analyse/classify the ison of three different cytometry platforms on charac- obtained Raman spectra. Machine learning (ML) can terising EVs from blood plasma regarding sensitivity, be a solution for this problem. ML is a widely used resolution and reproducibility: a conventional FCM, a strategy in the field of computer vision. It is used for high-resolution FCM and an imaging FCM. (2) Next, recognizing patterns and images as well as classifying we demonstrate how different pitfalls can influence the data. In this research, we applied ML to classify the interpretation of results on the different cytometry EVs’ Raman spectra. JOURNAL OF EXTRACELLULAR VESICLES 31

Methods: With Raman optical tweezers, we obtained Methods: Fabrication procedure of MEBS comprises Raman spectra from four EV subtypes – red blood cell, three main steps: first, biosensing surface was prepared platelet PC3 and LNCaP – derived EVs. To classify by immobilizing EPCAM binding aptamer (EBA) on a them by their origin, we used a convolutional neural nanostructured carbon electrode. The nanostructured network (CNN). We adapted the CNN to one-dimen- surface (NS) consists of 2-D nanomaterials including sional spectral data for this application. MoS2 nano-sheets, graphene nano-platelets, and a The ML algorithm is a data hungry model. The well-ordered layer of electrodeposited gold nanoparti- model requires a lot of training data for accurate pre- cles. The NS was well characterized with FESEM and diction. To further increase our substantial dataset, we EDX. FESEM analysis showed a well-ordered gold performed data augmentation by adding randomly nano-structuring for 50 nM of gold solution. generated Gaussian white noise. Furthermore, EDAX analysis confirmed >60% coverage The model has three convolutional layers and fully of gold nanoparticles on NS compared to bare carbon connected layers with five hidden layers. The Leaky electrode. At the second step, a herringbone structured rectified linear unit and the hyperbolic tangent are microfluidic channel, which is able to enrich BCE was used as activation functions for the convolutional designed and fabricated. Finally, microfluidic channel layer and fully connected layer, respectively. was integrated to biosensing surface. Different concen- Results: In previous research, we classified EV Raman trations of exosome solutions was introduced and spectra using principal component analysis (PCA). enriched to biosensing surface (SPCE/NS/GNP/EBA) PCA was not able to classify raw Raman data, but it using microchannel. After capturing BCEs on the sen- can classify preprocessed data. CNN can classify both sing surface a secondary aptamer labelled with silver raw and preprocessed data with an accuracy of 93% or nanoparticles (SNPs) as redox reporter was introduced higher. It allows to skip the data preprocessing and to the sensing surface. avoids artefacts and (unintentional) data biasing by Results: Direct electro-oxidation of SNPs was moni- data processing. tored as analytical signal. The unique design of micro- Summary/Conclusion: We performed Raman experi- channel in combining with high specific interaction ments on four different EV subtypes. Because of its between BCE and EBA provided a high sensitive detec- complexity, we applied a ML technique to classify EV tion of BCE as low as ~100 exosomes/μL. spectra by their cellular origin. As a result of this Summary/Conclusion: The unique design of MEBS approach, we were able to classify EVs by cellular provides a highly sensitive accurate platform for detec- origin with a classification accuracy of 93%. tion of ultra-low levels of cancer-derived exosomes. Funding: This work is part of the research programme This tool holds great potential for early cancer diagno- [Cancer-ID] with project number [14197] which is sis in clinical applications. financed by the Netherlands Organization for Scientific Research (NWO). OWP2.06=PS08.13

OWP2.05=PS08.12 A software suite allowing standardized analysis and reporting of fluorescent and scatter measurements from flow cytometers Joshua Welsh and Jennifer C. Jones Translational Nanobiology Section, Laboratory of Pathology, National Cancer Microfluidic electrochemical aptasensor for detection of breast Institute, National Institutes of Health, Bethesda, USA cancer-derived exosomes in biofluids Leila Kashefi-Kheyrabadi, Sudesna Chakravarty, Junmoo Kim, Kyung-A Hyun, Seung-Il Kim and Hyo-Il Jung Introduction: Single vesicle analysis using flow cyto- Yonsei University, Seoul, Republic of Korea metry is an extremely powerful technique to allow identification of unique proteins in biological samples, Introduction: Exosomes are nano-sized extracellular as well as enumerating the changes in concentrations. vesicles, which are emerging as potential noninvasive While small particle analysis (for viruses and large biomarkers for early diagnosis of cancer. However, the microparticles) using flow cytometry has been con- small size and heterogeneity of the exosomes remain ducted for several decades, there is no comprehensive significant challenges to their quantification in the bio- method for standardization of such studies. Therefore, fluids. In the present research, a microfluidic electro- we developed a suite of flow cytometry post-acquisition chemical biosensing system (MEBS) is introduced to analysis software (FCMPASS) tools that enable the con- detect ultra-low levels of breast cancer cell-derived version of scatter and fluorescent axes to standardized exosomes (BCE). units using appropriate controls, writing standardized 32 ISEV2019 ABSTRACT BOOK units to .fcs files for sharing upon publication with sequencing has shown that viral quasispecies existing open repositories, and exporting templates of obtained in PML patients contain mutations in the sialic acid data. binding pocket of the major viral capsid protein, ren- Methods: Standalone software packages for scatter and dering these virions incapable of binding LSTc. We fluorescent standardization were built using MATLAB. have recently demonstrated that JCPyV is packaged The scatter software is based upon Mie modelling and into extracellular vesicles (EVs) that can spread the is capable of predicting the optical collection angle of virus, potentially overcoming this paradox. Here, we the instrumentation and reporting the Mie modelling begin to characterize the biogenesis of this EV-virus criteria in a standardized way, making it possible to association by examining endosomal sorting complexes reproduce the models and flow cytometry settings. required for transport (ESCRT) proteins and neutral Fluorescent standardization data uses least-squares lin- sphingomyelinase 2 (nSMase2). ear regression to enable conversions of arbitrary unit Methods: Cambinol was used to specifically target scales to molecules of equivalent soluble fluorophore nSMase2 activity. Knockdown cell lines were created (MESF) using MESF calibration beads. with shRNA targeted against ALIX, TSG101 or Results: The FCMPASS software converts arbitrary SMPD3. SMPD3 was also targeted using CRISPR/ fluorescence units to MESF units and writes them to Cas9 genetic knockout in separate cell lines. data files for clearer reporting and sharing of data. Knockdown was confirmed by qPCR and/or Western FCMPASS also converts arbitrary scatter units to a mea- blot, and knockout by next generation sequencing. EV surement of scattering cross-section using modelling were concentrated by differential centrifugation and software that predicts the collection angle of the instru- evaluated by transmission electron microscopy, ments and normalizes the data automatically. Western blot, nanoparticle tracking analysis, infection Summary/Conclusion: Utilization of our FCMPASS and qPCR for protected viral genomes. Infection was software can help the EV flow cytometry more easily scored by immunofluorescence analysis with antibodies implement standardization into their experimental against the major viral capsid protein VP1. analysis and the use of the output templates can make Results: We found that depletion of nSMase2 by cam- reporting more consistent. While currently available binol, genetic knockdown or knockout caused a reduc- MESF controls can be further optimized for small tion in spread of JCPyV over time. Knockdown and particles, we believe their utilization along with the knockout SMPD3 cell lines produced less infectious other controls, can bring a new era to the reporting EV. In the absence of nSMase2, cells produced more of EV research using flow cytometry. This will be EV but there were fewer protected genomes associated particularly useful for future comparison and valida- with the EV. Knockdown of Alix or TSG101 had no tion of translational studies and will enable better effect on the infectivity of EV or the production of EV. understanding and utilization of EVs across a broad Summary/Conclusion: Overall, our studies found that range of disciplines. biogenesis of JCPyV associated EVs depends upon the enzymatic activity of nSMase2 and not the ESCRT- related proteins Alix or TSG101. OWP2.07=PF05.08 Funding: NIH R01NS043097.

Biogenesis of JC polyomavirus associated extracellular vesicles depends on neutral sphingomyelinase 2 OWP2.08=PF05.09 Jenna Morris-Lovea, Bethany O’Harab, Gretchen Geea, Aisling Duganb, Benedetta Assettac, Sheila Haleya and Walter Atwooda aBrown University, Providence, USA; bAssumption College, Worcester, USA; Exosomes mediate the antiviral activity of interferon-β against Zika cBrown Univerisity, Providence, USA virus infection Shuang Li, Shilin Li and Limin Chen Introduction: JC polyomavirus is a non-enveloped Provincial Key Laboratory for Transfusion-Transmitted Infectious Diseases, Institute of Blood Transfusion, Chinese Academy of Medical Sciences and virus that causes progressive multifocal leukoencepha- Peking Union Medical College, Chengdu, China (People’s Republic) lopathy (PML) in immunocompromised patients. JCPyV infects cells by first binding to the major attach- Introduction: IFNβ-induced exosomes (Exo-IFNβ) ment receptor lactoseries tetrasaccharide C (LSTc), fol- may impact on viral dissemination or antiviral immu- lowed by the serotonin receptor 5-hydroxytryptamine nity and therefore involve in the pathogenesis of many type 2 required for entry. In PML, JCPyV undergoes infectious pathogens. However, little is known about its lytic infection in oligodendrocytes and astrocytes, both underlying mechanisms. To better understand how of which have been shown to lack LSTc. Further, deep Exo-IFNβ perform its antiviral effect, we employed JOURNAL OF EXTRACELLULAR VESICLES 33

RNA sequencing analysis to explore the exosomal known that viruses incorporate viral material in extra- expression profiles of lncRNA and mRNA related to cellular vesicles (EVs) as a spreading strategy. These viral infections. We hypothesized that exosomes can membrane-enclosed vesicles play a vital role in inter- regulate viral infection through transmitting enclosed cellular communication. Currently, there is a lack of specific lncRNAs into neighbouring cells to inhibit knowledge on the possible involvement of EVs in viral replication. ZIKV pathogenesis. Our study aims to unravel the Methods: Exosomes were purified from A549 with/ role of EVs in ZIKV RNA transmission to the brain, without IFNβ treatment by serial centrifugation fol- via the BBB. lowed by sucrose density gradient purification, and Methods: Human brain microvascular endothelial cells characterized by TEM and Western Blot. ELISA assay (HBMEC/D3) were used in our study since they repre- were performed on purified exosome fractions to sent the BBB in vitro. Three different EV isolation demonstrate that they are free of IFNβ. Zika virus methods (precipitation kit, density gradient and size (ZIKV) replication was assayed by real-time PCR. exclusion chromatography combined with the density Results: ZIKV replication was significantly suppressed gradient) were performed. Western blot, Transmission in A549 cells pretreated with Exo-IFNβ followed by electron microscopy and Nanosight tracking analysis ZIKV infection. Moreover, we found that anti-ZIKV confirmed the presence of EVs in the supernatant of effect of Exo-IFNβ is IFN-independent because ZIKV HBMEC/D3 cells. The presence of ZIKV RNA in replication was also decreased in U5A cells (IFN-α/β infected-EVs (IEVs) was evaluated by immunofluores- receptor IFNAR deficient) pre-treated with Exo-IFNβ. cence and qPCR. In addition, the effect of IEVs on the Similar results were observed in Dengue virus and BBB was assessed using a label-free impedance-based HCV infections. RNA sequencing analysis found sev- biosensor (ECIS, Applied BioPhysics). eral lncRNAs and mRNAs were differentially expressed Results: We confirmed the presence of viral compo- and function annotation and pathway analysis demon- nents in our IEVs, including the NS1 and E proteins of strated that the differentially expressed genes were ZIKV. The obtained IEVs were able to reinfect suscep- involved in many functions and pathways, including tible cells, even after being pretreated with RNase A. antiviral infection. To validate the RNA sequencing This indicates that the viral RNA resides inside the analysis results, some lncRNAs were selected to test IEVs. Using impedance measurements on HBMEC/ their expression levels by qPCR. We are in the process D3 cell monolayers, we observed that IEVs, as well as of deciphering the mechanism employed by these exo- virus control caused similar and temporal disturbances somal lncRNAs in antiviral activity independent of on the monolayer’s integrity within 30 min post infec- inteferon. tion. No disturbances were seen upon addition of non- Summary/Conclusion: We believe that understanding infected EVs. the antiviral functional molecules wrapped in exosomes Summary/Conclusion: Our study demonstrates that may help design exosomes as efficient vehicles for EVs-derived from ZIKV-infected cells are able to trans- antiviral therapy. fer proteins and viral RNA to recipient cells. Since both Funding: Chinese Academy of Medical Sciences IEVs and viral particles can induce similar changes on Innovation Fund for Medical Sciences (2016-12M- barrier’s integrity it is possible that IEVs are involved 3-025) in an alternative mechanism of ZIKV transmission.

OWP2.09=PS02.09 OWP2.10=PF12.10

Deciphering the role of extracellular vesicles on the blood–brain HIV-specific antibody mediated targeting of ENV+ tissues by barrier during Zika virus infection exosomes Antonios Fikatas, Sam Noppen, Peter Vervaeke, Jordi Doijen, Mohammed Zou Xue, Yuan M’eng, Zheng Nan and Wu Zhiwei Benkheil, Christophe Pannecouque and Dominique Schols Nanjing University, Nanjing, China (People’s Republic) Laboratory of Virology and Chemotherapy, Rega Institute, KU Leuven, Belgium, Belgium Introduction: Antiretroviral therapy can effectively Introduction: The association of Zika virus (ZIKV) suppress HIV replication in the peripheral blood to with severe neurological disorders has gained increased an undetectable level. However, efforts to eradicate interest over the last decade. However, the mechanism the latent virus in reservoirs remain a challenge and by which ZIKV crosses the blood–brain barrier (BBB) are a major obstacle in the treatment of HIV patients. and reaches the brain remains to be elucidated. It is Exosomes exhibit huge promise as an endogenous drug 34 ISEV2019 ABSTRACT BOOK delivery nanosystem for delivering drugs to reservoir to the antigenic similarity between OMVs and the bacter- tissues given their unique properties, including low ial outer membrane, OMVs have proven to be promising immunogenicity, innate stability, high delivery effi- for the development of novel vaccines against bacterial ciency and mostly importantly the ability to penetrate pathogens. In this work, we describe the testing of OMV- solid tissues due to their lipophilic properties. based vaccine prototypes against Gallibacterium anatis,a Methods: In this study, we engineered and expressed Gram-negative pathogen of great veterinary interest. the ScFv of a high affinity HIV-specific monoclonal Methods: OMVs were isolated from a G. anatis hyper- antibody, 10E8, on exosome surface. Exosomes from vesiculating mutant using a modified version of the 293T cells were loaded with curcumin via saponin, Hydrostatic Filtration protocol described by Musante with efficient up to 34%. 10E8ScFv-expressing exo- et al. (2014). 120 16-week-old Lohmann-Brown chick- somes (10E8-Exo) showed highly efficient targeting of ens were divided in six groups and immunized twice and curcumin delivery to CHO cell that expresses a intramuscularly with different combinations of buffer trimeric gp140 on its surface (ENV+ cells) in vitro as (controls), OMVs and selected recombinant immuno- demonstrated by confocal imaging and flow cytometry. gens. Two weeks after second immunization, the effec- We showed that 10E8-Exo could effectively bind to tiveness of the immunization regimes adopted was CHO cell that expresses a trimeric gp140 on its surface. tested by challenging the animals intraperitoneally The exosomes loaded with curcumin, a chemical that with live CFUs from a heterologous G. anatis strain. was shown to kill HIV-infected cells, showed specific One week post-challenge, the animals were sacrificed killing of the trimeric gp140-expressing CHO cells. In and an established lesion score model was used during an NCG mouse model that was grafted with the necropsy to evaluate the clinical outcome of infection. tumorigenic gp140-CHO cells and developed solid tis- Results: Statistical analysis of the recorded lesion sue tumours intravenously injected 10E8-Exo targeted scores showed that the group immunized with G. ana- the ENV-expressing tissues and delivered curcumin to tis OMVs presented an average total score of 2.95, as induce a strong suppression of the ENV+ tumour opposed to an average total score of 8.77 in the control growth with a low toxicity. group. The approximately three-fold reduction in total Results: Our results demonstrated that engineered exo- average lesion score observed demonstrates that immu- somes can deliver anti-HIV agents to solid tissues by nization with G. anatis OMVs is able to effectively specifically targeting cells expressing viral env and decrease the morbidity of G. anatis infection in the induce cell killings. immunized animals. Summary/Conclusion: It suggesting that such an Summary/Conclusion: Our results show that G. anatis approach can be developed for eradicating virus- OMVs represent a promising candidate for the devel- infected cells in tissue reservoir. opment of cost-effective vaccination strategies for the Funding: This study was supported by The National prevention of G. anatis infections in a cross-serovar Key Research and Development Program of China manner. Accordingly, we hypothesize that dose/ (2016YFC1201000), Nature Science Foundation of response optimization and the enrichment of G. anatis Jiangsu Province (BY2015069-02) and National OMVs with selected immunogens should result in an Nature Science Foundation of China (81672020). The improvement of the effectiveness of the vaccination funders had no role in study design, data collection and regime proposed. analysis, decision to publish, or preparation of the Funding: This research project is being funded by a manuscript. grant from Huvepharma (https://www.huvepharma. com/). OWP2.11=PS02.10 OWP2.12=PT05.04

In vivo testing of OMV-based vaccine prototypes against Gallibacterium anatis Identification of a protein that presumably controls bacterial Fabio Antenuccia, Homa Arakb, Jianyang Gaob, Toloe Allahghadryb, Ida vesiculation in response to the extracellular environments Thøfnerb and Anders Miki Bojesenc Fumiaki Yokoyamaa, Jun Kawamotoa, Chen Chena, Tomoya Imaib and Tatsuo Kuriharaa aUniversity of Copenhagen, København S, Denmark; bUniversity of Copenhagen, Copenhagen, Denmark; cUniversity of Copenhagen, aInstitute for Chemical Research, Kyoto University, Uji, Japan; bResearch Copenhagen, USA Institute for Sustainable Humanosphere, Kyoto University, Uji, Japan

Introduction:Outermembranevesicles(OMVs)arepro- Introduction:Manybacteriautilizeextracellularmem- duced by the majority of Gram-negative bacteria. Thanks brane vesicles (EMVs) for survival in their growing JOURNAL OF EXTRACELLULAR VESICLES 35 environments through communication with others, patho- Research Laboratory, Dayton, USA; dU.S. Naval Research Laboratory, Washington, USA; eCalifornia Institute of Technology, Pasadena, USA; genesis, and biofilm formation. Therefore, the amounts fNational Research Council, Washington, USA and the components of EMVs should be tuned in response to the conditions. Although several vesiculation mechan- Introduction: Bin/Amphiphysin/RVS (BAR) domains isms are suggested, little is known how bacteria control belong to a superfamily of membrane-associated vesiculation in response to the environments. A bacterium coiled-coil proteins that influence membrane curva- Shewanella sp. HM13 has nine fold higher lipid-secretion ture. BAR domains are ubiquitous in eukaryotes and capability in EMV fractions than Escherichia coli,andits associated with membrane curvature formation, vesicle EMVs contain a major protein (P49), which is not required biogenesis/trafficking, protein scaffolding and intracel- for vesicle production. We used mutant EMVs that lack lular signalling. While advances in protein domain P49 to identify minor components of EMVs that may prediction have facilitated the identification of several control vesiculation. BAR domain proteins, they have yet to be characterized Methods: EMVs were subjected to 2D gel-based pro- in bacteria. Here we identified a putative BAR domain- teomics by peptide mass fingerprinting. Within the containing protein enriched in the outer membrane identified proteins, the function of a sensor protein vesicles (OMVs) of Shewanella oneidensis MR-1, a dis- homolog, HM1275, was analysed by swarming assay similatory metal-reducing bacteria known to produce and lipid-staining to quantify EMVs produced in var- outer membrane extensions (OMEs) that are suspected ious media. Changes in the number of EMVs depend- to facilitate long distance extracellular electron transfer ing on culture media were quantified by tunable (EET) but whose physiological relevance and mechan- resistive pulse sensing method. ism of formation remain unknown. Results:AproteinwithaPASdomainandamethyl- Methods:PurifiedS. oneidensis OMVs were prepared by accepting chemotaxis protein (MCP) sensing domain, filtration and ultracentrifugation for comparative proteo- HM1275, was identified in the EMVs. Although some mics with cell-associated outer membrane proteins or for MCPs are related to flagellar motility by binding some electrochemical measurements. Protein domains were pre- attractants, the flagellar motility of Delta-hm1275 was not dicted using HMMSCAN and CDD-search. OME forma- significantly different from that of WT. Although the tion and phenotype analyses were performed in situ by amounts of EMVs produced by WT were increased in confocal and cryo-electron microscopy. response to the concentration of casamino acids in poor Results: The putative BAR domain-like protein BdpA nutrient medium, those by Delta-hm1275 were not. was highly enriched in OMVs compared to cell-asso- Summary/Conclusion:Aputativesensorprotein, ciated outer membranes. During OME biogenesis, WT HM1275, was identified in EMVs and may recognize the S. oneidensis OMEs progress from elongated vesicle extracellular environments by binding signal molecules in chains to narrow, tubule-like extensions while ΔbdpA casamino acids to control vesiculation. Although further OMEs remain as disordered vesicle chains. Purified studies are required to reveal the signals and the sensing OMVs from these strains are electrochemically active, pathways, the results obtained in this study indicate that with redox signals consistent with multiheme outer bacterial vesiculation is controlled by extracellular envir- membrane cytochromes, supporting the role of OMEs onments, and artificial control of vesiculation with extra- in EET. Heterologous BdpA expression promotes OME cellular signals would be useful in applications such as formation in Marinobacter atlanticus and Escherichia suppression of vesicle-dependent pathogenicity. coli, suggesting BdpA membrane sculpting activity is Funding: Japan Society for Promotion of Science inducible and transferrable. Research Fellowship for Young Scientists Summary/Conclusion: The ability of BdpA to promote OME formation and maturation into tubules in vivo OWP2.13=PT05.05 supports BdpA as a comparator for BAR domain protein activity in bacteria. Funding: US DoD Synthetic Biology for Military Prokaryotic BAR domain-like protein BdpA promotes outer membrane extensions Environments (SBME) Applied Research for the Daniel A. Phillipsa, Lori Zacharoffb, Cheri Hamptonc, Grace Chongb, Brian Advancement of Science and Technology Priorities Eddied, Anthony Malanoskid, Shuai Xub, Lauren Ann Metskase, Lina Birdf, Grant Jensene, Lawrence Drummyc, Moh El-Naggarb and Sarah Glavend (ARAP) NSF Dimensions: DEB-1542527 aAmerican Society for Engineering Education – U.S. Naval Research Laboratory, Washington, USA; bUniversity of Southern California, Los US DOE: DE-FG02-13ER16415 Angeles, USA; cMaterials and Manufacturing Directorate, Air Force 36 ISEV2019 ABSTRACT BOOK

OWP2.14=PF07.10 OWP2.15=PT07.07

Diagnostic microRNA biomarkers from circulating extracellular vesicles Isolation of extracellular vesicles from extracellular matrix based for early detection of pneumonia and severe secondary complications hydrogel 3D cell cultures Stefanie Hermanna, Benedikt Kirchnera, Dominik Buschmannb, Melanie a b b Jens Luoto , Lea Sistonen and Eva Henriksson Märtec, Florian Brandesc, Stefan Kotschoted, Michael Bonine, Marlene Reithmairf, Matthias Kleing, Gustav Schellingc and Michael Pfafflh aCell Biology, Biosciences, Faculty of Science and Engineering, Åbo Akademi b University, Turku, Finland; Turku Centre for Biotechnology, University of aDivision of Animal Physiology and Immunology, School of Life Sciences Turku and Åbo Akademi University, Turku, Finland Weihenstephan, Technical University of Munich, Germany; bTUM School of Life Sciences Weihenstephan, Division of Animal Physiology and c Introduction:Cancer-derivedextracellularvesicles Immunology, Freising, Germany; Department of Anesthesiology, University Hospital, Ludwig-Maximilians-University Munich, München, (EVs) are commonly studied and isolated from two- Germany; dIMGM Laboratories GmbH, Planegg, Germany; eIMGM f dimensional (2D) cell cultures. Nevertheless, three- Laboratories GmbH, Planegg, Germany, Martinsried, USA; Institute of Human Genetics, University Hospital, Ludwig-Maximilians-University dimensional (3D) culture systems with extracellular Munich, München, Germany; gDepartment of Neurology, University matrix (ECM) provide physiologically more relevant Hospital, Ludwig-Maximilians-University Munich, München, Germany; hAnimal Physiology and Immunology, School of Life Sciences system to mimic in vivo tumour growth and progres- Weihenstephan, Technical University of Munich, Freising, Germany sion of invasion. However, there are currently no methods to efficiently isolate EVs from ECM-based Introduction: Pneumonia remains one of the most 3D cultures. For that purpose, we established a pro- deadly communicable diseases, causing three million tocol for isolating EVs from cancer cells growing in a deaths worldwide in 2016. Extracellular vesicles (EVs) 3D ECM-based hydrogel. are pivotal during signal transfer in the pathogenesis of Methods:HumanprostatecancerPC3cellswere inflammatory lung diseases. Since identifying pneumo- grown in 3D to form spheroids in a commercially nia is particularly challenging in high risk groups (e.g. available ECM-based hydrogel and the growth media the elderly or infants), which often present with atypi- was collected every two days for a period of 14 days, cal symptoms and are at high risk for secondary com- during which the spheroids grew invasive. The plications such as sepsis or acute respiratory distress respective media were differentially centrifuged at syndrom (ARDS), new approaches for early diagnosis 2, 10 and 100 Kg and the pellets were resuspended are required. In this study we identified EV in PBS. The EVs were analysed by western blotting microRNAs (miRNAs) as potential biomarkers for (WB) against the common EV markers CD81, CD63 inflammatory changes of the pulmonary tissue. and CD9. Methods: Our study included 13 patients with com- Results:Ourpreliminarydatashowsastep-wise munity-acquired pneumonia, 14 ARDS patients, 22 increase of the EV markers in the media as the PC3 patients with sepsis and 31 healthy controls. After pre- spheroids formed, expanded and invaded to the sur- cipitating EVs from 1 mL serum, total RNA was rounding 3D ECM. The EVs produced by non-inva- extracted. Subsequent to library preparation and small sive or invasive spheroids are currently being RNA-Seq, differential gene expression analysis was per- characterized with nano tracking analysis, electron formed using DESeq2. Data were filtered by mean microscopy and WB. miRNA expression of ≥50 reads, minimum twofold Summary/Conclusion: This study demonstrates that up or down regulation and adjusted p-value ≤0.05. EVs can be isolated from 3D ECM-based hydrogel Results: The mean relative miRNA frequency varied cell cultures, which recapitulate the tissue architecture slightly between the different groups and was highest in of solid tumours. Our results suggest that 3D cancer volunteers. Short sequences (<16 nucleotides), prob- cell cultures have dynamic EV secretion determined by ably degradation products from longer coding and the phenotype of the spheroids. Taken together, we non-coding RNA species, were predominantly detected present a novel protocol for EV isolation from a 3D in patients. Based on unsupervised clustering, patients culture system and provide a platform to investigate could be distinctly separated from healthy individuals. EVs from in vivo mimicking conditions. Although 21 miRNAs were significantly regulated in all Funding: This project is funded by Magnus Ehrnrooth patient groups compared to healthy controls, different Foundation, K. Albin Johansson Foundation and Åbo disorders showed unique miRNA expression profiles. Akademi University. Distinct miRNA subsets were identified, which are JOURNAL OF EXTRACELLULAR VESICLES 37 applicable to indicate disease progression from limited can be challenging, and secondary complications inflammation present in pneumonia to severe inflam- such as ARDS and sepsis might be prevented by matory changes as seen in ARDS and sepsis. early intervention and treatment. Summary/Conclusion:ThisstudyshowsthatEV Funding:ThisstudywassupportedbytheGerman miRNA biomarkers have potential for diagnosis of Federal Ministry for Economic Affairs and Energy pneumonia and to indicate disease progression under the programme “Zentrales Innovationsprogramm towards severe lung injury. Our findings are of clin- Mittelstand”. ical relevance, as the timely diagnosis of pneumonia 38 ISEV2019 ABSTRACT BOOK

Oral with Poster Session 3 Chairs: Michael Pfaffl; Ryuichi Ono Location: Level B1, Hall A 13:30–14:15

OWP3.01=LBT02.02 increase of miR-151a-3p and miR-3168 in NRES, and miR-22-3p in RES. These results were specific to the NDE fraction. Using plasma to identify neural biomarker for antidepressant Summary/conclusion response in a treatment resistant cohort : We have identified three poten- Corina Nagya, Saumeh Saeedib, Jean-Francois Therouxc, Marina Wakidc, tial biomarkers for ADT response which are uniquely c b Naguib Mechawar and Gustavo Turecki present in the neural-derived fraction of peripheral aDHRC- McGill University, Verdun, Canada; bMcGill University, Verdun, SEVs. c Canada; McGill University, Verdun, Canada Funding: Canadian Institutes of Health Research Introduction: Small extracellular vesicles (SEV) have emerged as candidate biomarkers in many complex OWP3.02=PT09.13 diseases. An important characteristic of SEVs is their ability to bidirectionally cross the blood-brain barrier. This is particularly important in the context of major Immunocapturing of tumour-derived extracellular vesicles on depressive disorder (MDD), where biomarkers are micropatterned and antibody-conjugated surfaces for individual correlative light, probe and electron measurements obtained from peripheral tissue and have been hard Pepijn Beekmana, Agustin Enciso-Martinezb, Cees Ottob and Séverine Le to relate to changes in brain functioning. 60% of Gacc MDD patients do not respond to their first antidepres- aWageningen University, Wageningen, Netherlands; bMedical Cell sant drug therapy (ADT) and treatment options are Biophysics, University of Twente, Enschede, Netherlands; cApplied Microfluidics for BioEngineering Research, University of Twente, The entirely at the discretion of the physician. Findings Netherlands, Enschede, Netherlands that can predict ADT response as well as provide insight into central mechanistic changes could revolu- Introduction: Tumor-derived extracellular vesicules tionize MDD treatment. The aim of this study is to (tdEVs) are promising biomarkers for cancer patient profile exosomal microRNA (miRNA) in the context of management. The screening of blood samples for ADT response in people with treatment-resistant tdEVs shows prognostic power comparable to screen- depression. miRNA can act as biomarkers and might ing of tumour cells. However, due to the overlap in size influence recipient cells to provide insight on disease- between tdEVs, non-cancer EVs, lipoproteins and cell relevant mechanistic changes. debris, new approaches, not only based on size, are Methods: This pilot uses plasma from 10 controls and required for the reliable isolation of tdEVs and their 10 patients with MDD (5 ADT responders (RES), and 5 quantification. We report an integrated analysis meth- non-responders (NRES)) from baseline (T0, before odology to study single tdEVs using correlative data treatment). SEVs were isolated using a size exclusion from scanning electron microscopy (SEM), Raman column from Izon Science (Christchurch, New imaging and atomic force microscopy (AFM) to obtain Zealand). Each isolation was divided into a “whole a comprehensive dataset allowing identifying features exosome” fraction and an immunoprecipitated unique to tdEVs. “(NDE)” fraction using neural marker L1CAM. Methods: Indium tin oxide (ITO)-coated fused silica Quantitation and size determination was done using was selected for its low Raman background. Substrates Tunable Resistive Pulse Sensing (TRPS) on the qNano (1 ×1 cm2) featuring position-dependent markings gold. RNA was also extracted from SEVs from both (“navigation marks”) patterned by photolithography fractions. The 4N-small RNA-Seq (Galas) protocol was were modified with a monolayer of amino dodecyl used for library preparation. phosphonic acid. The amine moieties were next reacted Results: We found that the range of SEVs in the NDE with poly(ethylene glycol) diglycidyl ether, forming an fraction was smaller than the pool of all exosomes anti-biofouling layer. Anti-EpCAM antibodies were combined. Further, SEVs from all depressed patients subsequently covalently bound on this surface. were significantly smaller than controls irrespective of Samples of both tdEVs obtained from LNCaP cell the fractions. Our sequencing results showed an lines and RBC-derived EVs were then introduced to JOURNAL OF EXTRACELLULAR VESICLES 39 the surfaces. Finally, non-specifically bound EVs were equipped to handle large data sets comprising hun- washed away before SEM, AFM and Raman measure- dreds/thousands of phenotypes and samples. Data can ments were performed. be visualized in a variety of ways along with clustering Results: Multiple objects were captured on the fully using multidimensional data analysis techniques. All functionalized ITO surfaces, according to SEM ima- software outputs can be exported in a standardized ging, while in negative control experiments (lacking templates containing metadata for reporting, as well functionalization or lacking antibody or using as uploaded into atlases such as Genboree, where mul- EpCAM-negative EVs), no object was detected. tiplex data can be stratified by RNAseq datasets. Principal component analysis of their Raman spectra, Analysis using this pipeline has been conducted using previously demonstrated to be able to distinguish human samples from a variety of mediums including tdEVs from RBC-derived EVs, revealed the presence CSF, serum and plasma comparing EV phenotypes. −1 of characteristic lipid bands (e.g. 2851 cm ) in the Results: Our multiplex approach and MPAPASS soft- captured tdEVs. AFM showed a surface coverage of ware allows the use of single cell -omics tools for EV ∽4×105 EVs per mm2 with a size distribution similar subset analysis in a manner that will elucidate the to that found by NTA. biological significance and function of different types Summary/Conclusion: A platform was developed for of EVs. This high-throughput pipeline evaluates hun- multi-modal analysis of selectively isolated tdEVs for dreds of EV protein profiles and will allow evaluation their multimodal analysis. In the future, the scope of of millions of RNA:protein profiles in an unprece- this platform will be extended to other combinations of dented manner. Integration of RNA sequencing with probe, light and electron microscopy techniques to protein characterization could provide an entirely new relate additional parameters describing the cap- way of understanding EV regulation and function. tured EVs. Summary/Conclusion: Our data show this form of EV Funding: Funded by NWO Perspectief. profiling provides a way to monitor clinical responses early in the course of treatment, which may ultimately improve patient care and outcomes. OWP3.03=PT09.14

The development of a scalable extracellular vesicle subset OWP3.04=PS04.13 characterization pipeline Joshua Welsha, Julia Kepleyb and Jennifer C. Jonesa a Translational Nanobiology Section, Laboratory of Pathology, National An integrated microfluidic device for selective exosome isolation Cancer Institute, National Institutes of Health, Bethesda, USA; from human plasma b Translational Nanobiology Lab, Laboratory of Pathology, National Cancer Hogyeong Gwaka, Junmoo Kimb, Leila Kashefi-Kheyrabadib, Seung-Il Kimb, Institute, National Institutes of Health, Bethesda, USA Kyung-A Hyunb and Hyo-Il Jungb

aSchool of Mechanical Engineering, Yonsei University, Seoul, Republic of Introduction: Liquid biopsies offer an important alter- Korea; bYonsei University, Seoul, Republic of Korea native to tumour biopsies that may be limited by the challenges of invasive procedures. We hypothesize that Introduction: Extracellular vesicles released by many circulating Extracellular Vesicles (EVs) and their cargo cell types circulate in blood vessel and play a key role in may provide a useful surrogate biopsy method. Due to intercellular communication. Exosomes are 30–150 nm their small diameter (30–1000 nm), EVs migrate from membrane vesicles and are also shed by both normal the tissue into the peripheral circulation and provide a and cancer cells. Cancer cells are known as very het- snapshot of the producing cells. Our lab has developed erogeneous, so exosomes are also heterogeneous and a first-in-class pipeline to use single cell – omics meth- have different surface expression markers. Cancer- ods to characterize EV heterogeneity with high-sensi- derived exosomes contain unique cargo determined tivity by combining multiplex assays and our custom by the molecular characteristics of cancer cells. MultiPlex Analysis post-acquisition analysis software Therefore, it is very important to selectively separate (MPAPASS), with subsequent high-resolution, single exosomes depending on surface expression for down- EV flow cytometric (FCM) methods. stream analysis. We designed an integrated microflui- Methods: A stan-dalone software package was devel- dic chip for selective exosome isolation. The oped in MATLAB to allow importation of multiplex microfluidic chip consists of Hoof Structure (HS) for flow cytometry output data. The package enables data mixing exosomes and two different sized aptamer- quality screening of detection antibodies, bead recovery coated particles and Multi-Orifice Flow Fractionation and data normalization methods. The software is (MOFF) for separating each particle. 40 ISEV2019 ABSTRACT BOOK

Methods: Biotinylated EpCAM aptamer was immobi- diagnostic ability was compared with conventional lized on the surface of 7 μm streptavidin-coated poly- diagnostic methods. styrene particle and HER2 on 15 μm. The HS has the Methods:Forty-twoprostatecancer(PCA)patients circular expansion channel on the 1st layer to generate and 20 benign prostate hyperplasia (BPH) patients’ expansion vortices and the two curvature channels on urine, plasma, saliva was collected and used for iden- the 2nd layer to make chaotic advection. It makes tifying EVs isolation ability of aqueous two-phase transverse flow and mixes two particles without parti- system (ATPS) and for comparing diagnostic ability cle focusing phenomenon. The 100-nm (exosome), 7- of ATPS with conventional diagnosis. and 15-μm fluorescence particles were used to test Results:WithanoptimizedATPS,EVswereisolatedwith mixing performance between exosomes and particles an efficiency of approximately 90%. In addition, the EV- in the HS. The MOFF was designed by a series of isolation time was within approximately 30 min, and the contraction/expansion microchannels for continuous purity of EVs in ATPS was approximately two times better size-based separation. Separation performance was than achieved with a conventional methods, ultracentrifu- tested by using the 7- and 15-μm fluorescence micro- gation and polymeric precipitation. After the ATPS iso- particles in the MOFF. lated EVs from patients’ body fluid, PCR and ELISA were Results: The mixing efficiency was the highest at the utilized to detect EVs derived from prostate cancer cells. flow rate 150 μL/min. Each exosome was continuously The expression levels of RNA and protein markers of captured by aptamer-conjugated particle in the HS prostate cancer were compared, and the relationship channel. The capture efficiency of EpCAM positive between expression levels and clinical data was analysed. exosome was 96.9% and HER 2 was 68.09%. Two The results demonstrated that diagnostic ability based on particles were separated in the integrated microfluidic ATPS was better than other conventional methods (serum device at the same flow rate. Also, 96.26% of 15-μm PSA and sediments). Moreover, sensitivity increased by at microparticles were positioned into the centre of the least 10%, and specificity was improved by at least 20% channel and 89.48% of 7 μm microparticles were sepa- compared to conventional methods. rated on both sides of the channel. Summary/Conclusion: High quality and quantity of Summary/Conclusion: Each exosome was continu- EVs can be obtained from patients’ body fluid using ously captured by mixing aptamer-conjugated particle ATPS. Using the abundant sources, which contains in the HS. Exosome-conjugated microparticles were cancer-related protein and genes, we can perform a successfully separated by inertial force in MOFF. This diagnosis with high specificity and sensitivity. analysis of each exosome will shed light on diagnosis Therefore, ATPS offers a powerful tool for more spe- and therapy of cancers. cific and sensitive diagnosis.

OWP3.06=PS05.11 OWP3.05= PF10.11

In vitro and in vivo investigation of extracellular vesicles (EVs) as biomarker carriers in the diagnosis of early Alzheimer’s disease Aqueous two-phase system to isolate extracellular vesicles for Soraya Moradi-Bachillera, Miriam Cianib, Roberta Zanardinib, Luisa Benussib, prostate cancer diagnosis Roberta Ghidonib, J. Mark Cooperc, Gianluigi Forlonia and Diego Albania Hyunwoo Shina, Jiyoon Kima, Mee Young Kimb, Yong Hyun Parkb, Yong c b d Goo Kim , Ji Youl Lee and Jaesung Park aDepartment of Neurosciences, Mario Negri Institute for Pharmacological Research IRCCS, Milan, Italy; bMolecular Markers Laboratory, IRCCS aPOSTECH, Pohang, Republic of Korea; bDepartment of Urology, Seoul St. Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy; Mary’s Hospital, The Catholic University of Korea, Seoul, Republic of Korea; cDepartment of Clinical Neurosciences, Faculty of Brain Sciences, cDepartment of Laboratory Medicine, Mary’s Hospital, The Catholic d University College London – Institute of Neurology, London, UK University of Korea, Seoul, Republic of Korea, Seoul; Department of Mechanical Engineering, POSTECH, Pohang, Republic of Korea Introduction: Extracellular vesicles (EVs) represent an Introduction: Analysing extracellular vesicles (EVs) is ideal source of biomarkers due to their role in cellular an attractive means in prostate cancer diagnosis. communication and their ability to carry protein aggre- However, existing methods of EVs isolation have low gates. The most investigated EVs are exosomes, active efficiency, purity and long process time, which induce entities secreted from cells and able to cross the blood low diagnostic ability. To approach the problems, we brain barrier. Several neurodegeneration-involved adapt a two-phase system to diagnose prostate cancer molecules may undergo intercellular spreading through by isolating EVs from patients’ urine. Using the two- exosome release. In Alzheimer’s disease (AD), before phase system, prostate hyperplasia (BPH) patients and clinical signs appear, several proteins implicated in prostate cancer (PCA) patients were diagnosed, and the exo- and endocytic pathways are altered. In this JOURNAL OF EXTRACELLULAR VESICLES 41 scenario, the identification of a correlation between comprise two main classes – exosomes and shed micro- variations in proteins carried by EVs and the progres- vesicles (sMVs, also termed microparticles and ectosomes) sion of AD is the main aim of our project. – with distinct modes of biogenesis. Within each EV class, Methods:Weperformedexosomeisolationandcharacter- subtypes exist that can be distinguished by their distinct ization from H4-SW glioma cells (a cell model featuring protein/ RNA signatures. Whilst much is known about mutated β-amyloid overexpression), as well as in mouse- exosome cargo content and functionality, sMVs are poorly (triple-transgenic mouse model for familial AD) and understood. human-plasma samples (Mild Cognitive Impairment Methods: Here, we compare protein/ RNA profiles and (MCI) and AD subjects). In every case, a differential cen- functionality of sMVs and exosomes secreted from trifugation protocol was applied and exosomes were then human primary (SW480) and metastatic (SW620) col- characterized using Nanoparticle Tracking Analysis with orectal cancer cell lines. Milligram amounts of EVs the NanoSight. We then explored exosome content, speci- were purified from cell culture media using a combina- fically Amyloid Precursor Protein (APP) and its proteolytic tion of differential ultracentrifugation/ isopycnic iodix- fragments, Microtubule Associated Protein Tau (tau), anol density centrifugation. Label-free quantitative Progranulin (PGRN protein), Soluble Triggering mass spectrometry was performed to obtain protein Receptor Expressed on Myeloid Cells 2 (sTREM2) and α- profiles for SW480-derived and SW620-derived sMVs. synuclein (α-syn), using Western blot and ELISA. L1CAM Results:WeshowthatsMVs,unlikeexosomes,areALIX-, and CD63 were evaluated to define the neural-derived TSG101-, CD63- and CD9- and contain a different suite of exosomes amount in human samples. key cancer progression modulators. Protein/ RNA signa- All the samples were collected after ethical committee tures for SW480-derived sMVs and exosomes differ from approval respecting Helsinki’s declaration. Informed each other and also from their SW620-derived counter- consents were provided by all the subjects. parts. SW480-derived sMVs are enriched in ITGA/B, Results: Our preliminary results show that APP, PGRN ANXA1, CLDN7, CD44 and EGFR/NOTCH signalling and sTREM2 are carried by H4- and human plasma- networks, while SW620-derived sMVs are enriched in derived EVs. H4-SW cell-culture medium and 3Tg PRKCA, MACC1, FGFR4 and MTOR/MARCKS signal- mouse plasma had a decrease in the EVs number ling networks. Fibroblast invasion capabilities of SW480- release (≈1*10e8 EVs/mL) in comparison to control derived and SW620-derived sMVs are comparable. (≈7*10e8 EVs/mL). This decrease was not found in Summary/Conclusion:Furthermore,wereportforthe human plasma samples. first time a comprehensive biochemical/ functional analy- Summary/Conclusion: EVs purified from H4-glioma sis of a hitherto undescribed subpopulation of sMVs. We cellular AD model, 3xTg mouse-, MCI- and AD- anticipate our in vitro findings will be a starting point for plasma samples carry proteins relevant for neurodegen- more sophisticated studies aimed at elucidating the bio- erative diseases (NDs). EVs release is reduced in cellu- chemical and functional properties of EV subtypes in vivo. lar and animal AD-models. The emerging roles of specific EV subtypes in the TME we Funding: Horizon 2020 Marie Sklodowska-Curie believe will alter our view of cancer biology and might Innovative Training Networks – Blood Biomarker-based present new targets for therapeutic intervention. Diagnostic Tools for Early Stage Alzheimer’s Disease. Funding: Funding support from La Trobe University, Melbourne, Australia. OWP3.07=PF12.07 OWP3.08=PF12.08

Shed microvesicles released from human primary and metastatic colorectal cancer cell lines contain key cancer progression proteins Mass spectrometry analysis of small extracellular vesicles isolated and RNA species from ovarian cancer ascites a a b c Wittaya Suwakulsiria, Alin Raia, Rong Xua, Maoshan Chena, David Greeningb Anna Kotrbová , Kristina Gomoryova , David Potesil , Vit Weinberger , Igor c d c b a and Richard Simpsona Crha , Eva Jandakova , Lubos Minar , Zbynek Zdrahal , Vítězslav Bryja and Vendula Pospíchalovaa aDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular a Science, La Trobe University, Melbourne, Australia, Melbourne, Australia; Department of Experimental Biology, Faculty of Science, Masaryk b bDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular University, Brno, Czech Republic; Core Facility Proteomics, Central Science, La Trobe University, Melbourne, Australia European Institute of Technology, Masaryk University, Brno, Czech Republic; cDepartment of Obstetrics and Gynecology, Faculty Hospital Brno, Brno, Czech Republic; dDepartment of Pathology, University Hospital Introduction:Extracellularvesicles(EVs)functionin Brno, Brno, Czech Republic bidirectional cell–cell communication and contribute to the sustained growth, invasion and metastasis of cancer Introduction:High-gradeserouscarcinomaoftheovaries, cells within the tumour microenvironment (TME). EVs fallopian tube and peritoneum (HGSC) is the deadliest 42 ISEV2019 ABSTRACT BOOK gynaecological malignancy with 5-year survival rate below Introduction: EVs derived from cancer cells play a role 30%. HGSC is frequently accompanied by ascites, a patho- in tumour cell proliferation, migration, invasion and logical accumulation of fluid in the peritoneum, which can metastasis. Their presence in body fluids, such as be exploited as a liquid biopsy containing not only cancer blood, makes them potential biomarkers for cancer cells, but also the tumour microenvironment including disease. However, the identification of single tdEVs extracellular vesicles (EVs). Tumour cells produce substan- can be challenging due to their heterogeneity, their tially more EVs than healthy cells, thus malignant ascites is ultra-small size, their size overlap with many other the source of enriched pool of EVs of HGSC origin. normal EVs and contaminants in body fluids and the Methods:Asciticfluidsdepletedofcellswerefractioned lack of knowledge on their chemical composition. using size-exclusion chromatography and two fractions – Methods: Synchronized optical tweezers and Raman containing and not containing EVs – were further ana- spectroscopy have enabled a study of individual EVs. lysed. In parallel, small EVs were also isolated from ascitic The new method detects individual trapping events fluids using differential ultracentrifugation followed by from Rayleigh scattering. The synchronous recording purification step in sucrose/D2O cushion. In total, 24 of Raman scattering enabled the acquisition of Raman malignant ascites and 5 non-malignant ascites were used spectra of both individual and multiple EVs, disclosing for EV isolation and further analysed using high-resolution their chemical composition. Furthermore, Mie light hybrid mass spectrometer Orbitrap Fusion Lumos Tribrid. scattering theory has been used to relate the Rayleigh The subsequent data visualization and statistical analyses scattering intensity to the size of trapped EVs. were performed using in-house-developed pipelines in Results: The light scattered of trapped EVs gave rise to KNIME environment. step-wise time traces that can be used to distinguish Results:Weidentified2441proteins,intotal,intheEVs individual trapping events from accumulative cluster from the ascites among which 21 were present in all 29 EV events due to the discrete nature of the steps which samples and not in non-vesicular fractions. Several of these correspond to single trapping events. Next, we con- proteins were specifically enriched in small EVs in malig- firmed the trapping of individual EVs derived from nant ascites in comparison with non-malignant ascites. PC3 cells, red blood cells, platelets and blood plasma These proteins are now being evaluated as biomarkers. by acquiring both, Rayleigh and Raman scattering sig- Summary/Conclusion: Using advanced mass spectro- nals. While the step-wise trend in the Rayleigh scatter- metry, we identified candidate proteins which are spe- ing signal suggests trapping of single particles, the cifically enriched in small EVs of HGSC. These Raman scattering signal demonstrates the nature of proteins warrant further investigation as they may act the trapped EVs. Through principal component analy- as important players in HGSC progression as well as sis (PCA), the main spectral variations among the four serve as potential prognostic/diagnostic/screening bio- EV types were identified. The principal component markers of HGSC. scores grouped the PC3-derived EVs in a separate Funding: Czech Science Foundation, Grant No. GJ17- cluster from the rest of the EVs. 11776Y. Summary/conclusion: We have developed an automated single particle optical tweezers – Raman and Rayleigh scattering setup to trap and release single OWP3.09=PT09.12 EVs over time. We demonstrated single-EV trapping by simultaneous acquisition of Rayleigh and Raman scattering. PCA enabled the identification of single-

Identification of single tumour-derived extracellular vesicles by EVs derived from the cancer cell line PC3. This dis- means of optical tweezers and Raman spectroscopy closes chemical information as a step towards the a b c Agustin Enciso-Martinez , Edwin van der Pol , Aufried Lenferink , Leon identification and characterization of single tumour- Terstappena and Cees Ottoa derived EVs in blood. aMedical Cell Biophysics, University of Twente, Enschede, Netherlands; bAmsterdam UMC, University of Amsterdam, Department of Biomedical Funding: Cancer ID – project number 14193, (par- Engineering and Physics, Amsterdam, Netherlands, Amsterdam, tially) financed by the Netherlands Organisation for c Netherlands; University of Twente, Enschede, Netherlands Scientific Research (NWO) JOURNAL OF EXTRACELLULAR VESICLES 43

Symposium Session 5: EVs in Infectious Diseases Chairs: Shilpa Buch; Vera Tang Location: Level B1, Hall A 14:15–15:00

OT05.01 IEVs contained more viral DNA and protein per vesicle. Summary/Conclusion: Given that adenovirus is a Extracellular vesicles provide a capsid-free vector for oncolytic DNA-virus that is assembled in the nucleus and adenoviral DNA delivery a b b c released via induced lytic cell death, the generation of Heikki Saari , Tiia Turunen , Mikko Turunen , Matti Jalasvuori , Sarah Butcherd, Seppo Ylä-Herttualab, Tapani Viitalaa, Vincenzo Cerulloa, Pia infective EVs as prescribed here suggests that part of e a Siljander and Marjo Yliperttula the produced viral DNA is secreted via IEVs before cell aDivision of Pharmaceutical Biosciences and Drug Research Program, Faculty lysis. As such, the role of EVs in the life cycle of of Pharmacy, University of Helsinki, Helsinki, Finland; bA.I.Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, adenoviruses may be an important part of the success- Finland; cDepartment of Biological and Environmental Science, ful infection and may also be harnessed for cancer- and d Nanoscience Center, University of Jyväskylä, Jyväskylä, Finland; Faculty of gene-therapy as vectors of viral DNA invisible to the Biological and Environmental Sciences, Molecular and Integrative Bioscience Research Programme, and Helsinki Institute of Life Sciences, Institute of immune system. Biotechnology, University of Helsinki, Helsinki, Finland; eEV-group, Molecular and Integrative Biosciences Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland OT05.02

Introduction: Extracellular vesicles (EVs) have been showcased as auspicious candidates for delivering ther- Bacterial growth stage regulates the size, composition and biological functions of membrane vesicles apeutic cargo, including oncolytic viruses for cancer Lauren Zavana, Natalie Bittoa, Ella Johnstona, David Greeningb and Maria treatment. Delivery of oncolytic viruses in EVs could Kaparakis-Liaskosa provide considerable advantages, hiding the viruses aDepartment of Physiology, Anatomy and Microbiology, La Trobe from the immune system and providing alternative University, Melbourne, Australia; bDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, entry pathways into cancer cells. Here we describe the Melbourne, Australia secretion and viral cargo of EVs secreted by cancer cells infected with an oncolytic adenovirus (IEVs, infected Introduction: Outer membrane vesicles (OMVs) are cell-derived EVs) as a function of time after infection. naturally released by all Gram-negative bacteria as Methods: IEV-containing cell culture medium was col- part of their normal growth and contain many of the lected from A549 and PC-3 cancer cell cultures every components found in their parent bacterium, including 24 h after being infected with an oncolytic adenovirus DNA, RNA and proteins. To date, few studies have and IEVs were isolated by iodixanol density gradient compared the proteome of OMVs to that of their centrifugation. IEVs were then characterized by cryo- parent bacterium and examined how it changes TEM, NTA, immunoblotting and qPCR for structural throughout bacterial growth. In this study, we aimed properties and viral components and their infectivity to elucidate the contribution of bacterial growth stage was confirmed by cytotoxicity assay and TEM of IEV- on the size, composition and biological functions of treated cells. Helicobacter pylori OMVs. Results: IEVs were secreted already before the lytic Methods: OMVs were purified from H. pylori cultures release of virions and their structure resembled nor- grown to early log, mid log or stationary phase of mally secreted EVs, suggesting that they were not just bacterial growth, and their size and protein composi- apoptotic fragments of infected cells. IEVs were able to tion were analysed using NTA and proteomics, respec- carry the viral genome and induce infection in other tively. The ability of OMVs isolated from various cancer cells. The amount of viral cargo associated with growth stages to stimulate an inflammatory response IEVs increased as the infection progressed, although no in human epithelial cells was determined by ELISA. intact virions were observed in any of the IEVs visua- Results: We found that OMVs became less heteroge- lized by cryo-TEM. The amount of viral cargo also neous in size throughout bacterial growth. We showed appeared to be density-dependent, in that heavier that the proteome of OMVs was vastly different to that 44 ISEV2019 ABSTRACT BOOK of their parent bacterium from each time point, sug- of its metabolic state, some of which are entrapped in gesting that there is preferential cargo packaging of exosomes and released from the host cell. Our ultimate bacterial proteins into OMVs. Gene ontology and goal is to improve upon the current diagnostics to enrichment analyses identified that bacterial growth facilitate early and rapid diagnosis of active disease, stage regulated the type of proteins packaged into which is a key to timely drug invention and further OMVs, as early log and stationary phase OMVs were spread of the disease. Using serum exosomes, we aimed enriched in proteins required for metabolic pathways, to determine if a proteomic fingerprint can be used to whereas late log phase OMVs contained proteins con- discriminate between individuals with TB from non- tributing to cell signalling. Finally, we identified that TB, as well as to classify TB suspects – individuals with bacterial growth stage affected the inflammatory pulmonary symptoms but without detectible mycobac- response mediated by OMVs in host epithelial cells, teria in their sputum. highlighting that bacterial growth stage regulates the Methods:HyperReactionMonitoringMassSpectrometry subsequent biological functions of OMVs. (HRM-MS) was applied to a sample set of serum exosomes Summary/Conclusion: Our findings identify that bac- isolated from four groups: TB negative (healthy non-ende- terial growth stage regulates the size, protein cargo mic and TB-suspect endemic) and TB positive (smear composition and biological functions of H. pylori negative or positive, all culture positive) individuals. This OMVs, and that therefore OMVs from various growth allowed us to decipher a host protein profile that is com- stages are not comparable. Collectively, these findings mon with exosomes from individuals who have sputum emphasise the importance of considering bacterial confirmed TB and distinct from those of whom do not. growth stage from which OMVs are isolated from, as Peptide intensities were normalized and differences were this will ultimately affect their protein content and in abundance, which were determined by t-test. biological functions. We are currently determining Results: Nine proteins – including FCGR3A and α-2- whether bacterial growth stage also regulates the com- HS-glycoprotein, show distinct patterns, either increas- position and functions of Gram-positive bacterial ing or decreasing with disease severity. Using adaptive membrane vesicles. least absolute shrinkage and selection operator (Lasso) Funding: Australian Research Council. we are able to discriminate TB patients from healthy and TB suspects using nine proteins. Application of the OT05.03 model to a test set of smear and culture negative TB suspect serum exosomes, we found that nine were consistent with the negative diagnosis, while one sur- Can exosomes be used to predict where patients are on the passed the threshold for positivity. When the same tuberculosis disease spectrum? Nicole Kruh-Garcia, Gustavo Diaz, Cristian Oliva Aviles and Karen Dobos sample was screened for the presence of mycobacterial peptides using our published targeted MS assays, it was Colorado State University, Fort Collins, USA positive for several Mtb peptides. Introduction: Mycobacterium tuberculosis (Mtb), the Summary/Conclusion: These results indicate that a causative agent of the disease tuberculosis (TB), is a novel assay detecting a combination of host and Mtb highly successful human pathogen. Mtb has the ability proteins can discriminate TB positivity with greater to survive within the host macrophage. In response to sensitivity than the current sputum diagnostics. the challenges of the intracellular environment, the Funding: Bill and Melinda Gates Foundation Fund #: bacteria secretes a dynamic subset of proteins reflective OPP1039688. JOURNAL OF EXTRACELLULAR VESICLES 45

Symposium Session 6: EV Engineering I Chairs: Hang Hubert Yin; Siyang Zheng Location: Level 3, Hall B 13:30–15:00

OT06.01 utilizing EV as therapeutic vectors. In order to achieve specific targeting to B16F10 malignant melanoma cells, we have decorated the EV surface with antibody target- Designed EVs for intracellular delivery of therapeutic antibodies Oscar Wiklandera, Dhanu Guptaa, Joel Nordina, Heena Sharmab, Xiuming ing surface proteins that are known to be displayed on Lianga, Giulia Corsoa, Dara Mohammada, Rim Jawada, André Görgensc and d B16F10 cells, which lead to cellular association of EV to Samir El Andaloussi these cells. a Clinical Research Center, Department for Laboratory Medicine, Karolinska Summary/Conclusion: Overall the Fc-EV platform Institutet, Stockholm, Sweden; bEvox Therapeutics Limited, Oxford, UK; cKarolinska Institutet, Department of Laboratory Medicine, Stockholm, offers the prospective of combining antibody and EV Sweden; dDepartment of Laboratory Medicine, Clinical Research Center, technology, with potential applications including tissue Karolinska Institutet, Stockholm, Sweden and cell targeting as well as intracellular delivery of Introduction: Extracellular vesicles (EV) can be engi- functional antibodies. neered to display various targeting and therapeutic moieties. Their ability to also act as a natural vector OT06.02 to shuttle cargo over biological barriers offers a unique platform for the development of a new class of therapeutics. Here, we introduce a novel concept consisting Extracellular vesicles derived from AT-MSCs mediated miR-424 delivery promote apoptosis via the PD-L1/PD-1 pathway in TNBC of antibody coupled therapeutic EV in order to target Yueyuan Zhoua, Nobuyoshi Kosakab, Zhongdang Xiaoc and Takahiro tissues or intracellular pathways. Ochiyab Methods: By engineering EV to express an Fc-binding [email protected], Tokyo, Japan; bDepartment of Molecular and moiety (Fc-EV), antibodies can be displayed on the Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Shinjyuku-ku, Japan; cSoutheast University, Nanjing, China (People’s surface of the vesicles. We have extensively evaluated Republic) the capacity of these EV to bind antibodies by immuno-electron microscopy, cellular uptake of Introduction: Extracellular vesicles (EVs) derived from labelled antibodies/EV and flow cytometry analysis, mesenchymal stem cells (MSCs) showed great potential which indicates that EVs can be decorated with anti- as the delivery vehicle of drugs including miRNAs bodies. As a proof of concept, antibodies bound to Fc- based on its low immunogenicity and natural homing EV, were assessed in inflammatory models as well as in ability. Triple-negative breast cancer (TNBC) is an cancer settings. aggressive and invasive subtype that has limited treat- Results: Delivery of anti-STAT3 antibodies in an in ment options. Meanwhile, TNBC is immunogenic with vitro STAT3 dependent inflammatory reporter model a greater percentage of tumour-infiltrating lympho- was assessed, with promising results showing inhibi- cytes and increased expression of the programmed tion of STAT3 transcriptional activity. Furthermore, death-ligand 1 (PD-L1) in the tumour microenviron- intracellular delivery of anti-STAT3 antibody using ment. The aim of our study is to apply MSC-EVs to Fc-EV displays a dose dependent growth inhibition in modulate the expression of PD-L1 via the delivery of pancreatic ductal adenocarcinoma (PDAC) cells. The miR-424 and contribute to the immunotherapy for Fc-EV platform can also be utilized for decorating EVs TNBC. with cancer targeting antibodies, a feature that can be Methods: EVs generated from adipose tissue-derived harnessed to address the differences in uptake dis- MSCs (AT-MSCs) were isolated by differential centri- played by different cancers. Certain cancer types are fugation and characterized by western blot, nanoparti- known to rapidly internalize EV, whereas other cancer cle tracking analysis and transmission electron types, such as malignant melanoma are known to take microscopy. Before EV collection, AT-MSCs were up EV to a very low extent, if taken up at all. Our modified to overexpress miR-424 through electropora- results show that antibodies targeting surface molecules tion, and miRNA mimics transfection. The miRNAs of cancer cells also aid the internalization of EV into targeting PD-L1 was predicted according to in silico cancer cells, thus further indicating the potential of analysis. The direct regulation of miR-424 on PD-L1 46 ISEV2019 ABSTRACT BOOK was verified via the 3’-UTR luciferase report assays. (1 ×1010) or naïve exosomes (exosomes derived The purified EVs were added to the recipient MDA- from HEK293T cells under normal culture conditions, MB-231 cells (MM-231). The expression of PD-L1 1×1010) every 2 h for a total of five injections. mRNA and protein was analysed via qRT-PCR and Treatment groups (Group 1-LPS+PBS; Group 2-LPS western blot, respectively. +SR; Group 3-LPS+naïve, and Group 4-PBS) were Results: We found that miR-424 directly regulated the monitored for preterm birth. Upon delivery of at least expression of PD-L1 through the binding to PD-L1 one pup in Group 1, mice were euthanized, and mater- 3’UTR. Furthermore, the expression of PD-L1 in nal plasma, uterus and cervix were collected for cyto- MM-231 cells was down-regulated and the expression kine analysis using Luminex (IL-1β, IL-8 and IL-10) of miR-424 in MM-231 was up-regulated after cocul- and Western blot for NF-B activation via RelA phos- ture with exosomes derived from normal AT-MSCs, phorylation (P-NF-B), respectively. Survival graphs and AT-MSCs with miR-424 overexpression. were created in GraphPad and one-way ANOVA was Moreover, the cell viabilities of MM-231 were performed to determine statistical significance decreased after coculture with exosomes or transfected (P < 0.05). with miR-424 mimics. Results: Animals injected with PBS delivered at the Summary/Conclusion: EVs derived from AT-MSCs expected gestational age (19.5 days). LPS and could transfer functional miR-424 to TNBC cell lines LPS + naïve-induced PTB within 10 h; however, injec- and promote the apoptosis via decreased immune- tion of SR exosomes prolonged delivery by an average negative PD-L1/PD-1 pathway. of 21 h in this model. Consistently lower levels of pro- Funding: This work was supported by Project for inflammatory cytokines, IL-1β and IL-8, were seen in Cancer Research and Therapeutic Evolution [P- maternal plasma of LPS + SR compared to LPS mice, CREATE; grant number:17cm0106402h0002], MEXT while anti-inflammatory cytokine, IL-10, levels were KAKENHI [Grant-in-Aid for Young Scientists (A); significantly increased in LPS + SR mice compared to grant number: 17H04991] and China Scholarship LPS (P = 0.01) and PBS controls (P < 0.0001). In the Council [grant number: 201706090122]. cervix and uterus, P-NF-B expression was significantly decreased in LPS + SR compared to LPS (P = 0.005, P = 0.03) (Figure 2B). OT06.03 Summary/Conclusion: Exosomes can be engineered to carry pharmaceutical agents that can dampen the infec-

Exosomal delivery of NF-κB repressor delays LPS-induced preterm tion-induced inflammation associated with PTB and birth in mouse models pPROM. Samantha Sheller-Millera, Kyungsun Choi, George Saade, Chulhee Choib and Ramkumar Menon aUniversity of Texas Medical Branch, Galveston, USA; bKAIST, Daejeon, OT06.04 Republic of Korea

Introduction: Intraamniotic infection and inflamma- Technologies for loading RNA-based therapeutics into extracellular vesicles for drug delivery tion are associated with spontaneous preterm birth Olga Shatnyevaa, Anders Gunnarssonb, Euan Gordonc, Elisa Lázaro-Ibáñezd, (PTB) and preterm premature rupture of the mem- Lavaniya Kunalingamc, Xabier Osteikoetxeae, Kristina Friisc, Marcello c b branes (pPROM). In this study, we tested engineered Maresca and Niek Dekker extracellular vesicles, or exosomes, cargoing an inhibi- aAstraZeneca, Molndal, Sweden; bAstraZeneca, Mölndal, Sweden; cAstrazeneca, Mölndal, Sweden; dAstraZeneca, molndal, Sweden; tor to pro-inflammatory transcription factor (NF-kB), eAstraZeneca, Macclesfield, UK called super-repressor (SR) IkB, to prolong gestation in an infection (LPS)-induced PTB mouse model. Introduction: Extracellular vesicles (EVs) have Methods: HEK293T (human embryonic kidney cell) emerged as a very potent new delivery system for derived exosomes were engineered to contain SR drug delivery. Recent advances in RNA-based thera- using a protein loading via optically reversible pro- peutics have broadened the scope of cellular targeting tein–protein interaction (EXPLORs) method (Yim, et of currently undruggable genes. Current approaches al 2016). In this method, SR is actively incorporated for RNA loading of EVs suffer from poor efficacy. into exosomes during biogenesis. These exosomes were Our study combines bioengineering of the therapeutic isolated, quantified and used for our studies. EVs with post-isolation RNA. We will here present Intraperitoneal (IP) injection of either LPS (100 g) or data showing (1) the use of RNA binding proteins PBS were performed in CD-1 mice on gestational day (RBP) fused to EV protein markers for in vitro loading 15 followed by injection of PBS, SR exosomes of EVs with tagged RNA cargo and (2) post-isolation JOURNAL OF EXTRACELLULAR VESICLES 47 incubation of EVs with RNA-loaded lipid nanoparti- inefficiency of exosome cargo transfer, such as trans- cles (LNP). fer of mRNA contained in exosomes, and lack of Methods: A library of targeted RNAs fused to a specific methods to create designer exosomes has hampered RNA binding protein (RBP) sequence was generated, the development of sophisticated therapeutic varying the position of recognition site. Surface plas- interventions. mon resonance was used to characterize the modified Methods: We have developed a set of synthetic-biol- sgRNAs for binding to the RBP. Activity of the hybrid ogy-inspired genetic devices that enable efficient cus- sgRNA was also confirmed for functional gene editing tomizable in situ-production of designer exosomes in with Cas9. Expi293F cells were co-transfected with the engineered mammalian cells, and pursued their thera- set of modified sgRNAs and RBP fused to EV proteins peutic applications. followed by EV purification by differential ultracentri- Results: The developed synthetic devices that can be fugation. EVs were characterized by nanoparticle track- genetically encoded in exosome producer cells (named ing analysis, Western blotting and single molecule “EXOtic (EXOsomal Transfer Into Cells) devices”) microscopy. Efficiency of sgRNA loading into EVs enhance exosome production, specific mRNA packa- was determined using qPCR. Post-isolation loading of ging and delivery of the mRNA into the cytosol of sgRNA with Expi293 EVs by co-incubation and func- recipient cells. Synergistic use of these devices with a tional delivery of sgRNA cargo in HEK293 cells were targeting moiety significantly enhanced functional also evaluated. mRNA delivery into recipient cells, enabling efficient Results: The introduction of RNA recognition ele- cell-to-cell communication without the need to con- ments into sgRNA sequence did not interfere with centrate exosomes. Further, the engineered exosome binding to RBP. Fusions between RBP and EV proteins producer cells implanted in living mice could consis- resulted into efficient incorporation of RBP in EVs. tently deliver mRNA to the brain. Moreover, therapeu- Co-expression of sgRNA resulted in selective targeting tic catalase mRNA delivery by designer exosomes of sgRNA to EVs. Additionally, EVs from cells co- attenuated neurotoxicity and neuroinflammation in expressing sgRNA and RBP contained 10-fold more both an in vitro and in vivo Parkinson’s disease model. sgRNA compared to EV from cells who only expressed Summary/Conclusion: These results indicate the sgRNA. Loading of synthetic sgRNA cargo with 40% potential usefulness of the EXOtic devices for RNA encapsulation efficiency was achieved by incubation of delivery-based therapeutic applications. (Nat. EVs with LNPs and the resulting particles led to func- Commun. 2018, 9, 1305) tional uptake in HepG2 cells. Funding: This work was supported by the European Summary/Conclusion: Here, we compare different Research Council (ERC) advanced grant [ProNet, no. techniques for therapeutic cargo loading and delivery 321381] and in part by the National Centre of into target cells. All approaches for RNA loading into Competence in Research (NCCR) for Molecular EVs demonstrates proof of principle. We envision that Systems Engineering (to M.F.). R.K. was supported by this approach will be useful for RNA loading for ther- a postdoctoral fellowship from the Human Frontier apeutic applications. Science Program.

OT06.05 OT06.06

Engineering designer exosomes produced efficiently by mammalian Protein engineering for loading of Extracellular Vesicles Xabier Osteikoetxeaa, Josia Steina, Elisa Lázaro-Ibáñezb, Gwen O´Driscollc, cells in situ and their application for the therapy of Parkinson’s d a c disease Olga Shatnyeva , Rick Davies and Niek Dekker a b c Ryosuke Kojima , Daniel Bojar and Martin Fussenegger aAstraZeneca, Macclesfield, UK; bAstraZeneca, molndal, Sweden; c d aGraduate School of Medicine, The University of Tokyo. JST PRESTO, AstraZeneca, Mölndal, Sweden; AstraZeneca, Molndal, Sweden Tokyo, Japan; bETH Zurich, Department of Biosystems Science and Engineering, Basel, Switzerland; cETH Zurich, Department of Biosystems Introduction: To date various reports have shown the Science and Engineering. University of Basel, Faculty of Science, Basel, Switzerland utility of extracellular vesicles (EVs) for delivery of therapeutic protein cargo. Currently, the most com- Introduction:Exosomesarecell-derivedextracellu- mon strategies for loading therapeutic cargoes occur lar nanovesicles 50–150 nm in size, which serve as after EV isolation mixing EVs with desired cargo and intercellular information transmitters in various bio- subjecting to passive incubation, electroporation, logical contexts, and are candidate therapeutic agents freeze-thaw cycling, sonication, extrusion, or mem- as a new class of drug delivery vesicles. However, brane permeabilization with saponin among various 48 ISEV2019 ABSTRACT BOOK techniques. An alternative approach is to modify aAstraZeneca, Mölndal, Sweden; bAstraZeneca, molndal, Sweden; c d releasing cells to secrete EVs containing the desired AstraZeneca, Molndal, Sweden; AstraZeneca, Vancouver, Canada; eAstraZeneca, Manchester, United Kingdom cargo with minimal impact on native EVs by post- isolation treatments. In this study, we designed differ- Abstract: Extracellular Vesicles (EVs) represent an ent constructs to compare Cre and Cas9 loading effi- exciting opportunity as biological delivery vehicle for ciency into EVs using (1) light-induced dimerization therapeutic cargo with excellent safety, low intrinsic systems (Cryptochrome 2 (CRY2), Phytochrome B immunogenicity, cell-specific tropism and biological (PHYB), and Vivid-based “Magnets” (Mag)), (2) fusion delivery efficiency. to EV associated proteins (CD9, CD63, CD81, Rab5), Methods: There are multiple approaches for the intro- (3) lipidation motifs (Myristoylation, Palmitoylation, duction of protein and RNA cargo into EVs, including Prenylation) and (4) a novel motif “mXO”. physical, chemical and cell engineering. We have engi- Methods: For EV production human Expi293F cell line neered Expi293F suspension cells with transient was transiently transfected with plasmid constructs expression of fusion proteins for reversible loading using PEI MAX 40K. EVs were then isolated by differ- with protein cargo with examples for Cre recombinase ential ultracentrifugation followed by iodixanol gradi- and Cas9 for CRISPR gene editing. ent and characterized using nanoparticle tracking Results: We have developed a single molecule fluores- analysis, western blotting and transmission electron cence microscopy technique to quantify cargo loading microscopy. Functionality of engineered Cre and Cas9 at a single particle level, showing excellent loading for cargo was assessed in reporter cell assays using fluor- GFP fusions with CD63 with on average 70 copies of escent microscopy, qPCR and Sanger sequencing fol- the fusion protein per particle. Functional delivery of lowed by TIDE analysis. Cre recombinase, as measured in a reporter cell line, Results:Light-induceddimerizationusingCRY2 was dependent on addition of small molecule or pep- resulted in better EV cargo loading for both Cre tide enhancers of endosomal escape. Using RNA-bind- and Cas9 than PHYB and Mag systems. Among the ing proteins fused to exosomal markers we were able to EV associated proteins and lipidation motifs tested enrich EVs with RNA cargo with up to 10-fold higher CD9, CD81, Myristoylation and mXO were most loading of sgRNA compared to loading from passive efficient at recruiting Cre and Cas9 cargo by light- mass redistribution. Human Expi293F cell-derived EVs induced dimerization. Using CRY2 in combination did not trigger any significant immune response in with CD9 or mXO we achieved loading efficiencies vitro in human blood. The in vivo assessment following of 10–34 Cre molecules per EV and 23–30 Cas9 a single intravenous administration of these EVs in molecules per EV. BALB/c mice did not reveal marked haematological Summary/Conclusion: Cre and Cas9 loading into EVs changes, cytokine induction or histopathological by producing cells is feasible using protein engineering effects. Labelling of EVs using fluorescent mCherry, with light-induced dimerization. Of the designs inves- luminescent NanoLuc or radio-isotope 111Indium tigated CRY2-induced light dimerization and CD9 and marker allowed for on line analysis of bio-distribution mXO motif were most effective resulting in multiple in vivo. copies of functional Cre and Cas9 loaded per EV. We Summary/conclusion: Opportunities of naïve and envision that this approach will be useful for protein engineered EVs for drug discovery and their potential loading in a variety of therapeutic applications. for therapeutic applications will be discussed. OT06.07

Engineered extracellular vesicles for drug delivery Niek Dekkera, Elisa Lázaro-Ibáñezb, Olga Shatnyevac, Nikki Heathd and Xabier Osteikoetxeae JOURNAL OF EXTRACELLULAR VESICLES 49

PT01: Cellular and Organ Targeting Thursday Poster Session Chairs: Charles Lai; Ikuhiko Nakase Location: Level 3, Hall A 15:30–16:30

PT01.01 PT01.03

Role of circulating extracellular vesicles in brain function and In vivo tracking and monitoring of extracellular vesicles with a new behaviour non-lipophilic dye Eisuke Dohi, Indigo Rose, Takashi Imai, Rei Mitani, Eric Choi, Dillon Muth, Sam Noppena, Gareth R Willisb, Antonios Fikatasa, Archana Guptac, Amirali Zhaohao Liao, Kenneth Witwer and Shinichi Kano Afsharic, Christophe Pannecouquea and Dominique Scholsa Johns Hopkins University School of Medicine, Baltimore, USA aLaboratory of Virology and Chemotherapy, Rega Institute, KU Leuven, Leuven, Belgium; bDepartment of Pediatrics, Harvard Medical School, MA, Boston, USA; cSystem Biosciences (SBI), Palo Alto, CA, USA Introduction: Accumulating evidence suggests that extracellular vesicles (EVs) circulate in the blood and Introduction: Extracellular vesicles (EVs) are gaining affect cellular functions in an organ distant from their increasing interest as drug delivery vehicles. However, origins. In neuroscience, systemic circulating factors there is still a lack of knowledge about the in vivo fate such as cytokines/chemokines, hormones and metabo- of exogenous delivered EVs. Noninvasive optical ima- lites have been shown to modulate brain function and ging is an important tool to analyse the biodistribution behaviour. They are also utilized as biomarkers to of EVs. Currently, one of the most popular techniques reflect brain disease status. Nonetheless, it remains is to directly label EVs with fluorescent lipophilic dyes. unclear whether circulating EVs modulate brain func- A major drawback is that the dye itself rather than EVs tion and behaviour. is detected. Hence, there is a need for other dyes that Methods: We used mouse models to study the effects overcome these limitations. A new non-lipophilic near of EVs from specific cell types on brain function and infrared (NIR) dye, ExoGlow-Vivo (SBI), was tested in behaviour. Because circulating EVs are extremely het- vivo in mice. erogeneous, we focused on immunodeficient mice that Methods:EVsfromhumanPBMC,HEKandMCF7cells lack specific lymphocytes (T and B cells). We assessed were labelled with ExoGlow-Vivo, precipitated with the changes in their circulating EVs and examined Exoquick-TC (SBI) and injected intravenously (i.v.) in their potential impact on the corresponding beha- adult SCID mice. Human mesenchymal stem cell vioural and neuronal dysregulation. (MSC)-derived EVs were labelled with ExoGlow-Vivo Results: As expected, immunodeficient mice lack the dye, washed via ultracentrifugation and injected i.v. in expression of T and B cell-related markers in the EV post-natal day-4 FVB mice. Fluorescent images were containing fractions from the peripheral blood. acquired with an IVIS® Spectrum (PerkinElmer). Immunodeficient mice also displayed social behavioural Results: Spectral unmixing of ExoGlow-Vivo revealed deficits, accompanying by enhance c-Fos immunoreac- optimal excitation/emission at 745/820 nm. tivity in the excitatory neurons in the medial prefrontal Biodistribution studies in SCID mice showed the liver cortex (mPFC). Notably, transfer of splenocytes from as main targeted organ. A high fluorescent signal was wild-type (WT) rescued the behavioural deficits, serum measured in the bladder but almost completely disap- EVs and brain c-Fos expression patterns in immunode- peared within 4 h. Ex vivo imaging of dissected organs ficient mice. Further analysis on the molecular mechan- confirmed the liver as the main organ, followed by isms is in progress. spleen and kidneys. No difference in biodistribution Summary/Conclusion: Our study has revealed a was observed between PBMC, HEK and MCF7-derived potential periphery-brain communication via EVs EVs. MSC-derived EVs accumulated preferentially in under physiological condition. Future studies are the liver of post-natal mice. Interestingly, ex vivo ima- required to identify the cellular targets of circulating ging revealed high positive staining in the lungs. This EVs and their ascending routes in the brain. may be associated with recent observations that found Funding: NIMH R01. 50 ISEV2019 ABSTRACT BOOK

MSC-derived EVs ameliorate core features of experi- was effectively delivered into the inside of cells, but mental bronchopulmonary dysplasia. did not suppress the target gene expression. By con- Summary/Conclusion: ExoGlow-Vivo has excellent trast, DOX-loaded Exolip-U251 significantly sup- NIR properties for in vivo imaging with almost negli- pressed the proliferation of U251 cells. Thus, the gible background. This non-lipophilic dye is ideal for encapsulation was critical for the agents to internalize biodistribution and kinetic studies of exogenous deliv- more effectively into cancer cells. ered EVs. However, uptake of most EVs by liver sup- Summary/Conclusion: The tropism of exosomes is plants the signal of other organs. partially regulated by their own lipid components and, mimicking this approach would lead to promising cancer-targeting technologies. PT01.04 Funding: MEXT-Supported Program for the Strategic Research Foundation at Private Universities

Exosomal lipids applicable to cancer targeting Yuki Todaa, Saeka Ukaia, Akari Kobayashia, Shin-ya Moritab, Shigekuni Hosogia and Eishi Ashiharaa PT01.05 aDepartment of Clinical and Translational Physiology, Kyoto Pharmaceutical University, Kyoto, Japan; bDepartment of Pharmacy, Shiga University of Medical Science Hospital, Kyoto, Japan The exosome that are released from mechanical stress-stimulated osteocyte induces osteoclastegenesis a b Introduction: Cancer-targeting technologies must be a Tomohiro Itoh and Yukihiro Akao crucial technology to develop diagnosis and therapy of aGraduate School of Bioresources, Mie University, Tsu, Japan; bGifu University Graduate School of Drug Discovery and Medical Information cancers. Many basic and clinical studies highlighted Sciences, Gifu, Japan lots of cancer-associated proteins and expected their ligands to honour the drug carrier with selectivity; Introduction: Osteocyte, which is the most abundant however, their targeting potential were not tolerable cell in bone tissues, is well known as a mechanical of advanced trials. We have previously identified glio- stress receiving cell. During bone remodelling, bone blastoma-derived exosomes (Exo-U251) that were resorptionby osteoclasts precedes bone formation by more effectively internalized into some types of cancer osteoblasts. However, its mechanism is still unknown. cell lines than into non-cancer cells. Because this trop- In this study, we examined whether exosome released ism was still maintained in the lack of protein ligand from osteocyte by MS stimulation are involved in interaction of the exosomes with cells, we focused on osteoclast differentiation. their lipid components. Methods: MC3T3-E1 cells or MLO-Y4 cells were Methods: Ultrapure exosomes (about 100 nm vesicles seeded on 3D scaffold and grown to 70–80% conflu- expressing CD63 in d = 1.16 mg/L fraction) were ence. The cells were exposed to pressure of 1.5 MPa for collected from cell culture media using density-gradi- 1 h at 37°C consisting a hydrostatic pressure system. ent ultracentrifugation. Exosomal lipids were extracted After cultivation, the cultured media harvested and by Bligh & Dyer method and reconstructed into lipo- then isolated then centrifuged at 8,000 ×g for 30 min somes (Exolip-U251) using extrusion method. Each at 4°C to remove cell debris. The extracellular exo- amount of lipid components was analysed by enzy- somes were pelleted in a final ultracentrifugation at matic fluorometric assay [Morita S.Y. and Terada T. 100,000 ×g for 1 h at 4°C. Pelleted exosomes were Sci. Rep., 5:11737, 2015]. Exosomes or reconstructed resuspended in PBS and ultracentrifuged again. The liposomes were fluorescent-labelled and applied to can- size distribution of exosomes was examined using a cer or non-cancer cells to evaluate the internalization NanoSight Tracking Analysis LM20 System. The efficiency using an image analysis of a laser scanning amount of osteoclast differentiation was estimated by microscopy. Exolip-U251 conjugating siRNA was pre- TRACP staining. The MLO-Y4 cell vesicle membrane pared by Exo-Fect reagent. Doxorubicin (DOX) was and vesicle internal protein profiles were analysed by encapsulated into liposomes using remote-loading nano-LC-MS/MS based shotgun proteomics. method. Results: The vesicles isolated from mechanical stress- Results: The enzymatic fluorometric assays revealed loaded MC3T3-E1 cells facilitated the mechanical the uniqueness of the exosomal lipid components stress-loaded osteoblast differentiation, but no effect according to the cells from which they are derived. against normal MC3T3-E1 cells. Though the vesicles The tropism of Exo-U251 lipid-reconstructed lipo- isolated from mechanical stress-loaded MLO-Y4 cells somes (Exolip-U251) partly mimicked that of the ori- had no effect against osteoblast differentiation, these ginal exosomes. The siRNA conjugated Exolip-U251 vesicles significantly induced osteoclast differentiation. JOURNAL OF EXTRACELLULAR VESICLES 51

To characterize the mechanisms by which mechanical HRP mimicking DNAzyme could catalyses the reduc- stress-loaded MLO-Y4 cell vesicles induces osteoclast tion of H2O2 and generate electrochemical signal. This differentiation in murine macrophage RAW264 cells, aptasensor exhibits high selectivity and sensitivity we analysed vesicle membrane and vesicle internal towards gastric cancer exosomes with a linear response proteins by nano-LC-MS/MS-based shotgun proteo- range from 4.8 ×103 to 4.8 ×106 exosomes/mL. mics. As a result, Protein X was only detected in Therefore, we expect this electrochemical apatasensor mechanical stress-loaded MLO-Y4 cell vesicles. to become a useful tool for the early diagnosis of gastric Summary/Conclusion: Our data indicated that cancer. mechanical stress-loaded MLO-Y4 cells vesicles are Methods: First of all, several gastric cancer cell or acting as one of osteoclast differentiation mechanisms. cancer overexpressed protein aptamers were screened Now, we are further investigating whether Protein X is in order to select gastric cancer exosome specific apta- involved in osteoclast differentiation. mer. Then different kinds of exosomes were captured Funding: This work was supported by a Grant-in-Aid in the anti CD-63 antibody modified gold electrode. for Scentific Research (C) [No. 18K11019] from Japan Among these exosomes, only gastric cancer exosomes Society for the Promotion of Science (JSPS). could trigger RCA to achieve the generation of large amount of G-quadruplex units. The products were PT01.06 then incubated with hemin to form hemin-G-quadruplex structures and catalysed H2O2 system to produce electrochemical signal. The aptasensor was also vali- A label-free aptasensor for electrochemical detection of gastric cancer dated in terms of the linearity and repeatability to exosomes demonstrate its potential in practice. lI Zhiyanga and He Nongyueb Results: Anti-CD63, which can bind to the exosome a Nanjing Drum Tower Hospital Clinical College, Nanjing, China (People’s surface marker was used as the capture probe. And the Republic); bSoutheast University, Nanjing, USA joint effects of hemin/G-quadruplex DNAzyme Introduction: Emerging evidence indicates exosomes towards H2O2 reduction and signal amplification pro- derived from gastric cancer cells enhances tumour duced by RCA reaction was used to generate signifi- migration and invasion through the modulation of cantly strong electrochemical and colorimetric tumour microenvironment. Here we represent a label- response. free electrochemical aptasensor for specific detection of Summary/Conclusion:Inthiswork,wedeveloped gastric cancer exosomes. This platform contains an an electrochemical and colorimetric aptasensor for anti-CD63 antibody modified gold electrode and a specific detection of gastric cancer exosomes. A spe- gastric cancer exosome specific aptamer. The aptamer cific gastric cancer exosome aptamer was selected is linked to a primer sequence which is complementary and used as the detection probe. The aptasensor to a G-quadruplex circular template. The presence of exhibits specificity towards target exosomes and target exosomes could trigger rolling circle amplifica- high sensitivity. tion and produce multiple G-quadruplex units. This 52 ISEV2019 ABSTRACT BOOK

PT02: EVs in reproduction and pregnancy Chairs: Nanbert Zhong, Qi Chen Location: Level 3, Hall A 15:30–16:30

PT02.01 death and DNA damage. Our data suggest placental EVs have the ability to protective cells against oxidative damage. In pregnancy this property of placental EVs Placenta extracellular vesicles: a potential protective may assist the function of maternal cells that are role against oxidative damage exposed to increased oxidative stress. Qi Chena, Chunlin Sub and Larry Chamleya aThe University of Auckland, Auckland, New Zealand; bFudan University of China, Shanghai, China (People’s Republic) PT02.02 Introduction: Extracellular vesicles (EVs) are lipid- enclosed packages of cellular contents including RNAs, protein and DNA that are produced by all Maternal serum miRNA biomarkers for detection of eukaryotic cells to facilitate intercellular communica- placenta accreta Rebecca R. Adamia, Louise Laurentb, Victoria Frattoc, Srimeenakshi tion and regulation. Upon reaching their target cells, Srinivasana, Cuong Trana, Peter DeHoffa, Melissa Westermannd, Allison EVs may deliver their cargo and can induce signalling O’Learye, Deborah Wingd and Gladys Ramosa to alter the behaviour of target cells. During pregnancy, aUCSD, San Diego, USA; bUCSD, La Jolla, USA; cNaval Medical Center, San a large number of EVs are extruded from placenta (a Diego, USA; dUCI, Irvine, USA; eUCSF, San Francisco, USA foetal organ) into maternal circulation. Placental EVs are implicated in maternal immunosuppression and Introduction: Failure to diagnose placenta accreta tissue repair. In this study we investigated whether spectrum (PAS) prior to delivery is associated with placental EVs can prevent cell damage. worse outcomes. However, use of ultrasound and mag- Methods: EVs were isolated from first trimester pla- netic resonance imaging for this diagnosis is costly and cental explants (range from 8–12 weeks of gestation) imprecise. We hypothesize that levels of specific cell- and separated into micro- and nano-EVs by differential free miRNAs in the maternal blood will differ among centrifugation. Human endometrium epithelial cells women with PAS, placenta previa and normal (HEE) were cultured for 18 h in the presence or placentation. absence of placental micro- or nano-EVs. After Methods: Women with suspected PAS, previa or nor- removal of excess EVs by washing with PBS, HEE mal placentation were prospectively recruited at three cells were treated with 1 mM H O for 30 min and academic centres in the UC foetal Consortium. PAS 2 2 was confirmed by pathologic evaluation. Maternal then the H2O2 was removed by washing. The culture was continued for 18 h and proliferation of HEE was serum was collected antenatally, and total RNA was measured by Alamar Blue assay. The expression of extracted, subjected to small RNA sequencing, and H2AX, a marker of DNA damage in HEE cells was mapped to the miRBase human miRNA database. measured by IHC. Groupwise differential expression analysis identified Results: The proliferation of HEE was significantly 13 candidate miRNAs, which were used to generate a reduced when HEE cells were treated with 1 mM support vector regression model for classification. The small RNA sequencing results for these candidate H2O2. However this reduction of proliferation was significantly reversed by pre-treatment with either micro- miRNAs were validated using qPCR. Results: 60 women were recruited: 18 PAS, 15 placenta or nano-placental EVs. In addition, the expression of previa and 27 normal placentation. The median gesta- H2AX was higher in HEE cells that had been treated tional age at sample collection was 30w3d (IQR 28w– with 1 mM H2O2, but higher expression of H2AX was reduced in HEE cells that had been pre-treated with 33w) and did not differ among groups (p = 0.13). The abundance of total miRNA reads as a percentage of all either micro- or nano-EVs. Summary/Conclusion: In this study, we found that reads in the small RNA sequencing data was highest pre-treatment with placental EVs can reduce the among women with PAS and lowest in normal placentation. Thirteen differentially expressed candidate adverse effects of H2O2 on HEE cell proliferation JOURNAL OF EXTRACELLULAR VESICLES 53 miRNAs were identified. Support vector regression processed in parallel showed continuous decline in accurately classified samples into the three categories. both DNA and protein quantities. Summary/Conclusion: The percent total miRNA was Summary/Conclusion: The CMPTX label incorpo- significantly higher in maternal serum in cases of PAS rated into placental EVs may be stable for 3 months compared to normal placentation. Thirteen candidate when stored at 4oC. However, the DNA of both micro miRNAs were differentially expressed among groups and nano-EVs was less stable with a rapid decline upon and were used in a training model to accurately classify storage. There was a marked difference in the stability samples. Our results suggest that maternal serum of EV-associated protein with the protein content of miRNAs have the potential to serve as biomarkers for nano-EVs being less stable than that of micro-EVs. accurate antenatal diagnosis of PAS. Studies in a larger Notably the total protein content of placental micro- independent cohort are needed for validation of these EVs was remarkably stable when the EVs were stored results. at 4oC. Further work is required to assess the intact- ness/functionality of placental EVs after storage. Funding: Marsden Fund of the Royal Society of New PT02.03 Zealand

Effects of medium term storage on placental extracellular vesicles Larry Chamley, Julie Wang and Cherie Blenkiron PT02.04

The University of Auckland, Auckland, New Zealand

Deciphering embryo-maternal communication; the dynamics of first Introduction: Studies on the function of isolated extra- contact between progenitor and progeny Kasun Godakumaraa, Masoumeh Es-haghib, Keerthie Dissanayakeb, Freddy cellular vesicles (EVs) are growing exponentially. b c d b However, as yet there is no consensus on how best to Lättekivi , Andres Salumets , Ülle Jaakma and Alireza Fazeli a store EVs. We hereby conducted a term study to exam- Department of Pathophysiology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia; bDepartment of ine the stability of various cargos carried by placental Pathophysiology, Institute of Biomedicine and Translational Medicine, EVs when stored at 4°C. University of Tartu, Estonia; cCompetence Centre on Health Technologies, Tartu, Estonia; dInstitute of Veterinary Medicine and Animal Sciences, Methods: First-trimester placental tissues were cul- Estonian University of Life Sciences, Tartu, Estonia tured for 24 h in medium supplemented with fluorescent cell tracker CMTPX (1 µg/mL). Debris was Introduction: Failure of implantation has long been removed by centrifugation at 2000 ×g. Micro EVs identified as a major challenge of assisted reproductive were harvested by centrifugation at 20,000×g and sub- technologies. It is hypothesized that the embryo alters sequently nano-EVs were harvested following centrifu- the endometrium to elevated receptivity by embryo- gation at 200,000×g. The EVs were resuspended in PBS maternal cross talk. In previous communications, we then aliquoted and stored at 4oC. CMTPX signal have shown that RNA originating from JAr (analogue strength was examined by flow cytometry (AriaII) for trophoblast) cell line, packaged in extracellular weekly. DNA was extracted, fortnightly, using vesicles (EVs) are transferred to RL95–2 (an analogue Purelink Genomic DNA kit and measured using a for endometrium) cell line and induce alterations in Qubit dsDNA assay; and total proteins were isolated, specific endometrial Zinc Finger Protein 81 (ZNF81) fortnightly, with RIPA and quantified using BCA assay. transcript. The objective of the current study was to Results: The proportions of micro and nano-EVs test the hypothesis that only EVs from viable embryo showing similar intensity of CMTPX signals did not alter ZNF81 transcript in the RL95-2 cell line. change significantly for 3 months (n > 5) but an incon- Methods: Human embryos were produced by classic in sistent and sample-dependent decline was observed vitro fertilization (IVF) or intracytoplasmic sperm thereafter. In contrast, the DNA content of EVs was injection (ICSI). They were cultured individually for stable for only 2 weeks. DNA quantities extracted from 20 h in Fert™ media (day 1), 48 h (day-3) in Cleav™ micro and nano-EVs declined by 40% and 60%, respec- media and additionally 48 h in Blast™ media (day-5). At tively, at week four compared to DNA extracted from day-3, embryos with equal size blastomeres and no freshly isolated EVs and thereafter remained stable fragmentation were considered as normal. At day 5, until 8 weeks. Total protein in micro EVs was stable embryos with identifiable inner cell mass, trophoblast for 2 months. Whereas there was a 20% decline in the and blastocyst cavity were considered normal while total protein extracted from nano-EVs by week 2 but embryos forming mass of degrading cells were consid- levels remained stable thereafter. Finally, the corre- ered degraded. Conditioned media was collected from sponding placental tissues also stored at 4oC and 6 normal day-3 embryos (three of which degraded by 54 ISEV2019 ABSTRACT BOOK day 5), day-5 normal (n = 3) and degraded (n =3) downregulated ZNF 81 expression in endometrial cells embryos, Cleav™ and Blast™ media. EVs were isolated compared to untreated controls, cells treated with using a sequential centrifugation and size-exclusion Cleave™ and Blast™ media EVs, cells treated with day-5 chromatography. A monolayer of RL95-2 cells (analo- degraded embryos and day-3 embryos degrading on gue for endometrium) was treated with isolated EVs. day-5 EVs. Control genes did not exhibit a significant The change of gene expression of ZNF 81 and control change of expression. genes (beta-actin, beta-2-microglobulin) in RL95-2 Summary/Conclusion: RL95-2 cells respond in differ- cells were measured using qPCR with absolute ent manners to EVs from normal and degraded human quantification. embryos. These findings can facilitate development of Results: Results exhibited that EVs derived both from biomarkers for differentiating viable and degraded day-5 normal blastocysts and day-3 embryos that embryos at early stages after IVF. undergo normal development significantly JOURNAL OF EXTRACELLULAR VESICLES 55

PT03: EV Nucleic Acid Biomarkers Chairs: Louise Laurent; Guoku Hu Location: Level 3, Hall A 15:30–16:30

PT03.01 asphyxial-induced brain injury were less abundant, including miR-30b-5p, miR-30a-5p, miR-27a, let-7f, miR-223/3p, miR-221, miR-22-3p, miR-151–3p, miR- Circulating exosomal miRNAs as potential biomarkers for evaluation 411–5p and miR-532 whereas only one miRNA (miR- of preterm brain injury Kenta HT Choa, Bing Xub, Nina Zengb, Randall F. D’Souzac, Cherie 455-3p) was more abundant. Blenkirond and Mhoyra Fraserb Summary/Conclusion: To the best of our knowledge, aDepartment of Physiology, Faculty of Medical and Health Sciences, The this study is the first to determine the usefulness of University of Auckland; bDepartment of Physiology, Faculty of Medical and plasma exosomal miRNAs as biomarkers for the pre- Health Sciences, The University of Auckland, Auckland, New Zealand; cDiscipline of Nutrition, Faculty of Medical and Health Sciences, The diction of preterm brain injury. Our data reveal a University of Auckland, Auckland, New Zealand; dThe University of unique plasma-derived exosomal miRNA profile, Auckland, Auckland, New Zealand which may aid the early diagnosis of preterm brain injury. Introduction: Insults such as oxygen deprivation Funding: Neurological Foundation of New Zealand. occurring in utero or during delivery have profound consequences on the neurological outcome of premature infants. This is a serious clinical problem, because PT03.04 treatment is a time-critical emergency and should be commenced within 6 h following injury. However, we Identification and Verification of Differentially Expressed MicroRNAs simply do not know which preterm infants to treat due in the plasma microvesicles for the Diagnosis of moyamoya Disease to the lack of sensitive biomarkers. Using our foetal Mi Jeong Oha, Eun Hee Kima, Yeon Hee Chob, Dong Hee Kimc, Ji Hee Sungb, a d sheep model of preterm brain injury, we sought to Eun Kyoung Shin and Oh Young Bang isolate exosomes from foetal plasma to establish asamsung medical center, Seoul, Republic of Korea; bsamsung medical center, seoul, Republic of Korea; cSungkyunkwan University, seoul, Republic of whether they contain miRNA biomarkers that are asso- Korea; dSamsung medical center, Seoul, Republic of Korea ciated with clinically significant neurologic outcomes. Methods: Chronically instrumented singleton foetal Introduction:Thereisnowell-recognizedmiRNAbio- sheep at 0.7 gestation (term 145 days) received marker for accurately predicting outcome in the presence asphyxia induced by umbilical cord occlusion for of moyamoya disease (MMD), a unique cerebrovascular 25 min. Size-exclusion chromatography (qEV) was per- occlusive disease of unknown etiology1,2.Weperformeda formed for isolation and purification of extracellular study of the significance of miRNAs expression in the vesicles (EVs) from plasma collected 4 h after occlusion plasma microvesicles (MVs) of MMD patients. from asphyxia (n = 10) and sham control (n = 10) Methods: The plasma MVs were purified from 38 foetuses. EV fractions were assessed for purity and healthy donors, 22 intracranial atherosclerotic stenosis quantity by nanoparticle tracking analysis and western (ICAS) patients and 40 moyamoya disease (MMD) blot against major EV protein markers. For biomarker patients. Plasma MVs were isolated using ultracentri- identification, miRNA expression profiles from plasma fugation. We perfomed miR expression analysis using EV fractions were determined by Affymetrix v4 miRNome miScript miRNA PCR Array. Specific microarrays. miRNAs were validated using real-time polymerase Results: Umbilical cord occlusion was associated with chain reaction, with normalization to an exogenous significant brain injury to areas commonly affected by control (cel-miR-39). The angiogenic effects were mea- asphyxia in preterm infants. Plasma EVs were charac- sured by over-expressing or inhibiting specific terised as rich in CD63 and HSP70, size ~100 nm, and miRNAs. with an exosome-like morphology by TEM. Profiling of Results: MiRNA profiles using miRNome miScript EV-miRNAs revealed significant differences (log2 fold miRNA PCR array of three pooled plasma MV samples change > 2 or < −2 and p value < 0.05) between the from patients with MMD, ICAS and controls revealed asphyxia and sham control foetal groups. Strikingly, 222 differentially expressed serum miRNAs, including the majority of miRNAs differentially abundant with 115 upregulated and 107 downregulated miRNAs. In 56 ISEV2019 ABSTRACT BOOK an independent MMD cohort, qRT-PCR confirmed Results: At the end of the study, DM + HCD had that miR-A was significantly upregulated. Hsa-miR-A significantly higher ACR while lower renal miR-451 in the MMD group exhibited greater performance than levels, relative to DM and DM + HCD + AT rats. In ICAS group (AUC 0.735) in ROC curve analysis. To addition, a strong negative correlation of renal miR- select target genes of specific miRNAs, we performed 451 was found with ACR. In contrast to kidney tissue, computational miR target prediction analysis UE analysis revealed a positive correlation of miR-451 (TargetScan) and found the seed sequence of CAV1 levels with ACR in these rats. Moreover, miR-451 in 3’-UTR interacting with hsa-miR-A. The deregulation UE was significantly higher DM + HCD rats relative to of miR-A by the transfection of HUVECs with pre- DM and DM + HCD + AT rats. In vitro studies con- miR-A was significantly decreased tube formation of firmed the direct effect of hyperglycaemia / mechanical HUVECs. In addition, miR-A inhibited tube formation on miR-451 expression. by suppressing the expression of CAV1at the posttran- Summary/Conclusion: Severity of kidney injury in rats scriptional level, respectively, resulting in defective is associated with lower renal miR-451 and higher UE angiogenesis and MMD pathogenesis. miR-451. Statin attenuated renal injury and miR-451 Summary/Conclusion: Hsa-miR-A is markedly ele- levels in UE, while improving renal levels. MiR 451 vated in plasma MV of patients with MMD. The poten- analysis in UE may assess therapeutic response of tial role of these microRNAs in the pathogenesis of reno-protective drugs non-invasively MMD can contribute to find new diagnostic and ther- Funding: Funded by ICMR and DBT, Govt. of India. apeutic target of MMD. Funding: This study was supported by a grant from the Korean Healthcare Technology R&D Project, Ministry PT03.06 of Health & Welfare (HI17C1256) and Basic Science Research Program, the Ministry of Science, ICT and

Future Planning (2018M3A9H1023675). Circulating miR-451a is a useful biomarker for haemolysis Yukichi Takada, Tatsuki Shibuta and Tsukuru Umemura

International University of Health and Welfare, Okawa City, Japan PT03.05 Introduction: Red blood cells (RBCs) are circulating enucleated cells, and its main component is haemoglo- Micro RNA- 451-5p in urinary exosomes for non-invasive monitoring bin carrying O2/CO2. RBCs contain erythroid lineage- of reno-protective response Manju Kumari, Aradhana Mohan, Ravi S. Singh, Rajni Sharma and Swasti specific microRNA (miRNA), miR-451a. Biogenesis of Tiwari miR-451a depends on the activity of Ago2, and also Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India circulating miR-451a is mainly Ago2-bound, non-exosome miRNA. For utilizing circulating miR-451a for Introduction: MicroRNA-451 in urinary exosomes diagnosis of anaemia which is caused by destruction of (UE) had been demonstrated as a sensitive predictor RBCs (haemolysis), it is important to evaluate distribu- of kidney injury in diabetic rats by us. Here, we deter- tion pattern of circulating miR-451a. mined whether miR-451 in UE could also assess sever- Methods: We analysed 120 remnant serum samples ity of kidney injury and/or reno-protective response to obtained from routine blood drawing for laboratory therapy. testing. The study was done under permission of The Methods: Streptozotocin-induced (STZ, 50 mg/kg of Ethical Committies of International University of body weight, i.p) type-1 diabetic rats were fed with Health and Welfare and Kohoukai Takagi Hospital. normal chow diet (DM) or high cholesterol diet Serum miRNAs were analysed using TaqMan (DM + HCD), to increase the severity of nephropathy. microRNA assay kits and RT-qPCR (ABI 7500fast). Half rats in DM + HCD group were treated with Exosomes were obtained using the ultracentrifugation atorvastatin (AT, 20 mg/kg body weight, method and the precipitation method. DM + HCD + AT) for 8 weeks and rest remained Results: We analysed 120 remnant serum samples untreated. After 8 weeks, urine was collected for exo- obtained from routine blood. The sensitive Hb detec- somes-enrichment and albumin to creatinine ratio tion method using 414 nm absorbance (ACR). Primary cultures of human proximal tubular (NanoDrop2000, ThermoFischer) showed that 100% cells (hPC) and their secreted exosomes were also stu- of blood sampling had haemolysis. The levels of died. Taqman-based real-time qPCR was done for serum miR-451a also increased in all samples. miR-451-5p. Ultracentrifugation method and precipitation method JOURNAL OF EXTRACELLULAR VESICLES 57 showed more than 90% of serum miR-451a is in the endogenous heparin from non-heparin spiked plasma non-exosome fraction. collected from catheters infused with low-dose heparin Summary/Conclusion: miR-451 is a unique miRNA and this effect was not improved by heparinise I. which is in the non-exosome fraction. The measure- Further, SEC partially removed contaminating effects ment of serum miR-451a is a sensitive and specific of heparin in heparin spiked whole plasma. biomarker for haemolysis. Summary/Conclusion: Treatment of foetal sheep Funding: This work was supported in part by a grant plasma with heparinase I enables utilisation of qRT- from the Japan Society for the Promotion of Science PCR for reliable miRNA quantification. SEC also (JSPS KAKENHI Grant Number: JP17K09020). enables removal of inhibitory heparin and therefore should be considered as a standard step for isolation and detection of EV-miRNAs in foetal plasma. PT03.07 Funding: Neurological Foundation of New Zealand.

Circumventing qPCR inhibition to improve amplification of exosomal PT03.09 miRNAs in preterm foetal sheep heparinised plasma Bing Xua, Mhoyra Fraser1 and Kenta HT Chob aDepartment of Physiology, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand; bDepartment of A particle-based multiplex RT-qPCR for measuring circadian rhythm- associated genes Physiology, Faculty of Medical and Health Sciences, The University of a a a b Auckland Kim Mi Yeon , Jung Seungwon , Kim Junsun , Lee Heon Jung and Kim Sangkyunga Introduction: Exosomal miRNAs have been identified aMolecular Recognition Research Center, Materials and Life Science Research Division, KIST, Seoul, Republic of Korea; bSleep-Wake Disorders Center, in plasma, which has led to an interest in their poten- Korea University Anam Hospital, Seoul, Republic of Korea tial as biomarkers of neural injury responses in survi- vors of prematurity. The chronically catheterized Introduction: Biological clocks which regulate the preterm foetal sheep model is a uniquely versatile expression of genes related to the circadian metabolism model to advance our understanding of the pathophy- of living organisms are directly linked to human health siological mechanisms underlying preterm brain injury and disease. Many studies also have suggested to ana- and develop new therapeutic strategies. However, to lyse mental health issues such as depression and bipo- ensure patency of catheters implanted into foetal ves- lar disorder through molecular analysis of circadian sels heparinised saline is infused continually. Heparin rhythm-associated genes recently. These genes are gen- can confound detection of plasma miRNAs. Here we erally analysed by reverse-transcription real-time PCR present an optimal procedure to collect and detect (RT-qPCR) but conventional methods require consid- exosomal miRNAs in foetal plasma that improves sen- erable time, cost and amount of the sample to figure sitivity and performance of qRT-PCR. out multiple genes at the same time. Methods: Size-exclusion chromatography (SEC; qEV) Methods: We introduce a sensitive and multiplex RT- and commercial ExoRNeasy were performed for isola- qPCR using hydrogel particles. One of the primer pair tion and purification of extracellular vesicles (EVs) on used in RT which captures and reverse-transcribes the foetal plasma collected in K2EDTA tubes in which target RNA is immobilized in the particle. The other varying amounts of heparin or enoxaparin (0–160 IU/ primer is stored not to react during RT and released mL) had been added post-thawing. RNA was extracted when amplification begins. This strategy can help the from qEV fractions (miRNeasy), ExoRNeasy EVs, or RT process to avoid non-specific products as well as from whole plasma (miRNeasy) and treated with or the amplification to occur effectively. without heparinase I to remove contaminating heparin. Results: In order to see RT-qPCR efficiency, PER3 Levels of endogenous miR-16 and let-7a were mea- RNA which is synthesized via in vitro transcription sured using qRT-PCR. (IVT) of complementary DNA, and total RNA Results: Important differences in EV-miRNA abun- extracted from LNCAP cell were tested with serial dance were observed between isolation methods. dilution. As a result, they showed high amplification Heparinase I treatment improved detectability of the efficiency of 95% and 98%, respectively. In addition, miRNAs in a dose-dependent manner in heparin/non- the expression level of genes was successfully measured heparin spiked whole plasma and when isolated using even from a single cell. Expression pattern of 8 circa- the ExoRNeasy kit. Strikingly, SEC removed dian rhythm-associated genes was acquired from total 58 ISEV2019 ABSTRACT BOOK

RNA of circadian rhythm-synchronized HeLa cells. In exosome and hair cells were confirmed particular addition, exosomal RNAs were monitored same cells patterns. Further study will emphasize to take a snap- every four hours for 48 h in order to obtain molecular shot of molecular circadian rhythm through the com- circadian rhythm cycles. Each gene showed unique bination of expression levels of associated genes pattern of expression. measured at a certain time point for diagnosis of the Summary/Conclusion:Fornon-invasivediagnosis, mood disorder. clinical sample of human epithelial cells, saliva JOURNAL OF EXTRACELLULAR VESICLES 59

PT04: EV Nucleic Acid Cancer Biomarkers Chairs: Christian Preußer; Harry Holthofer Location: Level 3, Hall A 15:30–16:30

PT04.01 selections was used in the further modelling. The results confirmed that few miRNAs are enough to implement early detection at high accuracy. Unveiling of extracellular exosomal miRNA profiles of breast cancer Summary/Conclusion: Through identifying these het- a a b c Shang-Che Kuo , Ko-Chien Chen , Takahiro Ochiya , Wen-Hung Kuo , erogeneous compositions of the cancer cells, under- King-Jen Changd, Kuo-Kan Liange and Tang-Long Shena standing of the molecular mechanisms underlying aNational Taiwan University, Taipei, Taiwan (Republic of China); bDepartment of Molecular and Cellular Medicine, Institute of Medical these identified biomarkers, which is essential in devel- Science, Tokyo Medical University, Shinjyuku-ku, Japan; cNational Taiwan oping effective treatments and translational research, University Hospital, Taipei, Taiwan (Republic of China); dTaiwan Adventist Hospital, Taipei, Taiwan (Republic of China); eAcademia Sinica, Taipei, could be established. Taiwan (Republic of China) PT04.03 Introduction: In an era of precision medicine, biomarker discovery is indispensable for early detection, therapeutic efficacy monitoring and outcome prediction. Hypoxia may promote tumour aggressiveness and extracellular vesicle-mediated cell-to-cell communication in multiple myeloma MicroRNAs within patient serum exosome have Kyung Ju Ryua, Ji Young Leeb, Sung Won Limb, Chaehwa Parkc, Kihyun emerged as significantly measurable biomarkers, Kimd and Seok Jin Kimc which abundantly existed in the form of liquid biop- aSungkyunkwan University, Seoul, Republic of Korea; bSamsung Medical sies, for several diseases, including cancers. They are Center, Seoul, Republic of Korea; cSungkyunkwan University, Samsung Medical Center, Seoul, Republic of Korea; dSungkyunkwan University, essential regulators of global mRNA expression in cells. Samsung Medical Center, Seoul, Republic of Korea Aberrant regulation of miRNA can enables resulting in tumour initiation, drug resistance and metastasis in Introduction: Hypoxia is one of important features of cancer. miRNA assays are convenient for large-scale tumour microenvironment, and tumour cells under studies covering multiple miRNA targets and realistic hypoxia can acquire aggressive characteristics including in screening across diverse breast cancer types for early drug resistance. Thus, tumour progression and resis- detection or factors that drive cancer progression. tance to therapy are associated with hypoxic tumour Methods: In this study, we collected patient serum microenvironment. Multiple myeloma (MM) is a neo- samples from four major molecular subtypes: luminal plasm of bone marrow plasma cells. Bone marrow is A, luminal B, HER2+ and triple negative types, and hypoxic compared to other organs, thus, tumour breast cancer patients with benign tumour and ductal aggressiveness of MM could be closely associated with carcinoma in situ (DCIS). Microarray analysis of hypoxic microenvironment. Extracellular vesicle is a miRNA expression was utilized and unique serum small vesicles containing a wide range of functional miRNA signatures between non-cancer (including proteins, mRNAs and miRNAs that are actively benign, DICS) and breast cancer patients were identi- secreted via exocytosis. In this study, we investigated fied with differential expression analysis and the com- the effect of hypoxia on the EV formation of MM cells mon features selection method – elastic net. All to identify the underlying mechanism of tumour combinations of selected miRNAs were modelled with aggressiveness in MM cells. three different approaches, including generalized linear Methods: We conducted a long-term culture of MM model (GLM), linear discriminant analysis (LDA) and cell lines under hypoxic conditions (1% and 2% of O2 support vector machine (SVM). for 4 weeks), and compared with same MM cell lines Results: To analyse more generally, we applied various cultured under normal oxygen concentration (20%). normalization methods and got different outcomes The RNA expression profiles of MM cells under after feature selection. All the selections were used to hypoxia were also compared with that of cultured fit prediction models. After applying the filter criteria cells under normal oxygen condition. The EV derived based on accuracy evaluation by 10-fold cross-valida- from MM cells was isolated using ExoQuick-TC solution, the top selected miRNAs showed the consistency tion and assessed by transmission electron microscopy, of different prediction methods, and the union of these Nanoparticle tracking analysis and Western blot. 60 ISEV2019 ABSTRACT BOOK

Results: The overexpression of HIF-1α was demon- association of candidates with bone metastasis. strated in MM cells under long-term hypoxia, and the Accuracy estimate of each candidate for the diagnosis expression of stem cell markers were more increased in of bone-metastatic PCa was quantified using the area MM cells under hypoxic condition compared to nor- under the receiver-operating characteristic curve (AUC). mal oxygen concentration The RNA sequencing Results: By miRNA-seq and miRNA-chip array, we showed up-regulation of gene associated with produc- found four prospective exosomal miRNAs including tion of EV in hypoxic cultured cells. When we mea- miR-181a-5p with significant differences between loca- sured EV from hypoxic cultured MM cells, the amount lized and bone-metastatic PCa groups (p＜0.05, fold of EV was significantly higher in hypoxic MM cells change ≥1.5 or ≤0.5). In the validation cohorts, logistic than normoxic control group. To identify specific regression analyses indicated that miR-181a-5p and alterations associated with hypoxic MM cells, we pro- miR-320a were significantly associated with bone- filed miRNAs derived from EV of hypoxic MM cell metastatic PCa. The AUC analyses identified miR- lines and those of normoxic MM cell lines. These 181a-5p as the best biomarker with the AUCs 93.1% results identified eight miRNAs with significantly dif- for diagnosis of PCa and 73.9% for that of tumour ferent expression between MM cells – derived EV. bone metastasis. Summary/Conclusion: We demonstrated the charac- Summary/Conclusion: Serum exosomal miR-181a-5p teristics of long-term hypoxic MM cell-derived EV. is a promising diagnostic biomarker for bone-meta- The EV-mediated cell-to-cell communication under static PCa. Further validation is needed. hypoxia might be associated with the content of Funding: National Natural Science Foundation of miRNA in MM cell-derived EV, and it might influence China (81630073 to W-QG, 81874097 to Y-XF, tumour aggressiveness of MM cells. 81672850 to BD, 81572536 and 81772742 to WX)

PT04.04 PT04.05

Deep sequencing identified serum exosomal miR-181a-5p as an Exosomal miRNAs and proteins signature as prognostic biomarkers indicator for bone-metastatic prostate cancer a b a b for early stage epithelial ovarian cancer Yanqing Wang , Yu-Xiang Fang , Baijun Dong , Wei-Qiang Gao and Wei a a b c a Shayna Sharma , Andrew Lai , Dominic Guanzon , Terry Morgan , Lewis Xue Perrind, John Hooperd and Carlos Salomonb a Department of Urology, Renji Hospital, School of Medicine, Shanghai Jiao a b Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Tong University, Shanghai, China (People’s Republic); State Key Laboratory Queensland Centre for Clinical Research, Royal Brisbane and Women’s of Oncogenes and Related Genes, Renji-Med X Clinical Stem Cell Research Hospital, The University of Queensland, Brisbane, Australia; bExosome Center, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Biology Laboratory, Centre for Clinical Diagnostics, University of Shanghai, China (People’s Republic) Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane, Australia; cDepartment Introduction: Prostate cancer (PCa) is the most com- of Pathology and Obstetrics, Oregon Health and Science University, Portland, OR, USA; dMater Health Services, South Brisbane, QLD, Australia, Brisbane, mon male malignancy worldwide with high heteroge- Australia neity from tumorigenesis to metastasis. Although bone metastasis is the most critical metastatic event, at pre- Introduction: Epithelial Ovarian Cancer (EOC) is the sent, there has been no specific and accurate biomarker leading gynaecological malignancy worldwide due to for its diagnosis or differentiation at an early stage of the limitations of current detection tests. The 5-year PCa. Given the fact that the profiling change of exoso- survival rate with early detection is 90% compared to mal miRNAs can work as a biomaker for metastasis in 20% with late detection. Unfortunately, only 30% of the multiple tumours, we seek to identify exosomal cases are detected early. Thus, it is essential to develop miRNAs in patient’s serum as indicators for bone- a novel and minimally invasive method to identify metastatic PCa. patients at an early stage. Exosomes have shown pro- Methods: The profiling change of serum exosomal mise as biomarkers as they encapsulate vital informa- miRNAs in patients with either benign prostatic hyper- tion. Therefore, the aims of this study were to (i) plasia (BPH) or localized or bone-metastatic PCa was determine the content of circulating exosomes at early detected by miRNA-seq and miRNA-chip array, respec- stages of EOC, and (ii) to determine the prognostic tively. Prospective miRNAs were further confirmed performance of an early-ovarian cancer screening test using TaqMan miRNA assay in two independent valida- to identify women at risk of developing EOC. tion cohorts of total 127 patients with either BPH or Methods: Exosomes were isolated from the plasma of localised or bone-metastasic PCa. Logistic regression patients with either benign disease (n = 50) or Stage I/ analysis was performed to evaluate the diagnostic II EOC (n = 28), through differential centrifugation JOURNAL OF EXTRACELLULAR VESICLES 61 and size exclusion chromatography. Exosomes were Methods: This study aimed to determine the circulat- characterized using Nanoparticle Tracking Analysis, ing EV-miR profile of tumour-bearing animals com- Western Blot and Electron Microscopy. Exosomal pro- pared to healthy controls, and relate this to the EV-miR teins were profiled using Liquid Chromatography– profile secreted by tumour cells in vitro. EVs were Mass Spectrometry (LC-MS/MS) and SWATH analysis. isolated from the supernatant of HCC-1954 breast An Illumina TrueSeq Small RNA Library Prep kit was cancer cells expressing luciferase (HCC-luc). EVs used for exosomal miRNA profiling. A binomial clas- were also harvested from healthy BALB/c nude mice sification algorithm was generated using a boosted or those bearing mammary fat pad HCC-luc tumours. logistic regression analysis (WEKA machine learning Serum and media EVs were isolated by differential software (ver 3.6.12)) of the results obtained from the centrifugation, followed by microfiltration and ultra- benign and Stage I/II samples. The algorithm was built centrifugation. EVs were characterised by Nanoparticle using 5 miRNAs and 5 proteins identified through Tracking Analysis (NTA), western blot and circulating exosome profiling. The expression of speci- Transmission Electron Microscopy (TEM). Next- fic miRNAs was confirmed using RT-qPCR to validate Generation Sequencing (NGS) targeting miRNA was the miRNA sequencing results. preformed to compare the HCC-luc EV-miR profile Results: miRNAs and proteins were identified as being in vitro and in vivo. differentially expressed across EOC progression. The Results: EVs were successfully isolated and demon- algorithm that we built delivered discrimination strated to express CD63, CD9 and CD81. Using NTA, between women with EOC (Stage I/II) compared to size distribution was confirmed to be of the predicted benign. The classification efficiency was assessed by range of 30–120 nm. A range of miRNA was detected ROC curve analysis (area under the curve (AUC) was in the HCC-luc EVs in vitro. Interestingly, nine of 0.785 ± 0.091 (p = 0.0106)) with positive and negative these miRNAs were present at significantly higher predictive values of 75% and 76%, respectively. levels in the EVs than the cells from which they were Summary/Conclusion: We propose that the combined released, e.g. miR-184 and let-7c. Initial analysis measurement of exosomal miRNAs and proteins might revealed that a number of miRNAs packaged into allow for the early identification of women with EOC, EVs by HCC-luc cells In Vitro were also detectable in distinguishing between patients with benign disease circulating EVs isolated from tumour-bearing animals. and patients with Stage I/II EOC. Future directions Summary/Conclusion: EVs are thought to represent a involve the validation of the proposed miRNAs and fingerprint of the cell from which they are released, and proteins in a larger cohort. hold great potential as biomarkers for breast cancer Funding: OCRF. detection. Further understanding of miRNA trafficking and transfer in EVs will shed light on their true potential in the cancer biomarker and therapeutic setting. PT04.06 Funding: I am funded by the National Breast Cancer Research Institute (NBCRI).

Circulating Extracellular vesicle (EV)-encapsulated microRNAs as a PT04.07 biomarker of breast cancer Clodagh O’Neilla, Róisín Dwyerb, Sonja Khana, Katie E. Gilliganc and Peter Dockeryb Role of exosomal microRNA as a biomarker for extranodal NK/T-cell a b National University of Ireland, Galway, Galway, Ireland; NUI Galway, lymphoma 3 Galway, Ireland; National University of Ireland Galway, Galway, Ireland Kyung Ju Ryua, Chaehwa Parkb, Won Seog Kimb and Seok Jin Kimb aSungkyunkwan University, Seoul, Republic of Korea; bSungkyunkwan Introduction: Early detection of breast cancer is the University, Samsung Medical Center, Seoul, Republic of Korea key to improve patient survival. There is currently no robust biomarker available to detect breast cancer. Introduction: Extranodal natural killer (NK)/T-cell Extracellular vesicle encapsulated microRNAs (EV- lymphoma (ENKTL) is one of aggressive subtype of miR) provide novel potential in this field. EVs are non-Hodgkin lymphoma, and all tumour cells are inar- tiny nanoparticles released by all cells in the body guably infected with Epstein–Barr virus. The cell to cell that contain various bioactives including miRNA, interaction and association with tumour microenviron- believed to reflect the characteristics of the parent ment could be important for this disease entity. cell. Evidence suggests that tumour associated EVs Exosomes are small membrane vesicles of 30–150-nm have a distinct miRNA expression profile from normal diameter that plays an important role in the tumour cells, and could hold novel biomarker potential. microenvironment, and they are actively secreted by 62 ISEV2019 ABSTRACT BOOK most cell types, including cancer cells. In particular, the Introduction: Tumour-derived molecular signatures of intra-exosomal microRNA is known as being impor- breast cancer (BCa) have accelerated personalized med- tant for intercellular communications. However, the icine as prognostic and predictive indicators leading to clinical significance of exosomal miRNAs in ENKTL improved clinical outcomes. Currently, molecular pro- has not been examined. Thus, we characterized exoso- filing is performed on biopsied breast tumour tissue mal miRNAs in ENKTL and analysed their effect on but our goal of “liquid biopsy” is to obtain disease- the outcomes of patients. relevant genetic material non-invasively by capturing Methods: We isolated exosomes from ENKTL patient exosomes, cfDNA, or protein from bodily fluids. serum and lymphoma cell lines using ExoQuick and Unfortunately, a major limitation of liquid biopsy analysed by transmission electron microscopy, stems from the scarcity of disease-relevant material Nanoparticle tracking analysis (NTA) and Western compared to background. Here we describe an enrich- blot. We performed exosomal microRNA profiling via ment process in plasma capable of isolating cancer the nCounter miRNA expression assay on exosomes specific exosomal subpopulations originating from from 45 ENKTL patients and lymphoma cell lines. early stage breast tumours. Results: We isolated and characterized exosomes from Methods: Tumour-specific surface markers on exo- NKTL patient serum and cell lines using ExoQuick, somes were targeted and enriched from plasma and analysed by TEM, NTA and Western blot. The obtained from stage I/II ER positive / HER2 negative serum-derived exosomes had a diameter of BCa patients and age-matched controls. RNA-sequen- 95.84 ± 11.37 nm and exosome concentrations ranged cing was performed on total RNA isolated from 15 BCa from 0.25 to 14 ×1012/mL. We verified exosomes tumour tissues (FFPE) and 15 patient-matched plasma morphology and size using TEM, and detected exoso- exosome samples (with and without exosome enrich- mal markers, including Alix, and CD63 by western ment). We also sequenced RNA from 12 healthy breast bolt. We performed miRNA microarrays to compare tissues (FFPE) and plasma exosomes from 10 healthy exosomal miRNAs of patients with ENKTL having post-menopausal women (with and without tumour good and bad prognosis. As shown in the microarray exosome enrichment). RNA-seq data were used for results, we found various miRNAs that were differen- gene-level differential abundance analysis. tially contained in the serum – derived exosomes of Results:Tumour-derivedexosomeenrichmentwas NKTL poor relative to good subjects. These results observed in 63% of the BCa patients with detectible levels identified 30 miRNAs with significantly different of the target antigens in their plasma. RNA-seq gene expression between NKTL samples. Five of these expression profiles of these enriched exosomes were highly miRNAs were up-regulated and 25 ware down-regu- correlated with those of the breast tumour FFPE samples. lated in the serum-derived exosomes of NKTL bad Tumour-enriched exosomal RNA abundance clustered compared to the good subjects (p value < 0. 05). We most tightly with the FFPE tissue derived from the same identified two exosomal miRNA signatures, has-miR- patient; even more so than BCa FFPE samples correlated to 320e and miR-4516, that were associated with poor each other. The strength of the correlation between BCa outcomes with regard to OS and PFS. enriched plasma exosomes and matched patient tissue was Summary/Conclusion: Our study provides that exoso- sufficient to enable correct tumour subtyping (by both mal miRNA, miR-320e and miR-4516, may serve as PAM50 & IntClust gene targets) using only the enriched potential diagnostic and prognostic biomarker in plasma exosomal RNA. NKTL. Summary/Conclusion: Tumour-specific exosome enrichment improved plasma-derived exosomal RNA signal to noise and revealed RNA profiles that closely PT04.08 reflect the donor tumour, thus enabling the detection and characterization of early stage breast cancers.

Cancer-derived exosomes enriched from patient plasma strongly mirror parent tumour and enable subtyping of early stage breast PT04.09 cancer via liquid biopsy Christine Coticchiaa, Robert Kitchenb, Sudipto Chakraborttyb, Douglas Robertsa, Lisa Bedfordc, Sunita Badolac, Sylvie Vincentc, Seth YuB and Johan Skogd Exosomes: the same team for hepatocellular carcinoma development a b on the background of HCV and ergotism? Exosome Diagnostics, Waltham, USA; Exosome Diagnostics, Inc, Waltham, Alisa Petkevich, Alexandr Abramov, Mohamed Kadle and Vadim Pospelov USA; cTakeda, Cambridge, USA; dExosome Diagnostics, Inc., Waltham, MA, USA Peoples’ Friendship University of Russia (RUDN University), Moscow, Russia JOURNAL OF EXTRACELLULAR VESICLES 63

Introduction: Hepatocellular carcinoma (HCC) may Republic); cZhenjiang, China (People’s Republic); dZhenjiang Key Laboratory of High Technology Research on Exosomes Foundation and be caused by a wide variety of reasons, two possible Transformation Application, Jiangsu Key Laboratory of Medical Science of them are hepatitis C virus infection (HCV) and and Laboratory Medicine, School of Medicine, Jiangsu University, alkaloids contained in the ergot (Claviceps). Anyway, ZhenJiang, China (People’s Republic) not all of the individuals infected with HCV or living in Introduction:Exosomesarecriticalmediatorsof regions endemic for ergot develop HCC so it is reason- intercellular communication and promising biomar- able to develop biomarker panel for identification of ker for cancer. However, whether the release of exo- risk groups for HCC. Exosomes seem to be an ideal somes has an effect on donor cells has not been well source of such biomarkers as far as they contain exactly investigated. In this study, we aimed to identify the the information molecules packed by cells during its clinical values of exosomal circRNAs in gastric cancer physiological (or pathological) functioning. (GC). Meanwhile, we exploredthebiologicalrolesand Methods: 48 plasmas of patients with HCC from mechanisms during GC cells selectively sorting exo- Somalia (from a region with a high degree of ergot somal circRNAs. alkaloides in food), and 18 plasmas of HCC (Russia) on Methods:Databasecirc2TraitsandstarBasev2.0 the background of cirrhosis due to HCV. Exosomes were used to screen potential GC related circRNAs were isolated from plasma by differential ultracentrifu- and validated their expression levels in over 50 gation following free-flow electrophoresis. MiRNA let- paired serum or exosomes from GC patients and 7a-5p, −224-5p, −106b-3p, −126-5p, −122-5p, −16-5p healthy volunteers, or 100cancertissuesandadja- and −34a-5p were determined in exosomes by qPCR- cent normal tissues from GC patients. Receiver RT. Same free miRNA from plasma were determined. operating characteristic curve, clinicopathological PD-L1 expression was assessed on the surface of exo- features analysis and overallsurvival(OS)anddis- somes by TEM and HR-FCM. PD-L1 expression was ease-free survival (DFS) curve were made to evalu- also assessed on the surface of exosomes isolated from ate the clinical relevance of these circRNAs and plasma of healthy donors (n = 8). select circ1477 as potential biomarker for further Results: There was a slight difference in exosomal studies. Circ1477 overexpression and knockdown miRNA profile of plasma from HCC on the back- experiments were conducted to assess the effects ground of HCV and on the background of HCV and on GC progression in vitro and in vivo.RIP,luci- living in ergot region. PD-L1 expression on the surface ferase assay, RNA FISH and rescue experiments of exosomes from HCC plasmas were higher (MV were applied to demonstrate its molecular 35,8% for both HCC groups, MV 5% for healthy mechanism. donors group). Plasma free miRNA profiles were dif- Results: We found that the level of circ1477 in serum ferent inside every HCC group. or serum exosomes of GC patients was significantly Summary/Conclusion: According to our results, exo- higher than that in healthy volunteers. Whereas, the somal miRNA identification in HCC patients seem to level of circ1477 in cancer tissues and was remarkably be more accurate than plasma free miRNAs, further lower compared to adjacent normal tissues of GC research is needed in order to identify whether it is patients, which was associated with lymph node metas- reasonable to use both free and exosomal miRNAs. The tasis and prognosis. Also, the expression of circ1477 difference in miRNA profiles of HCC patients on the was observed to be decreased in GC cell lines while background of HCV or alkaloids of ergot may allow increased in their derived exosomes, suggesting that supposing different epigenetics dysregulation happen circ1477 could be selectively sorted into exosomes by in HCC depending on the trigger factor. GC cells. Furthermore, cytoplasmic circ1477 could suppress the migration and invasion of GC cells acting as miRNA sponges while knockdown of it could PT04.10 reverse these effects. Summary/Conclusion: Taken together, our findings Exosomal sorting of circRNA promotes cancer progression and serves indicate that tumour suppressive circ1477 could be as a novel biomarker for gastric cancer selectively sorted into exosomes to promote tumour Rong Lia, Junyi Wangb, Xu Zhangb, Hui Qianc and Wen Rong Xud progression and serve as a potential biomarker for a Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School gastric cancer. of Medicine, Jiangsu University, Zhenjiang, China, Zhenjiang, China (People’s Republic); bZhenjiang Key Laboratory of High Technology Funding: National Natural Science Foundation of Research on Exosomes Foundation and Transformation Application, China: (81572075); Technology Development Project Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of Medicine, Jiangsu University, Zhenjiang, China (People’s of Jiangsu University (20180361) 64 ISEV2019 ABSTRACT BOOK

PT04.11 localised in extracellular vesicles (EVs) than in cellular material. A simple and reliable process was optimised to concentrate urinary EVs and a novel method was Development of non-invasive tests for prostate cancer developed to specifically isolate the EV’s of prostatic a a a a David J. Whittaker , Bianca Dobson , Clare Elton , Damian White , Nancy origin with high efficiency. Subsequently a clinical Frédérickxa, Nicola Jacksona, Niha Phukana, Mike Herberta, Rebecca Girvana, Lia Boottena, Genevieve Johnstonb, Dug Yeo Hana, Ben Currana, Jennifer study was performed using qRT-PCR to quantify Barnesa, Robert Mitchella and Keith Hudsonb RNA biomarkers in approximately 300 urine samples aCaldera Health, Auckland, New Zealand; bCaldera Health, Auckland, USA collected from men scheduled for prostate biopsy tests. The clinical study participants provided informed con- Introduction: Prostate cancer (PC) is the most common sent and the study was approved by recognised medical non-skin cancer in males and is fast becoming the most ethics committees in New Zealand and Australia. frequently diagnosed cancer in men. Despite significant Results: Comparison of the qPCR data for prostate, advances in diagnosis and treatment PC remains a lead- bladder and kidney-specific genes indicated our pros- ing cause of cancer deaths. Analysis of PSA in blood has tate vesicle isolation method successfully reduces con- long been used for early diagnosis and monitoring but is tamination with vesicles from both kidney and bladder. flawed by low sensitivity and a high rate of false posi- The clinical study data was used to develop accurate tives, with negative health consequences including the prostate cancer diagnostic models. overtreatment of many indolent prostate cancer Summary/Conclusion: Caldera Health has identified tumours. Caldera Health is developing non-invasive EV RNA biomarkers associated with prostate cancer liquid biopsy tests for prostate cancer to improve upon and developed a novel method to specifically isolate and replace the controversial serum PSA test. prostate-derived EVs from urine. We have tested multi- Methods: Through a series of clinical studies, Caldera ple biomarkers and developed gene signatures identify- Health has identified promising RNA biomarkers for ing prostate cancer with high sensitivity and specificity. PC diagnosis. Preliminary experiments indicated that in urine a far greater proportion of prostate RNA is JOURNAL OF EXTRACELLULAR VESICLES 65

PT05: EV Biogenesis Chairs: Imre Mager, Hollis Cline Location: Level 3, Hall A 15:30–16:30

PT05.01 Summary/Conclusion: Optimising EVs may generate highly efficacious and cost-effective treatments in comparison to those based on the producer cell line. Uncovering the role of heparan sulphate proteoglycans in extracellular vesicle biogenesis: potential tools for improved Alterations to the HS structures on syndecan could be therapies an ideal method for optimisation. Rebecca L. Morgana, Rebecca Holleyb, Jason Webberc, David Oniond, Cathy Merryd and Oksana Kehoee Funding: This PhD project is funded by EPSRC aKeele University, Nottingham, UK; bThe University of Manchester, and MRC. Manchester, UK; cCardiff University, Cardiff, UK; dUniversity of Nottingham, Nottingham, UK; dKeele University, Oswestry, UK PT05.02 Introduction: Many cell types deliver therapeutic effects by secreting extracellular vesicles (EVs). Augmentation by GnRH of ectosome containing annexin A5 Therefore, EVs could be used as an alternative formation by blebbing of pituitary gonadotropes and its biological approach to cell-based therapies, overcoming many effect Mitsumori Kawa “a” minamia, Fungbun Numfab, Makoto Sugiyamac, Ryota cell-associated challenges. EVs could be optimised to Terashimad and Shiro Kurusue generate potent therapies through manipulating the aVeterinary Physiology, Faculty of veterinary medicine, Okayama University mechanisms driving EV biogenesis. We aim to prove of Science, Imabari, Ehime, Japan; bKhon Kaen University, Towada, Japan; this concept by altering the heparan sulphate (HS) cKitasato University, Towada, Japan; dVeterinary Physiology, Kitasato University, Towada, Japan; eVeterinary Physiology, Kitasato University, chains found on syndecan, a key component in the Towada, Japan syndecan-syntenin-ALIX mechanism. We predict that HS is involved in cargo selection due to its ability to Introduction: We have demonstrated that gonadotro- form interactions with a wide range of factors. In pin releasing hormone (GnRH) stimulates the synthesis addition, the structure of HS influences the activity of of annexin A5 (ANXA5), a member of annexin family heparanase, a regulator in the rate of EV production. protein, in the pituitary gonadotropes and ANXA5 Therefore, structural alterations to HS could allow the augments GnRH stimulation of gonadotropin secre- cargo (thus therapeutic activity) to be modulated whilst tion. It is, however, obscure how ANXA5 augments simultaneously increasing EV yields. gonadotropin release at gonadotropes. As ANXA5 Methods:MCF-7smutatedtoalterexpressionofHS was demonstrated both in and out of cells, in the biosynthetic enzymes were generated using CRISPR- present study, we examined translocation of ANXA5 Cas9. Wild type and mutant MCF-7s were cultured in in response to GnRH stimulation in relation to the bioreactors using media containing EV-depleted release of luteinizing hormone (LH). Knockout Serum Replacement. EVs were isolated by Methods: Rat pituitary tissues, primary pituitary cells differential ultracentrifugation and characterised and LβT2 gonadotrope cells were used. The condi- using Transmission Electron Microscopy (TEM), tioned medium was sequentially centrifuged at 20,000 Nanoparticle Tracking Analysis (NTA) and Western ×g and 110,000 ×g to obtain ectosome and exosome Blot. respectively. Immunochemistry for ANXA5 and LHβ Results: A FACS-based method has been developed to were performed. Transmission electron-microscope characterise and sort EVs based on their displayed HS. (TEM) was also used. The cargo and functional activity of the sorted popula- Results: GnRH agonist (GnRHa) administration tions was then assessed. Since heparanase influences showed the formation of blebs containing ANXA5 on EV production rates, MCF-7s were incubated with a LβT2 cells and primary pituitary cells after only 10 and heparanase inhibitor (OGT2115). Subsequent altera- 30 min incubation. Hemi-pituitary gland was cultured tions to soluble, cellular and vesicular HS composition with GnRHa and TEM showed that the boundary of was analysed by fluorescent labelling and SAX-HPLC GnRHa stimulated gonadotrope-like cell became identification. EV size and concentration was assessed obscure with many bubble like particles after 30 min using TEM and NTA. incubation. The 20,000 ×g and 110,000 ×g particles 66 ISEV2019 ABSTRACT BOOK were increased by the GnRHa treatment. ANXA5 was Mass spectrometry EV identification: Western Blot, detected dominantly in 20,000 ×g pellet after treatment Co-immunoprecipitation with GnRHa. It increased until 180 min. ANXA5 in Results:CypAisfoundtobeenrichedincancer-derived 110,000 ×g pellet was also shown at 180 min. GnRHa EVs in a range of solid and haematopoietic malignancies. treated 20,000 ×g particulate fraction significantly sti- In addition, CypA is predominantly found in EVs within a mulated LH release in a dose dependent manner. specific density range. Moreover, homozygous loss of Extracellular vesicle fraction prepared from plasma of CypA expression reduces the number of EVs within a one-week ovariectomized rats, in which GnRH secre- specific size range. Investigation of CypA interacting pro- tion was expected to be augmented, showed significant teins by mass spectrometry reveals potential functions in increase of ANXA5 in the 20,000 ×g pellet. The bleb- EV cargo loading. bing induced by GnRH was inhibited by H89, protein Summary/Conclusion: This study reveals a potential kinase A inhibitor. It is suggested that Gαs signalling is role for CypA in EV biogenesis, and highlights its necessary for GnRH stimulation of blebbing. potential as a novel EV target for the prevention of Summary/Conclusion: Present study clearly demon- tumour progression. Significance of this study is that strates a hormonal regulation of ectosome formation CypA could be a potential target for EV release. This and a novel mechanism of cell–cell communication by work contributes to the understanding of CypA-depen- means of ANXA5 including ectosome. dent EV subtype for its biology and function during cancer metastasis and may reveal novel strategies for the generation of targeted EV subtype therapeutics. PT05.03 Funding: UCD-CSC Scholarship (not include travel funding).

Investigation into a novel role for the prolyl isomerase cyclophilin A during Extracellular vesicle signaling in cancer Yunjie Wua, Kieran Brennanb and Margaret M. Mc Geea PT05.04=OWP2.12 aUCD School of Biomolecular & Biomedical Science, Conway Institute, University College Dublin, Dublin, Ireland; bUniversity College Dublin, Ireland Identification of a protein that presumably controls bacterial Introduction: Extracellular vesicles (EVs) released vesiculation in response to the extracellular environments Fumiaki Yokoyamaa, Jun Kawamotoa, Chen Chena, Tomoya Imaib and – a from cells mediate local and systemic cell cell commu- Tatsuo Kurihara nication via the horizontal transfer of functional pro- aInstitute for Chemical Research, Kyoto University, Uji, Japan; bResearch tein, DNA and RNA into recipient cells. Evidence Institute for Sustainable Humanosphere, Kyoto University, Uji, Japan reveals that tumour-derived EVs mediated intercellular communication between tumour cells and normal cells Introduction: Many bacteria utilize extracellular mem- within the tumour microenvironment to initiate meta- brane vesicles (EMVs) for survival in their growing static niche formation. Thus, disruption of EV- environments through communication with others, mediated tumour-niche interactions is a novel strategy pathogenesis and biofilm formation. Therefore, the for metastasis prevention. However, significant chal- amounts and the components of EMVs should be lenges in EV biology must be overcome for the transla- tuned in response to the conditions. Although several tion of EVs into the clinic; in particular, in vesiculation mechanisms are suggested, little is known understanding their biogenesis and mechanism of how bacteria control vesiculation in response to the action within the tumour microenvironment. The pro- environments. A bacterium Shewanella sp. HM13 has lyl isomerase Cyclophilin A is overexpressed in a large 9-fold higher lipid-secretion capability in EMV frac- variety of cancers and is associated with an aggressive tions than Escherichia coli, and its EMVs contain a phenotype of metastasis and chemoresistance. major protein (P49), which is not required for vesicle Unpublished data from our lab revealed that loss of production. We used mutant EMVs that lack P49 to CypA expression significantly reduced tumour growth identify minor components of EMVs that may control and metastasis in vivo supporting a role in tumour vesiculation. progression. In this study, potential functions of Methods: EMVs were subjected to 2D gel-based pro- CypA in EV biology and function are investigated. teomics by peptide mass fingerprinting. Within the Methods:EVIsolation:DifferentialUltracentrifugation, identified proteins, the function of a sensor protein Optiprep Density Gradient homolog, HM1275, was analysed by swarming assay EV characterization: Nanosight Tracking Analysis, and lipid-staining to quantify EMVs produced in var- Flow cytometry, Transmission Electron Microscopy, ious media. Changes in the number of EMVs JOURNAL OF EXTRACELLULAR VESICLES 67 depending on culture media were quantified by tunable intracellular signalling. While advances in protein resistive pulse sensing method. domain prediction have facilitated the identification Results: A protein with a PAS domain and a methyl- of several BAR domain proteins, they have yet to be accepting chemotaxis protein (MCP) sensing domain, characterized in bacteria. Here, we identified a putative HM1275, was identified in the EMVs. Although some BAR domain-containing protein enriched in the outer MCPs are related to flagellar motility by binding some membrane vesicles (OMVs) of Shewanella oneidensis attractants, the flagellar motility of Delta-hm1275 was MR-1, a dissimilatory metal-reducing bacteria known not significantly different from that of WT. Although to produce outer membrane extensions (OMEs) that the amounts of EMVs produced by WT were increased are suspected to facilitate long distance extracellular in response to the concentration of casamino acids in electron transfer (EET) but whose physiological rele- poor nutrient medium, those by Delta-hm1275 vance and mechanism of formation remain unknown. were not. Methods: Purified S. oneidensis OMVs were prepared Summary/conclusion: A putative sensor protein, by filtration and ultracentrifugation for comparative HM1275, was identified in EMVs and may recognize proteomics with cell-associated outer membrane pro- the extracellular environments by binding signal mole- teins or for electrochemical measurements. Protein cules in casamino acids to control vesiculation. domains were predicted using HMMSCAN and Although further studies are required to reveal the CDD-search. OME formation and phenotype analyses signals and the sensing pathways, the results obtained were performed in situ by confocal and cryo-electron in this study indicate that bacterial vesiculation is con- microscopy. trolled by extracellular environments, and artificial Results: The putative BAR domain-like protein BdpA control of vesiculation with extracellular signals was highly enriched in OMVs compared to cell-asso- would be useful in applications such as suppression of ciated outer membranes. During OME biogenesis, WT vesicle-dependent pathogenicity. S. oneidensis OMEs progress from elongated vesicle Funding: Japan Society for Promotion of Science chains to narrow, tubule-like extensions while ΔBdpA Research Fellowship for Young Scientists OMEs remain as disordered vesicle chains. Purified OMVs from these strains are electrochemically active, PT05.05=OWP2.13 with redox signals consistent with multiheme outer membrane cytochromes, supporting the role of OMEs in EET. Heterologous BdpA expression promotes OME Prokaryotic BAR domain-like protein BdpA promotes outer formation in Marinobacter atlanticus and Escherichia membrane extensions Daniel A. Phillipsa, Lori Zacharoffb, Cheri Hamptonc, Grace Chongb, Brian coli, suggesting BdpA membrane sculpting activity is Eddied, Anthony Malanoskid, Shuai Xub, Lauren Ann Metskase, Lina Birdf, inducible and transferrable. Grant Jensene, Lawrence Drummyc, Moh El-Naggarb and Sarah Glavend Summary/conclusion: The ability of BdpA to promote aAmerican Society for Engineering Education – U.S. Naval Research Laboratory, Washington, USA; bUniversity of Southern California, Los OME formation and maturation into tubules in vivo Angeles, USA; cMaterials and Manufacturing Directorate, Air Force supports BdpA as a comparator for BAR domain pro- d Research Laboratory, Dayton, USA; U.S. Naval Research Laboratory, tein activity in bacteria. Washington, USA; eCalifornia Institute of Technology, Pasadena, USA; fNational Research Council, Washington, USA Funding: US DoD Synthetic Biology for Military Environments (SBME) Applied Research for the Introduction: Bin/Amphiphysin/RVS (BAR) domains Advancement of Science and Technology Priorities belong to a superfamily of membrane-associated (ARAP) coiled-coil proteins that influence membrane curva- NSF Dimensions: DEB-1542527 ture. BAR domains are ubiquitous in eukaryotes and US DOE: DE-FG02-13ER16415 associated with membrane curvature formation, vesicle biogenesis/trafficking, protein scaffolding and 68 ISEV2019 ABSTRACT BOOK

PT06: EV Cancer Immunology Chairs: Jason Webber; Koichi Furukawa Location: Level 3, Hall A 15:30–16:30

PT06.01 demonstrated by increased IL-2 secretion from melanoma antigen gp100-specific T cells. Summary/Conclusion: It was shown that CD40L- Development of CD40L-modified small extracellular vesicles for the modified sEVs can be used to induce effective anti- effective induction of anti-tumour immune response tumour immune response. Wen Liua, Yuki Takahashib, Masaki Morishitac, Makiya Nishikawad and Yoshinobu Takakurab aKyoto University, Kyoto, Japan; bGraduate School of Pharmaceutical PT06.02 Sciences, Kyoto University, Kyoto, Japan; cKyoto Pharmaceutical University, kyoto, Japan; dTokyo University of Science, Noda, Japan Development of Interferon γ-loaded tumour cell-derived extracellular Introduction: Tumour-derived small extracellular vesi- vesicles applicable to cancer vaccine therapy a b b cles (sEVs) are anticipated to be a novel cancer vaccine Naoki Nakagawa , Yuki Takahashi and Yoshinobu Takakura because of their inherent encapsulation of tumour anti- aGraduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan; bGraduate School of Pharmaceutical Sciences, Kyoto University, gens. In this research, tumour-derived sEVs were mod- kyoto, Japan ified with CD40 ligand (CD40L), which is a ligand for CD40 expressed on dendritic cells (DCs) and can acti- Introduction: Extracellular vesicles (EVs) contain var- vate DCs, in order to induce effective tumour antigen- ious substances such as proteins and nucleic acids specific immune response. derived from their producing cells. As tumour cell- Methods: B16-BL6 murine melanoma cells were derived EV (TEV) contains tumour antigens, TEV is selected as sEVs-producing cells. Plasmid vector expected to be used as a cancer vaccine. However, since encoding a fusion protein of CD40L and lactadherin the immune activation ability of TEV is low, it is (LA), named as CD40L-LA, was constructed. B16-BL6 difficult to induce effective anti-tumour immunity by cells were transfected with the CD40L-LA-expressing simple administration of TEV alone. Hence, in this plasmid vector and CD40L-modified sEVs (CD40L- study, we attempted to enhance the immune activation sEVs) were collected from the culture medium of the ability of TEV by loading Interferon (IFN)-γ. transfected cells. The collected sEVs were characterized Methods: A plasmid vector encoding a fusion protein by using western blot, zeta sizer and transmission elec- of lactadherin that specifically bind to phosphatidylser- tron microscope (TEM). CD40L-sEVs labelled with ine contained in EV membrane and mouse IFN-γ was PKH67 were added to DCs and the uptake of CD40L- prepared and the vector was transfected into a mouse sEVs was evaluated by flow cytometer. CD40L-sEVs melanoma cell line B16BL6 cells. Then, IFN-γ-loaded were added to DCs and the cytokine release from the TEV (γ-TEV) was collected from the supernatant of cells was measured by ELISA. Presentation of mela- the transfected cells by ultracentrifugation. IFN-γ noma antigens contained in sEVs were evaluated by loaded on the collected TEVs was detected by measuring cytokine release from melanoma antigen Western blotting and ELISA. IFN-γ biological activity gp100-specific T cells, which were co-incubated with of IFN-γ loaded on γ-TEV was evaluated by a reporter CD40L-sEVs added DCs. The concentrations of cyto- assay. In addition, γ-TEV was added to the mouse kines in the culture medium were determined using dendritic cell line, DC 2.4, and mRNA and protein ELISA. expression levels of antigen presentation-related genes Results: The negatively charged sEVs with a diameter were analysed using RT-qPCR and FACS analysis. of approximately 100 nm were successfully modified Finally, splenocytes of mice that had received intrader- with CD40L. CD40L-sEVs were more efficiently taken mal administration of γ-TEV were collected and the up by DCs than unmodified sEVs. DCs added with amount of IFN-γ produced from the splenocytes incu- CD40L-sEVs produced more TNF-alpha and IL-12 bated with B16BL6 antigens was measured. than those added with unmodified sEVs. Moreover, Results: It was confirmed that IFN-γ was successfully CD40L-modification of sEVs improved the melanoma loaded to TEV. In addition, the reporter assay con- antigen presentation efficiency of DCs, which was firmed that the biological activity of IFN-γ was retained JOURNAL OF EXTRACELLULAR VESICLES 69 in γ-TEV. Addition of γ-TEV to DC 2.4 increased Summary/Conclusion: Although MSCs are commonly mRNA and protein expression of MHC class I and known to have an immunosuppressive function, after CD86 compared to TEV alone group, which suggests the uptake of EVs derived from apoptotic neuroblas- that immune activation ability of TEV was increased by toma, MSC was able to switch to an immunostimula- loading IFN-γ. Furthermore, in the splenocytes assay, tory phenotype and decreasing Treg differentiation. the amount of IFN-γ production was significantly Dying tumour cells may package danger signals and increased in the γ-TEV administration group com- alarmins in their EVs thereby activating immune pared with the group administered with simple mixture response in the tumour microenvironment. of IFN-γ and unmodified TEV. Funding: The Edward & Yolanda Wong Research Summary/Conclusion: These results indicated that Fund IFN-γ loading to TEV is an effective approach for cancer immunotherapy using TEV. PT06.04 PT06.03 Chronic Lymphocytic Leukaemia-derived small extracellular vesicles: a potential strategy for immune escape Ernesto Gargiuloa, Sandrine Piersonb, Bassam Janjia, Jérôme Paggettia and Apoptotic neuroblastoma derived extracellular vesicles can prime Etienne Moussaya mesenchymal stem cells to decrease regulatory T cells differentiation a Anita KY. Li and Godfrey Chan Luxembourg Institute of Health (LIH), Department of Oncology, Luxembourg, Luxembourg; bLuxembourg Institute of Health (LIH), The University of Hong Kong, Hong Kong, Hong Kong Department of Oncology, Luxembourg, Luxembourg

Introduction: There are many ongoing studies investi- Introduction: Chronic Lymphocytic Leukaemia (CLL) gating tumour derived extracellular vesicles (EVs). Yet is the most common adult leukaemia and characterized in cancer patients receiving chemotherapy, a majority of by the accumulation of abnormal B lymphocytes. CLL the tumour are undergoing apoptosis and the difference cell survival and proliferation are highly dependent on between health cancer and dying cancers EVs are still interactions with the microenvironment. Thus, to iden- unknown. Apoptotic tumour cells can secrete EVs con- tify effective strategies to impair tumour proliferation, taining different messages to the tumour microenviron- it is essential to understand the communication ment and effect the surrounding cells in a different way. between CLL and surrounding tissues. Mesenchymal stem cell (MSC) is a heterogeneous multi- Methods: To obtain a biological representation of small potent stem cell found within the tumour microenvir- extracellular vesicles (small Evs) in the tumour micro- onment and can regulating the immune system. The environment, we established a new protocol allowing aim of this study is to investigate the role of apoptotic us to isolate highly pure small Evs directly from the EVs on mesenchymal stem cell immunomodulatory spleen of leukemic mice. Small Evs quality and sample function in a tumour microenvironment. purity were evaluated with qNano (TRPS principle), Methods: EVs were obtained from both healthy SK-N- western blot and conventional bead-based flow cyto- LP neuroblastoma cell line and those treated with the metry. Next, we screened a wide range of immune chemo drug cisplatin for 24 h. EVs were isolated from checkpoint ligands on the surface of CLL-derived ultracentrifugation at 16,000 g for larger EVs and small Evs and corresponding receptors on the surface 100,000 g for smaller EVs. The characterization of the of T cells. different populations of EVs was performed by western Results: We have succeeded in isolating small Evs blot and nanoparticles tracking analysis. Neuroblastoma generated by CLL cells in vivo. Our screen suggested derived EVs were then co-cultured with immortalized the presence on immune checkpoint ligands directly human MSC (hTMSC) for 48 h. The immunomodula- anchored on tumour-derived small Evs. Furthermore, tory function of hTMSC was determined by their effect we identified a promising pair ligand-receptor poten- on T cells isolated from PBMC. tially implicated in immune escape. Validation of can- Results: T cells co-cultured with hTMSC have an didates from the screen is currently being performed increase in FoxP3 expression whereas hTMSC that through FACS, iFACS and EM. These techniques will has been primed with apoptotic EVs from neuroblas- allow us to better define tumour-derived small Evs toma showed a significant decrease in FoxP3 expres- populations presenting different immune checkpoints sion. The DAMP molecule HMGB1 was found to be and to visualize single small Evs with high resolution. present in apoptotic EVs, whilst being absent in healthy Summary/Conclusion: In this project, we aimed to neuroblastoma EVs. isolate and characterize CLL-derived small Evs to 70 ISEV2019 ABSTRACT BOOK define their involvement in tumour development, with regulatory T cells (Treg) that were sorted from normal focus on the evaluation of their impact on CLL peripheral blood. The exosomes were detected in cyto- immune escape. sol of Treg by fluorescent microscopy. Microarray ana- Altogether, this study will give insight into the spe- lysis of miRNAs in Treg intaking MDS-exosomes cific immune and stromal cells involved in CLL devel- showed that significant increases of 9 miRNAs in opment, with emphasis on their involvement in MDS-exosomes. The conditioned medium of MDS- tumour-derived small Ev-mediated tumour immune exosomes treated Treg culture reduced the population escape. of activated CD4 cells (CD38 positive cells was 39%; Funding: This project is funded by the Fonds National control 68%). de la Recherche (FNR) INTER/DFG/16/11509946/EV- Summary/Conclusion: Our data suggested that exo- RNA/Moussay. somes from MDS cells affected the function of regula- Sandrine Pierson and Jérôme Paggetti are supported tory T cells via miRNA transfer. MDS exosomes may by the FNR INTER/DFG/16/11509946/EV-RNA/ effect on immune cells to avoid the exclusion from Moussay. cancer-immune system, and may be a target for the Ernesto Gargiulo is supported by the grant FNR new therapies or diagnostic methods. Luxembourg PRIDE15/10675146/CANBIO. Funding: This work was supported in part by a grant from the Japan Society for the Promotion of Science (JSPS KAKENHI Grant Number: JP17K09020 and PT06.05 17H07059).

Interaction via exosome miRNAs between myelodysplatic cell and PT06.06 normal Treg Tatsuki Shibuta, Yukichi Takada and Tsukuru Umemura

International University of Health and Welfare, Okawa City, Japan Mechanism of antitumor immunity activation by ‘artificial neoantigen’-presenting exosomes Introduction: Myelodysplastic Syndrome (MDS) is a Yoshiyuki Koyamaa, Tomoko Itoa, Masazumi Eriguchia, Aya Hasegawab, c b b clonalhematopoietic disease and develops leukaemia in Wakana Ouchi , Toshio Inaba and Kikuya Sugiura some cases. Thus, MDS is a malignant hematopoietic aJapan Anti-tuberculosis Association, Shin-Yamanote Hospital, Tokyo, Japan; bOsaka Prefecture University, Osaka, Japan; cOsaka Prefecture University, disease and its prevalence ratio is increasing in Japan. Tokyo, Japan Hematopoietic microenvironment such as bone marrow niche is a crucial factor for maintaining leukaemic Introduction: Tumour-derived exosomes are known to stem cells. To understand mechanisms of interactions have same antigens as the parent tumour cells, and between leukaemic stem cells and microenvironment is were expected as cancer vaccines. However, treatment important for the treatment of hematopoietic with those exosomes often failed to elicit antitumor malignancies. immune responses, probably owing to a weak immu- In this study, to develop the new therapies and nogenicity of the tumour-associated antigens (TAAs). diagnostic methods for MDS, we focused on the effect TAAs can be divided into two categories, overex- of exosomes released from MDS cells on peripheral T pressed self-antigens and tumour-specific mutated lymphocytes. neoantigens. Recently, it was found that immunother- Methods: MDS cell line (MDS-L) was kindly provided apy was effective only in patients whose tumours had by Kasawaki Medical University and normal peripheral neoantigens with high immunogenicity. However, most blood mononuclear cells were obtained from healthy tumours do not have such efficient neoantigens. To volunteer donors. Exosomes from MDS cells were pur- overcome the disadvantage, we have developed an ified by using miRCURY Exosome Cell/Urine/CSF Kit “artiﬁcial neoantigen strategy“. Previously, we prepared and labelled by PKH67. Extracted miRNAs were ana- the exosomes expressing strong bacterial antigen, as an lysed by microarray method (Genopal, Mitsubishi “artificial neoantigen” by transformation of the original Chemical, Japan). Cell surface antigens were analysed cultured cells with a gene of the Mycobacterium tuber- by FACS Aria II and fluorescence conjugated culosis antigen, early secretory antigenic target-6 antibodies. (ESAT-6). Injection of the exosomes induced signifi- Results: miRNA-microarray analysis showed that nine cant antitumor activity in tumour bearing mice. Here miRNAs were abundant in exosomes from MDS cells we investigated the mechanism of antitumor immunity and were not detected in MDS cells. Exosomes labelled activation by the ”artificial neoantigen”-presenting with PKH67 dye were added to liquid culture of exosomes. JOURNAL OF EXTRACELLULAR VESICLES 71

Methods: Mouse B16 melanoma cells were transfected differentiated with PMA and HGFs were treated with with ESAT-6, and secreted ESAT-6 antigen-presenting T. forsythia and OMVs at various does. The expression exosomes (ESAT-Ex) were isolated. Cultured mouse of caspase-1/4 and pro-inflammatory cytokines was DCs were treated with the exosomes and expression determined by immunoblotting and ELISA, respec- of costimulatory molecules and cytokines was assessed. tively. Cell death was measured by LDH cytotoxicity Antitumor activity of the ESAT-Ex-stimulated DCs was assay kit. evaluated in tumour bearing mice. In vivo immune Results: T. forsythia OMVs activated caspase-1 and −4, activating ability of ESAT-Ex was investigated by co- resulting in increased IL-1α and IL-1β release and incubation of the lymphocytes taken from the ESAT- inflammatory cell death. The OMVs also induced the Ex-treated mice with B16 tumour cells. Immuno-sti- expression of IL-6 and IL-8. mulation by the fibroblasts-secreted ESAT-Ex was also Summary/Conclusion: The results indicated that T. studied. forsythia OMVs may play an important role in inflam- Results: The DCs stimulated with ESAT-Ex showed matory response in T. forsythia-infected cells. enhanced CD80 and CD86 presentation, and exhibited Funding:ThisworkwassupportedbyNationalResearch significantly improved antitumor activity in tumour- Foundation of Korea grants (No. NRF-2018R1A5A bearing mice. When the lymphocytes harvested from 2024418 and NRF-2018R1A2A2A05018558). the mice injected with ESAT-Ex were co-incubated with B16 cells, they intensely accumulated around the PT06.09 tumour cells, and secreted higher level of IFN-gamma. Increased uptake of thymidine was also observed. Summary/Conclusion: “Artificial neoantigen”-pre- Specific decrease of CD19+extracellular vesicles enhances post- chemotherapeutic CD8+T cell responses senting exosomes effectively stimulated DCs and Fanghui Zhang, Xuefeng Fei and Zhijian Cai evoked antitumor immunity. They are expected as Institute of Immunology, School of Medicine, Zhejiang University, novel cancer vaccines. Hangzhou, China (People’s Republic) Funding: This work was supported by JSPS KAKENHI Grant Number 16K01394. Introduction: Chemotherapy has long been related with induction of systemic immunosuppression. PT06.07 Systemic immunosuppression greatly affects chemotherapeutic antitumor effect. Therefore, amelioration of systemic immunosuppression following Outer membrane vesicles of Tannerella forsythia induce inflammatory chemotherapy is necessary to improve post-chemother- response in periodontal tissue cells Sunjin An apeutic antitumour immunity. Methods:CD19+EVswereisolatedandidentifiedbyEM, Department of Oral Microbiology and Immunology, School of Dentistry, Seoul National University, Seoul, Republic of Korea NTA, FACS and western blotting. CD19+EVs functions on degrading ATP and their immunosuppressive func- Introduction: Tannerella forsythia, a Gram-negative tions were assessed in vitro and in vivo.Theeffectsof oral bacterium, is one of the major periodontal patho- CD19+EVs on antitumour effect of chemotherapy were gens which can cause inflammatory responses. detected by transfer of exogenous EVs into tumour mice Inflammasome is crucial for host defence against or in Rab27a or Hif1a conditional knockout tumour pathogens, but excessive inflammasome activation can mice. The effects of CD19+EVs on antitumor effect of lead to tissue damage. Outer membrane vesicles chemotherapy were also evaluated in humanized NSG (OMVs) are derived from the cell envelope of Gram- mice by knocking down Rab27a with inactivated EBVs negative bacteria. OMVs can contain DNA, RNA, lipo- loading with Rab27a siRNA. polysaccharide, proteins, toxins and peptidoglycan. T. Results:CD19+extracellularvesicles(EVs)fromBcells forsythia induces maturation of IL-1α/IL-1β and cell hydrolyse ATP from chemotherapy-treated tumour cells death via activation of caspase-1/4 in THP-1 macro- into adenosine by CD39 and CD73, resulting in impaired phages and human gingival fibroblasts (HGFs). The CD8+T cell responses. Serum CD19+EVs increase in aim of this study was to investigate whether T. forsythia tumour mice and patients. Patients with fewer serum OMVs are involved in inflammasome activation which CD19+EVs have a better prognosis following chemother- may contribute to periodontitis, a chronic inflamma- apy. Up-regulated HIF-1α promotes B cells releasing tory disease. CD19+EVs by inducing Rab27a mRNA transcription. Methods: T. forsythia OMVs were isolated using Exo- Rab27a or HIF-1α deficiency in B cells inhibits CD19 bacteria OMVs isolation kit. THP-1 macrophages +EV production and strikingly improves 72 ISEV2019 ABSTRACT BOOK chemotherapeutic antitumor effect. Knockdown of Funding: This work was supported by grants from the Rab27a in B cells by inactivated Epstein-Barr viruses National Key Basic Research Program of China carrying Rab27a siRNA greatly improves chemotherapeu- (2015CB943301), the National Key R&D Program of tic efficacy in humanized NSG mice. China (2016YFA0501800), and the National Natural Summary/Conclusion: Our findings unravel a mechan- Science Foundation of China (31770951 and ism underlying systematic immunosuppression after che- 31670877). motherapy. Combination of chemotherapy and iEBVs/ Rab27a siRNA holds high potential for cancer treatment. JOURNAL OF EXTRACELLULAR VESICLES 73

PT07: EVs in Acute and Chronic Inflammatory Disorders Chairs: Eric Boilard; Aleksandra Gasecka Location: Level 3, Hall A 15:30–16:30

PT07.01 340 (−13.5, p = 0.006), miR-29b (−12.8, p = 0.001), miR-744 (−7.1, p = 0.05), miR-618 (−4.0, p = 0.02), miR-598 (−3.8, p = 0.035), miR-1260 (−2.5, p = 0.035); Circulating microvesicles as potential biomarkers of Acute Respiratory Distress Syndrome in Sepsis and miR-885-5p is expressed at higher levels (9.5; Marcelo Fernando do Nascimentoa, Ana Cláudia Aparecida Santos p = 0.028). In paired samples, the set of altered b c d Nussbaum , Ludhmila Abrahao Hajjar , Luciano Cesar Pontes Azevedo , miRNAs is generally different (p < 0.05) between José Mauro Vieira Juniord, Daniel Deheinzelind, Roger Chammasc, Juliana Monte. Realc sepsis + ARDS (miR-148a, −193a-5p, 199a-3p, −222, aInstituto do Servidor Publico Estadual, Santo André, Brazil; bHospital do −25, −340, 744) or sepsis only (miR-1183, −1267, Coracao, sao paulo, Brazil; cInstituto do Cancer do Estado de Sao Paulo, Sao −1290, −17, −192, −199a-3p, −25, −485-3p, d Paulo, Brazil; Hospital Sirio-Libanes, Sao Paulo, Brazil −518d, −720). Summary/Conclusion: Circulating EV-miRNAs cargo Introduction: Acute respiratory distress syndrome could be potential biomarkers of lung inflammation (ARDS) is a clinical condition of sudden respiratory during sepsis in patients who will require MV. failure in critically ill patients. ARDS-related mortality Funding: FAPESP. rate is higher when is associated with Sepsis (>50%). Recently, we screened 754 miRNAs and discovered a different cargo transported by circulating extracellular PT07.02 vesicles (EVs) and exosomes from patients with sepsis, remarkably in those who progressed to death. The early sequence of events of respiratory failure after the onset Innate/ inflammatory cross talk between macrophages (Mps) and RPE of sepsis are still unknown. Our hypothesis is that lung cells are mediated by exosomes secreted by RPE cells: Proposal of new trait for the pathogenesis of age-related macular should signal through EVs that it is being affected degeneration (AMD) by SIR. Atsushi Mukaia, Eiko Itoa, Morio Uenoa, Shigeru Kinoshita, Chie Sotozonoa a Methods: Blood samples were obtained from septic and Junji Hamuro patients with (n = 8) and without ARDS (n =5)at aDepartment of Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan; bDepartment of Frontier Medical Science and Technology for 24 h of intensive care unit (ICU) admission and 3 days Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan later at Sirio-Libanes Hospital. Pulmonary originated sepsis was not considered. Eight patients under Introduction: The pathogenesis of AMD is aggravated mechanical ventilation (MV) without pulmonary dis- by chronic inflammation. Intact RPE down-regulates ease and 12 healthy volunteers were used as controls. the production of TNF-alpha by choroid-infiltrating Plasma was 0.22 µM filtered, EVs were isolated by Mps, whereas degenerated RPE by oxidative stress ultracentrifugation and analysed by nanoparticle track- were devoid of this regulatory function. Subsequently, ing analysis. Based on our previous data, 48 miRNAs locally produced TNF-alpha induces the production of were measured by Taqman Low Density PCR array and some pro-inflammatory cytokines and angiogenic fac- normalized by RNU6. tor VEGF by RPE (Yamawaki et al., 2016). This implies Results: The main population of EVs peaked at size of that innate/inflammatory cross talk between Mps and 155–165 nm with no difference in the mean concen- RPE may be the indispensable trait for AMD tration between groups. Patients with sepsis + ARDS pathogenesis. showed a significant decrease in plasma EVs 3 days The purpose of this study is to elucidate the signal after ICU stay (234 to 137 x 10e8/mL, p = 0.0175). that causes up-regulated TNF-alpha production in con- Compared to healthy donors, sepsis promotes an even genital / inflammatory crosstalk between Mps and RPE. significant alteration of EVs-miRNAs when it is asso- Methods: Mps cell line RAW 264.7(RAW) was co- ciated with ARDS. Comparing all samples from cultured with primary RPE taken from C57BL/6 mice. patients with sepsis + ARDS to sepsis only, nine Some cytokines in the culture supernatants (CSs) were miRNAs are transported in smaller amounts: miR-766 quantified by ELISA. The expression profiles of com- (−35.7, p = 0.002), miR-127 (−23.8, p = 0.001), miR- plement-associated genes, TNF-alpha, and 74 ISEV2019 ABSTRACT BOOK angiogenesis-associated genes (VEGF & PEDF) were signalling pathway by its dephosphorylation function. analysed by qRT-PCR. For the preparation of exosomes Here we reveal that Shp2 inhibits the biogenesis of (Exo), CSs were harvested after co-cultures of RAW epithelial exosomes which have proinflammatory with primary RPE, then Exo in each CSs were purified effects on macrophages during ALI. It’s uncovered in by either EVsecondTM or ultracentrifugation. The our study that Shp2 is a protective factor of ALI by incorporation of the Exo either into RPE or RAW inhibiting release of proinflammatory epithelial was histologically quantified using Qdot 655 streptavi- exosomes. din conjugated biotinylated Exo. Methods: Exosomes were isolated by differential ultra- Results: Elevated levels of CD63 positive Exo in co- centrifugation and filtration, and they were character- cultures were detected by western blot or FACS analy- ized by nanoparticle tracking analysis (NTA), sis. The produced Exo in co-culture CSs were incorpo- transmission electron microscopy (TEM) and western rated solely into RAW, but not into RPE. The semi- blot (e.g. CD9, CD63, CD81, ALIX, TSG101). In vitro purified Exo, but not the Exo depleted residual CSs transwell system for exosome transfer model indicated enhanced the secretion of MCP-1 and IL-6 in co-cul- the direction of exosome transfer. Nanoscale flow cyto- ture of Mps and RPE, while the enhancement of VEGF metry (CytoFLEX) was used for detecting exosome are similarly detected by the Exo deprived residual CSs. subpopulation. Most remarkable elevation was observed in TNF-alpha Results: Exosomes were increased in Bronchoalveolar production by RAW in a dose-dependent manner even Lavage Fluid (BALF) of LPS-induced ALI murine in the absence of RPE. The down-regulated TNF-pro- model. In vitro transwell system revealed that exosomes duction by RAW in the presence of RPE was not were transferred from epithelial cells to macrophages reconstituted by the addition of Exo even in the co- in inflammation environment. Shp2 was revealed to culture. inhibit the biogenesis of epithelial exosomes without Summary/Conclusion: Exosome displays a critical role changing their size and subpopulation. Adaptor protein in the triggering of vicious inflammatory cytokines Gab2, which can bind Shp2, was found to interact with cycle through the elevation of TNF- production Syntenin. It suggests that with the help of adaptor by Mps. Gab2, Shp2 was involved in dephosphorylating synte- Currently, in order to construct an experimental nin whose phosphorylation can facilitate exosome biosystem closer to the pathology of AMD, we are study- genesis. Shp2-disruption derived epithelial exosomes ing a co-culture system using human Mps and human promoted macrophage inflammation, thus aggravat- iPS-derived RPE. ing ALI. Summary/Conclusion: Our study shows that phospha- PT07.03 tase Shp2 inhibits proinflammatory epithelial exosome release, which can promote M1-macrophage polarization. It offers a potential target for ALI mechanism Epithelial exosomes regulated by phosphatase Shp2 promote study and treatment. macrophage activation Yuefei Zhanga, Yiqing Lib, Dacheng Gongb, Hongqiang Chengb, Xue Zhangc b and Yuehai Ke PT07.04 aZhejiang University, Hangzhou, China (People’s Republic); bZhejiang University, School of Medicine, Hangzhou, China (People’s Republic); cZhejiang University, School of Medicine, Hangzhou, China (People’s Republic) Detection of CD11b-expressing exosomes in plasma of mice with sepsis Yasunori Fujita, Kyojiro Kawakami and Masafumi Ito Introduction: Acute lung injury (ALI) and its more Research Team for Mechanism of Ageing, Tokyo Metropolitan Institute of severe form, acute respiratory distress syndrome Gerontology, Itabashi-ku, Japan (ARDS), are life-threatening diseases that are associated with high mortality rates due to treatment lim- Introduction: Cells communicate with each other itations. Increasing researches suggest exosomes play through extracellular vesicles including exosomes, an important role in pathogenesis, diagnosis and treat- which contain host cell-derived molecules such as pro- ment of ALI. However, it’s not clear how exosomes are teins, lipids and nucleic acids. Secreted exosomes formed, secreted, transferred during ALI. migrate not only to neighbouring cells but also to Phosphorylation of signalling proteins are reported to distant organs. Monocyte and macrophage have been control exosome biogenesis (e.g. syntenin phosphory- reported to secret exosomes that modulate immune lation promotes exosome formation). Shp2 is a widely responses. However, the characteristics of monocyte/ expressed cytoplasmic phosphatase which can regulate macrophage-derived exosomes in blood during JOURNAL OF EXTRACELLULAR VESICLES 75 systemic immune response remain largely unknown. In inflammatory responses. In addition, proteomic com- this study, we characterized exosomes released from positions of fEVs were further investigated. monocyte/macrophage-like cells and determined the Methods: The faeces of wild-type mice were utilized to temporal change in monocyte/macrophage-derived isolate fEVs. The fEVs were characterized with trans- exosomes in plasma of mice with sepsis. mission electron microscopy, dynamic light scattering, Methods: Exosomes collected by ultracentrifugation ELISA, and Western blot. The fEVs were intraperito- from the conditioned medium of lipopolysaccharide neally administered into the mice, and the number of (LPS)-stimulated murine monocyte/macrophage-like infiltrated cells as well as the concentrations of TNF-α RAW264.7 cells were subjected to quantitative proteo- and IL-6 were measured from the peritoneal lavage mic analysis using iTRAQ labelling and LC-MALDI- fluid, serum, and bronchoalveolar lavage fluids. TOF/TOF. Plasma exosomes isolated from LPS- Proteomic analyses on the fEVs were conducted by injected mice were analysed by Western blot analysis. the combination of one-dimensional SDS-PAGE and CD11b-expressing exosomes in plasma were measured LC-MS/MS. by sandwich ELISA. Plasma TNF-α level was deter- Results: Significant amounts of fEVs were isolated mined by ELISA. from mouse faeces, and the fEVs were derived from Results: Proteomic analysis showed that monocyte/ bacteria and host cells. Upon intraperitoneal adminis- macrophage marker proteins such as CD11b, CD14 tration, the fEVs mediated peritoneal, systemic, and and F4/80 were detected in exosomes from pulmonary inflammation by increasing the numbers RAW264.7 cells. Glucose metabolism-related proteins of infiltrated immune cells and the pro-inflammatory including GLUT1, PKM2 and GAPDH increased in cytokines such as TNF-α and IL-6 in the peritoneal exosomes from LPS-stimulated cells compared with lavage fluid, serum, and bronchoalveolar lavage fluid. those from non-treated cells. Western blot analysis Proteomic analyses on the fEVs identified a total of 295 demonstrated that GLUT1 and CD11b were signifi- proteins, comprising 222 bacterial proteins and 73 cantly increased in plasma exosomes from LPS-injected murine proteins. mice. After LPS stimulation, TNF-α transiently Summary/Conclusion: The fEVs derived from bacter- increased, whereas CD11b-expressing exosomes ial and host cells could mediate local and systemic increased and remained high in plasma of mice with inflammation, and composed of bacterial and host sepsis. proteins. These results shed lights on the roles of com- Summary/Conclusion: We characterized monocyte/ mensal bacterial EVs in the pathogenesis of inflamma- macrophage-derived exosomes in plasma of mice with tory diseases. sepsis and developed a sandwich ELISA for detection Funding: National Research Foundation of Korea of CD11b-expressing exosomes in plasma, which could (NRF) Herman Krefting Foundation for Allergy and be a novel marker for systemic immune response as Asthma Research, Lundberg Foundation well as sepsis. Funding: JSPS KAKENHI Grant Number JP17K01888. PT07.06 PT07.05 Opioid-mediated release of astrocytic EV miR-23 induces pericyte migration and blood-brain barrier breach Systemic inflammatory activity and proteome analysis of extracellular Shilpa Buch, Ke Liao, Fang Niu and Guoku Hu vesicles from faeces University of Nebraska Medical Center, Omaha, USA Kyongsu Parka, Jaewook Leeb, Yein Juna, Daekyum Kima, Jungwook Kima and Yong Song Ghoc Introduction: Pericytes are important constituents of aPohang University of Science and Technology (POSTECH), Pohang, Republic of Korea; bDepartment of Life Sciences, Pohang University of the cerebrovascular unit and play a key role in main- Science and Technology (POSTECH), Pohang, Republic of Korea; taining the integrity of the blood-brain barrier. It is cDepartment of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea well recognized that drugs of abuse such as opioids can result in breach of the BBB, ultimately leading to Introduction: Substantial quantities of bacteria reside enhanced monocyte transmigration and ensuing neu- in the gastrointestinal tract. Severe inflammatory roinflammation. Mechanism(s) by which pericytes con- responses are induced when the bacteria went through tribute to morphine-mediated neuroinflammation, the peritoneum from the gastrointestinal tract. In this however, remains less understood. study, extracellular vesicles isolated from faeces (fEVs) Methods: EVs were isolated from morphine-stimulated were assessed to see whether they could mediate mouse/human primary astrocytes using the standard 76 ISEV2019 ABSTRACT BOOK differential ultracentrifugation method and character- Introduction: Pneumonia remains one of the most ized by transmission electron microscopy, NanoSight & deadly communicable diseases, causing three million western blot analyses. Among the various miRs dysre- deaths worldwide in 2016. Extracellular vesicles (EVs) gulated in morphine-stimulated astrocyte EV cargo, are pivotal during signal transfer in the pathogenesis of miR-23 was found to be upregulated by real-time inflammatory lung diseases. Since identifying pneumo- PCR. Confocal microscopy identified uptake of astro- nia is particularly challenging in high risk groups (e.g. cytic EVs by pericytes. Functional assessment of astro- the elderly or infants), which often present with atypi- cytic EV uptake by pericytes involved cell migration cal symptoms and are at high risk for secondary com- using Boyden chamber and wound healing assays. plications such as sepsis or acute respiratory distress Additionally, an in vitro 3D model comprising of peri- syndrome (ARDS), new approaches for early diagnosis cytes and human endothelial cells was also used to are required. In this study we identified EV assess astrocyte EV-mediated migration of pericytes microRNAs (miRNAs) as potential biomarkers for in presence of morphine. inflammatory changes of the pulmonary tissue. Results: Exposure of astrocytes to morphine induced Methods: Our study included 13 patients with com- the expression and secretion of miR-23 in the EVs, munity-acquired pneumonia, 14 ARDS patients, 22 which, upon uptake by the pericytes resulted in their patients with sepsis and 31 healthy controls. After pre- migration. Additionally, in the pericytes that had taken cipitating EVs from 1 ml serum, total RNA was up morphine stimulated astrocyte EVs, there was extracted. Subsequent to library preparation and small downregulation of phosphatase and tensin homologue RNA-Seq, differential gene expression analysis was per- (PTEN), a target of miR-23. formed using DESeq2. Data were filtered by mean Summary/Conclusion: Our findings indicate that miRNA expression of ≥ 50 reads, minimum twofold morphine-mediated dysregulation of miRNA expres- up or down regulation and adjusted p-value ≤ 0.05. sion in the CNS involves astrocyte-pericyte communi- Results: The mean relative miRNA frequency varied cation via the extracellular vesicles, leading, in turn, to slightly between the different groups and was highest in loss of pericyte coverage at the BBB. volunteers. Short sequences (< 16 nucleotides), prob- Funding: This work was supported by grants ably degradation products from longer coding and DA040397, MH112848 (S.B.) and DA042704, non-coding RNA species, were predominantly detected DA046831 (G.H.) from the National Institutes of in patients. Based on unsupervised clustering, patients Health. The support by Nebraska Center for could be distinctly separated from healthy individuals. Substance Abuse Research is acknowledged. Although 21 miRNAs were significantly regulated in all patient groups compared to healthy controls, different disorders showed unique miRNA expression profiles. PT07.07=OWP2.15 Distinct miRNA subsets were identified, which are applicable to indicate disease progression from limited Diagnostic microRNA biomarkers from circulating extracellular inflammation present in pneumonia to severe inflam- vesicles for early detection of pneumonia and severe secondary matory changes as seen in ARDS and sepsis. complications Stefanie Hermanna, Benedikt Kirchnerb, Dominik Buschmannc, Melanie Summary/conclusion: This study shows that EV Märted, Florian Brandesd, Stefan Kotschotee, Michael Boninf, Marlene miRNA biomarkers have potential for diagnosis of g h d d Reithmair , Matthias Klein , Gustav Schelling and Michael Pfaffl pneumonia and to indicate disease progression towards aDivision of Animal Physiology and Immunology, School of Life Sciences severe lung injury. Our findings are of clinical rele- Weihenstephan, Technical University of Munich, Freising, Germany; bAnimal Physiology and Immunology, School of Life Sciences vance, as the timely diagnosis of pneumonia can be Weihenstephan, Technical University of Munich, Freising, Freising, challenging, and secondary complications such as Germany; cTUM School of Life Sciences Weihenstephan, Division of Animal Physiology and Immunology, Freising, Freising, Germany; ARDS and sepsis might be prevented by early inter- dDepartment of Anesthesiology, University Hospital, Ludwig-Maximilians- vention and treatment. University Munich, München, Germany; eIMGM Laboratories GmbH, Planegg, Martinsried, Germany; fIMGM Laboratories GmbH, Planegg, Funding: This study was supported by the German Germany, Martinsried, USA; gInstitute of Human Genetics, University Federal Ministry for Economic Affairs and Energy Hospital, Ludwig-Maximilians-University Munich, München, Germany; under the program “Zentrales Innovationsprogramm hDepartment of Neurology, University Hospital, Ludwig-Maximilians- University Munich, München, Germany Mittelstand”. JOURNAL OF EXTRACELLULAR VESICLES 77

PT08: EVs in Metabolism and Metabolic Diseases Chairs: Sophie Rome; Alena Ivanova Location: Level 3, Hall A 15:30–16:30

Summary/Conclusion: Elevated levels of PEVs and PT08.01 EEVs found in patients with type 1 diabetes are unrelated to clinical microangiopathy, indicating that type 1 diabetes is a procoagulant state per se. Comparison of Elevated levels of platelet and endothelial extracellular vesicles in type 1 diabetes, a cohort study of 236 patients PEV levels between the sexes showed a more favour- Karin Bergena, Katherina Aguilera Gaticab, Fariborz Mobarrezc, Gun able phenotype in healthy women compared with d b d Jörneskog , Håkan Wallén and Sara Tehrani healthy men, while no sex differences were found a Karolinska Institutet, Department of Clinical Sciences, Danderyd University among patients. This could be linked to the loss of Hospital, Division of Nephrology, Stockholm, Sweden; bKarolinska Institutet, Department of Clinical Sciences, Danderyd University Hospital, Division of female protection against cardiovascular disease in Cardiology, Stockholm, Sweden; cKarolinska Institutet, Department of type 1 diabetes. Medicine, Rheumatology Unit, Karolinska University Hospital, Sweden, Stockholm; dKarolinska Institutet, Department of Clinical Sciences, Funding: Berth von Kantzow Foundation, Swedish Danderyd University Hospital, Division of Internal Medicine, Stockholm, Diabetes Foundation, Wallenius Foundation, Swedish Sweden, Stockholm, Sweden Heart-Lung Foundation, Foundation of Women and Health Introduction: We have recently presented data on increased levels of circulating extracellular vesicles (EVs) in patients with type 1 diabetes. In the same cohort, we have now analysed subpopulations of plate- PT08.02 let- and endothelial EVs in relation to diabetic microangiopathy and sex. Methods: Two hundred and thirty-six patients (107 Role of extracellular vesicles in the regulation of inflammation and metabolism in obesity women) and 100 healthy controls matched for age, Takahisa Nakamuraa, Ahlee Kimb, Esam Salemb, Kazutoshi Murakamib and sex and body mass index (BMI) gave written informed Vishnupriya Borrab consent to the study. Plasma platelet EV (PEV) and aCincinnati Children’s Hospiltal Medical Center, Cincinnati, USA; endothelial EV (EEV) levels were assessed using flow bCincinnati Children’s Hospital Medical Center, Cincinnati, USA cytometry with labelled antibodies directed against platelet (CD42a and CD61) and endothelial specific Introduction: The worldwide prevalence of obesity has (CD144 and CD62E) antigens. EV expression of pro- reached pandemic proportions. Obesity has strong coagulant phosphatidylserine (PS) and tissue factor inflammatory underpinnings, which are associated (TF) were assessed using lactadherin (lac) and CD142 with the development of type 2 diabetes (T2D) and antibody. The study was approved by the local ethics non-alcoholic steatohepatitis (NASH). However, the committee. mechanisms by which obesity provokes aberrant Results: PEV and EEV levels with or without expres- inflammation have yet to be clearly defined. sion of procoagulant PS and TF were statistically Extracellular vesicles (EVs), including exosomes and higher among patients than in controls (p < 0.05 for microvesicles, are a novel mode of tissue-to-tissue all). The patients had about 50% higher PEV levels and communication. Recent studies indicate that EVs are up to a 50-fold increase in EEV levels compared to involved in many pathophysiological events including controls. No statistically significant differences were inflammatory responses and metabolic dysfunctions. found between PEV or EEV levels in patients with or We hypothesize that EVs play critical roles in the without clinical microangiopathy. Healthy women had induction of obesity-associated aberrant inflammation lower PS+, PS− and total PEV levels compared to and the development of metabolic diseases. healthy men (p < 0.05 for all), whereas no differences Methods: To investigate the role of EVs in the patho- between sex were found in the patients. PEV and EEV genesis of obesity, we have taken systematical levels in patients did not correlate with glycaemic con- approaches including novel computational methods, trol (HbA1c), BMI, blood pressure, blood lipids or analyses of EVs collected from human obese patients diabetes duration. undergoing bariatric surgery, utilization of novel 78 ISEV2019 ABSTRACT BOOK mouse models monitoring cell type-specific EVs, and exosomal material, we performed proteomic profiling cellular-based EV functional assays. using data independent acquisition (DIA) on an Results: Using novel computational methods, we have OrbitrapTM Fusion Lumos instrument. Spectronaut identified strong associations with EV-related genes in TM Pulsar software was used to integrate spectral metabolic syndrome associated with T2D. Our analyses libraries and perform quantitative proteomic profiling of EVs from adolescent obese patients undergoing bar- of exosomes derived from different human primary iatric surgery have shown that serum EV concentration cells as well as human serum and plasma. is inversely correlated to metabolic improvements in Results: EPS stimulated the release of exosomes from glucose metabolism and inflammation post-surgery, hSkMC and regulated the release of 408 exosomal with unique EVs’ extracellular RNA (exRNA) profiles. proteins. Ingenuity pathway analysis (IPA) revealed Further, our newly established mouse models monitor- significant regulation of, e.g. integrin, vascular ing specific cell type-derived EVs in vivo indicates that endothelial growth factor, Liver X receptor/Retinoid X in obesity, EVs from metabolic tissues behave like a receptor and PI 3-kinase/Akt signalling by these pro- pathogen and induce inflammation. teins. HIIT regulated the amount of 62 exosomal Summary/Conclusion: While the research of EVs has plasma proteins in vivo, of which nine candidates attracted much attention, therapeutic targeting and were also similarly regulated by EPS in vitro. IPA significance of EVs in metabolic diseases are still a links these exosomal proteins to pathways involved in controversial area of research. By utilizing our novel metabolic diseases and lipid metabolism. mouse models coupled with access to human samples, Summary/Conclusion: We identified various exercise- our systematical approaches allow to propose novel regulated exosomal proteins with predicted metabolic mechanisms by which pathologic EVs induce aberrant effects. Further analysis of these novel tissue-specific inflammation and deteriorate metabolism in obesity. candidates will help to better understand systemic metabolic effects of exercise. PT08.03 PT08.04

Characterization of exosomal proteins derived from contracting skeletal muscle as potential mediators of beneficial metabolic effects Transcriptome and proteome of extracellular vesicles derived from of exercise cellular targets of diabetic kidney disease a b c d d Henrike Sell, Elisabetta De Filippo, Sonja Hartwig, Lucia Mastrototaro, Maria Karina A. Barreiro , Abigail Lay , Wei Liang , Xiaomeng Xu , Sihui Luo , c e f g Apostolopoulou, Dominik Pesta, Julia Szendrödi, Hadi Al-Hasani, Stefan Tillmann Bork , Richard Coward , Denis Delic , Tobias B. Huber and Harry h Lehr and Michael Roden Holthöfer a b German Diabetes Center, Düsseldorf, Germany FIMM, University of Helsinki, Helsinki, Finland; University of Bristol, Bristol, UK; cUniversity Hospital Freiburg, Freiburg, Germany; dInstitute for Molecular Medicine Finland (FIMM), University of Helsinki, Helsinki, Finland; eBristol Introduction: Exercise training improves glucose Univ Medical School, Bristol, UK, Bristol, UK; fBoehringer Ingelheim Pharma metabolism and insulin sensitivity supporting the con- GmbH & Co. KG, Biberach a.d. Riss, Germany; gIII. Department of Medicine, University Medical Center Hamburg-Eppendorf, Hamburg, Germany, cept that lifestyle modification is useful for patients to Hamburg, Germany; hProfessor prevent and treat type 2 diabetes. Skeletal muscle also releases circulating factors following exercise affecting Introduction: Kidney disease (DKD) is common, metabolism of other organs probably involving the costly and the most feared complication of long stand- release of exosomes. However, little is known about ing diabetes. Its root causes remain unknown. muscle-specific release of exosomes and the differential Interestingly the characteristic changes in circulating protein content of exosomes during exercise. Thus, we glucose levels in this disease appear to alter the signa- aimed to identify exosomal proteins regulated by exer- ture of extracellular vesicles as found in the urine. Here cise in vitro and in vivo and to discover pathways we wanted to explore the EV secretion pattern by key regulated by exosomal cargo. DKD target cells in the glomerulus (podocytes, Methods: Exosomes from human skeletal muscle cells endothelial and mesangial cells) and proximal tubular (hSkMC), contracted by electrical pulse stimulation cells by harvesting the entire EV repertoire using (EPS), were isolated by differential ultracentrifuga- Hydrostatic Filtration Dialysis (HFD). tion/ultrafiltration. Exosomes were also isolated by Methods: We used cell culture media from podocytes, size exclusion chromatography from plasma of patients proximal tubule, mesangial and glomerular endothelial with and without type 2 diabetes on high intensity cells in four conditions: (1) Insulin sensitive, (2) interval training (HIIT). In order to allow reliable Insulin resistant, (3) Insulin receptor transfected and label-free quantification of the very limited muscle insulin sensitive, (4) Insulin receptor transfected and JOURNAL OF EXTRACELLULAR VESICLES 79 insulin resistant. EVs were isolated from 50 mL of cell Aims: To characterize extracellular vesicles (EVs) in culture media, respectively, by HFD. Quality of the EV obese (F/M = 8/8; age = 21.0 ± 8.5 years, yield was verified with negative staining Electron BMI = 37.9 ± 6.0 kg/m2) and normal-weight (F/ Microscopy (EM) and Western blotting (WB). Vesicle M = 4/4; age = 25.1 ± 8.2 years, BMI = 20.9 ± 1.5 kg/ concentration was determined by Nanoparticle m2) subjects who underwent a moderate-intensity Tracking Analysis (NTA). Isolated RNAs were profiled (60% VO2max for 30 min or until exhaustion) exercise with Bioanalyzer Pico kit and subjected to miRNAseq on a treadmill and RNAseq. EV proteins were analysed using tandem Methods: Blood samples were drawn before, at the end mass tag labelling. and during post-exercise recovery period (3 and 24 h). Results: The isolated EVs appeared typical at EM and EVs were analysed by Nanosight and flow cytometry were positive for the EV-marker TSG101 in WB. RNA after labelling with the following markers: CD14+ quantity and quality proved appropriate for both (monocyte), CD61+ (platelet), CD62E+ (activated miRNA and RNAseq. Different treatments affected endothelium), CD105+ (resting endothelium), characteristically the vesiculation from the investigated HERVW+ (human endogenous retrovirus W), SCG+ target cells of diabetes. Ninety-six EV miRNAs could (muscle) and FABP+ (adipose tissue). characteristically discriminate between the cell types Results: After exercise, 100–200 nm EVs significantly and special treatments studied. Some EV miRNAs decreased (p < 0.01). There was a significantly higher showed treatment effects and the analysis of their target post-exercise release of these EVs in normal-weight genes using KEGG disease database showed a clear link than obese subjects (p = 0.025). Considering the 30– to kidney diseases. Integrated miRNA-mRNA and pro- 130 nm size range, there was a significant lower release tein analysis was also performed. of EVs in females than males (p < 0.01). After exercise, Summary/Conclusion: EV analysis provides a novel the 130–700 nm EVs significantly decreased approach to reveal valuable pathophysiology, pathway (p = 0.016). There was a higher release of these EVs and signalling information of cultured disease target in females than males (p = 0.05). After exercise, CD61 cells. Changes in EV miRNAs, mRNA and proteomics + EVs significantly decreased in all subjects (p = 0.02). may thus give valuable insight into mechanisms and SCG+ EVs were increased after exercise (p = 0.06). targets to insulin resistance on DKD target cells. There were no significant associations of other Funding: BEAt-DKD, Paulo Foundation and Novo biomarkers Nordisk Foundation. Summary/Conclusion: An acute exercise induces changes in the release of plasma EVs, which are tissue-, gender- and BMI-specific, suggesting that the PT08.05 exercise-related benefits might depend upon a complex interaction of tissue, endocrine and metabolic factors Funding: The study was supported by Progetti di

Effects of an acute exercise on circulating extracellular vesicles: Ricerca Corrente, Istituto Auxologico Italiano, IRCCS, tissue-, gender- and BMI-related differences Milan, Italy. Jacopo Mariania, Antonello Rigamontia, Silvano Cellaa, Alessandra De Colb, Federica Rotac, Sabrina Cicolinib, Gabriella Tringalib, Roberta De Michelib, a b d Valentina Bollati , Sartorio Alesandro and Mario Barilani PT08.06 aUniversity of Milan, Department of Clinical Sciences and Community Health, Milan, Italy; bIstituto Auxologico Italiano, Experimental Laboratory for Auxo-endocrinological Research, Verbania and Milan, Italy; cEPIGET LAB, Department of Clinical Sciences and Community Health, Università Profiling of extracellular vesicles from human hepatic stellate cells degli Studi di Milano, Milan, Italy; dUnit of Regenerative Medicine – Cell Cristina Zivkoa, Gina Valentinoa, Kathrin Fuhrmannb, Gregor Fuhrmannc Factory, Department of Transfusional Medicine and Hematology, and Paola Luciania

Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano a b (MI), Italy; Università degli Studi di Milano, Milan, Italy Friedrich Schiller University Jena, Jena, Germany; HIPS, Saarbrücken, Germany; cHelmholtz-Institut for Pharmaceutical Research Saarland (HIPS), Saarbrücken, Germany Introduction: Exercise is recognized to evoke multi- systemic adaptations that, particularly in obese sub- Introduction:Theroleofextracellularvesicles(EVs) jects, reduce body weight, improve gluco-metabolic in intercellular communication makes them particu- control, counteract sarcopenia and lower the risk of larly interesting in the research of many pathologies, cardiometabolic diseases. Understanding the molecular especially those diagnosed invasively. In this study we mechanisms of exercise-induced benefits is of great evaluated differences in the EVs shed from human interest due to the ensuing therapeutic implications hepatic stellate cells (HSCs) in their different pheno- against obesity. typical states, since the activation of HSCs plays a 80 ISEV2019 ABSTRACT BOOK pivotal role in the progression of hepatic fibrosis, for when significant damage from diabetic retinopathy has which liver biopsy is still the diagnostic gold standard. occurred. Early signs of damage typically remain unno- Methods: Protocols were optimized as to induce the ticed until it has reached advanced stages of disease. different activation states in the cells; untreated HSCs Identifying early biomarkers of disease will allow clin- were compared to their activated and quiescent coun- icians to detect the progression of disease before the terparts. EVs were isolated from conditioned cell cul- onset of complications. Circulating microRNA con- ture medium (CCM) after differential centrifugation tained in extracellular vesicles such as exosomes are followed by an ultracentrifugation (UC) step, and potential early biomarkers and can be targeted to pre- they were purified by size exclusion chromatography vent diabetes from progressing. The aim of our project (SEC). The purification was evaluated by protein con- is to validate and determine the role of miR-15a as a tent determination by bicinchoninic acid (BCA) assay. potential early biomarker in diabetic retinopathy. The concentration and size distribution profiles of EVs Methods: This project was approved by the University in the SEC fractions were determined by nanoparticle of Malaya Medical Centre (UMMC) Medical Research tracking analysis (NTA). EV-morphology was observed Ethical Committee. A total of about 100 subjects (con- by scanning and cryogenic transmission electron trols and patients with Type 2 DM) was recruited from microscopy (SEM and cryo-TEM). UMMC, Kuala Lumpur. All subjects underwent com- Results: Purification by SEC resulted in a distinct reso- plete eye examination and graded for diabetic retino- lution between EVs and protein aggregates as deter- pathy. Clinical information collected included HbA1C, mined by BCA assay. Protein content associated with renal function testing, hypertension and smoking. EVs was only detectable in the SEC-fractions with the Extracellular vesicle (EV) isolation was performed highest EV-yields and was comparable in all groups. using differential ultracentrifugation and quantified. Differently treated HSCs yielded EVs in similar Results: In this study, we analysed miR-15a concentra- amounts and size distributions. Quantile subtraction tions in plasma and exosomal-enriched fractions using of the distribution curve obtained from untreated droplet digital and real-time PCR. There was no differ- cells shows that activated HSCs produce smaller EVs ence in microRNA levels in plasma observed. However, (80–150 nm) more prominently than quiescent cells. there was a significant increase in exosomal concentra- SEM imaging confirmed the polydispersity in the tion (average diameter <130nM) in patients with dia- samples. betic retinopathy compared to controls (p < 0.05). Summary/Conclusion: EVs originating from differ- There was also an increasing trend of miR-15a level ently treated HSCs were isolated and characterized in among diabetic patients compared to controls. terms of yield, size, morphology and general protein Summary/Conclusion: The findings from this study content. Our results were consistently indicative of corroborated with our previous findings of increase in differences in EV populations originating from the miR-15a levels in diabetes prior to the onset of retino- same cells in either healthy or diseased state (quiescent pathy compared to controls. This suggests that miR- or activated respectively), thus creating an important 15a is involved in the early development of diabetic basis for the potential non-invasive detection of liver microvascular complications and may be a potential diseases. biomarker for early complications of diabetes. Funding: The Phospholipids Research Center is grate- Funding: 1. Bayer Global Ophthalmology Award fully acknowledged for its support to the project and Program Grant. 2. University of Malaya Special Lipoid GmbH for the endowment to the University of Research Fund (BKS056-2017). 3. BioRad Institutional Jena. Funding (Materials and Lab consumables). 4. Fulbright Visiting Research Scholar Grant PT08.07 PT08.08

Role of exosomal miR-15a in diabetic retinopathy Tengku Ain Kamalden, Anne Macgregor-Das, Nurliza Khaliddin, Nur The effects of outer membrane vesicles delivered from Musfirah Mahmud, Adib Redzuan, Adil Mohamed, Hayatun Syamila Jamil, Porphyromonas gingivalis on hepatic glucose metabolisms Nadia Hanib, Nur Hasyimah Azemi and Samarjit Das Kaya Yoshidaa, Mariko Seyamab, Natsumi Fujiwarab, Hirohiko Okamurac d University of Malaya, Kuala Lumpur, Malaysia and Kazumi Ozaki aDepartments of Oral Healthcare Education, Institute of Biomedical Sciences, Introduction: Diabetic retinopathy is a debilitating Tokushima University Graduate School, Tokushima, Japan; bDepartments of Oral Healthcare Promotion, Institute of Biomedical Sciences, Tokushima complication of diabetes mellitus which results in irre- University Graduate School, Tokushima, Japan; cDepartment of Oral versible blindness. Currently treatment is only initiated Morphology, Okayama University Graduate School of Medicine, Dentistry JOURNAL OF EXTRACELLULAR VESICLES 81 and Pharmaceutical Sciences, Tokushima, Japan; dDepartments of Oral insulin signalling in HepG2 cells is analysed by western Healthcare Promotion, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan blot. The organ distribution of OMV was analysed by IVIS sectrum after injecting intraperitoneally Cy7- Introduction: The outer membrane vesicles (OMVs) of labelled Pg OMV. We also estimated the insulin sensi- Porphyromaons gingivalis (Pg), a gram-negative bacteria tivity using glucose tolerance test (GTT), insulin toler- known as a major pathogen of periodontal diseases, ance test (ITT) in mice treated with Pg OMV for 3 include its virulence factors and regulate the aetiology weeks. of periodontal diseases by affecting microbial environ- Results: Pg selectively sorted its specific proteases such ment and the host cells in the oral cavity. However, it is as arginine-specific gingipain (Rgp) and lysine-specific unknown whether Pg OMVs in oral cavity could trans- gingipain (Kgp) into OMVs. The treatment with Pg locate to distant organ and affect the systemic diseases, OMV attenuated the insulin signalling in HepG2 whereas periodontal diseases are well known to influ- cells, and its effects were eliminated by OMVs from ence the develop of diabetes mellitus. To elucidate the gingipain-deleated Pg. A Cy7 fluorescent signal was mechanisms by which periodontal diseases progress dia- detected in the liver in mice injected with Cy7- betes mellitus, we identified Pg OMV cargo proteins and labelled-Pg OMVs. The exposure of Pg OMVs for 3 verified its effects on the insulin signalling in vitro.We weeks slightly increased casual blood glucose and insu- also analysed the translocation of Pg OMVs to the lin tolerance level in mice. organ, and assessed the changes of hepatic glucose mata- Summary/Conclusion: Pg OMVs packaging gingipains bolisms in Pg OMV-treated mice. were delivered to the liver, resulting in the reduction of Methods: We identified the OMV cargo proteins by insulin sensitivity. These capabilities of Pg OMVs may LC-MS/MS analyses. The effects of Pg OMV on the contribute to the progress of diabetes mellitus. 82 ISEV2019 ABSTRACT BOOK

PT09: Advances in EV Quantification and Characterization Chairs: Randy Carney; Edwin van der Pol Location: Level 3, Hall A 15:30–16:30

PT09.01 platelet-derived EV contains approx. 15 CD61 glycoproteins in average. Summary/Conclusion: By the combination of MRPS Extracellular vesicle concentrations in human plasma and serum as and fluorescence SEC we quantified the overall particle revealed by microfluidic resistive pulse sensing and size exclusion concentrations in serum and plasma, and using a pla- chromatography coupled with on-line fluorescence detection Diana Kitkaa, Zoltan Vargab, Gergo Bartab, Judith Mihalya and Jean-Luc telet-specific fluorescently labelled antibody, we deter- Fraikinc mined the average number of CD61 glycoproteins on aRCNS HAS, Budapest, Hungary; bResearch Centre for Natural Sciences, platelet-derived EVs formed during blood clotting. c Hungarian Academy of Sciences, Budapest, Hungary; Spectradyne LLC, Funding: This work was supported under grant num- Torrance, USA bers PD 121326 and NVKP_16-1-2016-0007 by NKFIH Introduction: Blood is one of the most important (Hungary). ZV was supported by the János Bolyai sources of EVs in biomarker applications. However, Research Fellowship. there is a huge variation in the reported values of EV concentrations in plasma and serum in the current PT09.02 literature. Therefore, there is a continuous demand for new techniques for accurate determination of EV concentration. The aim of this study was to character- The nanobioanalytical platform, a tuneable tool for a sensitive detection & characterization of extracellular vesicles subsets from ize EVs in normal plasma and serum using novel biological samples techniques such as microfluidic resistive pulse sensing Balasubramaniam Namasivayama, Yu-Wen Wub, Liling Delilab, Annie Frelet- a b a a (MRPS) and size exclusion chromatography (SEC) Barrand , Thierry Burnouf , Celine Elie-Caille and Wilfrid Boireau coupled with on-line fluorescence detection. aFEMTO-ST Institute, UBFC, CNRS, Besançon, France; bCollege of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan Methods: To obtain cell free serum and plasma, blood (Republic of China) was collected from healthy volunteers using serum activator and EDTA vacutainer tubes, respectively. Introduction: The NanoBioAnalytical (NBA) platform Cells were removed by centrifugation at 2500 x g is an established, calibrated and label-free system to twice. Samples were further purified with a Sepharose characterize Extracellular Vesicles (EVs), without lim- CL-2B gravity column and analysed by MRPS using the itation in size, in different biological samples [1, 2]. nCS1 instrument (Spectradyne LLC, USA). For the NBA benefits were recently highlighted in latest fluorescence SEC experiments, samples were labelled MISEV guidelines [3]. The NBA platform combines with PE-conjugated anti-CD61 and analysed with a biodetection and phenotyping of EVs subsets by JASCO (Japan) liquid chromatography system supple- immunocapture monitored by Surface Plasmon mented with an FP-2020 fluorescence detector and Resonance (SPR) on biochip, followed by EVs quanti- using a 1 mL column filled with CL-2B gel. tation and sizing thanks to metrological evaluation by Results: The particle concentrations of serum and Atomic Force Microscopy (AFM). Our aim is to push plasma determined by MRPS in the 65–250 nm size the limit of the NBA to address clinical studies invol- range were 2.06E+10 1/mL and 1.77E+10 1/mL, respec- ving EVs. tively. In the 250–2000 nm range, we found 2.22E+8 1/ Methods: We emphasise here the performance of the mL and 5.50E+7 1/mL for serum and plasma. These NBA platform for establishing its dynamic range and concentrations correspond to 0.29 E+10 1/mL increase limit of detection (LOD) for blood derived EVs. for the smaller size range, and 1.67E+8 1/mL for the Concentration of EVs was first determined in solution larger size range, which can be accounted for the EVs by Tunable Resistive Pulse Sensing; NBA sensitivity produced during clotting. Fluorescence SEC experi- and reliability was then studied by SPR on biochips ments with PE-CD61 revealed that the percentage of presenting a-CD41 antibody arrays. Finally, even on CD61 bound to EVs increased from 2.25% (plasma) to 1000-fold diluted samples, reliable and complementary 36% (serum). Using these data, we obtained that one information to SPR measurements on size distribution, JOURNAL OF EXTRACELLULAR VESICLES 83 counting and shape deciphering could be obtained The calculated fluorescence detection limit approaches by AFM. single fluorescence sensitivity established using fluor- Results: Optimizing different factors (flow rate, density escent polystyrene nanoparticles (20–200nm diameter) of receptors on the surface, etc.) enabled detection of corresponding to 180–110,000 MESF. blood derived EVs at dynamic range from 106 to 109 Results: A tetraspanin assay was developed on the particles /mL on a-CD41 surface. The determination of ExoView™ platform for the detection of CD81, CD63, the LOD of EVs and their subsets size distribution at CD9 positive vesicles directly from cell culture samples different capture levels are currently in progress. without the need for purification. We can also permea- Summary/Conclusion: The NBA platform is modular bilize the vesicles to probe the cargo of individual and capable of detecting EVs reliably even in highly vesicles. To validate the detection of tetraspanins and diluted samples. Such characterization and correlation internal cargo proteins we used knockout cell lines as studies are crucial for accurate and comprehensive negative controls. The assay can also be used for detec- characterization of EVs in biological samples with tion of vesicles from other biological fluids like urine, good reproducibility. plasma, CSF, and saliva. We demonstrated that most tetraspanin positive vesicles have a diameter around References 50 nm, which agrees with TEM, versus the widely 1. Obeid et al., B&B. 2017. 93:250–259 reported diameter of 100nm in the literature. 2. Obeid et al., NBM. 2019 (in revision) Summary/Conclusion: The ExoView platform is a 3. Thery et al., JEV. 2018. 8;1535750 scalable single vesicle analysis platform that can size, enumerate and run multi-parametric co-localization Funding: Region Franche-Comté 2017–2020. experiments directly from sample. The platform can be applied for basic research as well as biomarker PT09.04 discovery for liquid-biopsy applications.

Multi-parametric single vesicle analysis using an interferometric imaging platform PT09.05 George Daaboula, Veronica Sanchezb, Aditya Dhandeb, Chetan Soodb, Gregg Lithgowb, Francis Fordjourc, Stephen Gouldc and David Freedmanb aNanoView Biosciences, Brighton, USA; bNanoView Biosciences, Boston, Quantification of circulating extracellular vesicles from human USA; cJohn Hopkins University, Baltimore, USA plasma by utilizing a membrane-based microfluidic system Yi-Sin Chena, Gwo-Bin Leea and Chihchen Chenb

Introduction: Current single vesicle analysis techni- aDepartment of Power Mechanical Engineering, National Tsing Hua ques like electron microscopy and atomic force micro- University, Taiwan, Hsinchu, Taiwan (Republic of China); bInstitution of NanoEngineering and MicroSystems, National Tsing Hua University, scopy require high expertise and are limited in Hsinchu, Taiwan (Republic of China) throughput. Flow cytometry (FC), which is regularly used to for single cell analysis and sorting, has limited Introduction: Extracellular vesicles (EVs) have served sensitivity in light scatter mode for detection of highly as biomarkers for cancer diagnosis and prognosis based abundant populations of EVs smaller than a 100 nm. on their carried cargos such as proteins and nucleic Recent publications show that the exosome average acids. To accurately and specifically quantify tumour- diameter is around 50 nm, which has been measured derived EVs from complex biofluids such as human by super-resolution imaging, nanoFCM, and TEM. The plasma is potentially significant for precise diagnosis. more sensitive fluorescence-based detection of EVs is Many techniques for EVs quantification have been also difficult because EVs could have much less than 10 developed in the past decade, including nanoparticles epitopes of the marker of interest, a limit for most FC tracking analysis, total internal reflection fluorescence systems. microscopy, flow cytometry and enzyme-linked immu- Methods: To address the limitation in single vesicles nosorbent assays (ELISA). However, bulky and expen- analysis we have developed a technique that can size, sive instruments are required for these approaches. enumerate, and co-localize 4 markers (surface and Therefore, this study provides a simple and low-cost cargo) on single vesicles across 10 different subpopula- approach to quantify circulating EVs from human tions on a single sensor surface. The technique is plasma by using the ELISA method and a fluorescent termed SP-IRIS and commercialized as ExoView™ by microscope on a membrane-based integrated micro- NanoView Biosciences. EvoView™ relies on a bilayer fluidic platform. substrate (silicon/silicon dioxide) that forms a common Methods: In this study, a membrane-based integrated path interferometer for enhanced nanoparticle analysis. microfluidic platform was used for EVs collection, 84 ISEV2019 ABSTRACT BOOK enrichment and fluorescent detection process. A track- volume and reagent consumption. To solve several etched membrane filter with a pore size of 0.03 μm that technical problems involving the generation of electro- could enrich EVs and deplete small molecules during lysis gas on the electrodes, most of the micro-FFE washing steps was packaged in a polydimethylsiloxane- devices reported in the past were fabricated using ela- based microfluidic platform. After EVs enriching, an borate micromachining process on silicon or glass sub- on-chip ELISA assay was performed involving the fol- strates. However, high-cost micromachining processes lowing steps including (1) anti-CD63 antibody were required, and these were not suitable for mass (EPR5702) incubation, (2) horseradish peroxidase production. (HRP) conjugated anti-rabbit antibody incubation, Results: Based on these backgrounds, we recently and (3) tetramethylrhodamine-labelled tyramide incu- developed a polymer-based easy-to-fabricate micro- bation. It is worth noting that tyramide molecules FFE device and overcame the problems mentioned could be accumulated on the surface of EVs to amplify above. In this presentation, we will introduce the appli- the fluorescent signal and observed under a fluorescent cation of this device to EV separations in this presenta- microscope. With this approach, absolute quantification. Electrophoretic separation of Sk-Br-3 derived tion of EVs with high specificity could be achieved. exosomes expressed with HER2 antigen were demon- Results: The experimental results showed that CD63- strated with and without the combination use of the positive circulating EVs in human plasma could be anti-HER2 antibody for molecular specific separation. individually observed under a fluorescent microscope. Summary/Conclusion: The present method will be By using imaging software (ImageJ) to perform image one of the promising candidates for separating favour- analysis, the total number of EVs could be quantified able types of EVs from heterogeneous samples. such that the concentration of EVs in plasma could be Funding: Center of Innovation Program (COI measured. STREAM) from Japan Science and Technology Summary/Conclusion: The developed method could Agency (JST) be used to quantify EVs with high specificity and could be widely used in most general laboratory for PT09.07 precise diagnosis of circulating EVs from human plasma. Funding: Ministry of Science and Technology of Size distribution of extracellular vesicles by microfluidic resistive pulse sensing and small-angle neutron scattering Taiwan (MOST 106–2221-E-007–001, MOST 107– Zoltan Vargaa, Bence Feherb, Diana Kitkac, Vitaliy Pipichd and Jean-Luc 2221-E-007–013-MY3) Fraikine aResearch Centre for Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary; bEötvös Loránd University, Budapest, Hungary; cRCNS PT09.06 HAS, Budapest, Hungary; dJülich Centre for Neutron Science JCNS, Garching, Germany; eSpectradyne LLC, Torrance, USA

Electrophoretic separation of EVs using a microfluidic platform Introduction: Accurate size determination of extracel- Takanori Ichiki and Hiromi Kuramochi lular vesicles (EVs) is still challenging because of the The University of Tokyo, Tokyo, Japan detection limit and sensitivity of the methods used for their characterization. In this study, we used two novel Introduction: Absence of adequate tools for analysing techniques such as microfluidic resistive pulse sensing and/or identifying mesoscopic-sized particles ranging (MRPS) and small-angle neutron scattering (SANS) for from tens to hundreds of nanometres is the potential the size determination of reference liposome samples obstacle in both fundamental and applied studies of and red blood cell derived EVs (REVs) and compared extracellular vesicles (EVs), and hence, there is a grow- the obtained mean diameter values with those mea- ing demand for a novel analytical method of nanopar- sured by dynamic light scattering (DLS). ticles with good reproducibility and ease of use. Methods: Liposomes were prepared by extrusion using Methods: In the last several years, we reported the polycarbonate membranes with 50 and 100 nm pore usefulness of electrophoretic mobility as an index for sizes (SSL-50, SSL-100). REVs were isolated from red typing individual EVs based on their surface properties. blood cell concentrate supernatant by centrifugation at To meet the requirement of separation and recovery of 16.000 x g and further purified with a Sepharose CL-2B different types of EVs, we demonstrate the use of gravity column. MRPS experiments were performed micro-free-flow electrophoresis (micro-FFE) devices with the nCS1 instrument (Spectradyne LLC, USA). for this purpose. Since the 1990s, micro-FFE devices SANS measurements were performed at the KWS-3 have been developed to allow for smaller sample instrument operated by Jülich Centre for Neutron JOURNAL OF EXTRACELLULAR VESICLES 85

Science at the FRMII (Garching, Germany). DLS mea- chamber (horizontal connection type co-culture plate; surements were performed using a W130i instrument HTCP). HTCP made it possible to analyse intracellular (Avid Nano Ltd., UK). kinetics and changes in surface markers of exosomes. Results: MRPS provided particle size distributions with Methods: To examine the essential interactions of exo- mean diameter values of 69, 96 and 181 nm for SSL-50 somes, we evaluated the uptake of extracellular exo- and SSL-100 liposomes and for the REV sample, somes using this HTCP. Culturing cells with GFP- respectively. The values obtained by SANS (58, 73 labelled exosomes in only one container and detecting and 132 nm, respectively) are smaller than the MRPS the presence of GFP in cells in the adjoining container. results, which can be explained by the fact that the Also, various chemicals were added, and analysis hydrocarbon chain region of the lipid bilayer gives was made on changes in the kinetics of exosome and the highest scattering contribution in case of SANS, changes in surface markers. which corresponds to a smaller diameter than the over- Results: It was possible to confirm the exosome passed all size determined by MRPS. In contrast, DLS pro- through the filter and to identify the origin of exo- vided the largest diameter values, namely 109, 142 and somes and to analyse the distribution of the exosome 226 nm, respectively. in the cells. We found that the amount of exosome Summary/Conclusion: Size determination methods secreted by cells increased by an agent. As a result of based on different physical principles can result in the analysis, although the amount of CD63 per one large variation of the reported mean diameter of lipo- exosome was decreased, the amount of CD63 per one somes and EVs. Optical methods are biased due to cell was increased. their size-dependent sensitivity. SANS can be used for Summary/Conclusion: This fact indicates that there mono disperse samples only. In case of resistive pulse may be no point in comparing the amount of protein sensing, the microfluidic design overcomes many prac- or miRNA contained in exosomes. Detailed data will be tical problems accounted with this technique, and as a presented at this workshop. single particle, non-optical method, it is less affected by the above-mentioned drawbacks. Funding: This work was supported under grant num- PT09.10 bers PD 121,326 and NVKP_16-1–2016-0007 by NKFIH (Hungary). ZV was supported by the János Protease biomarker detection using functionalized bioplastic-based Bolyai Research Fellowship. biosensors Richard Kelwicka, Alexander Webbb, Yizhou Wanga, Fiona Allanb and Paul Freemontc PT09.09 aImperial College London, London, UK; bNatural History Museum London, London, UK; cThe London DNA Foundry, Imperial College London, London, UK

Analysis of intracellular dynamics of exosomes and changes of surface markers Introduction: Extracellular vesicles (EVs) are poten- Takeo Shimasaki and Satoko Yamamoto tially the “seeds”, that were famously metaphorized by Kanazawa Medical University, Uchinada, Japan Dr Stephen Paget in 1889 when he noted that particular primary tumours preferentially metastasized to par- Introduction: In the biological study, a standard ticular organs. EV-associated metalloproteinases method for observing natural interactions between conceivably play important roles in priming metastatic cells is co-culturing technique. The existing co-culture sites. Indeed, many studies demonstrate the complex research method is generally classified into two main roles that metalloproteinases have in cancer biology. groups depending on the state of adhesion between EVs can be readily accessed from patient liquid biop- cells: direct co-culture or indirect co-culture. In indir- sies and an analysis of EV-associated metalloproteinase ect co-culture, standard methods for filter separation of biomarkers may enable early-stage cancer detection. cells include methods using vertical-insert type co-cul- Methods: In order to detect EV-associated metallopro- ture plate (VTCP) named after the structure or trade- teinases we developed a library of biosensors. These mark (i.e. cell-culture insert, Transwell). These biosensors utilize PhaC-reporter fusion proteins that methods have been used in many studies thus far, its are bound to microbially manufactured bioplastic application to exosomes research has been limited. It is beads. These PhaC-fusions also incorporate specific difficult to obtain high-quality images of cells in the metalloproteinase cleavage sites. In the presence of a upper culture chamber due to the short focal length of specific metalloproteinase, the reporter protein is the microscope. We developed a novel cell culturing cleaved off the bioplastic bead – resulting in a loss of 86 ISEV2019 ABSTRACT BOOK bead fluorescence that can be measured using high- resolution imaging on the same device. Specifically, throughput flow cytometry. These biosensors were the surface of the imaging chamber is passivated with assayed using either recombinant proteinases or iso- anti-CD 63 to capture the DiD stained vesicles. The lated EVs from in vitro cancer models. acquisition of the raw image series was done using total Results: Human metalloproteinase recognition motifs internal reflection fluorescence microscopy (TIRF) were identified in the literature and a total of 70 dif- with a 642-nm diode laser for excitation. Two types ferent metalloproteinase biosensors were designed. A of super-resolution techniques were tested including control biosensor (PhaC-112L-T-G) detected 0.5 U of super resolution radial fluctuations (SRRF) and sto- tobacco etch virus protease (AcTEV) activity and the chastic optical reconstruction microscopy (STORM). PhaC-112L-P14-G biosensor, despite some background Results: The size of single exosomes in the final images off-target activity, was able to detect 0.033 mU of were estimated by the full-width at half-maximum recombinant MMP14 activity. Membrane-bound (FWHM) of Gaussian fitted to the distribution of single metalloproteinases MMP14 and ADAM10 were also molecules. We have found that the resolution limit of detected in EVs isolated (ultracentrifugation) from in the single particle is reduced to 70 nm. The preliminary vitro cancer models. data from SRRF and STORM showed the particle size Summary/Conclusion: Our biosensors detected EV- and size distribution were compared to nanoparticle associated metalloproteinases and could serve as useful tracking analysis (NTA) results. research tools for EV-biomarker discovery. Summary/Conclusion: This method provides in-depth Funding: Dr Richard Kelwick is funded by a Royal size analysis of single exosomes below the diffraction Society of Edinburgh Enterprise Fellowship and an limit. Additionally, capturing exosomes from coarsely Imperial Confidence in Concept 2018 grant. We also isolated samples via specific antibodies would reduce acknowledge the support of Engineering and Physical the time required for sequential ultracentrifugation, the Science Research Council (EPSRC) grants [EP/ current standard technique for exosome isolation. L011573/1; EP/P028519/1] and the Biotechnology and Finally, this imaging chamber presents a versatile plat- Biological Sciences Research Council (BBSRC) form for protein profiling as the captured exosomes Foundry grant [BB/L027852/1]. can be labelled with specific antibody-dye conjugates to reveal the surface proteins contents.

PT09.11 PT09.12=OWP3.09

Single exosome size analysis using super resolution microscopy a b a Identification of single tumour-derived extracellular vesicles by Xia Li , Mina Hoorfar and Isaac Li means of optical tweezers and Raman spectroscopy a b c a b Agustin Enciso-Martinez , Edwin van der Pol , Aufried Lenferink , Leon University of British Columbia Okanagan, Kelowna, Canada Department of a a Chemistry, University of British Columbia Okanagan, Kelowna, Canada Terstappen and Cees Otto aMedical Cell Biophysics, University of Twente, Enschede, Netherlands; Introduction: Exosomes are a type of extracellular bAmsterdam UMC, University of Amsterdam, Department of Biomedical Engineering and Physics, Amsterdam, Amsterdam, Netherlands; cUniversity vesicle (EV) with diameters of 30–150 nm and are of Twente, Enschede, Netherlands secreted from most cell types. Owing to their significant role as cellular messengers and potential applica- Introduction: EVs derived from cancer cells play a role tions in disease detection, treatment and targeted in tumour cell proliferation, migration, invasion and delivery, growing efforts have been made in this rela- metastasis. Their presence in body fluids, such as tively new field. However, exosome research is hin- blood, makes them potential biomarkers for cancer dered by significant challenges including inefficient disease. However, the identification of single tdEVs separation methods, difficulties in characterization can be challenging due to their heterogeneity, their and lack of definitive biomarkers. Particularly, exo- ultra-small size, their size overlap with many other somes are difficult to visualize since their small size normal EVs and contaminants in body fluids and the falls below the resolution limit of conventional micro- lack of knowledge on their chemical composition. scopes (~200 nm). Methods: Synchronized optical tweezers and Raman Methods: Recent progress in super-resolution has pro- spectroscopy have enabled a study of individual EVs. vided novel tools in exosome characterization. In this The new method detects individual trapping events study, we present a single platform to capture pre- from Rayleigh scattering. The synchronous recording coarsely isolated exosomes onto an imaging flow cham- of Raman scattering enabled the acquisition of Raman ber through specific anti-bodies and perform super- spectra of both individual and multiple EVs, disclosing JOURNAL OF EXTRACELLULAR VESICLES 87 their chemical composition. Furthermore, Mie light methodology to study single tdEVs using correlative scattering theory has been used to relate the Rayleigh data from scanning electron microscopy (SEM), scattering intensity to the size of trapped EVs. Raman imaging (RI) and atomic force microscopy Results: The light scattered of trapped EVs gave rise to (AFM) to obtain a comprehensive dataset allowing step-wise time traces that can be used to distinguish identifying features unique to tdEVs. individual trapping events from accumulative cluster Methods: Indium tin oxide (ITO)-coated fused silica events due to the discrete nature of the steps which was selected for its low Raman background. Substrates correspond to single trapping events. Next, we con- (1 x 1 cm2) featuring position-dependent markings firmed the trapping of individual EVs derived from (“navigation marks”) patterned by photolithography PC3 cells, red blood cells, platelets and blood plasma were modified with a monolayer of amino dodecyl by acquiring both, Rayleigh and Raman scattering sig- phosphonic acid. The amine moieties were next reacted nals. While the step-wise trend in the Rayleigh scatter- with poly(ethylene glycol) diglycidyl ether, forming an ing signal suggests trapping of single particles, the anti-biofouling layer. Anti-EpCAM antibodies were Raman scattering signal demonstrates the nature of subsequently covalently bound on this surface. the trapped EVs. Through principal component analy- Samples of both tdEVs obtained from LNCaP cell sis (PCA), the main spectral variations among the four lines and RBC-derived EVs were then introduced to EV types were identified. The principal component the surfaces. Finally, non-specifically bound EVs were scores grouped the PC3-derived EVs in a separate washed away before SEM, AFM and Raman measure- cluster from the rest of the EVs. ments were performed. Summary/conclusion: We have developed an auto- Results: Multiple objects were captured on the fully mated single particle optical tweezers – Raman and functionalized ITO surfaces, according to SEM ima- Rayleigh scattering setup to trap and release single ging, while in negative control experiments (lacking EVs over time. We demonstrated single-EV trapping functionalization or lacking antibody or using by simultaneous acquisition of Rayleigh and Raman EpCAM-negative EVs), no object was detected. scattering. PCA enabled the identification of single- Principal component analysis of their Raman spectra, EVs derived from the cancer cell line PC3. This dis- previously demonstrated to be able to distinguish closes chemical information as a step towards the tdEVs from RBC-derived EVs, revealed the presence identification and characterization of single tumour- of characteristic lipid bands (e.g. 2851 cm−1) in the derived EVs in blood. captured tdEVs. AFM showed a surface coverage of Funding: Cancer ID – project number 14193, (par- ,4×10^5 EVs per mm2 with a size distribution tially) financed by the Netherlands Organisation for similar to that found by NTA. Scientific Research (NWO) Summary/conclusion: A platform was developed for multi-modal analysis of selectively isolated tdEVs for their multi-modal analysis. In the future, the scope of PT09.13=OWP3.02 this platform will be extended to other combinations of probe, light and electron microscopy techniques to Immunocapturing of tumour-derived extracellular vesicles on relate additional parameters describing the cap- micropatterned and antibody-conjugated surfaces for individual tured EVs. correlative light, probe and electron measurements Pepijn Beekmana, Agustin Enciso-Martinezb, Cees Ottob and Séverine Le Funding: Funded by NWO Perspectief Gacc aWageningen University, Wageningen, Netherlands; bMedical Cell Biophysics, University of Twente, Enschede, Netherlands; cApplied PT09.14=OWP3.03 Microfluidics for BioEngineering Research, University of Twente, The Netherlands, Enschede, Netherlands

The development of a scalable extracellular vesicle subset Introduction: Tumour-derived extracellular vesicles characterization pipeline. (tdEVs) are promising biomarkers for cancer patient Joshua Welsha, Julia Kepleyb and Jennifer C. Jonesa management. The screening of blood samples for aTranslational Nanobiology Section, Laboratory of Pathology, National tdEVs shows prognostic power comparable to screen- Cancer Institute, National Institutes of Health, Bethesda, USA; bTranslational Nanobiology Lab, Laboratory of Pathology, National Cancer ing of tumour cells. However, due to the overlap in size Institute, National Institutes of Health, Bethesda, USA between tdEVs, non-cancer EVs, lipoproteins and cell debris, new approaches, not only based on size, are Introduction: Liquid biopsies offer an important alter- required for the reliable isolation of tdEVs and their native to tumour biopsies that may be limited by the quantification. We report an integrated analysis challenges of invasive procedures. We hypothesize that 88 ISEV2019 ABSTRACT BOOK circulating Extracellular Vesicles (EVs) and their cargo templates containing metadata for reporting, as well may provide a useful surrogate biopsy method. Due to as uploaded into atlases such as Genboree, where mul- their small diameter (30-1000 nm), EVs migrate from tiplex data can be stratified by RNAseq datasets. tissue into the peripheral circulation and provide a Analysis using this pipeline has been conducted using snapshot of the producing cells. Our lab has developed human samples from a variety of mediums including a first-in-class pipeline to use single cell – omics meth- CSF, serum, and plasma comparing EV phenotypes. ods to characterize EV heterogeneity with high-sensi- Results: Our multiplex approach and MPAPASS soft- tivity by combining multiplex assays and our custom ware allows the use of single cell -omics tools for EV MultiPlex Analysis post-acquisition analysis software subset analysis in manner that will elucidate the biolo- (MPAPASS), with subsequent high-resolution, single gical significance and function of different types of EV flow cytometric (FCM) methods. EVs. This high-throughput pipeline evaluates hundreds Methods: A standalone software package was devel- of EV protein profiles and will allow evaluation of oped in MATLAB to allow importation of multiplex millions of RNA:protein profiles in an unprecedented flow cytometry output data. The package enables data manner. Integration of RNA sequencing with protein quality screening of detection antibodies, bead recovery characterization could provide an entirely new way of and data normalization methods. The software is understanding EV regulation and function. equipped to handle large data sets comprising hun- Summary/conclusion: Our data show this form of EV dreds/thousands of phenotypes and samples. Data can profiling provides a way to monitor clinical responses be visualized in a variety of ways along with clustering early in the course of treatment, which may ultimately using multidimensional data analysis techniques. All improve patient care and outcomes. software outputs can be exported in a standardized JOURNAL OF EXTRACELLULAR VESICLES 89

PT10: EVs and Stem Cells Chairs: Takashi Asada; Myung-Shin Lee Location: Level 3, Hall A 15:30–16:30

PT10.01 higher than the spheroids in HS medium and the cells from 2D culture. Vesicles isolated from DPPSC 3D culture of dental pulp pluripotent-like stem cells (DPPSC) spheroid in SR1 conditioned medium from Day 1–12 improves their pluripotency and provides a serum-free culture and Day 13–24 of culture showed sizes that fall within condition for exosome production Farid N. Faruqua, Khuloud Al-Jamala, Shuai Zhoub, Noor Samia, Fatemeh the exosomal size range, and are positive for the exo- Gheidaric and Maher Atarid somal markers CD81, CD9 and CD63. Vesicle yield for aKing’s College London, London, UK; bKing’s College London, XuZhou, Day 13–24 was higher than that of Day 1–12, but a China (People’s Republic); cKing’s College London, Tehran, Iran; larger percentage of particles from the latter were posi- dUniversitat Internacional de Catalunya, Barcelona, Spain tive for the three exosomal markers. Introduction: Exosomes from stem cells have been Summary/Conclusion: 3D spheroid culture of DPPSC identified as a novel cell-free therapeutic for regenera- in SR1 medium showed improvement in pluripotency, tive medicine. Culturing them in a serum-free condi- and allows for a serum-free culture for exosome tion for exosome isolation still poses a major challenge. production. This work focused on the establishment of a 3D culture of Dental Pulp Pluripotent-like Stem Cells (DPPSC) – a PT10.02 newly characterized pluripotent-like stem cell from adult tissue, for exosome production. Methods: DPPSC were initially cultured in monolayer Increased exosome secretion is essential for myeloma stem cells to survive in hypoxic condition (2D) in their basal medium with four different supple- Sayaka Nakayama, Yuki Toda, Shigekuni Hosogi and Eishi Ashihara mentations: human serum (HS), exosome-depleted Department of Clinical and Translational Physiology, Kyoto Pharmaceutical human serum (ED-HS), and two different serum repla- University, Kyoto-shi, Japan cements (SR1 & SR2). Morphology and growth rate of cells were analysed by bright-field microscopy and Introduction: Cancer stem cells (CSCs) of the highly regular cell counting. DPPSC were then transferred to tumorigenic cell population are critically associated a microwell culture plate for 3D culture in the four with the poor prognosis of patients in various types differentially supplemented media and maintained for of cancer. In our previous study, the multiple myeloma 24 days. Spheroid formation and morphology was (MM) cells which were chronically cultured in a observed throughout culture using bright-field micro- hypoxic condition (over 6 months, 1% oxygen) exhib- scopy. Spheroids were harvested on Day 24 and the ited stem cell characteristics. It suggests that MM stem expression of pluripotency genes Oct4A and Nanog cells are capable of adapting to hypoxic stress although were analysed by qPCR. Vesicles isolated from the adaptation mechanism remains unclear. We DPPSC conditioned-medium were characterized for focused on the excessive secretion of exosomes from size, yield and exosomal markers using Nanoparticle hypoxia-adapted MM cells (HA-MM cells). Exosomes Tracking Analysis (NTA) and dot blot. are considered as a garbage bin to remove unnecessary Results: In 2D culture, only DPPSC cultured in the molecules from the cytoplasm to maintain cellular default HS medium proliferated and showed the homeostasis, as well as a novel intercellular commu- expected morphology. In 3D culture, DPPSC in SR1 nication tool. medium formed spheroids of similar morphology and Methods: GW4869, an inhibitor of the ceramide- size to that of HS medium. Significantly smaller spher- mediated inward budding of the multivesicular bodies oids were formed by DPPSC in ED-HS medium, while for exosome biogenesis, was applied to analyse the DPPSC barely formed spheroids in SR2 medium. qPCR response to a deficiency of exosome secretion from analysis showed that while expression of Oct4A gene in their reduced production in HA-MM cells. DPPSC cells from 2D and 3D culture (both in HS and Results: GW4869 increased the rate of Annexin V SR1 media) was similar, expression of Nanog in positive (apoptotic) cells and induced the expression DPPSC spheroids in SR1 medium was significantly of fragmented PARP in HA-MM cells, but not in 90 ISEV2019 ABSTRACT BOOK parental cells cultured in a normoxic condition (20% EV-mediated gene regulation in the retinal microenvir- oxygen). With the addition of HA-MM-derived exo- onment remains undefined. In this study, we charac- somes, GW4869-induced apoptosis was not attenuated. terize the microRNA, tRNA, and piRNA composition From these results, HA-MM cells are likely to release of EVs secreted from human induced pluripotent stem exosomes to maintain the intracellular environment in cell (hiPSCs) – derived 3D retinas at three develop- a state of homeostasis, but not to receive them for mental time points that correlate with hallmarks of autocrine signal. Hexokinase 2 (HK2) generates glu- retinal cell differentiation and lamination in vivo. cose-6-phosphate, which is further metabolized by both Methods: Retinal organoids were generated from the glycolytic pathway and the pentose phosphate path- hiPSCs. We selected three developmental time points way (PPP). PPP plays a major role in supplying (day 42, 63 and 90) that represent distinctive stages NADPH for detoxification of intracellular reactive oxy- during normal retinal cell fate specification and lami- gen species (ROS). The upregulated HK2 protein nation. We analysed the release rate, concentration, expression in HA-MM cells was diminished by morphology and content (miRNA, tRNA and pi- GW4869. With dichlorodihydrofluorescein staining RNA) of EVs released from human hiPSCs-derived assay, GW4869 increased intracellular ROS production 3D retinas. in HA-MM cells. Thus, the failure of exosome secretion Results: The genetic signalling, developmental time might alter the energy metabolism leading to ROS- course and morphogenesis of these retinal organoids associated apoptosis. were comparable to those of developing human retinas Summary/Conclusion: Enhancement of exosome in vivo. According to Gene Ontology analysis, miRNA secretion is a survival strategy for hypoxic adaptation targets at the earliest stage of development were more in MM stem cells. This study could provide a critical relevant to early differentiation and cell morphogen- insight to develop a novel strategy for CSC-targeted esis, whereas miRNA targets at the later stages were therapy. more relevant to cell proliferation, cell differentiation, Funding: MEXT-Supported Program for the Strategic and cell migration. Research Foundation at Private Universities Summary/Conclusion: For the first time, this work demonstrates the rate of release and concentration of PT10.04 EVs from developing hiPSC-derived 3D retinal tissue. We report a large variety of small RNAs in EVs from hiPSC-derived 3D retinas, including miRNAs, tRNAs Extracellular vesicles released from human iPSC-derived 3D retinas and piRNAs. The full range of small RNAs detected in contain small RNAs with roles in development and differentiation Miguel Flores-Bellvera, Jing Zhoub, Xiufeng Zhongc, Alberto Benito-Martínd, our EVs may act as regulatory elements to modulate Jason Mightyb, Jiang Qiane, Jianbo Pane, Hao Wub, Bo-Juen Chenf, Alice gene activity and may serve as biomarkers of normal g h i j Liang , Héctor Peinado , Maria Valeria Canto-Soler and Stephen Redenti development. This work represents the first sequencing aCellSight – Ocular Stem Cell and Regeneration Program, Department of analysis of small RNA species contained in hiPSC- Ophthalmology, University of Colorado, Denver, USA; bLehman College, Biology Doctoral Program, The Graduate School and University Center, derived 3D retinas and their released EVs. City University of New York, New York, USA; cState Key Laboratory of Funding: NIH (R21 EY026752, SC3 GM113782); Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China; dChildren’s Cancer and Blood Foundation Laboratories, Research to Prevent Blindness; Challenge Grant to the Departments of Pediatrics, Weill Cornell Medicine, New York, NY, USA; Department of Ophthalmology, CU; NIH/NEI eWilmer Eye Institute. Johns Hopkins University School of Medicine, f g R01EY022631. Baltimore, USA; New York Genome Center, New York, USA; New York University Langone Medical Center, New York, USA; hMicroenvironment and Metastasis Laboratory, Molecular Oncology Programme. Spanish National Cancer Research Center (CNIO), Madrid, Spain; iCellSight – Ocular Stem Cell and Regeneration Program, Department of PT10.05 Ophthalmology, University of Colorado, Aurora, USA; jLehman College, Biology & Biochemistry Doctoral Program, The Graduate School and University Center, City University of New York, New York, USA Xeno-free manufacturing of MSC-EVs in scalable bioreactor culture Katrina Adlerza, Josephine Lembonga, Tim Olsena, Jon Rowleya and Taby Introduction: Noncoding small RNAs in the retina Ahsanb regulate gene expression by targeting and repressing aRoosterBio. Inc., Frederick, USA; bRoosterBio, Inc., Frederick, USA mRNA. Small RNAs are secreted in extracellular vesicles (EVs). Analysis of EVs released from developing Introduction: There have been over 800 clinical trials retinal tissue is an essential step in elucidating the role using mesenchymal stem/stromal cells (MSCs) for of EV molecular cargo and signalling during retinogen- therapeutic applications. Due to their similar therapeu- esis. A number of canonical genes are associated with tic effects to MSCs and potential as a key bioactive retinal cell fate determination during development, but agent in regenerative medicine applications, MSC- JOURNAL OF EXTRACELLULAR VESICLES 91 derived extracellular vesicles (MSC-EVs) are being brain area and increased the proliferation rate and the increasingly investigated as a clinical therapy for a neurorestorative capacity of MSCs. Our previous study broad range of indications. It was recently found that confirmed that intravenous infusion of MSC-EVs is the number of exosomes released from 2M MSCs in 48 more advantageous than MSCs as a safe treatment. h is equivalent to a single dose for a rodent. Hence, EVs are lipid enclosed vesicular structures that contain most indications would require a MSC production lot bioactive RNAs, DNA and proteins, which possess size that is hardly achievable in 2D culture. Therefore, therapeutic molecules similar to MSCs. Therefore, we larger scalable bioreactor systems will be crucial to hypothesized that the certain proteins containing EV generate enough EVs for clinical doses. This study released from stroke serum cultured MSCs may affect developed a protocol for xeno-free (XF) scalable the neurogenesis and angiogenesis of recipient cells. MSC-EV manufacturing and compared MSC-EV char- Methods: EVs were purified from conditioned media acteristics from 2D culture and various bioreactor of MSC cultured with FBS (FBS-MSC) and MSC cul- scales. tured with stroke serum (SS-MSC). These EVs were Methods: Human Bone Marrow-Derived MSCs (hBM- characterized by nanoparticle tracking analysis. EV MSC) were cultured on microcarriers in suspension protein profiling in conditioned media was systemati- using 0.1, 3 and 15L bioreactors. After cell inoculation, cally compared through utilizing LC-MS/MS-based a bioreactor feed was added on Day 3, and cultures label-free quantification. Real-time PCR was performed switched to an EV collection media on Day 4. After 1, 2 to determine the difference in the gene expression in and 3 days, the collection medium was analysed for each cell. The protein concentration in the EV was metabolites, particle size, and particle concentration. confirmed by ELISA. The MSC-EVs in the conditioned medium was also Results: A total of 1068 proteins were identified from evaluated for protein expression of Alix, CD63, and SS-MSC-EV and FBS-MSC-EV through LC-MS. CD81, RNA expression and wound healing capability. According to statistical analysis, 22 proteins were Results: A protocol was developed for manufacturing found to be more than 2-fold (p < 0.05) upregulated MSC-EVs in an XF fed-batch bioreactor culture, result- in SS-MSC_EV. ITGA5, CLU, and CTSB were signifi- ing in cell yield of >0.5M cells/mL within 4 days. We cantly increased of SS-MSC gene expression levels demonstrated that this process was directly scalable compared to FBS-MSC. Among the candidate proteins, from the small (0.1L) to development (3L) and pilot clusterin (CLU) was found to be upregulated in EVs scale (15L) bioreactors, maintaining similar cell den- from SS-MSC compared to those from FBS-MSC. sity. To remove the residual particles from the expan- Summary/Conclusion: These results suggest that SS- sion media, an additional wash step before the switch MSC_EVs containing clusterin may promote intercel- to the collection media was required in bioreactors. lular communication and affect neurogenesis and With a similar collection process and comparable cell angiogenesis of recipient cells. density, we expect to see consistent EV production per Funding: This study was supported by a grant from the cell at these different scales of manufacturing systems. Korean Healthcare Technology R&D Project, Ministry Summary/Conclusion: Optimizing EV yield will of Health & Welfare (HI17C1256) and Basic Science become increasingly important as EVs become used Research Program, the Ministry of Science, ICT and in the clinic. We have developed a scalable fed-batch Future Planning (2018M3A9H1023675). process for large scale expansion of hMSCs and a protocol for EV production in suspension bioreactors. PT10.07 Funding: This work was funded by RoosterBio, Inc.

Adipose-derived Stem/Stromal Cell secretome, containing both PT10.06 soluble factors and extracellular vesicles, exerts chondroprotective effects in vitro Chiara Giannasia, Stefania Niadaa, Sara Casatib and Anna Brinic

a b Proteomic analysis of extracellular vesicles from MSC cultured with IRCCS Istituto Ortopedico Galeazzi, Milano, Italy; Department of stroke serum Biomedical, Surgical and Dental Sciences, University of Milan, Milano, c Yeon Hee Choa, Eun Hee Kima, Dong Hee Kimb, Ji Hee Sunga, Mi Jeong Oha, Italy; Department of Biomedical, Surgical and Dental Sciences, University Eun Kyoung Shina and Oh Young Banga of Milan. IRCCS Istituto Ortopedico Galeazzi, Milano, Italy aSamsung Medical Center, Seoul, Republic of Korea; bSungkyunkwan University, Seoul, Republic of Korea Introduction: Up to now several clinical trials have shown the safety and efficacy of the intra-articular Introduction: Serum from stroke patients increases injection of Adipose-derived Mesenchymal Stem/ mesenchymal stem cells trophism towards the infarcted Stromal Cells (ASCs) in contrasting osteoarthritis. 92 ISEV2019 ABSTRACT BOOK

Since ASCs act predominantly through paracrine lineage are likely candidates, thus indicating that MSCs mechanisms, their secretome represents a promising and the OS stroma cells may be related cell types. cell-free alternative. Here we identified anti-hyper- Therefore, this study set out to examine the contribu- trophic and anti-catabolic effects of ASC conditioned tion of EV-mediated intercellular crosstalk of MSC medium (ASC-CM) on TNFα-stimulated human pri- and OS. mary articular chondrocytes (CHs). Methods: MSCs and pre-osteoblasts were treated with Methods: CHs were treated with 10 ng/mL TNFα and/ OS-EVs at different time points, and the epigenetic or ASC-CM administered at a 1:5 recipient:donor cell signature of OS-EVs was assessed by LINE-1 and ratio. Cell viability was assessed up to day 9. The tumour suppressor genes methylation analysis. In addi- activity, expression and/or release of hypertrophy mar- tion, surface markers and gene and expression of spe- kers (MMP-13, Collagen X and Osteocalcin), catabolic cific genes related to bone microenvironment mediators (MMP-3) and cartilage-protective factors remodelling (MMP1, VEGF-A, ICAM1) were also were assessed up to day 3 by enzymatic assays, qRT- evaluated. PCR, Western Blot and multiplex immunoassays. Results: Our data indicated that OS-EVs mediated Results: ASC-CM blunted TNFα-induced hypertrophy, LINE-1 hypomethylation in MSCs, whereas an oppo- reducing the enhanced levels of MMP-13 activity site effect was seen in pre-osteoblast, indicating that (−61%), Osteocalcin (−37%) and Collagen X (−18%). MSCs but not pre-osteoblasts were more susceptible to In addition, also MMP-3 activity was diminished by epigenetic transformation. Thus, OS-EVs dictated the −59%. We associated the observed reduction of MMP- fate of MSCs by modulating the epigenetic status, and 3 and MMP-13 activity to the abundancy of TIMPs also influenced the expression of genes related to bone (Tissue Inhibitors of MMPs) in ASC secretome, rather microenvironment remodelling. than to a direct down-modulation of their expression Summary/Conclusion: Overall, this study provided and/or release. Moreover, ASC-CM contains high evidence that epigenetic regulation may appear to be levels of OPG and DKK-1, other known chondropro- an early event in the transformation of MSCs. tective factors. Elucidating the mechanisms of EV-mediated commu- Summary/Conclusion: ASC-CM is rich in cartilage- nication may lead to new avenues for therapeutic protective factors and exerts anti-hypertrophic and exploitation. anti-catabolic effects on TNFα-stimulated CHs. These Funding: This research was supported by University of evidences open the way for its possible clinical use as a Helsinki project funding (WBS490302, WBS73714112), cell-free approach in contrasting osteoarthritis. We are Helsinki University Hospital State funding for univer- currently investigating through a differential proteomic sity-level health research (Y1014SUL05, TYH2016130), analysis if the recognized chondroprotective effectors the Finnish Dental Society Apollonia, Egyptian are enriched in the vesicular rather than the soluble Ministry of Higher Education (MoHE) and Selma and component of the secretome. Maja-Lisa Selander foundation.

PT10.08 PT10.09

Comprehensive proteomics and microRNA analyses of adult neural Epigenetic alterations in mesenchymal stem cells by osteosarcoma stem cell derived exosomes after stroke derived extracellular vesicles Xianshuang Liua, Michael Choppb, Chao Lia, Wan Long Pana, Bao Yan Fana, Roman Kornilov, Sippy Kaur, Bettina I. Mannerström, Ahmed Abu-Shahba, Albert M Levinc, Rui Lan Zhanga and Zheng Gang Zhanga Iftekhar Chowdhury, Snehadri Sinha and Riitta Seppänen-Kaijansinkko aDepartment of Neurology, Henry Ford Health System, Detroit, MI, USA; Department of Oral and Maxillofacial Diseases, University of Helsinki and bDepartment of Physics, Oakland University, Rochester, MI, USA; Helsinki University Hospital, Helsinki, Finland cDepartment of Public Health Sciences, Center for Bioinformatics, Henry Ford Health System, Detroit, MI, USA Introduction: Extracellular vesicles (EVs) are central to intercellular communication and play an important Introduction: Neural stem cells (NSC) are known to role in cancer progression and development. facilitate healing of ischemic brain tissues. Recent stu- Osteosarcoma (OS) is an aggressive bone tumour, dies show that NSC derived exosomes function as characterized by presence of malignant mesenchymal paracrine effectors to promote neurovascular remodel- cells. Specific tumour-driving genetic alterations asso- ling including angiogenesis and axonal outgrowth after ciated with OS development are poorly understood. stroke; nevertheless, the contents of the non-stroke and The cell of origin for OS also remain unknown but post stroke NSC exosome proteome and miRNA cargo cells of the mesenchymal stem cell (MSC) osteogenic have not been determined. JOURNAL OF EXTRACELLULAR VESICLES 93

Methods: NSC derived exosomes were purified from clinical application in the future. We sought to apply a conditioned media of cultured NSCs harvested from novel anion chromatography for the isolation of iPSC- the subventricular zone of non-ischemic and ischemic MSC EVs, and explored the effects and mechanisms of rats, respectively. Liquid chromatography mass spec- iPSC-MSC EVs in the therapy for asthma. trometry (LCMS) and miRNA array were employed to Methods: The EV-enriched supernatants were col- comprehensively characterize the protein and miRNA lected for the isolation of the iPSC-MSC EVs using contents of NSCs and their derived exosomes after the anion chromatography. The morphologies of EVs stroke. Bioinformatic analyses were performed using were characterized by transmission electron micro- Ingenuity Pathway Analysis (IPA). scope, the markers of EVs were assayed by western Results: Exosome markers including CD63, CD9, Alix blot and flow cytometry. The anti-inflammatory effects and size distribution (50–200nm) were verified with of the EVs were determined using the macrophage Western blot, transmission electron microscopy assay. Also, the uptake activities of macrophages on (TEM) and Nanosight, respectively. In total, proteo- RPF-iPSC-MSC EVs were determined. Finally, the mics analysis yielded 2409 and 1770 proteins identified asthma mouse model was developed and the iPSC- in ischemic NSC and NSC derived exosomes, respec- MSC EVs were administrated intravenously. The lung tively. Bioinformatics analysis identified that 52, 39 and pathology, the levels of inflammatory cytokines in the 31 proteins in the NSCs-derived exosomes were related bronchoalveolar lavage fluids (BALF) and the polariza- to regulating neuronal cell proliferation, migration and tion of lung macrophages were evaluated. differentiation, respectively. In addition, 318 miRNAs Results: We successfully obtained concentrated iPSC- were identified in ischemic NSCs with 26% of miRNAs MSC EVs after the isolation and the final concentration (84 miRNAs) overlapped with parent NSCs. Gene of EVs was about 200 µg/mL (Bradford) and 10– ontology analysis showed that up- and down-regulated 15*1011/mL (Nanosight). The iPSC-MSC EVs were miRNAs with the fold change above 1.5 were highly morphologically intact and were positive for the mar- related to inflammation, invasion, cell proliferation, kers including CD9/63/81, Alix and TSG101. Most of cell cycle, cell death, differentiation, etc. The top three the preparations of iPSC-MSC EVs could significantly upregulated miRNAs were validated in ischemic NSC- decreased the level of IL-6 in the macrophage assay. exosomes using real-time RT-PCR. The Raw 264.7 macrophages began to uptake iPSC- Summary/Conclusion: Collectively, the results of our MSC EVs at 4 h and the uptake activities peaked at proteomic and miRNA analysis, to our knowledge, 12 h and then receded at 24 h. Also, our in vivo study demonstrate for the first time that NSC derived exo- showed that splenic macrophages started to uptake somes contain a robust profile of protein and miRNA iPSC-MSC EVs at 4 h and the uptake activities were effectors. These data provide a platform for beginning augmented at 24 h. In addition, the iPSC-MSC EVs to understand the mechanism by which NSCs are acti- significantly reduced the inflammatory infiltration, the vated after cerebral ischemia, and may lead to a deeper epithelial goblet cell numbers, the levels of inflamma- mechanistic understanding of their role in tissue repair tory cytokines and inflammatory cells in the BALF as after neural injury. well as the polarizations of pulmonary macrophages. Funding: NIH RO1 DK102861, American Heart Summary/Conclusion: Our results showed that the Association (AHA) grant 18IPA34170331, NINDS anion exchange chromatography was a promising RO1 NS075156 and RO1 NS088656. method for the large preparation of iPSC-MSC EVs, which could possibly be an alternative therapy for PT10.10 asthma in the future. Funding: NSFC (81322012, 81373174, 81471832, 81671882 and 81770984) Anion exchange chromatographic isolation of iPSC-MSC derived extracellular vesicles ameliorated allergic asthma in mice Shubin Fang, Hongyu Zhang, Yongdong Lin and Qingling Fu PT10.11 Otorhinolaryngology Hospital, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China (People’s Republic) Characterization and miRNA expression profiles of exosomes from HLA homozygous haplotype dental pulp cells and iPS cells Introduction: The extracellular vesicles (EVs) derived Yuta Shimizua, Tomoko Kawaguchib, Shuhei Otaric, Izumi Kurodad, Yuki from mesenchymal stem cells have been shown to Kuranagae, Hideya Kawasakif, Takahiko Hariyamaf, Toshiyuki Shibatab, Takahiro Kunisadad, Toshiaki Shibutanic, Yukihiro Akaoe and Ken-ichi elicited similar therapeutic effects to their patent cells Tezukag in many diseases. However, the difficulties in the large- aAsahi University School of Dentistry, Department of Periodontology, scale preparation of high-purity EVs largely limited its Mizuho, Japan; bGraduate School of Medicine Department of Oral and 94 ISEV2019 ABSTRACT BOOK

Maxillofacial Science, Gifu, Japan; cAsahi University School of Dentistry serum-free conditions. Exosomes were purified from Department of Periodontology, Gifu, Mizuho, Japan; dGifu University Graduate School of Medicine Department of Tissue and Organ culture supernatants by ultracentrifugation. Purified Development Regeneration and Advanced Medical Science, Gifu, Japan; exosomes were subjected to particle size determination eGifu University Graduate School of Drug Discovery and Medical Information Sciences, Gifu, Japan; fHamamatsu University School of with a nanoparticle analysis system (Nanosight LM10), Medicine Depatment of Preeminent Medical Photonics Education & exosome markers and HLA class I evaluation by g Research Center Institute for NanoSuit Research, Hamamatsu, Japan; Gifu Western blotting (WB), and miRNA expression analy- University Graduate School of Medicine Department of Tissue and Organ Development Regeneration and Advanced Medical Science, Gifu, Japan and sis, and results were compared. HHH-iPS cell exo- Center for Highly Advanced Integration of Nano and Life Sciences, Gifu somes were also examined if teratomas were formed University (G-CHAIN), Gifu, Japan in immunodeficient mice. Introduction: Human leucocyte antigen (HLA) has Results: Nanosight LM10 confirmed that the particle played an important role to distinguish between self size peaks were nearly identical at ~100 nm. WB and non-self in the immune system. HLA homozygous revealed that both DP cell exosomes and iPS cell exo- cell of the multi-locus has been considered to be less somes expressed CD81 and HLA class I, but expression likely to be rejected in the allograft. In recent years, the levels of CD81 and HLA class I were lower in iPS cell diameter to 100 nm of the extracellular vesicles called exosomes. The miRNA analysis showed that some exosomes, to cellular functions secreted from iPS cells miRNAs differed between cells and between exosomes. or tissue stem cells, partially responsible, such an In assessment of teratoma forming ability, no tumour immune response and during communication tools it formation was observed. has been reported to play a role as a tissue repair. In Summary/Conclusion: HHH-DP cell exosomes and this study, we compared the exosomes from HHH-DP HHH-iPS cell exosomes were found to have different HLA homozygous haplotypes from cell-derived HHH- surface antigens and miRNA expression profiles. iPS cells (HHH) pulp (DP) cells and exosomes. HHH-iPS cell exosomes showed a reduced level of Methods: Three lines of HHH-DP cells established at HLA expression and no teratoma formation, and thus Gifu University and HHH-iPS cells derived from these are potentially useful for therapeutic purpose. cells were used. DP and iPS cells were cultured in JOURNAL OF EXTRACELLULAR VESICLES 95

PT11: EV Based Cancer Therapeutics Chairs: AC Matin; Eva Rhode Location: Level 3, Hall A 15:30–16:30

PT11.01 biological processes that could potentially be regulated by the EV-miR profile detected such as tumour prolif-

Cellular and secreted extracellular vesicles-encapsulated miRNAs in eration and bone cell resorption. the 4T1 murine model of breast cancer Summary/Conclusion: Analysis of EVs from animals a b c b Katie E. Gilligan , Róisín Dwyer , Clodagh O’Neill , Eimer o’Connell and bearing 4T1 tumours is ongoing to determine whether Peter Dockeryb the EV-miR profile could serve as a biomarker of dis- aNational University of Ireland Galway, Galway, Ireland; bNUI Galway, Galway, Ireland; cNational University of Ireland, Galway, Ireland ease. The data presented demonstrates the selective packaging of tumour associated miRNAs into EVs Introduction: Extracellular vesicles (EVs) are secreted which could play an important role in disease by all cells and are known to contain a range of genetic progression. material such as microRNAs (miRNAs). EVs have been Funding: Irish Research Council, Government of implicated in mediating intercellular communication Ireland Postgraduate Scholar 2016 GOIPG/2016/978. to support breast cancer progression and also highlighted as a potential biomarker of disease. This study aimed to investigate the miRNA profile of EVs released PT11.03 by 4T1 breast cancer cells in vitro and to relate this to the circulating EV profile of an animal model of this disease. Delivery of miR-185 enriched EVs from MSCs inhibits the progression Methods: 4T1 cells were cultured in EV-depleted of OPMD Lin Wanga, Yuanyuan Wangb, Jiaqi Wangb, Congcong Miaob, Haimei Sunb, media, and secreted EVs isolated through sequential Yu Zhouc and Xiaobing Guana differential centrifugation, micro-filtration and ultra- aCapital Medical University, Beijing, USA; bCapital Medical University, centrifugation. EVs were also isolated from the sera of Beijing, China (People’s Republic); cBeijing Ludaopei Institute of balb/c mice bearing 4T1 tumours. EVs were character- Haematology, Beijing, China (People’s Republic) ized by Nanoparticle Tracking Analysis (NTA), Western Blot and Transmission Electron Microscopy Introduction: Oral leucoplakia is one of the most (TEM). RNA was extracted from all cells and EVs common oral potentially malignant disorders using the MagNA pure compact and Next-Generation (OPMD) and its malignant transformation is asso- Sequencing (NGS) targeting miRNA was performed. ciated with chronic inflammation. It is clear that the Targets of interest were validated by Polymerase tumour microenvironment, which is largely orche- Chain Reaction (PCR). strated by inflammatory cells, is an indispensable par- Results: EVs were successfully isolated from all sam- ticipant in the fostering proliferation, survival and ples with the majority of vesicles falling within the migration. Extracellular vesicles (EVs) shuttle complex range of exosomes (30–120 nm). Western blot analysis molecular cargo between producer and recipient cells confirmed the presence of tetraspanins CD63, CD81 resulting in epigenetic regulation of cell function. EVs and CD9. The characteristic size and shape (cup) of derived from mesenchymal stem cells (MSCs) have EVs were visualized by TEM. Over 380 previously been found to promote therapeutic activities that are annotated miRNAs were detected in the 4T1 secreted comparable to MSCs themselves. EVs, with 11 novel putative miRNA sequences identi- Methods: Bone marrow derived MSCs were transfected fied. Twenty-five miRNAs were found to be differen- with high copy numbers of miR-185 mimics and EVs tially expressed between the cells and their secreted were harvested using Genexosome Isolation kit. EVs. Interestingly, of these, 14 miRNAs were present miR185 enriched EVs were characterized and applied at a significantly higher level in the EVs compared to on the buccal mucosa in the OPMD model exposed to the cells. Including a range of miRNA previously asso- 7,12-dimethylbenz anthracene (DMBA). Pathological ciated with cancer progression, e.g. miR-486-5p. Gene analysis of the buccal mucosa was studied, and the ontology enrichment identified a range of key topical and serum levels of inflammatory cytokines 96 ISEV2019 ABSTRACT BOOK and chemokines were measured. In addition, the protein expression of RAB27A in various cancer cell expression levels of caspase 3 and 9 were examined. lines. Moreover, migration and invasion activity of Results: EVs released from genetically modified MSCs cancer cells were markedly suppressed by drug, sug- had ~25-fold higher expression levels of miR-185 than gesting that this drug has possibility to be used for anti- the control. Confocal microscopic imaging revealed cancer therapy. that the PKH26 fluorescence labelled EVs principally Summary/Conclusion: These findings demonstrate localized in the buccal mucosa after administration. that a drug to inhibit exosome secretion selectively in After treatment with miR-185 enriched EVs for 3 or cancer cells could be used for the therapy of various 5 weeks, the topical inflammation severity in buccal cancers. Importantly, our study offers a new mechan- mucosa was remarkably attenuated, the levels of IL-6, istic insight into drug development by the inhibition of IL-1β, JE, MIP-1a, MIP-2 and TREM-1 were decreased, exosome secretion. and the numbers of inflammatory cells were reduced as Funding: This work was supported by the National well. Pathological analysis of the buccal tissue showed Research Foundation of Korea (NRF) grant funded by significantly decreased numbers of cells with hyperpla- the Korea government (2014R1A5A2009242) sia or dysplasia after treatment. In addition, miR185 This research was supported by the Bio &Medical enriched EVs led to significantly increased levels of Technology Development Program of the National caspase 3 and 9 in the buccal tissue, indicating Research Foundation (NRF) funded by the Ministry miR185 promotes the activation of apoptotic pathway. of science & ICT (2017M3A9G8083382) Summary/Conclusion: miR-185 enriched EVs from MSCs are anti-inflammatory and anti-proliferative, PT11.05 and promote apoptosis. Genetically modified MSC- derived EVs have signiﬁcant potential as a novel therapy for oral leucoplakia. Platelet-derived microparticles as an oriented bullet for cancer treatment Yu-Wen Wu and Thierry Burnouf

College of Biomedical Engineering, Taipei Medical University, Taipei, Taiwan PT11.04 (Republic of China)

Introduction: Platelets (PLTs) and PLTs-derived Identification of exosome secretion inhibitor for cancer therapy Jong-In Kim, Eun-Ju Im, Chan-Hyeong Lee and Moon-Chang Baek microparticles (PMPs), released by PLTs upon thrombin activation, interact closely with cancer cells in the School of medicine, Kyungpook National University, Daegu, Republic of Korea tumour microenvironment. Some researchers have been used synthetic nanoparticles loaded with antic- Introduction: Exosomes are nanosize secreting vesicles ancer agents and coated with whole PLT membranes that can internalize and interact with other cells to for cancer therapy. However, isolating PLT membranes initiate physiological and pathological signalling path- and synthesizing nanoparticles coatings sufficient for ways. Especially, tumour-cell derived exosomes (TDXs) translational applications. Further, procedures for iso- activate tumour-related mechanism such as prolifera- lating PLT membranes may denature proteins, which tion, metastasis and drug resistance. We hypothesized may alter targeting specificity and incur an adverse risk that inhibition of exosome secretion may have benefi- of immunogenicity in patients. Therefore, our aim is to cial effects in the treatment of cancer. Here, we found isolate and evaluate the ability of PMPs to serve as an old drug which inhibits exosome secretion from Trojan Horse carriers of anticancer drugs for cancer various cancer cells. treatment. Methods: Human breast cancer and Human melanoma Methods: PLT concentrates were centrifuged at cancer cell lines were cultured. Immunoblotting was 3000 ×g for 15 min at 24 ± 3°C and the pellet performed with primary antibodies against RAB27A (PLTs) was suspended in thrombin in Tyrode’s buffer and beta-actin. Cells were seeding in 24 well plates (0.1U/mL) to induce activation and incubated at 37°C then treated candidate drugs for 24 h. Cell viability for 1 h. The solution was then centrifuged at 3000 ×g was measured by MTT assay. Exosomes were isolated for 10 min at 24 ± 3°C to remove PLTs and the super- by serial centrifugation method, then resuspended in natant (PMPs) was centrifuged at 20,000 ×g for 90 min PBS for further experiments. Exosome concentration at 18°C. The PMPs pellet was resuspended in platelet was analysed by NTA. additive solution (PAS) and stored at −80°C. PMPs Results: Exosome secretion was significantly decreased were thawed at 37°C then incubated with 100 μM by drug treatment. In addition, this drug affected doxorubicin (DOX) in PAS at 37°C for 1 h. The JOURNAL OF EXTRACELLULAR VESICLES 97 supernatant was centrifuged at 20,000 ×g for 90 min at characterized with transmission electron microscopy 18°C. The pellet of PMPs loaded with DOX (PMPDs) (TEM), atomic force microscopy (AFM), flow cytome- was resuspended in PAS. The sizes and the concentra- try, Western Blot and dynamic light scattering. The tions of PMPs and PMPDs were measured using a interaction of PvD1 and vCPP2319 ACPs with the nanoparticle tracking analysis (NTA). Data were ana- breast cells and respective exosomes was also followed lysed using NTA software. Transportation of DOX with surface plasmon resonance (SPR) as to detail from PMPDs to breast cancer cell lines was observed peptide’s binding to the different exosomes. by deconvolution microscopy. Results: Results suggests an intracellular target for Results: NTA results revealed that the mean size of vCPP2319 cytotoxic activity on breast cancer cells. PMPDs (234.1 ± 48.01 nm) was slightly larger com- The binding of the peptides to both membranes of pared with that of PMPs (200.1 ± 57.71 nm) and that human cells and exosomes results in cell death and in DOX incorporation did not influence the quantifica- strong binding, respectively, pointing to the potential tion of PMPs. The concentration of them was no sig- ability of these breast exosomes in transporting ACPs, nificant difference. The size distributions and images of which in turn are highly effective towards tumour cells. PMPs and PMPDs indicated the absence of aggregated Summary/Conclusion: Even though more studies are PMPs associated with DOX loading. When incubated currently in development, the combination of potential with MCF-7 and MDA-MB-231 cells, PMPDs trans- ACPs with human-derived exosomes are shown as a ferred DOX to the nuclei of cancer cells within 30 min. potential source for a highly selective and effective Summary/Conclusion: These results support the DDS aiming to attack breast tumour cells located in potential clinical use of PMPDs as novel cell-based the brain. “Trojan Horse” anti-cancer therapeutic strategy. Funding: Fundação para a Ciência e a Tecnologia Funding: This study was supported by the Ministry of (FCT I.P., Portugal) is acknowledged for funding – Science and Technology. PTDC/BBBBQB/1693/2014. F. O., J. S. and T. F. acknowledge FCT I.P., Portugal for fellowships PD/ BD/135046/2017, PD/BD/114177/2016 and SFRH/BD/ PT11.06 5283/2013, respectively. Marie Skłodowska-Curie Research and Innovation Staff Exchange (RISE) is Design of an exosome-based drug delivery system transporting acknowledged for funding: call H2020-MCA-RISE- anticancer peptides for targeting breast metastases in the brain 2014, Grant agreement 644, 167, 2015–2019. Filipa Oliveiraa, Julia Skalskaa, Tiago Figueiraa, Patrícia Napoleãoa, Érica Mellob, David Andreuc, Valdirene Gomesb, Miguel Castanhoa and Diana Gaspara aInstituto de Medicina Molecular João Lobo Antunes, Faculdade de PT11.07 Medicina, Universidade de Lisboa, Lisboa, Portugal; bLaboratório de Fisiologia e Bioquímica de Microrganismos do Centro de Biociências e Biotecnologia da Universidade Estadual do Norte Fluminense Darcy Ribeiro, Rio de Janeiro, Brazil; 3Department of Experimental and Health Sciences, Pompeu Fabra University, Barcelona Biomedical Research Park, Embryonic stem cells-derived exosomes endowed with targeting Barcelona, Spain properties as chemotherapeutics delivery vehicles for glioblastoma therapy Xiaozheng Ling, Qingwei Zhu, Yunlong Yang, Yang Wang, Zhifeng Deng Introduction: The treatment of breast cancer brain Shanghai Jiao Tong University Affliated Sixth People’s Hospital, Shanghai, metastases can be addressed with the effective delivery China (People’s Republic) of anti-tumoural drugs into the brain. The development of a drug delivery system (DDS) that can physio- Introduction: Glioma treatment is severely hindered logically match the cell membrane, decrease the by blood brain barrier (BBB) which leads to very lim- development of immune responses and that crosses ited on-target activity of therapeutic agents. Exosomes biological barriers is significantly valuable for treating are nanosized extracellular vesicles with efficient BBB metastatic breast cancer (MBC). When compared to penetration ability and presents a promising drug car- other nanoparticle delivery vehicles, exosomes repre- rier for glioma treatment. However, several reports sent an interesting approach to conventional DDS. In have demonstrated that injected exosomes mainly dis- the present work, exosomes from breast cells were tribute in liver and spleen rather than brain. In this isolated and biophysically characterized. In addition, study, we find embryonic stem cell derived exosomes their interaction with anticancer peptides (ACPs) was (ES-Exos) show broad spectrum anti-tumour ability unravelled envisioning the design of a DDS for MBC. including glioma, and thus we further use ES-Exos as Methods: Exosomes from breast cell lines were isolated paclitaxel (PTX) carrier and modify them with tumour using a commercially available kit and biophysically targeting ligand cRGD. 98 ISEV2019 ABSTRACT BOOK

Methods: CCK-8 analysis and flow cell analysis were Results:Thisstudyreportsanenzymaticexosome,which used to test the anti-tumour ability of ES-Exos. cRGD harbours native PH20 hyaluronidase (Exo-PH20), which was incorporated onto the surface of ES-Exos by post- is able to penetrate deeply into tumour foci via hyalur- insertion methods with cRGD-DSPE-PEG2000 (cRGD- onan degradation, allowing tumour growth inhibition Exos), and PTX was loaded into cRGD-Exos by co- and increased T cell infiltration into the tumour. This incubation to get cRGD-Exos-PTX. In situ glioma exosome-based strategy is developed to overcome the model of mice was built by injecting glioma cells in immunosuppressive and anticancer therapy-resistant brain. In vivo imaging was used to test the biodistribu- tumour microenvironment, which is characterized by an tion of cRGD-Exos-PTX. Further, subcutaneous overly accumulated extracellular matrix. Notably, this tumour of mice was also built to evaluate the anti- engineered exosome with the native glycosylphosphatidy- tumour ability of ES-Exos and cRGD-Exos-PTX. linositol-anchored form of hyaluronidase has a higher Results: Our results showed that ES-Exos could inhibit enzymatic activity than a truncated form of the recombi- tumour cell proliferation of broad spectrum, including nant protein. In addition, the exosome-mediated codeliv- U87, U251, A549, HCC, HepG2, B16, MDA-MB-231 ery of PH20 hyaluronidase and a chemotherapeutic and DU145. Flow cell analysis showed that ES-Exos (doxorubicin) efficiently inhibits tumour growth. This induced tumour cell apoptosis. Furthermore, after exosome is designed to degrade hyaluronan, thereby aug- cRGD modification, cRGD-Exos showed enhanced menting nanoparticle penetration and drug diffusion. tumour cell uptake compared with ES-Exos. And in Summary/Conclusion: Here, we developed the engi- vivo imaging analysis demonstrated that more cRGD- neered exosome that facilitates its own penetration into Exos distributed in glioma site in mice brain. And mice the HA-containing tumour ECM. Enabling chemical with in situ glioma treated with cRGD-Exos-PTX lived drugs, nanoparticles, and immune cells to penetrate more longer than the group treated with Exos-PTX. deeply into tumour foci is a challenging goal of studies Finally, cRGD-Exos-PTX showed the beat anti-tumour aimed at achieving antitumor therapeutic efficacy. The ability in subcutaneous tumour model. exosome-triggered infiltration of cytotoxic T cells into Summary/Conclusion: In this study, we demonstrate tumour tissues, which was observed in the present that ES-Exos is antineoplastic, and their tumour site work, could induce an adaptive immune response to distribution is enhanced by cRGD modification. help combat cancer. Moreover, we provide a general cRGD-Exos-PTX is an efficient therapeutic agent for strategy that may be used to decorate exosomal sur- glioma treatment. faces with natural-state membrane-bound proteins. Funding: NSFC Project No. 81671209 and No. 81471243. PT11.09 PT11.08 Surface engineering of exosomes to block HIV infection Exosome as a vehicle for delivery of membrane protein therapeutics, Pooja Bhardwaja, Shivani Desaia, Ali Danesha, Amirali Afsharib, Archana PH20, for enhanced tumour penetration and antitumor efficacy Guptab and Satish K. Pillaia Yeonsun Hong, Yoon Kyoung Kim and Yoosoo Yang aVitalant Research Institute, San Francisco, USA; bSystem Biosciences (SBI), Korea Institute of Science and Technology, Seoul, Republic of Korea Palo Alto, CA, USA

Introduction: As biochemical and functional studies of Introduction: While lifelong antiretroviral therapy has membrane protein remain a challenge, there is growing dramatically reduced the morbidity and mortality of interest in the application of nanotechnology to solve HIV infection, treated individuals still experience the difficulties of developing membrane protein ther- immune dysregulation and chronic inflammation, driv- apeutics. Exosome, composed of lipid bilayer enclosed ing interest in alternative therapeutic and curative stra- nanosized extracellular vesicles, is a successful platform tegies. Exosomes, extracellular membrane vesicles 30– for providing a native membrane composition. 100 nm in size, have shown promise as engineerable Methods: Exosome Preparation and Characterization – therapeutic agents for a broad range of diseases. We DLS, western blot, TEM Enzymatic Activity Assay in aimed to engineer exosomes with the capacity to block vitro and in vivo HA Depletion Analysis Tumour Blood HIV infection as a novel antiviral approach. Flow Biodistribution Imaging of Dox Fluorescence Methods: Exosomes were isolated from 1 mL of Distribution in Tumours Evaluation of Anti-tumour healthy donor plasma using polymer-based precipita- Effect in Mouse Model. tion and column purification. Nanoparticle tracking JOURNAL OF EXTRACELLULAR VESICLES 99 analysis was used to determine the abundance and size disease. Exosomes (Exo) as cell-derived vesicles are of particles. Exosomes were quantified by fluorometer, widely used as natural nanocarriers for drug delivery. and 200 µg protein equivalents were decorated with P21-activated kinase 4 (PAK4) is oncogenic when over- single-chain variable fragment (scFv)-C1C2 fusion pro- expressed, promoting cell survival, migration and teins with complementarity determining regions target- anchorage-independent growth. In this study, we vali- ing the HIV envelope protein. The HIV-1 NL4-3 EGFP date PAK4 as a therapeutic target in an in vivo PC reporter virus was incubated with decorated exosomes tumour mouse model using Exo nanocarriers following for 2 h at 1:1, 1:2 and 1:4 ratios. Virus was incubated intra-tumoural administration. with no exosomes, undecorated exosomes, or anti-PD- Methods: PC derived Exo were firstly isolated by ultra- 1 scFv-decorated exosomes as negative controls. Jurkat centrifugation on sucrose cushion and characterized E6.1 cells and primary human CD4+T cells were for their surface marker expression, size, number, pur- infected with virus-exosome preparations via spinocu- ity and shape. siRNA was encapsulated into Exo via lation, and GFP fluorescence was measured by flow electroporation and dual uptake of Exo and siRNA was cytometry to determine infection levels after 72 h. investigated by flow cytometry and confocal micro- Results: Our engineered anti-HIV scFv-decorated exo- scopy. In vitro siPAK4 silencing in PC cells was somes significantly inhibited HIV infection in Jurkat assessed by western blotting, flow cytometry, and in cells with respect to all negative controls (n =3; vitro scratch assay. In vivo efficacy (tumour growth p < 0.05, paired t-test). Anti-HIV scFv-decorated exo- delay and mouse survival) of siPAK4 was evaluated in somes potently inhibited HIV infection in primary PC bearing NSG mouse model. Ex vivo tumours were human CD4 + T cells (n = 2 donors) in a dose-depen- examined using Haematoxylin and eosin (H&E) stain- dent manner, suppressing up to 87% of infection in the ing and immunohistochemistry. absence of toxicity. Results:HighqualityPCderivedPANC-1Exowere Summary/Conclusion: Engineering exosomes ex vivo obtained. siRNA was incorporated in Exo with 16.5% represents a promising therapeutic approach for HIV loading efficiency. Exo and siRNA co-localization in infection. Future work will test the capacity of our cells was confirmed by in vitro imaging. PAK4 designer exosomes to inhibit HIV replication in vivo knock-down was successful at 30 nm Exo-siPAK4 at in humanized mouse models. Beyond viral suppres- 24 h post-incubation in vitro.Intra-tumoraladmin- sion, we will determine if designer exosomes can accel- istration of Exo-siPAK4 (1 µg siPAK4 and 7.7 ×1011 erate the clearance of HIV latently-infected cells, the Exo, each dose, two doses) reduced PC tumour main obstacle to a cure for HIV infection. growth and enhanced mice survival (p <0.001), Funding: NIH P01AI131374 and R01GM117901 with minimal toxicity observed compared to polyethylenimine (PEI) used as a commercial transfection reagent. H&E staining of tumours PT11.10 showed significant tissue apoptosis in siPAK4 treated groups. Exosome-mediated RNAi of PAK4 prolongs survival of pancreatic Summary/Conclusion:PAK4interferenceprolongs cancer mouse model after loco-regional treatment survival of PC bearing mice suggesting its candidacy Lizhou Xua, Julie Wangb, Farid N. Faruqub, Kee Limb, Adam Waltersb, Claire Wellsb and Khuloud Al-Jamalb as a new therapeutic target in PC. PANC-1 Exo a demonstrated comparable efficacy but safer profile School of Cancer and Pharmaceutical Sciences, King’s College London, London, UK; bKing’s College London, London, UK than PEI as in vivo RNAi transfection reagent. Funding: The K. C. Wong Education Foundation and Introduction: Pancreatic cancer (PC) remains one of The Marie Skłodowska-Curie actions, “Horizon 2020” the most aggressive and devastating malignancies, pre- project, EU (H2020-MSCA-IF-2016). dominantly due to the absence of a valid biomarker for diagnosis and limited therapeutic options for advanced 100 ISEV2019 ABSTRACT BOOK

PT12: EV Based Therapeutics Chairs: Mario Gimona; Saara Laitinen Location: Level 3, Hall A 15:30–16:30

PT12.02 Summary/Conclusion: The exosomes from ADSC were able to reduce the oxidative stress through manipulating mitochondrial functions. The effect is specu- Exosomes from adipocyte-derived stem cells reduce the oxidative lated to achieve by the modulation of genetic stress through the mitochondrial uncoupling in pantothenate kinase 2 mutation in vitro models expression in the recipient cells. Chien Tai Honga and Ruey Meei Wub Funding: Minister of Science and Technology, Taiwan aShuang Ho Hospital-Taipei Medical University, New Taipei City, Taiwan (MOST 107–2314-B-038 −086 -MY2) (Republic of China); bNational Taiwan University, Taipei, Taiwan (Republic of China)

Introduction: The exosome is a promising novel ther- PT12.03 apy for human diseases. Exosomes-derived from mesenchymal stem cell is thought to contain plenty of unique microRNA, which is specific for boosting cel- Extracellular vesicle-secretion system based on agarose gel encapsulation of cells for cell therapy lular repair and regeneration. Neurodegenerative dis- Mami Hiranoa, Masaya Hagiwarab, Nahoko Bailey Kobayashic, Tetsuhiko eases are characterized by the neuronal pre-mature Yoshidac, Eiichi N. Kodamad and Ikuhiko Nakasea apoptosis and the lack of the ability of regeneration. aGraduate School of Science, Osaka Prefecture University, Sakai-shi, Japan; It is hypothesized that the supplement of exosomes- bNanoSquare Research Institute, Osaka, Japan; cInstitute for Advanced Sciences, Toagosei Co., Ltd., Tsukuba, Japan; dTohoku University School of derived from the stem cells could activate the expres- Medicine, Sendai, Japan sion of neuroprotective gene/protein expression, resume the impaired cellular function and reverse the Introduction: Extracellular vesicles (exosomes, EVs, degenerative process. 30 ~ 200 nm in diameter) are released from various Methods: Patient with pantothenate kinase 2 (PANK2) types of cells. Because EVs carry functional molecules mutation-related neurodegeneration with brain iron such as e.g. microRNAs and enzymes, EVs play crucial accumulation was recruited and the leukocytes were roles in cell-to-cell communication. On the other hand, immortalized to establish the in vitro models. The EVs have pharmaceutical advantages as carriers for adipose-derived stem cell (ADSC) was obtained from intracellular delivery of therapeutic molecules, including, the healthy donors and the exosomes were isolated e.g. encapsulation of natural and/or artificial therapeu- from the culture medium at confluent. tic/diagnostic molecules, controlled immunoreaction, Results: The PANK2 mutation resulted in the elevated effective usage of cell-to-cell communication routes, infi- oxidative stress and depolarization of mitochondria. nite secretion and expression of functional proteins in Exosome treatment (5 μg of exosome suspension EV membranes. We are currently developing cell encap- upon 3*106 leukocytes) for 24 h up-regulated the pro- sulated gel system for secretion of functional EVs in cell tein level of mitochondrial uncoupling protein 2 and 3, therapy. In this research, agarose gels, which has been as well as boosted the mitochondrial biogenesis, widely used in cell culture and chamber, is used for assessed by the protein level of PGC-1α and encapsulation of cells that secrete functional EVs from TOMM20. Those proteins were undatable in the exo- the gels. We here demonstrate our methods for cell somes themselves. Mitochondrial uncoupling proteins encapsulation in the gels and cellular uptake efficacy of are responsible for the dissipating of mitochondrial secreted EVs from the gels. membrane potential and down-regulation of the oxi- Methods: CD63 (EV marker protein)-GFP stably dative phosphorylation from respiration, which is the expressing HeLa cells were encapsulated using collagen major source of free radical and oxidative stress. and agarose gels. Secreted EVs from the gel system were Exosome treatment for 24 h led to the obvious mito- separated using ultracentrifuge and analysed by western chondrial depolarization, assessed by JC-1 and further blotting, zeta potential, DLS and electron microscope reduction of the oxidative stress, assessed by the (TEM). Cellular uptake of secreted EVs from the gels H2DCFDA. was observed using confocal laser scanning microscope. JOURNAL OF EXTRACELLULAR VESICLES 101

Results: In the experimental optimization for encapsu- lung injury. Murine fibroblast (NIH3T3) EVs, which lation of cells in gels, we successfully attained CD63- do not contain abundant miRNA-126, did not provide GFP stably expressing HeLa cells-encapsulated agarose these beneficial effects. In human small airway epithe- (1.5%) gels (e.g. 5 ×104 cells can be encapsulated in lial cells, we found that overexpression of miRNA-126- approx. 2 mm ×25 mm ×25 mm sheet-like gel). DLS 3p can target phosphoinositide-3-kinase regulatory analysis showed 30 ~ 100 nm EVs secreted from the subunit 2, while overexpression of miRNA-126-5p gels, and zeta potential of the EVs was average −17 mV. inhibits the inflammatory cytokine HMGB1 and per- Western blotting confirmed expression of exosomal meability factor VEGFα. Interestingly, both miR-126- marker proteins (e.g. CD63 and CD81). A431 cells 3p and 5p increase the expression of tight junction (human epidemoid carcinoma) were cultured with the proteins suggesting a potential mechanism by which CD63-GFP stably expressing HeLa cells-encapsulated miRNA-126 may mitigate LPS-induced lung injury. agarose gels for 24 h, and efficient cellular uptake of Summary/Conclusion: Our data demonstrated that secreted EVs (CD63-GFP-EVs) from the gels were human EPC EVs are beneficial in LPS-induced ALI observed using confocal laser scanning microscope. mice, in part through the delivery of miRNA-126 into Summary/Conclusion: Although we have to conduct the injured alveolus. further optimization in this system as next step to Funding: 1R01GM113995 (HF), 1R01GM130653 (HF), obtain sophisticated methodology, these experimental 1K23HL135263-01A1 (AG), UL1TR001451 (PVH) techniques and findings will contribute to development for cell therapy based on EVs as basic studies. PT12.05 PT12.04 Hsa_circ_0000077-overexpressing extracellular vesicle: a new tool to prevent cartilage degeneration Shi-Cong Tao and Shang-Chun Guo Extracellular vesicles from endothelial progenitor cells improve outcomes of the lipopolysaccharide-induced acute lung injury Shanghai Jiao Tong University Affiliated Sixth People’s Hospital, Shanghai, Yue Zhou, Pengfei Li, Andrew Goodwin, James Cook, Perry Halushka, China (People’s Republic) Eugene Chang and Hongkuan Fan

Medical University of South Carolina, Charleston, USA Introduction: Trauma and degeneration of articular cartilage (AC) could trigger the morbidity of one of Introduction: The acute respiratory distress syndrome the leading disabling disease, osteoarthritis (OA). One is characterized by disruption of the alveolar-capillary of the most difficult issues in treatment is the poor self- barrier resulting in accumulation of proteinaceous healing ability of AC. Extracellular vesicle (EV) trans- oedema and increased inflammatory cells in the alveo- plantation has received more and more attention as lar space. We previously found that extracellular vesi- potential cell-free therapeutic approaches to promote cles (EVs) from endothelial progenitor cells (EPCs) tissue healing. In our preliminary study, we found that prevent endothelial dysfunction and lung injury in decreased expression of hsa_circ_0000077 (circ77) was sepsis due to their encapsulation of miRNA-126. closely related to OA. And circ77-overexpression in However, the effects of EPC EVs in acute lung injury chondrocytes can prevent the chondrocyte degenera- (ALI) remains unknown. tion. In this study, EVs derived from circ77-overex- Methods: To determine if EPC EVs would have bene- pressing synovium mesenchymal stem cells (SMSC-77- ficial effects in ALI, intratracheal administration of EVs) were used to promote cartilage regeneration. lipopolysaccharide (LPS) was used to induce ALI in Methods: CCK-8, qPCR and western blotting (WB) mice. Lung permeability, inflammation and the role were used to investigate the biological functions of of miRNA-126 in alveolar epithelial barrier function SMSC-77-EVs on the proliferation and cartilage regen- were examined. eration. Furthermore, interleukin 1β (IL-1β) were used Results: The intratracheal administration of EPC EVs to simulate the inflammatory conditions of OA, and reduced lung injury following LPS-induced ALI at 24 then, the protective effects of SMSC-77-EVs were con- and 48 h. Compared to placebo, intratracheal adminis- firmed by CCK-8, qPCR and WB. tration of EPC EVs significantly reduced the cell num- Results: CCK-8 assay confirmed that SMSC-77-EVs ber, protein concentration and cytokines/chemokines enhanced the proliferation of chondrocytes, compared in the bronchoalveolar lavage fluid, indicating a reduc- with normal control and EVs derived from synovium tion in permeability and inflammation. Further, EPC mesenchymal stem cells which were transfected by EVs reduced myeloperoxidase activity and reduced the empty vectors (SMSC-Empty-EVs). WB and qPCR lung injury score, demonstrating protection against assays confirmed that SMSC-77-EVs enhanced the 102 ISEV2019 ABSTRACT BOOK expression levels of cartilage related proteins including The incubation time was 48 h in proliferation assay, Type II collagen (Col-II), aggrecan (ACAN) and SOX9, 16 h in migration assay, 8 h in tube formation assay compared with normal control and SMSC-Empty-EVs. and 12 and 24 h in qRT-PCR. IL-1β significantly inhibited the proliferation and car- Results: ADSC-EVs group showed almost one point tilage regeneration-related proteins (Col-II, ACAN and five to twice increase of proliferation, migration and SOX9). SMSC-77-EVs could observably restrain the tube formation function compared to PBS group. harmful effects of IL-1β, while SMSC-Empty-EVs Furthermore, gene expressions for lymphatic markers showed limited ability. such as VEGFR-3, Lyve-1, Podoplanin, Prox-1 were Summary/Conclusion: These findings suggest that the also shown almost two to five times increase in the novel SMSC-77-EVs provides the preferable function ADSC-EVs group. in promoting the repair of cartilage damage. The use of Summary/Conclusion: The present study showed lym- SMSC-77-EVs would represent a development trend of phangiogenic effects of EVs derived from ADSCs, cell-free therapies, using engineered EVs (or modular- which lead to new treatment options for chronic lym- ized EVs), for promoting cartilage regeneration. phedema. Further studies are needed to elucidate what Funding: The National Natural Science Foundation of kind of molecular in ADSC-EVs works in LEC. In vivo China [Nos. 81871834, 81802226 and 81301589], and studies using mouse lymphedema model are also Shanghai Jiao Tong University K.C.Wong Medical needed to confirm the biological function of ADSC- Fellowship Fund supported this work. EVs. EVs for cell free therapy are less potential risk compared to stem cell transplantation and could be promising tool for patients suffering from PT12.06 lymphedema. Funding: JSPS Kakenhi; Takeda Science Foundation.

Lymphangiogenesis induced by exosomes derived from adipose- derived mesenchymal stem cells Kensuke Tashiroa, Yusuke Yoshiokab and Takahiro Ochiyab PT12.07 aJichi Medical Unversity, Tochigi, Japan; bDepartment of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Embryonic stem cell-derived extracellular vesicle-mimetic Shinjyuku-ku, Japan nanovesicles rescue erectile function by enhancing penile neurovascular regeneration in the streptozotocin-induced diabetic Introduction: Lymphedema is chronic oedema of mouse Kang-Moon Songa, Mi-Hye Kwona, Guonan Yina, Kalyan Ghataka, Nguyen limbs caused by the accumulation of lymphatic fluid Nhat Minha, Min Ji Choia, Jiyeon Ocka, Yong Song Ghob, Ji-Kan Ryua and and characterized by a progressive disorder of the Jun-Kyu Suha smooth muscle cells of the lymphatic channels. aNational Research Center for Sexual Medicine and Department of Urology, Transplantation of adipose-derived mesenchymal Inha University School of Medicine, incheon, Republic of Korea; bDepartment of Life Sciences, Pohang University of Science and stem cells (ADSCs) has been reported to improve the Technology, Pohang, Republic of Korea severity of lymphedema, however, the detailed mechanism has not been elucidated yet. Extracellular Introduction: Extracellular vesicles (EV)-mimetic vesicles(EVs) derived from mesenchymal stem cells nanovesicles (NVs) contains a variety of protein, have been reported to have functions such as cancer mRNA and miRNA and is known to play an important development, angiogenesis, suppression of inflamma- role in intercellular communication as a bio-nanopar- tion, regeneration of damaged organs and treatment of ticle with a diameter of 40 to 100 nm. Recent studies degenerative disease. ADSCs are thought to be promis- have demonstrated the therapeutic potential of EV- ing source of regenerative medicine, and EVs derived mimetic NVs in a variety of animal models for cardi- from ADSCs are thought to have similar effects as well. ovascular diseases and neuropathies. The aim of this Here, we analysed lymphangiogenesis induced by EVs study was to investigate effectiveness of embryonic derived from ADSCs for treatment of chronic stem cell (ESC)-derived EV-mimetic NVs in restoring lymphedema. erectile function in diabetic mice. Methods: EVs derived from ADSCs were isolated by Methods: Diabetes was induced by intraperitoneal ultracentrifugation. The effect of EVs to lymphatic injection of streptozotocin into 8-week-old C57BL/6 endothelial cells (LECs) were analysed in proliferation male mice. At 8 weeks after the induction of diabetes, assay, migration assay and tube formation assay. Gene the animals were distributed into 7 groups: control expression analyses were also performed by qRT-PCR. non-diabetic mice and diabetic mice receiving two LECs were treated with PBS as control, VEGF-C(10 ng/ successive intracavernous injections of HEPES-buf- ml) and ADSC-EVs(100 μg/ml) one time in each assay. fered saline (HBS, days −3 and 0; 20 μL) or ESC- JOURNAL OF EXTRACELLULAR VESICLES 103 derived EV-mimetic NVs (ESC-NVs, days −3 and 0; of A-exo was determined by BCA assay. The effect of 0.1 μg, 0.5 μg, 1 μg, 2 μg, or 5 μgin20μL of HBS, A-Exo on the expression level of α-SMA was evaluated respectively). Two week after treatment, we measured by IF analysis. Mice were received thioacetamide intra- erectile function by electrical stimulation of the caver- peritoneally. Fluorescently labelled A-exo was adminis- nous nerve. The penis was then harvested for histolo- tered to mice and whole-body fluorescence was gical and biochemical studies. We also examined the observed in order to evaluate the in vivo distribution. effects of ESC-Exo in primary cultured mouse caver- The therapeutic efficacy of A-exo was determined by nous endothelial cells (MCEC) and pericytes (MCP) in measuring the level of ALT, ALP, TBIL and TP in vitro; and in cultured aortic ring and major pelvic blood of mice. A-exo was injected intravenously three ganglion (MPG) ex vivo. times and blood was collected after final injection. Results: Intracavernous injections of ESC-NVs signifi- Results: When hepatic stellate cells were activated with cantly improved erectile function in diabetic mice, TGF-β1, the expression level of α-SMA was signifi- which reached up to 90% of control values. ESC-NVs cantly increased. While, the level was remarkably induced significant restoration of cavernous contents decreased depending on the treatment concentration of endothelial cells, smooth muscle cells, pericytes, and of A-Exo. A-exo treatment significantly decreased neuronal cells in diabetic condition. Moreover, ESC- expression mRNA of pro-fibrogenic marker: α-SMA, NVs promoted micro-vascular sprouting from aortic Collagen I and MMP-2. After systemic administration ring and accelerated tube formation in primary cul- of exosome, a substantial accumulation of A-Exo at tured MCEC and MCP mono-culture or co-culture liver was observed in both the normal and mice system in vitro. model of liver fibrosis. Furthermore, liver function of Summary/Conclusion: ESC-NVs successfully restored A-exo treated group was restored to normal. These erectile function through enhanced cavernous angio- results showed A-exo had the high therapeutic efficacy. genesis and neural regeneration in diabetic mice. It will Summary/Conclusion: In this study, we investigate the be a better strategy to use ESC-NVs than ESCs for the potential of stem cell-derived exosome as the new treatment of retractable erectile dysfunction although it therapeutic approach for liver fibrosis treatment. A- remains to be solved for future clinical application of exo has similar bioactive capacity to its origin cell, ESCs. mesenchymal stem cell. The beneficial effect of A-exo was confirmed in vitro and in vivo tests. The superior therapeutic efficacy was displayed in A-exo treated PT12.08 mouse group.

Human adipose tissue-derived mesenchymal stem cell exosomes for PT12.09 the treatment of liver fibrosis Sol Shin, Soyoung Son, Gyeongtaek Lim, Seunglee Kwon and Jaehyung Park HucMSC exosome confer protection against ultraviolet radiation aSungkyunkwan University, Suwon, Republic of Korea induced acute photodamage via modulation of SIRT1 pathway Peipei Wua, Hui Qianb and Wen Rong Xuc Introduction: Liver fibrosis, often leading to liver cir- aJiangsu university, Zhenjiang City, China (People’s Republic); 2ZhenJiang, rhosis and cancer, is a challenging health issue for China (People’s Republic); cZhenjiang Key Laboratory of High Technology which there are no effective medicines. Conventional Research on Exosomes Foundation and Transformation Application, Jiangsu Key Laboratory of Medical Science and Laboratory Medicine, School of therapies are merely symptomatic treatment. Hence, Medicine, Jiangsu University, Zhenjiang, China (People’s Republic) there have been lots of efforts to develop better therapeutic strategies. Cell therapy based on mesenchymal Introduction: Exosomes are nano-sized membrane stem cells (MSCs) is one of the attractive options. vesicles secreted by most cells, including human umbi- MSCs hold immunomodulatory properties and multi- lical cord mesenchymal stem cells (hucMSC). hucMSC potency. However, cell transplantation has limitations derived exosomes (hucMSC-ex) have been reported to such as teratoma formation and low cell viability in significantly facilitate skin regeneration, resembling the vivo condition. In order to overcome these limitations, effect of parental cells. However, the role of hucMSC- we suggest MSCs derived exosome-based therapy for ex in ultraviolet radiation (UV) induced skin damage treating liver fibrosis. and the underlying mechanisms are largely unknown. Methods: Exosome-derived from ADSCs (A-exo) was Methods: Herein, we examined the benefit of hucMSC- purified using a tangential flow filtration system. The ex in a rat acute skin photodamage model. physical characteristics of A-exo were investigated Results: We found that the subcutaneous injection of using DLS, TEM and NTA. The protein concentration hucMSC-ex (1 mg) elicited noted antioxidant and anti- 104 ISEV2019 ABSTRACT BOOK inflammatory effects against UV induced DNA damage epidermal model was used to evaluate the inhibition of and apoptosis in vivo. Further studies shown that sir- melanogenesis. tuin1(SIRT1) expression level in skin keratinocytes Results: The leaves and stems-derived exosome-like (HaCaT) decreased in a time- and dose-dependent nanovesicles are able to suppress cellular melanin con- manner under oxidative stress in vitro, however, the tent melanoma cells. Also, melanogenesis protein treatment of hucMSC-ex reverses this phenomenon. expression was reduced with leaves- and stems-derived Activation of SIRT1 significantly attenuated UV and exosome-like nanovesicles. These results suggest that H2O2-induced cytotoxic damage by inhibiting oxida- leaves- and stems-derived exosome-like nanovesicles of tive stress and promoting autophagy activation. the D. morbifera could be a candidate of natural sub- Furthermore, we also discovered that the cytoprotec- stances for anti-melanogenic agents. tion function provided by hucMSC-ex carried 14–3-3ζ Summary/Conclusion: The leaves and stems-derived was potential associated with modulation of SIRT1 exosome-like nanovesicles are able to suppress cellular dependent antioxidant response. melanin content melanoma cells. Also, tyrosinase activ- Summary/Conclusion: Collectively, our findings indi- ity and melanogenesis protein expression were reduced cated that hucMSC-ex is a new potential agent for with leaves- and stems- derived exosome-like nanove- preventing and/or treating UV-induced skin photo- sicles. These results suggest that leaves- and stems- damage and ageing. derived exosome-like nanovesicles of the D. morbifera Funding: Zhenjiang Key Laboratory of Exosomes could be a candidate of natural substances for anti- Foundation and Transformation Application High- melanogenic agents. tech Research, china: (ss2018003). National Natural Funding: This work was supported by the Basic Science Foundation of China: (BE2016717). Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (NRF- PT12.10 2016R1C1B2013345).

Anti-melanogenic effect screening for natural plant-derived exosome- like nanovesicles PT12.11 Ruri Leea, Yeon Juhunb, Kim Kimina, Min seo yunc and Kim Jung Ahc aUniversity of brain education, Cheon-an, Republic of Korea; buniversity of c brain education, cheon-an, Republic of Korea; korea basic science institute, Stem cell extracellular vesicles as therapeutics for autoimmunity ochang, Republic of Korea Weian Zhaoa, Milad Riazifarb, Rezaa Mohammadib and Jan Lotvallc

aUniversity of California, Irvine, Irvine, USA; bUC Irvine, IRVINE, USA; Introduction: Demand for whitening agents is increas- cUniversity of Gothenburg, Gothenburg, Sweden ing due to their anti-melanogenic effects by improving skin darkness and decreasing melanin production in Introduction: Stem cells including mesenchymal stem the cosmetics industry. However, there have been side cells (MSC) hold great potential in treating autoim- effects and high toxicity issue as well as poor skin mune disorders. However, their clinical translation penetration. Therefore, many researchers have focused has been hindered due to incomplete understanding on natural plants as an alternative chemo-therapeutics of mechanisms of action (MOA) and potential safety agent to avoid various side effects. Recently, it is known concerns. Recent evidence revealed that some of the that exosome-like nanovesicles have biocompatibility MSC MOA may be associated with extracellular vesi- and excellent drug delivery capacity. In this study, cles (EV), leaves and stems-derived exosome-like nanovesicles Methods: We investigated MSC derived exosomes in were isolated from Dendropanax Morbifera and we immune modulation in a multiple sclerosis experimen- have found that inhibition of those nanovesicles on tal autoimmune encephalomyelitis (EAE) a mouse melanin products. model in vivo as well as in T cell proliferation suppres- Methods: Exosome-like nanovesicles from leaves and sion and Treg induction in vitro. stems were isolated and identified size using DLS and Results: Our results indicated that that intravenous NTA. These shapes were observed by TEM. The anti- administration of exosomes produced by MSCs stimu- melanogenic effect was verified by evaluating the mel- lated by IFNγ (IFNγ-Exo) (i) enhanced the mean clin- anin content and tyrosinase activity on melanoma cell. ical score of EAE mice compared to PBS control, (ii) Also, western blot was used to observe melanogenesis- home into the spinal cords and reduced demyelination, related protein expression. In addition to, cellular mel- (iii) decreased neuroinflammation and (iv) upregulated anin formation was confirmed using TEM. The human the number of CD4+/CD25+/FOXP3+regulatory T JOURNAL OF EXTRACELLULAR VESICLES 105 cells (Tregs). In addition, we found that IFNγ-Exo Results: Both OMVs did not induce cytotoxicity or significantly reduced the proliferation of T-cells in negatively influenced the viability of THP-1 and A549 vitro and reduced production of proinflammatory fac- cells even at concentrations of 10,000 OMVs/cell. tors including IL-6, IL-17 and IL-22 while enhanced Cbv34 and Cbfe23 OMVs showed a bactericidal activ- the production of Indoleamine 2,3-dixygenase (IDO), a ity against E. coli and S. aureus. The antibacterial effect key player in MSC-mediated immunosuppression. of Cbv34 OMVs remained potent upon storage at 4°C Summary/Conclusion: Our findings suggest that stem for 4 weeks. The presence of cystobactamid in Cbv34 cell derived EVs can serve as promising candidates in OMVs was confirmed by MS. Using flow cytometry treating autoimmune and neurodegenerative diseases. and labelled OMVs, we observed 60% positive cells for Funding: National Institute of Health (1DP2CA195763- Cbv34 OMVs and 40% positive cells for Cbfe23 OMVs 01; #NS082174). with THP-1, and 50% positive A549 cells for Cbv34 OMVs after 4 h of incubation. PT12.12 Summary/Conclusion: The biocompatibility, inherent bacterial activity and uptake into mammalian cells may be promising for the treatment of infections, especially Biocompatible myxobacteria-derived outer membrane vesicles show those induced by intracellular S. aureus. However, their inherent antibacterial activity against gram-negative and gram- positive microbes mechanism of action needs further investigation. Adriely Goesa,EilienSchulza,PhilippLapuhsa,RobertRichterb,FabianPanterc, Funding: This work was supported by the Federal Marcus Kochd,RolfMüllerc,KathrinFuhrmanne and Gregor Fuhrmanne Ministry for Research and Education through the aHelmholtz Institute for Pharmaceutical Research Saarland, Biogenic Nanotherapeutics (BION), Saarbrücken, Germany; bHelmholtz-Institute for NanoMatFutur programme. Pharmaceutical Research Saarland, Department of Drug Delivery (DDEL), Saarbrücken, Germany; cHelmholtz Institute for Pharmaceutical Research Saarland, Department of Microbial Natural Products (MINS), Saarbrücken, PT12.13 Germany; dLeibniz Institute for New Materials (INM), Saarbrücken, Germany; eHelmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken, Germany; 6Helmholtz-Institut for Pharmaceutical Research Saarland (HIPS), Saarbrücken, Germany Placental MSCs and their exosomes as vehicles for the Na/I symporter (hNIS): a new theragnostic agent Alejandra Crespo-Barredaa, Miguel Quintanillab, Maite Iglesiasc, Antonio de Introduction: Myxobacteria are gram-negative bacteria la Viejad and Pilar Martin-Duquee that live in the soil and can be found in different aInstituto Aragonés de Ciencias de la Salud/ Universidad Francisco de habitats and they have been explored for their capabil- Vitoria, Majadahonda, Spain; bInstituto de Investigaciones Biomedicas ity of providing natural products with great potential Alberto Sols, Madrid, Spain; cUniversidad Francisco de Vitoria, Madrid, Spain; dInstituto de Salud Carlos III, Madrid, Spain; 5Instituto Aragonés de for antimicrobial therapy. We investigated the antimi- Ciencias de la Salud/ IIS Aragón/ Fundación Araid, Zaragoza, Spain crobial activity of OMVs isolated from the myxobac- terial strains Cystobacter velatus (Cbv34) and Introduction: The Na > I symporter gene (hNIS) is Cystobacter ferrugineus (Cbfe23) against E. coli DH5- expressed in the thyroid and allows the accumulation alpha and S. aureus Newman. Cbv34 and Cbfe23 are of iodine from the diet, to form T3 and T4 hormones. producers of the antibacterial compound cystobacta- Moreover, it is widely used (i) as a reporter gene for mid that we hypothesized to be naturally loaded into molecular imaging (when the positron emitter isotope the OMVs they shed. is I124 for PET or Tc99 for SPECT) or (ii) as a ther- Methods: The OMVs were isolated by differential cen- apeutic gene for cancer therapy, mediated by the accu- trifugation and size-exclusion chromatography. Particle mulation of 1131. An unresolved challenge is how to size, distribution, concentration and morphology were direct this gene specifically to the tumoral area. analysed by nanoparticle tracking analysis and cryo- Previously, our group demonstrated the migratory transmission electron microscopy. Particle interaction capacity of placental mesenchymal stem cells (MSCs), and uptake into macrophages (THP-1) and lung alveo- carrying an adenovirus expressing hNIS to tumours, lar epithelial cells (A549) was investigated by confocal with good results as a theragnostic tool. However, as laser scanning microscopy and flow cytometry. hNIS is expressed at the placental tissue (because it Cytotoxicity and viability assays were performed transfers iodine to the foetus from the maternal using PrestoBlue and lactate dehydrogenase assays. blood), in this work we decided to study whether The antibacterial activity of the OMVs was assessed placental MSCs and their derivatives (exosomes) (1) by overnight incubation with E. coli or S. aureus, fol- express hNIS endogenously and therefore transfers lowed by optical density measurement and CFU deter- the imaging and therapeutic potentials when adminis- mination. The OMVs content was investigated by tered with radioactive iodine (2) are capable to reach liquid chromatography coupled mass spectrometry. the tumoral areas when they are intravenously injected 106 ISEV2019 ABSTRACT BOOK due to the tumoral tissues extravasation.The aim of this migrate specifically to the tumour and their endo- research was to develop a new anti-tumoural therapy genous expression of NIS is enough to visualized in by the combination of the advantages of both NIS and vivo cells and exosomes accumulation and to see the human placental MSCs (hPMSCs). significant therapeutic effect on cancer treatment Methods: Here, we used two approaches using the with 131I. endogenous hNIS expression, first in hPMSCs but also Summary/Conclusion: Our findings highlight the pos- on its exosomes. For both cases, we determined in vitro sibility to use endogenous expression of NIS as therapy NIS location and functionality but also we followed and opening a wide range of new possibilities to treat those vectors by SPECT CT and studied their antitu- cancer. moral effect after radioactive iodine injection. Funding: This work has been funded by Universidad Results:WeprovedthathumanplacentaMSCsand Francisco de Vitoria, Instituto de Salud Carlos lll and their exosomes have endogenous expression of NIS, Instituto Aragonés de Ciencias de la Salud JOURNAL OF EXTRACELLULAR VESICLES 107

LBT01: Late Breaking- Technological advances Chairs: M. Selim Unlu, Olga Shatnyeva Location: Level 3, Hall A 15:30–16:30

LBT01.01=OWP1.09 Summary/conclusion: EVs from autologous blood products represent a novel and cell free regeneration approach. We observed that the clotting cascade Coagulation influences properties of extracellular vesicles isolated (plasma versus serum) has an influence on concentra- from autologous blood derived products Andrea De Lunaa, Alexander Otahala, Olga Kutenb, Zsombor Laczac and tion, size and miRNA expression patterns of EVs. Stefan Nehrera These differences might have an impact on the biolo- aDanube University Krems, Krems, Austria; bOrthoSera GmbH, Krems, gical mode of action of blood derived products used in Austria; cOrthosera GmbH, Krems, Austria clinics. Funding: Financial support was received from the Introduction: Platelet rich plasma (PRP) is the most European Fund for Regional Development (EFRE) commonly used blood derivative in clinics due to its and the Science Fund of Lower Austria. miRNA high concentration of platelets and perceived high expression analysis was performed by TAmiRNA growth factor levels. Drawbacks of using PRP are dis- GmbH. Cryo-electronmicroscopy was conducted at crepancies among preparation protocols and the pre- the Core Facility of the Vienna Bio-Center. sence of cells (platelets, leuxcocytes) which can evoke cellular processes (e.g. inflammation) when injected into the host. One possibility is to isolate only the LBT01.02=OWP1.11 active components of blood derivatives which may overcome this problem. In the current study we Ev-avogadro project: towards a liposomal concentration standard for focused on extracellular vesicles (EVs) isolated from extracellular vesicle research two autologous blood derivatives, PRP and hyperacute Gergo Bartaa, Diana Kitkaa, Andras Wachaa, Judith Mihalya, Attila Botaa, a a b c serum and investigated whether the clotting cascade Krisztina Nemeth , Pal Szabo , Jean-Luc Fraikin and Zoltan Varga influences EV properties. aResearch Centre for Natural Sciences HAS, Budapest, Hungary; bSpectradyne LLC, Torrance, USA; cResearch Centre for Natural Sciences, Methods: EVs were isolated from citrate-anticoagu- Hungarian Academy of Sciences, Budapest, Hungary lated PRP (CPRP) and hyperacute serum using differential ultracentrifugation followed by a size exclusion Introduction: There is an unmet need for standardiza- chromatography. Particle concentration and size were tion of concentration measurements in the field of determined by nanoparticle tracking analysis (NTA). extracellular vesicles (EVs). Liposomes may serve an Cryo-electronmicroscopy was performed to visualize ideal reference system for EVs, but the determination isolated EVs. Expression of miRNAs transported of the number concentration of liposomes from first within EVs as well as in their respective input material principles was not attempted so far. Inspired by the was analysed by qPCR. International Avogadro project, we aimed to determine Results: NTA revealed higher particle concentrations the concentration of liposomes with well-defined size and bigger sized EVs within CPRP compared to hyper- and composition via counting the number of phospho- acute serum. These findings were confirmed by cryo- lipid molecules in these “nanospheres”. electronmicroscopy. Profound differences were Methods: Liposomes composed of phosphocholine and detected regarding miRNA expresion between the two phosphoglycerol were prepared by the extrusion blood derivatives. 126 miRNAs were identified which method. Wide-angle X-ray scattering (WAXS) was were expressed both in input material as well as in the used to determine the area-per-lipid value. The size corresponding EVs. The correlation between miRNAs distribution of the liposomes was determined by in EVs and input material was higher in CPRP com- microfluidic resistive pulse sensing (MRPS) and pared to hyperacute serum meaning that in hyperacute freeze-fracture combined TEM. Small-angle X-ray scat- serum miRNAs were identified which were higher tering (SAXS), differential scanning calorimetry (DSC), expressed in EVs than in the corresponding input and infrared spectroscopy (IR) were used to prove the material. unilamellarity, the ideal miscibility of the lipids and the 108 ISEV2019 ABSTRACT BOOK ordered packing of the hydrocarbon chains of the cells (RBCs) and platelets (PLTs), and from cultured lipids, respectively. Concentration of the lipids was cell lines using centrifugation and ultrafiltration. EV determined by liquid chromatography–mass spectro- size and number were evaluated using microfluidic metry (LC-MS). resistive pulse spectroscopy (MRPS), nanoparticle Results: The prepared liposomes proved to be unila- tracking analysis (NTA), cryo-electron microscopy mellar with narrow size distribution (83 nm avg.), as (cryo-EM), conventional light scatter-based flow cyto- obtained by MRPS and TEM. DSC and IR measure- metry (FC), and fluorescence-based vesicle flow cyto- ments confirmed that the phospholipid bilayer of these metry (VFC). EV surface markers were measured using liposomes is in the liquid-ordered phase, hence the VFC with well-characterized fluorescence-labelled anti- area-per-lipid of 0.41 nm2 was determined from bodies and calibrated using fluorescence intensity and WAXS measurements. Using the concentration of antibody binding standards. phospholipids from LC-MS measurements, the number Results: Cell-derived EVs are stable for months at concentration of liposomes was determined (8E+13 −80C and weeks at 4C, as assessed by measurement 1/mL). of number, size distribution, and surface markers. RBC Summary/conclusion: Liposomes containing saturated EVs had a median diameter of 115 nm and expressed a phospholipids are in the liquid-ordered phase, which median of ~2700 anti-CD235ab binding sites per EV, can be utilized to determine the area-per-lipid using while PLT EVs had a median diameter of 145 nm and WAXS. This value, together with the independently expressed a median of ~1200 anti-CD41 binding sites determined size, and lipid concentration can be used per EV. to calculate the number concentration of liposomes. As Summary/conclusion: EV standards that are well char- the light scattering properties of liposomes matches acterized at the single EV level in terms of number, that of EVs, liposome based standards for optical mea- size, and molecular cargo can facilitate assay validation, surements of EVs can be obtained with the presented sharing of data and results between labs, and support techniques. the development of new analysis technologies with Funding: This work was supported under grant num- improved sensitivity, resolution, and throughput. bers PD 121326 and NVKP_16-1-2016-0007 by NKFIH Funding: Supported by the US National Institutes of (Hungary). ZV was supported by the János Bolyai Health. Research Fellowship. LBT01.04 LBT01.03

Cell-specific EV tetraspanin expression Standards for EV research John Nolan and Erika Duggan a a b b c John Nolan , Erika Duggan , Ngoc Do , Franklin Monzon , Jean-Luc Fraikin Scintillon Institute, San Diego, USA and Tom Maslanikd aScintillon Institute, San Diego, USA; bSpectradyne, Torrance, USA; Introduction: Tetraspanins (TSs) are integral mem- cSpectradyne LLC, Torrance, USA; dCellarcus Biosciences Inc, San Diego, USA brane proteins present on plasma and internal membranes and are thought to affect membrane Introduction: Progress in understanding the origins, organization and function. Tetraspanins can also be composition, and effects of extracellular vesicles (EVs) found in extracellular vesicles released from cells and depends on the reproducibility and rigor of experimen- have been considered canonical EV markers. To gain tal results. Standards can improve experimental rigor insight into the significance of TS expression on EVs, and reproducibility and promote data sharing. To we used single vesicle flow cytometry (VFC) to mea- address the needs for standards for single EV analysis, sure the TS expression on individual EVs from differ- we have developed a set of standardized vesicle pre- ent cell sources. parations and characterized them with respect to num- Methods: EVs were prepared from 10 different cell ber, size, and cargo using a suite of single EV lines cultured in seru-free media and enriched by ultra- characterizations methods. centrifugation or ultrafiltration. EVs from washed red Methods: We prepared synthetic lipid vesicles with a blood cells (RBCs) and platelets (PLTs) by were iso- lipid composition approximating that of a mammalian lated by centrifugation, and characterized by nanopar- cell plasma membrane and extruded through a nucleo- ticle tracking analysis (NTA), microfluidic resistive pore membrane (100 nm mean pore diameter). We pulse spectroscopy (MRPS), cryo-electron microscopy prepared cell-derived EVs from washed red blood (cryo-EM), and vesicle flow cytometry (VFC). TS JOURNAL OF EXTRACELLULAR VESICLES 109 expression was measured using a panel of phycoery- high density lipoproteins (HDL) and low/very low thrin-conjugated monoclonal antibodies against CD9, density lipoproteins (LDL/VLDL). CD63, CD81, CD82, CD151, CD53 and CD231. The Methods: EVs, HDL and LDL/VLDL fraction were fluorescence scale was calibrated using intensity stan- collected from 12 plasma or serum samples obtained dard meads and expressed as PE MESF (mean equiva- from young healthy African Americans using commer- lent soluble fluorochromes). cially available isolation kits. Written informed con- Results: The “canonical” TS EV markers CD9, CD63, sents were obtained from all participating donors. and CD81 were expressed on EVs from all cells except Protein marker expression of each fraction was ana- RBCs, which expressed detectable amounts (LOD ~25 lysed by Western blotting. Lipidomic analysis was per- MESF) of no TS, but the relative and absolute amounts formed using LC-MS operating in negative ion mode. varied drastically from cells which expressed primarily Results: Successful EVs, HDL and LDL/VLDL isola- CD9 molecules on EVs (PLT and A431), to those that tions were validated by confirming corresponding mar- expressed predominantly CD63 (MCF7, U87) to those ker proteins (CD9; EVs, apoA-I; HDL, apoB; LDL/ that expressed predominately CD81 (293T, iPSC- VLDL). As a result of lipidomic analysis, we identified derived neurons). Moreover, EVs from most cells 264 lipids in plasma EVs, HDL and LDL/VLDL frac- expressed some level of CD151, while CD82 was tions. We also found that EVs showed strikingly higher detected on EVs from A431 and U87MG cells. levels of lyso-glycerophospholipids than HDL and Summary/conclusion: Tetraspanins appear to be LDL/VLDL. Additionally, compared with EVs, higher involved in many different cellular processes and sphiongolipid species levels were observed in LDL/ their specific roles in EV-related physiology is not VLDL, while polyunsaturated phosphatidylcholine understood. Single vesicle analysis of TS expression were highly detected in HDL. Similar profiles were using VFC reveals the diversity in TS expression and also observed in each fraction derived from human abundance on EVs from different cell types. serum. Understanding the tetraspanin expression on EVs Summary/conclusion: Lipidomic profiling demon- may provide information about the cellular origin of strates that EVs has a unique lipid profile compared EVs, their effects on recipient cells, or both. with lipoprotein particles, although the biological Funding: Supported by the US National Institutes of meaning of these differences should be further evalu- Health. ated in future studies. Nevertheless, the method presented in this study can be useful for lipid biomarker screening for EVs as well as lipoprotein particles LBT01.05 derived from both plasma and serum for human diseases.

Characterization of lipid profile of extracellular vesicles and Funding: Japan Agency for Medical Research and lipoproteins in human plasma and serum Development Yuchen Suna, Kosuke Saitob and Yoshiro Saitob aDivision of Medical Safety Science, National Institute of Health Sciences, Kanagawa, Japan; bDivision of Medical Safety Science, National Institute of LBT01.06 Health and Sciences, Kawasaki, Japan

Introduction: Extracellular vesicles (EVs) are lipid Enhancing extracellular vesicle isolation of human plasma verified by high resolution lipidomics bilayer nano-vesicles existing in various biofluids, and Amani M. Batarseha, Alex Chenb, Kim Ekroosc, Susannah Hallald, Kimberley regarded as valuable sources for biomarker. To data, Kaufmane and Michael Marianif the main target field of previous biomarker studies on aBCAL Dx, Eveleigh, NSW, Australia 2015, Eveleigh, Australia; bThermo Fisher Scientific, Scoresby, VIC, Australia 3179, Scoresby, Australia; EVs are proteome and transcriptome. Meanwhile, cLipidomics Consulting Ltd., Esbo, Finland 02230, Esbo, Finland; liquid chromatography coupled with high resolution dDiscipline of Pathology, Brain and Mind Centre, Sydney Medical School, mass spectrometry (LC-MS) has recently been University of Sydney, Camperdown, NSW, Australia 2050, Camperdown, Australia; e1-Department of Neurosurgery, Chris O’Brien Lifehouse, employed to study comprehensive lipid profiles of in Camperdown, NSW, Australia 2050, 2-Discipline of Pathology, Brain and vitro EVs and their parental cells. However, lipid pro- Mind Centre, Sydney Medical School, University of Sydney, Camperdown, NSW, Australia 2050, Camperdown, Australia; fThermo Fisher Scientific, file of EVs in biolfluids, especially blood specimens North Ryde, NSW, Australia 2113, North Ryde, Australia such as plasma and serum, has not been well-characterized. To use control data for EVs, we aimed to Introduction: Extracellular vesicles (EVs) are secreted characterize lipid profile of EVs in human healthy from many cell types and play important roles in plasma and serum, and to compare their lipid profile intercellular communication. EVs carry a range of bio- with that of other lipid-containing particles in blood, molecules that reflect the identity and molecular state 110 ISEV2019 ABSTRACT BOOK of their parental cell and are found in biological fluids. aDepartment I of Internal Medicine, University Hospital of Cologne, b Omics studies have extensively focused on character- University of Cologne, Cologne, Germany; Experimental Tumor Research, Center for Tumor Biology and Immunology, Department of Hematology, isation of the protein and nucleic acid cargo of EVs Oncology and Immunology, Philipps University Marburg, Marburg, while lipids are less studied. EVs are increasingly being Germany; cInstitute for Clinical Chemistry and Clinical Pharmacology, University of Bonn, Bonn, Germany; dDepartment I of Internal Medicine, utilised in disease diagnosis as they are considered to University Hospital of Cologne, University of Cologne, Cologne, Germany, carry valuable information about the disease state. São Paulo, Brazil; eCECAD Center of Excellence on ‘‘Cellular Stress Responses in Aging-Associated Diseases’’, University of Cologne, Cologne, Thus, novel disease biomarkers might be identified Germany EV lipidomes. Methods: EVs were enriched from 1ml normal human Introduction: The expanding field of extracellular vesi- plasma samples using ultracentrifugation (UC), consid- cle (EV) research needs reproducible and accurate ered the gold standard approach for EV enrichment, methods to characterize single EVs. Nanoparticle and size exclusion chromatography (SEC) using qEV Tracking Analysis (NTA) is commonly used to deter- original columns (Izon, NZ). Lipids extracted accord- mine EV concentration and diameter. As the EV field ing to Matyash et al. (2008) were loaded on a C30 is lacking methods to easily confirm and validate NTA Acclaim column (Thermo, AU) using a Vanquish data, questioning the reliability of measurements liquid chromatography (LC) system and analysed remains highly important. In this regard, a comparison using a Fusion orbitrap mass spectrometer (MS) addressing measurement quality between different using targeted and untargeted lipidomics approaches. NTA devices such as Malvern’s NanoSight NS300 or LipidSearch software was used to annotate and quan- Particle Metrix’ ZetaView has not yet been conducted. tify lipid species. Methods: To evaluate the accuracy and repeatability of Results: More than 250 lipid species were identified size and concentration determinations of both devices, and quantified in the plasma EVs following both we employed comparative methods including transmis- enrichment methods. The two methods also generated sion electron microscopy (TEM) and single particle highly similar lipid profiles, indicating that SEC may be interferometric reflectance imaging sensing (SP-IRIS) a viable alternative to the cumbersome UC method. by ExoView. Multiple test measurements with nano- Interestingly, the SEC approach yielded less lysopho- spheres, liposomes and ultracentrifuged EVs from sphatidylcholine (LPC) lipids, which may be related to human serum and cell culture supernatant were per- a more homogenous vesicle population captured by formed. Additionally, serial dilutions and freeze-thaw SEC. Various literature reviews refer to glycerolipids, cycle-dependent EV decrease were measured to deter- likely originating from co-isolating vesicles such as mine the robustness of each system. low-density lipoproteins, as contaminants in the EV Results:Strikingly,NanoSightNS300exhibiteda2.0–2.1- fractions. We detected these lipids and propose that if fold overestimation of polystyrene and silica nanosphere they are differentially expressed in states of disease, concentration. By measuring serial dilutions of EV sam- they can be used as biomarkers independent of their ples, we demonstrated higher accuracy in concentration origin. determination by ZetaView (% BIAS range: 2.7–8.5) in Summary/conclusion: This study presents a workflow comparison to NanoSight NS300 (% BIAS range: 32.9– for comprehensive lipidomics of EVs using two isola- 36.8). The concentration measurements by ZetaView were tion methods that are compatible with downstream also more precise (% CV range: 0.0–4.7) than measure- state-of-the art LCMS, improving our ability to study ments by NanoSight NS300 (% CV range: 5.4–10.7). On the lipid components of EVs and identifying new dis- the contrary, quantitative TEM imaging indicated more ease biomarkers. As lipidome profiles were similar accurate EV sizing by NanoSight NS300 (% DTEM range: between the two isolation methods, large scale diagnos- 79.5–134.3) compared to ZetaView (% DTEM range: tic assays should consider employing the SEC, which is 111.8–205.7), while being equally repeatable (NanoSight by far the more efficient, scalable approach. NS300% CV range: 0.8–6.7; ZetaView: 1.4–7.8). However, both devices failed to report a peak EV diameter below LBT01.07 60 nm compared to TEM and SP-IRIS. Summary/conclusion: Taken together, NTA devices differ strongly in their hardware and software affecting Extracellular vesicle measurements with nanoparticle tracking measuring results. ZetaView provided a more accurate analysis – An accuracy and repeatability comparison between NanoSight NS300 and ZetaView and repeatable depiction of EV concentration, whereas Daniel Bachurskia, Maximiliane Schuldnerb, Phuong-Hien Nguyena, NanoSight NS300 supplied size measurements of Alexandra Malzb, Katrin S. Reinersc, Patricia C. Grenzid, Felix Babatze, Astrid C. Schausse, Hinrich P Hansena, Michael Halleka and Elke Pogge higher resolution. von Strandmannb JOURNAL OF EXTRACELLULAR VESICLES 111

LBT01.09 isolation of EVs from whole-blood. The device offers a simple, fast and efficient means of intact EV isolation in a reproducible manner, from small sample volumes Exodisc for fast and robust isolation of extracellular vesicles from measuring as small as 30 µL of whole-blood. whole-blood Funding: This work was supported by grants A121994 Vijaya Sunkaraa, Chi-Ju Kimb, Juhee Parkc, Hyun-Kyung Wood, Dongyoung Kima and Yoon-Kyoung Chod and IBS-R020-D1 funded by the Korean Government. aCenter for Soft and Living Matter, Institute for Basic Science (IBS), South Korea, Ulsan, Republic of Korea; bUlsan National Institute of Science and Technology (UNIST), South Korea, Ulsan, Republic of Korea; cCenter for soft and living matter, institute for basic science (IBS), South Korea, Ulsan, LBT01.10 Republic of Korea; dUlsan national institute of science and technology (UNIST), South Korea, Ulsan, Republic of Korea

Optimization and characterization of low vacuum filtration procedure Introduction: The circulating nano-vesicles, known as – novel method for the isolation of extracellular vesicles extracellular vesicles, are abundant in most of the body Anna Elżbieta. Drożdża, Agnieszka Kamińskaa, Magdalena Surmanb, c d d fluids and play vital roles in regulation of various Agnieszka Gonet-Surówka , Andrzej Wróbel and Ewa Łucja Stępień biological processes, including signalling in the tumour aFaculty of Physics, Astronomy and Applied Computer Science of the Jagiellonian University, Kraków, Poland; bInstitute of Zoology and microenvironment. They possess significant potential Biomedical Research of the Jagiellonian University, Kraków, Poland; for disease diagnosis and treatment monitoring, how- cFaculty of Chemistry of the Jagiellonian University, Kraków, Poland; dFaculty of Physics, Astronomy and Applied Computer Science of the ever, their use in clinical settings is limited due to lack Jagiellonian University, Kraków, Poland of simple and robust isolation methods. To address this, earlier we have developed Exodisc for isolation Introduction: Despite recent developments in the field and analysis of the EVs from urine. In this study, lab- of extracellular vesicles (EVs) isolation methods, the on-a-disc for the isolation of EVs from whole blood, process remains challenging, mainly due to the low Exodisc-B, is demonstrated. isolation yield, co-precipitation of proteins, changes in Methods: Exodisc-B comprises of blood separation and biophysical properties of EVs and time consuming filtration chambers connected with individually addres- procedures. Answering these problems, we created sable diaphragm valves for the automatic control of and validated new EVs isolation method – Low sequential transfer of liquid samples. The device con- Vacuum Filtration (LVF) and compared it with two sists of two nano-porous membrane filters with pore most commonly applied procedures – differential cen- sizes of 600 nm (track-etched PC membrane) and trifugation (DC) and ultracentrifugation (UC). 100 nm (AAO membrane). First, the plasma was sepa- Methods: The main element of the isolation system is rated and passed through two filters sequentially to dialysis membrane (MWCO = 1,000 kDa) combined concentrate the EVs on filter-II. Then the EVs were with the low vacuum pump, assuring the high yield of washed and transferred to a collection chamber for isolation and short procedure time. EVs isolated from retrieval. The performance of the device in comparison endothelial cells culture media have been characterized to ultracentrifugation (UC) was evaluated by analysing by (a) transmission electron microscopy (TEM) (b) yield, purity, RNA and protein content of the iso- nanoparticle tracking analysis (NTA), (c) western blot lated EVs. and (d) Fourier-Transform Infrared Spectroscopy Results: Compared with the UC technique, the (FTIR). Exodisc-B is capable of isolating at least an order of Results: TEM measurement visualized EVs with size of magnitude higher number of EVs with about 30-fold (a) LVF: 201 ± 136 nm, (b) DC: 256 ± 140 nm and (c) higher mRNA count within 40 min. Sandwich ELISA UC: 78 ± 25 nm. For LVF and DC EVs size was of EV-specific membrane proteins – CD9-CD81 – con- confirmed by NTA, for UC estimated size was higher firmed that it can isolate EVs with a capture efficiency (224 ± 112 nm). NTA showed substantial increase in >75%. The device also facilitates temporal monitoring EVs concentration, compared to the initial sample: (a) of tumour progression within live mouse xenograft LVF: 22 fold, (b) DC: 13 fold, (c) UC: 35 fold. Western models over a period of 13 weeks while using minimal blot analysis confirmed the presence of exosome’s volumes of weekly collected blood samples. Further, in (hsp70) and ectosome’s (Arf6) markers in (a) LVF – ELISA analyses of multiple cancer-related proteins CHsp70 = 0.48 ± 0.14 AU and CArf6 = 0.05 ± 0.02 AU, extracted from EVs isolated from human plasma, 43 (b) DC – CHsp70 = 0.04 ± 0.01 AU and patients were differentiated from 30 healthy donors. CArf6 = 0.07 ± 0.02 AU) and (c) UC Summary/conclusion: We have demonstrated the per- (CHsp70 = 0.23 ± 0.12 AU and CArf6 = 0.07 ± 0.04 formance of Exodisc-B for label-free and automatic AU). We observed correlation between ATR-FTIR 112 ISEV2019 ABSTRACT BOOK spectra quality (amid I:lipids ratio) and the EVs and concentrated between 170 nm to 220 nm (ranging proteins concentration. 100 nm to 370 nm). eMV CaCl2 showed a greater Summary/conclusion: LVF method is an easy and fast concentration between 150 nm to 250 nm (ranging EVs isolation method which allows for isolation of 100 nm to 600 nm) and eMV NHS was between both ectosomes and exosomes from high volume 120 nm to 350 nm (ranging 100 nm to 750 nm). sources and could be an efficient alternative for com- Summary/conclusion: Here, the FC size beads proved monly applied methods. to be useful not only for the measurement of MV sizes, Funding: The authors acknowledge financial support but also for the concentration as MV samples fitted from National Science Centre Poland [grant no. 2017/ with the created gate. In relation to MV concentration 25/N/ST5/00831]. distribution, the qNano proved to be a helpful appara- tus by accurately distributing the concentration of MV LBT01.11 according to their size spectrum. A combination of the different approaches may be able to provide useful information on the amount of MV released from Heterogeneity of erythrocytes derived microvesicles: their size and inducements. concentration Roberta Cordeiro Freezora and Sheelagh Heughb aLondon Metropolitan University, London, United Kingdom; bline manager, London, United Kingdom LBT01.12

Introduction: Extracellular Vesicles (EV) are heterogeneous populations of vesicles with different compo- Pentapartite fractionation of particles in oral fluids by differential centrifugation sitions, physiochemical properties and sizes. Random Chiho Hiragaa, Tamiko Minamisawab, Sachiko Matsumurac and Kiyotaka sampling is convenient when the population members Shibad are similar to one another on variables, ensuring a high aDivision of protein Engineering, Japanease Foundation for Cancer Research, degree of representativeness because it can critically Koto-ku, Japan; bJapanese Foundation for Cancer Research, Tokyo, Japan; cJapaese Foundation for Cancer Research, Koto-ku, Japan; dJapaese construct generalizability of results. These key concepts Foundation for Cancer Research, Tokyo, Japan can be misconstrued and blended, concealing the general drawbacks of validity. Here, erythrocytes MV Introduction: Novel diagnostic methods are being (eMV) samples were derived from the same blood developed for various oral maladies by using extracel- donor and are not to be presented as representative lular vesicles (EVs) contained in oral fluids because data of the general type of MV population, but to EVs carry condensed diagnostic information. compare their heterogeneity (induced and non- However, our knowledge on the comprehensiveness induced). of oral EVs is still very limited. In particular, cross- Methods: Blood cells were purified and isolated utilis- contamination of desquamated epithelial cells in oral ing the Ficoll-Paque PLUS method. Erythrocytes con- fluids with EV fractions is our current interest because centrate pellets were used in 3 different ways (in this contamination could interfere with the diagnostic triplicates): 1. eMVc- erythrocyte pellet with 10 mL information. To understand the possible interference of 200 nm filtered PBS pH 7.4; 2. eMV CaCl2- As in 1 desquamated epithelial cells in oral EVs, we fractio- plus 20 µL of 2 mM of CaCl2 and; 3. eMV NHS- as in 2 nated human oral fluids into 5 fractions by differential plus 1 mL 20 nm filtered normal human serum (NHS). centrifugation and analysed the protein markers and Samples were incubated for 45mins at 37°C and eMV nucleic acids in the fractions. were isolated by ultracentrifugation and suspended in Methods: We obtained oral fluids from three healthy 200 nm filtered PBS then analysed using the Guava volunteers with informed consent. Each sample was flow cytometry (FC) EasyCyte HT system and qNano separated into 5 fractions (0.3K, 2K, 10K, 160K and instrument. supernatant) by differential centrifugation. The num- Results: FC analyses showed similar characteristics in bers and the sizes of the particles in the fractions were sizing and FC position (>800nm), the majority of sam- analysed by nanoparticle tracking analysis (NTA). The ples laying within the MV gate created (size beads expression levels of the protein markers were estimated used), but eMV CaCl2 (3.4x10 6/mL) and eMV NHS by western blotting (WB). The amounts of mitochon- (2.9x 10 7/mL) showed a significant increase in their drial and bacterial DNAs were quantified by PCR- amount of release (P value/mL). qNano analyses based methods targeting the ND1 gene and rRNA showed an increase in concentration distribution for gene, respectively. The numbers of cells were estimated induced samples (100nm to 800nm). eMVc peaks were by Trypan blue and Papanicolaou staining. JOURNAL OF EXTRACELLULAR VESICLES 113

Results: Trypan blue staining showed that the 0.3K and activation, and in the degradation of senescent proteins 2K fractions contained 1.35 ×105 and 2.22 ×102 cells/ for recycling of components and displaying for mL of nucleated cells, respectively, while no intact cell immune surveillance. During stressful situations, HSP was observed in the 10K and 160K fractions by quantities and/or activities are increased as cells and Papanicolaou staining. NTA showed that the average tissues seek protection from insults. On occasion, these diameters of the particles in the 10K, 160K, and the insults can result in the cell surface display of HSPs, supernatant were 206.1 ± 17.0 nm, 122.1 ± 9.2 nm and which can then lead to the surface display of HSPs on 139.4 ± 29.4 nm, respectively. WB analyses showed that exosomes, membrane-enclosed vesicles released extra- CD81, CD9, Alix, and Aquaporin 5 were mostly cellularly after passage through the endosomal system. enriched in the 160K fraction, whereas HSP70, Ago2, HSPs present on the cell surface or in the extracellular and ATP5A were the most abundant in the 0.3K frac- space are regarded as “danger signals” in an ancient tion. Mitochondrial DNA was abundant in the 0.3K biologic paradigm. HSP-accessorized exosomes may act fraction, and bacterial ribosomal DNAs were present in as “danger boli”, carrying not only the HSPs, but hun- the 0.3K and 2K fractions. dreds of components of the stressed parental cell, cap- Summary/conclusion: The WB suggested that HSP70, able of prompting immune responses, or possibly Ago2, and ATP5A can be used as markers of whole immune suppression, depending on the status of the cells (mostly desquamated cells). Because the expres- recipient cell. sion levels of these markers in 10K and 160K were very Methods: Exosomes from the plasma of patients suf- limited, we concluded that cross-contamination of des- fering from neurological maladies (glioblastoma grade quamated epithelial cell-derived particles in 10K and IV, traumatic brain injury, multiple sclerosis) are pre- 160K would be very less, if any. cipitated by peptides designed to bind HSPs and analysed by metabolomics. Results: The metabolome of exosomes purified by HSP LBT01.13 peptides from plasma of patients with various neurological disorders is distinct from that of blood exo-

Heat shock protein-accessorized exosomes: presence in states of somes from healthy donors (>80 distinct compounds danger, disease, and disruption in GBM exosomes, and TBI exosomes; >30 compounds Xiaoli Yua, Mary Wanga, Anthony Fringuelloa, Steve Griffithsb and Michael Granera in MS exosomes; all are unique to those groups). There are also numerous lipid and metabolic pathways linked aUniversity of Colorado Denver, Aurora, USA; bminervagen biotechnologies corporation, tucson, USA to those compounds. Summary/conclusion: Such HSP-accessorized exo- Introduction: Heat shock proteins (HSPs) function as somes thus possess metabolites with possible ties to chaperones under both normal and pathologic condi- the different CNS pathologies that may represent dis- tions. As chaperones they assist in protein folding, in ease-specific biomarkers in a “liquid biopsy” setting. holding protein complexes for current or future 114 ISEV2019 ABSTRACT BOOK

LBT02: Late Breaking- EV Biomarkers Chairs: Maja Mustapic; Dakota Gustafson Location: Level 3, Hall A 15:30–16:30

LBT02.01 LBT02.02=OWP3.01

Cancer stem cell-derived exosomes：potential for early detection in Using plasma to identify neural biomarker for antidepressant pancreatic cancer a b c d response in a treatment resistant cohort Haobin Wang , Yingshu Luo , Margot Zoeller and Shijing Yue Corina Nagya, Saumeh Saeedib, Jean-Francois Therouxc, Marina Wakidc, c b aThe third people‘s hospital of Chengdu/Affiliated hospital of Southwest Naguib Mechawar and Gustavo Turecki b Jiaotong University, Chengdu, China (People‘s Republic); University of a b DHRC- McGill University, Verdun, Canada; McGill University, Verdun, ‘ Electronic Science and Technology of China, Chengdu, China (People s Canada; cMcGill University, verdun, Canada Republic); cHeidelberg University, Heidelberg, Germany; dNankai University, Tianjin, China (People‘s Republic) Introduction: Small extracellular vesicles (SEV) have Introduction: Pancreatic cancer (PaCa) is the most emerged as candidate biomarkers in many complex deadly malignancy, due to late diagnosis and early diseases. An important characteristic of SEVs is their metastatic spread, which prohibits surgery. it is ability to bidirectionally cross the blood-brain barrier. urgently for reliable, early detection. Research shows This is particularly important in the context of major that tumour-derived exosomes, which had been present depressive disorder (MDD), where biomarkers are in the blood in the early stage of tumour formation and obtained from peripheral tissue and have been hard before metastasis, is the vanguard forces of tumour to relate to changes in brain functioning. 60% of formation and metastasis; Cancer stem cell-derived MDD patients do not respond to their first antidepres- exosomes (CSC-Exos) has stronger migration ability, sant drug therapy (ADT) and treatment options are so the detection of blood CSC-Exos for early diagnosis entirely at the discretion of the physician. Findings and monitoring of progress for PaCa has great research that can predict ADT response as well as provide potential and the value of application. insight into central mechanistic changes could revolu- Methods: Protein markers were selected according to tionize MDD treatment. The aim of this study is to expression in exosomes of PaCa cell line culture super- profile exosomal microRNA (miRNA) in the context of natants, but not healthy donors’ serum- exosomes. ADT response in people with treatment-resistant According to these preselections, serum-exosomes depression. miRNA can act as biomarkers and might were tested by flow cytometry for the pancreatic cancer influence recipient cells to provide insight on disease- stem cell marker Tspan8. relevant mechanistic changes. Results: The majority (95%) of patients with PaCa and Methods: This pilot uses plasma from 10 controls and patients with nonPa-malignancies reacted with anti- 10 patients with MDD (5 ADT responders (RES), and 5 Tspan8. Serum-exosomes of healthy donors’ and non-responders (NRES)) from baseline (T0, before patients with nonmalignant diseases were not reactive. treatment). SEVs were isolated using a size exclusion Recovery was tumour grading and staging independent column from Izon Science (Christchurch, New including early stages. Zealand). Each isolation was divided into a “whole Summary/conclusion: Thus, the evaluation of pan- exosome” fraction and an immunoprecipitated creatic CSC-derived exosomes awaits retrospective ana- “(NDE)” fraction using neural marker L1CAM. lyses of larger cohorts, as it should allow for a highly Quantitation and size determination was done using sensitive, minimally-invasive PaCa diagnostics. Tunable Resistive Pulse Sensing (TRPS) on the qNano Funding: Supported by the National Natural Science gold. RNA was also extracted from SEVs from both Foundation of China (No. 81702963) fractions. The 4N-small RNA-Seq (Galas) protocol was used for library preparation. JOURNAL OF EXTRACELLULAR VESICLES 115

Results: We found that the range of SEVs in the NDE computational analysis of gene expression and proteo- fraction was smaller than the pool of all exosomes mics data. We have applied this framework to the combined. Further SEVs from all depressed patients isolation of neuron-specific EVs in human biological were significantly smaller than controls irrespective of fluids. We envision these methods being broadly the fractions. Our sequencing results showed an applicable to the development of novel diagnostic bio- increase of miR-151a-3p and miR-3168 in NRES, and markers for a variety of diseases. miR-22-3p in RES. These results were specific to the NDE fraction. LBT02.04 Summary/conclusion: We have identified three potential biomarkers for ADT response which are uniquely present in the neural-derived fraction of peripheral Labelling and tracking extracellular vesicles using a RNA-targeting SEVs. AIE fluorogen Bo Situ, Xiaojing He and Lei Zheng Funding: Canadian Institutes of Health Research Nanfang hospital, southern medical university, guangzhou, china (people‘s republic) LBT02.03=OWP1.08 Introduction: Extracellular vesicles (EVs) are considered as crucial carriers in cell-to-cell communication, Isolation of neuron-specific extracellular vesicles immune response, tumourigenesis and metastasis. To Dmitry Ter-Ovanesyana, Maia Kipmanb, Emma Kowalc, Ju Hyun Leeb, Wendy Trieub, Aviv Regevd, David Waltb and George Churchb gain direct insights into EVs functions, it is necessary to observe their intracellular localizations and biodis- aHarvard, Cambridge, USA; bWyss Institute, Boston, USA; cMIT, Cambridge, USA; dBroad Institute, Cambridge, USA tribution. Given the fact that EVs carry various RNA species, fluorescence labelling of RNA in EVs is one of Introduction: Human biological fluids contain extra- the most high-profile strategies. However, ideal probes cellular vesicles (EVs) from different cell types. It are still lacking. would be incredibly useful to be able to isolate EVs Methods: In this work, we report that a commercial that originated from specific cell types for diagnostic cell-permeant dye HSP may serve as a simple and facile purposes as a way to gain molecular information probe for staining RNA within EVs. The good perfor- (RNA, protein) from inaccessible cell types non- mance of HSP allows EVs to be analysed and imaged invasively. by nano-flowcytometry and structured illumination Methods: We have developed a general framework for microscopy (SIM), respectively. Additionally, for the identifying EV surface markers that can be used for first time we uncover that HSP exhibits typical AIE immuno-isolation of cell type specific EVs. As a proof (aggregation-induced emission) property. The labelling of principle, we have applied this framework to the procedure can thus be performed in a wash-free man- isolation of neuron-derived EVs from human cere- ner due to the low fluorescent background of HSP in brospinal fluid or plasma. In addition to the computa- water before binding to RNA, which greatly avoid EVs tional analysis, we have developed an in-vitro system of losing during the experiment. human neurons differentiated from human induced Results: HSP shows advantages over traditional pluripotent (iPS) cells. We performed mass spectro- SytoRNASelect in labelling EVs RNA in terms of its metry on EVs isolated from these neurons to identify superior brightness, high specificity and excellent neuron-specific proteins. We also used this system to photostability. develop a robust immune-isolation method for neuron Summary/conclusion: HSP may serve as a new probe EV markers. for EVs labelling and shows great potential in studying Results: We have characterized the proteins present in behaviours and bio-distributions of EVs in a wide neuron exosomes by mass spectrometry and then used range of research fields. computational analysis of published gene expression and proteomics data to come up with a list of candidate neuron-specific EV markers. After developing methods LBT02.05 for immuno-isolation of neuron EVs with these markers, we applied our methods to human cerebrospinal The identification of extracellular vesicles proteins in glioblastoma fluid and plasma. diagnosis Szu-Yi Choua, Che-Chang Changb and Shun-Tai Yangc Summary/conclusion: We have developed a frame- aGraduate Institute of Neural Regenerative Medicine, Taipei Medical work for the isolation of cell type specific EVs through University, Taipei, Taiwan (Republic of China); bGraduate Institute of the combination of an experimental in vitro system and Translational Medicine, Taipei Medical University, Taipei, Taiwan 116 ISEV2019 ABSTRACT BOOK

(Republic of China); cDivision of Neurosurgery, Shuang Ho Hospital, Taipei, aAnimal Physiology and Immunology, School of Life Sciences Taiwan (Republic of China) Weihenstephan, Technical University of Munich, Freising, Germany, Freising, Germany; bDepartment of Biochemistry and Cell Biology, Utrecht University, Utrecht, The Netherlands, Utrecht, Netherlands Introduction: Glioblastoma multiforme (GBM) is a highly malignant type of brain tumour in humans. Introduction: Due to their importance in intercellular GBM cells reproduce quickly and the median survival communication, extracellular vesicles (EV) have time for patients is about 1 ~ 2 years. Current diag- emerged as important sources of biomarkers for pro- nostics and treatments for GBM are limited. Recently, and diagnostic purposes. With the advent of RNA-seq many studies used proteomic analyses of GBM extra- as the tool of choice for unbiased biomarker screening, cellular vesicles (EVs) or secretomes have been helpful a major focus has been laid on miRNAs, important in identifying biomarkers and potential treatment stra- regulators of post-transcriptional gene expression. tegies for GBM. Feasibility of RNA biomarkers presently still relies on Methods: Herein, our study used mass spectrometry validation and analysis by RT-qPCR which in turn is – (MS) to analysis the EV proteins from GBM cell lines depending on stably expressed reference transcripts for – U87 and A172, and normal human astrocyte SVG- normalization. To assess whether a set of universal p12 cultures. IPA analysis identified several proteins reference miRNA transcripts for normalization exists, from GBM cell lines EVs are significantly different a meta-analysis on blood derived EV samples was from the normal astrocytes cultures. EVs from 30 conducted. patients plasma with different grades of glioma were Methods:Fromeightdifferentresearch studies, we isolated and analysed to conform the findings from analysed small RNA-seq reads of 531 EV samples IPA analysis that were isolated from various pathological condi- Results: We identified several signalling pathways have tions or healthy controls and enriched by standar- been changed in different GBM cultures. Further vali- dized methods (SEC, UC or precipitation). To dation with 30 different grade of glioma patients, we account for the variety of commonly utilized RNA- – identified three proteins chaperonin containing seq analysis methods, a standardized big-data analysis TCP1 subunit 8 (CCT8), Glypican (GPC1) and pipeline was established, that combined robust filter- Periostin (POSTN) which levels in plasma EVs are ing by six different normalization methods and three associated to GBM but not plasma which also have algorithms to detect suitable reference transcripts. Sets been reported associated to GBM progression. of stably expressed transcripts were finally compared Database analysis also found the EVs level of CCT8, across different studies, isolation methods and data GPC1 and POSTN in different grade of glioma can analysis combinations. represent the RNA level in tumour from microarray. Results:Resultsofourpipelineshowedsubstantial Additionally, we also found some specific signalling overlap for miRNAs ranked by stability for different pathways changes in different GBM lines such as trans- normalizations and algorithms over all samples albeit forming growth factor beta induced (TGFB1) in U87 compromised by high variances in general. EVs and prosaposin (PSAP) in A172 EVs. The eleva- Contrarily reference miRNAs determined within a tion of different molecules in EVs provides specific single research study showed much higher stability characters to individual GBM. values and were consistent over multiple analysis – Summary/conclusion: We found EV contents CCT8, combinations. GPC1 and POSTN were associated in GBM which Summary/conclusion: Although first results suggest could be used for clinical diagnosis; also some different the possibility that blood EVs contain a common set – GBM EV proteins TGB1 and prosaposin could be of miRNAs that may be used as universal reference used in characterization and targeting therapy of GBM transcripts, different EV isolation methods, pathophy- in the further. siological conditions and sequencing methodology – Funding: Ministry of Science Technology MOST have a major influence on expression profiles. With 105-2628-B-038-005-MY3 the availability of additional small RNA-seq data sets in the future, robustness and validity of miRNA refer- LBT02.06 ences will continue to grow, but success will ultimately be depending on further standardization of EV isolation and data analysis. Based on the current data, we Universal reference transcripts for miRNA normalization – a meta- analysis on human blood extracellular vesicle RNA sequencing data recommend analysing reference transcripts in each sets study individually. Alexander Hildebrandta, Benedikt Kirchnera, Chenna R. Galivetib, Esther N. Nolte-‘t Hoenb and Michael Pfaffla JOURNAL OF EXTRACELLULAR VESICLES 117

LBT02.07 LBT02.08

Small extracellular vesicle content in porcine blood plasma, Quantitative proteomic profiling of tissue-exudative EVs identified a cerebrospinal fluid and seminal plasma for proteomic analyses in novel diagnostic antigen for early detection of colorectal cancer biomarker discovery Makoto Konishia, Makoto Sumazakia, Satoshi Nagayamab and Koji Uedaa Helena Kupcova Skalnikovaa, Jakub Cervenkaa, Karolina Turnovcovab, a a a a Bozena Bohuslavova , Jana Juhasova , Stefan Juhas and Petr Vodicka aCancer Proteomics Group, Cancer Precision Medicine Center, Japanese Foundation for Cancer Research, Tokyo, Japan; bDepartment of aCzech Academy of Sciences, Institute of Animal Physiology and Genetics, Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation Libechov, Czech Republic, Libechov, Czech Republic; bCzech Academy of for Cancer Research, Tokyo, Japan Sciences, Institute of Experimental Medicine, Prague, Czech Republic, Prague, Czech Republic Introduction:Developmentofbiomarkersforearly Introduction: Extracellular vesicles (EVs) released to detection of colorectal cancer (CRC) is demanded as body fluids carry molecules of the source cells and are the number of CRC patients is increasing. Recent subjects of intensive research of protein and nucleic acid studies exhibit that extracellular vesicles (EVs) are biomarkers of diseases. Pig represents a valuable experi- expected as biomarker carriers in any body fluids. mental biomedical model to study human diseases due to To explore CRC-specific antigens, we isolated EVs close anatomic and physiologic similarity to human. The from viable CRC or adjacent normal tissues aim of this work was to compare suitability of porcine (n = 17), followed by global quantitative proteome blood plasma, cerebrospinal fluid and seminal plasma for analysis. EV isolation for proteomic analyses and optimize sample Methods: Tissue-exudative EVs (Te-EVs) were purified preparation for mass spectrometry. from serum-free media of freshly resected CRC and Methods: EVs were isolated from porcine body fluids adjacent normal tissues, using the sequential ultracen- by differential centrifugation and ultracentrifugation trifugation method (n = 17). Purified Te-EVs were and characterized by transmission electron microscopy, analysed by Orbitrap Fusion Lumos LC/MS system flow cytometry and western blotting. Three different (Thermo Scientific). Protein identification, label-free lysis buffers (RIPA, Triton X100 and SDS) were com- quantification, and statistical analysis were performed pared in efficacy to extract EV proteins in combination on MaxQuant and Proteome Discoverer softwares. A with filter-aided sample preparation (FASP) for LC- statistically valid biomarker candidate protein MS/MS analysis (triple TOF). (TMAM) was further evaluated by plasma exosome Results: Seminal plasma yielded largest amount of EVs, sandwich ELISA (n = 357). Additional clinical and followed by blood plasma. In cerebrospinal fluid, the EV functional assessments were also performed including content was very low. Proteomic analysis of seminal IHC staining and cell growth assays. plasma-derived EVs enabled identification of approxi- Results: Among 6,149 identified Te-EV proteins, 393 mately 1200 proteins, including 76 of the top 100 mainly proteins were significantly overexpressed (p < .05 and identified proteins in EVs (Exocarta). Approximately 550 fold change > 4.0) in EVs from CRC tissues compared proteins were quantified by SWATH-MS. In contrast, to those from normal mucosa. We especially focused only 200 proteins were identified in the crude seminal on transmembrane protein TMAM (p = 3.62 E-5, fold plasma used for EV isolation. change = 7.0) which was known to be a key regulator Summary/conclusion: We have optimized techniques of cell growth and also overexpressed in CRC cells. for the EV enrichment from porcine body fluids and Exosome sandwich ELISA confirmed significant eleva- for characterization of their protein content by mass tion of TMAM level in plasma EVs even in stage-I spectrometry. Such techniques may be applied to bio- CRC patients (n = 72) compared to healthy donors marker discovery in porcine model of diseases as well (n = 72, p = .040). IHC staining analysis also showed as adopted to other species, including human. that TMAM was specifically overexpressed in CRC Funding: This study was supported by Czech Science tissues. Interestingly, TMAM-overexpressed EVs Foundation (reg. No. 19-01747S), Operational decoyed its inhibitory ligand away from cancer cells, Programme Research, Development and Education (reg. resulting in their outgrowth. No. CZ.02.1.01/0.0/0.0/16_019/0000785), and National Summary/conclusion: These results indicate that Sustainability Programme I. of the Czech Ministry of TMAM on EVs should have great potential as a novel Education, Youth and Sports (reg. No. LO1609). target for CRC diagnosis and therapy. 118 ISEV2019 ABSTRACT BOOK

LBT02.09 schemes, the quantity and PPI strengths of EGFRs derived from EVs and the original lung adenocarcinoma cells are determined. Single-molecule co-Immunoprecipitation reveals functional Results: It is found that the microvesicles exhibit inheritance of epidermal growth factor receptors in extracellular higher correlations with the original cells than the vesicles Mi Sook Sunga, Jik Han Jungb, Tae-Young Yoonc, Ji-Ho Parkb and Cherlhyun exosomes in terms of the EGFR levels and their PPI Jeonga patterns. In spite of these detailed differences between aCenter for Theragnosis, Korea Institute of Science and Technology, Seoul, the microvesicles and exosomes, the EGFR PPI Republic of Korea; bDepartment of Bio and Brain Bioengineering, Korea strengths measured for EVs generally show a tight Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea; cSchool of Biological Sciences and Institute for Molecular Biology correlation with those determined for the original cells. and Genetics, Seoul National University, Seoul, Republic of Korea Summary/conclusion: With epidermal growth factor receptor (EGFR) in lung adenocarcinoma cells as a Introduction: Cancer cells actively release extracellular model oncoprotein, it is studied how distinct types of vesicles (EVs) as important carriers of cellular informa- EVs, microvesicles and exosomes, represent their ori- tion to tumour microenvironments. Although the com- ginal cells at the protein and protein–protein interac- position and quantity of the proteins contained in EVs tion (PPI) level. The results suggest that EGFRs are characterized, it remains unknown how these pro- contained in EVs closely reflect the cellular EGFR in teins in EVs are related to those in the original cells at terms of their downstream signalling capacity. the functional level. Ultimately, the question should be Moreover, it gives a possibility that EGFRs derived resolved to ensure the use of EVs in diagnosing the from different types of EVs may work as a biomarker status of cancer patients by liquid biopsy. for the intensity of the EGFR signalling pathway in the Methods: Using the recently developed single-molecule parental cancer tissue. immunolabelling and co-immunoprecipitation JOURNAL OF EXTRACELLULAR VESICLES 119

LBT03: Late Breaking- EVs and Stem Cells Chairs: Sicheng Wen; Hiroaki Tateno Location: Level 3, Hall A 15:30–16:30

LBT03.01 Summary/conclusion: We have shown that transfection of MSC by siRNA anti miR-146a decrease the biologic effect of MSC-EVs on migratory capacity of Regenerative potential of extracellular vesicles-derived from epithelial cells. It could be a direct effect of the absence mesenchymal stem cells on epithelial wound healing Tatiana Lopatinaa, Chiara Gaib, Giovanni Camussic and Giuseppe of miR-146a in MSC-EVs or consequence of the miR- Montrucchiod 146a signalling pathway disruption. aPostdoc, Turin, Italy; bDepartment of Medical Sciences, University of Turin, Funding: CAMG_PRIN_2015_16_01 Turin, Italy; cDepartment of Medical Sciences, University of Turin, Turin, Italy; dUniversity of Turin, Turin, Italy LBT03.02 Introduction: Wound healing is a complex process involving cell death, migration, proliferation, differentiation, inflammation, and extracellular matrix remo- Intravenous administration of xenogenic adipose-derived mesenchymal stem cells (ADMSC) and ADMSC-derived exosomes delling. A key role in this context is played by resident markedly reduced brain infarct volume and preserved neurological function in rat after acute ischemic stroke stem cells. Mesenchymal stem cells (MSCs) favour a b c wound healing via extracellular vesicles (EVs), which Shun-Cheng Wu , Pei-Lin Shao and Hon-Kan Yip a transfer transcription modulators and nucleic acid, Orthopaedic Research Center, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan (Republic of China); bDepartment of including mRNA and micro-RNA. Nursing, Asia University, Kaohsiung, Taiwan (Republic of China); cDivision Methods: We found that MSC-derived EVs favour of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, epithelial wound healing in vitro, and that EVs regulate Kaohsiung, Taiwan (Republic of China) the EGFR/PI3K/Akt/mTOR pathway, a key player in keratinocyte stem cells biology, glucose homeostasis Introduction: We tested the hypothesis that combined and aging. Moreover we have characterized the xenogenic (from mini-pig) adipose-derived mesenchy- mRNA and miRNA content of MSC-derived EVs and mal stem cell (ADMSC) and ADMSC-derived exosome our analysis revealed several miRNA potentially therapy could reduce brain-infarct zone (BIZ) and involved in wound healing. To identify potential miR enhance neurological recovery in rat after acute candidates, we clustered miRNAs expressed by EVs ischemic stroke (AIS) induced by 50-min left middle into families, according to their seed sequence and cerebral artery occlusion. scanned the 3ʹ-UTR of keratinocyte expressed genes Methods: Adult-male Sprague-Dawley rats (n = 60) for perfect seed-match occurrences. To account for were divided equally into group 1 (sham-control), potential cooperative action of different miRNAs, we group 2 (AIS), group 3 [AIS-ADMSC (1.2 ×106 will restrict our research to those genes targeted by at cells)], group 4 [AIS-exosome (100 μg)], and group 5 least 2 expressed miRNA families. (AIS-exosome-ADMSC). All therapies were provided Results: We selected several miRNAs which target intravenously at 3h after AIS procedure. wound healing cellular pathways and carried by MSC- Results: BIZ determined by histopathology (by day-60) EVs (miR-let-7a-5p, miR-10a-5p, miR-10b-5p, miR-21- and brain MRI (by day-28) were highest in group 2, 5p, miR-22-3p, miR-100-5p, miR-143-3p, miR-146a, lowest in group 1, higher in groups 3 and 4 than in miR-191-5p, miR-181a-5p, miR-27b-3p). We have group 5, but they showed no difference between groups found that miRNA146a is a key activator of the 3 and 4 (all p < .0001). By day-28, sensorimotor func- Notch1/Akt pathway. Notably, Notch1 levels are ele- tional results exhibited an opposite pattern to BIZ vated in limbal-corneal epithelial stem cells relative to among the five groups (p < .005). Protein expressions their migratory cell progenies, and abnormally elevated of inflammatory (inducible nitric oxide synthase/ miRNA146a levels are implicated in defective corneal tumour necrosis factor-α/nuclear factor-κB/interleu- wound healing in diabetes. Thus, miRNA146a may kin-1β/matrix metalloproteinase-9/plasminogen activa- regulate the balance between LESC self-renewal versus tor inhibitor-1/RANTES), oxidative-stress (NOX-1/ migration/differentiation via Notch/Akt regulation. NOX-2/oxidized protein), apoptotic (caspase-3/Poly- 120 ISEV2019 ABSTRACT BOOK

ADP-ribose polymerase), and fibrotic (Smad3/trans- show by comparing both media, where methionine and forming growth factor-β) biomarkers, and cellular cysteine are decreasing to produce cystine. expressions of brain-damaged (γ-H2AX+/XRCC1- Summary/conclusion: Glutamate-Glutamine cycle and CD90+/p53BP1-CD90+), inflammatory (CD11+/CD68 Methionine-Cysteine cycles were shown in the meta- +/glial fibrillary acid protein+) and brain-oedema bolism of UC-MSC proliferation during cell culture. (aquaporin-4+) markers showed a similar pattern of This cause changes in amino acid concentration BIZ among the groups (all n < 0.0001). between fresh and post-culture media. Summary/conclusion: In conclusion, xenogenic Funding: All the research were funded by PT. Prodia ADMSC/ADMSC-derived exosome therapy was safe StemCell Indonesia and offered the additional benefit of reducing BIZ and improving neurological function in rat AIS. LBT03.04 LBT03.03 Combination of adipose-derived mesenchymal stem cells (ADMSC) and ADMSC-derived exosomes for protecting kidney from acute ischemia-reperfusion injury Changes in amino acid concentration of umbilical cord mesenchymal Shun-Cheng Wua, Pei-Lin Shaob and Hon-Kan Yipc stem cell culture medium a Angliana Chouw, Geofanny Facicilia, Cynthia Retna Sartika, Emilia Orthopaedic Research Center, College of Medicine, Kaohsiung Medical b Rahmadania Utami, Julia Riswandani, Yanni Dirgantara, Dwi Ajeng University, Kaohsiung, Taiwan (Republic of China); Department of c Permata Dewi and Endah Dianty Pratiwi Nursing, Asia University, Kaohsiung, Taiwan (Republic of China); Division of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung PT prodia stemcell Indonesia, Jakarta, Indonesia Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan (Republic of China) Introduction: Mesenchymal Stem Cell is a multipotent cell that works in two ways, replacing the injured cells Introduction: In this study, we tested the hypothesis and releasing active biomolecules that work as a para- that a combined adipose-derived mesenchymal stem crine signal for cell migration and proliferation. Recent cell (ADMSC) and ADMSC-derived exosome therapy discoveries suggest that potential cytokine, growth fac- protected rat kidney from acute ischemia-reperfusion tors, and many different soluble factors are released by (IR) injury (i.e., ligation of both renal arteries for 1h MSCs during the culturing process into its environ- and reperfusion for 72h prior to euthanization). ment. In this study, we aim to analyse that changes of Methods: Adult-male SD rats (n = 40) were equally amino acid concentration from the fresh complete categorized into group 1 (sham control), group 2 (IR), growth medium and post-culture medium from umbi- group 3 [IR+exosome (100 μg)], group 4 [IR+ADMSC lical cord mesenchymal stem cell (UC-MSC) cultured. (1.2 ×10(6) cells)] and group 5 (IR-exosome-ADMSC). Methods: UC-MSC was cultured with the seeding den- All therapies were performed at 3 h after IR procedure sity of 5000 cells/cm2 in tissue culture plasticware. from venous administration. When the cells, reached 70–80% confluency, the cul- Results: By 72h, the creatinine level and kidney injury ture medium was collected and centrifuged to remove score were the lowest in group 1 and the highest in the unwanted debris. Collected medium was stored in group 2, significantly higher in group 3 than in groups −80°C until the amino acid concentration was analysed 4 and 5, and significantly higher in group 4 than in using Mass Spectrophotometry. group 5 (all P < .0001). The protein expression of Results: The fresh and post-culture media contains inflammatory (TNF-α/NF-κB/IL-1β/MIF/PAI-1/Cox- both essential and non-essential amino acid. The 2), oxidative-stress (NOX-1/NOX-2/oxidized protein), post-culture culture media contains higher amino acid apoptotic (Bax/caspase-3/PARP) and fibrotic (Smad3/ compared to the fresh medium. In this study, there is TGF-β) biomarkers showed an identical pattern, an increasing concentration of glycine, l-arginine, l- whereas the anti-apoptotic (Smad1/5, BMP-2) and phenylalanine, l-histidine, l-leucine, l-lysine, l-serine, angiogenesis (CD31/vWF/angiopoietin) biomarkers l-threonine, l-tyrosine and l-valine concentration. The and mitochondrial cytochrome-C showed an opposite concentration of L-glutamine from post-cultures is pattern of creatinine level among the five groups (all decreasing compared to fresh medium while the con- P < .001). The microscopic findings of glomerular- centration of L-glutamic acid (+959 mg/ml) is increas- damage (WT-1), renal tubular-damage (KIM-1), ing. This due to the regulation of glutamate synthase DNA-damage (γ-H2AX), inflammation (MPO/MIF/ which changes the L-glutamine into L-glutamate (L- CD68) exhibited an identical pattern, whereas the glutamic acid). The methionine and cysteine cycle also podocyte components (podocin/p-cadherin/ JOURNAL OF EXTRACELLULAR VESICLES 121 synaptopodin) displayed a reversed pattern of creati- Summary/conclusion: In conclusion, HMSCEXO nine level (all P < .0001). might be superior to AMSCEXO for improving survi- Summary/conclusion: Combined exosome-ADMSC val and suppressing the inflammatory reactions in rats therapy was superior to either one for protecting kid- after SS. ney from acute IR injury. LBT03.06 LBT03.05 Bile acids hybrid extracellular vesicles derived from mesenchymal stem cells for cartilage tissue regeneration Yoshie Araia, Hyoeun Parka, Sunghyun Parkb, Alvin Belloc, Jinsung Ahna, Adipose-derived mesenchymal stem cell-derived exosomes alleviate a a d e overwhelming systemic inflammatory reaction and organ damage Dohyun Kim , Byoung Ju Kim , Hansoo Park and Soo-Hong Lee and improve outcome in rat sepsis syndrome a b a b c Dongguk University, Goyang-si, Republic of Korea; CHA University, Pei-Lin Shao , Shun-Cheng Wu and Hon-Kan Yip Goyang-si, Republic of Korea; cChung-Ang University, Goyang-si, Republic d e aDepartment of Nursing, Asia University, Kaohsiung, Taiwan (Republic of of Korea; Chung-Ang University, Seoul, Republic of Korea; Dongguk China); bOrthopaedic Research Center, College of Medicine, Kaohsiung University, goyang, Republic of Korea Medical University, Kaohsiung, Taiwan (Republic of China); cDivision of Cardiology, Department of Internal Medicine, Kaohsiung Chang Gung Introduction: Tauroursodeoxycholic acids (TUDCA) Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan (Republic of China) has been known as an amphiphilic therapeutic drug for a variety of diseases such as cholestasis, amyo- Introduction: This study tested the hypothesis that trophic lateral sclerosis, type 1 diabetes and so on. healthy adipose-derived mesenchymal stem cell Recently, we reported TUDCA has a role in bone and (ADMSC)-derived exosomes (HMSCEXO) and apopto- cartilage regeneration through leading to osteogenic or tic (A) (induced by 12 h hypoxia/12 h starvation)- chondrogenic differentiation of mesenchymal stem ADMSC-derived exosomes (AMSCEXO) were compar- cells (MSCs). In addition, TUDCA is also able to ably effective at alleviating sepsis syndrome [SS; form a nano-sized micelle, penetrate and incorporate induced by cecal-ligation and puncture (CLP)]-induced into the membrane of cells depending on the concen- systemic inflammation and reduced organ damage and tration, therefore, suggesting that TUDCA would be a unfavourable outcomes in rats. useful drug to modify cell membrane and extracellular Methods: SD rats were divided into sham control (SC), vesicles (EVs). SS only, SS + HMSCEXO (100 µg intravenous admin- Methods: In this study, we investigated whether the istration 3 h after CLP), and AMSCEXO. EVs derived from the amphiphilic bile acids-treated Results: By day 5 after CLP procedure, the mortality cells could produce hybrid EVs composed with cell rate was significantly higher in SS than in SC and membrane and bile acid and also they include HMSCEXO (all P < .01), but it showed no significant mRNA, micro RNA and proteins at the core of EVs. different between SC and HMSCEXO, between To aim this, we isolated EVs from TUDCA-treated AMSCEXO and HMSCEXO or between SS and mesenchymal stem cells (MSCs) and identified their AMSCEXO (P > .05). The levels of inflammatory med- characteristics. In addition, the regenerative effect of iators in circulation (CD11b/c/Ly6G/MIF), bronchioal- EVs was also evaluated using degenerated chodnro- veolar lavage (CD11b/c/Ly6G) and abdominal ascites cytes (DC) derived from the knee cartilage of osteoar- (CD11b/c/CD14/Ly6G/MIF) were highest in SS, lowest thritis (OA) patients. in SC and significantly higher in AMSCEXO than in Results: It was found that TUDCA-hybrid EVs HMSCEXO (all P < .001). The circulating/splenic levels (TUDCA-EVs) was successfully isolated and the total of immune cells (CD34+/CD4+/CD3+/CD8+) were amount of EVs was increased with the treatment of expressed in an identical pattern whereas the T-reg+ TUDCA. We also tried to characterize the size and cells exhibited an opposite pattern of inflammation zeta-potential of TUDCA-EVs as well as the expression among the groups (all P < .001). The protein expres- of genes which are contained in TUDCA-EVs com- sions of inflammation (MMP-9/MIF/TNF-α/NF-κB/IL- pared to control EVs. Next, TUDCA-EVs were treated 1β) and oxidative stress (NOX-1/NOX-2/oxidized pro- to DC. TUDCA-EVs treatment on degenerated chon- tein), and cellular expressions (CD14+/CD68+) in drocyte (DC) increased IL-6 expression associated with lung/kidney parenchyma exhibited an identical pattern the differentiation of M2 macrophage and anti-inflam- of inflammatory mediators (all P < .001). The kidney/ matory signalling. In addition, TUDCA-EVs increased lung injury scores displayed an identical pattern of COL2 expression while they decreased RUNX2 and inflammatory mediators among the groups (all MMP13, as a hypertrophic marker gene, expression P < .001). in DC. 122 ISEV2019 ABSTRACT BOOK

Summary/conclusion: These indicated that TUDCA- and verified let-7 target genes, including α-SMA, col- EVs have the potential to restore the chondrogenic lagen a1, p16, CCl2, TLR4, MMP-2, MMP-9 and properties of degenerated chondrocytes. Therefore, it TIMP-3, were significantly altered in liver specimens is concluded that TUDCA-EVs would be a useful sys- and LCM isolated HSCs from lin28 knockout mice temic drug for OA therapy. In ongoing in vivo study, with ethanol feeding relative to WT ALD mice we are confirming whether TUDCA-EVs indeed have controls. an influence on cartilage tissue regeneration and alle- Summary/conclusion: Our findings provide new viate OA symptoms. insight into the function of specific miRNAs in stem cell-derived EVs regulating HSC senescence, and their LBT03.07 therapeutic potentials in alcoholic liver injury and fibrosis.

Role of stem cell-derived extracellular vesicles and their enriched microRNAs during alcoholic liver injury LBT03.08 Nan Wua, Heather Francisa, Gianfranco Alpinib and Fanyin Menga aIndiana University School of Medicine, Indianapolis, USA; bIndiana University School of Medicine, Pittsburgh, USA Interferon-gamma priming, but not hypoxia, modifies the miRNA landscape of human mesenchymal stromal cells (MSC) extracellular Introduction: Senescence of activated stellate cells lim- vesicles (EV) Juliette Peltzera, Kyle Lundb, Philippe Mauduitc, Jean-jacques Latailladeb and its hepatic fibrogenesis. Stem cell-derived extracellular Sebastien Banzetd vesicles (EVs) and their related microRNAs mediate aInstitut de Recherche Biomédicale des Armées, INSERM UMR-MD-1197, genetic changes that promote recovery of liver disor- Clamart, USA; bInstitut de Recherche Biomédicale des Armées, INSERM ders. The present study was aimed to characterize the UMR-MD-1197, Clamart, France; cINSERM UMR-MD-1197, Villejuif, France; dInstitut de Recherche Biomédicale des Armées, INSERM UMR- functional role of liver stem cell-derived EVs and spe- MD-1197, CLAMART, France cific miRNAs in the regulation of hepatic stellate cell senescence during alcohol induced liver injury. Introduction: MSC-based cell therapy has received Methods: microRNA expression was assessed using great interest in the past years, especially in regenera- microarray and real-time PCR assays in isolated EVs tive medicine and tissue repair. The concept of priming from human mesenchymal stem cells (MSCs) and liver consists in preconditioning the cells during the culture stem cells (LSCs). HSCs were also isolated from lin28 phase (often with cytokines or hypoxia) to improve knockout mice with or without ethanol feeding for their effects. The literature shows that MSC EVs can 5 weeks by laser capture microdissection (LCM) and recapitulate a substantial part of the beneficial effects of the senescence and fibrosis genes are evaluated by the cells they originate from, and that miRNAs are key Western blot and real-time PCR analysis. players in EVs action. Therefore, in the present work, Results: We found that expression of several miRNAs our aim was to determine if IFN or hypoxia priming of were consistently up-regulated in both MSCs and LSC- MSC could modify their EVs miRNA content. derived EVs compared to normal hepatocyte-derived Methods: Human bone marrow MSC from five healthy EV controls, including let-7 family members. donors were isolated and cultured at 20% of O2 in Treatment of human HSCs with TGF-β/LPS (20 ng/ MEM-alpha/FBS medium until 60–70% confluence, ml) for 72 h induced a significant decrease of let-7a and then with (IFN) or without (CONT) interferon- let-7b in both activated and control states. Transfection gamma (25ng/ml, 48 h) or in hypoxia (3% O2 through- of let-7a and let-7b precursors in human HSCs mark- out the duration of the culture process). Then the cells edly induced the expression of cellular senescence mar- were rinced with PBS and placed in serum free MEM kers p16 and CCl2, and blunted the enhanced for 48 h. The conditioned media was collected and EV expression of α-SMA, collagen a1, MMP-2 and MMP- were isolated by ultracentrifugation (100 000g for 9 (key genes involved in the activation of HHSCs) by 1h10). Total RNA was isolated and reverse transcribed. TGF-β/LPS treatment. Treatment with MSC/LSC Pools of CONT, IFN and HYP cDNA were prepared, derived EVs (30 μg/ml, 72 h) phenocopied the senes- miRNA profiling was performed using Exiqon cence/anti-fibrosis effects of let-7 overexpression in miRnome PCR panel I and II. Then, selected activated HHSCs by TGF-β/LPS. A complementary miRNAs were measured on each sample. mass spectrometry-based proteomics approach with Results: A set of 89 miRNAs was detected (quantifica- luciferase reporter assay identified TLR4, the key LPS tion cycle < 35) in at least one of the pools of MSC receptor, as putative let-7 cluster target. Furthermore, EVs. They were measured on each individual sample. the expressions of senescent hepatic stellate markers 41 miRNAs were measured in all samples; results were JOURNAL OF EXTRACELLULAR VESICLES 123 normalized with 5 endogenous miRNAs. Hypoxia Summary/conclusion: MSC priming can modify the induced no significant modification of EVs miRNA miRNA landscape of their EVs. IFN priming modifies content. IFN priming induced a significant increase in MSCs EVs miRNA involved in biological pathways hsa-miR-106a-5p, 25-3p, 126-3p, 451a and 665. Their relevant to tissue repair. Functional analysis of these validated targets were determined with miRTarBase EVs with selected miRNAs inhibition is needed to and the proteins were analysed with Panther classifica- evaluate the biological effects of such an approach. tion system. Among the most cited pathways, we found Funding: This work has been funded by the french p53, inflammation, Wnt signalling, Apoptosis signal- Direction Générale de l’Armement, Biomedef PDH-1- ling and Angiogenesis. SMO-1–218 124 ISEV2019 ABSTRACT BOOK

Industry Poster Session Thursday 25 April 2019 Location: Level 3, Hall A

IP.01 IP.03

Standardizing F-NTA measurements: evaluation of four-wavelengths Double tangential flow filtration and size exclusion chromatography nanoparticle tracking analysis with cell-line derived EVs for scalable and reproducible EV isolation and size fractionation a b Clemens Helmbrecht and Paolo Guazzi Elina Aleksejevaa, Julia Gavrilovaa, Maija Puhkab, Karina A. Barreiroc, Clemens Helmbrechtd and Paolo Guazzie aParticle Metrix GmbH; bHansaBioMed Life Sciences aHansaBioMed Life Sciences; bInstitute for Molecular Medicine Finland and EV Core, University of Helsinki; cInstitute for Molecular Medicine Finland Introduction: Nanoparticle tracking analysis (NTA) (FIMM), University of Helsinki, Finland; dParticle Metrix; eHansaBioMed has emerged to a vital and fast characterization tech- Life Sciences nology for exosomes, microvesicles or viruses. In combination with fluorescence detection (F-NTA), NTA Introduction: The purification of Extracellular Vesicles enables the user to perform biomarkers detection on (EVs) for industrial processes is still missing of repro- the single particle level, thus enhancing real EV con- ducible, scalable and high throughput method, applic- centration measurement. Classic NTA instruments are able to multiple sources of material (cell conditioned equipped with one laser, requiring phenotyping in media, biofluids, plant extracts). HansaBioMed Life sequence. Multi-fluorescence detection of four biomar- Sciences (HBM-LS) has developed a scalable EV pur- kers in one sample by NTA is shown for the first time. ification process combining two tangential flow filtra- Methods: A four-laser NTA instrument (ZetaView tion steps followed by size exclusion chromatography. PMX-420) equipped with excitation wavelengths of We set a standardized procedure which easily allows 405, 488, 520 and 640 nm and dedicated long-pass the isolation and the collection of big EVs (>200 nm), filters was evaluated. Concentration and particle size the fluid concentration and the removal of small mole- measurements were performed with fluorescent stan- cules (< 500 kDa) with minimal loss of EVs, finally dard beads and proprietary labelled sub-micrometre purified by SEC. The quality of vesicles has been sized vesicles. Phenotyping was performed on EVs assessed in terms of particle size distribution, morphol- from HCT116 cell line (HansaBioMed Life Sciences). ogy, concentration, phenotyping and storage stability. Results: The efficiencies of the individual laser chan- Methods: EVs were isolated from cell conditioned nels were determined by fluorescently labelled vesicles. media combining 2 TFF steps (HBM-TFF: HBM-TFF- SOPs for conjugation of EVs were optimized regarding MV) and SEC (maxiPURE-EVs HBM-LS). EV mor- antibody to vesicle ratio and incubation time. phology and phenotype was analysed by NTA Phenotyping by single and multi-wavelength NTA for Zetaview (Particle Metrix), ExoTEST ELISA (HBM- wash and no-wash strategies were compared regarding LS), and electron microscopy. background and efficiency. Results: Analysing different purifications performed Summary/conclusion: Standardization of SOPs is a combining the double TFF and SEC we defined quality key to improve repeatability for concentration mea- parameters for EVs in term of size distribution, con- surements. Using four wavelengths, phenotyping of centration and maker expression, showing high repro- EVs was performed with four-fold reduction of sample ducibility and EV stability under defined storage amount in shorter time compared to sequential one conditions. laser measurements. NTA delivers total particle count, Summary/conclusion: The combination of two TFF biomarker count and/or vesicle count on one sample steps and SEC allows an efficient fractionation of dif- including size distributions. Cross-validation with ferent EV sizes and works as a scalable and reproduci- complementary techniques such as ELISA and FC/ ble method for EV production from large quantity of IFC becomes imperative. different fluids. JOURNAL OF EXTRACELLULAR VESICLES 125

IP.04 and minimizes samples processing related reproducibility issues for clinical studies.

Development of an automated, high-precision, standardizable IP.05 extracellular vesicle isolation platform for clinical studies Anoop Pala, Shayne Harrela, Robert Vogelb and Murray Broomb aIzon Science US Ltd; bIzon Science Ltd Faster, More Reproducible Exosomes Data – Hands Free! Kohei Shiba, Pauline Carnell-Morris, Matthew McGann and Agnieszha Siupa Introduction: Extracellular Vesicles (EVs) derived from biological fluids possess extensive heterogeneity Malvern Panalytical with regards to size, number, membrane composition Introduction: In analytical data collection, the most and cargo. Tremendous research interest exists towards common form of error is that generated by human development and use of EV fraction of bio-fluids as error. From simple pipetting to manually adjusting rich sources of diagnostic and prognostic biomarkers. optical settings on an instrument all these sources of High precision fractionation of the nanobiological con- error result in data sets that are less reproducible and tent of biofluids can dramatically reduce background, increasingly difficult to interpret. The introduction of increase purity and inform on the biology of the bio- the NanoSight Sample Assistant for the NS300 brings markers and therapeutic biomolecules. about a new level of repeatability and reproducibility in Methods: Size exclusion chromatography (SEC) is the analysis of Extracellular Vesicle (EV) samples. This most standardizable technique, already widely used for work will examine the benefits of using the sample the purification of EVs from biofluids. Significant assistant for sample handling including time saving, improvement to the use of SEC is possible through and improved data quality. automation and precision. Here, we developed a Methods: The particle size distribution and concentra- range of SEC columns of various sizes, with 2 resin tion of exosome samples isolated from urine (20 x types, separating down to 35 nm or 70 nm. We also 1 mL) and SKOV3 cells (96 x 1 mL) was determined developed a low-cost prototype automatic fraction col- using the NanoSight NS300 system (Malvern lector (AFC) that adds high precision, improves repeat- Panalytical, UK) integrated with the NanoSight ability, speeds up workflow. RFID tags are proposed to Sample Assistant (1mL). All samples were analysed ensure high quality of data capture and transfer. under the same capture and process settings and the Moreover, Tunable Resistive Pulse Sensing technology total time of analysis recorded. A series of experiments was used for accurate, high-resolution particle analysis were also completed using SKOV3 samples, acquired (size, size range, concentration, and electrophoretic manually on the NanoSight NS300 system to compare mobility) and normalization. repeatability, reproducibility of data to that acquired by Results: SEC columns provide a convenient, reprodu- the sample assistant. cible and highly effective means of eliminating >99% of Results: Analysis of the data shows that data acquisi- non-vesicular protein from biological fluid samples, tion of 96 EV samples can be completed in approxi- and separating exosomal and non-exosomal volumes mately 15 h using the Sample Assistant, a 70% for further downstream analysis. 35 nm pore sized SEC improvement compared to an estimated 50 h of man- gel leads to increased resolution, higher yield and one ual acquisition. Setup time of the instrument however fraction earlier elution of EVs from plasma compared was approximately 30 min, reducing hands on instru- to the 70 nm pore size. Use of AFC allowed precise ment time by ~99%. An additional dataset of EV sam- mass-based measurements and tunability within 30 ul ples was measured as a dilution series, both manually of volume exiting the column. and using the Sample Assistant. Data showed a mea- Most importantly, due to the additional functionality surable improvement in both repeatability of the con- provided by AFC, the EV field needs to revisit the way centration as well as linearity of the series. fraction numbers, post-SEC are used. That will be Summary/conclusion: The new NanoSight sample replaced with a more logical framework, wherein the assistant accessory for NS300 provides size and con- void volume is measured and disposed of, and precise centration data measurements of up to 96 samples in as volumes are used instead of the somewhat arbitrary little as 15 h, including under 30 min of set-up time. fraction numbers. Data quality is typically improved by the elimination of Summary/conclusion: Thus, the qEV-AFC platform user error and subjectivity. The Sample Assistant is allows for QA, high-precision EV volume collection compatible with many sample types, and generates 126 ISEV2019 ABSTRACT BOOK key exosome characterization data, whilst freeing up IP.07 valuable scientist time to work on other tasks. Funding: This project received funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement No 646,002 Microfluidic Resistive Pulse Sensing (MRPS) Measurements of EVs and EV Standards Franklin Monzona, Jean-Luc Fraikinb, Ngoc Doa, Tom Maslanikc, Erika Duggand and John Noland IP.06 aSpectradyne; bSpectradyne LLC; cCellarcus Biosciences Inc; dScintillon Institute

Introduction: As science-based on EVs advances, it is Identifying, characterizing and quantifying extracellular vesicles important to be able to compare measurements of using multispectral imaging flow cytometry Haley R. Pugsley, Sherree Friend, Bryan Davidson and Phil Morrissey vesicles across different manufacturing sites and man-

Amnis part of Merck KGaA ufacturing methods. To isolate differences or drifts in EV formulations, it is necessary to have stable metrol- Introduction: Extracellular vesicles (EV) are a hetero- ogy so that these differences can be properly attributed geneous group of membrane derived structures that to changes in the formulation and not the metrology. include exosomes, microvesicles and apoptotic bodies. Establishing stable metrology in turn relies on the Quantifying and characterizing EVs in a reproducible development of standards measured by multiple ortho- and reliable manner has been difficult due to their gonal methods. With this goal in mind, this paper small size (down to 30 nm in diameter). Attempts to discusses measurements of EVs and EV standards analyse EVs using traditional PMT based flow cyt- using Microfluidic Resistive Pulse Sensing (MRPS) ometers has been hampered by the limit of detection and other measurement techniques. of such small particles, their low refractive index and Methods: The size distribution and concentration of the swarming effect. To overcome these limitations, we EV standards and EVs derived from various sources have employed multispectral imaging flow cytometry were characterized by MRPS, Nanoparticle Tracking that has the advantage of high throughput flow cyto- Analysis (NTA), cryo-Electron Microscopy (EM), and metry with higher sensitivity to small particles due to Vesicle Flow Cytometry (VFC). In some cases, EVs the CCD based, time-delay-integration image capturing were destroyed by lysing agents and measurements system. Several recent publications have reported using were repeated to demonstrate this effect. multispectral imaging flow cytometry to identify and Results: MRPS measurements gave high resolution size characterize EVs; however, the collection settings and and concentration information down to ~ 50 nm dia- gating strategies used to identify and characterize EVs meter for all samples. Because MRPS is an electrical is not consistent between publications. technology, it did not suffer from sensitivity limitations Methods: Here we demonstrate the optimal collection related to the low index of refraction contrast between settings, parameters and gating strategy to identify, the nanoparticles (be they EVs or standards) and the characterize and quantify a variety of EVs using multi- surrounding liquid. MRPS could not distinguish parti- spectral imaging flow cytometry. EVs obtained from cles based on type (in contrast to VFC), however it was commercial sources are identified using a combination more sensitive to the presence of non-EV nanoparticles of CD markers, membrane stain and 405 nm SSC. In in the samples. Concentration reproducibility was in each case, the membrane stain and 405 nm SSC initi- the range of ± 20% and sizing reproducibility in the ally identify an EV and CD markers are used for range of ± 5% independent of particle material. characterization and immunophenotyping the EV. Summary/conclusion: Quantifying the purity of an EV Results: Data will be presented using the ImageStream population is important. Techniques such as VFC do multispectral imaging flow cytometer to identify, char- an excellent job in quantifying the EV population of acterize and quantify a variety of EV samples. interest but are not necessarily sensitive to contamina- Strategies for optimal collection and analysis of the tion or the presence of non-target EVs. MRPS, on the multispectral imaging flow cytometry EV data will other hand, gives high resolution information on all also be discussed. nanoparticles present in a mixture. From a process Summary/conclusion: Multispectral imaging flow development standpoint, this information is critical to cytometry is able to characterize and quantify EVs the improvement of a formulation. The orthogonal with very high sensitivity due to the CCD based time- nature of MRPS measurements, compared to optical delay-integration image capturing system. methods, is therefore an important part of the JOURNAL OF EXTRACELLULAR VESICLES 127 development of robust EV standards, and the asso- yield by immune-isolation approaches and facilitate ciated measurement protocols, that will be required the analysis of enriched EV subpopulations. for the successful wide deployment of EV-based diag- Funding: The project is funded under the Marie nostics and therapeutics. Skłodowska-Curie grant agreement No. 765,492 “ELBA – European Liquid Biopsies Academy” and internal Exosomics R&D Funds. IP.08

IP.09

Development of EV-targeting tools for novel liquid biopsy approaches in Lung Cancer Diogo Fortunatoa, Laura Bianciardib, Gaia Papinic, Mattia Criscuolic, Davide Zoccod and Nataša Zarovnib Side scatter module for enhanced detection of Extracellular Vesicles aExosomics/University of Siena; bExosomics; cExosomics Siena, University of by flow cytometry. Siena; dExosomics Siena Tina Van Den Broecka, Ludovic Monheimb, Ihor Berezhnyya, Oleg Guryeva, Tatyana Chernenkoa, Marybeth Sharkeya and Geoffrey Osbornea Introduction: Mounting clinical evidence suggests that aBD Biosciences; bBD Life Sciences, Erembodegem, Belgium liquid biopsy may revolutionize the way cancer patients are currently managed. Within this context, our study Introduction: EVs are nanosized (20 ~ 5000 nm) aims to assess and reinforce unique and complemen- membrane vesicles released from cells that can trans- tary advantages of EV/exosome-based approaches, port cargo – including miRNA and proteins – between through identification and quantitative detection of cells as a powerful way of intercellular communication. non-small cell lung cancer (NSCLC) EV biomarkers. Currently, flow cytometry is the only high throughput Current technology and methods for exosome isolation technique capable of single particle cell surface pheno- from complex biological samples (i.e. plasma), have typing and sorting with the possibility of concentration shown to be unreliable. There is a need to substantially determination. Unfortunately, the drawback of stan- improve them to enable multiparameter EV analysis. dard flow cytometry is lack of sensitivity to detect Therefore, in addition to EV-biomarker discovery, we smallest particles, especially for those with a size less are testing plasma processing and preanalytical tools, than or equal to the dimensions of the excitation laser devices and optimized immunoaffinity protocols that wavelength. tackle fundamental obstacles, such as complex matrix Methods: BD has developed an accessory side scatter effects. Our goal is to provide an EV immunocapture (SSC) module for enhanced scatter detection of small approach with enough sensitivity, specificity and particles by flow cytometry: the SP SSC module. The robustness for clinical grade diagnostic applications. SP SSC module should be used in combination with a Methods: Size-based vs. immunocapture methods for laser power of at least 100 mW. Small particle detection exosome isolation. Enzymatic and immunological enhancement is achieved by significantly increasing the assays for plasma pre-clearing; Flow cytometry, signal-to-noise ratio of the SSC. ELISA, nanoparticle tracking analysis, Western Blot, Results: The SP SSC module can be installed on most SPR and ddPCR for antibody and exosome commercially available BD flow cytometers, which characterization. have sufficient laser power, as an additional option. Results: Exosomes derived from NSCLC cell lines dis- The normal SSC detector remains in place and the SP play distinct membrane markers recognized by a panel SSC module has minimal impact on regular SSC and of proprietary Abs, screened by flow cytometry, SPR, fluorescent performance therefore use of the system for IP, ELISA and PCR. We developed and tested a screen- cell analysis applications is still possible. Initial results ing platform based on endogenously labelled EVs to using the SP SSC module were obtained using a BD ™ ™ identify NSCLC EV antigens. Selected antibodies will FACSCelesta SORP and a BD FACSAria Fusion, be used to develop an immune-isolation protocol, respectively having a 100 and 200 mW 488 laser. coupled to state-of-the-art analytics for a rapid and Side-by-side comparison of the regular SSC detection sensitive readout, thus enabling a comparative evalua- vs. SP SSC detection was done using polystyrene beads, tion of a repertoire of plasma pre-analytical protocols. silica beads, EV reference material and antibody- Summary/conclusion: Different plasma pre-analytical stained EV material. protocols are ranked and orthogonally combined to Summary/conclusion: Utilization of the SP SSC mod- optimally counteract matrix effects, increment EV ule for sorting of natural (plasma EVs) and artificial 128 ISEV2019 ABSTRACT BOOK

(liposomes) membrane particles is currently being IP.11 undertaken.

Benchmarking of established exosome isolation methods (density IP.10 gradient centrifugation, size-exclusion chromatography and immune- bead separation) with glycan recognizing EXÖBead Dapi Meng Lin. Chianga, Chin-Sheng Linb and Michael Pfafflc

aBiovesicle; bDivision of Cardiology, Tri-Service General Hospital, Taiwan & National Defense Medical Center, Taiwan; cAnimal Physiology and Quantitative imaging and phenotyping of EVs with 20 nm resolution Immunology, School of Life Sciences Weihenstephan, Technical University Andras Miklosi, Zehra Nizami, Blanka Kellermayer and Mariya Georgieva of Munich, Freising, Germany

ONI (Oxford Nanoimaging ltd) Introduction: Exosomes are small vesicles (30– Introduction: Complex extracellular vesicle (EV) phe- 150 nm) found in various human biofluids, such as notyping is a major technical challenge that hinders plasma. OptiPrep density gradient centrifugation clinical translation. Single-molecule localization micro- (DGC) is widely accepted as a pure exosome isolation scopy (SMLM) is a Nobel-Prize winning technique that method. Size-exclusion chromatography (SEC) is a fast allows quantitative imaging below the diffraction limit exosome isolation method, but exhibit contaminations necessitating only simple and fast sample preparation. such as lipoprotein or aggregated proteins. Immuno- The data presented here constitutes one of the first beads (HBM) are based on high specific recognition of accounts of single-molecule imaging used to success- exosome CDs, but uses a harsh elution procedure to get fully resolve the structure, protein (CD9, CD63, and intact exosome. EXÖBead (Biovesicle) are glycan CD81) and nucleic acid content of EVs with 20 nm recognition magnetic beads and show high exosome resolution. specificity by FACS, NTA and TEM analysis. In this Methods: EV isolation was performed from keratino- study, we compared these four isolation methods based cyte culture media. EV suspensions were stained using on FACS established exosomal markers, intact exo- fluorescently labelled primary antibodies raised against some size/number and lipoprotein contamination. known exosome markers, and commercially available Methods: Mix plasma samples were collected from membrane and nucleic acid labels. Characterization of healthy donors (n = 5) and patients undergoing cor- the molecular content and structural properties of sur- onary angiography (n = 6). Exosomes were isolated face-immobilized EVs was performed using the SMLM from 250 μl plasma by SEC and DGC, fractions were mode of the ONI Nanoimager. Sizing of EVs in solu- collect from SEC (7 ~ 10) or DGC (6 ~ 8), and then tion was performed using the dual-colour single-parti- covalent-coated on 1 μm magnetic beads (followed cle tracking mode of the ONI Nanoimager. Chemicell). We also covalent-coated 1 ml 10% exo- Results: Multicolour super-resolution microscopy ima- some free (EF) FBS in PBS as a negative control. We ging of purified EVs revealed the phenotypic and struc- directly incubated 250 μl plasma with 1 μm glycan tural properties of hundreds of individual vesicles at a recognition magnetic beads EXÖBead (37°C, 1 h) or time. The membrane staining allowed to visualize EVs 1 μm latex HBM immunobeads (4°C, 16h). As a nega- with sizes ranging from ~20 nm to >250 nm, and sizing tive control 1 ml (EF) FBS was incubated. Universal by tracking confirmed this distribution and a mean size antibody mix (PE-Cy7-CD63, FITC-CD81 and APC- of 120 nm. For EVs of >40 nm the membrane appeared CD9) was used for all isolation methods. The negative as a ring and was a confirmation of their intact struc- control reduced fluorescence data are presented by ture. CD63, CD9 and CD81 co-localized with the median fluorescence intensity (MFI). NTA data were membrane staining at the nm scale, thus allowing to collected only from intact exosomes. determine the molecular ID of EV subpopulations and Results: EXÖBead represents highest MFI of CD63 correlate the protein marker levels with the size of EVs. (247.9) compared to SEC (232.42), DGC (25.72) and Summary/conclusion: The quantitative nature of sin- HBM (5.13). EXÖBead also showed highest MFI of gle-molecule imaging and tracking significantly CD9 (475.4) compared to SEC (42.3), DGC (5.1) and improves EV characterization. This work provides evi- HBM (0). Only SEC (88.9) and EXÖBead (41.1) could dence of the use of SMLM imaging as a novel and detect CD81. Experiment processing time for EXÖBead powerful tool for rapid and multiplexed EV character- is 2h, SEC is 4h, HBM is 19h, and DGC even 22h. SEC ization with unique combination of structural and represents highest intac phenotypic insight. t exosomes/ml (4.9E+10), EXÖBead (1.7E+9), HBM (1.9E+8), and DGC (1.5E+8), measured by NTA. JOURNAL OF EXTRACELLULAR VESICLES 129

Median exosome sizes are EXÖBead 72.0 nm, SEC cross the blood brain barrier, and to further explicate 107.0 nm, DGC 89.6 nm and HBM 96.1 nm. these findings, we investigated the uptake of ExoPr0 at Summary/Conclusion: EXÖBead serves as a new time- the cellular level using live-cell imaging techniques. saving plasma isolation method with high exosome Methods: We employed live-cell confocal microscopy yield and specificity. to directly visualize uptake of fluorescently labelled exosomes. A quantitative image analysis protocol was developed and applied to assess the uptake of exosomes IP.12 in a number of cell types. Results: Time course incubations of cells treated with ExoPr0 produced data that revealed heterogeneity in uptake between cell types. ExoPr0 was compared to Characterizing the cellular uptake of neural stem-cell derived exosomes derived from a control producer cell line, exosomes using live-cell imaging techniques highlighting source-specific differences in uptake Samuel Jonesa, Thomas Cawsb, Anthony Hayesa, Victoria Marsh Durbanb, Randolph Cortelingb and Peter Watsona kinetics. Uptake was observed to occur through more aSchool of Biosciences, Sir Martin Evans Building, Cardiff University, than one pathway resulting in trafficking through Museum Avenue, Cardiff, Wales, UK; bReNeuron Limited, Pencoed endo-lysosomal compartments. The effect of cell cycle Business Park, Pencoed, Bridgend, Wales, UK on the uptake of ExoPr0 was investigated, but was not observed as having a significant influence. Introduction: Neural stem cell derived exosomes Summary/conclusion: Findings from this study have (“ExoPr0”); purified from the conditioned medium of eluded to the specificity of ExoPr0 towards different a GMP manufactured, conditionally-immortalized cell types and work is ongoing to further elucidate the human neural stem cell line (“CTX0E03”), demon- delivery mechanism of ExoPr0 and understand the strates a unique biodistribution profile in mice com- subcellular trafficking in recipient cells. pared to exosomes derived from a control producer cell line. We have previously shown that ExoPr0 is able to 130 ISEV2019 ABSTRACT BOOK

Symposium Session 7: Advances in EV Isolation in Cancer Chairs: Leonora Balaj; Johan Skog Location: Level B1, Hall A 17:00–18:00

5 7 OT07.01 is estimated to be 4.07 ×10 –1.83 ×10 exosomes/μL with a detection of limit (LOD) of 3.43 ×105 exosomes/µL. We further successfully applied it for exo- Aggregation-induced emission probe/graphene oxide aptasensor for somes quantification in plasma samples from prostate label-free and “turn-on” fluorescent aptasensor for cancerous cancer patients. exosomes Bo Li, Weilun Pan, Chunchen Liu and Lei Zheng Summary/Conclusion: This aptasensor is expected to Clinical Laboratory Department, Nanfang Hospital, Southern Medical become a powerful tool for rapid and simple cancer University, Guangzhou, China (People’s Republic) liquid biopsy. Funding:Thisstudywasfinancedbygrantsfromthe Introduction: Exosomes are the smallest subset (30– National Natural Science Foundation of China 150 nm) of extracellular vesicles (EVs), a heteroge- (81371901, 81702100), the Science and Technology neous population of vesicles originate from all types Planning Project of Guangdong Province of tissue cells, which can freely pass through the blood (2017A020215123). vessel wall and distribute in various body fluids. Exosomes carry different macromolecules, such as nucleic acids, proteins and lipids for intercellular communication. In the last decade, numerous researches OT07.02 demonstrated that exosomes’ cargo is affected in the progression of malignant tumours, positioning exo- Single extracellular vesicle (EV) profiling and EV subpopulation somes as potential sources for the discovery of novel analysis of cancer related EVs in human plasma biomarkers. For example, it is confirmed that PSMA is Yanling Caia, Zesong Lia and Di Wub enriched in the membrane of exosomes from prostate aShenzhen Second People’s Hospital, First affiliated hospital of Shenzhen cancer cells. So, PSMA positive exosomes subpopula- University, Shenzhen, China (People’s Republic); bDepartment of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm tion is regarded as the diagnostic biomarker for pros- University, Solna, Sweden., Solna, Sweden tate cancer. But conventional methods can hardly quantify low-concentration PSMA positive exosomes Introduction: Extracellular vesicles (EV) carry impor- subpopulation in small volumes of clinical samples tant information of their parental cells, and are there- rapidly. fore promising biomarkers for liquid biopsy and early Methods: In this work, we constructed the label-free diagnosis of multiple diseases including cancer. and “turn-on” aptasensor for the detection of the However, the detection of disease specific EV among PSMA positive prostate cancer exosome based on huge numbers of EVs in the clinical sample, e.g. plasma PSMA aptamer as the recognition element, remains a challenge, which makes single EV and EV Aggregation-Induced Emission (AIE) probes: TTAPE subpopulation analysis preferable to bulk analysis. as fluorescent indicators and Graphene Oxide (GO) as Methods: In the presented work, in order to recognize fluorescent quencher. In the absence of PSMA positive the cancer cell line specific EVs, we utilized a proximity exosomes, the fluorescence of TTAPE aggregated in the barcoding assay (PBA) to analyse the surface protein aptamer would be quenched efficiently by GO. composition of single EVs and investigated the EV However, in the presence of PSMA positive exosomes, subpopulation. A pool of hundred-plex oligonucleo- the specific and stronger binding between aptamers tide-conjugated antibodies against reported cancer bio- and PSMA positive exosomes could weaken the bind- markers candidates was employed to recognize the ing interaction between aptamer and GO. So the fluor- surface proteins of individual EVs. Then all the oligo- escence of TTAPE aggregated in the aptamer would nucleotides on the same EV obtained an unique EV tag recover, which could appear “turn-on” fluorescent in a PBA. The pool of extension products can be property. amplified and sequenced by next generation sequen- Results: Under optimal conditions (37°C, 15 min), the cing. After sorting the reads, we could reconstruct the linear range of detection for prostate cancer exosomes surface protein composition of individual EVs. JOURNAL OF EXTRACELLULAR VESICLES 131

Results: We applied PBA to analysed EVs purified donor plasma exosomes. Ingenuity Pathway Analysis from cancer cell lines and from human plasma. We showed that these differentially expressed miRNAs tar- could identify different subpopulation EVs, that are get mRNAs that are associated with distinctive GBM specific for certain cell lines and human plasma. We and cancer pathways. In order to test the diagnostic then spiked in different amount cancer cell-line derived accuracy of the proposed technique, ROC analysis was exosomes in the plasma derived EVs from healthy performed based on the top 33 differentially expressed donors in different ratio. We could observe en expected miRNA samples. The area under the ROC curve (AUC; increase of certain population of exosomes in the a figure of merit to determine the optimal miRNA human plasma. signature) was 0.968. In addition, multiple novel Summary/Conclusion: In summary, PBA is a multi- miRNAs and other short non-coding RNA species plexed and high throughput approach to analyse sur- (Y-RNA, piRNA, snoRNA) were found with some dif- face proteins of individual EVs. The cancer cell line ferential expression. EVs mixed into healthy control plasma were success- Summary/Conclusion: In conclusion, miRNA sequen- fully detected, indicating this technique can be applied cing from plasma exosomes shows marked differential to search for rare population of EVs in the plasma miRNA expression between healthy donors and GBM samples of patients. patients. These findings as well as additional differen- Funding: National Natural Science Foundation of tially expressed short non-coding RNA species suggest China, project 81802052 plasma EVs may serve as a robust platform to develop GBM liquid biopsies. OT07.03 Funding: Mayo Clinic Center for Individualized Medicine (CIM) Brains Together For a Cure miRNA signature derived from GBM plasma exosomes as a diagnostic biomarker Luz M. Cumba Garciaa, Pritha Chananab and Ian Parneyc OT07.05 aMayo Clinic Graduate School of Biomedical Sciences, Department of Immunology, Rochester, USA; bMayo Clinic, Department of Health Sciences Research- Division of Biomedical Statistics and Informatics, Isolation of extracellular vesicles by nanoDLD lab-on-a-chip Rochester, USA; cMayo Clinic, Department of Neurologic Surgery, technology for clinical applications Department of Immunology, Rochester, USA Stacey M. Gifforda, Joshua Smitha, Benjamin Wunscha, Navneet Dograa, Mehmet Ahsenb, Kamlesh Yadavc, Ashutosh Tewarid, Carlos Cordon- Introduction: Gliomas including glioblastoma (GBM) Cardoe and Gustavo Stolovitzkya are the most common malignant brain tumours. aIBM T.J. Watson Researc Center, Yorktown Heights, NY, USA; bDepartment Glioma extracellular vesicles (EVs), especially plasma of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY, USA; cDepartment of Urology, Icahn School of Medicine at exosomes, have biological effects such as mediating Mount Sinai, New York, NY, USA; dDepartment of Urology, Icahn School of immunosuppression and contain signature tumour- Medicine at Mount Sinai, New York, NY, USA; eDepartment of Oncology Sciences and Pathology, Icahn School of Medicine at Mount Sinai, New York, specific cargo that could serve as liquid biopsies. NY, USA Increasing interest in molecular biomarkers to determine patient prognosis in GBM has suggested that EV Introduction: There is great interest in exosome isola- miRNA-based signatures may be able to predict pro- tion and analysis to develop non-invasive “liquid biop- gression-free and overall survival, differentiate normal sies” for diagnosis, prognosis, and surveillance of donors from GBM patients, and distinguish true pro- diseases. However, current exosome isolation methods gression from treatment-related pseudo-progression. lack purity, yield and reproducibility and the inability Methods: We have established a simple technique, to rapidly and reliably separate exosomes hinders clin- using density gradient ultracentrifugation, to isolate ical application. Thus, there is an urgent need to plasma exosomes from glioma patients and normal develop novel tools to isolate exosomes as a promising donors. Purification of total RNA, including miRNA, source of new biomarkers. was performed on plasma exosomes from normal Methods:Wehavedevelopedalab-on-a-chiptech- donors (n = 8) and GBM patients (n = 7) using the nology based on deterministic lateral displacement at miRNeasy kit (Qiagen). Next generation short non- the nanoscale (nanoDLD) which separates and con- coding RNA sequencing was performed by Illumina centrates particles in continuous flow and in specific HiSeq 4000. size ranges, going to scales as small as 20 nm. We used Results: RNA sequencing revealed many differentially nanoDLD to isolate EVs from urine and serum and expressed miRNAs in GBM patients with high fold characterized these EVs by NTA and RNA change/low false discovery rates compared to normal sequencing. 132 ISEV2019 ABSTRACT BOOK

Results:BenchmarkingstudiesofnanoDLDisola- improved yield, concentration and processing time tion of exosomes show comparable or improved compared to existing isolation methods. This technol- yield and concentration compared to standard tech- ogy has enabled high-resolution temporal studies of niques such as SEC and UC at volumes suitable for urinary EVs to better understand the impact of preclinical applications. We isolated EVs from the analytical challenges on EV studies. Finally we used urine and serum of prostate cancer (PCa) patients. nanoDLD to isolate EVs from prostate cancer patient Our preliminary data show PCa patient serum exo- samples and detect an enrichment of known mRNA somes are enriched in known PCa biomarkers. prostate cancer markers in serum EVs. Our nanoDLD Screening for an EV RNA panel associated with technology enables frequent, rapid isolation of EVs at aggressiveness could aid detection of clinically sig- improved yield and concentration enabling the use of nificant PCa and reduce unnecessary radical smaller sample volumes. prostatectomies. Funding: Work was funded by IBM Research and the Summary/Conclusion: We have developed a chip- Icahn School of Medicine at Mount Sinai. based tool for EV separation and demonstrated JOURNAL OF EXTRACELLULAR VESICLES 133

Symposium Session 8: Mechanisms of Delivery Chairs: Lorraine O’Driscoll; Carlos Salomon Location: Level 3, Hall B 17:00–18:00

OT08.01 unclear or as a novel cell function control technique using exosome.

Magnetically navigated intracellular delivery of extracellular vesicles using nanogels OT08.02 Yoshihiro Sasaki, Ryosuke Mizuta and Kazunari Akiyoshi

Kyoto University, Kyoto, Japan Tissue distribution of extracellular vesicle-binding proteins after in vivo gene transfer into mice Introduction: Extracellular vesicles can control impor- Yoshihiko Shimazawaa, Kosuke Kusamorib, Yuki Takahashic, Yoshinobu c b tant biological phenomena such as cell differentiation Takakura and Makiya Nishikawa and cell death. In addition, extracellular vesicle is also aFaculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Japan; bTokyo University of Science, Noda, Japan; cKyoto University, regarded as a promising material for biomedical appli- Kyoto, Japan cation. However, due to their low efficiency of intracellular uptake, development of effective intracellular Introduction: Successful application of extracellular delivery method has been remained challenging issue. vesicles (EVs) as delivery systems for bioactive mole- We report here the complexation of extracellular vesicules, including miRNAs and tumour antigens, cles and magneto-responsive nanogels, and efficient requires the understanding and control of their tissue intracellular delivery of extracellular vesicles into cells distribution. Our previous studies demonstrated that by magnetic guidance for induction of differentiation the exogenously administered EVs of about 100 nm in of stem cells by delivered extracellular vesicles. diameter quickly disappeared from the systemic circu- Methods: Magnetic nanogels were prepared by mixing lation after intravenous injection into mice. Despite oleic acid-coated iron oxide nanoparticles dispersed in these results, endogenous EVs might have different an organic solvent to nanogels composed of cholesteryl tissue distribution properties from exogenously admi- group-substituted pullulan. Magnetic nanogel-exosome nistered ones. To test this hypothesis, it is important to complexes were prepared by isolating exosomes from develop a method to analyse the properties of endo- culture supernatants of myoblasts and nerve cells by genous EVs. In this study, as a first step, we selected ultracentrifugation and mixing this exosome with mag- Gaussia luciferase (gLuc) and lactadherin (LA) as a netic nanogels. The resulting magnetic nanogel-exo- reporter protein and an EV-binding protein, respec- some complex was delivered to the cells by magnetic tively, and examined whether the fusion of LA to gLuc induction and its intracellular dynamics were investi- could alter the tissue distribution of gLuc after in vivo gated using a confocal laser microscope and flow gene transfer into mice. cytometry. Methods: pcDNA3.1 plasmid vectors encoding gLuc, a Results: In 24 h, 90% of exosome could be complexed fusion protein of gLuc and LA (gLuc-LA), or a fusion with magnetic nanogel. The obtained magnetic nano- protein of gLuc and a mutated LA which has low gel-exosome complex was delivered to adipose-derived affinity to EVs (muLA) were constructed (pCMV/ mesenchymal stem cells (ADSC) by magnetic induc- gLuc, pCMV/gLuc-LA and pCMV/gLuc-muLA). Each tion. As a result, the introduction of magnetic nanogel plasmid was injected into 4-week-old male ddY mice and exosome into the cytoplasm was confirmed. From using the hydrodynamic injection method, and blood the results of immunostaining, expression of the differ- was collected at several time points to obtain plasma. entiation marker was confirmed in which the complex Then, EVs in plasma were separated and collected by was introduced to ADSC by magnetic induction for the ultracentrifugation method. The characteristics of both myoblasts and nerve cells. the EVs were evaluated by western blotting and Summary/Conclusion: Differentiation was induced to dynamic light scattering. The luciferase activity of the ADSC by efficient magnetic delivery of exosome. This plasma and the EVs was measured in a luminometer. magnetic nanogel introduction method is expected to Results: In all the cases examined, the luciferase activ- be used as analysis of exosomes whose function is ity in the plasma was very high soon after 134 ISEV2019 ABSTRACT BOOK hydrodynamic injection of the plasmid vectors, then it Results: Data revealed an homogeneous exosome- decreased with time. No significant luciferase activity enriched sample in terms of exosome-like morphology was detected in the EVs when pCMV/gLuc or pCMV/ and size. Exosome-sperm binding to the head, mid-piece gLuc-muLA was injected. By contrast, about 5% of and tail was confirmed with up to two exosomes/sperm luciferase activity of the plasma was recovered in the cell. No statistically significant differences were found in EV fraction when mice received an injection of pCMV/ terms of viability, MMP and MF for any of the tested gLuc-LA. ratios at each time point, compared to controls. Summary/Conclusion: These results indicate that Summary/Conclusion: HEK293T cell-derived exo- gLuc-LA binds to EVs in mouse blood through LA somes bound to all sperm parts soon after the incuba- after in vivo gene transfer, which suggests that gLuc- tion began. A high exosome concentration did not LA can be used to analyse the tissue distribution of compromise the viability nor the response of boar endogenous EVs. spermatozoa to induced capacitation and acrosome- exocytosis in vitro. In conclusion, HEK293T cell-exosomes have shown to have potential as a future clinical OT08.03 delivery system in the context of male infertility. Funding: SRF and St. Peter’s College (University of Oxford). Capabilities of HEK293T cell-exosomes as a non-invasive delivery tool for mammalian sperm Teresa Vilanovaa, Celine Jonesa, Rebecca Dragovica, Kevin Cowarda and Marc Yestea OT08.04 aUniversity of Oxford, Oxford, UK; bUniversidad de Gerona, Girona, Spain

Extracellular vesicles from de-differentiated human adipose tissue Introduction: Male infertility accounts for 35–50% of endothelial cells have potential to disseminate angiostatic signals in human infertility, and 2.5–12% of men present some human obesity Anca D. Dobriana, Bronson Haynes, Ryan Huyck, Lifang Yang, Vanessa form of infertility. Several non-invasive approaches to Correll, William McPheat and O. John Semmesb treat sperm-borne aberrations are being developed aEastern Virginia Medical School, Norfolk, USA; bLeroy T. Canoles Jr. Cancer such as exosomes for compound delivery. Human Research Center, Eastern Virginia Medical School, Norfolk, USA Embryonic Kidney (HEK)293T cell-exosomes appear to be safe and versatile in terms of their targeting Introduction: Endothelial-to-mesenchymal transition abilities. However, the safety aspects for gametes need (EndoMT) characterized by endothelial cell (EC) de- to be investigated. In this study we developed differentiation into a mesenchymal phenotype is a focal HEK293T cell-exosomes for in vitro co-incubation event present in the vasculature of obese adipose tissue with boar sperm. Exosome binding and exposure (AT) and has been shown to contribute to various effects (for viability, mitochondrial membrane poten- vascular pathologies. EC from human AT impacted tial (MMP) and membrane fluidity (MF)) were by EndoMT are angiostatic and have a quiescent meta- examined. bolic phenotype. We hypothesize that extracellular Methods: HEK293T-exosomes were characterised by vesicles (EV) produced by such EC may lead to propa- Nanoparticle Tracking Analysis, Western Blotting and gation of angiostatic signals which may contribute to Transmission Electron Microscopy. Boar sperm sam- hypoxia and insulin resistance in obese AT. ples (n = 3) were in vitro co-incubated at an exosome: Methods: We modelled EndoMT in vitro by treatment sperm ratio of 10:1 (4h pH<7). Sperm aliquots at 0, 2 of human AT ECs with pro-inflammatory cytokines and 4h post-incubation were analysed for exosome and prepared EV from conditioned media by ultracen- binding. Moreover, boar sperm (n = 5) was in vitro trifugation. Uptake of EVs by naïve EC was measured co-incubated at different ratios (1:1, 10:1 and 100:1) by flow cytometry; angiogenesis by in vitro tube for- under capacitating and progesterone-induced hyper- mation; and mitochondrial energetics with Seahorse activating conditions. Analysis at 0h, 2h, 4h, 4h 10 bioanalyzer. The miRNA cargo of the EVs was analysed min, 4h 30 min and 5h post-incubation by flow cyto- using the Nanostring platform and the proteome was metry for viability, MMP and MF of exosome-treated determined using LC/MS/MS. samples was performed by staining with SYBR-14/PI, Results: EV from EndoMT cells produced a dramatic JC-1 and YO-PRO-1/Merocyanine-540, respectively. angiostatic effect on recipient EC without affecting Data were analysed with a mixed model (between-sub- migration or proliferation. Recipient EC became quies- jects factor: treatment; within-subjects factor: incuba- cent and had lower ATP production compared to con- tion time) followed by the post-HOC Sidak test. trols. Pathway analysis of EV cargo showed significant JOURNAL OF EXTRACELLULAR VESICLES 135 targeting of fatty acid synthesis and oxidation in reci- their angiostatic effect. miR-155 delivered via EV may pient EC. We found abundant miR-155-3p in EV and be key for metabolic quiescence via inhibition of CPT1 reduced expression of its metabolic enzyme targets and ACLY. We report a novel mechanism by which CPT1a and ACLY in recipient EC. Treatment of EC EndoMT in EC produces EVs that may propagate with the CPT1a inhibitor etomoxir recapitulated the angiostatic effects throughout the AT vasculature in angiostatic effect of the EVs. The EV proteome was obesity. also enriched in peptide signatures for VEGFR1, Funding: NIHR15NHLBI, American Heart Association- VEGFR2 and neuropilin. AIREA. Summary/Conclusion: We show that the metabolic shift produced by EV from EndoMT cells may explain 136 ISEV2019 ABSTRACT BOOK

Symposium Session 9: EV Biogenesis II Chairs: Bong Hwan Sung; Graca Raposo Location: Level B1, Hall B 17:00–18:00

OT09.01 Funding: This work was funded by Cancer Research UK [C19591/A19076], the CRUK Oxford Centre Development Fund [C38302/A12278], BBSRC [BB/

Different exosome subtypes have distinct ESCRT-associated biology K017462/1, BB/N016300/1, BB/R004862/1], John Fell and control tumour cell adaptation in vivo Fund, Oxford, Wellcome Trust [MICRON; #091911, Shih-Jung Fana, Benjamin Kroegerb, Pauline Mariea, Esther Bridgesa, Kristie a a a a #107457], Royal College of Surgeons. McCormick , John Mason , Helen Sheldon , Claudia Mendes , Mark Wainwrighta, John Morrisa, Adrian Harrisa, Clive Wilsona and Deborah C I. Goberdhana OT09.02 aUniversity of Oxford, Oxford, UK; bPeter MacCallum Cancer Centre, Melbourne, Australia

Emerging role of L-type calcium channel-mediated calcium influx in Introduction: Determining the function of specific regulating apoptotic bodies formation extracellular vesicle (EV) and exosome subtypes has Thanh Kha Phana, Bo Shib, Niall Geogheganc, Kelly Rogersd and Ivan Poone proved challenging, in part due to the difficulty in aDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular b untangling the mechanisms leading to their generation. Science, La Trobe University, Bundoora, Australia; Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Methods: We investigated the cell biology behind exo- Trobe University, Melbourne, Australia; cCentre for Dynamic Imaging, some formation using the large endosomal compart- Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia; dCentre for Dynamic Imaging, Walter and Eliza Hall Institute of Medical ments offered by an in vivo fly model, and analysis in Research, Melbourne, Australia; eLa Trobe University, Bundoora, Australia human HCT116 and other cancer cell lines. EV preparations were also tested in vivo following injection in Introduction: Dying cells often break into smaller to human xenografts in mice. We analysed different EV membrane-bound fragments, called apoptotic bodies preparations by mass spectrometry using Tandem (ApoBD), via apoptotic cell disassembly (ACD), an Mass Tag labelling to identify changes in protein essential physiological or pathophysiological event cargo of EVs in response to microenvironmental stress. downstream of apoptosis. Emerging evidence implies Results: Using these complementary approaches, we the importance of ApoBD formation in mediating effi- show that microenvironmental stress, such as gluta- cient phagocytic removal of apoptotic debris and facil- mine depletion, leads to a switch in membrane traffick- itating intercellular communication through trafficking ing from the classic late endosomal multivesicular of biomolecules and pathogen-derived materials. In endosomes to Rab11a-positive recycling endosomes contrast to long-lasting belief, our recent findings and the production of Rab11a-positive exosomes, have demonstrated that apoptotic cell disassembly is a which promote cell growth under stress conditions. tightly regulated and temporally-controlled three-step This activity is suppressed by blocking Rab11a-depen- process: (i) membrane blebbing, (ii) formation of thin dent trafficking and ESCRT function. Our proteomics membrane protrusion promoting bleb separation and and fly data suggest that some ESCRTs are differen- (iii) protrusion fragmentation to form ApoBD. tially involved in these two exosome-generating pro- However, detailed insights to the underlying mechan- cesses. Furthermore, mouse xenografts highlight roles ism, particularly ion channels and chemical signalling, for stress-induced EVs in increasing the turnover of undoubtedly require further investigations. tumour cells, leading to an increase in hypoxic stress, Methods: To identify ion channel(s) involved in ACD associated with selection for aggressive cells that can process, cells were treated channel blockers prior to promote tumour progression. These stress-induced UV irradiation. ApoBD formation was monitored vesicles also have a potent effect on blood vessel growth using DIC microscopy and quantified by our in vivo. recently-developed multi-parametric flow cytometry Summary/Conclusion: We conclude that stress- analysis using TOPRO-3 dye and Annexin V. Lattice induced EVs and exosomes made in Rab11a-positive light sheet microscopy allowed us to obtain high-reso- recycling endosomes are involved in tumour lution imaging of calcium-mediated ACD in presence adaptation. of various fluorescent stains. JOURNAL OF EXTRACELLULAR VESICLES 137

Results: Our data showed that calcium influx preceded those from wild-type mice. To identify the types of disassembly step of apoptotic cell, blockade of which, proteins that are modified by UBL3, we perform com- using calcium channel inhibitors, abolished ApoBD prehensive proteomics analysis and find 1,241 UBL3- formation. Strikingly, calcium channels contain a ten- interacting proteins depending on the two C-terminal tative caspase cleavage site, immediately preceding cal- cysteine residues. Among these, 369 proteins are anno- modulin-binding IQ motif which mediates calcium- tated as “extracellular vesicular exosome” by Gene dependent feedback inactivation of the channels. Ontology (GO) analysis, and there are at least 22 dis- Thus, maximised calcium influx by caspase-cleaved ease-related molecules, including Ras. To investigate calcium channels could be a novel regulatory mechan- whether UBL3 modification affects protein sorting to ism of ACD. Furthermore, we could monitor the sEVs, we choose Ras as a model protein. We show that detailed progression of the process, from cytosolic cal- Ras and oncogenic RasG12V mutant are post-transla- cium accumulation to form electrochemical force, driv- tionally modified by UBL3, and that increased sorting ing protrusion formation and ACD process. of RasG12V to sEVs by UBL3 modification enhances Summary/Conclusion: Our findings therefore provide activation of Ras signalling in the recipient cells. further molecular insights into dying cell disassembly Summary/Conclusion: Collectively, these results indi- and calcium-induced ApoBD-associated pathogenesis, cate that a novel PTM by UBL3 influences the sorting particularly vascular calcification. of proteins to sEVs. UBL3 modification could be a novel therapeutic target for sEV-related disorders. OT09.03 OT09.04 A novel UBL3 modification influences protein sorting to small extracellular vesicles Hiroshi Agetaa, Natsumi Ageta-Ishiharab, Keisuke Hitachia, Takanori Onouchia, Hisateru Yamaguchia, Yusuke Yoshiokac, Nobuyoshi Kosakad, Tomihiko Idea, Makoto Kinoshitab, Takahiro Ochiyad, Mitsutoshi Setoue Stringent small extracellular vesicle purification and ligation- and Kunihiro Tsuchidaa independent small RNA-seq: new insights into released RNA populations aFujita Health University, Toyoake, Japan; bNagoya University, Nagoya, Kenneth W. Witwera, Tine Schøyena, Yiyao Huanga, Andrey Turchinovichb, Japan; cTokyo Medical University, Shinjyuku-ku, Japan; dDepartment of Senquan Liua, Linzhao Chenga and Vasiliki Machairakic

Molecular and Cellular Medicine, Institute of Medical Science, Tokyo a b e Johns Hopkins University School of Medicine, Baltimore, USA; SciBerg, Medical University, Shinjyuku-ku, Japan; Hamamatsu University School of c Medicine, Hamamatsu, Japan Heidelberg, Germany; Johns Hopkins University, Baltimore, USA

Introduction: Small extracellular vesicles (sEVs) are Introduction: MicroRNAs are a major focus of exRNA nanometre-sized vesicles secreted from various cell and EV studies. Many publications report miRNAs as types. Exosomes, a type of sEVs, derived from multi- the plurality or majority of released small RNAs. vesicular bodies (MVBs), mediate cell-to-cell commu- However, legacy sRNA profiling methods are biased nication by transporting proteins, mRNAsand towards miRNAs. Abundant RNAs outside vesicles miRNAs. The delivery of proteins between cells by also contaminate many EV preparations. We sEVs, including exosomes, is related to tumour pro- sequenced exRNA from induced pluripotent stem gression and neurodegenerative diseases. However, the cells (iPSCs) with a ligation-independent method: molecular mechanism by which proteins are sorted to ultra-low-input capture and amplification by tailing sEVs is not fully understood. and sequencing (CATS). Methods: By using immunoprecipitation, immunocy- Methods: Culture conditioned medium (CCM) was tochemical, electron microscopic and proteomics ana- collected from 4 lines of count-normalized iPSCs over lysis, we report that ubiquitin-like 3 (UBL3)/ 3 passages (> 200 mL/passage). Fractions were: cells membrane-anchored Ub-fold protein (MUB), an evo- (washed/lysed); “whole releasate" = clarified CCM (300 lutionarily conserved protein, acts as a novel posttran- x g, 2k x g); “large EVs (lEVs)" = pellet of 10k x g spin; slational modification (PTM) factor that regulates “small EVs (sEVs) = preparation by tangential flow protein sorting to sEVs. filtration (100 kDa cutoff) and size exclusion chroma- Results: We find that UBL3 modification is through tography (Izon); and “soluble" = flow-through from cysteine residues only under non-reducing conditions sEV preparation. Particles were counted by and is indispensable for sorting of UBL3 to MVBs and ParticleMetrix, visualized by TEM, and tested for up sEVs. Furthermore, we observe a 60% reduction of to 7 positive or negative markers per MISEV2014/18. total protein, but not RNA, levels in serum sEVs pur- lEVs and sEVs were treated with nucleases. CATS ified from UBL3-knockout (KO) mice compared with sRNA libraries were analysed for contribution of 138 ISEV2019 ABSTRACT BOOK

RNA classes. Statistics were corrected for multiple Summary/Conclusion: miRNAs are found at lower comparisons; significance = corrected p < 0.01. relative levels in cells and releasate than indicated by Results: Using CATS, miRNAs mapped at only a small % legacy sequencing methods. miRNAs also tend to be of total sRNA reads; typically less than 1%. Nuclease- excluded from sEVs vs. cells or other releasate frac- treated sEVs had significantly lower relative miRNA tions. While this study uses iPSCs, similar results levels than cells or soluble releasate. tRNAs/fragments would likely be obtained with other cells. We do not had highest relative abundance in whole releasate and discount the role for miRNAs in cell-cell communica- soluble fractions, albeit with substantial variability. tion but suggest that sEVs may not be a vastly superior Significantly different in most releasate fractions vs cells source of miRNAs. were sno/scaRNA, mRNA, and lncRNA. Cellular distri- Funding: This work was supported by the US NIH: bution differed only from lEV and sEV for RNU RNAs, NIA (AG057430), NIDA (DA040385 and DA047807) and only from sEV for Y RNAs. rRNAs/fragments did and NIMH (MH118164). not differ significantly. Medium-only process controls had only a small per cent of human mapping. JOURNAL OF EXTRACELLULAR VESICLES 139

Symposium Session 10: EVs in Blood and Blood Disorders Chairs: Ai Kotani; Rienk Nieuwland Location: Level B1, Lecture Room 17:00–18:00

to reveal the nature of this phenomenon. Our findings OT10.01 suggest that analysis of ATT enriched in EV preparations might be useful to gain insights into the patho- Different ATT isoforms are associated to EVs from ATT type II genesis and be of support in the diagnostic algorithm deficient patients of ATT deficiency. Annalisa Radeghieria, Silvia Alacquab, Giuliana Martinic, Eugenio Montib and Paolo Bergesed Funding: This work acknowledges FFABR (Fondo finanziamento attività Base di ricerca from MIUR, aDepatment of Molecular and Translational Medicine and CSGI, Università degli Studi di Brescia, ITALY, Brescia, Italy; bDepartment of Molecular and Ministry of Education, Universities and Research, Translational Medicine, Università degli Studi di Brescia, Brescia, Italy; Italy) for financial support. cSpedali Civili of Brescia, Clinical Chemistry Laboratory, Brescia, Italy; dDepartment of Molecular and Translational Medicine and CSGI, Università degli Studi di Brescia, Italy OT10.02 Introduction: Antithrombin (AT) is a glycoprotein involved in the regulation of blood coagulation. It Search for EV signature in sickle cell disease belongs to the family of serine-protease inhibitors and Sisareuth Tana, Celine Gounoua, Marc Romanab, Stephane Mornetc, Alain R. d acts as the most important antagonist of different clot- Brisson ting factors. Two types of inherited AT deficiency can aUMR-CBMN, Pessac, France; bUMR-1134 INSERM-Université Antilles- Guyanne, Pointe à Pitre, France; cUMR-5026-ICMCB, Pessac, USA; be distinguished: Type I (quantitative deficit), and dUniversity of Bordeaux, Pessac, France Type II (qualitative deficit). The latter is characterized by an impaired inhibitory activity related to dysfunc- Introduction: Sickle cell disease (SCD) is a hereditary tional domains of the protein. Three Type II subtypes haemoglobinopathy characterized by the production of can be defined: Type IIa (reactive site defect), Type IIb sickled red blood cells (RBC), anaemia and vascular (heparin binding site defect) and Type IIc (pleiotropic occlusion crises. The presence of extracellular vesicles defect). This classification has clinical importance since (EV) in blood from SCD patients has long been recog- these subtypes have a different thrombotic risk. No nized, yet with a large divergence of results (1). Our functional routine diagnostic assay, however, can be objective was to characterize in details EV in plasma assumed to detect all forms of Type II deficiencies from SCD patients, by combining flow cytometry and since false-negative results may hamper the diagnosis. immuno-gold cryo-electron microscopy (2,3). We Methods: We analysed the biochemical/biophysical focused on two EV populations: 1) EV exposing phos- association of ATT to EVs. We separated EVs from phatidylserine (PS), because the increased exposure of plasma of healthy or Type II affected patients or from PS at the RBC surface is a hallmark of SCD (4), and 2) cultured hepatocytes through differential ultracentrifu- exosomes exposing CD71 (CD71-Exo), because the gation followed by sucrose density gradient and/or reticulocyte count is a marker of anaemia and CD71- immunoprecipitation. We next combined dot blot ana- Exo are released during the maturation of reticulocytes lysis, WB, 2D electrophoresis and enzymatic assays to into erythrocytes (5). reveal the nature of ATT association to EVs. Methods: Platelet-free plasma (PFP) was obtained from Results: We evidenced that ATT is associated to the 11 SCD patients and 18 control individuals. Annexin-5, external leaflet of EVs. We also found that specific ATT anti-CD235a- and anti-CD71-IgGs, either fluorescently isoforms are enriched in EV preparations in respect to labelled or conjugated to gold particles, were used to total plasma and that those isoforms are selectively detect PS+ EV, RBC-derived EV and CD71-Exo, associated to EVs when comparing healthy or ATT respectively, by flow cytometry and immuno-cryo- type II deficient patients. EM (2,3). Summary/Conclusion: ATT selective association pat- Results: By flow cytometry, seven populations of RBC- tern to EVs might be related either to mutations in the derived EV were identified in SCD plasma, based on primary sequence of the protein or alterations in the the presence vs. absence of PS, EV size and morphol- glycosylation process, hence experiments are ongoing ogy. The main difference between SCD and control 140 ISEV2019 ABSTRACT BOOK

PFP was the presence in SCD PFP of large amounts of also studied by phagocytosis and TaqMan® assays. PS+ EV of small size (100 to 200 nm, as determined by Flow cytometry was also performed after saline wash- immuno-cryo-EM) (250,000 ± 20,000 / µL for SCD ing and protease digestions. All experiments were per- PFP vs. 30,000 ± 10,000/µL for control PFP). formed in accordance with the Declaration of Helsinki. In addition, CD71-Exo were detected in SCD PFP by Infomed consent was obtained from all participants. immuno-cryo-EM, while they are almost absent in Results: A significantly higher number of proteins was control PFP. As expected, CD71-Exo were highly found in the plasma+EV samples compared with the homogeneous in size, ranging from 50 to100 nm. summed number of proteins found in the only plasma Their concentration was determined by fluorescence- and the only EV samples (p < 000.1). Subtracting the triggered flow cytometry: 70,000 ± 40,000 / µL for SCD proteins that were found in pure EV samples from the PFP vs. 7,000 ± 5,000 / µL for control PFP. list of proteins of EV samples incubated in plasma for Summary/Conclusion: We have identified two EV 30 min and washed 2 times, a high number of proteins populations present in large amounts in SCD plasma, were found, out of which several were more character- while they are almost absent in control plasma. Further istic of rheumatoid arthritis samples and only a few study is needed to evaluate the use of these EV as were more prevalent in healthy samples. Interactions biomarkers of the coagulation or endothelium activa- between fibrinogen, haptoglobin, complement protein tion states in SCD. C3. and EVs were also confirmed by flow cytometry and immune electron microscopy. 1. Hebbel & Key. Brit J. Haem 2016 174:16 Summary/Conclusion: Our data suggest the existence 2. Arraud et al., J. Thromb Haemost 2014 12:614 of a protein corona on EVs of blood plasma. The 3. Arraud et al., Cytometry A. 2016 9:184 differences in protein coronas found between healthy 4. Chiu et al., Blood 1981 58:398 controls and patients with rheumatoid arthritis suggest 5. Harding et al., J. Cell Biol 1983 97:329 that EV surface-associated proteins may play a role in disease pathology and may serve as biomarkers. Funding: Labex GR-Ex Funding: NKFIH-OTKA PD 104369; PD 112058; 111958; 120237, NVKP_16, MTA-SE Immun- OT10.03 Proteogenomikai EV Kutatócsoport, VEKOP-2.3.2-16- 2017–000002, VEKOP-2.3.3-15-2017-00016 H2020 MSCA ITN TRAIN-EV, SE STIA-2017 Surface protein cargo of extracellular vesicles in blood plasma; the effect of an inflammatory disease on the vesicle surface protein interactome Eszter Á. Tóth, Katalin É. Szabó-Taylor, Tamas Visnovitz, György Nagy and OT10.04 Edit I Buzás Semmelweis University, Department of Genetics, Cell and Immunobiology, Budapest, Hungary Oxidized LDL stimulates production of inflammatory extracellular vesicles by platelets Maarit Neuvonena, Katariina Öörnib, Erja Kerkeläc, Kati Hyvärinenc, Saara Introduction: Extracellular vesicles (EVs) are endogen- Laitinenc and Pia Siljanderd ous nanoparticles produced by cells. Artificial nanopar- aEV-Group, Faculty of Bio- and Environmental Sciences and Faculty of ticles used for targeted therapy have been found to Pharmacy, Helsinki, Finland; bWihuri Research Institute, Helsinki, Finland; develop a protein corona altering their biodistribution cFinnish Red Cross Blood Service, Helsinki, Finland; dEV-group, Molecular and Integrative Biosciences Research Programme, Faculty of Biological and and bioavailability in biological media. Here we set the Environmental Sciences, University of Helsinki, Helsinki, Finland aim to study if a similar protein corona is formed at the surface of EVs in biofluids and if inflammation had an Introduction: Platelets may become activated under effect on the protein corona formation. hyperlipidemic conditions and are thought to promote Methods: Blood plasma depleted in both platelets and atherosclerotic plaque development. Platelets can gen- EVs was generated from blood samples of healthy erate a diverse mixture of extracellular vesicles (EVs) subjects (n = 12) and rheumatoid arthritis patients when they are activated through different signalling (n = 10). Nascent EVs of THP1 cells and platelets pathways. In this study, we investigated in detail the were isolated and incubated in the plasma samples for EV-generating capacity of different lipoproteins and 30 minutes. EVs were then washed and were studied by compared the cellular effects of the resulting EVs on mass spectrometry (MS/MS), immune electron micro- macrophage differentiation. scopy and flow cytometry. Controls included i) plasma Methods: Platelets (isolated by gel-filtration from fresh without the addition of EVs; ii) EVs incubated in concentrates) were stimulated by LDL, oxidized LDL or buffer. The effect of different protein coronas was HDL together with thrombin + collagen co-stimulus, a JOURNAL OF EXTRACELLULAR VESICLES 141 potent EV-inducing signal. After careful platelet conditions. Oxidized LDL increased EV formation by removal, EVs were isolated by serial ultracentrifuga- platelets, whereas co-incubation with HDL inhibited tion. Platelet activation was monitored by P-selectin this effect. Platelet derived EVs modulated the macro- exposure in flow cytometry. EVs were analysed by an phage differentiation as seen by the changes in their EV-dedicated high-resolution flow cytometer and pro-inflammatory cytokines and surface marker Western blotting, and quantified by protein concentra- profiles. tion and particle number. Macrophage differentiation Summary/conclusion: In conclusion, hyperlipidemic was induced by GM-CSF in the presence or absence of lipoprotein profiles in plasma can manifest in (1) the different EVs. After 6 days, macrophages were altered platelet EV generation which in turn (2) may activated by IFN-γ and LPS, and after a further program macrophage differentiation in a manner rele- 3 days, the macrophages were profiled by flow cytome- vant for atherosclerotic plaque development. try and their secreted cytokines. Funding: Academy of Finland grant 287089, Finnish Results: Lipoproteins induced platelet EV production Foundation for Cardiovascular Research in a concentration- and time-dependent manner at concentration levels relevant to hyperlipidemic 142 ISEV2019 ABSTRACT BOOK

Symposium Session 11: EV Therapeutics I Friday 26 April 2019 Chairs: Andre Gorgens; Sai Kiang Lim Location: Level B1, Hall B 08:30–10:00

OF11.01 by 50–72%. In sciatic nerve tissues, CEC-exos in combination with platinum drugs significantly increased miR-15b, −26a, and −214, and substantially reduced Exosomes from cerebral endothelial cells suppress chemotherapy- induced peripheral neuropathy and sensitize anti-tumour effects of axonal damage protein levels of PTEN, SARM1, and platinum drugs TRPV1. In tumour tissues, the combination treatment a b c d Yi Zhang , Zheng Gang Zhang , Michael Chopp and Chao Li significantly increased miR-15b and −26a, and reduced a b Henry Ford Health System, Detroit, USA; Department of Neurology, Henry their target chemoresistant protein levels of P-gp and Ford Hospital, Detroit, MI, USA, Troy, USA; cDepartment of Neurology, Henry Ford Health System, Detroit, MI, Department of Physics, Oakland ABCC1. University, Rochester, MI, USA; dDepartment of Neurology, Henry Ford Summary/Conclusion: Our data demonstrate that Health System, Detroit, MI, USA CEC-exos reduce CIPN by reversing the platinum- Introduction: Platinum-based drugs are commonly inactivated neuroprotective network and that CEC- used to treat cancers. However, peripheral neuropathy exos suppress a chemoresistant network of miRNAs/ is a common adverse effect of platinum-based che- protein-coding genes to enhance the anti-tumour effect motherapy. Neurotoxicity often requires platinum of platinum drugs. drug dose reduction thereby, compromising therapeu- Funding: NIH R01CA219829; NIH R01NS088656; tic efficacy of platinum drugs to suppress tumour AHA 16SDG29860003. progression. Methods: Using differential ultracentrifugation, we iso- OF11.02 lated exosomes from cultured human primary cerebral endothelial cells (CEC-exos). Ovarian tumour was induced in mice by implantation of human ovarian EV-mediated in vitro transcribed (IVT) mRNA-based gene delivery for cancer cells. Platinum-induced CIPN start from distal specific pro-drug activation in the tumour treats breast cancer in mice with no offsite toxicity axons. Thus, we examined the direct effect of platinum Alexis Forterrea, Jing-Hung Wanga, Alain Delcayreb, Kyuri Kimc, Carol drugs on distal axons of dorsal root ganglia (DRG) Greenc, Mark Pegramd, Stefanie Jeffreye and Ac Matinf neurons using a microfluidic device that separates dis- aDepartment of Microbiology & Immunology, Stanford University School of b c tal axons from their parent cell bodies. Medicine, Stanford, USA; ExoThera LLC, Menlo Park, USA; SRI Biosciences, Menlo Park, USA; dDepartments of Medicine and Oncology, Results: We found that addition of oxaliplatin or car- Stanford University School of Medicine, Stanford, USA; eDepartment of boplatin into the axonal compartment significantly Surgery, Stanford University School of Medicine, Stanford, USA; fDepartment of Microbiology & Immunology Stanford University School of suppressed axonal elongation, whereas application of Medicine, Stanford, USA CEC-exos into the axonal compartment completely abolished oxaliplatin-inhibited axonal growth. In vivo, Introduction: We previously reported EV-mediated treatment of tumour-bearing mice with platinum drugs specific delivery of a gene (as mRNA) encoding the (n = 7/group) induced CIPN characterized by tactile HChrR6 enzyme to orthotopically implanted HER2+ and cold allodynia, reduction of sensory nerve conduc- breast cancer tumours in mice. HChrR6 activated the tion velocity, and decreases of the number of epidermal pro-drug CNOB and completely inhibited tumour nerve fibres compared to the control mice (n = 7/ growth. EVs and CNOB were delivered systemically. group). However, tumour-bearing mice treated with A new plasmid loaded the EVs with the mRNA; they platinum drugs along with CEC-exos (n = 7/group) displayed the EVHB protein containing anti-HER2 exhibited a significant reduction of platinum-drug scFv (“PEVs”). However, no direct evidence was pre- induced peripheral neuropathy. Moreover, CEC-exos sented that the treatment was free of off-target toxicity. in combination with platinum drugs significantly Here, three aspects, relevant to clinical transfer of the decreased tumour size by 80–91% compared to plati- regimen, are explored: IVT- instead of plasmid-based num drugs alone which reduced tumour growth only mRNA loading; use of the pro-drug CB1954 whose safe JOURNAL OF EXTRACELLULAR VESICLES 143 dose in humans is known (HChrR6 also activates Introduction: On the road towards the use of extra- CB1954); haematological and histopathologic studies cellular vesicles (EVs) for regenerative medicine, tech- to detect off-target toxicity. nological hurdles remain unsolved: high-yield, high Methods: Exogenous mRNA delivery using exosomes purity and cost-effective production of EVs. is difficult because of its fragmentation. To ensure the Methods: Pursuing the analogy with shear-stress mRNA integrity (at synthesis, loading, and delivery), induced EV release in blood, we are developing a we used the “MCHB” test. MCHB is the product of mechanical-stress EV triggering cell culture approach CNOB activation and is strongly fluorescent, providing in scalable and GMP-compliant bioreactors for cost- a facile method to test this. The mRNA was transcribed effective and high yield EV production. The third gen- in vitro from the HChrR6 gene (standard kits) and was eration setup allows the production of up to 300,000 tested by translating it into the enzyme and ascertain- EVs per Mesenchymal Stem Cell, a 100-fold increase ing the enzyme’s ability to produce fluorescence upon compared to classical methods, i.e physiological spon- CNOB addition. mRNA copy number in the EVs and taneous release in depleted media (around 2000 EVs/ the recipient cells was determined by qRT-PCR. The cell), with a high purity ratio 1 ×10e10 p/µg loaded EVs were HER2 targeted as above, using EVHB Results:Weinvestigatedin vitro the regenerative poten- (“IEVs”). HER2+ BT474 breast cancer tumours were tial of high yield mechanically induced MSC-EVs by orthotopically implanted in mice. demonstrating an equal or increased efficiency compared Results: While 5,000 PEVs were needed to deliver one to classical EVs with the same amount of EVs. The copy of the mRNA, <50 IEVs delivered this. BT474 cells regenerative properties of mechanically induced MSC- transfected in vitro with IEVs showed significantly higher EVs was confirmed in vivo in a murine model of chronic mRNA expression than those transfected with PEVs. heart failure demonstrating that high, medium shear Expression of the latter peaked at ca. 72 h; of the former stress EVs and serum starvation EVs or mMSCs had the continued throughout the length of the experiment same effect using local injection. We later on tested the (120 h). We found that CB1954 (20 mg/kg) completely effect of the injection route and the use of xenogenic arrested tumour growth with 220-fold fewer IEVs (3.4 x hMSC-EVs on their efficiency in the same model of 10^8) than PEVs; experiments in progress suggest that murine chronic heart failure. Heart functional para- even lower IEVs may be sufficient. Extensive haematolo- meters were analysed by ultrasound 2 months (1 month gical and histopathologic studies showed no significant post EV injection) post infarction. Interestingly, hMSC- toxicity. EVs had the same effect compared to mMSC-EVs in local Summary/Conclusion: IEV + CB1954 therapy comple- injection, showing that xeno-EVs in immunocompetent tely arrests tumour growth at a much lower IEV dose mices was well tolerated. Moreover, hMSC EV IV injec- and without introducing potentially harmful plasmid tion was as efficient as local intra-myocardium muscle DNA and off-target toxicity. The findings move this injection with an increase in the left ventricular ejection approach closer to clinical transfer. fraction of 26% compared to pre-treatment values, Funding: NIH NCATS UH3TR000902. whereas PBS injected controls lost 13%. Summary/Conclusion: We demonstrated an equal or OF11.03 superior regenerative effect of high yield mechanically produced EVs compared to spontaneously released EVs or parental cells in vitro and in vivo, and good High yield hMSC derived mechanically induced xenografted tolerance and efficacy of hMSC EV both with local and extracellular vesicles are well tolerated and induce potent regenerative effect in vivo in local or IV injection in a model of IV injection. This unique technology for EV produc- chronic heart failure tion combines decisive assets for clinical translation of a b c d Max Piffoux , Iris Marangon , Nathalie Mougenot , Claire Wilhelm , EV-based regenerative medicine : a GMP-compliant Florence Gazeaue, Onnik Agbulutf and Amanda Brun-Silvag setup, high density cell culture, high yield release of aLaboratoire Matière et Systèmes Complexes, CNRS UMR 7047 Université Paris Diderot, Paris, France; bUniversité Sorbonne Paris Cité, Laboratoire EVs per cell, high purity EVs. Matière et Systèmes Complexes, CNRS UMR 7047 Université Paris Diderot, France; cSorbonne Universités, Université Pierre et Marie Curie Paris 6, Plateforme PECMV, UMS28, Paris, France; dlaboratoire Matière et Systèmes Complexes, paris, France; eUniversité Sorbonne Paris Cité, OF11.04 Laboratoire Matière et Systèmes Complexes, CNRS UMR 7047 Université Paris Diderot, Paris, France; fUniversité Sorbonne Paris Cité, Laboratoire Matière et Systèmes Complexes, CNRS UMR 7047 Université Paris Diderot, Prolongation of allograft survival via donor MHC chimerism induced 7 Paris, France; Université Sorbonne Paris Cité, Laboratoire Matière et by extracellular vesicles Systèmes Complexes, CNRS UMR 7047 Université Paris Diderot, Paris, Bruno Adonai Gonzalez Nolascoa, Mengchuan Wanga, William Orenta, France Aurore Prunevieillea, Jane Oa, Kaitlan Ahrensa, Joren C Madsenb and Gilles Benichoua 144 ISEV2019 ABSTRACT BOOK aDepartment of Surgery, Center for Transplantation Sciences, Massachusetts General Hospital and Harvard Medical School, Boston, USA; bDepartment of OF11.05 Surgery, Center for Transplantation Sciences and Division of Cardiac Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, USA Proteomic and transcriptomic characterization of exosomes-mimetic nanovesicles reveals their relevance as a therapeutic delivery system Introduction: Achieving robust and durable host Amirmohammad Nasiri Kenaria, Kenneth Kastaniegaardb, Mitch C. immune tolerance of allogeneic transplants is the ulti- Shambrooka, David Greeninga, Allan Stensballeb, Lesley Chenga and Andrew Hillc mate goal in clinical transplantation. Mixed chimerism aDepartment of Biochemistry and Genetics, La Trobe Institute for induced via donor bone marrow transplantation and Molecular Science, La Trobe University, Melbourne, Australia; host non-myeloablative conditioning has reliably bDepartment of Health Science and Technology, Faculty of Medicine, c achieved tolerance of allogeneic organ transplants in Aalborg University, Denmark, Aalborg, Denmark; The Department of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La mice and humans. Tolerance in this model is believed Trobe University, Bundoora, Australia to rely essentially on the presentation of donor MHC molecules in the host’s thymus. In this study, we inves- Introduction:Thepotentialofutilizingexosomes tigated whether donor MHC chimerism could be (endosomal derived vesicles) as a therapeutic delivery achieved via donor extracellular vesicles (EVs) injec- system of biological and chemical drugs are an active tions and subsequent cross-dressing of recipient cells in area of clinical phase investigation. However, the field the host’s thymus. is currently facing challenges such as the insufficient Methods: Conditioned SJL (CD45.1+, H2-Ks+) recipi- release of exosomes, their heterogeneity and reprodu- ent mice received a single IV dose of purified bone cibility of isolation. These issues may be overcome marrow derived exosome-enriched EVs (BM-EVs) iso- through the development of artificial extracellular lated from C57BL/6 (CD45.2+, H2-Kb+) donors vesicles (EVs). Cell-derived mimetic nanovesicles through sequential centrifugation or using a commer- (M-NVs) can be generated from numerous cell lines cially available exosome isolation kit. Nanoparticle with advantages such as, reproducibility, large scale tracking showed vesicles of approximately 100nm in production, uniformity, cost effectiveness and a sim- size in the BM-EVs preparation and Western Blot ple purification method. Although several studies showed the presence of MHCI. Image flow cytometry have shown that M-NVs have similar morphology, was used to detect the presence of cross-dressed cells size and therapeutic potential to exosomes, compre- from day 10 through 100 after exosome injection. For hensive characterization and to what extent these NHP studies, MHC class I H38+ BM-EVs were artificial EV components mimics exosomes remain injected into a H38- conditioned cynomolgus macaque elusive. prior to a combined heart and kidney transplant. Methods: In this study, M-NVs generated by subject- PBMCs, thymus, spleen and mesenteric lymph nodes ing cells to the extrusion, were comprehensively char- were collected for image flow cytometry. acterized and compared to the exosomes by proteomic Results: Intravenous injection of BM-EVs into condi- and transcriptomic analysis. tioned mice resulted in the presentation of donor MHC Results: We analysed the proteome between M-NVs and CD45.1 molecules by host’s thymic and splenic and exosomes to provide key insights into key mem- cells. Similarly, H38+cross-dressed cells were detected brane surface features of exosomes for cargo sorting at D33 after exosome injection in all of the NHP and therapeutics delivery are preserved in M-NVs (158 recipient tissues collected. In mice, donor but not syn- proteins). Furthermore, our study highlighted differ- geneic or third-party BM-EVs significantly prolonged ences in protein post-translational modifications skin allograft survival (median survival = 17 VS among M-NVs, as distinct from exosomes, using a 11 days, p < 0.001). non-targeted informatic approach, specifically showing Summary/Conclusion: These results show that deliv- phosphorylation, ubiquitination, and thiophosphoryla- ery of donor-derived extracellular vesicles can induce tion as enriched protein modifications in M-NVs. donor MHC chimerism via cross-dressing of recipient Small RNA analysis reveals that unlike exosomes, the APCs with allogeneic MHC molecules in the host’s RNA cargo of M-NVs is similar to that of the parental thymus. This suggests that donor EVs could be used cells. In addition, we found that M-NVs could be useful in place of bone marrow cells to induce chimerism and for packaging proteins or RNA which are globally allograft survival with minimal conditioning and no enriched in cells. Indeed, this may overcome the chal- risk of graft versus host disease (GVHD). lenges involved in selective packaging of therapeutic Funding: NIH R01DK115618. proteins or RNAs into exosomes. JOURNAL OF EXTRACELLULAR VESICLES 145

Summary/Conclusion: In summary, results from this potential of exosomes from human PDL-MSCs to sti- study provides key insights into omics of M-NVs cargo mulate wound healing. in comparison to exosomes and ultimately its potential Methods: We used ultrafiltration and size-exclusion as therapeutic delivery system. chromatography to isolate vesicles from serum-free Funding: Grants from the Australian Research conditioned media. BCA-assay and nanoparticle track- Council, Lundbeck Foundation and the Danish ing analysis (NTA) was used to determine yield. We National Mass Spectrometry Platform for Functional performed Western Blot for positive and negative Proteomics. extracellular vesicle-markers, and transmission electron microscopy was used to evaluate morphology. We then performed wound healing assay in immunocompetent OF11.06 rats. Each rat received two full-thickness wounds, treated with either topical application or perilesional injections, and PBS was used in control rats. The animal

Exosomes from periodontal ligament-derived cells promote weights were measured and wounds were photo- cutaneous wound healing and topical application is superior to local graphed every other day. The animals were sacrificed injection on day 7 and the tissue was collected for histopatholo- Sebastian Sjoqvista, Azela Gladyb, Ryo Okadac, Akiko Takahashid, Taichi Ishikawae, Satoru Onizukaf, Nobuo Kanaif and Takanori Iwataf gical analysis. aKarolinska Institutet/Tokyo Women’s Medical University, Tokyo, Sweden; Results: Exosome yield was on average 0.83 µg proteins bDepartment of Pharmacology, Keio University School of Medicine, Tokyo, per million cells per 24 h. The exosomes had a mean Japan; cProject for Cellular Senescence, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo, Japan; dProject for Cellular size of 130 nm, showed positivity for CD9 and Senescence, Cancer Institute, Japanese Foundation for Cancer Research, Flotillin-1 and negativity for GRP94 and had a spheri- e Koto-ku, Japan; Division of molecular microbiology, Iwate Medical cal morphology. The exosomes were applied to wounds University, Iwate, Japan; fInstitute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Tokyo, Japan and rats receiving exosomes gained significantly more weight than controls. Topical application proved to be Introduction: Periodontal ligament-derived mesench- superior to injections based on macroscopic wound ymal stromal cells (PDL-MSCs) represent an attractive evaluation and histopathology. source of cells for regenerative medicine due to four Summary/Conclusion: PDL-MSC-derived exosomes reasons; 1) similarly to other MSCs, they exhibit pro- stimulate wound healing in a xenogeneic setting and regenerative properties, 2) accessibility is great due to topical application is superior to local injections. abundance of extracted teeth, 3) they can easily be Funding: This work was supported by The Swedish expanded and 4) they can be used in an allogeneic Society of Medicine, Erik och Edith Fernströms stiftelse fashion. We are currently investigating the use of för medicinsk forskning, Misao-Yanagihara-Grant for sheets produced from these cells to regenerate period- regenerative medicine research and JSPS KAKENHI ontal tissue. In this study we aimed to investigate the Grant Number JP18H02985. 146 ISEV2019 ABSTRACT BOOK

Symposium Session 12: Protein Biomarkers in Human Disease Chairs: Malene Møller Jørgensen; Koji Ueda Location: Level B1, Lecture Room 08:30–10:00

OF12.01 Summary/Conclusion: Our results confirm the potential of analysing PDE-EV as biomarkers of PM alteration allowing improved monitoring of PD patients Biomarkers of peritoneal membrane alteration in dialysis efflux- extracellular vesicles: a longitudinal study in patients under compared to PET. peritoneal dialysis treatment Funding: The IGTP is member of the CERCA network Laura Carreras-Planellaa, Jordi Soler-Majoralb, Cristina Rubio-Esteveb, Míriam Morón-Fontc, Marcella Franquesac, Jordi Bonald, Maria-Isabel of institutes. LCP is sponsored by the FPU scholarship Troya-Saboridob and Francesc E. Borràsc (FPU17/01444) from the Ministerio de Ciencia, aReMAR-IVECAT group, Health Science Research Institute Germans Trias i Innovación y Universidades of the Spanish Pujol (IGTP), Badalona, Spain; bNephrology Department, Badalona, Spain; Government. MF is sponsored by the PERIS contract cREMAR-IVECAT Group, “Germans Trias i Pujol” Health Science Research Institute, Can Ruti Campus, Badalona, Spain; dNephrology Department, SLT002/16/00069, from the Generalitat de Catalunya. “Germans Trias i Pujol” University Hospital, Can Ruti Campus, Badalona, F.E.B. is a researcher from Fundació Institut de Recerca Spain en Ciències de la Salut Germans Trias i Pujol sup- Introduction: Peritoneal dialysis (PD) is considered ported by the Health Department of the Catalan the best renal replacement therapy for patients waiting Government (Generalitat de Catalunya). for a kidney transplant. Many patients eventually suffer ultrafiltration failure of the peritoneal membrane (PM), leading to severe clinical complications and the urgent OF12.02 need to change the dialysis technique. Currently, PM functionality is monitored by the peritoneal equilibra- Proteomics of urine-derived extracellular vesicles to identify tion test (PET), a tedious technique that only shows biomarkers of prostate cancer risk groups changes when the PM damage is advanced. We Amanda Khooa, Meinusha Govindarajanb, Vladimir Ignatchenkoc, Vincent Huangd, Julius O. Nyalwidhee, O. John Semmese, Paul Boutrosf, Stanley Liug hypothesized that peritoneal dialysis efflux (PDE)- and Thomas Kislingerc extracellular vesicles (EV) may contain biomarkers of aDepartment of Medical Biophysics, University of Toronto, Toronto, PM state. In a previous study (Carreras-Planella, et al., Burlington, Canada; bDepartment of Medical Biophysics, University of c PLoS One 2017), we showed for the first time that Toronto, Toronto, Canada; Princess Margaret Cancer Centre, University Health Network, Toronto, Canada; dOntario Institute for Cancer Research, PDE-EVs could be isolated and their protein content Toronto, Canada; eLeroy T. Canoles Jr. Cancer Research Center, Eastern showed differences between newly enrolled and long Virginia Medical School, Norfolk, USA; fUCLA, Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA; gOdette Cancer Research Program, term PD patients. Here, we present the results of a Sunnybrook Research Institute, Toronto, Canada longitudinal study in a new cohort of PD patients. Methods: PDE was collected from 12 PD patients every Introduction: Prostate cancer (PCa) is the most com- 6 months (coincident with PET controls) up to mon cancer in men and can be detected early through 24 months follow up. PDE-EV were isolated by size- screening of asymptomatic men. Most prostate cancers exclusion chromatography and characterized by are indolent at time of diagnosis and current prognos- expression of classical tetraspanin EV markers. EV tic protocols do not accurately predict disease aggres- proteome was analysed by Mass Spectrometry (LC- sion and clinical outcome, which limits optimal patient MS/MS). management. For example, high serum prostate-speci- Results: In accordance with our previous study, PDE- fic antigen (PSA) levels could be indicative of meta- EV proteome showed reduced expression of several static cancer or benign prostatic conditions, while proteins at longer timer points (>12 months) of treat- needle biopsies are invasive and can undersample the ment. In addition, statistical analysis revealed confi- prostate, resulting in uncertainty of cancer grading. We dently identified proteins – potentially involved in hypothesize that small extracellular vesicles (sEVs) iso- fibrotic processes – that are significantly deregulated lated from post-digital rectal exam urine (pDRE-urine) in patients showing alterations in PET monitoring at contain protein biomarkers will allow for non-invasive 12 months of treatment. PCa risk stratification. JOURNAL OF EXTRACELLULAR VESICLES 147

Methods: We have performed deep proteomics analy- matched Controls who remained cognitively normal. sis on pDRE-urine-derived sEVs from 105 treatment- The earliest samples preceded AD symptom onset by a naïve, richly annotated patients (age, T-stage, Gleason median of 4.1 years. We precipitated total particles score, PSA, etc.). sEVs were isolated by differential using Exoquick and then immunoprecipitated neuro- ultracentrifugation and processed for proteomics (LC- nal-enriched EVs using antibody against neuronal cell MS). Size and morphology of sEVs were verified by adhesion molecule L1CAM. We lysed isolated EVs and nanoparticle tracking analysis and TEM. quantified proteins by immunoassays. We adjusted Results: We detected 3,688 proteins in sEVs, 80% of values for EV concentration and diameter to normalize which are shared with the prostate cancer tissue pro- for EV yield. We compared cross-sectional and long- teome (unpublished). sEV proteins were enriched in itudinal trajectories of EV biomarkers between future cytoplasmic and membrane proteins and depleted in AD and Control participants and performed stepwise nuclear proteins. Interestingly, sEVs were also enriched logistic regression with internal cross-validation and for prostate-specific proteins compared to the pro- receiver operating characteristic analysis to assess the teome of urine that was analysed in parallel, suggesting ability of EV biomarkers to discriminate future AD enrichment for low-abundance tissue-originating pro- cases from Controls. tein cargo in sEVs. Samples clustered into three groups Results: Future AD cases had cross-sectionally and based on global protein expression, suggesting that longitudinally higher p181-Tau, p231-Tau, pSer312- there may be subtypes of sEVs within pDRE-urine. IRS1, pY-IRS1 and EV diameter than Controls but Summary/Conclusion: We are currently applying similar Aβ42, total Tau, TSG101 and EV concentration. machine learning approaches to identify biomarkers A model optimally combining longitudinal data for that could supplement current diagnostic tests and multiple biomarkers achieved 90.2% sensitivity (95% improve stratification of patient risk groups. In the confidence interval [CI], 81.2–95.4%), 83% specificity future, we will confirm differential protein expression (95% CI, 76–88%) and 91.6% area under-curve (95% by targeted proteomics assays using an active surveil- CI, 87.9–95.4%) for predicting AD. Preclinical levels of lance cohort and perform parallel profiling of sEV several EV biomarkers were associated with cognitive RNA cargo. Ethics approval at University Health performance. Network. Summary/Conclusion: We validated several neuronal- Funding: National Cancer Institute-Early Detection enriched EV biomarker candidates and further demon- Research Network. strated that their preclinical longitudinal trajectories predict AD diagnosis with high sensitivity. These OF12.03 findings motivate further development of EV biomarkers towards a clinical blood test for AD. Funding: This research was supported entirely by the Extracellular vesicle biomarkers predict Alzheimer’s disease in the Intramural research Program of the NIH, National baltimore longitudinal study of ageing Maja Mustapica, Michelle Shardella, Sean Berkowitzb, Thomas Diehlc, Ryan institute on Aging Spanglerd, Joyce Trane, Michael Lazaropoulosc, Sahil Chawlaa, Seema Gulyania, Erez Eitand, Yang Ana, Chiung-Wei Huanga, Susan Resnika, Edward Goetzlf, Luigi Ferruccia and Dimitrios Kapogiannisg OF12.04 aNIH/National Institute on Aging (NIA), Baltimore, USA; bNIH/NIA, Nashville, USA; cNIH/NIA, Philadelphia, USA; dNIH/NIA, Boston, USA; eNIH/NIA, San Diego, USA; fDepartment of Medicine, University of CD315 (PTGFRN) – a new biomarker for tumour-derived extracellular California, San Francisco, CA; ‡Jewish Home of San Francisco, San vesicles Francisco, San Francisco, USA; gNational Institute on Aging, Baltimore, USA Kathrin Gärtnera, Corinna Hülsa, Gabor Gondia, Judith Dünzkoferb and Reinhard Zeidlerc Introduction: It was recently reported that plasma aHelmholtz Center Munich German Research Center for Environmental neuronal-enriched extracellular vesicles (EVs) of Health, Research Unit Gene Vectors, Munich, Germany; bDepartment of Alzheimer’s disease (AD) patients exhibit elevated Otorhinolaryngology, Klinikum der Universität (KUM), Munich, Germany; cHelmholtz Center Munich German Research for Environmental Health, levels of phosphorylated tau, Aβ42, and phosphory- Research Unit Gene Vectors, Munich, Germany, Munich, Germany lated insulin receptor substrate-1 (IRS1). To validate them as AD predictors, we interrogated preclinical Introduction: Extracellular vesicles (EVs) represent samples from Baltimore Longitudinal Study of Ageing important mediators of cell-cell communication and participants. are secreted by many types of cells, including tumour Methods: We blindly analysed 931 longitudinal plasma cells, into the extracellular milieu. Tumour-derived samples from 138 cognitively normal participants who EVs hold a lot of promise for non-invasive diagnostic eventually developed AD (cases) and 233 age and sex- tests, also known as liquid biopsy, because they are 148 ISEV2019 ABSTRACT BOOK present in all kind of biological fluids and carry a large Methods: First morning void urine and citrate blood variety of proteomic and genetic information. There is from the same donor were centrifuged at 4,600 g for 30 now an ever-growing need for new specific biomarkers, and 15 min, respectively. The supernatant was centri- which allow for the isolation of distinct EV subclasses fuge at 20,000g to collect urinary (uEVs) and plasma in order to improve EV-based diagnostics. We show (pEVs) which were stained with the same commercial for the first time that CD315 (also known as PTGFRN, clone antibody (3D3) anti podocalyxin (PODXL) con- EWI-F or CD9P-1) may represent a new potential jugated with 3 different fluorescent dyes: Alexa Fluor® biomarker for tumour-derived EVs. 405 (AF405), Alexa Fluor® 488 (AF488) and Alexa Methods: The expression of CD315 was studied in cell Fluor® 647 (AF647). Stained EVs were acquired with lines, primary tumour samples and corresponding EVs. both imaging flow cytometry and spectral flow cyto- CRISPR/Cas9 CD315 knockout cells were used to metry. Gate strategy was based on the low scatter of the investigate the impact of CD315 on cell proliferation unstained uEVs and the negative control was the fluor- and EV secretion. Furthermore, we generated a escent probe alone in buffer. CD315-specific monoclonal antibody to elucidate the Results: Acquisition of uEVs alone showed auto-fluor- diagnostic potential of CD315+EVs in blood samples of escence emission in channel 2 (λex 488 nm; λem 480– cancer patients. 560 nm) camera 1 and channel 11 (λex 658 nm; λem Results: We demonstrated that CD315 is highly 660–740 nm) but not channel 7 (λex 405 nm; λem 420– expressed on a large variety of tumour cells and is 505 nm) for camera 2 for the imaging flow cytometry present on the surface of tumour-derived EVs. In meanwhile the spectral flow cytometry revealed a spec- vitro knockout of CD315 hampered proliferation and tral fingerprint spanning from the violet to the red migration of tumour cells and affected cellular EV emission. Autofluorescence was detected for uEVs but production. Moreover, our CD315-specific antibody not pEVs. Podocalyxin-AF405 conjugated stained both was successfully used to capture and isolate CD315 uEVs and pEVs with a double staining for the auto- +EVs by immunoaffinity. fluorescence and PODXL on the same uEV. While Summary/Conclusion: We identified CD315 as a pro- PODXL-AF488 and AF647 stained pEVs both the anti- mising new biomarker with diagnostic potential. body conjugated failed to detect the uEVs as per Although its specific function still remains to be eluci- PODXL-AF405. Same results were obtained for both dated, we were the first to show that CD315 is highly flow cytometry instruments. abundant in tumour-derived EVs. Additionally, we Summary/Conclusion: While imaging flow cytometry generated a CD315-specific antibody as a valuable represent a major advancement in the identification of tool for immunoisolation of distinct EV subclasses. uEVs, our results showed an unexpected additional complication of the analysis originated from the autofluorescence of the uEVs fraction. In fact, The auto- OF12.05 fluorescence quenched the emission of PODXL-AF488 and AF647 but not AF405. uEVs auto-fluorescence needs to be taken into account especially when simul- Analysis of urinary extracellular vesicles auto fluorescence in imaging taneous co-detection of uEVs markers of podocyte flow cytometry and spectral flow cytometry. origin is planned with particular emphasis on the cri- Luca Musantea, Sabrina La Salviaa, Joanne Lanniganb and Uta Erdbrueggerc tical selection of the antibody conjugated fluores- aDepartment of Medicine/Nephrology Division, University of Virginia, Charlottesville, USA; bSchool of Medicine, Flow Cytometry Core, University cent dye. of Virginia, Charlottesville, USA; cUniversity of Virginia Health system, Charlottesville, USA OF12.06 Introduction: Urinary extracellular vesicles (uEVs) provide a source of valuable biomarkers for kidney and urogenital diseases. Analysis of uEVs in imaging Serum vs. plasma: a comparative study in EV composition Razieh Dalir Fardoueia, Rossella Crescitellib, Aleksander Cvjetkovica, Jan flow cytometry is challenging for its intrinsic natural Lötvallc and Cecilia Lasserd auto fluorescence emission across the whole electro- aKrefting Research Centre/University of Gothenburg, Gothenburg, Sweden; magnetic spectrum. To date it is not known what the bKrefting Research Centre, Dept of Internal medicine and clinical nutrition, Institute of Medicine, University of Gothenburg, Sweden, Gothenburg, rate of the autofluorescence interference is with respect Sweden; cKrefting Research Centre, Dept of Internal medicine and clinical to the detection of specific marker uEVs markers nutrition, Institute of Medicine, University of Gothenburg, Sweden, JOURNAL OF EXTRACELLULAR VESICLES 149

Gothenburg, Sweden; 4Krefting Research Centre/University of Gothenburg1 Results: As determined by NTA and protein measure- Krefting Research Centre, Dept of Internal Medicine and Clinical Nutrition, Institute of Medicine, University of Gothenburg, Sweden, Gothenburg, ment more EVs could be isolated from plasma. This Sweden result was supported by experiments were labelled EVs were spiked in to blood, which demonstrated that less Introduction: The ability to isolate extracellular vesi- labelled EVs could be retrieved from serum compared cles (EVs) from blood is paramount in the develop- to plasma. Enough plasma EVs could be isolated for ment of EVs as disease biomarkers. However, this is proteomic analysis from 12 ml blood, which was not complicated by the profuse presence of plasma proteins possible for serum-derived EVs from the same amount and lipoprotein particles, making blood one of most of blood. When larger amount of serum and plasma difficult body fluids to isolate EVs from. We have was used as starting material 1789 proteins could be previously developed a method to isolate EVs from identified in plasma-derived EVs, while only 628 pro- blood with minimal contamination of lipoprotein par- teins could be identified in serum-derived EVs. Both ticles (Karimi et al 2018). The aim of this study was to proteomes were strongly associated with the GO term compare the amount of EVs and their protein cargo “Extracellular exosome”, while the serum derived EVs isolated from plasma and serum. were more associated with “Complement activation”. Methods: Blood was collected from healthy subjects, Summary/Conclusion: This study shows that a larger from which plasma and serum were isolated. EVs were amount of EVs could be isolated from plasma com- isolated using a combination of density cushion and pared to serum. We currently don’t have the explana- size exclusion chromatography (SEC). Purity and yield tion why this is so, however it might be due to the fact of EVs were determined by nanoparticle tracking ana- that EVs get trapped in the clot during serum forma- lysis (NTA), Western blot, electron microscopy (EM), tion. Future studies are needed to answer how this and mass spectrometry (LC-MS/MS). Additionally, affects the use of blood-derived EVs as biomarkers Cy7-labelled cell line-derived EVs were spiked in to from serum and plasma. blood prior to isolation of plasma and serum to compare the recovery. 150 ISEV2019 ABSTRACT BOOK

Symposium Session 13: Stem Cell Derived EVs Chairs: Qingling Fu; Tatiana Lopatina Location: Level 3, Hall B 08:30–10:00

OF13.01 damage, gain replating competency and show increased in vivo repopulation. Summary/Conclusion: Altogether, our studies suggest Extracellular vesicles confer DNA damage on residual long-term HSC that EV miRNA dysregulate proteostasis and confer in the AML niche HSC quiescence in the AML BM. We uncover evidence Sherif Abdelhameda, John Butlerb and Peter Kurrec of long-lasting DNA damage in residual LT-HSC via aKnight Cancer Institute, Oregon Health & Science University, Portland, USA; bMedical Scientist Training Program, Oregon Health & Science AML EV. University, Portland, USA; cChildren’s Hospital of Philadelphia, Funding: Institutional,; Hyundai Hope on Wheels Philadelphia, USA Foundation. Introduction: Acute Myeloid Leukaemia (AML) is a hematopoietic cancer that arises from mutations in hematopoietic stem cells (HSC). Genomewide sequen- OF13.02 cing has revealed that patients harbour multiple leukaemic clones operating in dynamic succession. Extracellular vesicles contribute to the development of ionizing Molecular abnormalities have also been uncovered in radiation-induced late bone marrow pathologies phenotypically normal residual HSC from AML Dávid Kisa, Rita Hargitaib, Nikolett Sándora, Eszter Persaa, Tünde Szatmárib, a b b patients. Independently, several groups showed Enikő Kis , Géza Sáfrány and Katalin Lumniczky enforced quiescence in residual long-term (LT-) HSC aNational Public Health Center, Budapest, Hungary; bNational Public Health Center, Division of Radiobiology and Radiohygiene, Department of Radiation in the AML microenvironment. Neither observation is Medicine, Budapest, Hungary fully understood. Methods: Our previous studies in AML xenografts Introduction: Bone marrow (BM) is a particularly showed that extracellular vesicles (EV) contribute to radiosensitive organ; haematological malignancies, the erosion of hematopoietic progenitor function. myelodysplastic syndrome and chronic bone marrow Here we hypothesized that AML EV may similarly insufficiency are considered long-term consequences of shape fate and function of residual HSC in the AML bone marrow irradiation. Ionizing radiation (IR) niche. We used a combination of in vivo, ex vivo and damages the stem and progenitor cells and alters sig- in vitro approaches and utilizing both AML cell lines nalling between the stem cell compartment and the BM and primary AML patient cells. stroma. The major objective of our work was to inves- Results: We confirmed the relative enrichment of resi- tigate extracellular vesicles (EVs) mediated IR effects in dual HSC in the BM due to gains in quiescence even at the BM and stroma at low and high irradiation doses low leukaemic burden, or following AML EV injec- and to study possible underlying mechanisms using an tions. We also observed in vivo AML-EV trafficking in vivo murine model. to LT-HSC associated with p53 hyperphosphorylation, Methods: C57Bl/6 mice were irradiated with 0.1 Gy or but not apoptosis or senescence. Next, a proteomic 2 Gy and EVs isolated from the BM supernatant were screen of EV-exposed HSPC identified the systematic injected systemically into naive animals. EV-mediated suppression of ribosome biogenesis as the most highly phenotypical changes were determined by flow cyto- enriched Gene Ontology category. The mTOR pathway metry in the stem and progenitor cell compartment governs ribosome biogenesis and protein synthesis, and and in the BM stroma. Apoptosis in various cellular we went on to show that AML-EV trafficking of micro subpopulations was measured by Tunnel assay, DNA RNA-1246 targets Raptor, a pathway component. damage by immunostaining using the γH2AX assay, Translational suppression of Raptor In turn caused senescence by β-gal staining. Oxidative damage was ribosomal protein S6 hypo-phosphorylation and sup- evaluated in the BM cells by measuring protein oxida- pressed protein synthesis. Quiescent HSC are known to tion and lipid peroxidation and systemically by deter- rely on error prone mechanisms of DNA repair, and mining the level of 8-hydroxy-2’ -deoxyguanosine in we demonstrate that residual HSC accrue DNA the urine. JOURNAL OF EXTRACELLULAR VESICLES 151

Results: Treatment of naïve mice with BM-derived EVs Results: Histopathological analysis of Csf1r.iCre; from irradiated animals induced apoptosis in certain Porcnfl/fl mice rectum demonstrated no differences in cellular subpopulations, led to local and systemic oxi- epithelial morphology compared to wild type mice. dative damage, decreased the number of haematopoie- However, exposure to PIR which depletes all RSCs tic and mesenchymal stem cells and of lymphoid demonstrated higher radio-sensitivity and significant progenitors, changed the ratio between the long term damage in rectum epithelium in Csf1r.iCre;Porcnfl/fl and short term stem cells, increased systemic release of mice compared to wild type mice. EV purified from immature progenitors into the circulation. Stroma was Mφ conditioned medium demonstrated presence of less affected; endothelial cells were the most sensitive. functionally active WNT ligands and improve regen- Summary/Conclusion: BM-derived EVs mediated IR- erative capacity of RSCs in both human and mice rectal induced damage in the bone marrow and stroma, organoid model ex-vivo. Treatment with Mφ condi- which raise the role of BM-derived EVs in the devel- tioned medium containing EV promote regenerative opment of IR-induced late BM pathologies. capacity of Lgr5+ ve RSCs in Lgr5/GFP-IRES-Cre- Funding: Euratom research and training programme ERT2 knock-in mice exposed to PIR. However, treat- 2014–2018 under grant agreement No 662287 ment with EV depleted condition medium failed to (CONCERT) rescue RSCs against irradiation. Summary/Conclusion: Homeostasis of rectal epithelium is not dependent on Mφ derived EV packaged OF13.03 WNT. However, Mφ derived EV packaged WNT is critical for regenerative response of RSCs against

Myeloid derived extracellular vesicular WNT induces rectal stem cell injury. regeneration Payel Bhanjaa, Felipe Rodrigueza, Giselle Sanchez Guerreroa and Subhrajit Sahab OF13.04 aKUMC, Kansas City, USA; bDepartment of Radiation Oncology, University of Kansas Medical Center, Kansas City, USA Glycome analysis of extracellular vesicles derived from stem cells using lectin microarray Introduction: Rectal epithelial injury is the major lim- Sayoko Saito, Keiko Hiemori, Kayo Kiyoi and Hiroaki Tateno iting factor for pelvic radiotherapy. Activation of National Institute of Advanced Industrial Science and Technology, Tsukuba, regenerative response of rectal stem cells (RSCs) is Japan critical to mitigate radiation injury. Wnt β catenin signalling plays a critical role in homeostasis and Introduction: In addition to proteins, nucleic acids regeneration of intestinal stem cell (ISC). Both epithe- and lipids, extracellular vesicles (EVs) are also com- lium and stroma are the major source of WNT ligands. posed of glycans. EV glycome may provide vital clues Intestinal stroma consists of several cell types including for a better understanding the biogenesis, release and mesenchymal cells and myeloid/macrophages (Mφ). transfer of vesicles. However, little is known regarding Genetic or pharmacological inhibition of WNT release glycans on EVs. Do glycans on EVs change depending from mesenchymal stromal cells did not affect the ISC on cell types and cellular conditions? More specifically, homeostasis or regeneration. In the present study we do stem cell-derived EVs carry stem cell glycan mar- have examined the effect of Mφ derived extracellular kers? Such basic questions remain unclear. vesicle (EV) packaged WNT in homeostasis and repair Methods: Here, we performed glycome analysis of EVs of RSCs. derived from stem cells including human induced plur- Methods: Csf1r.iCre;Porcnfl/fl mice deficient in Mφ ipotent stem cells (hiPSCs) and human messenchymal derived WNT due to Mφ-restricted ablation of stem cells (hMSCs) using high-density lectin microar- Porcupine, a gene essential for WNT synthesis were ray and flow cytometry. used to determine effect of Mφ derived in EV-WNT in Results: Detailed analysis of the results obtained by RSC homeostasis and regeneration. Mice were exposed lectin microarray and flow cytometry revealed that to lethal dose of pelvic irradiation (PIR) (18Gy) to hiPSC-derived EVs carry characteristic features of cell deplete RSCs and therefore evaluate the regenerative surface glycans. rBC2LCN, a specific lectin for hPSCs, response following treatment with Mφ derived EV bound to hiPSC-derived EVs, but not to non-hiPSC- packaged WNT. Effect of Mφ-EV WNT on RSCs derived EVs. One of the glycoprotein ligands of were also examined in ex-vivo rectal organoid system rBC2LCN on EVs was identified as podocalyxin, developed from Lgr5/GFP-IRES-Cre-ERT2 knock-in which is a cell surface glycoprotein ligand of for visualization and quantification of Lgr5+ve RSCs. rBC2LCN. Other hiPSC surface glycan markers were 152 ISEV2019 ABSTRACT BOOK also detected on the surface of EVs. Finally, we devel- muscle. Additionally, monocyte and macrophage oped a sandwich assay to specifically detect hiPSC- depletion through clodronate treatment completely derived EVs using rBC2LCN and Tim4, which binds abrogated the pro angiogenic effect of huMSC-Exo. to phosphatidylserine (PS). rBC2LCN is useful for the Summary/Conclusion: This study demonstrates, for specific detection of hiPSC-derived EVs. the first time, that huMSC derived exosomes enhance Summary/Conclusion: The EV glycome reflects its the angiogenic process in the radiation-induced cellular origin, which could be a novel target for the ischemic tissue by stimulating the mobilization and development of the quality control system of stem cells recruitment of innate cells to the lesion and nurturing used for regenerative medicine. neovascularization. These results highlight the concept Funding: JST CREST that huMSC-Exo administration represents a suitable innovative approach for therapeutic angiogenesis in irradiated tissue. OF13.05

Exosomes derived from human MSC mediate monocyte mobilization OF13.06 to orchestrate neovascularization in radiation-induced skin injury Alexandre Ribaulta, Bruno Lhommea, Celine Loinarda, Marc Benderittera, Stephane Flamanta, Ruenn Chai Laib, Sai Kiang Limc and Radia Tamarata hucMSCs derived exosomes enhance lymphangiogenesis in aIRSN, Paris, France; bIMB ASTAR, Singapore, USA; cInstitute of Medical experimental lymphedema via exosomal transfer of Ang-2 and Tie2 Biology, Agency for Science, Technology and Research, Singapore, Singapore Ting Zhao and Yongmin Yan Jiangsu University, Zhenjiang, China (People’s Republic) Introduction: Emerging evidences indicate that extracellular membrane vesicles, such as exosomes, could Introduction: Exosomes are small biological mem- recapitulate the therapeutic effects of huMSC. Of brane vesicles secreted by cells, including MSCs. Here, note, exosomes displayed marked pro-angiogenic activ- we evaluated lymphangiogenic potential and key exo- ity, however a better understanding of their underlying somal prolymphangiogenic factors of human umbilical mechanisms of action remained to be defined. This cord MSC-derived exosomes (hucMSC-Ex) to provid- study aims to investigate the mechanisms governing ing a mechanistic basis for optimizing future hucMSC- the pro-angiogenic effects of huMSC derived exosomes Ex-based lymphedema therapies. (huMSC-Exo) in a mouse model of radiation-induced Methods: hucMSC-Ex were extracted from condition musculo-cutaneous injury. medium of hucMSCs. Using a murine lymphedema Methods:Micelowerlimbwasexposedto80GyX-ray model, we evaluated oedema at various time points irradiation to induce radiation injury. After 14 days, mice post hucMSC-Ex injection. HE stain and received an intramuscular injection of 106 human MSCs, Immunohistochemical stain were applied to analyse 400 µg MSC-EXO, or PBS. Angiogenesis was estimated the lymphaniogenesis. In vitro, human dermal lympha- by skin perfusion (laser Doppler imaging), immunohis- tic endothelial cells (HDLECs) were treated with tochemistry (CD 31 endothelial marker) and microangio- hucMSC-Ex, and cell proliferation, migration and graphy (barium sulphate). Mice were sacrificed at several tube formation were assayed using cell counting Kit-8 time points, and tissues of both irradiated and contral- (CCK-8), transwell chamber inserts, and matrigel- ateral limbs were harvested for histological and biochem- based tube formation assays, respectively. Western ical analyses. Bone marrow, spleen and blood were blot and immunofluorescence stain were performed collected for analysis of inflammatory cells and circulating to test the expression level of proteins which were factors. In vitro assays were used to validate the pro associated with lymphaniogenesis after co-cultured angiogenic effet of HuMSC- exo. with hucMSC-Ex in HDLECs. Results: The huMSC-Exo stimulated vascular growth Results: Mice treated with hucMSC-Ex showed signifi- as revealed by the increase in cutaneous blood perfu- cantly decreased oedema formation and restored drai- sion, capillary density and angiographic score with nage of intradermally injected methylene blue after 6 stimulation of pro-angiogenic factor levels such as weekly injections. HE stain showed subcutaneous VEGF-A and eNOS. In vitro, huMSC-Exo fostered oedema of tail faded obviously after hucMSC-Ex injec- endothelial cells and fibroblast migration in a PI3K/ tion. Immunohistochemical analysis revealed that mice AKT and TGF-β/SMAD2 dependent pathways. Finally, tails receiving hucMSC-Ex injections had enhanced huMSC-Exo triggered the mobilization of both Ly6Chi lymphangiogenesis compared to the PBS-treated and Ly6Clo monocytes from the spleen and the bone groups as determined by staining of lymphatic marker marrow and their recruitment into the irradiated LYVE-1. The proliferation, migration, and tube JOURNAL OF EXTRACELLULAR VESICLES 153 formation of HDLECs were significantly increased by Summary/Conclusion: Ang-2 and Tie2 are essential hucMSC-Ex. Also, the expression level of Ang-2, for hucMSC-Ex effects on lymphangiogenesis in vitro Lyve1, Prox1, VEGFR3, p-Akt in HDLECs was and in vivo. up-regulated both in western blot and Funding: Zhenjiang Key Laboratory of Exosomes Immunofluorescence stain. Mechanically, hucMSC-Ex Foundation and Transformation Application High- derived Ang-2 and Tie2 proteins were transferred to tech Research,china: (ss2018003);National Natural HDLECs. Ang-2 controlled the proliferation, migration Science Foundation of China: (81670549) and tube formation of HDLECs. And hucMSC-Ex delivered Ang-2 and Tie2 activated the expression of lymphangiogenic factors. 154 ISEV2019 ABSTRACT BOOK

Symposium Session 14: Parasite and Bacterial EVs Chairs: Yong Song Gho; Mariko Ikuo Location: Level B1, Hall A 08:30–10:00

OF14.01 on our -omics study, we identify Salmonella antigens and other pro-inflammatory molecules in exosomes isolated from Salmonella infected-macrophages from Macrophage-derived exosomes encapsulate Salmonella antigens and stimulate the activation of Type 1 T-helper cells in vivo 24 and 48 h infections. Hence, the cargo plays a critical Winnie W. Huia, Mark Oub, Beata Clappc, David Pascualc and Mariola a role in intercellular communication in response to Edelmann infection as naïve macrophages treated with these exo- a University of Florida Dept of Microbiology and Cell Science, Gainesville, somes result in M1 polarization. USA; bUniversity of Florida Dept of Microbiobiology and Cell Science, Gainesville, USA; cUniversity of Florida Dept of Infectious Disease, Summary/Conclusion: Our data support the hypoth- Gainesville, USA esis that exosomes isolated from Salmonella infected macrophages carry Salmonella antigens as a cargo and Introduction: Salmonella enterica serovar stimulates the activation of Type 1 effector T Typhimurium is a Gram-negative, intracellular bacter- lymphocytes. ium which invades macrophages and leads to the production of pro-inflammatory exosomes. S. Typhimurium is the causative agent of salmonellosis OF14.02 affecting 1.2 million people annually in the USA. There are no FDA approved vaccines against non- typhoidal Salmonella infections for human thus show- Extracellular vesicles from Leishmania donovani infected macrophages contain infection-specific cargo that contribute to ing a significant limitation in current prevention meth- lesion development ods. Exosomes are a subclass of extracellular vesicles Anna E. Gioseffi and Peter Kima characterized by their size, morphology and biogenesis. University of Florida, Gainesville, USA The cargo, including protein, nucleic acids and metabolites, carried by exosomes vary depending on the Introduction: Extracellular vesicles (EVs) have physiological conditions present and the origin of the emerged as important mediators of cell-to-cell commu- cell. We hypothesize that during Salmonella infections, nication and have been shown to contribute to the exosomes transport Salmonella antigen to alert neigh- pathogenesis of infectious microorganisms. bouring cells which can lead to the stimulation of naïve Leishmania is an intracellular eukaryotic parasite and T-lymphocytes. causative agent of leishmaniasis. This work aims to Methods: We focus on the release of exosomes by S. evaluate EVs in the context of Leishmania donovani Typhimurium-infected macrophages and their function infection. in stimulating an adaptive immune response in vivo. Methods: To better understand the properties and To determine if exosomes have any effect on the adap- function of EVs produced by L. donovani infected tive immune response, mice were given doses of exo- RAW264.7 macrophages (iEVs), we used a series of somes derived from S. Typhimurium infected approaches, including comparative proteomics of macrophage. Fluorescent activated cell sorting was iEVs or EVs derived from uninfected RAW 264.7 used to monitor T- lymphocyte response. macrophages, pathway analysis to infer activity, and Results: Exosomes stimulate a distinct cytokine secre- functional assays such as in vitro migration assays tion pattern among CD4+T lymphocytes in vivo. The and flow cytometry to evaluate endothelial cell activa- cytokines milieu, including IFN-, TNF- and IL-2, tion after EV treatment. expression by T-lymphocytes suggest that the CD4 T- Results: We obtained a profile of host and parasite lymphocytes differentiated in to Type 1 T-helper set proteins in iEVs, EVs from uninfected macrophages, producing pro-inflammatory cytokines. Additionally, and EVs from macrophages infected with Centrin mouse serum was taken to analyse for antibody pro- knockout (CenLd) parasites. CenLd parasites are duction against Salmonella in which we observe exo- unable to mature into the amastigote form within somes derived from Salmonella infected cells provide a macrophages. In addition to host derived molecules similar antibody production to the live vaccine. Based previously identified by others in exosome JOURNAL OF EXTRACELLULAR VESICLES 155 preparations, we identified host and parasite derived Results: Altered DBH mRNA expression was identified molecules, such as parasite PI3K, vasohibin, and serine/ during T. gondii infection in rat catecholaminergic and threonine protein phosphatase, and mouse histone 2B, human neuronal cells. This down-regulation of DBH annexin A3, and galectin-3 within iEVs. Our results was identified in de novo RNA, suggesting that regula- showed that EVs from macrophages infected with tion occurs at the transcriptional level. Using MSRE- CenLd parasites have a molecular composition that is qPCR, hypermethylation in the 5‘ upstream region of qualitatively different from iEVs released by macro- the DBH promoter was identified in infected rat cate- phages infected with wild type parasites. Pathway ana- cholaminergic cells and human neuronal cells. lysis of the host-derived proteins in iEVs suggested Surprisingly, DBH mRNA down-regulation and their involvement in cell migration and neovasculariza- methylation in the 5’ promoter were globally altered tion. In vitro cell migration assays validated the obser- in vivo, despite the fact that only a small number of vation that intact iEVs enhance cell migration, which is cysts can be identified in the host brain. DBH silencing in contrast to EVs from Cen-/-Ld infected macrophage was observed in cells only exposed to infected cells. or disrupted iEVs that do not induce cell migration. In Extracellular vesicles purified from infected rat cate- addition, flow cytometry analysis of endothelial cells cholaminergic cells induced transcriptional silencing of treated with iEVs suggested that EVs from infected DBH. This effect could also be generated in vivo when macrophages activate endothelial cells. The latter rats received intracererbral injections with purified observations were surrogate indicators of pathogenic extracellular vesicles from infected cells. This repre- neovascularization. sents a new perspective of the host-pathogen Summary/Conclusion: Taken together, we present a interaction. comprehensive molecular profile of the molecules Summary/Conclusion: Through this mechanism T. released in EVs from L. donovani-infected macro- gondii may be able to induce many of neurophysiolo- phages which includes infection-specific molecules gical changes associated with chronic infection. In that contribute to lesion development. addition, T. gondii is a unique model to study mam- Funding: NIH. malian neurological function, by examining the influence the parasite is able to exert over cell-cell communication, we may be able to further understand OF14.03 the mechanisms governing CNS function and dysfunction. Funding: The University of Leeds.

The role of extracellular vesicles in neurophysiological changes induced by chronic Toxoplasma gondii infection Ellie Tedforda and Glenn McConkeyb OF14.04 aUniversity of Cambridge, Cambridge, UK; bUniversity of Leeds, Leeds, USA Bacterial membrane vesicles as vaccines in aquaculture Introduction: During chronic Toxoplasma gondii Julia Tandberga, Leidy Lagosb, Alexander Kashulin-Bekkelundc, Petter a a infection, parasites become encysted in neurons in the Langlete and Hanne C. Winther-Larsen host brain. Infection is associated with a plethora of aUniversity of Oslo, Oslo, Norway; bNorwegian University of Life Sciences, Ås, Norway; cNorwegian University of Life Sciences, Oslo, Norway behavioural and neurophysiological changes despite only a small number of neurons infected. Introduction: Infections by two Gram-negative intra- Perturbations in dopamine metabolism have been cellular bacterial pathogens Piscirickettsia salmonis and observed with infection. We have previously described Francisella noatunensis, are causing major problems in parasite induced down-regulation of dopamine β- aquaculture world-wide. F. noatunensis sp hampers the hydroxylase. In this study, the role of extracellular development of fish farming based on cod in and is vesicles during chronic T. gondii infection was deleterious to tilapia. P. salmonis infections have been investigated. devastating for salmon aquaculture. As of today no Methods: Primarily transwell culturing and extracellu- effective treatments are available against the diseases. lar vesicle purification via ultracentrifugation were Both P. salmonis and F. noatunensis secrete membrane used during this study. Extracellular vesicles were col- vesicles (MV). Bacterial MV has been reported as lected from chronically infected human and rat cate- potential vaccine candidates for a range of host includ- cholaminergic cells. Purification via a 12 step sucrose ing humans, mice and fish against infection caused by gradient was performed prior to conditioning in vitro intracellular pathogenic bacteria as they induce both a and in vivo. humoral and cellular immunity. 156 ISEV2019 ABSTRACT BOOK

Methods: We have isolated MVs from both Francisella resistance and apical-basolateral polarity of normal and Piscirickettsia by the ultracentrifugation Method. epithelium. For this, colonic epithelial cells of the T84 The MVs were characterized by their size distribution, line were grown on Transwell filters to generate trans- by transmission electron microscopy (TEM) and pro- epithelial electrical resistance (TEER), a measure of teomics. Their toxicity were tested by injecting MVs epithelial monolayer integrity. The cells were then co- into both our zebrafish vaccine and challenge model as cultured with Alexa Fluor-labelled OMVs from the well as in cod, tilapia and salmon. A vaccine trail was gastric pathogen, Helicobacter pylori. performed first in our zebrafish model, and then in Results: We showed that H. pylori OMVs readily cod, tilapia and salmon. entered polarised epithelial cells, but had no effect on Results: The MV size analysis showed that the MVs the TEER nor permeability of these monolayers. OMVs size distribution ranged from 20–250 nm in size with induced the basolateral secretion of the neutrophil most ranging from 70–100 nm. Both single and double chemoattractant, interleukin-8 (IL-8) and expression membrane MV were found in the population as inves- of human leukocyte antigen class I and II molecules. tigated by TEM. Further, immune-gold labelling In exosomes isolated from the basolateral compartment revealed the presence of DNA in both populations. of OMV-stimulated cells, we identified peptides Proteomics analysis revealed that the MV content var- derived from eight H. pylori proteins, of which seven ied between bacterial strains. Immunization with MV are surface- or membrane-associated and are known to gave protection against disease caused by both P. sal- localise within OMVs. monis and F. noatunensis in our zebrafish model, how- Summary/Conclusion: Collectively, the data show that ever, did not protect cod, tilapia nor salmon. OMVs can enter polarised epithelial cells and deliver Summary/Conclusion: The MVs from P. salmonis and their protein cargo to exosomes. We propose that these F. noatunensis revealed a similar size distribution and exosomes may directly or indirectly present antigen to that the content contains various bacterial virulence immune cells and even transport bacterial proteins to factors as well as DNA that can be transferred to the other tissue sites. host. As for their immunogenic properties this seems Funding: This project was supported by funding from to vary between the vaccine and challenge model com- the National Health and Medical Research Council pared to the natural hosts. The use of the MVs as (NHMRC), the Australian Research Council, The vaccines in their natural hosts such as strain-specificity Juvenile Diabetes Research Foundation and the and cross-immunity need further investigation. Victorian Government’s Operational Infrastructure Funding: Research Council of Norway (RCN) and Support Program. R.L.F. is supported by an NHMRC University of Oslo. Senior Research Fellowship. N.S. is funded through a Canadian MSFHR Research Trainee Fellowship and an NHMRC Early Career Fellowship. L.T. was funded by OF14.05 an Australian Postgraduate Award and an Excellence Award from Monash University FMNHS.

Bacterial membrane vesicles enter polarised epithelial cells and deliver their protein cargo to exosomes Lorinda Turnera, Nestor Solisb, Georg Rammc, Viola Oorschotc, Amanda De OF14.06 Paolia, Hassan Chaudhrya, Stuart Manneringd, Stuart Cordwellb, Maria Kaparakis-Liaskose and Richard Ferreroa Bacterial Extracellular Vesicles: intercellular package or intracellular aHudson Institute of Medical Research, Melbourne, Australia; bThe c garbage? The example of RNAs associated to Salmonella enterica EVs University of Sydney, Sydney, Australia; Monash University, Melbourne, a a b a d Antoine Malabirade , Janine Habier , Anna Heintz-Buschart , Patrick May , Australia; St. Vincent’s Institute of Medical Research, Melbourne, c a d d a 5 Julien Godet , Rashi Halder , Alton Etheridge , David Galas , Joëlle V. Fritz Australia; Department of Physiology, Anatomy and Microbiology, La and Paul Wilmesa Trobe University, Melbourne, Australia aLuxembourg Centre for Systems Biomedicine, University of Luxembourg, b Introduction: Gram-negative bacteria use outer mem- Esch-sur-Alzette, Luxembourg, Belval, Luxembourg; Department of Soil Ecology, Helmholtz-Centre for Environmental Research – UFZ, Halle, brane vesicles (OMVs) to deliver a range of factors to Germany, Belval, Luxembourg; cUMR CNRS 7021, Laboratoire de host cells. Although OMVs are highly effective at enter- BioImagerie et Pathologies, Université de Strasbourg, Strasbourg, France, Strasbourg, France; dPacific Northwest Research Institute, Seattle, WA, ing simple non-polarised cell monolayers, it is not United States, Seattle, USA known whether these nano-sized vesicles can penetrate an intact epithelial barrier and, potentially, disseminate Introduction: Bacteria have developed many ways of their protein cargo to tissues. communicating with one another and with other pro- Methods: We have addressed this question using a cell karyotic or eukaryotic species. The secretion of culture model that reproduces the transepithelial Extracellular Vesicles (EVs) is one of them. Bacterial JOURNAL OF EXTRACELLULAR VESICLES 157

EVs are small spherical containers filled with a wide comparison with the intracellular RNA composition, range of biomolecules originating from the mother cell, our data demonstrate that a proportion of RNAs including RNAs. The protection conferred by the phy- exported through EV secretion were enriched. This sical envelope of EVs to these delicate components is of export is depending on the environmental conditions prime importance for message delivery to other cells. and reflects the adaptation to each infection step. Some However, this idea of EVs being mail carriers competes transcripts were confirmed to be in their native state with the concept of a simple trash bin used by bacteria and not degradation products, opening the possibility to get rid of unnecessary components. for a functional RNA delivery to surrounding cells. Methods: Taking Salmonella enterica as an example, Finally, we show by a digestion protection assay that we purified EVs and sequenced their RNA content. vesicles prevent enzymatic degradation of given full- The strain was cultivated in different conditions length transcripts (SsrS, CsrC, 10Sa and rnpB). mimicking separate stages of a gut infection. Growth Summary/conclusion: These results reinforce the idea until stationary phage in Lysogeny Broth (LB) medium of a complex interaction network existing in the gut induces Salmonella pathogenicity island 1 (SPI-1), microbiome and more generally in microbial which is required for virulence during the intestinal ecosystems. phase of infection. Growth in acidic and phosphate- Funding: Luxembourg National Research Fund (FNR) depleted medium triggers the expression of Salmonella (CORE Junior/14/BM/8066232, CORE/15/BM/ pathogenicity island 2 (SPI-2) and is comparable to the 10404093, CORE/16/BM/11276306), NIH Common macrophage environment. Fund Extracellular RNA Communication Consortium Results: Every type of RNA was exported, including (1U01HL126496), Baylor subaward (5U54DA036134). ribosomal, messenger and non-coding RNAs. By 158 ISEV2019 ABSTRACT BOOK

Plenary Session 2: Therapeutics Chairs: Edit Buzás; Uta Erdbrügger Location: Level 3, Hall B 11:54–11:55

Self-assembled supramolecular nanosystems for Smart diagnosis and reagents were developed, opening a new avenue for targeted therapy of intractable diseases the novel type of theranostic nanomedicines12. Kazunori Kataoka Furthermore, nanosysems hold promise for the treat- Innovation Center of NanoMedicine, Kawasaki Institute of Industrial Promotion, Kawasaki 210-0821, Japan; Institute for Future Initiatives, The ment of intractable diseases other than cancer. Very University of Tokyo, Tokyo113-0033 recently, we developed nanosystems decorated with [email protected] glucose to crossing blood-brain barrier by recognizing glucose-transporter overexpressing on brain endothe- Nanotechnology-based medicine (Nanomedicine) has lial cells, indicating a novel route to deliver versatile received progressive interest for the treatment of drugs into brain for the treatment of neurodegenerative intractable diseases, such as cancer, as well as for the diseases, including Alzheimer’s disease13. non-invasive diagnosis through various imaging mod- [1] H. Cabral and K. Kataoka, J. Control. Rel. 190 alities. Engineered polymeric nanosystems with smart (2014) 465–476; [2] Y. Matsumoto, et al, Nature functions play a key role in nanomedicine as drug Nanotech. 11 (2016) 533–538; [3] H. Cabral, K. carriers, gene vectors and imaging probes. This presen- Miyata, K. Osada, and K. Kataoka, Chem. Rev. 118 tation focuses present status and future trends of (2018) 6844–6892; [4] H. Cabral, et al, Nature supramolecular nanosystems self-assembled from Nanotech. 6 (2011) 815–823; [5] Y. Miura, et al, ACS designed block copolymers for therapy and non-inva- Nano 7 (2013) 8583–8592; [6] H. Kinoh, et al, ACS sive diagnosis of intractable diseases. Nanosystems with Nano 10 (2016) 5643–5655; [7] N. Nishiyama, et al, 10 to 100 nm in size can be prepared by programmed Cancer Sci. 107 (2016) 867–874; [8] K. Miyata, et al, self-assembly of block copolymers in aqueous entity. Chem. Soc. Rev. 41 (2012) 2562–2574; [9] Y. Yi, et al, J. Most typical example is polymeric micelle (PM) with Control. Rel. 295 (2019) 268–277; [10] K. Katsushima, distinctive core-shell architecture. PMs have several et al, Nature Commun. 7 (2016) 13616; [11] B.-S. Kim, properties relevant for nanosystems, including con- et al, J. Amer. Chem. Soc. 141 (2019) 3699; [12] P. Mi, trolled drug release, tissue penetrating ability, and et al, Nature Nanotech. 11 (2016) 724–730; [13] Y. reduced toxicity1,2. Furthermore, smart functionalities, Anraku et al, Nature Commun. 8 (2017) 1001. such as pH- and/or redox potential responding properties, can be integrated into the PM structure3. These smart PMs loaded with various chemotherapy reagents MSC-sEV translation: back to basics were evidenced to have a significant utility in the Sai Kiang Lim treatment of intractable and metastatic cancers, includ- Institute of Medical Biology (IMB) ing pancreatic cancer4, glioblastoma5 and tumours harbouring recalcitrant cancer stem cells (CSCs)6. Mesenchymal stromal cell (MSC) are presently the Eventually, five different formulations of the PMs most used cell type in clinical testing and are being developed in our group have already been in clinical tested against a wide spectrum of diseases. Their ther- trials world-wide, including Japan, Asia, USA and apeutic efficacy is increasingly shown to be mediated European countries7. by their secretion and in particular, the secreted extra- Versatility in drug incorporation is another relevant cellular vesicles (sEVs) of 50–200 nm. In this talk, I will feature of supramolecular nanosystems for drug deliv- elaborate on the development of clinical applications ery. Nucleic acid-based medicine can be assembled into for MSC sEVs and the associated challenges. A major nanosytstems through the electrostatic interaction with challenge is the complexity of a typical MSC-sEV pre- oppositely-charged polycationic block copolymers8.In paration. As the size ranges of many EV types such as this way, siRNA- or antisense oligo (ASO)-loaded exosomes, microvesicles, and ectosomes overlap signif- micellar or vesicular nanosystems were prepared, and icantly and most EV types include EVs of 50–200 nm, their utility in molecular therapy of cancer has been the term “sEVs” essentially describes a complex popu- revealed9-11. Recently, nanosystem-based imaging lation of similarly sized EVs consisting of many known JOURNAL OF EXTRACELLULAR VESICLES 159 and possibly unknown EV types. This complexity is the ISCT, ISEV, ISBT and SOCRATES have recently further compounded by the heterogeneity in the source proposed several metrics to quantify distinctive fea- and culture of MSCs, and in the downstream proces- tures of a MSC-sEV preparation that will identify the sing of MSC secretion. Together, this poses a challenge cellular origin of the sEVs in a preparation, presence of to data sharing by the research community and to the lipid-membrane vesicles, and the degree of physical regulation of MSC sEVs as therapeutic products. and biochemical integrity of the vesicles. Such metrics Unfortunately, resolution of this conundrum through will facilitate comparison among different MSC-sEV process standardization or purification of specific EV preparation as differences could then be mapped to type is presently not practical and/or technically chal- quantified differences in the features. The biological lenging. To circumvent this, about 20 members from significance of such mapping would then be testable. 160 ISEV2019 ABSTRACT BOOK

Featured Abstracts- Session 1 Chairs: Edit Buzás; Uta Erdbrügger Location: Level 3, Hall B 11:55–12:30

Summary/Conclusion: Our findings provide a concep- FA1.01 tual advance in the understanding of the biogenesis and function of EVs, identifying BAG6 as an ESCRT- associated protein and a molecular switch for the for- Molecular basis for contradictive roles of melanoma-derived EVs in mation of anti- versus pro-tumourigenic EVs in metastasis Maximiliane Schuldnera and Elke Pogge von Strandmannb tumour immune surveillance. aExperimental Tumor Research, Center for Tumor Biology and Immunology, Clinic for Hematology, Oncology and Immunology, Philipps University of Marburg, Marburg, Cologne, Germany, Germany; bExperimental Tumor FA1.02 Research, Center for Tumor Biology and Immunology, Department of Hematology, Oncology and Immunology, Philipps University Marburg, Marburg, Germany Development of a live-cell imaging technique for secretion activity of extracellular vesicles of individual cells Introduction: Recent studies have highlighted the role Yoshitaka Shirasakia, Keisuke Tsukadab, Nobutake Suzukic, Tamiko Minamisawad, Mai Yamagishie, Nobuyoshi Kosakaf, Takahiro Ochiyaf, of melanoma cell-derived EVs in the formation of pre- Osamu Oharag, Kiyotaka Shibah and Sotaro Uemurai metastatic niches or, on the contrary, in tumour aJST PRESTO, Tokyo, Japan; bThe University of Tokyo, bunkyo, Japan; cThe immune surveillance. The molecular machinery and university of Tokyo, Bunkyo-ku, Japan; dJapanese Foundation For Cancer e mechanisms directing distinct cargo loading, regula- Research, Koto-ku, Japan; Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Japan; fDepartment of tory release and function of stress-induced EVs remain Molecular and Cellular Medicine, Institute of Medical Science, Tokyo unknown. Medical University, Shinjyuku-ku, Japan; gRIKEN Institute for Integrative Medical Sciences, Yokohama, Japan; hJapaese Foundation for Cancer Methods: EV release was quantified by NTA. EVs were research, Tokyo, Japan; iThe University of Tokyo, Tokyo, Japan isolated by ultracentrifugation and analysed by proteomics and transcriptomics. EV function was investigated Introduction: The cells in our body exchange their in vivo by intravenous injections followed by lung information using various methods to control the transcriptomics and by using an experimental metas- expression of functions, to form higher order systems tasis transplantation model. The mechanistic release of and to maintain homeostasis. Particularly in the com- EVs was analysed using diverse molecular, cell biologi- munication between spatially separated cells, mediation cal, spectroscopic and microscopic techniques. of humoral factors such as cytokines can be mentioned. Results: Our study reveals a crucial role of the chaper- Addition to this, extracellular vesicles (EVs) have been one and NK cell ligand BAG6 for the formation and reported to participate in intercellular communication. reprogramming of pro- and anti-tumour EVs. Loss of The EVs classically include exosomes, microvesicles BAG6 led to an increase in EV production and a and apoptotic bodies. Also, vesicular autophagosome decrease in EV size. In contrast to the melanosome- and transport vesicles in the intercellular space might like protein signature observed for WT-EVs, BAG6KO- be released during necrosis-like cell death (pyroptosis, EVs showed an exosome-like profile and induced a necroptosis etc.). Although conventional biochemical neutrophil gene signature in the lungs of mice. methods can classify these EVs by size, density or Education with B-16V WT-EVs, but not BAG6KO- antigens on their membrane, it is difficult to distin- EVs, suppressed lung metastasis concomittant with guish individual vesicles depending on their biogenesis. the accumulation of anti-tumour Ly6Clow patrolling Methods: We have developed LCI-S (Live Cell Imaging monocytes. Mechanistically, the formation of anti- for Secretion activity), which is a time-resolved micro- tumour EVs was dependent on BAG6 mediating the scopic observation technology of secreted humoral fac- nucleo-cytoplasmic shuttling of CBP/p300 acetyltrans- tors from individual cells. LCI-S utilises sandwich ferases to acetylate p53. We have identified a late fluoroimmunoassay and in situ detection of the immu- endosomal P(S/T)AP motif in BAG6 which mediated nocomplex by total internal reflection microscopy. its direct recruitment to the ESCRT machinery, thereby LCI-S reveals intercellular variability of secretion activ- providing a molecular link between the regulatory role ity and time-correlation with the cellular state such as of BAG6 to EV cargo loading. intracellular enzymatic activity, cell shape or viability. JOURNAL OF EXTRACELLULAR VESICLES 161

In this study, we focused on the release of EVs having was not uniform nor constant. Some cells showed a the same topology as cell membranes such as exo- burst secretion mode, and some showed an acceler- somes, targeting membrane surface antigens such as ated secretion mode. The cells bursting free-EVs CD63 and CD9. tended to be associated with membrane blebbing, Results:WehaveevaluatedEVssecretionfrom suggesting that they were in an excessive stress kinds of cancer cell lines and succeeded in detecting state. vesicle-like adherent plaque under the cells and also Summary/Conclusion: In this study, we demonstrated free-diffusion type vesicles (free-EVs). We also that LCI-S has the potential to distinguish individual found that the minor population showed higher EVs depending on their biogenesis. secretion activity of free-EVs in our experimental Funding: This research was supported by JST, condition. Furthermore, free-EVs secretion activity PRESTO Grant Number JP17940748, Japan. 162 ISEV2019 ABSTRACT BOOK

Symposium Session 15: EVs in Cancer Chairs: Takahiro Ochiya; Carolina Soekmadji Location: Level 3, Hall B 13:30–15:00

OF15.01 and dramatically enhance the growth of tumour colonoids. Summary/Conclusion: This study describes a simpli- Transfer of functional cargo in exomeres fied method for exomere isolation and provides the a b a a Qin Zhang , James Higginbotham , Dennis Jeppesen , Yu-Ping Yang , Wei first demonstration of transfer of functional cargo by Lib, Ramona Graves-Deala, Jie Pinga, Colleen Britainc, Kaitlyn Dorsettc, Celine Hartmand, David Fordd, Ryan Allena, Kasey Vickersa, Qi Liua, exomeres and underscores the functional heterogeneity Jeffrey Franklina, Susan Bellisc and Robert Coffeya of secreted nanoparticles. aVanderbilt University Medical Center, Nashville, USA; bVanderbilt University Medical Center, Nashville, USA; cUniversity of Alabama at Birmingham, Birmingham, USA; dSaint Louis University School of Medicine, Saint Louis, USA OF15.02 Introduction: There is an increasing appreciation that secreted nanoparticles are a heterogeneous mixture of Exosomes secreted from senescent cells provoke chromosomal distinct entities. One newly identified nanoparticle, instability exomeres (< 50 nm), were recently identified by the Kenichi Miyataa, Kazuhiro Hitomia, Tomoka Misawaa, Ryo Okadaa and Akiko Takahashib Lyden lab using asymmetric flow field-flow fractiona- aProject for Cellular Senescence, Cancer Institute, Japanese Foundation for tion (AF4), a method that is not widely available. No b Cancer Research, Tokyo, Japan; Project for Cellular Senescence, Cancer known biological function has been assigned to these Institute, Japanese Foundation for Cancer Research, Koto-ku, Japan nanoparticles. In this study, we employed a simplified ultracentrifugation method to isolate and characterize Introduction: Cellular senescence is the state of irre- subpopulations of exomeres and distinguish them from versible cell cycle arrest that can be induced by a exosomes. variety of potentially oncogenic stimuli and is therefore Methods: A two-step ultracentrifugation method was considered to act as an important tumour suppression used to separate exomeres from exosomes. Purified mechanism in vivo. However, cellular senescence is exomeres were characterized by NTA, TEM, proteo- also associated with the increasing expression and mics, lipidomics, DNA and RNA analysis Cell surface secretion of inflammatory and pro-proliferative factors. target sialylation by exomeres was measured by flow This phenotype, termed the senescence-associated cytometry using fluorescence-labelled SNA lectin. secretory phenotype (SASP), contributes to cancer Subpopulations of exosomes were purified by fluores- development. In addition to inflammatory proteins, cence-activated vesicle sorting (FAVS) and analysed for we reported that exosome secretion has dramatically distinguishing cargos. Normal and neoplastic mouse increased in senescent cells, acting as harmful SASP colonic organoids were used for functional studies factors. Recently, we found that senescence-associated comparing exosome and exomere activities. non-coding RNAs (SA-ncRNA) are enriched in exo- Results: Our analysis of the content of exomeres lar- somes and these exosomes provoke chromosomal gely confirms what has been reported by Lyden and co- instability in normal cells. workers. We identify distinct functions of exomeres Methods: Pre-senescent normal human diploid fibro- mediated by two of their cargos, the β-galactoside α2, blasts were rendered senescent by either serial passage, 6-sialyltransferase 1 (ST6Gal-I) that α2,6- sialylates N- ectopic expression of oncogene or X-ray irradiation. glycans, and the EGF Receptor (EGFR) ligand, amphir- Then we collected the exosomes secreted from young egulin (AREG). Functional ST6Gal-I in exomeres can or senescent cells and checked the component of exo- be transferred to recipient cells resulting in hypersialy- somes. To analyse the biological function of these exo- lation of cell surface proteins, including β1-integrin. somes, colony formation analysis and karyotype AREG-containing exomeres elicit prolonged EGFR analysis were performed. Additionally, we manipulated and downstream signalling in recipient cells, modulate SA-ncRNA to load into exosome using Exotic devise, EGFR trafficking in mouse-derived colonic organoids, then investigated the biological roles of them. JOURNAL OF EXTRACELLULAR VESICLES 163

Results: We found that epigenetic de-regulation of Results: Preliminary experiments with PKH67-stained genomic DNA induces the aberrant expression of NB-derived EV injected i.v. showed that after 24 h 0.9– non-coding RNA in senescent cells and SA-ncRNAs 1% of CD 45+cells in the BM, 6.7–20.3% of CD105 are enriched in exosomes secreted from senescent + cells in the bone, and 0.2–8.2% of CD45+ in the liver cells. Surprisingly, these exosomes cause anchorage- and lung contained green vesicles. In mice orthotopi- independent growth of normal cells and change the cally implanted with NB cells producing GFP-labelled number of chromosomes. It is therefore possible that EV, we observed an increasing amount of GD2- /GFP+ the overexpression of SA-ncRNA in old mice may cells in the BM (0.2%) between week 2 and 6. The eventually promotes tumorigenesis. These results indi- expression of CD45, CD11b, and CD105 in these cate that senescence-associated epigenetic dysregula- GD2- cells suggests their myeloid, monocytic, and tion is likely to contribute to tumour development mesenchymal origin. In the liver, a similar capture by not only through SASP but also exosomes during CD45+ and CD11b+ was observed (up to 0.2%). We aging process. also observed an increasing amount of GD2- /GFP+ Summary/Conclusion: Here we show a novel function cells that were negative for CD45, CD11b, and CD105 of exosomes secreted from senescent cells on chromo- at week 6–8. No GFP+ cells were detected in the lung, somal instability. These data suggest that senescence- spleen and kidney. associated exosome secretion may contribute to age- Summary/Conclusion: Tumour-derived exosomes are related increase of cancer incidence. specifically captured by a small percentage (within the Funding: PRESTO, JST. limits of FACS detection) of myeloid and stromal cells in the BM and the liver in the early stages of tumour development before NB cells home to these organs. OF15.03 The data which used an orthotopic model rather i.v. injection, support the concept that exosomes contribute to the pre-metastatic niche. Orthotopic neuroblastoma tumour model generating GFP-labelled Funding: RO1 CA 207983 from the National Institutes extracellular vesicles (EV) reveals specific capture of GPF EV by monocytes/macrophages and mesenchymal cells in liver and bone of Health, USA. marrow Yves A. DeClercka, Laurence Blavierb and Rie Nakatac aUniversity of Southern California, Los Angeles, CA, USA; bChildren’s OF15.04 Hospital Los Angeles, LosAngeles, CA, USA; cChildren’s Hospital Los Angeles, Los Angeles, CA, USA ExoBow – a transgenic strategy to study CD63+extracellular vesicles Introduction: EV released by tumours reaches target in vivo Bárbara Adema, Nuno Bastosa, Carolina Ruivoa, Maxwell Goodrichb, Zhang cells at distant sites. The study of their capture in vivo Xiaojingc, Barbara Seidlerd, David W Goodriche, Jose L Costaf, José has been limited by methods relying on intravenous Machadof, Dieter Saurg, Dawen Caih and Sónia Melof injection (i.v.) of EV isolated in vitro. Using human ai3S – Instituto de Investigação e Inovação em Saúde, Porto University, tumour cells producing GFP-labelled EV, we have Portugal; IPATIMUP – Instituto de Patologia e Imunologia Molecular da Universidade do Porto; ICBAS – Instituto de Ciências Biomédicas Abel examined the capture of tumour-derived EV in distant Salazar da Universidade do Porto, Porto, Portugal; bDepartment of organs in vivo. Pharmacology & Therapeutics, Roswell Park Comprehensive Cancer Center, New york, NY, USA; 34Department of Pharmacology & Therapeutics, Methods: Luciferase expressing NB cell lines (SK-N-BE Roswell Park Comprehensive Cancer Center, New York, NY, USA; (2), CHLA-136, CHLA-255) were transduced with a dGerman Cancer Research Center (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany, heidelberg, Germany; eDepartment of lentivector targeting the GFP protein to the exosomal Pharmacology & Therapeutics, Roswell Park Comprehensive Cancer Center, membrane (CMV-XP-GFP-EF1 aka XPack). The ana- New York, NY, USA; fi3S – Instituto de Investigação e Inovação em Saúde, lysis of EV produced by XPack NB cells by differential Porto University, Portugal; IPATIMUP – Instituto de Patologia e Imunologia Molecular da Universidade do Porto; FMUP – Faculdade de Medicina da ultracentrifugation followed by OPDG confirmed the Universidade do Porto, Porto, Portugal; gGerman Cancer Research Center presence of GFP in fractions containing exosomes. (DKFZ) and German Cancer Consortium (DKTK), Heidelberg, Germany, Heidelberg, Germany; hUniversity of Michigan Medical School, Ann Arbor, Mice orthotopically implanted with XPack NB cells MI, USA. were sacrificed at week 2, 4, 6 and 8, and the bone marrow (BM), liver, lung, kidney, and spleen were Introduction: The whereabouts of extracellular vesicles examined by FACS and immunofluorescence imaging (EVs) inside a multicellular organism following their (BM and liver) for the presence of GFP+ cells. The spontaneous natural flow and the identification of their presence of the disialoganglioside 2 (GD2) was used to recipient cells is still elusive. A comprehensive map of distinguish positive tumour cells from host cells having the network of communication established by EVs in captured EV. vivo requires the development of new tools. 164 ISEV2019 ABSTRACT BOOK

Methods: We have developed a CD63 multireporter with bone morphogenic protein-2 (BMP2) to activate transgenic mouse model to determine the spatiotem- BMP/Smad pathway and induce osteoblastic differen- poral biodistribution of tissue/cell specific derived tiation. Alkaline phosphatase (ALP) induction and cal- CD63-enriched EVs, exosomes, that we termed cium deposition were used as indicators of ExoBow. Using organ-specific promoters we have differentiation. The promoter activities of Smad’s target mapped the network of communication mediated by genes were quantified by luciferase reporter assays. pancreas and intestine derived exosomes within the Results: In BMP2-treated MC3T3-E1, MM-EV respective organ microenvironment, and also with repressed ALP induction and calcium deposition. neighbour and distant organs. The ExoBow transgene MM-EV fractions were collected by Total Exosome allows a stochastic Cre recombination that determines Isolation Reagent (Invitrogen) or ultracentrifugation. the expression of one of the fusion proteins CD63- The ALP suppression activity of the MM-EV collected mCherry, -phyYFP, -eGFP or -mTFP, and secrete col- by the kit and MM-EV collected by ultracentrifugation our-coded CD63+ EVs. We have used genetically engi- were correlated with the vesicle quantity and exosomal neered mouse models of pancreatic cancer crossed with marker protein quantity. The suppression of ALP our ExoBow to determine the flow of cancer exosomes induction by MM-EV was inhibited by macropinocy- during disease progression. tosis inhibitor 5-(N-Ethyl-N-isopropyl) amiloride. In Results: We demonstrate that communication from the mouse cell MC3T3-E1 and human cell SaOS-2, MM- pancreas occurs more frequently upon cancer-asso- EV did not suppress Smad signal transduction. ciated transformation when compared to a healthy Contrary, these MM-EV inhibited promoter activation setting. of genes targeted by Smad. This suppression activity Summary/Conclusion: Our work is the first attempt to required Smad binding elements (SBEs) of the promo- dissect the spontaneous flow of exosomes in a multi- ter sequence. On Smad target promoters, a transcrip- cellular organism and to understand their involvement tion factor X co-represses Smad’s activity and inhibit in several processes that occur in non-pathological and osteoblast differentiation. The factor X was translo- in pathological conditions. The ability of the ExoBow cated in the nucleus and its target genes’ expressions model to conditionally label any unique organ/tissue/ were changed in the cells treated with MM-EV. cell within a mouse, opens an unprecedented opportu- Summary/Conclusion: MM-EV suppresses osteoblast nity to determine the connectome established by the differentiation by inhibiting promoter activation of flow of exosomes in vivo, unravelling their biological Smad. This finding will lead a novel drug development significance in health and disease. strategy for the bone defects of MM. Funding: NORTE-01-0145-FEDER-000029. Fundacao Funding: Research Support Foundation of Tokushima Ciencia Tecnologia: IF/00543/2013/CP1184/CT0004, University and TAIHO Pharmaceutical Co., LTD, JSPS PTDC/BIM-ONC/2754/201, POCI01-0145-FEDER- Grant-in-Aid for Young Scientists (B) (ID 26860037), 32189. FAZ Ciencia Astrazeneca and JSPS Grant-in-Aid for Early-Career Scientists (ID 18K15213). OF15.05 OF15.06 BMP2-dependent osteoblast differentiation is suppressed by multiple myeloma-derived extracellular vesicles Mariko Ikuoa,b, Kei Sugisakib, Jumpei Teramachib, Ryou-u Takahashia, Tumour-derived extracellular vesicles require β1 integrins to promote Masahiro Abeb, Kohji Itohb and Hidetoshi Taharaa anchorage-independent growth aHiroshima University, Hiroshima, Japan; bTokushima University, Tokushima, Lucia R. Languino, Rachel DeRita, Aejaz Saeed, Vaughn Garcia, Shiv Ram Japan Krishn, Christopher Shields, Andrea Friedman and Srawasti Sarker Thomas Jefferson University, Philadelphia, PA, USA Introduction: Multiple myeloma (MM) suppresses osteoblast differentiation and destroys bones. Cancer- Introduction: While the significance of extracellular derived extracellular vesicles (EVs) such as exosomes vesicles (EVs) in disease progression is known, it is control microenvironments, but little is known about not clear whether “tumour-derived” EVs are detectable EVs and exosomes secreted from MM cells (MM-EV). in vivo and are active. EVs contain different integrins; We examined whether and how MM-EV affects osteo- the β1 integrins, which are expressed in different cell blastic differentiation. types, contribute to cancer progression, and are known Methods: The mouse pre-osteoblast MC3T3-E1 cells to signal through endosomes. In this study, we inves- and human osteosarcoma SaOS-2 cells was stimulated tigated whether prostate cancer (PrCa) EVs affect JOURNAL OF EXTRACELLULAR VESICLES 165 anchorage-independent growth and whether β1 integ- are functional and co-express β1, src, as well as CD9, rins in EVs are required for this effect. CD63 and TSG101; in contrast, EVs from β1pc-/- Methods: We used EVs separated by ultracentrifuga- /TRAMP or wild-type mice lack β1 as well as the tion and density–gradient from TRAMP mice, which other markers listed above. develop PrCa (TRAMP, transgenic adenocarcinoma of Summary/Conclusion:Inthisstudy,wedemonstrate the mouse prostate). We also used a cell line-based that tumour-derived epithelial EVs require β1integ- genetic rescue approach. For this study, we selected rins to stimulate anchorage-independent growth of EVs with 1.14g/ml density and 100nm mean size. recipient cells. Overall, this study opens new perspec- Results: We show that EVs from either cancer cells in tives in cancer treatment based on inhibition of cir- vitro or from blood of tumour-bearing TRAMP mice culating β1integrin-containingEVsshedbycancer promote anchorage-independent growth of PrCa cells. cells. In contrast, EVs from cultured cells harbouring a Funding: This study was supported by NIH R01 CA- shRNA to β1, from wild-type mice or from β1pc-/- 224769, P01 CA-140043; Thomas Jefferson University /TRAMP mice carrying a β1 conditional ablation in Dean’s Transformational Science Award. This project the prostatic epithelium, do not. Additionally, we show is also funded, in part, under a Commonwealth that genetic rescue of β1 restores the stimulatory func- University Research Enhancement Program grant tion of secreted EVs on anchorage-independent with the Pennsylvania Department of Health (H.R.); growth. We demonstrate that EVs isolated through the Department specifically disclaims responsibility density–gradients from cancer cells or TRAMP blood, for any analyses, interpretations or conclusions. 166 ISEV2019 ABSTRACT BOOK

Symposium Session 16: Central Nervous System EVs Chairs: Lesley Cheng; Dimitrios Kapogiannis Location: Level B1, Hall A 13:30–15:00

OF16.01 separated AD from non-AD controls. Stratifying by APOE genotype several differences were observed. In contrast with a recent report on APOE4, counts of ε4- Brain tissue-derived extracellular vesicles of Alzheimer’s disease associated bdEVs were not lower than those of brains patients with different apolipoprotein E genotypes Yiyao Huanga, Vasiliki Machairakib, Lesley Chengc, Olga Pletnikováa, Juan with other genotypes. Indeed, liberated particle counts Troncosoa, Andrew Hilld, Lei Zhenge and Kenneth W. Witwera were highest for ε4/ε4. Fragment Analyser revealed aJohns Hopkins University School of Medicine, Baltimore, USA; bJohns abundant sRNAs in sEVs. Total RNA and miRNA Hopkins University, Baltimore, USA; cDepartment of Biochemistry and abundance from highest to lowest by source was: BH, Genetics, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Australia; dThe Department of Biochemistry and Genetics, La lEVs, and sEVs. Trobe Institute for Molecular Science, La Trobe University, Bundoora, Summary/Conclusion: Our results suggest ε4/ε4 gen- Australia; eClinical Laboratory Department, Nanfang Hospital, Southern Medical University, Guangzhou, China (People’s Republic) otype in AD associates with greater bdEV recovery than for other genotypes or non-AD brain. Ongoing Introduction: Sporadic Alzheimer’s disease (AD) evaluation of protein and RNA from these samples associates with Apolipoprotein E (APOE) genotype. may reveal correlates or mechanisms of EV release. The ε4 allele is associated with increased risk vs. the Funding: US NIH: NIA (AG057430), NIMH more common ε3, while ε2 is protective. Recently, (MH118164). Vella, et al. (JEV, 2017) reported efficient enrichment of EVs from brain by differential and gradient density OF16.02 ultracentrifugation. Importantly, the method was carefully evaluated by levels of proteins presumed to be depleted in EVs vs. artefacts of tissue processing, per Murine CNS-Derived extracellular vesicles originate from astroctyes and neurons and carry misfolded proteins MISEV. Using a modification of this rigorous method, Judith Maxwell. Silverman, Sarah Fernando, Catherine Cowan, Luke we extracted brain-derived EVs (bdEVs) of AD McAlary, Leonard Foster and Neil R. Cashman patients with different APOE alleles and non-AD University of British Columbia, Vancouver, Canada brain tissues for quantitive and qualitative evaluation of EVs and their cargo. Introduction: Extracellular vesicles (EVs) are secreted Methods: Brain of AD patients with different APOE by myriad cells in culture and unicellular organisms, genotypes [ε2/ε3(n = 5), ε3/ ε3 (5), ε3/ε4 (6), ε4/ε4 (6)] and their identification in mammalian biofluids sug- and non-AD controls (n = 7) was obtained from the gests that vesicle release occurs at the organism level Johns Hopkins Alzheimer’s Disease Research Center. also. However, despite clear importance to the under- Tissue was processed per Vella et al. (JEV, 2017) standing of EVs in organismal biology, EVs in solid through 10k x g centrifugation. Subsequently, SEC tissues have received little attention. Amyotrophic lat- was followed by UC to concentrate bdEVs. Protein eral sclerosis (ALS) is a fatal neurodegenerative disease and particle concentration, morphology, and protein resulting in the progressive loss of motor neurons in markers were examined by BCA, nano-flow cytometry the brain, brainstem and spinal cord. The disease is (NanoFCM), TEM, and Western blotting. RNA and characterized by progressive propagation of pathology protein from brain homogenate (BH), 10k x g large spreading from the CNS foci in which symptoms first EVs (lEVs) and small EVs (sEVs) were extracted for appear. proteomics and small RNA QC (Fragment Analyser) Methods: To better understand to role of EVs in an and sequencing. ALS-affected central nervous system, we employed a Results: bdEVs of acceptable purity were obtained method of whole tissue vesicle isolation. We applied a using the modified method. No remarkable differences protocol for primary neural cell culture and modified it in bdEV morphology or size distribution were for the collection of EVs from frozen whole murine observed between AD and non-AD material. and human neural tissues by serial centrifugation and Similarly, no significant differences in particle counts purification on a sucrose gradient. JOURNAL OF EXTRACELLULAR VESICLES 167

Results: Quantitative proteomics found that brain- manipulation, single MVs in suspension were trapped derived EVs contain canonical exosomal markers, by an infra-red laser collimated into the optical path of with enrichment in synaptic and RNA binding pro- the microscope, and delivered to neuron surface. The teins. The brain EVs contained numerous proteins MV-neuron dynamics were monitored by collecting implicated in ALS, and SOD1G93A transgenic EVs bright-field images. were significantly depleted in myelin-oligodendrocyte Results: Analysis of time-lapse recordings revealed that glycoprotein compared to non-transgenic animals. MVs efficiently adhered to neurons and about 70% Brain and spinal cord EVs are positive for the astrocyte showed a displacement along the surface of neurites. marker GLAST and the synaptic marker SNAP25, Interestingly, the MVs velocity (143 nm/sec) is in the while CD11b, a microglial marker, was largely absent, same range of retrograde actin flow, which regulates suggesting that microglia do not contribute to the membrane diffusion of receptors linked to actin. tissue EV population under these conditions. EVs Accordingly, we found that MV movement is highly from SOD1G93A transgenic ALS mouse model brains dependent on neuron energy metabolism. Indeed, only and spinal cords, as well as human SOD1 familial ALS 33% of MVs were able to move on energy depleted patient spinal cord, possess abundant misfolded and neurons treated with rotenone. Moreover, inhibiting non-native disulfide-crosslinked aggregated SOD1. neuron actin cytoskeleton rearrangements (polymeriza- Summary/Conclusion: We established a phenotypic tion and depolymerization) with cytochalasin D, which profile of vesicles from whole mouse brains and spinal binds fast growing end of actin, the percentage of EVs cords, and investigated how model motor neuron dis- able to move on neuron surface was significantly ease modifies this phenotype. The data demonstrates reduced from 79% to 54%, revealing that neuronal that intra-organ CNS-EVs from disease affected ani- actin cytoskeleton is involved in EV-neuron dynamics. mals and humans contain pathogenic disease-causing Unexpectedly, we found by cryo-electron micro- protein, and suggests that in the brain and spinal cord, scopy that a subpopulation of MVs contains actin fila- astrocytes and neurons, as opposed to microglia, are ments, suggesting an intrinsic capacity of MVs to the main source of EVs. move. To address this hypothesis, we inhibited actin Funding: A Bernice Ramsay ALS Canada grant sup- rearrangements in EVs with Cytochalasin D and ported the work, along with funding from the Paul observed a significant decrease, from 71% to 45%, of Heller Memorial Fund for JMS. MVs able to drift on neuron surface. Summary/Conclusion: Our data support two different OF16.03 way of MV motion. In the first case, MV displacement could be driven by the binding with neuronal receptors linked to the actin cytoskeleton. In the second, actin Investigating microvesicle motion on neuron surface through optical rearrangements inside MVs could drive the motion tweezers Giulia D’Arrigoa, Martina Gabriellib, Dan Cojocc, Giuseppe Legnamed and along a gradient of molecules on neuron surface. Claudia Verderioe aInternational School for Advanced Studies of Trieste, Varese, Italy; bCNR Institute of Neuroscience, Milano, Italy; cCNR – Institute of Materials, OF16.04 Trieste, Italy; dInternational School for Advanced Studies of Trieste, Trieste, Italy; eCNR Institute of Neuroscience, Trieste, Italy P2RX7 Inhibitor suppresses tau pathology and improves hippocampal Introduction: Microvesicles (MVs) play an essential memory function in tauopathy mouse model Seiko Ikezu, Zhi Ruan, Jean Christophe Delpech, Mina Botros, Alicia Van role in intercellular communication. Exposing adhe- Enoo, Srinidhi Venkatesan Kalavai, Katherine Wang, Lawrence Hu and sion receptors, they can interact with target cells and Tsuneya Ikezu deliver complex signals. It has been shown that MVs Boston University School of Medicine, Boston, USA also cover a crucial role in the spreading of pathogens in neurodegenerative disorders, but almost nothing is Introduction: Microglia, the innate immune cells in known about how MVs can transport messages moving the central nervous system, could spread pathogenic in the extracellular microenvironment exploiting neu- tau protein via secretion of extracellular vesicles, such ronal connections. as exosome. P2X7 receptor (P2RX7) is an ATP-gated Methods: In order to investigate the interaction of cation channel and highly expressed in microglia and MVs with the plasma membrane of neurons, MVs triggers exosome secretion. We hypothesize that released from cultured astrocytes and isolated by dif- P2RX7 inhibitor could alleviate tauopathy in PS19 tau ferential centrifugation, were added to the medium of transgenic mice by inhibiting the exosome secretion by cultured hippocampal neurons. Using optical microglia. 168 ISEV2019 ABSTRACT BOOK

Methods: BV-2 murine microglial cell lines were trea- released in response to IL-1β (ADEV-IL-1β) contain ted with GSK1482160, a specific inhibitor of P2RX7, cargo that regulate APP processing in neurons. prior to ATP stimulation. Exosomes were enriched Methods: Neurons were stimulated with constitutively from the conditioned media and quantified using released ADEV or ADEV-IL-1β. APP, and BACE1 co- Nanoparticle Tracking Analysis and CD9 ELISA. localization in membrane microdomains was measured Three-months old P301S Tau (PS19) and control by immunofluorescence staining. APP and BACE1 wild-type mice were treated with GSK1482160 protein and mRNA levels measured by western blot (20 mg/kg) or vehicle by oral gavage for 30 days. The and RT-QPCR. The binding of APP mRNA and het- animals were tested for hippocampal memory function. erogeneous nuclear ribonuclear protein C (hnRNP C) The accumulation of pathogenic Tau was determined was measured by immunoprecipitation. Casein kinase by immunohistochemistry and ELISA. 1 (CK1) efficiency was blocked using pharmacological Results: ATP stimulation of BV-2 cells significantly inhibition and genetic knockdown. Isolation of human increased secretion of exosomes (30–150 nm), which plasma ADEV was achieved by GLAST-1 pull-down. was significantly inhibited by GSK1482160 treatment Results: Neurons exposed to ADEV-IL-1β promoted the in a dose-dependent manner. Daily administration of co-localization of APP and BACE1 into membrane GSK1482160 over 30 days had no effect on body weight microdomains (p < 0.001), and increased production of PS19 mice. Interestingly, GSK1482160 treatment of Aβ1-42. Protein expression of APP (p =0.0051)but enhanced spontaneous alteration in Y-maze and not BACE1 was also increased. APP mRNA was not improved prepulse inhibition as compared to vehicle- increased following exposure of neurons to ADEV-IL- treated group. In addition, pTau level in the hippocam- 1β, suggesting a post-translational event. APP transla- pal tissue was significantly reduced in GSK1482160- tion is regulated by a competitive interaction of the treated PS19 mice as compared to vehicle-treated fragile X mental retardation protein (FMRP), and group as determined by neuropathology and ELISA. hnRNP C with the APP coding region. hnRNP C can Summary/Conclusion:GSK1482160treatmentsup- displace FMRP and increase APP translation. The asso- presses ATP-induced exosome secretion from BV-2 ciation of hnRNP C with APP mRNA increased follow- cells. Its oral administration to PS19 mice improved ing exposure to ADEV-IL-1β. Compared with ADEV- hippocampal memory function and reduced the accu- CR, ADEV-IL-1β selectively carried CK1. CK1 is known mulation of pathogenic tau. These data demonstrate that to activate hnRPC. Knockdown of CK1 in astrocytes targeting P2RX7 is a novel target for suppressing the reduced CK1 in ADEV-IL-1β (p =0.0061),prevented exosomal spread of pathogenic tau protein by microglia, the upregulation of APP protein expression (p =0.007), which may be applicable to Alzheimer’s disease. and co-localization of APP with BACE1 (p <0.001). Funding: Grant/Other Support: NIH 1RF1AG054199- Overexpression of CK1 in astrocytes increased CK1 in 01 ADEV-IL-1β, increased APP protein expression Grant/Other Support: 1R01AG054672-01 (p = 0.0026), and co-localization of APP with BACE1 Grant/Other Support: 1R56AG057469-01 (p = 0.0015). We confirmed in ADEVs isolated from Grant/Other Support: DVT-14–320835 human plasma that AD patients, but not healthy age- Grant/Other Support: Cure Alzheimer’s Fund. matched controls contain CK1 (p =0.0467). Summary/Conclusion: These data suggest that neuroinflammatory stimuli modify ADEV cargo to OF16.05 enhance amyloidogenic processing of APP by delivering CK1 to regulate the association of hnRNPC with

Astrocyte-derived extracellular vesicles shed in response to IL-1beta APP mRNA. up-regulate amyloidogenic processing in neurons Zhigang Lia, Raha Dastgheyba, Seung-Wan Yooa, Carlos Nogueras-Ortizb, b a Dimitrios Kapogiannis and Norman Haughey OF16.06 aJohns Hopkins University, Baltimore, MD, USA; bNational Institute on Aging, Baltimore, MD, USA

The role of human choroid plexus-derived extracellular vesicles in Introduction: Chronic inflammation is thought to viral neuroinvasion contribute to the pathogenesis of Alzheimer’s disease Bethany O’Hara, Jenna Morris-Love, Gretchen Gee, Walter Atwood and by upregulating amyloidogenic processing of APP. Sheila Haley Based on previous findings that inflammatory stimuli Brown University, Providence, RI, USA modify the cargo of astrocyte derived extracellular Introduction:ThehumanpolyomavirusJCPyVcausesthe vesicles (ADEV), we sought to determine if ADEVs fatal disease progressive multifocal leukoencephalopathy JOURNAL OF EXTRACELLULAR VESICLES 169

(PML) in immunocompromised individuals and patients Infection was evaluated by immunofluorescence analy- undergoing immunomodulatory therapy. A critical ques- sis with antibodies against the major viral capsid pro- tion in JCPyV pathogenesis is understanding how the virus tein VP1. Uptake was evaluated by flow cytometric is transported from the periphery to the CNS to infect glial analysis of PKH67 fluorescently labelled particles and cells and cause demyelination. An additional paradox is confocal imaging. that the target glial cells do not express known virus Results: EV from CPE cells display characteristic mar- receptors. Previously, we analysed JCPyV infection of the kers and morphology, contain intact JCPyV virions, choroid plexus (CPE), a functional barrier to the CSF and and are infectious to both CPE cells and human glial showed CPE cells are permissive to JCPyV infection and cells. EV-mediated infection is receptor independent. express viral attachment and entry receptors. Here, we Infection and uptake of EV cannot be inhibited by investigate the role of CPE-derived extracellular vesicles neutralizing antisera and internalization is via endocy- in receptor-independent infection of glial cells by JCPyV. tosis. EV from CPE contains significantly more VP1 Methods: In addition to analysing primary human CPE than other glial cell lines. cells, we also developed an immortalized human CPE Summary/Conclusion: Primary and transformed CPE line. CPE were transformed using hTERT lentiviral produce similar EVs that are able to deliver significant transduction and verified by STR profiling. EV from amounts of JCPyV in a receptor independent manner both cell types were concentrated by differential cen- to target cells in the CNS. The choroid plexus may be trifugation and evaluated by transmission electron an entry point by which JCPyV accesses the brain microscopy, Western blot, nanoparticle tracking analy- leading to the development of PML. sis, infection, and qPCR for protected viral genomes. Funding: NIH RO1NS043097. 170 ISEV2019 ABSTRACT BOOK

Symposium Session 17: EVs in Tissue Injury and Repair Chairs: Benedetta Bussolati; Dominique de Kleijn Location: Level B1, Hall B 13:30–15:00

OF17.01 36.21 ± 3.63%) after 4 weeks transplantation, respectively. The similar restorations of engraftment were also seen in 12 weeks post-transplantation. These data Mesenchymal stem cell derived extracellular vesicles restore the engraftment capacity of stem cells in radiation exposed mice suggested that EVs have early and late mitigating Sicheng Wena, Mark Doonerb, Laura Goldbergc, Elaine Papac, Michael Del effects on peripheral blood cytopenias and BMSCs. Tattoc, Mandy Pereirac, Yang Chenc, Theodor Borgovand and Peter Quesenberryb No toxic effect was observed in bone marrow, kidney, liver, spleen, lung and heart up to 53 weeks post-EV aBrown University/Rhode Island Hospital, Providence, RI, USA; bBrown University Department of Hematology Oncology; Rhode Island Hospital, injection. Providence, RI, USA; cRhode Island Hospital, Providence, RI, USA; dRhode Summary/Conclusion: Our data suggest that there is a Island Hospital/ Brown University, Providence, RI, USA long-term effect of MSC-EVs on the restoration of engraftment of BMSCs in radiation-exposed mice, Introduction: We have shown that pretreated irra- and MSC-EV treatment is a safe therapeutic strategy. diated murine bone marrow stem cells (BMSCs) with Funding: NIH grants 5UH2TR000880 and mesenchymal stem cells-extracellular vesicles (MSC- 5T32HL116249. EVs) in vitro, could significantly improve the engraftment capacity of radiation damaged BMSCs with a predominant reversal effect in later periods of post- OF17.02 transplant from 12 weeks up to 36 weeks. This indicates a long-term effect of MSC-EVs on reversal of Connexin43-positive exosomes released by osteoarthritic radiation damage of BMSCs. chondrocytes favours osteoarthritis progression by spreading Methods: In this study, we investigated the long-term senescence and inflammatory mediators to nearby tissues Marta Varela-Eirína, María D. Mayán Santosb, Adrián Varela-Vázqueza, effect of MSC-EVs on the restoration of engraftment of Amanda Guitián-Caamañoa, Susana B. Bravo-Lópezc, Carlos Paínod, Raquel BMSCs in radiation-exposed mice in vivo up to Largoe, Eduardo Fonsecaa, Mustapha Kandouzf, Trond Aaseng, Arantxa Taberneroh, Alfonso Blancoi, José R. Caeiroj and María D. Mayána 53 weeks. Moreover, the safety and toxicity of MSC- a EVs treatment were also evaluated. CellCOM research group. Instituto de Investigación Biomédica de A Coruña (INIBIC), Servizo Galego de Saúde (SERGAS), A Coruña, Spain; Results: 500cGy radiated mice were injected with bTranslational Research in Cell Communication and Signalling (CellCOM), human MSC-EVs by tail vein injection at 24, 48 and Instituto de Investigación Biomédica de A Coruña (INIBIC), A Coruña, Spain; cProteomics laboratory. Instituto de Investigación Sanitaria de 72h post-radiation. We followed the peripheral blood Santiago de Compostela (IDIS), Complexo Hospitalario Universitario de cell counts up to 53 weeks post-EV injection. There Santiago de Compostela (CHUS), A Coruña, Spain; dUnit of Experimental Neurology-Neurobiology., Madrid, Spain; eBone and Joint Research Unit, was a significant RBC, HGB and platelet restoration in Rheumatology Department, IIS-Fundación Jiménez Díaz UAM, Madrid, EV treated radiated mice compared to untreated mice Spain; fDepartment of Pathology, School of Medicine, Wayne State University, Detroit, MI, USA; gTranslational Molecular Pathology research in the early period (before day 35). For the evaluation group. Vall d’Hebron Research Institute (VHIR), Universitat Autònoma de of reversal effect on BMSCs, bone marrow, harvested at Barcelona, Barcelona, Spain; hDepartamento de Bioquímica y Biología 6, 12, 26 and 53 weeks. post-EV injection, were trans- Molecular, Instituto de Neurociencias de Castilla y León (INCYL), Universidad de Salamanca, Salamanca, Spain; iFlow Cytometry Core planted into 950 cGy exposed B6.SJL mice. The Technologies, UCD Conway Institute, University College Dublin, Dublin, engraftment was evaluated at 4 and 12 weeks post- Ireland; jDepartment of Orthopaedic Surgery and Traumatology, Complexo Hospitalario Universitario de Santiago de Compostela (CHUS). Universidade transplantation. In those transplanted mice at 6 weeks de Santiago de Compostela (USC), Santiago de Compostela, Spain post-EV injection, there was a slight increase in the restoration of engraftment rate in EV treated mice Introduction: Chondrocytes in articular cartilage (17.58 ± 2.32%) compared to untreated mice undergo phenotypic changes and senescence, restrict- (13.80 ± 1.41%) after 1 month post-transplantation. ing cartilage regeneration and favouring osteoarthritis However, for those mice transplanted at 12, 26, and (OA) progression. Like other wound healing disorders, 53 weeks post-EV injection, there were significantly chondrocytes from OA patients show a chronic higher restorations of engraftment in EV treated mice increase in the transmembrane channel protein con- (40.48 ± 6.03%, 33.93 ± 3.76%, and 56.62 ± 3.63) com- nexin43 (Cx43). Extracellular vesicles (EVs), including pared to untreated mice (12.39 ± 1.30%, 15.14 ± 2.21%, exosomes, have been show to harbour connexin JOURNAL OF EXTRACELLULAR VESICLES 171 channels that allow the formation of gap junctions Introduction: Cellular senescence has evolved from an between the exosome and the target cell. However, in vitro model system to study aging to a multifaceted the role of these vesicles and exosomal-Cx43 in OA phenomenon of in vivo importance since senescent progression has not been studied yet. The objective of cells in vivo have been identified and their removal this study was to investigate the role of EVs released by delays the onset of age-associated diseases in a mouse osteoarthritic chondrocytes (OACs) in cellular plasti- model system. In order to understand how senescent city and senescence of surrounding tissues. cells that accumulate within organisms with age nega- Methods: EVs were isolated from OA/healthy chon- tively impact on organ and tissue function, we have drocytes by ultracentrifugation and their protein con- started to characterize secreted miRNAs within extra- tent was analysed by LC-MS/MS using 6600 triple cellular vesicles that are differentially expressed in early TOF. RNA levels, protein activity and cellular senes- passage versus senescent cells and their functional role cence were analysed by RT-qPCR, western blot, immu- in the context of cellular and organismal aging. nofluorescence and flow cytometry. Methods: We performed next generation sequencing as Results: Our results indicate that OACs contain well as qPCR on extracellular vesicles from senescent increased levels of Cx43 within their EVs in compar- versus control cells, after characterizing the EVs in ison to the EVs isolated from healthy donors. detail. In addition, open flow microperfusion experi- Overexpression of Cx43 in chondrocytes increased ments were used to proof the presence of EVs in the senescence and the total content of Cx43 in the EVs. interstitial fluid of the skin. The treatment of target cells with EVs containing Cx43 Results: We identified extracellular vesicle contained led to a significant increase in Cx43 mRNA and protein miRNAs as bona fide members of the senescence asso- levels. The increase of Cx43 lead to dedifferentiation in ciated secretory phenotype (SASP) that are transferred the recipient cells via EMT by activation of Twist-1, from senescent cells to their microenvironment or even with increased levels of the mesenchymal markers the systemic environment. These miRNAs, among CD105 and CD166. The phenotypic changes detected them miR-23a-5p, are transported via extracellular in OACs lead to a decrease in the main cartilage vesicles also in organotypic human skin equivalents markers Col2A1 and ACAN expression, and increased and recipient cells taking them up are altered in their the levels of cellular senescence and SASP in target cells cell fate, including altered wound healing and apopto- via p53/p16 and NF-kß. These results were corrobo- totic behaviour. In addition, miR-31 is transferred to rated by analysing the protein cargo of these Cx43 mesenchymal stem cells, inhibiting osteogenic positive EVs, where we found enrichment in proteins differentiation. related with the catabolic, senescence and wound-heal- Summary/Conclusion: In summary, we present evi- ing pathways dence of the importance of specific miRNAs and high- Summary/Conclusion: Together, these results suggest light their potential use as biomarkers of aging and age- that Cx43-positive EVs released by OACs may be associated diseases, or even as therapeutic tools and involved in the spread of cellular senescence, inflam- targets to prevent age-associated diseases. mation and reprogramming factors involved in wound Funding: This work was funded by the Christian healing failure to neighbouring tissues in the joint. Doppler Society. The financial support by the Further understanding of the role of exosomal Cx43 Austrian Federal Ministry of Economy, Family and in OA will help to halt the disease spread and Youth; the National Foundation for Research, progression. Technology and Development is also gratefully acknowledged, as is funding by the Austrian Science OF17.03 Fund (FWF: I2514 to JG) and the PhD Programme BioToP –“Biomolecular technolgy of proteins”.

Extracellular vesicles in ageing: from skin to bone Lucia Terlecki-Zaniewicza, Madhusudan Reddy Bobbilia, Matthias Hacklb, OF17.04 Regina Grillaric, Ingo Lämmermanna, Vera Pilsd, Florian Grubere and Johannes Grillarif a Human embryonic stem cells derived exosomes promote tissue BOKU – University of Natural Resources and Life Sciences Vienna, Vienna, regeneration in aged mice by rejuvenating senescent endothelial Austria; bTAmiRNA GmbH, Vienna, USA; cEvercyte GmbH, Vienna, d cells Austria; BOKU – University of Natural Resources and Life Sciences Bi Chen, Liangzhi Gonga and Yang Wangb Vienna, Vienna, USA; eChristian Doppler Laboratory on Biotechnology of Skin Aging; Medical University of Vienna, Vienna, USA; fChristian Doppler aShanghai Jiaotong University Affiliated Sixth People’ Hospital, Shanghai, Laboratory on Biotechnology of Skin Aging, University of Natural Resources China (People’s Republic); bShanghai Jiao Tong University Affiliated Sixth and Life Sciences, Vienna (BOKU), Vienna, Austria People’s Hospital, Shanghai, China (People’s Republic) 172 ISEV2019 ABSTRACT BOOK

Introduction: Angiogenesis plays a crucial role in tis- aAnesthesiology and Program in Neuroscience, University of California, San b sue repair. This process is significantly impaired by Diego, CA, USA; Department of Anesthesiology, School of Medicine, University of California, San Diego, CA, USA; cUniversity of California, age-related dysfunction of vascular endothelial cells in San Diego, San Diego, CA, USA; dDepartment of Orthopaedic Surgery, aged bodies. Exosomes from embryonic stem cells Chiba University, Chiba, Japan; eDepartment of Anesthesiology and Program in Neuroscience, School of Medicine, University of California, San (ESCs) contain primitive molecules (proteins, miRNA, Diego, CA, USA etc.) from their parent cells. Therefore, our hypothesis is that ESCs derived exosomes (ES-Exos) would influ- Introduction: Exosomes (Exs) are small extracellular ence and rejuvenate aging endothelial cells and lead to vesicles originally known to be secreted from multi- enhanced tissue repair in aged bodies. vesicular endosomes in dendritic cells. We now know Methods: Six- to eight-week-old C57BL/6 mice were that Exs are secreted from many cell types and are daily subcutaneous injection of D-gal (1000 mg/kg) to essential for autocrine/paracrine communication. In establish aged mice model. Pressure ulcers were created the peripheral nervous system (PNS), Exs derived on the back of each mouse, followed by pipetting ES- from primary Schwann cells (SC) appear to facilitate Exos (1*1011/mL) suspension or PBS one time per day. axon growth after injury, however their effects on SC Mice were sacrificed at 3, 7, 14, and 21 days after physiology and pain outcomes are unknown. intervention. In addition, a group of young mice with Methods: Exs were purified from primary SC condi- pressure ulcer was also set. Samples from each mouse tioned media by ultracentrifugation (SC-Ex) and char- were evaluated in the aspect of vascular formation and acterized by immunoblotting and NanoSight In aging condition. Furthermore, we induced HUVEC cultures of SC, TNFa robustly activated proinflamma- senescence in vitro by D-gal treatment and investigate tory cell signalling and migration. SC-Ex (50–500 ng/ the function and mechanism of ES-Exos in restoring mL) were added to TNFa treated SC, and phosphoryla- function and rejuvenation of senescent endothelial cells tion of p38MAPK and JNK1/2 were measured. by qRT-PCR, WB, and immunofluorescent staining. Transwells were used to evaluate SC migration. To Results: Our results showed that ES-Exos treated aged determine if SC-Ex regulate neuropathic pain, we per- mice exhibit faster repairing than PBS treated group. formed intraneural injections of SC-Ex (500–1500 ng) The angiogenesis condition of ES-Exos treated group or vehicle into sciatic nerves during partial nerve liga- was similar as that of young mice and was better than tion (PNL) surgeries in adult male rats (n = 12). Tactile PBS treated senescent mice. The number of SA-β-gal- allodynia was assessed using von Frey filaments. positive cells and the expression level of P16 and P21 in Results: Nanoparticle tracking of SC-Ex showed the ES-Exos treated group were significantly lower than expected size distribution with a mean peak diameter that in PBS treated group. In vtiro experiments showed of 121 nm. Immunoblotting of SC-Ex revealed that that ES-Exos could also downregulate senescent related exosome markers, TSG101 and flotillin-1, and SC protein expressions and enhance tube formation of marker, P0 protein, were expressed. The golgi marker, senescent endothelial cells. In addition, our results GM130, and GFAP were not. In cultured SC, the SC-Ex also showed that ES-Exos could greatly decreased the signalling response was distinguished from the cell expression level of MDA and increase the activity of signalling signature elicited by TNF alone, which SAD, CAD, and GSH, molecules tightly related with robustly activated p38MAPK and JNK1/2 by > 6 and endogenous anti-oxidative condition. Further investi- 4-fold (p < 0.01), respectively. When SC-Ex were gation demonstrated that ES-Exos could activate NRf2 added, p38MAPK and JNK1/2 activation were dose pathway by inhibiting Keap1, leading to rejuvenative dependently and significantly inhibited (p < 0.05). function on senescent endothelial cells. TNF increased SC migration 3-fold after 4 h that was Summary/Conclusion: We demonstrate that ES-Exos blocked by SC-Ex at low doses. Local injections of can accelerate wound healing and promote angiogen- SC-Ex modified tactile allodynia associated with PNL esis in aged mice by rejuvenating endothelial compared to saline injected controls. senescence. Summary/Conclusion: We demonstrated that SC uti- Funding: NSFC Project No. 81871833 and 81672254. lizes autocrine secretion of Exs for regulating SC signalling and migration. SC-Ex act as cell independent OF17.05 entities, carrying bioactive substances capable of inhibiting pro-inflammatory signalling in SCs that may contribute to the extent and magnitude of chronic Schwann cell derived exosomes regulate Schwann cell activation and pain. Future studies will elucidate SC-Ex cargo driving neuropathic pain related behaviours a b b b autocrine/paracrine activities after PNS injury. Naoya Hirosawa , HyoJun Kwon , Haylie Romero , John Kim , Coralie Brifaultc, Seiji Ohtorid and Wendy Campanae Funding: VA. JOURNAL OF EXTRACELLULAR VESICLES 173

OF17.06 were intravenously injected and 48 h later mice were sacrificed. Results: Our data showed that administration of uEVs Urinary extracellular vesicles improve the recovery of renal function in AKI mice resulted in the acceleration of renal recov- in an Acute Tubular Injury model restoring Klotho levels Elli Papadimitrioua, Benedetta Bussolatib, Cristina Grangec, Veronica ery in a MSC EV-treatment comparable manner. Dimuccioc and Giovanni Camussid Functional and histological abnormalities, observed aDepartment of Molecular Biotechnology and Health Sciences; University of upon AKI, were alleviated, cell proliferation was stimu- Turin, Turin, Italy; bDepartment of Molecular Biotechnology and Health lated, while the expression of renal tissue injury and Sciences, University of Turin, Turin, Italy; cUniversity of Turin, Turin, Italy; dDepartment of Medical Sciences, University of Turin, Turin, Italy inflammation markers was reduced. The analysis of uEV miRNA cargo showed the presence of several Introduction: Extracellular vesicles present in urine miRNAs possibly involved in tissue repair. miR-30 (uEVs), are considered a non-invasive source of infor- and miR-151, previously described present in MSC mation regarding the pathophysiology of the whole EVs, were further found transferred in renal tissue of kidney. Mainly secreted by renal cells lining the uEV-injected mice. In addition, the reno-protective nephron, uEVs have been studied as biomarkers for factor Klotho, was found present in uEVs at both diagnosis of renal diseases. However, their possible protein and mRNA level. The administration of uEVs therapeutic use has not been addressed yet. In the in AKI mice resulted in the restoration of Klotho current study, we investigated the potential therapeutic protein levels in renal tissue, significantly lowered effect of uEVs, in a murine model of acute kidney upon damage. Of interest, ineffective fibroblast-derived injury (AKI). While the beneficial effect of mesenchy- EVs loaded with recombinant Klotho exhibited a reno- mal stromal cell-derived EVs (MSC EVs) for AKI treat- protective effect, suggesting a possible Klotho-mediated ment has been extensively described, we here tested the mechanism in the amelioration of AKI. possible therapeutic use of uEVs as more “renal com- Summary/Conclusion: Overall, our results reveal a mitted” source. novel potential therapeutic approach for AKI treat- Methods: uEVs were isolated by ultracentrifugation of ment, using renal cell-derived EVs present in urine human urine provided by healthy subjects. AKI was and indicate common, as well as unique, uEV and performed by intramuscular injection of 8 ml/kg MSC EV mechanisms of action. hypertonic glycerol. Next day, 2 ×108 uEVs /mouse Funding: FP7 NephroToolsproject and by Miur ex60% 174 ISEV2019 ABSTRACT BOOK

Symposium Session 18: EV Function in Health and Disease Chairs: David Carter; Jacky Goetz Location: Level B1, Lecture Room 13:30–15:00

OF18.01 in plasma of patients diagnosed with HIV, inflammatory bowel disease and cancer therapy-induced intestinal mucositis compared to respective controls (Mann- Increased levels of systemic LPS-positive bacterial extracellular Whitney U test, p < 0.01). These bacterial EV are able vesicles in patients with intestinal barrier dysfunction Joeri Tulkensa, Glenn Vergauwena, Jan Van Deuna, Edward Geeurickxa, Bert to induce immune activation and significantly correlate Dhondta, Lien Lippensa, Marie-Angélique De Scheerderb, Ilkka Miinalainenc, with impaired barrier integrity of the patient Pekka Rappud, Bruno G De Geeste, Katrien Vandecasteelef, Debby Laukensg, Linos Vandekerckhoveb, Hannelore Denysh, Jo Vandesompelei, Olivier De (Spearman’s ρ = 0.4241, p = 0.0245). Wevera and An Hendrixa Summary/Conclusion: Pathologies with an intestinal aLaboratory of Experimental Cancer Research, Department of Human barrier dysfunction open the door for bacterial EV to Structure and Repair, Ghent University, Ghent, Belgium; bDepartment of enter the circulation and to induce immune activation. Internal Medicine, Ghent University Hospital, Ghent, Belgium; cBiocenter Oulu, University of Oulu, Oulu, Finland; dDepartment of Biomolecular Their systemic presence correlates with impaired gut medicine, University of Turku, Turku, Finland; eDepartment of barrier integrity. These data deliver novel opportunities Pharmaceutics, Ghent University, Ghent, Belgium; fDepartment of Radiation Oncology, Ghent University Hospital, Ghent, Belgium; to advance our knowledge of PAMP-induced systemic gDepartment of Gastroenterology, Ghent University Hospital, Ghent, reactions and biomarker development. Belgium; hDepartment of Medical Oncology, Ghent University Hospital, Ghent, Belgium; iCenter for Medical Genetics, Ghent University, Ghent, Funding: This work was supported by concerted Belgium research action from Ghent University and Krediet aan Navorsers from the Research Foundation Introduction: Bacterial extracellular vesicles (EV) are Flanders (FWO). JVD, EG, LV and AH are supported secreted by gut bacteria and contain nucleic acids, by fellowships from FWO. proteins, metabolites and endotoxins. Consequently, bacterial EV that enters the systemic circulation may deliver and elicit a variety of immunological and meta- OF18.02 bolic responses in different organs. However, the systemic presence and activity of bacterial EV in patients Milk exosomes accumulate in the intestinal mucosa and peripheral with intestinal barrier dysfunction have not been tissues in wild-type pups nursed by exosome and cargo tracking investigated. dams Janos Zemplenia, Bijaya Upadhyayaa, Mengna Xiaa, Hideaki Moriyamaa and Methods: Size exclusion chromatography and density Masato Ohtsukab gradient centrifugation were combined to fractionate aUniversity of Nebraska-Lincoln, Lincoln, USA; bTokai University, faeces and plasma in two dimensions separating bac- Kanagawa, USA terial EV-associated LPS from other LPS products and eukaryotic EV. Bacterial EV-associated LPS levels of 49 Introduction: Exosomes and their cargos may be subjects with a compromised or intact intestinal barrier obtained from dietary sources such as milk. were measured by Limulus Amebocyte Lysate and Toll- Hypothesis: Milk exosomes accumulate in the intestinal like receptor four reporter assays and confirmed by mucosa and peripheral tissues in suckling mice. Aims: immunoelectron microscopy. Plasma zonulin was mea- 1) Develop an Exosome and Cargo Tracking (ECT) sured to assess intestinal barrier integrity and Caco-2 mouse to assess the origin, destination and cargo of transwell systems were used for in vitro validation exosomes. 2) Assess the bioavailability and distribution experiments. of milk CD63-positive EVs (“exosomes”) labelled with Results: We calculate that the human gut harbours fluorescent proteins in suckling pups. approximately 100 trillion bacterial EV which may Methods:ECTmiceweredevelopedbyrandomintegra- serve as a substantial source of systemic pathogen- tion of an ECT plasmid. The plasmid encodes a fusion associated molecular patterns (PAMP), evidenced by protein of CD63 and green fluorescent protein (eGFP) plus proteomic analysis of faeces-derived bacterial EV. We stop codon, flanked by two loxP sites (ORF-1). A second demonstrate that bacterial EV can translocate the ORF, coding for a CD63/near-infrared protein (iRFP) intestinal epithelial layer in a paracellular way. LPS- fusion protein, follows downstream of ORF-1. A trans- positive bacterial EV levels are significantly increased membrane domain is fused to the C-terminus of the iRFP JOURNAL OF EXTRACELLULAR VESICLES 175 to create an extra-exosomal C-terminus, followed by a with the host immune system remain poorly under- second iRFP and a stop codon. In the presence of Cre, stood. Similarly, the interaction of eukaryotic immune the CD63/eGFP/Stop insert is removed and the mice EVs with pneumococcus remains unexplored. In this switch from expressing eGFP-labeled exosomes to iRFP- study, EVs from pneumococci and pneumococci-con- labeled exosomes, including an extra-exosomal iRFP for ditioned macrophages were isolated and reciprocal collection with anti-iRFP and magnetic beads. Wild-type interaction between the host and the microbe was (WT) pups were fostered to ECT dams to assess the explored. bioavailability of milk exosomes. Methods: EVs from pneumococci and pneumococci- Results: ECT mice showed no disease phenotypes. In conditioned macrophages were isolated using size the absence of Cre, the mice expressed eGFP-labeled exclusion chromatography and characterized by trans- exosomes, whereas the offspring of mice mated with mission electron microscopy, dynamic light scattering, Cre mice expressed iRFP-labelled exosomes. The same tunable resistive pulse sensing and western blotting. patterns were obtained when HEK-293 cells were Effects of pneumococcal EVs on different immune transfected with the ECT plasmid in the absence of cell subsets were assessed using flow cytometry, wes- presence of Cre plasmid. Fluorescent proteins localized tern blotting and immunostaining. Effects of EVs on S. to exosomes but not to other complexes secreted by pneumoniae were assessed by evaluating their growth HEK-293 cells. The particles had the size expected for kinetics and biofilm development. exosomes (131 ± 49 nm), stained positive for CD9, Alix Results: Pneumococcal EVs were internalized by var- and TSG101, stained negative for histone H3, and were ious immune cells and conversely, eukaryotic EVs from captured by anti-CD63. When WT pups were nursed J774A.1 cells were taken up by both Gram-positive and by ECT dams for three weeks, exosomes accumulated Gram-negative bacteria. In a reporter murine macro- primarily in the intestinal mucosa, brain and kidneys. phage cell line (RAW 264.7), pneumococcal EVs Summary/Conclusion: We have developed a mouse induced NF-kB expression in a dosage-dependent that permits tracking CD63-positive exosomes and manner and induced p65 nuclear translocation in their cargos. CD63-positive exosomes are bioavailable human primary macrophages. Pneumococcal EVs also in suckling mice. activated primary human CD4+ and CD8 + T cells as Funding: NIFA, NIH, Gates Foundation, Gerber evidenced by their CD69 upregulation in a dose and Foundation, USDA. JZ serves as consultant for time-dependent manner. Moreover, EVs secreted by S. PureTech Health, Inc. pneumoniae promoted their own planktonic growth and biofilm development in a dose-dependent manner. OF18.03 Summary/Conclusion: For the first time, we show that eukaryotic EVs are taken up by Gram-positive bacteria. Our data also indicate that pneumococcal EVs have Deciphering extracellular vesicle mediated host-pathogen interaction immunomodulatory effects on the host immune cells in streptococcus pneumoniae Saigopalakrishna Yernenia, Rory Eutseyb, Sarah Wernerb, Surya Aggarwalb, and represents a sophisticated communication system Changjin Huangc, K. Jimmy Hsiac, Luisa Hillerb and Phil Campbelld that needs further investigation. aDepartment of Biomedical Engineering, Carnegie Mellon University, Pittsburgh, PA, USA; bDepartment of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA; cSchool of Mechanical and Aerospace Engineering, College of Engineering, Nanyang Technological University, OF18.04 Singapore, Singapore; dDepartment of Biomedical Engineering and Engineering Research Accelerator, Carnegie Mellon University, Pittsburgh, PA, USA Calpain carried by platelet-derived microparticles mediates protease- Introduction: Extracellular vesicles (EVs) represent a activated receptor 1-dependent vascular inflammation in diabetes. highly sophisticated cell-to-cell mailing system. Both Anastasia Kyselova, Amro Elgheznawy, Ingrid Fleming and Voahanginirina Randriamboavonjy eukaryotic and prokaryotic cells selectively pack signal- Goethe University, Frankfurt, Germany ling cargo into and onto EVs and deliver them to the extracellular milieu. However, it was not until recently, Introduction: Diabetes mellitus is a major risk factor for that Gram-positive bacteria including the major cardiovascular diseases and platelet hyperactivation in diabetes is linked to the release of platelet-derived respiratory pathogen Streptococcus pneumoniae (pneu- b+ mococcus), were shown to produce EVs, likely origi- microparticles (PMPs) that carry the Ca -activated pro- nating from the plasma membrane to be released into tease calpain 1 (CAPN1). Here we determined whether the extracellular milieu. The role of Gram-positive EVs CAPN1 could target proteins on the vascular wall that in cell-to-cell communication, and their interactions could precipitate the development of vascular disease. 176 ISEV2019 ABSTRACT BOOK

Methods:MassspectrometryandELISAwereusedto Germans Trias i Pujol (IGTP), Badalona/Barcelona, Spain, and Institució analyse proteins in the culture medium. Protein levels on Catalana de Recerca i Estudis Avançats (ICREA), Barcelo, Barcelona, Spain the surface of endothelial cells were measured by FACs and Introduction: Using a reticulocyte-prone rodent en-face immunostaining was used to assess protein levels malaria model, resembling P. vivax, our group demon- on intact aorta. Diabetes was induced with streptozotocin. strated cytoadherence of infected reticulocytes to Results: In vitro treatment of human endothelial cells spleen blood barrier cells of fibroblastic origin with PMPs or recombinant calpain 1(CAPN1) led to a (Martin-Jaular et al., 2011). Here, as extracellular vesi- decrease in endothelial protein C receptor (EPCR) levels cles (EVs) play a role in intercellular communication, on the cell surface and an increase in its levels in the we hypothesized that plasma-derived EVs from natural culture medium. In agreement, EPCR levels were vivax infections (PvEVs) signal human spleen fibro- increased in plasma from diabetic patients. Also, dia- blasts facilitating adherence of P. vivax, a reticulocyte- betes induction in mice led to a similar increase in prone human malaria parasite. plasma EPCR levels, an effect prevented by treatment Methods: Upregulation of ICAM1 and other targeted with the calpain inhibitor. At the molecular level, genes upon uptake of PvEVs in human spleen fibro- CAPN1 did not directly target the EPCR but rather blasts (hSF) was determined by qRT-PCR. Expression of cleaved the protease-activated receptor 1 (PAR-1), indu- ICAM1 was validated by FACS. NF-kB nuclear translo- cing an intracellular signalling cascade i.e the phosphor- cation analysis was determined by confocal microscopy. ylation of protein kinase C (PKC) and the extracellular The binding capacity of P. vivax-infected reticulocytes regulated kinase (ERK) as well as the activation of the from infections upon uptake of PvEVs was tested after tumour necrosis factor (TNF)- converting enzyme maturation and purification of frozen estabilates of iso- (TACE). The latter was responsible of EPCR shedding lates from Mae Sot (Thailand). P. vivax-infected reticu- as well as of the increased local TNF- levels. TNF- locytes were incubated with hSF previously stimulated triggered the phosphorylation of the p65 subunit of with PvEVs, hEVs or PBS, and the number of binding NFκB, increased ICAM-1 expression and enhanced parasites determined by microscopy. monocyte adhesion. The same phenomenon was evident Results: ICAM-1, a known receptor for binding of in vivo as en-face preparations of aortae from diabetic malaria, was specifically upregulated by EVs from mice revealed a loss of PAR-1 but induction of ICAM-1 infections in a dose-dependent manner at mRNA and which could be prevented by CAPN inhibitor or by protein levels. NF-κ B was observed both in the cyto- specific knocking out of CAPN1 in platelets (PF4- plasm and the nucleus on non-stimulated and hEVs- Capn1-/-). All of the effects of PMPs or CAPN1 were stimulated hSF, whereas PvEVs stimulation induced abolished in PAR-1-deficient endothelial cells. nuclear translocation of NF-κ B on hSF. By comparing Summary/Conclusion:Thesedatademonstratethatpla- the binding of iRBCs to hSF, we last demonstrated telet-derived calpains contribute to diabetes-associated significant higher binding to the cells after uptaken of vascular inflammation by targeting the PAR-1 receptor. exosomes from infections. Funding: Deutsche Forschungsgemeinschaft RA 2435/ Summary/Conclusion: These results suggest that cir- 3-1. culating exosomes from vivax malaria infections have spleen-tropism signalling spleen fibroblasts to induce OF18.05 ICAM-1 through NF-kB and facilitate adherence of infected reticulocytes. Thus, unveiling molecular

Plasma-derived extracellular vesicles from P. vivax patients increase insights of cytoadherence in P. vivax infections. ICAM-1 expression of human spleen fibroblasts facilitating adherence Funding: Funded by Generalitat de Catalunya, of infected reticulocytes Haruka Todaa, Wanlapa Roobsoongb, Miriam Díaz-Varela1a, Barbara Baroc, Ministerio Español de Economía y Competitividad, Marcus VG Lacerdad, Pilar Armengole, Jetsumon Sattabongkotb, Carmen REDiEX, and Fundación Ramón Areces. HT is recipi- f g Fernandez-Becerra and Hernando A del Portillo ent of an AGAUR PhD fellowship aISGlobal Institute for Global Health, Hospital Clínic – Universitat de Barcelona, Barcelona, Spain; bMahidol Vivax Research Unit, Fac. of Tropical Medicine, Mahidol University, Bangkok, Thailand; cFundaçao de OF18.06 Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), Manaus, Brazil; dFundaçao de Medicina Tropical Dr. Heitor Vieira Dourado (FMT-HVD), Manaus, Brazil, and Instituto Leônidas & Maria Deane (ILMD), Fiocruz, Oxidative stress alert by extracellular vesicles, in vitro study in ocular Manaus, Amazonas, Brazil., Manaus, Brazil; eInstitut d’Investigació drainage system Germans Trias i Pujol (IGTP), Badalona/Barcelona, Spain; 6ISGlobal Natalie Lernera, Sofia Schreiber-Avissara and Elie Beit-Yannaib Institute for Global Health, Hospital Clínic – Universitat de Barcelona, a Barcelona, and Institut d’Investigació Germans Trias i Pujol (IGTP), Clinical biochemistry and Pharmacology department, Ben-Gurion b Badalona/Barcelona, Spain; 7ISGlobal Institute for Global Health, Hospital University, Beer-Sheva, Israel; Ben-Gurion University, Beer-sheva, Israel Clínic – Universitat de Barcelona, Barcelona, and Institut d’Investigació JOURNAL OF EXTRACELLULAR VESICLES 177

Introduction: The ocular drainage system is chroni- cytosolic increase was found when N-EV treatment cally exposed to oxidative stress (OS) contributing to was done. The most pronounced significant increase cataract and primary open angle glaucoma (POAG) cytosolic and nucleic expression was found following development. Classical markers of OS were found in Ox-EV treatment. Antioxidant gene expression showed patients ocular drainage tissues. The ability of EVs to a significant increase following Ox-EV treatment in deliver OS alert messages between the aqueous humor SOD2, GPx, HOX1 and NRF2 an effect that was not producing cells named non pigmented ciliary epithe- archived following N-EV treatment. SOD1 gene lium (NPCE) end the Trabecular Meshwork (TM) cells expression decreased following N-EV treatment but draining the aqueous humor was studied. did not change when Ox-EV were used. Using the Methods: NPCE cells were exposed to OS and their DCF-DA analytical method for total antioxidant capa- released EVs were collected (Ox-EV). Non-stressed city of TM cells we found that OX-EV treatment NPCE derived EVs (N-EV) were used as control. TM resulted in significantly higher antioxidant capacity vs cells exposed to the same OS were treated with Ox-EV N-EV or untreated TM cells. The two major antioxi- or N-EV and non-stressed TM cells were use as con- dant enzymes, SOD and Catalase activity was signifi- trol. The EV treatment effect was measured by Nrf2- cantly higher following OX-EV treatment. Keap1 signaling pathway changes including Nrf2 Summary/Conclusion: EVs are capable of OS alert to expression, related antioxidant gene expression, SOD other cell resulting in a better antioxidant capacity. and Catalase activity and TM cell antioxidant capacity. This phenomenon is relevant probably for all cells, Results: TM cells exposed to OS caused a significant can be the result of EVs cargo modification under OS 25% reduction in viability. When treated with Ox-EV including proteins and miRNAs or/and oxidized pro- the viability decrease was abolished. This cell rescue teins, lipid and nucleic acids carried by the EVs as effect was not shown with N-EV treatment. Increase in cargo or on their surface. Nrf2 cytosolic and nucleic expression was found fol- Funding: ISRAEL SCIENCE FOUNDATION (grant lowing TM oxidative stress. Some nucleic but not No. 1315/ 14). 178 ISEV2019 ABSTRACT BOOK

PF01: EVs Immune System Friday Poster Session Chairs: Wilfrid Boireau; Saara Laitinen Location: Level 3, Hall A 15:30–16:30

PF01.01 markers, but no accumulation of EVs with senescent features was found with age. The co-culture of plasma EVs with PBMCs influences diverse aspects of T cells. From adults to centenarians: characterization of T cell They enhance cell viability. Besides, under PHA stimu- immunosenescence markers on plasma extracellular vesicles and their influence on T cell activation, viability and interleukin secretion lation, EVs influence T cell activation and interleukin Ainhoa Alberroa, Iñaki Osorio-Querejetaa, Leire Iparraguirrea, Susana secretion. Interestingly, the effect of plasma EVs is Carregalb, Natalia Elguezabalc, Itziar Vergaraa, Adolfo López de Munaind, Matías Sáenz-Cuestaa and David Otaeguia distinct depending on the age of the donor. aBiodonostia Health Research Institute, Donostia – San Sebastián, Spain; Summary/Conclusion: Plasma EVs bear characteristic bCIC biomaGUNE, Donostia – San Sebastián, Spain; cNeiker Tecnalia, tetraspanins and T cell markers and can be detected by d Derio, Spain; Donostia University Hospital and Biodonostia Health FC. However, EVs with senescent features do not accu- Research Institute, Donostia – San Sebastián, Spain mulate with age. Moreover, EVs can influence T cell Introduction: Aging is a universal, complex and het- viability activation and IL secretion in vitro. erogeneous process that leads to reduced adaptation Funding: AA, IOQ and LI are supported by a fellow- capacity and increased vulnerability. Two of the hall- ship from the Dept. of Education of the Basque marks of aging are cellular senescence and altered Government intercellular communication. Specifically, the dysfunction and accumulation of senescent cells of the PF01.02 immune system is called immunosenescence. Regarding intercellular communication, the term senescence-associated secretory phenotype (SASP) is Profiling tetraspanin expression patterns on the surface of retroviruses and extracellular vesicles by nanoscale flow cytometry used and while inflammaging has been broadly studied, Vera Tang, Tyler Renner; Anna Fritzsche; Mariam Maltseva and Marc-André the role of extracellular vesicles (EVs) remains unclear. Langlois In the present work, we investigated the senescent University of Ottawa, Ottawa, Canada features of plasma EVs and their role in T cell activation, viability and interleukin (IL) secretion. Introduction: Tetraspanins are membrane-associated Methods: All participants (24–104 years) gave proteins with variable functions. Given that extracellu- informed consent and the study was approved by the lar vesicles (EVs) bud from the membranous structures Donostia University Hospital Ethics Committee. of the cell, whether it be at the cell surface or through PBMCs were isolated with Ficoll-Hypaque method the endosome, they serendipitously uptake these mem- and EVs by differential centrifugation as described brane-spanning proteins, most notably CD9, CD63 and before by our group (Saenz-Cuesta et al., 2015). T CD81. Retroviruses share numerous biophysical and cells were characterized by flow cytometry (FC) biochemical features with EVs, and especially small (FACSCanto II). Isolated EVs were detected by EVs in the 80 – 150nm size range. They also have a cryoEM, NTA and FC. The immunosenescence mar- membrane-derived envelope that captures tetraspanins, kers of EVs were also assessed by FC (CytoFLEX). as they too can egress from the cell surface and from Coculture experiments of PBMCs and EVs were per- the endosomal pathway. Here we attempt to distin- formed and activation of T cells was induced by PHA. guish EVs from retroviruses according to their surface Cultured cells were evaluated by FC and the super- tetraspanin expression profiles. natants by Luminex for IL measurement. Methods: Using nanoscale flow cytometry (NFC), we Results: Senescent T cells accumulate with age, and assessed the expression of tetraspanins CD9, CD63 and CD8 cells are more affected than CD4 cells. Most of CD81 by optimized antibody labelling on different isolated EVs are 100–200 nm. They carry characteristic fluorescent retroviruses including murine leukemia EV markers (CD63, CD81, CD9) as well as T cell virus (MLV), avian leukosis virus (ALV), and the JOURNAL OF EXTRACELLULAR VESICLES 179 human immunodeficiency virus-1 (HIV-1). These ret- smallest number of particles. Two million HOK-16B roviruses were engineered to express either eGFP or cells released 10 billion EVs for 24 h. The EVs superfolder GFP (sfGFP) on their surface as a fusion expressed general EV markers such as CD9, CD63, protein with the viral envelope glycoprotein. We also Flotillin-1 and Alix. According to the NTA the EVs analysed endogenous retroviruses released from mur- were heterogeneous in size. ine T cell lines EL-4 and BW5147, and from primary Summary/Conclusion: HOK-16B cells released EVs splenocytes from various mouse strains. that have general EV markers. The EVs derived from Results: Differential levels of CD9, CD63 and CD81 HOK-16B infected with periodontopathogen need to expression was observed among the retrovirus strains. analyse and confirm the biological function to other Also, the type of fluorescent reporter present influ- cells. enced tetraspanin capture on the surface of these Funding:ThisworkwassupportedbyNationalResearch viruses. Overall, these expression patterns appear dis- Foundation of Korea grants (No. NRF-2018R1A5A2 similar to those present on EVs released from unin- 024418 and NRF-2018R1A2A2A05018558). fected cells. Analysis of murine T cells lines and primary splenocytes also exhibit release of endogenous retroviruses, which was confirmed by Western Blot PF01.04 analysis of the p30 capsid protein. Summary/Conclusion: Taken together, these findings Air pollution effects on the clinical course of autoimmune diseases: suggest that caution must be exercised in the use of the role of extracellular vesicles tetraspanins as identifying markers for EVs as they are Mirjam Hoxhaa, Tommaso Schioppob, Simona Iodicea, Laura Pergolia, Nicola b a b a also highly expressed on retroviruses, which are known Ughi , Luca Ferrari , Francesca Ingegnoli and Valentina Bollati to be prevalent in most animal species including birds, aUniversity of Milan, Department of Clinical Sciences and Community Health, Milan, Italy; bDivision of Clinical Rheumatology, G. Pini Hospital, mice and humans. Furthermore, mouse studies should Milano, Italy control for the presence of endogenous retroviruses, which may contaminate EV sample preparations. Introduction: Autoimmune diseases (ADs) are charac- Funding: Natural Sciences and Engineering Research terized by the body’s intolerance to self-antigens. The Council of Canada (NSERC) cause of autoimmunity is still unknown. However, it is generally accepted that ADs might be triggered by PF01.03 environmental factors able to increase inflammation. In recent years, extracellular vescicles (EVs) have been described to play an important role both in ADs patho- Isolation of EVs derived from human oral keratinocytes genesis and environmental toxicants, such as particu- Younggap Lim and Bong-Kyu Choi late matter (PM). The aim of our study is to evaluate Department of Oral Microbiology and Immunology, School of Dentistry, PM effects on EV release in ADs. Seoul National University, Jongno-gu, Seoul, Republic of Korea, Seoul, Republic of Korea Methods: We recruited 24 patients with ADs (12 Rheumathoid Arthritis, RA and 12 Systemic Sclerosis, Introduction: Oral keratinocytes are the first defense SSc) and 12 patients with Osteoarthritis (OA), a non- line against external environments such as chemical autoimmune inflammatory disease taken as control. agents, microbes and physical factors. Stimulated oral Plasma EVs were analysed by Nanosight and flow keratinocytes produce cytokines/chemokines to modu- cytometry after labelling with the following markers: late local inflammatory status. Based on recent CD14+ (monocyte), CD61+ (platelet), CD25+ (T-reg), researches, not only cytokines/chemokines but extra- ERVWE1+ (human endogenous retrovirus W), HLAG cellular vesicles (EVs) also regulate immune response. + (human leukocyte antigen G). PM10 and PM2.5 Therefore, we hypothesized that oral keratinocytes concentrations at the residency of each subject were release EVs and those EVs could modulate immune obtained from the regional air quality monitoring response in the gingival tissue. network. Methods: EVs were isolated from human oral kerati- Results: The increase of PM2.5 led to a decrease of nocytes (HOK-16B) by ultracentrifugation (UC) and MVs CD14+ (β = −0.13; p < 0.01) and CD61+ commercial EVs isolation kit and analysed by western (β = −0.08; p = 0.05) in RA, of ERVWE1+ in both blotting and Nanoparticle Tracking Analysis (NTA). SSc (β = −0.10; p = 0.01) and OA (β = −0.09; p = 0.01), Results: To exclude EVs originated from cell culture and of HLA+ (β = −0.12; p < 0.01) only in SSc. Similar medium, we compared three different keratinocyte cul- results were observed analyzing PM10 exposure. ture media, then we chose medium that contained the Analysis of EVs concentration according to their 180 ISEV2019 ABSTRACT BOOK dimensions showed a negative association in the size could be partially impaired by the endosomal pathway range of exosomes (63–92 nm) in RA and SSc com- inhibitor, GW4869. In the CD4+ population, pared to OA (p < 0.05). Finally, we observed a negative HUCPVC-derived EVs promoted both the prolifera- association between exosomes and C-reactive protein tion of Treg and Teff. Notably, the ratio of proliferating (β = −1.99; p = 0,03), and a positive association Treg/proliferating Teff is increased by HUCPVC- between ERVWE1+ and Erythrocyte Sedimentation derived EVs treatment when compared to no cell-CM Rate (ESR) (β = 0.53; p = 0,06) and HLA+ and ESR control isolation, which eventually resulted in an (β = 0.29; p = 0,01). increase of Treg/Teff ratio. In the CD8+ population, Summary/Conclusion: Our findings showed that PM administration of HUCPVC-derived EVs significantly exposure could be a further risk factor of autoimmu- shifted the CD8+ population towards a CD8low popu- nity through a modulation of EV release. lation. We found no significant difference in the effect of EVs derived from inflammatory primed and unprimed HUCPVCs. PF01.05 Summary/Conclusion:HUCPVC-derivedEVsdemon- strated immunomodulatory effects by increasing Treg/ Teff ratio in the CD4 T helper cells and shifting the The immunomodulatory effects of human umbilical cord perivascular cell-derived extracellular vesicles on T lymphocyte differentiation cytotoxic T cell phenotype towards CD8low. We suggest Ching-Po Huanga, Lianet Lopezb, Daniel Ngb, Ansar Khanb, Peter Szarazc, that HUCPVC-derived EVs represent a promising cell-free b b d Denis Gallagher , Andrée Gauthier-Fisher and Clifford Librach immunomodulatory therapy. aCReATe Fertility Centre, Toronto, Ontario, Canada, National Yang Ming University, Taiwan., Hsinchu City, Taiwan (Republic of China); bCReATe Fertility Centre, Toronto, ON, Canada; cCReATe Fertility Centre, Toronto, ON, Canada; dCReATe Fertility Centre, Toronto, ON, Canada. Department PF01.06 of Obstetrics & Gynecology, University of Toronto, Toronto, Canada. Institute of Medical Sciences, University of Toronto, Toronto, Canada Cytokine and miRNA profiling of plasma extracellular vesicles in Introduction: We have characterized human umbilical individuals with myalgic encephalomyelitis/chronic fatigue syndrome Ludovic Giloteauxa, Adam O’Neala, Jesus Castro-Marrerob, Jennifer Grenierc, cord perivascular cells (HUCPVC) as a promising Maureen Hansona source of mesenchymal stromal cells (MSC). Our pre- aDepartment of Molecular Biology and Genetics, Cornell University, Ithaca, vious data from in vitro and in vivo models of myo- USA; bCFS/ME Unit, Vall d’Hebron University Hospital Research Institute cardial infarction and neurovascular injury support (VHIR), Universitat Autònoma de Barcelona, Barcelona, Spain, Barcelona, Spain; cRNA Sequencing Core, Department of Biomedical Sciences, College that HUCPVCs have potent immunomodulatory prop- of Veterinary Medicine, Cornell University, Ithaca, USA erty, and in many cases, are superior to bone marrow MSCs. The immunomodulatory effects of HUCPVCs Introduction: Myalgic encephalomyelitis/chronic fati- are thought to be contributed by paracrine factors. gue syndrome (ME/CFS) is a debilitating disease of However, the role of HUCPVCs in immunomodula- unknown aetiology lasting for 6 months or more, tion is still unknown. Here, we reveal the immunomo- with features including fatigue, cognitive impairment, dulatory effects of HUCPVC-derived extracellular myalgias, postexertional malaise, and immune system vesicles (EV) on T cell differentiation in vitro. dysfunction. Given the possibility that abnormal pro- Methods: Conditioned medium (CM) was obtained tein signaling could be altering cellular function in ME/ from sub-confluent first trimester (FTM) and term CFS, we aimed to characterize the cytokine/chemokine HUCPVCs cultured for 48 hrs in serum-free RPMI profile and miRNA content of blood-derived medium with or without cytokines (10 ng/mL of IFN- Extracellular Vesicles (EVs) in ME/CFS individuals γ, 15 ng/mL of TNF-α). HUCPVC-derived EVs were and healthy controls. enriched from CM using the Qiagen exoEasy Maxi kit, Methods: We included 35 ME/CFS patients and 35 followed by a Vivaspin 100k MWCO buffer exchange. sedentary controls matched for age, sex and BMI. Human peripheral blood mononuclear cells (PBMC) EVs were enriched from plasma by precipitation and were isolated by Ficoll gradient with written informed characterized by NTA, TEM and western blotting. A consent from healthy donors. PBMCs stimulated with 43-plex immunoassay was used to determine cytokine/ anti-CD3/CD28 beads were co-culture with HUCPVCs chemokine concentrations in both plasma and isolated or their EVs for five days. T cell differentiation and EVs. Total RNA was isolated from EVs and small RNA proliferation were analyzed by flow cytometry. libraries were prepared and sequenced for miRNA Results: HUCPVCs elicited an immunomodulatory profiling. effect by increasing the T regulatory cells/T effector Results: ME/CFS patients had significantly higher cell (Treg/Teff) ratio in a paracrine manner, which levels of EVs than controls and morphological analysis JOURNAL OF EXTRACELLULAR VESICLES 181 showed a homogeneous population of vesicles in both Results: Our evidence shows that immortalized T cells groups. Comparison of cytokine concentrations in treated with exosomes isolated from explanted 40 µm plasma and isolated EV for cases and controls yielded PTS significantly down regulate TNF-α expression few significant results. Cases had higher plasma levels without other transcriptomic effects. Moreover, pri- of MCP-1 and IP-10 and lower levels of RANTES, EGF mary co-stimulated (CD3/CD28) T cells treated with and GROβ. In isolated EVs, EGF and GROβ were also exosomes from macrophages (MØ) resident to 100 µm significantly lower in patients while concentrations of PTS exhibited broad transcriptional upregulation and IL-12p70, IL-2 and GM-CSF were higher. Correlations significantly upregulated proportional expression of analysis revealed that CD40L was a negative driver of several pro-inflammatory genes: TNFα, TBX21, TRAIL and IP-10 in patients but had no inverse asso- GATA3 and TGF-β1. When treated with exosomes ciations with other cytokines within the control group. from MØ in 40 µm PTS, these T cells exhibited no We also generated on average over 800,000 miRNA- broad transcriptional upregulation but proportionally mapped reads per EV sample. A total of 316 mature upregulated immunomodulatory T cell phenotypic miRNAs sequences were detected and no significant markers FoxP3 and IL-10. differences between groups after multiple corrections Summary/Conclusion: These data show that exosomal were found. PCA analysis of all ME/CFS and control signalling of PTS resident cells is controlled by pore miRNAs revealed a separation of the groups based size, thus influencing T cell differentiation and host on PC4. response. Particularly, exosomes from cells in 100 µm Summary/Conclusion: Higher levels of pro-inflamma- PTS proportionally upregulate T cell markers asso- tory cytokines were found in plasma and EVs from ciated with Th1, Th2 and Th3 T cell subpopulations ME/CFS patients and inter-cytokine correlations and transcriptomic stimulation, whereas exosomes revealed unusual regulatory relationships among cyto- from 40 µm PTS induce a proportional upregulation kines in the ME/CFS group that were not apparent in of T cell markers associated with immunomodulatory the control group. Studies with expanded cohorts are Tregs, without broad transcriptomic stimulation. Our needed to increase power of analysis. next experiments will examine the ability of exosomes Funding: This project was funded by NIH NIAID R01 generated in 40 µm PTS to recapitulate a healing AI107762. response in implants known to otherwise promote the foreign body response.

PF01.08 PF01.09

Immunomodulatory exosomal signalling mediated by porous Extracellular vesicles in systemic sclerosis as potential mediator for templated scaffolds pulmonary vascular disease a b b b Thomas Hady, Billanna Hwang and James Bryers Federica Rota , Alessandro Albini , Marco Vicenzi , Rosa Casella , Santaniello Alessandrob, Lorenzo Berettab, Jacopo Marianic, Laura University of Washington, Seattle, USA Cantonea, Laura Dionia, Valentina Bollatic and Federico Lombardib aEPIGET LAB, Department of Clinical Sciences and Community Health, Introduction: Porous templated scaffolds (PTS) with Università degli Studi di Milano, Milan, Italy; bFondazione IRCCS Ca’ pores 40 µm in diameter drive healing upon implanta- Granda Ospedale Maggiore Policlinico, Milan, Italy; cUniversity of Milan, Department of Clinical Sciences and Community Health, Milan, Italy tion by reducing inflammation and foreign body rejection while increasing local angiogenesis. Macrophage Introduction: Pulmonary vascular disease (PVD) is recruitment and polarization are known to play roles in characterized by media muscular hypertrophy/hyper- this phenomenon, but the mechanism driving this plasia. Recently, the deregulation of EVs in some forms healing response is poorly understood. We believe of pulmonary hypertension studies has been reported, 40 µm PTS resident immune cells are releasing exo- but data on pulmonary vascular disease are still lack- somes containing unique cargo that modulates healing + ing. We investigated whether EVs from SSc patients by influencing CD4 T cell subsets. with or without established PVD can induce hypertro- Methods: We quantified the cellular origin and inter- phy and/or hyperplasia in in vitro smooth muscle cells nal composition of exosomes isolated from explanted and to study vesicular miRNAs expression. 40 µm and 100 µm PTS using a Cre-Lox double trans- Methods: We isolated plasma EVs from: 3 SSc-PAH genic mouse model and qPCR, respectively. We then patients with established PVD under target therapy quantified the cellular response to these exosomes in [PH+]; 3 SSc patients with high clinical risk without vitro using qPCR, ELISA and cell proliferation assays. PVD [PH−]; 3 early SSc patients with low clinical risk 182 ISEV2019 ABSTRACT BOOK

[Ea]; and 3 healthy control subjects. Smooth muscle those that were associated with EVs, remain upregu- cells were cultured in RPMI complete medium lated in spite of complete suppression of HIV replica- enriched with EVs purified from each study subject. tion. Cardiovascular disease: Myocardial infarction Real-time cell growth was analysed with xCELLigence (STEMI) is associated with increased production of RTCA. miRNAs from both plasma and medium cell various cytokines of which IL-2, IL-6, IL-18, Gro-α EVs were characterized and target prediction was per- and MIG were selectively increased in EV-associated formed via Diana Tools mirPath 2.0. form. Logistic regression analysis of a cohort of 110 Results: Real-time analysis of cellular growth showed a individuals demonstrated that healthy controls and brisker growth in every aliquot exposed to EVs with STEMI patients can be discriminated solely based on respect to the control. The intergroup comparison the analysis of EV-associated cytokines with 82.3% showed that EVs from controls induced an inferior sensitivity and 85.4% specificity. Pregnancy complica- growth in terms of cell index and doubling time. PH− tions: Amniotic fluid from pregnancies with intra- showed the greatest effect on cell growth with respect amniotic infection (IAI) demonstrated significantly to Ea and PH+ treated subjects. The most deregulated increased concentrations of almost all cytokines in miRNA was miR-324-3p which was strongly downre- EV-associated form, as well as an increase of cytokine gulated in PH−, weakly downregulated in PH+ and fraction released in EV-associated form. upregulated in Ea. Bioinformatics prediction for 324- Summary/Conclusion: The increase of EV-associated 3p showed it to target lipids synthesis and metabolism cytokines is a common denominator for diverse human pathways. diseases associated with chronic immune activation. Summary/Conclusion: These results provide evidence For three diverse human pathologies, we observed that EV content may predispose to PVD. The observed increased cytokines packaging in EVs. In HIV infection miRNA is potentially linked with the effect on cellular EV-associated cytokines failed to return to baseline growth, suggestive of a role in subjects with high risk to with ART, increases in STEMI allow for discrimination develop PVD. The potential implication of deregulated from controls, and IAI increases EV-associated cyto- miRNAs, especially 324-3p, on lipids metabolism indi- kines. EV-associated cytokines may contribute to var- cates that this pathway could be involved in the patho- ious disease progressions and can be developed into genesis of SSc-PVD. diagnostic tools. Funding: NIH/NICHD Intramural Program. PF01.10 PF01.11 Extracellular vesicles-associated cytokines in human pathologies Leonid Margolis

Section of Intercellular Interactions, Eunice Kennedy Shriver National Isolation and characterization of serum exosomes from Cystic Fibrosis Institute of Child Health and Human Development, National Institutes of patients receiving lung transplant Health, Bethesda, MD, USA Ruying Chen, Billanna Hwang, Erika D. Lease, Ryan V. Abbaszadeh, James Bryers, Michael S. Mulligan

Introduction: Many human diseases progress due to University of Washington, Seattle, USA low-level chronic immune activation associated with the release of cytokines. Recently, we found that Introduction: Lung transplantation is an important many cytokines are released in association with extra- therapeutic option for Cystic fibrosis (CF) patients cellular vesicles (EVs) rather than in the soluble form. with end-stage lung diseases. Long-term survival has Here, we investigated these EV-associated cytokines in been impaired by various post-transplant complica- three human diseases that are known to be facilitated tions. There is a critical need for development of meth- by improper immune activation. ods to noninvasively monitor and evaluate patient Methods: Multiplexed bead-based assays of 33 free and recovery. Exosomes are the key mediator of intercellu- EV-associated cytokines in culture supernatants, plate- lar communication, regulator of immune system and let poor plasma and amniotic fluid. have been considered a promising candidate for liquid Results: HIV disease: Infection of human lymphoid biopsy. Here, we developed imaging flow cytometry tissue by HIV-1LAI.04 resulted in the increased release method that enables easy quantification and character- of cytokines of which IL-7, IL-21, IFN-, MIP-1 and ization of the exosomes surface markers in clinical RANTES were selectively increased in EV-associated samples. We profiled circulating exosome in serum form. After 13 days of tissue treatment with combined isolated from a cohort of CF lung transplant patients anti-retroviral therapy (ART) cytokines, in particular, with two macrophage and T cell-associated panels. JOURNAL OF EXTRACELLULAR VESICLES 183

Methods: Serum samples were acquired from 30 CF University Hospital of Cologne, Center for Integrated Oncology Cologne- patients 0, 1, 3, 6 and 12 months following lung trans- Bonn, CECAD Center of Excellence on “Cellular Stress Responses in Aging- Associated Diseases”,Center for Molecular Medicine Cologne, University, plantation. Upon isolation, exosomes were labelled Köln, Germany with CFSE and a macrophage (CD68, CD86, CD163) or T cell panel (CD3, CD4, CD8, CD45RO). Flow Introduction:Macrophagesarekeyeffectorcellsofthe cytometry analysis was carried out with Amnis chemo-immunotherapy (CIT) response in B-cell malig- Imagestream X Mark II. Results were analysed with nances. We have previously shown that loss of TP53 in patients’ clinical indicators including pulmonary func- leukemic B-cells diminishes the anti-tumour capacity of tion tests (PFTs) and chronic lung allograft dysfunction macrophages upon CIT. Here, we investigated the poten- (CLAD) diagnosis. tial p53-dependent mechanisms in leukemic B-cells that Results: Using optimized staining protocol, exosomes could alter the phagocytic capacity of macrophages isolated from a wide range of patient samples were upon CIT. labelled consistently. All seven selected surface markers Methods: The proteomic profile of control and TP53- were detected on circulating exosomes. Results indicate deficient leukemic B-cells, untreated or treated with that markers were selectively expressed on exosomes mafosfamide, was analysed by mass spectrometry. and do not follow the classic immune cell subsets. EVs were isolated from control and TP53-deficient Results indicated that pan markers (CD68, CD3) leukemic B cells by differential ultracentrifugation and expressions were lower than some subset markers, their proteomic content was evaluated by mass spectro- especially pro-inflammatory CD86. Patients diagnosed metry. Validation of protein expression was performed with CLAD showed higher percentage of macrophage by Western Blot and flow cytometry. The measure- surface markers consistently than the ones without ments of exosomes concentration and size distribution CLAD. Additionally, patients not diagnosed with were performed by NanoSight NS300 and ZetaView. CLAD demonstrated a higher percentage of CD45RO Results: 244 of 5785 proteins were observed to be expressing exosomes than those with CLAD at significantly different between TP53-deficient and con- 6 months. trol leukemic B-cells, with 159 independent of mafos- Summary/Conclusion: Detection and profiling of famide treatment, 147 associated to mafosfamide and macrophage and T cell associated surface markers on 86 modifications shared between DMSO and mafosfa- circulating exosomes provide additional insights into mide treatment. Enrichment analysis for GO terms patients’ recovery and immune processes. Imaging flow showed that TP53-deficient leukemic B-cells exhibited cytometry-based exosome profiling represents a novel mainly altered expression of proteins associated with and promising strategy in CF patients post-transplant EVs. We confirmed that TP53-deficient leukemic B- follow-up. cells produced higher concentration of EVs and that Funding: 265-2368 LTR Training Funds Mulligan (PI) the EV-protein content differed from control leukemic 09/01/2010-12/1/2025. B-cells. Notably, 1239 of 2663 proteins were significantly different between TP53-deficient and control PF01.12 leukemic B-cells, 68 were exclusively detected in the control-derived EVs and 128 proteins were only found in the TP53-deficient-related EVs Loss of TP53 modifies the quantity and protein load of extracellular Summary/Conclusion: The loss of TP53 drastically vesicles in leukemic B-cells Elena Izquierdoa, Daniela Vorholtb, Janica Wiedersteinc, Liudmila modifies the proteomic profile of leukemic B-cells Lobastovab, Stuart Blakemoreb, Marcus Krügerc, Hinrich Peter Hasenb, and influences the protein expression of leukemic B- d b Michael Hallek and Christian Pallasch cells upon mafosfamide treatment. Especially, the loss aDepartment I for Internal Medicine and CECAD, University Hospital of of TP53 regulates the EV-related protein expression Cologne, Bonn, Germany; bDepartment I for Internal Medicine and CECAD, University Hospital of Cologne, Köln, Germany; cGermany Institute for and EV production in leukemic B-cells Genetics and CECAD, Köln, Germany; dDepartment I of Internal Medicine, 184 ISEV2019 ABSTRACT BOOK

PF02: EVS in the Central and Peripheral Nervous System Chairs: Sowmya Yelamanchili; Elena Batrakova Location: Level 3, Hall A 15:30–16:30

PF02.01 concentrations of UC and EQ are 8.16 ×10^10 and 5.77 ×10^10 particles/mL. The AUC (area under curve) in identifying AD was 1.0, 1.0, and 0.875 by The effect of exosome purification method on the detection of amyloid β in exosomes with Photooxidation-Induced Fluorescence UC, MB and EQ, respectively. The result showed it Amplification (PIFA) could clearly identify AD from NC. Youhee Heoa, Min Cheol Parkb, SangYun Kimc, Kwanwoo Shind and Ji Yoon Kange Summary/Conclusion: Exosome isolations using the magnetic beads, the exosomes can be extracted even aKorea Institute of Sceince and Technology, Seoul, Republic of Korea; bIntekBio, seoul, Republic of Korea; cSeoul national university bundang in a small amount of less than 50 μl. Therefore, it is hospital, seoul, Republic of Korea; dsogang university, soeul, Republic of advantageous that the sample is used less and the Korea; eKorea Institute of Science and Technology, Seoul, Republic of Korea exosome can be isolated quickly. We believe that the Introduction: Blood-based diagnosis of disease using reliability of human samples will be improved by an exosomes sometimes demands a highly sensitive bioas- additional number of testing samples and optimization say to detect rare protein biomarkers. New assay meth- of PIFA assay. ods were suggested to overcome the limitations of a conventional ELISA system such as digital ELISA or PF02.02 plasmonic ELISA. However, these methods need a special expensive equipment with the long process. We Bioinformatic and biochemical evidence for extracellular vesicle have developed a photo-oxidation-induced fluores- remodelling in Huntington’s disease cence amplification (PIFA) that can measure less than Francesca Farinaa, françois-Xavier Lejeuneb, Satish Sasidharan Nairb, Frédéric Parmentierb, Jessica Voisinb, Lorena Martin-Jaularc, Clotilde Theryc and 1 pg/mL by continuous irradiation on resorufin for the Christian Nerib photooxidation of chemi-fluorescent substrate amplex aSorbonnes Université, Centre National de la Recherche Scientifique, red. This paper demonstrated it can identify Research Unit Biology of Adaptation and Aging, Team Brain-C, Paris, Alzheimer’s disease (AD) patient from normal control France; bSorbonnes Université, Centre National de la Recherche Scientifique, Research Unit Biology of Adaptation and Aging, Team Brain- (NC) by measuring a low level of amyloid beta(Aβ)in C, Paris, France; cInstitue Curie, Paris, France the neuronal exosome from plasma samples. Methods: The level of resorufin was measured by PIFA Introduction: Intercellular communication mediated to compare with conventional ELISA. The oligomer Aβ by extracellular vesicles (EV) is emerging as a mechan- was detected by same antibody system whose capture ism that is important to neuronal development and antibody is same as detection antibody to exclude the survival. Here, we investigated the features of EV sig- signals from monomer Aβ. We isolated exosomes from nalling in response to Huntington’s disease (HD), a plasma samples (AD:4, NC:4) by three methods: ultra- neurodegenerative disease that is caused by CAG centrifuge(UC), CD9 antibody-coated magnetic beads expansion in the Huntingtin gene and that shows a (MB) and ExoQuick with agarose precipitation (EQ). significant degree of clinical heterogeneity. Exosomes were lysed with RIPA buffer and Aβ as a Methods: We applied an integrated approach in which cargo protein in exosomes were measured by PIFA. we combined bioinformatic analysis of public HD data- ELISA was performed by an automated machine sets and biological analysis in cellular models of HD using polypropylene tip. After removing the tip with pathogenesis. HRP-tagged detection antibody, the fluorescence was Results: Using network methods to integrate high- measured continuously to amplify the fluorescence. dimensional HD transcriptomic data, we built a com- Results: The LOD of PIFA in measuring oligomer Aβ putational model of the transition between different was less than 100 fg/mL that was lower than 2 orders of phases of the HD process: from cell differentiation magnitude than commercialized ELISA kit. The (early phase) to dysfunctional striatum (intermediate dynamic range of PIFA assay is more than 5 decades. phase) and finally advanced neurodegeneration (late The volume of plasma sample was 150 uL and the final phase). This model evidenced the deregulation of a volume of exosome was almost the same. The set of genes associated with the biology of EVs from JOURNAL OF EXTRACELLULAR VESICLES 185 the earliest to latest phases of the disease. To test this Summary/Conclusion: We demonstrate that exposure hypothesis experimentally, we analysed EVs in mouse of astrocytes to HIV protein Tat mediates the induc- and human neuronal cell models of HD pathogenesis. tion and release of EV-miR-7 that is taken up by To this end, we analysed different EV subtypes, testing neurons, leading in turn, to downregulation of neuro- for changes in secreted level and protein cargo compo- nal NLGN2 and ensuing synaptic alterations. sition. The results suggest that EV subtypes, especially Importantly, synaptic impairment could be reversed small EVs, possibly including exosomes, may be altered by pretreatment of neurons with a neurotropic factor in these cells. PDGF-CC. Summary/Conclusion: Collectively, these data point to Funding:ThisworkwassupportedbygrantsDA040397, EV remodelling in the course of HD. Biological and MH112848 (S.B.) and DA042704, DA046831 (G.H.) from clinical implications will be discussed. the National Institutes of Health. The support by Nebraska Funding: ANR, France Center for Substance Abuse Research is acknowledged.

PF02.03 PF02.04

The pericytes-derived extracellular vesicle-mimetic nanovesicles HIV-1 Tat-induced astrocytic extracellular vesicle miR-7 impairs rescues erectile function by enchancing penile neurovascular synaptic architecture regeneration in a mouse model of cavernous nerve injury. Guoku Hu, Fang Niu, Ke Liao and Shilpa Buch Jiyeon Ocka, Guonan Yinb, Mi-Hye Kwona, Kang-Moon Songa, Kalyan Ghataka, Nguyen Nhat Minha, Min-Ji Choic, Yong Song Ghod, Ji-Kan Ryua University of Nebraska Medical Center, Omaha, USA and Jun-Kyu Suha

a Introduction:Althoughcombinationantiretroviralther- National Research Center for Sexual Medicine and Department of Urology, Inha University School of Medicine, incheon, Republic of Korea; bNational apy (cART) has improved the health of millions of those Research Center for Sexual Medicine and Department of Urology, Inha living with HIV, the penetration into the CNS of many University School of Medicine, Incheon, Republic of Korea; cinha university urology, incheon, Republic of Korea; dDepartment of Life Sciences, Pohang such therapies is limited, thereby resulting in residual University of Science and Technology, Pohang, Republic of Korea neurocognitive impairment, commonly referred to as NeuroHIV. Additionally, although cART can successfully Introduction: Extracellular vesicles (EVs) contains var- suppress peripheral viremia, there is a continuous persis- ious proteins, mRNA and miRNA, that have many tence of the cytotoxic viral Transactivator of transcription regulatory effects on recipient cells. However, most (Tat) protein in tissues such as the brain, thereby con- mammalian cells release low quantities of EVs, there- tributing to neuronal injury. fore, we use bioengineered method and extract extra- Methods:Transmissionelectronmicroscopy,NanoSight cellular vesicle-mimetic nanovesicles from mouse and western blot analyses were used to characterize astro- cavernous pericyte. The aim of this study was to inves- cyte-derived EVs (ADEVs). Among the various dysregu- tigate effectiveness of pericytes-derived extracellular lated miRs in the ADEV cargo, miR-7 levels were found vesicle-mimetic nanovesicles (PC-NVs) in restoring to be upregulated by real-time PCR. Uptake of ADEVs by erectile function in cavernous nerve injury (CNI) mice. neurons was assessed by confocal microscopy. Rodent Methods: Twelve-week-old C57BL/6J mice were used hippocampal neurons were exposed to Tat-ADEVs and and divided into into groups (N = 5 per group): sham assessed for inhibitory (GAD65 and gephyrin) and exci- operation group and bilateral cavernous nerve crushing tatory (vGlut1 and PSD95) synapses by immunostaining group receiving a single intracavernous injection of and confocal microscopy. PBS (20 µl) or PC-NVs (0.1 µg, 1 µg, 5 µg/20 µl, Results: Expression level of miR-7 was upregulated in respectively). One week later, erectile function was the astrocytes from SIV+/HIV+ brains. In addition, measured by electrical stimulation of the cavernous Tat-stimulated astrocytes also demonstrated upregu- nerve (N = 5 per group). The penis was then harvested lated expression and release of miR-7 in the EVs, that and stained with antibodies to CD31, NG2 and βIII were taken up by neurons, resulting in synaptic injury. tubulin. We also determined the neurotrophic and Furthermore, our results also demonstrated that expo- angiogenic potential of PC-NVs in an ex vivo major sure of hippocampal neurons to Tat-ADEVs resulted in pelvic ganglion (MPG) culture experiment. decreased expression of neuronal NLGN2, which in Results:ThePC-NVswasextractandcharacterizedby turn, led to loss of both excitatory and inhibitory several EVs markers, such as CD63, CD81 and TSG101. synaptic densities. Moreover, we also demonstrated a Intracavernous injections of PC-NVs significantly neuroprotective role of PDGF-CC in rescuing Tat- improved erectile function in CNI mice, which reached ADEV-mediated synaptic loss. up to 84% of control values. PC-NVs induced significant 186 ISEV2019 ABSTRACT BOOK restoration of cavernous contents of endothelial cells, PF02.06 pericytes, and neuronal cells in CNI condition. Moreover, PC-NVs promoted major pelvic ganglion neurite sprouting in ex vivo culture experiment. Comprehensive study of vesicular and non-vesicular-associated Summary/Conclusion: In light of critical role of neu- miRNAs from a volume of cerebrospinal fluid compatible with the clinical practice ropathy in the pathogenesis of radical prostatectomy- Endika Prieto Fernándeza, Ana Maria Aransayb, Felix Royoc, Esperanza induced ED, pericytes-derived extracellular vesicle- Gonzalezc, Juan Jose Lozanod, Borja Santos-Zorrozuae, Nuria Macias- Camarab, Monika Gonzalezb, Raquel Perez Garayf, Javier Benitog, Africa mimetic nanovesicles successfully restored erectile Garcia-Orada and Juan Manuel Falcon-Perezc function in cavernous nerve injury mice. Further stu- aDepartment of Genetics, Physical Anthropology and Animal Physiology, dies are needed to clarify mechanism by which PC- Faculty of Medicine and Nursery, University of The Basque Country (UPV/ NVs induces neuropathy repair. EHU), Leioa, Spain; bGenome Analysis Platform, CIC bioGUNE, Derio, Spain; cExosomes Lab, CIC bioGUNE, CIBERehd, Derio, Spain; Funding: This work was supported by the National dBioinformatics Unit, Centre Esther Koplovitz (CEK), CIBERehd, Research Foundation of Korea (NRF) grant funded by Barcelona, Spain; eDepartment of Genetics, Physical Anthropology and Animal Physiology, Faculty of Medicine and Nursery, University of The the Korea government (MSIP) (Guo Nan Yin, Basque Country (UPV/EHU), Leioa, Spain; fBiochemistry service, Cruces 2018R1C1B6003829). University Hospital, Barakaldo, Spain; gDepartment of Pediatric Emergency, Cruces University Hospital, Barakaldo, Spain

PF02.05 Introduction: Cerebrospinal fluid (CSF) miRNAs have emerged as a potential low invasive diagnostic tool for central nervous system malignancies. However, they Neuronal EVs and microglial activation in hypoxia Yvonne Couch and Kate Lambertsen have not yet been implemented in the clinic since University of Oxford, Oxford, UK there is no a reliable and simple method established to analyse the limited amount of CSF obtained from Introduction: There is significant data suggesting that patients, especially from infants. EVs play a key role in CNS injuries such as stroke. Methods:Wehavecomparedsixexistingprotocolsfor During these types of injury the penumbral tissue characterizing miRNAs from a clinically rational volume around the core of the injured area is hypoxic and (i.e. 200 µl) of CSF. Four of the methods incorporated an inflammatory. Here, we aimed to test the hypothesis extracellular vesicles (EVs) enrichment step and the other that hypoxia affects EV production in cells of the two aimed to extract miRNAs directly from cleared CSF. neurovascular unit. The efficiency of each method was assessed by real-time Methods: Using endothelial cells, microglia and neu- PCR (qPCR) and smallRNA sequencing (smallRNAseq). rons, we subjected cells to short periods of oxygen and Additionally, by size-exclusion chromatography, we glucose deprivation (OGD) measuring both the EVs determined the distribution of miRNAs among different released into the medium, and the downstream effects CSF components. of these EVs on cells. Results: We found that NOR and INV protocols were Results: Brain-derived endothelial cells (b.END3) and the most efficient. According to our results, NOR was microglia (BV-2) do not produce significant numbers very reproducible by qPCR, showed a good miRNA of EVs in response to hypoxia. However, N2A neuronal levels correlation between techniques, and presented a cells do, both under normal and differentiated condi- user-friendly protocol starting from low volumes of tions. These EVs appear to be pro-inflammatory when CSF. In addition, we identified a set of microRNAs applied to microglia in culture. enriched in CSF exosomes that are involved in neuro- Summary/Conclusion: Together, these data suggest developmental pathways. that EVs produce pro-inflammatory EVs during peri- Summary/Conclusion: We found that different proto- ods of hypoxia, capable of activating microglia. cols purify specific miRNAs subpopulations and CSF Characterizing these neuronal EVs more fully may exosomes isolated by size-exclusion chromatography enable us to determine whether they are also able to contain miRNAs involved in neurodevelopment. escape the CNS during brain injury and activate per- Funding:ThisworkwassupportedbytheBasque ipheral macrophage populations. Government [IT989-16]; the Spanish Ministry of Funding: MRF University of Oxford, Rose Trees Trust Economy and Competitiveness MINECO (SAF2015- 66312), and the Ramon Areces Foundation (FRA-17- JOURNAL OF EXTRACELLULAR VESICLES 187

JMF). We also thank MINECO for the REDIEX (Spanish observed changes in the injured L5 nerve. This suggests Excellence Network in Exosomes) and the Severo Ochoa that the plasma-derived EVs can potentially serve as Excellence Accreditation (SEV-2016-0644). key regulators, biomarkers and targets in the progression and treatment of neuropathic pain. Funding:ThisresearchwassupportedbyCongression- PF02.07 ally Directed Medical Research Programs-Applied Pain Research (MR157005C). Research was conducted in com- Identifying plasma-derived extracellular vesicle (EV) contained pliance with the Animal Welfare Act, and all other biomarkers in the development of chronic neuropathic pain Federal requirements. The views expressed are those of Natasha Sosanyaa, Raina Kumarb, John Cliffordc, Roger Chavezd, George Dimitrove, Seshamalini Srinivasanf, Aarti Gautamg, Alex Trevinoa, Rasha the authors and do not constitute endorsement by the U. Hammamiehh, Bopaiah Cheppudirai, Robert Christya, Stephen Crimminsj S. Army. aUSAISR, San Antonio, USA; bUSACEHR/FNLCR, Fort Detrick, USA; cUS Army Center for Environmental Health Research, Frederick, USA; dCo- Author, Floresville, USA; eUSCEHR; FNLCR, Fort Detrick, USA; fUS Army Medical Research and Materiel Command, USACEHR, Frederick, USA; gUS PF02.08 Army Center For Environmental Health Research, Frederick, USA; hUSACEHR, Frederick, USA; iUS Army Institute of Surgical Research, San Antonio, USA; jUS Army Institute of Surgical Research, San Antonio, USA A pilot study to evaluate serum miRNA from extracellular vesicles of neural origin for insight into neurological disorders Introduction: Over 100 million Americans suffer from Michelle M. Barnetta, Michael Geaghana, Ebrahim Mahmoudia, Judith b c chronic pain. However, research into potentially novel Weidenhofer and Murray Cairns biomarkers and therapeutics for chronic pain is lack- aUniversity of Newcastle, Australia, Callaghan, Australia; bUniversity of Newcastle, Australia, Ourimbah, Australia; cUniversity of Newcastle, ing. microRNAs (miRNAs) are attractive candidates as Callaghan, Australia biomarkers due to their conservation across species, stability in liquid biopsies, and variation that corre- Introduction: miRNA are important regulators of gene sponds to a pathologic state. miRNAs can be sorted expression and can be informative biomarkers of dis- into extracellular vesicles (EVs) within the cell and ease state. Alteration of miRNA expression in the brain released from the site of injury. EVs transfer cargo has been observed in several neurological and psychia- molecules between cells thus affecting key intercellular tric disorders postmortem, but they are almost impos- signalling pathways. The focus of this study was to sible to non-invasively biopsy. These molecules, determine the plasma-derived EV miRNA content in however, have been observed in serum and serum a chronic neuropathic pain model. vesicles and have application as trait biomarkers. Methods: Male Sprague-Dawley rats underwent either While it is possible that brain disorders also have spinal nerve ligation (SNL; n = 6) or sham (n =6) systemic changes that contribute to the serum profile surgery, while under anaesthesia. Mechanical allodynia it is reasonable to suspect that extracellular vesicles (MA) was assessed and plasma-derived EV RNA was (EV) that originated in the brain would have more isolated at baseline and days 3 and 15 post-injury specificity and relevance to the neuropathology. In followed by small RNA sequencing. Differentially this pilot study we investigated strategies for capturing expressed (DE) miRNAs and gene target enrichment/ and profiling the miRNA content from extracellular signalling pathways analysis was performed using R vesicles derived from the brain using an immunoaffi- packages and TargetScan/IPA, respectively. nity approach. Results:SNLratsdisplayedatime-dependentincrease Methods:Wholebloodfromahealthymalevolunteer in MA from day 3 to day 15 post-injury. Our results was collected in serum separator tubes, immediately indicate that 22 miRNAs at day 3, 74 miRNAs on day processed and stored at −80°C. Antibody-coupled 15, and 33 miRNAs at both day 3 and 15 were magnetic beads were incubated with serum and cell uniquely differentially expressed between the SNL culture EV. Enriched and non-enriched fractions were and sham groups. The plasma-derived DE EV recovered using a commercial spin column kit for EV miRNAs were also found to regulate processes impor- purification and RNA extraction. Small RNA libraries tant in the development and maintenance of neuro- were prepared for 75 cycles of single end multiplex pathic pain states. sequencing. Raw reads were converted to fastq, adap- Summary/Conclusion: The key findings from this pro- tors trimmed, reads mapped, aligned and counted. posal include (1) the majority of the DE EV miRNAs, Results: miRNA were identified in both total human which normally function to suppress inflammation, serum and the neural fractions. Functional annotation were downregulated, and (2) twenty-one of the of the 37 miRNA present in the neural EV revealed plasma-derived DE EV miRNAs reflect previously that neuron projection was the most enriched cellular 188 ISEV2019 ABSTRACT BOOK component in predicted targets, suggesting that they alterations in the expression of these proteins have may serve to augment the synaptic regulatory environ- been associated with synaptic plasticity, intellectual ment. In support of this hypothesis, we found over disability and autism spectrum disorder. representation of all four cytoplasmic polyadenylation Summary/Conclusion: Serum EV can be enriched for element binding proteins (CPEB1-4) among targets those of neuronal origin and this strategy may provide predicted to be regulated by the most abundant insight into the brain’s regulatory environment and miRNA. CPEB proteins affect translation of mRNA ultimately more sensitive biomarkers of neurological bearing polyadenylation element sequences and and psychiatric disorders. JOURNAL OF EXTRACELLULAR VESICLES 189

PF03: EVs Cancer Metastasis Chairs: Ryou-u Takahashi; Irina Nazarenko Location: Level 3, Hall A 15:30–16:30

PF03.01 metastasis in vivo model. Exosome miR-1246 levels in blood of melanoma and oral cancer patients were significantly higher than those in healthy subjects. Promotion of metastasis via alteration of vascular endothelium by Summary/Conclusion: Thus, it was suggested that tumour exosome miRNA Masahiro Morimotoa, Nako Maishia, Aya Matsudaa, Tetsuya Kitamuraa, miR-1246 in tumour-derived exosomes promotes lung Fumihiro Higashinoa, Yoshimasa Kitagawab, Yasuhiro Hidac and Kyoko metastasis by inducing the adhesion of cancer cells to a Hida ECs and destroying the EC barrier. aVascular Biology and Molecular Pathology, Hokkaido University Graduate School of Dental Medicine, Sapporo, Japan; bOral Diagnosis and Medicine, Hokkaido University Graduate School of Dental Medicine, Sapporo, Japan; cCardiovascular and Thoracic Surgery, Hokkaido University Graduate School PF03.02 of Medicine, Sapporo, Japan

Introduction: We have reported that tumour endothe- Patient-derived circulating exosomes enhance cancer and stemness properties through polymeric immunoglobulin receptor in liver lial cells acquire diversity by cancer secreting factors. cancer Cancer cells secrete exosomes to create a suitable envir- Sze Keong Tey and Judy Yam onment for themselves. miRNA (miR) is transported The University of Hong Kong, Hong Kong, Hong Kong by exosome from cell to cell. We have identified miR- 1246 that is more abundant in high metastatic mela- Introduction: Hepatocellular carcinoma (HCC) is a noma (A375SM) exosomes compared with in low primary malignancy of liver which often diagnosed at metastatic melanoma (A375) exosomes by miRNA an advanced stage accompanied by extrahepatic metas- array analysis. In this study, we investigated the role tasis. Emerging evidences have demonstrated cancer of miR-1246 in alteration of the character of endothe- cells derived exosomes play an important role in inter- lial cells (ECs). In addition, we addressed the mechan- cellular communication in tumour microenvironment ism of cancer metastasis induced by miR-1246. during metastasis. Thus, exosome research may solve Methods: We focused on the adhesion between ECs the mystery of metastatic organotropism in HCC. and between cancer cells and ECs. Changes in adhesion Methods: Exosomes were isolated from HCC patients. molecule expression and endothelial permeability were The biological effects of exosomes were studied using examined. We analysed the effect of the administration transwell and matrigel invasion assays. The in vivo of A375SM exosome and miR-1246 knockdown on effect of exosomes were analysed in mice by intrave- lung metastasis in vivo. In addition, exosome miR- nous injection of exosomes together with hepatoma 1246 levels in blood of melanoma and oral cancer cells. The in vivo lung pulmonary vasculature and patients were compared with healthy subjects. vascular leakiness were revealed by FITC-lectin stain Results: miR-1246 transfection in ECs increased the and presence of Texas Red-dextran, respectively. Stem- expression of adhesion molecule, ICAM-1 via activa- like properties were examined by spheroid assay and tion of STAT3, and increased the number of adherent flow cytometry. Proteomic profiling and expression cancer cells to ECs. Furthremore, A375SM exosome level of exosomal proteins were analysed by mass spec- treatment enhanced endothelial permeability. Also, trometry and enzyme-linked immunosorbent assay the expression of VE-Cadherin, which is intracellular (ELISA), respectively. adhesion molecule of EC and function as EC barrier, Results: We recruited representative individuals of dif- was downregulated by miR-1246 transfection. The tar- ferent stages of HCC development for the collection of get gene prediction database and reporter assay showed circulating exosomes, namely, exosomes of healthy that VE-Cadherin is the target gene of miR-1246. individual (Normal-Exo), patient with cirrhosis Intravenous administration of A375SM exosome- (Cirrhosis-Exo), HCC patients at early stage (E-HCC- induced cancer cell colonization in the lung, resulting Exo) and late stage with distant metastasis to lungs (L- in establish of lung metastasis. In contrast, miR-1246 HCC-Exo). Among these exosomes, L-HCC-Exo knockdown in A375SM caused in decrease of lung exhibited the highest effect in promoting HCC cell 190 ISEV2019 ABSTRACT BOOK migration and invasion in vitro and colonization of Results: We identified 892, 311 and 331 proteins hepatoma cells in lungs. L-HCC-Exo also modulated including ATP synthase subunits in apoptotic bodies, tumour microenvironment by enhancing pulmonary microvesicles and exosomes, respectively. We further vascular permeability. Along with cancerous beha- confirmed that ATP synthase was indeed localized on viours, L-HCC-Exo induced stem-like properties such EV membrane. Additionally, we observed the release of as increased self-renewal, expressions of hepatic CSC these three subtypes of EVs was decreased after starva- markers and drug resistance. Elevated level and activity tion stress and an eATP synthase inhibitor citreoviridin of β-catenin were also detected in cells stimulated by L- treatment. However, we did not measure the signifi- HCC-Exo. Proteomic profiling of exosomal proteins cantly different ATP production between control and identified polymeric immunoglobulin receptor (pIgR) citreoviridin treatment in apoptotic bodies, microvesi- to be markedly upregulated in L-HCC-Exo when com- cles and exosomes, indicating that ATP synthase on the pared with exosomes of other subjects. EVs may not be for ATP synthesis. We observed that Summary/Conclusion: HCC patient-derived circulat- eATP synthase was transferred from cancer cells to ing exosomes augment cancer and stemness properties normal cells via EVs, indicating eATP synthase plays through the activation of β-catenin signalling by exo- an important role for cell-to-cell communications and pIgR, leading to a more aggressive phenotype in HCC eventually promotes cancer metastasis. tumourigenesis and metastasis. Summary/Conclusion: Our findings suggest that ATP synthase indeed exists on the membrane of EVs and PF03.03 enhances cancer cells to release EVs for cell-to-cell communications. Funding:MinistryofScienceandTechnology(MOST Ectopic ATP synthase induces extracellular vesicle release for cell-to- 106-2320-B-002-053-MY3) and National Health cell communications Yi-Chun Kaoa, Nai-Wen Changb, Yi-Wen Changa, Charles P. Laic, Hsuan- Research Institutes. (NHRI-EX107-10709BI) in Taiwan. Cheng Huangd and Hsueh-Fen Juane aDepartment of Life Science, National Taiwan University, Taipei, Taiwan (Republic of China); bInstitute of Molecular and Cellular Biology, National PF03.04 Taiwan University, Taipei, Taiwan (Republic of China); cInstitute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan (Republic of China); dInstitute of Biomedical Informatics, National Yang-Ming University, Taipei, Taiwan (Republic of China); eNational Taiwan Anesthesia-dependent changes in vesicular miRNA profiles during University, Taipei, Taiwan (Republic of China) colorectal cancer surgery Dominik Buschmanna, Anja Lindemannb, Florian Brandesc, Gustav Schellingc, Michael Pfaffld and Marlene Reithmaire Introduction: Ectopic Adenosine triphosphate aTUM School of Life Sciences Weihenstephan, Division of Animal Physiology synthase (eATP synthase) is defined as ATP synthase and Immunology, Freising, Germany; bInstitute of Human Genetics, located on cell surface instead of inner membrane of University Hospital, LMU Munich, Germany, Munich, Germany; c mitochondria. Our previous studies showed eATP Department of Anesthesiology, University Hospital, Ludwig-Maximilians- University Munich, Germany, München, Germany; dAnimal Physiology and synthase located on lung cancer cell surface and gen- Immunology, School of Life Sciences Weihenstephan, Technical University of erated ATP to extracellular space. Recently, several Munich, Freising, Germany, Freising, Germany; eInstitute of Human Genetics, University Hospital, Ludwig-Maximilians-University Munich, reports indicated that the subunits of ATP synthase Germany, München, Germany were identified in extracellular vesicles (EVs) using proteomics approach. However, where does ATP Introduction: Colorectal cancer (CRC) is one of the synthase in EVs come from and what are the functions most common and most deadly cancer types world- of it are still unclear. We proposed the hypothesis: ATP wide, leading to 50,000 annual deaths in the US alone. synthase in EVs may be conveyed from cell surface for Even though surgical resection presents the best chance cell-to-cell communications. of survival for CRC patients, accumulating evidence Methods: Here we used immunochemistry to detect demonstrates that removal of primary tumours can eATP synthases and serial high-speed centrifugation foster disease progression and metastasis. Recent out- to isolate EVs including apoptotic bodies, microvesicles come-based studies described differential effects of the and exosomes which were further confirmed by trans- type of anaesthesia used during CRC surgery on metas- mission electron microscopy (TEM) and nano tracking tasis as well as overall and recurrence-free survival. As analysis (NTA). Furthermore, we used quantitative mechanistic data on how anaesthesia impacts cancer proteomics by dimethyl labelling to profile the pro- progression are sparse, we assessed the potential invol- teomes in extracellular vesicles, and dot blot analysis vement of extracellular vesicles (EVs) in the process. to elucidate whether ATP synthase was localized on the Methods:Serumwassampledfrom18CRCresection EV membrane. patients before induction of anaesthesia (pre) using JOURNAL OF EXTRACELLULAR VESICLES 191 propofol (n =8)orsevoflurane(n =10)andaftersurgery characterized the proteomic and miRNAs cargo of (post). EVs were precipitated from 1 ml serum, and EXO isolated from NB cell lines cultured at different associated microRNAs (miRNAs) were profiled by oxygen concentrations to identify an exosomal signa- Next-Generation Sequencing. The anaesthesia-dependent ture associated with NB metastatic dissemination. impact on miRNA profiles in paired EV samples was Methods: SKNAS and SKNDZ NB cell lines were cul- assessed using DESeq2. Next, we performed pathway tured for 48 h in standard (20% O2) and hypoxic (1.5% analyses based on differentially regulated miRNAs. O2) conditions. EXO were purified from the media Additionally, deregulated candidates selected from NGS using Ultra spin tubes 100K MWCO and characterized data were validated by RT-qPCR. by scanning electron microscopy (SEM) and qNANO. Results:NGS-basedprofilingofEVsresultedin Proteome and miRNA cargo profiles were analysed by 3.79E6 ± 1.58E6 (propofol pre), 3.09E6 ± 1.81E6 (propo- quantitative mass spectrometry and FirePlex Discovery fol post), 3.40E6 ± 1.65E6 (sevoflurane pre) and Panel (on 405 miRNAs), respectively, and surface mar- 3.34E6 ± 1.32E6 (sevoflurane post) mean miRNA reads kers were evaluated using MACSplex technology. per sample. As evidenced by Principal Component Results: SEM and qNANO size distribution analysis Analysis, samples from pre- and post-operative sera clus- gave populations of round particles within the expected tered into distinct groups for both types of anaesthesia. diameters (50–120 nm). Surface markers analysis Differential expression analysis revealed 64 and 44 revealed that NB hypoxia-derived EXO express an miRNAs significantly regulatedbypropofolandsevoflur- increase of proteins associated with angiogenesis, adhe- ane, respectively. Despite substantial overlap in the sion, stemness and immune function such as CD105, intraoperative miRNA changes, a set of 31 (propofol) CD29, CD49e, SSEA4, HLA-DR and HLA-ABC. We and 11 (sevoflurane) miRNAs specifically responsive to characterized the proteomic cargo of EXO isolated either drug was also identified. In silico analyses indicated from cultures in normal and hypoxic conditions reveal- adifferentialeffectofanaesthesia-responsivemiRNAson ing differential expression of about 90 proteins. These cancer-relevant pathways such as proliferation, apoptosis preliminary results highlight relevant changes in the and migration. expression of several markers of EXO derived from Summary/Conclusion: Previous studies have demon- cultures exposed to different oxygen concentrations. strated distinctive effects of propofol and sevoflurane Summary/Conclusion: We successfully isolated and on tumour cells, host immunity and survival in CRC. purified exosomes from NB cell lines and assessed Anaesthesia-induced changes in circulating miRNAs their protein composition. These promising results might mediate disease progression and impact post- are the starting point for the identification of predictive surgical outcome. biomarkers to be used to detect and monitor metastatic spread in NB. PF03.05 Funding: ERC Starting Grant 2017 to Elisa Cimetta.

The role of hypoxia-derived exosomes in determining Neuroblastoma PF03.06 dissemination and aggressiveness Pina Fuscoa, Maria Rosaria Espositob, Giulia Borilec, Marcello Manfredid, Emilio Marengod and Elisa Cimettaa HNSCC exosomes drive tumour angiogenesis via ephrin reverse signalling aDepartment of Industrial Engineering (DII), Padova University – Shinya Sato and Alissa Weaver Fondazione Istituto di Ricerca Pediatrica Città della Speranza (IRP), b Padova, Italy; Department of Industrial Engineering (DII), Padova Department of Cell and Developmental Biology, Vanderbilt University University – Fondazione Istituto di Ricerca Pediatrica Città della Speranza School of Medicine, Nashville, USA (IRP), Padova, Italy; cUniversity of Padova, Department of Physics and Astronomy, Padova, Italy; dUniversity of Piemonte Orientale, Department of Science and Technological Innovation, Alessandria, Italy Introduction: Exosomes are small extracellular vesicles (EVs) that are secreted upon fusion of multivesicular Introduction: Neuroblastoma (NB) is a heterogeneous endosomes (MVE) with the plasma membrane and paediatric malignancy of the sympathetic nervous sys- carry bioactive protein and RNA cargoes. A number tem accounting for up to 10% of childhood cancers of studies have identified key roles for exosomes in with a strong tendency to metastasize. Hypoxia is a key promoting tumour angiogenesis; however, the mechan- feature of solid tumours and is specifically known to (i) isms are unclear. Our goal is to identify the role of head favour NB metastasis and dedifferentiation towards and neck squamous cell carcinoma (HNSCC) exo- immature stem cell-like phenotypes and to (ii) stimu- somes in tumour angiogenesis. late release of exosomes (EXO), facilitating intercellular Methods: EVs were collected from the conditioned communication at distant sites. In this study, we media of HNSCCs and purified through cushioned 192 ISEV2019 ABSTRACT BOOK density gradient ultracentrifugation. An orthotopic cholesterol using high density lipoprotein mimetic mouse model was used for the assessment of tumour nanoparticles (HDL NPs). angiogenesis. Angiogenic potential of EVs was assessed Methods:Exosomeswereisolatedviaultracentrifuga- by tube formation assays with Human Umbilical Vein tion of conditioned media from EnzR CWR-R1 pros- Endothelial Cells (HUVECs). tate cancer cells. Murine bone marrow macrophages Results: In HNSCC tumours, the microvessel density were obtained by culturing total bone marrow in M- correlated with exosome secretion rates of original CSF for 7 days. For in vitro experiments, cells were HNSCC lines. In vitro, CM and purified exosomes treated with exosomes derived from EnzR CWR-R1 but not exosome-depleted CM from HNSCC cells cells (1–10 ug/mL exosomal protein) with or without drove tube formation of HUVECs and human lympha- HDL NPs (50–150 nM). For in vivo experiments, 10 tic endothelial cells. Proteomics analysis of HNSCC ug exosomal protein were injected via tail vein with or exosomes revealed multiple potential angiogenic pro- without HDL NPs (1 uM, 100 ul). Confocal micro- teins, including EphB2 and EphB4. The addition of scopy and flow cytometry were used for uptake purified HNSCC exosomes to HUVECs-induced experiments. Osteoclast differentiation assays were reverse ephrin-B signalling in endothelial cells, as performed using a commercially available TRAP assessed by Western blot analysis. To test whether staining kit (Sigma Aldrich). NF-kB activation assays reverse ephrin-B signalling might account for exowere performed using the human monocyte reporter some-induced angiogenesis, we pre-incubated purified cell line, THP-1 Dual. HDL NPs were synthesized exosomes with Fc-ephrin-B2 to block the interaction using 5 nm gold nanoparticle templates, phospholi- between exosomal EphB2 and ephrin-B2 on endothe- pids, and apolipoprotein A-1. Mechanistic studies lial cells. We found that low concentrations of this were performed using transgenic, SR-B1 knockout reagent had little effect on endothelial tube formation mice. in the absence of exosomes but blocked the pro-angio- Results:Resultsshowedthatmyeloidcelluptakeof genic effect of the exosomes. In addition, EphB2-KD EnzR CWR-R1 exosomes was inhibited in vitro and HNSCC derived exosomes significantly reduced in vivo upon treatment with HDL NPs. Furthermore, endothelial tube formation compared to parental functional inhibition was observed via reduced osteo- HNSCC derived exosomes. clast differentiation and reduced stimulation of NF- Summary/Conclusion: We find that HNSCC-derived kB signalling. Finally, experiments conducted using exosomes can induce reverse ephrin-B signalling and SR-B1 knockout mice revealed that nanoparticle angiogenesis. This mechanism may be important in the inhibition is dependent upon the scavenger receptor, HNSCC microenvironment. SR-B1. Funding: This work was funded by the National Summary/Conclusion: Our findings demonstrate that Institutes of Health grant R01CA163592. exosome-mediated signalling between prostate cancer cells and myeloid cells can be inhibited using HDL NPs. Furthermore, our results strongly suggest that PF03.07 exosome-mediated crosstalk between prostate cancer cells and myeloid cells are dependent upon cholesterol

Nanoparticle mediated inhibition of intercellular communication homeostasis. between enzalutamide resistant prostate cancer cells and myeloid Funding: This work was supported by the National cells Stephen Henricha, Kaylin McMahona, Michael Plebanekb and C. Shad Institutes of Health and the Prostate Cancer Thaxtona Foundation. aNorthwestern University, Chicago, USA; bDuke University, Durham, USA PF03.08 Introduction: Crosstalk between neoplastic cells and myeloid cells has emerged as an axis of communication High-grade bladder cancer cells secrete extracellular vesicles which drives tumour progression and metastasis. containing MiRNA-146a-5p and promotes angiogenesis Recently, our group and others have shown that cancer Marta Prieto Vilaa, Wataru Usubab, Nobuyoshi Kosakac, Fumitaka Takeshitad, Hideo Sasakib, Tatsuya Chikaraishib and Takahiro Ochiyac exosome-mediated intercellular signalling is depen- a dent, in part, upon target cell cholesterol homeostasis. Division of Mollecular and Cellular Medicine, National Cancer Center Research Institute, Japan, Tokyo, Japan; bSt. Marianna University, School of In this study, we investigated whether exosome signal- medicine., Tokyo, Japan; cDepartment of Molecular and Cellular Medicine, ling between enzalutamide resistant (EnzR) prostate Institute of Medical Science, Tokyo Medical University, Shinjyuku-ku, Japan; dDivision of functional analysis, National Cancer Center Research Institute, cancer cells and myeloid cells could be effectively Tokyo, Japan inhibited by targeted reduction of myeloid cell JOURNAL OF EXTRACELLULAR VESICLES 193

Introduction: High recurrence is one of the major facilitate their brain metastasis. Long distance of pri- issues in bladder cancer (BCa). The classical method mary breast cancer site to the brain may require the for detecting recurrence is cystoscopy, a highly invasive communication mediator to deliver cancer favourable technique. Thus, novel methodologies with high relia- information to the brain. However, the exact role of bility and low-invasiveness are needed. To overcome breast cancer-derived exosome during brain metastasis this problem, biomarkers in the urine such as is not well understood. In this study, we observed the microRNA (miRNA) in extracellular vesicles (EVs) phenomenon that breast cancer-derived exosomes are been proposed. We previously detected high urin- directly activate primary astrocytes and the co-culture ary levels of miR-146a-5p in patients with BCa related condition of these activated astrocytes with microglia to tumour grade. However, the function of this miRNA cells enhances cancer cell proliferation and invasion. in EVs from BCa had not been elucidated yet. Here, we Methods:Totracetheextracellularvesicle(EV)including show that miRNA-146a-5p in EVs promoted angiogen- exosome movement, Palm-tandem dimer tdTomato esis in BCa. (Palm-tdTomato) lentiviral vector was transduced into Methods:High-gradeBCacellline,UMUC-3,withmiR- MDA-MB-luc-D3H2LN (D3H2LN) breast cancer cells. 146a overexpression, was established and orthotopically EVs isolated from D3H2LN-Palm-tdTomato cell lines transplanted in SCID mice. Immunohistochemical analy- showed the increased Palm-tdTomato fluorescence inten- sis was performed to evaluate angiogenesis. Cellular pro- sity and were stably internalized into astrocytes. After liferation, migration, and invasion were assessed in human astrocytes were treated by the EVs, we checked the level umbilical vein cell (HUVEC) after the addition of EVs of Glial Fibrillar Acidic Protein (GFAP), vimentin, MCP- from BCa. The target gene of miR146-5p was identified 1/CCL2 and IL-6 expression. Astrocytes and microglia by microarray and in silico analyses, and downregulation are co-cultured under the EVs containing media. was further confirmed by qPCR and western blot. Results: We found that astrocytes taken up by cancer- Results: Urinary miR-146a-5p level was higher in derived exosomes were activated, showing the increase patients with high-grade BCa and the tumours pre- in GFAP, vimentin, MCP-1/CCL2 and IL-6 expression. sented high levels of angiogenesis. Similarly, miR- Also, we found that co-culture glial cells of astrocytes 146a overexpressed BCa cells orthotopically injected and microglia significantly increased cytokine IL-6 pro- into mice generated tumours with high levels of angio- duction. The co-cultured medium from cancer exo- genesis. HUVEC cell proliferation was significantly somes-stimulated astrocytes and microglia increases increased, both under transfection of miR-146a mimic invasion and proliferation of cancer cells and inhibits and treatment with miR-146a-enriched EVs. Moreover, tumour suppressor gene in breast cancer cells. the target of miR-146a was found to be TET2, which Summary/Conclusion: These results indicate that has been reported to regulate cell growth in other breast cancer-derived exosomes participate in activat- malignancies. Consequently, TET2 was downregulated, ing astrocytes and the activated astrocytes and micro- both at RNA and protein level, under miRNA-146a- glia induce breast cancer proliferation and invasion enriched EVs treatment. during brain metastasis. Summary/Conclusion: Our findings indicate that EVs containing miR-146a-5p from BCa, previously described as BCa biomarker, promoted the prolifera- PF03.10 tion of endothelial cells that supported tumour growth. These results demonstrate that miRNAs in EVs may The glycosylation status affects the biodistribution of cancer become not only a diagnosis tool but also a target extracellular vesicles Akiko Kogurea, Nao Nishida-Aokib, Naoomi Tominagac, Nobuyoshi Kosakad molecule for cancer therapy. and Takahiro Ochiyad

aDivision of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan; bHuman Biology Division, Fred PF03.09 Hutchinson Cancer Research Center, Seattle, USA; cDepartment of Biology, Massachusetts Institute of Technology, Massachusetts, USA; dDepartment of Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Activated glial cells stimulated by breast cancer-derived exosomes Medical University, Shinjyuku-ku, Japan enhance proliferation of brain metastatic breast cancer cells Dayi Jeonga, Oh Jinhyea, Dowon Hwangb and Dongsoo Leeb Introduction: It has been shown that extracellular vesicles (EVs) from cancer cells delivered to the meta- aSeoul National University, Seoul, Republic of Korea; bSeoul National University Hospital, Seoul, Republic of Korea static site, and promoted metastasis by communicating with microenvironmental cells, although molecules, Introduction: Brain metastatic breast cancer cells have which are indispensable for cancer progression, has been known to stimulate glial cells in the brain to been investigating yet. It is well known that aberrant 194 ISEV2019 ABSTRACT BOOK glycosylation is a hallmark of cancer, and is related to fluorescence intensities ofeachorganweremeasuredin the cancer malignancy; however, the role of the glyco- order to determine the amount of the EVs remained at sylation of EV surface proteins in cancer progression the organs. has not been clarified yet. In this study, we investigated Results: We found that the glycosylation level of EVs the role of glycosylation of the EVs from metastatic from metastatic cancer cells was higher than that from cancer cells in the biodistribution. non-metastatic cancer cells. Moreover, enzymatic Methods:Weperformedlectinblotanalysisinorderto digestion of N- and O-linked glycans on EVs increased compare the glycan level of the EVs among metastatic their incorporation to the endothelial cells in vitro. cancer cell lines and non-metastatic cancer cell lines. Furthermore, we found that the glycosylation status Then, we investigated whether glycosylation of EVs of EVs from cancer cells influenced their direction to affects their incorporation rate to endothelial cells by the organs in mice. enzymatic deglycosylation in vitro.DiR-labelledEVs Summary/Conclusion: These findings suggest that the were employed to analyse the location of EVs in vivo by glycosylation of EVs from cancer cells involved in the intravenous injection. After 24 h of injection, the biodistribution of EVs. JOURNAL OF EXTRACELLULAR VESICLES 195

PF04: EV-mediated inter-organism communication Chairs: Chitose Oneyama; Kyoko Hida Location: Level 3, Hall A 15:30–16:30

PF04.01 PF04.02

Preferential packaging of tRNA fragments into extracellular vesicles released by protozoan parasite Trichomonas vaginalis The endothelial PlGF is upregulated by exosomes from activated a b a Anastasiia Artuyants , Anthony Phillips and Augusto Simoes-Barbosa kidney fibroblast a b c c a Noritoshi Kato , Fumitoshi Nishio , Yoshio Funahashi , Hiroki Kitai , School of Biological Science, The University of Auckland, Auckland, New c c b Shintaro Komatsu and Shoichi Maruyama Zealand; Department of Surgery, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand aNagoya University Graduate School of Medicine, Nagoya, Japan; bTushima City Hospital, Tushima, Japan; cNagoya University Graduate School of Introduction: Extracellular vesicles (EVs) are impor- Medicine, Nagoya, USA tant mediators of cell-to-cell communication. Delivery Introduction: It is well known that patients with of EVs is known to modulate the response of the chronic kidney disease (CKD) are at risk of cardiovas- recipient cells. EVs are produced by most if not all cular diseases, but the mechanism of this distant organ organisms and are involved in communication between crosstalk is not fully understood. Recently, placental host and pathogen. Trichomonas vaginalis is a unicel- growth factor (PlGF) received attention in pathogenesis lular eukaryotic pathogen, known to produce EVs with of cardio-renal syndrome (CRS). Under the hypothesis proteins and RNA cargo. This parasite colonizes the that exosomes are involved in pathophysiology of CRS, mucosal surface of the human genitourinary track the aim of this study is to explore the role of exosomes extracellularly. In this study, we hypothesised that the from kidney fibroblasts, which actively proliferate in RNA cargo of parasite EVs is an important element of diseased kidney, on vascular endothelial cells. this host-pathogen communication. Methods: Clinical samples; HUVECs were stimulated Methods: As the first step of this investigation, we by serum exosomes from stage G5 CKD patients and isolated and characterised EVs from T. vaginalis strain healthy donor. Exosomes tracking; Primary culture of B7RC2. Small RNAs present in these vesicles were activated kidney fibroblasts were obtained from experi- identified by deep-sequencing and specificity of these mental renal fibrosis model mice. These exosomes were molecules as EVs cargo was evaluated. labelled by microRNA of C. elegance (Cel-miR-39) then Results: Our results show that T. vaginalis releases labelled exosomes were injected to the mice through membrane-bound vesicles with an average size of tail vein. Effects of exosomes on endothelial cells; We ~100 nm that are taken up by host cells. These vesicles purified exosomes from culture media of TGF-b sti- are depleted of DNA but enriched with RNAs of small mulated kidney fibroblasts cell line (NRK-49f), and size. These RNAs are physically protected from exo- then primary culture of vascular endothelial cells genous RNases. The population of small RNAs was (RAOEC) was stimulated using these exosomes. By consistent among libraries, with tRNA being the most qPCR, we evaluated the expression of PlGF genes. abundant RNA biotype in all samples. We identified Results: (1) Not only the serum but also exosomes individual sequences from the top 30 transcript clusters from CKD stage G5 patients stimulated PlGF expres- as being mostly tRNA fragments, particularly 5’-tRNA sion on HUVECs. (2) Injected labelled exosomes from halves. The presence of the identified fragments was activated kidney fibroblast distributed mainly in lung, validated and compared with total cells by digital dro- liver and aorta. (3) RAOEC stimulated with exosomes plet PCR, showing the preferential packaging for these form TGF-b activated rat kidney fibroblast showed tRNAs into EVs. higher expression of PlGF than control. Summary/Conclusion: Our study indicates that tRNA Summary/Conclusion: So far, CRS is considered to be fragments from T. vaginalis EVs (particularly tRNA caused by uremic factor, RAS system, chronic inflam- halves) might play a role in communication with host mation and so on. From this study, both serum and cells. Work to confirm their bioactivity continues. exosomes from CKD patients stimulated PlGF 196 ISEV2019 ABSTRACT BOOK transcription on endothelial cells. Exosomes from acti- cargo of EVs, but does not affect their uptake. Our vated kidney fibroblast had same tendency. We specu- study helps to disclose the radiation-related mechan- lated that exosomes from diseased kidney have some isms involved in EV signalling and the role of EV roles in atherosclerotic change by modulating the signalling in systemic response of organisms to IR. expression of PlGF on endothelial cells. Farther studies Funding: The Euratom research and training pro- are needed to elucidate the degree of contribution gramme 2014–2018 (CONCERT, grant agreement to CRS. number 662287) and a Hungarian Scientific Research Funding: N/A. Fund–OTKA (124879).

PF04.03 PF04.04

The effect of in vivo irradiation on the extracellular vesicle’s cargo UVB induced-release of bioactive microvesicle particles in and uptake keratinocytes carry platelet-activating factor Tünde Szatmária, Dávid Kisa, Nikolett Sándora, Eszter Persab, Rita Hargitaia, Ji Bihl, Langni Liu, Christine Rapp and Jeffrey Travers Enikő Kisa, Katalin Balázsa, Géza Sáfránya and Katalin Lumniczkya Wright State University, Dayton, USA aNational Public Health Center, Division of Radiobiology and Radiohygiene, Department of Radiation Medicine, Budapest, Hungary; bNational Public Health Center, Budapest, Hungary Introduction: Ultraviolet B radiation (290–320 nm; UVB) has profound effects upon skin and generates Introduction: Recent studies suggest that ionizing systemic consequences. As UVB only penetrates the radiation (IR), as a stress agent, induces changes in epidermis, a major question in photobiology is how the release, uptake and composition of extracellular UVB-treated skin sends systemic signals. Recent stu- vesicles (EVs). EVs were shown to play a role in radia- dies have indicated that small membrane-bound vesi- tion-related signalling and radiation induced bystander cles known as microvesicle particles (MVP) released effects (RIBE). We have recently shown that EVs from cells in response to various stressors can act as released by bone marrow (BM) cells of mice irradiated potent signalling agents due to their ability to carry with X-rays mediate RIBE, such as DNA damages, nuclear and cytoplasmic components. Our lab has pre- chromosomal aberrations or phenotypical changes in viously determined that UVB induces the production certain cellular subpopulations of the BM. The aim of of the lipid mediator, platelet-activating factor (PAF), this study is to investigate the mechanism of these which is involved in mediating both acute pro-inflam- functional changes. matory and immunosuppressive UVB responses. More Methods: In order to follow the uptake of irradiated recently, we discovered that UVB generates MVP EVs, we isolated EVs from BM of total-body irradiated release (UVB-MVP) from epithelial cells and skin in a (TBI) mice, labelled them with a selective RNA stain PAF-PAFR dependent way. However, the contents of and co-incubated them in vitro for 3 h with BM cells UVB-MVP have not identified and whether UVB-MVP extracted from nonirradiated mice. We quantified the carry PAF is not known. uptake of EVs in different BM subpopulations by flow Methods: In this study, we determined the kinetics of cytometry and fluorescence microscopy. To test PAF production in cell- vs. MVP over time. IL-8 whether in vivo irradiation affects the miRNA cargo release assay was further used to confirm the PAF-R- of EVs, total RNA was isolated from the same EVs, agonist activity in KBP cells using PAF as positive subjected to miRNA profiling and assessed by bioin- control. Moreover, we verified the PAF-R-agonist formatical tools. Significantly altered miRNAs were activity in UVB-MVP in animal models. validated by qRT-PCR in EVs, BM cells of EV donor Results: The kinetics of PAF agonist production fol- and recipient mice. lowing UVB suggest that PAF-R agonists generated in Results: There were differences in EV uptake capacity response to UVB were cell-associated early, then, were of different BM cell subpopulations but irradiation did found predominantly in MVP. The PAF-R-agonist not change the extent of EV uptake. We identified a activity found in MVP of HaCaT cells 2 h post UVB. panel of miRNAs differentially expressed in the EVs UVB-MVP contain approximately 20 ng of PAF activ- following TBI of mice with involvement in DNA ity per 1E+10 MVP. However, PAF agonistic activity damage repair, immune system regulation and acute was not found in control MVP, and UVB-MVP did not leukaemia. generate IL-8 release in PAFR- negative KBM cells. Summary/Conclusion: We proved that EVs transmit Topical application of lipid extracts from UVB-MVP certain radiation related signals; IR alters the miRNA derived from HaCaT cells onto ears of WT mice JOURNAL OF EXTRACELLULAR VESICLES 197 resulted in an increase in ear thickness at 2 h, however, interpreted as the euclidean distance between cells. In there was no effect on PAF-R Knock-out (KO) mice order to understand the main features of the model, it Summary/Conclusion: This study suggests that UVB- is enough to isolate one designated cell, called the root, MVP contain bioactive PAF agonists involved in acute and understand how effective is its communication UVB-induced inflammation. This is the first study with the other cells. demonstrating that UVB-MVP carry PAF. Results: We regard the EV as signals sent to other cells. Funding: National Institutes of Health (NIH): R21 At each stage the root sends a signal to another cell AR071110. chosen with probability proportional to the weight associated to the connecting it to the root. Each time PF04.05 an edge is traversed, its weight is updated. This allows learning during the communication. In other words, the root has preference in communicating with cells A mathematical model for extracellular vesicles, as a communication that has been already contacted before. Each signal tool between cells. Anna Concetta Berardia and Andrea Collevecchiob contains a task. Once a cell receives a task, it will activate in order to complete it. On the other hand, aospedale Santo Spirito Pescara, Pescara, Italy; bMonash University, Melbourne, Australia the completion of the task has a random duration. If during this time the cell is contacted too frequently by Introduction: The main goal of the present work is to the root cell (that is above a certain threshold), it will introduce a mathematical model for extracellular vesi- abort the task. cles (EV), as a communication tool between cells. Summary/Conclusion:Ourgoalistounderstand Methods: Our basic model has a graph theoretical what are the phases transitions of this model with representation in terms of weighted graphs and sto- respect to its parameters as the number of vertices chastic processes that take values on the vertices of the grow to infinity. In other words, if the threshold graph, which play the role of cells. More specifically associated to the abortion is large enough, we expect consider a complete graph, where each vertex commu- to have a positive proportion of the cells to accom- nicates with any other vertex. To each edge of the plish the task. graph associate a positive number, which might be 198 ISEV2019 ABSTRACT BOOK

PF05: EVs in Infectious Diseases and Vaccines Chairs: Tsuneya Ikezu; Maja Mustapic Location: Level 3, Hall A 15:30–16:30

PF05.01 Foundation of Korea (NRF-2017R1A2B1006373, NRF- 2017R1A2B4002405).

Extracellular vesicles from KSHV-infected cells stimulate antiviral immune response through mitochondrial DNA PF05.02 Hyungtaek Jeon, Jisu Lee, Suhyuk Lee, Su-Kyung Kang, Sang June Park, Seung-Min Yoo and Myung-Shin Lee Exosomes secreted by platelets infected with Hepatitis E virus can Eulji University School of Medicine, Daejeon, Republic of Korea mediate transmission of HEV Lishan Chenga, Yu Liub, Ping Fuc, Bingting Wuc and Ling Kec Introduction: Interferon-stimulated genes (ISGs) are aChinese Academy of Medical Sciences and Peking Union Medical College, vital in controlling viral infections. As many antiviral Chengdu, China (People’s Republic); bChinese Academy of Medical Sciences ISGs continue to be identified, their roles in viral and Peking Union Medical College, 石家庄市, USA; cChinese Academy of Medical Sciences and Peking Union Medical College, chengdu, China pathogenesis are also being explored in more detail. (People’s Republic) Kaposi’s Sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi’s sarcoma, which is the most Introduction: Evidences suggested that exosomes can common cancer in acquired immune deficiency syn- transfer genetic material between cells, including viral drome patients. Because KSHV contains numerous nucleic acids, proteins or miRNAs which can mediate viral proteins that modulate antiviral response, type 1 transmission of viruses such as HBV or HCV. It is Interferon response is strongly suppressed in KSHV- known that platelet-derived exosomes constitute the infected cells. However, the antiviral effects of extra- major fraction in the circulating plasma which can cellular vesicles (EVs) during de novo KSHV infection participate in haemostasis, immunity and development. have not been investigated to our best knowledge. Whether the virus infected platelet-derived exosomes Methods: EVs were isolated from KSHV-infected cells can also promote the transmission of virus has not at 24 h of postinfection and characterized. The expres- been reported. The hepatitis E virus (HEV) is one of sion of ISGs in these EVs-treated human endothelial the most common causes of acute hepatitis worldwide. cells was investigated and underlying mechanisms were Recent studies have shown that the exosomes secreted analysed. by HEV-infected cells were infectious. Our studies have Results: In this study, we showed that KSHV-infected confirmed that HEV can infect platelets, thus we con- cells induce ISG response in uninfected bystander cells ducted this study to prove if exosomes secreted by using EVs. mRNA microarray analysis indicated that platelets infected with HEV are also infectious, thereby ISGs and IRF-activating genes were prominently acti- further promoting the transmission of HEV. vated in EVs from KSHV-infected cells (KSHV EV)- Methods:Anin vitro model of HEV-infected platelets treated human endothelial cells, which were validated were established by HEV-G3 virus strain and washed by RT-qPCR. Mechanistically, mitochondrial DNA on human platelets and the exosomes were isolated from the surface of KSHV EVs was presumed to be asso- HEV-infected and uninfected platelet by differential cen- ciated with ISG response via the cGAS-STING path- trifugation and magnetic bead separation. Exosomes were way. In addition, KSHV EV-treated cells showed lower characterized by Western Blot and TEM, and quantitated infectivity for KSHV and viral replication activity than by NTA. qRT-PCR and ELISA were used to detect HEV mock EV-treated cells. RNA and proteins in exosomes. Positive exosomes were Summary/Conclusion: Our results indicated that EVs used to infect PLC/PRF/5 cells, observing the changes of from KSHV-infected cells would be an initiating factor HEV RNA and proteins within one month. for the innate immune response against viral infection, Results: The in vitro model of HEV-infected platelets which would be helpful to expand our understanding was successfully established. The concentration of exo- of the microenvironment of virus-infected cells. somes secreted by HEV-infected platelets was higher Funding:ThisworkwassupportedbytheBasicScience than uninfected platelets. Exosomes isolated from Research Program through the National Research HEV-infected platelets contained HEV RNA and JOURNAL OF EXTRACELLULAR VESICLES 199 proteins. HEV RNA and proteins were detected in cells roles in HBV hepatitis through the multiorgan association and supernatant of PLC/PRF/5 cells infected with posi- of liver, bone marrow and gut. tive exosomes, and the concentration of which Funding: AMED hepatitis grant. increased after the culture of one month. Summary/Conclusion: Our study showed that HEV can promote the secretion of platelet exosomes and PF05.04 these vesicles can establish a productive infection which suggested that the exosomes secreted by platelets HIV-1 Nef mediated Hck kinase activation triggers loading of TACE not only play a role in haemostasis, immunity and into EVs in a ceramide-dependent manner development, but also play a non-negligible role in Zhe Zhao, Riku Fagerlund and Kalle Saksela the transmission of the virus. University of Helsinki, Helsinki, Finland Funding: 1.CAMS Innovation Fund for Medical Sciences (CIFMS2016-I2M-1-018) Introduction: TNF-α converting enzyme (TACE) 2. Supported by the Fundamental Research Funds for exists in circulating EVs collected from HIV-infected the Central Universities(Item No:3332018125) individuals. In addition to its role in TNF-α shedding, TACE is responsible for the proteolytic maturation of PF05.03 numerous cytokines, cytokine receptors and extracellular matrix components involved in inflammation. Thus, EV-associated TACE could be a key player in Multi organ association mediated by extracellular vesicles secreted the chronic immune activation that drives AIDS patho- from HBV positive hepatocyte Ai Kotania and Masatoshi Kakizakib genesis and persistent viral replication. Although uploading of TACE into EVs is promoted by the aTokai University, Isehara, Japan; bTokai University, School of Medicine, Department of Gastroenterology, Iisehara, Japan HIV-1 pathogenicity factor Nef, the mechanism of TACE secretion remains incompletely understood. Introduction: Hepatitis B virus (HBV) infected hepa- Methods: EVs were isolated by ultracentrifugation and tocytes secreted extracellular vesicles such as virion, analysed by western blotting. Gene knock-out by exosome and incomplete virions such as hallow parti- CRISPR was used to study TACE loading into cles which have only HBs viral antigens but neither exosomes. capsid and HBV genome. We found that the EVs are Results: We have previously shown that uploading of taken by monocyte/macrophage which upregulates PD- TACE into EVs is promoted by HIV-1 pathogenicity L1, immune checkpoint molecule. (Kakizaki et al PLOS factor Nef via its interaction with cellular protein one in press). It suggests that the EVs play critical roles kinases. Herein, we have studied the molecular in viral immunology. mechanisms of TACE loading into EVs in more Methods: PKH labelled EVs are injected into mice then depth and show that active Src family tyrosine kinases those biodistribution are analysed by tissue sections (SFKs) Lck, Hck, Lyn, Fyn and Fgr can trigger TACE and confocal microscopy and flow cytometry then secretion. Among the SFKs Hck is unique by displaying multiorgan association are analysed by use of bone two isoforms p59 and p61. We found that only acti- marrow transplantation. The HBV hepatitis model vated p59, but not p61, could trigger TACE secretion. mice was established to analyse the significance of the In contrast to the myristoylated and palmitoylated EVs on HBV hepatitis. SFKs and p59Hck the p61 isoform lacks the palmitoy- Results: In almost all the organs we analysed the PKH lation signal and these differing signals have been signal were detected which suggest the biodistribution shown to direct p59 and p61 to the plasma membrane of the EVs are systemic. Especially in the bone marrow and lysosomes, respectively. Our observations show monocytes, PKH positive cells are abundant. Then the that catalytic activity and proper membrane association bone marrow cells which were treated by PKH labelled domain of Hck is required for TACE secretion into the EVs secreted from HBV infected hepatocytes were EVs. We characterized the origin of the EVs in Hck- transplanted. The organ where the most PKH positive activated TACE secretion by nSmase2 knock-out, and cells were detected was gut. These cells showed CD103 found that Hck-mediates the loading of TACE into positive which suggest these cells are regulatory DCs. vesicles that similar to classical exosomes are generated The HBV hepatitis model mice treated by the EVs in a manner requiring nSmase2-mediated ceramide showed persistent HBV infection. synthesis. Summary/Conclusion:Basedontheresults,theEVs Summary/Conclusion: We conclude that HIV-1 Nef secreted from HBV infected hepatocytes have significant mediated Hck p59 kinase activation triggers the 200 ISEV2019 ABSTRACT BOOK loading of TACE into exosome-like vesicles in a cer- show that the release of gp120 is followed by the amide dependent manner. increase in syncytia formation in the macrophage Funding: Jane and Aatos Erkko Foundation cultures. Summary/Conclusion: We conclude that chronic PF05.05 Meth abuse interferes with EV biogenesis and cargo release in HIV infected cells. These results can uncover the role of chronic Meth abuse in progression of HIV Role of Extracellular Vesicles in HIV and Methamphetamine induced pathogenesis. neurotoxicity Sowmya V. Yelamanchilia, Dalia Moorea, Catherine DeMarinob, Farah Funding: NIH/NIMH/NIDA Shahjinc and Fatah Kashanchib aDepartment of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, USA; bGeorge Mason University, PF05.06 Manassas, USA; cDepartment of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, OMAHA, USA

Extracellular vesicle-associated cytokines in HIV infected human Introduction: The advent of combined antiretroviral lymphoid tissue ex vivo treatments (cART) has markedly decreased the preva- Vincenzo Mercurioa, Wendy Fitzgeraldb and Leonid Margolisc lence of HIV-associated dementia. However, there aDepartment of Biomedical and Clinical Sciences ‘L. Sacco’, University of b remains a high prevalence rate of the milder forms of Milan, Milan., Bethesda, USA; Section of Intercellular Interactions, Eunice Kennedy Shriver National Institute of Child Health and Human HIV-associated neurocognitive disorders (HAND). Development, National Institutes of Health, Bethesda, MD, USA; cSection Although many contributing factors have been studied, of Intercellular Interactions, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, the role of drugs of abuse has remained elusive. Bethesda, MD, USA Methamphetamine (Meth) and related amphetamine compounds, which are potent psychostimulants, are Introduction: Cytokines play an important role in HIV among the most commonly used illicit drugs. Long- infection. Some of these cytokines are present on the term Meth abuse is associated with a host of systemic surface or encapsulated in extracellular vesicles (EVs). and neurological maladies. Neurologically, Meth abu- We investigated the modulation of EV-associated cyto- sers exhibit cognitive and psychomotor impairment, kines during HIV infection and antiretroviral therapy and have shown increased risk for HIV infection. (ART) in human ex vivo tonsils. However, the mechanisms underlying Meth and HIV Methods: Ex vivo tonsils were infected with HIV-1 neurotoxicity are still not known. This study focuses strains, X4-LAI04 or R5-SF162. HIV was either allowed extracellular vesicles (EVs) and their role in HIV infec- to replicate for 15 days, or tissues were treated with tion and chronic Meth abuse. Our results presented ART (3TC and AZT) at day 2 post-infection. 33 cyto- here, indicate that Meth can not only increase EV kines in soluble or EV-associated forms were evaluated biogenesis and release but also change the composition with multiplexed bead-based assays. of EV cargo. Results: Infection with HIV-1 led to increases in many Methods: EV isolations, EV quantification by soluble cytokines, with the highest increases in IL-2, IL- Nanoparticle tracking analysis, Immunoflurescence 12, IL-15, IFN-γ, MIP-1α, MIP-1β and RANTES by and structural illumination microscopy, transmission both viruses. These same, and additional, cytokines, electron microscopy, Taqman RT-PCR, In situ hybri- were elevated in EV-associated form, often increasing dization, in vitro primary macrophage cultures. in both EV surface and internal compartments. Results: Nanoparticle tracking analysis and transmis- Likewise, soluble cytokines unaffected by HIV infection sion electron microscopy revealed that Meth changed tended to also be unchanged in EVs. EV dynamics in uninfected and HIV infected macro- ART treatment halted HIV-1 replication, but cyto- phage cultures. Our investigation revealed that the kines increased by HIV infection remained elevated. genes involved in the endosomal sorting complexes After 13 days of ART, five of the above seven soluble required for transport (ESCRT) are responsible are cytokines remained high for X4, and 4 for R5. EV- significantly increased upon Meth treatment. Further, associated cytokines were less likely to be restored: our data reveals that Meth increases the release of HIV 13 days after ART, for X4 all 10 of the most upregu- accessory protein, myristoylated Nef (Myr-Nef), that lated cytokines remained high, and for R5 7 of 10. plays a critical role in HIV/AIDS progression. Myr- Summary/Conclusion: Cytokine levels increased dur- Nef is N-terminally myristoylated, which acts as a ing HIV infection in both soluble and EV-associated membrane anchor. Furthermore, we also reveal that forms; the same cytokine is often upregulated in both gp120 is released in the EVs along with Myr-Nef. We forms. Many soluble cytokines upregulated by HIV did JOURNAL OF EXTRACELLULAR VESICLES 201 not decrease even after 13 days of ART, and EV-asso- HCC, COF of miR122 was 0.04175 (sensitivity and ciated cytokines were even less likely to decrease. X4 specificity of 86% and 75%, respectively) and COI of induced increases were less likely to return to control let-7a was 0.0166 (sensitivity and specificity of 71% and levels. ART-treated infected human tissues provide a 87%). The cumulative incidence rate of HCC was sig- new model to study tissue activation after HIV replica- nificantly different miR-122 ≥ 0.04175 and > 0,04175 tion is suppressed, in particular, the role of EVs in this groups or let-7a ≥ 0.01666 (p = 0.050 and 0.00054) phenomenon. These studies will assist in deciphering Summary/Conclusion: Serum miR-122 and let-7a mechanisms of pathologies that develop in ART-trea- values appear to have superior ability to predict HCC ted patients. development in patients with chronic HCV infection, which implies the possibility that they have crucial role in HCC development. PF05.07

Circulating MiR-122 and let-7a may predict progression to PF05.08=OWP2.07 hepatocellular carcinoma in patients with chronic hepatitis C virus infection Yuki Ichikawaa, Masashi Sakakib and Hitoshi Yoshidac aShowa University, Ito, Japan; bShowa University, Tokyo, Japan; cShowa Biogenesis of JC polyomavirus associated extracellular vesicles University, Tokyo, Japan depends on neutral sphingomyelinase 2 Jenna Morris-Lovea, Bethany O‘Haraa, Gretchen Geea, Aisling Duganb, Benedetta Assettac, Sheila Haleya and Walter Atwooda Introduction: At the bedside, circulating microRNAs aBrown University, Providence, USA; bAssumption College, Worcester, USA; in human body liquids are noted as `liquid biopsy’ to cBrown Univerisity, Providence, USA evaluate the disease stages, especially in Liver diseases, `actual biopsy’ is used in patients because of its inva- Introduction: JC polyomavirus is a non-enveloped siveness. In chronic liver disease, many circulating virus that causes progressive multifocal leukoencepha- miRNAs are recently reported such as miR-122, miR- lopathy (PML) in immunocompromised patients. 192, miR-223 to diagnose HCC patients and HCV- or JCPyV infects cells by first binding to the major attach- HBV-infected patients, in this study. To identify the ment receptor lactoseries tetrasaccharide C (LSTc), fol- miRNAs which can predict HCC (hepatocellular cell lowed by the serotonin receptor 5-hydroxytryptamine carcinoma) development, we first focused on the type 2 required for entry. In PML, JCPyV undergoes microRNAs associated with the liver fibrosis, because lytic infection in oligodendrocytes and astrocytes, both liver fibrosis stages are one of the most important of which have been shown to lack LSTc. Further, deep factors to predict Hepatocellular cell carcinoma sequencing has shown that viral quasispecies existing (HCC) development and evaluated those predictable in PML patients contain mutations in the sialic acid ability among other noninvasive fibrosis markers to binding pocket of the major viral capsid protein, ren- clarify our hypothesis that those miRNAs are patholo- dering these virions incapable of binding LSTc. We gically related to carcinogenesis development. have recently demonstrated that JCPyV is packaged Methods:SerumcirculatingmiR-122 and let-7a were into extracellular vesicles (EV) that can spread the retrospectively evaluated using RT-PCR in 35 virus, potentially overcoming this paradox. Here, we patients with HCV-related chronic hepatitis and cir- begin to characterize the biogenesis of this EV-virus rhosis who undergone DAA treatment. HCC had association by examining endosomal sorting complexes developed in 8 patients afterwards in the observation required for transport (ESCRT) proteins and neutral period. Informed consent was obtained and the study sphingomyelinase 2 (nSMase2). was approved by a recognized medical ethics com- Methods: Cambinol was used to specifically target mittee in our hospital. nSMase2 activity. Knockdown cell lines were created Results: Serum miRNA miR122and let-7a levels were with shRNA targeted against ALIX, TSG101, or significantly higher in liver chirrosis than chronic SMPD3. SMPD3 was also targeted using CRISPR/ hepatitispatients. (miR122, p = 0.00836 let-7a Cas9 genetic knockout in separate cell lines. p = 0.01595). For the predictable ability of HCC, Knockdown was confirmed by qPCR and/or Western AUROC of miR122 was 0.85606 and le1-7a was blot, and knockout by next generation sequencing. EV 0.76667,which showed highest ability compared with were concentrated by differential centrifugation and other non-invasive fibrosis markers, such as APRI, evaluated by transmission electron microscopy, FIB-4. (AUROC = 0.5023, 0.66697, respectively) Western blot, nanoparticle tracking analysis, infection, Based on our ROC results to predict complicating and qPCR for protected viral genomes. Infection was 202 ISEV2019 ABSTRACT BOOK scored by immunofluorescence analysis with antibodies specific lncRNAs into neighbouring cells to inhibit against the major viral capsid protein VP1. viral replication. Results: We found that depletion of nSMase2 by cam- Methods: Exosomes were purified from A549 with/ binol, genetic knockdown or knockout caused a reduc- without IFNβ treatment by serial centrifugation fol- tion in spread of JCPyV over time. Knockdown and lowed by sucrose density gradient purification, and knockout SMPD3 cell lines produced less infectious characterized by TEM and Western Blot. ELISA assay EV. In the absence of nSMase2, cells produced more were performed on purified exosome fractions to EV but there were fewer protected genomes associated demonstrate that they are free of IFNβ. ZIKV replica- with the EV. Knockdown of Alix or TSG101 had no tion was assayed by real-time PCR. effect on the infectivity of EV or the production of EV. Results: ZIKV replication was significantly suppressed Summary/conclusion: Overall, our studies found that in A549 cells pre-treated with Exo-IFNβ followed by biogenesis of JCPyV associated extracellular vesicles ZIKV infection. Moreover, we found that anti-ZIKV depends upon the enzymatic activity of nSMase2 and effect of Exo-IFNβ is IFN-independent because ZIKV not the ESCRT-related proteins Alix or TSG101. replication was also decreased in U5A cells (IFN-α/β Funding: NIH R01NS043097 receptor IFNAR deficient) pre-treated with Exo-IFNβ . Similar results were observed in Dengue virus and PF05.09=OWP2.08 HCV infections. RNA sequencing analysis found several lncRNAs and mRNAs were differentially expressed and function annotation and pathway analysis demon- Exosomes mediate the anti-viral activity of interferon-β against zika strated that the differentially expressed genes were virus infection Shuang Li, Shilin Li and Limin Chen involved in many functions and pathways, including

Provincial Key Laboratory for Transfusion-Transmitted Infectious Diseases, anti-viral infection. To validate the RNA sequencing Institute of Blood Transfusion, Chinese Academy of Medical Sciences and analysis results, some lncRNAs were selected to test Peking Union Medical College, Chengdu, Sichuan, 610052, China, Chengdu, their expression levels by qPCR. We are in the process China (People‘s Republic) of deciphering the mechanism employed by these exo- Introduction: IFNβ-induced exosomes (Exo-IFNβ) somal lncRNAs in anti-viral activty independent of may impact on viral dissemination or antiviral immu- inteferon. nity and therefore involve in the pathogenesis of many Summary/conclusion: We believe that understanding infectious pathogens. However, little is known about its the anti-viral functional molecules wrapped in exo- underlying mechanisms. To better understand how somes may help design exosomes as efficient vehicles Exo-IFNβ performs its anti-viral effect, we employed for antiviral therapy. RNA sequencing analysis to explore the exosomal Funding: Chinese Academy of Medical Sciences expression profiles of lncRNA and mRNA related to Innovation Fund for Medical Sciences (2016-12M- viral infections. We hypothesized that exosomes can 3–025) regulate viral infection through transmitting enclosed JOURNAL OF EXTRACELLULAR VESICLES 203

PF06: Advances in EV Quantification and Characterization Chairs: Estefanía Lozano-Andrés; Kenneth Witwer Location: Level 3, Hall A 15:30–16:30

the exosomes and control samples were shown by PF06.01 CellStream flow cytometer. The robust sensitivity of ELISA and CellStream flow cytometer with use of the

Exosome quantification by ELISA and Flowcytometry using anti-CD9 validated CD9 antibody would provide an informative antibody platform for measuring exosomes. a a b b Naoki Hata , Hiroyuki Kogure , Hikaru Sonoda and Chihiro Okada Funding: No fundings. aLuminex corporation, Tokyo, Japan; bHakarel Inc, Osaka, Japan

Introduction: Quantifying and characterizing exo- PF06.02 somes in a reproducible and reliable manner has been challenging due to their small sizes, of which the ranges Characterizing the light-scatter sensitivity of the CytoFLEX flow are from 30 to 150 nm in diameter. The analysis used Cytometer to be mainly performed with either the electric micro- George Brittain, Sergei Gulnik and Yong Chen scopy or the nanoparticle tracking analysis; however, Beckman Coulter Life Sciences, Miami, USA these techniques are low throughput and not enough for the quantification especially in the large and het- Introduction: Extracellular vesicles (EVs) and other erogeneous populations. Also, attempts to analyse exo- biological nanoparticles (NPs) generally fall within the somes using traditional PMT-based flow cytometers optical noise of light-scatter-based detection methods, has been hampered by the limit of detection of such and most flow cytometers are not sensitive enough to small particles and low refractive index. Here, to over- effectively detect NPs less than 300 nm in diameter. come these limitations, we used the highly qualified The CytoFLEX is a notable exception to this: it is so and validated monoclonal antibodies for CD9 on the sensitive that the SSC detector actually has an attenua- surface of exosome to employ ELISA and the high tion filter to reduce 95% of the scatter signal, adjusting sensitive flow cytometry. In this study, we would like it to a range useful for cells. As an alternative, the to show and discuss more reliable and robust platforms Violet SSC (VSSC) signal is unfiltered and can be for the quantification of exosomes with use of ELISA used to bring the CytoFLEX sensitivity well into the and flow cytometry. nanoparticle range. However, the added VSSC layer Methods: Malignant cell line-derived exosome was can confuse individuals, and a few instrument compar- prepared by the ultracentrifugation isons have even been published by users unfamiliar ↓ with the use of VSSC on the CytoFLEX. Diluted the samples in PBS at 1:60, 1:120, 1:240, 1:480 Methods: In order to better characterize the biological and 1:960 threshold sensitivity of the CytoFLEX using VSSC, we ↓ analysed a variety of NPs of different compositions, Measured the samples either by CD9-based ELISA including viruses and purified plasma EVs. The plasma (Hakarel Inc) or Flow cytometer (CellStream, EVs were prepared from fresh human blood using Luminex Corporation) centrifugation, size filtration, and column chromato- Results: The quantifications of exosomes were per- graphy, followed by size characterization using DLS. formed by ELISA and CellStream flow cytometer with After acquisition on the CytoFLEX, we converted the use of anti-CD9 monoclonal antibody median scatter intensity for each sample to either their Summary/Conclusion: In this study, the quantifica- size or refractive index (RI) using Mie theory tions of exosomes were performed by ELISA and approximations. CellStream flow cytometer with use of anti-CD9 anti- Results: We found that the CytoFLEX could fully body. Tumour cell-derived exosomes were labelled resolve 70 nm polystyrene and 100 nm silica (Si) NPs, with CD9-PE. The average concentration of the exo- including Si with a RI of 1.43 at 405 nm. We could fully somes was measured by CD9 ELISA whereas the mean resolve both 110 nm MLV viruses and 90 nm fluorescence intensity and the objects per microlitre for Adenoviruses with RIs of 1.47–1.50. And, we were 204 ISEV2019 ABSTRACT BOOK able to detect plasma EVs at least as small as 80 nm in market. However, with such great power comes great diameter using only a VSSC trigger, though immuno- responsibility to properly prepare the instrument and fluorescence was necessary to fully resolve the smallest samples for effective nanoscale flow cytometry of these EVs from noise. experiments. Summary/Conclusion: Ultimately, the CytoFLEX is The CytoFLEX is for Research Use Only. Individual highly sensitive for NP detection. Moreover, unlike results may vary. The Beckman Coulter product and dedicated microparticle analysers, the CytoFLEX is a service marks mentioned herein are trademarks or regis- full-fledged flow cytometer with a biological dynamic tered trademarks of Beckman Coulter, Inc. in the USA range extending from approximately 80 nm–50 µm. and other countries. The CytoFLEX is for research use only. Individual results may vary. The Beckman Coulter product and service marks mentioned herein are trademarks or regis- PF06.04 tered trademarks of Beckman Coulter, Inc. in the USA and other countries. Improved scatter sensitivity of a flow cytometer for detection of extracellular vesicles Leonie de Ronda, Edwin van der Polb, Ludovic Monheimc, Ton van Leeuwend PF06.03 and Frank Coumanse

aAmsterdam University Medical Centers, Amsterdam, USA; bAmsterdam UMC, University of Amsterdam, Department of Biomedical Engineering Preparing a CytoFLEX for Nanoscale flow Cytometry and Physics, Amsterdam, Netherlands; cBD Life Sciences, Erembodegem, George Brittain, Sergei Gulnik and Yong Chen Belgium; ddAmsterdam UMC, University of Amsterdam, Department of Biomedical Engineering and Physics, Amsterdam, Netherlands, ; Beckman Coulter Life Sciences, Miami, USA eAmsterdam UMC, University of Amsterdam, Laboratory of Experimental Clinical Chemistry, Amsterdam, Netherlands, Introduction:Builtaroundsemiconductortechnology, with a number of innovations to enhance light capture, Introduction: To investigate the biomarker potential of reduce noise and prevent signal losses, the CytoFLEX is extracellular vesicles (EVs), EV subtypes are studied by capable of detecting biological nanoparticles (NPs) as flow cytometry. A flow cytometer detects fluorescence, small as 80 nm by light scatter, and has a linear fluores- forward (FSC) and side scattered (SSC) light of single cence range that extends down into the single digits for EVs. However, the scatter intensities of the majority of fluorophores like FITC. However, in order to properly EVs are below the detection limit of common flow setup the CytoFLEX for NP analyses, a variety of con- cytometers because EVs are small and have a low siderations need to be taken into account, some of which refractive index. We aim to improve the scatter sensi- are extraordinary to conventional flow cytometry. tivity of a common flow cytometer 450-fold for SSC Methods: In this poster, we will demonstrate how to and 107-fold for FSC, which will allow detection of properly setup and clean a CytoFLEX flow cytometer 100 nm EVs. Improved scatter sensitivity enables us for NP analyses. First, we will explore the different to derive the size of EVs from the scatter signal and to threshold options and sensitivity ranges. Next, we will increase the fraction of EVs that can be characterized show how to clean the instrument and reduce noise. using immunofluorescence as well as scatter-based And finally, we will discuss several important issues sizing. that affect proper sample analyses. Methods: A FACSCanto (Becton Dickinson) was Results: The three primary detection methods on the adapted by replacing the 20 mW laser with a 20– CytoFLEX are FSC, SSC and Violet-SSC (VSSC). FSC 200 mW adjustable power laser (both 488 nm on the CytoFLEX utilizes comparative signal analyses Sapphire, Coherent). Confocal detection was achieved rather than traditional small-angle scatter, and is accu- by replacing the standard 1000 µm pinhole on SSC by a rate for sizing events from 500 nm to 50 µm, indepen- 200 µm pinhole, and the standard photodiode on FSC dent of the refractive index or membrane integrity. The by a 350 µm pinhole and PMT. The improvements in biological threshold sensitivities for SSC and VSSC on scatter sensitivity were quantified by calculating the the CytoFLEX range roughly between 250 nm–20 µm scatter stain index (SI) (median intensity of a bead and 80 nm–2 µm, respectively. In order to take full minus median intensity of the noise divided by two advantage of the lower end of these scatter ranges, times the standard deviation of the noise) of a 500 nm cleaning the instrument and thoughtful sample pre- polystyrene bead and the robust coefficient of variation paration are very important. (rCV) of a 100 nm polystyrene bead (both BioCytex). Summary/Conclusion: Ultimately, the CytoFLEX is Ideally the SI is as high as possible and rCV as low as one of the most sensitive flow cytometers on the possible. JOURNAL OF EXTRACELLULAR VESICLES 205

Results: A 10-fold increase in laser power increased the phenotypes. From 0–2 FT cycles, ApoB bound to PS SI on SSC 2.9-fold and on FSC 20-fold, whereas the +CD41+ and PS+CD41+ CD36+ phenotypes tended to rCV improved (reduced 0.67-fold and 0.97-fold, decrease (p > 0.05). Moreover, ApoB bound to PS respectively). The improved confocal detection +CD36+ increased 4.9-fold from 0–2 FT cycles for increased the SI on SSC 6.4-fold and on FSC 550- (p < 0.05). Interestingly, this progression mirrored fold, while the rCV slightly worsened (increased 1.1- that of PS+CD36+ (2.0–2.5-fold, p < 0.05), bulk CD36 fold and 1.02-fold, respectively). Combining both + (1.8–2.4-fold, p < 0.05) and ApoB+ (4.1–5.0-fold, increased laser power and confocal detection resulted p < 0.01). Finally, in line with previous reports, PS+ in a 20-fold increase in SI for SSC and 2 · 10^4-fold for tended to increase following FT (1.5-2.1-fold, p > 0.05). FSC, and improved the rCV (reduced 0.39-fold and Contrary to previous reports, certain EV phenotypes 0.24-fold, respectively). decreased from 0–2 FT cycles (PS+CD41+ and PS Summary/Conclusion: Adaption of the optical config- +CD41+ CD36+, both 2.6-fold, p < 0.05) suggesting uration of the FACSCanto by increasing the laser that EV phenotypes might perish following FT further power and confocal detection improved the scatter confirmed on bi-variable plots of data. sensitivity 20-fold for SSC and 2 · 10^4-fold for FSC. Summary/Conclusion: This study demonstrates that Next, we will evaluate the influence of increased mea- ApoB can be detected on hFCM and thereby interfere surement time and reduction of the number of parti- with EV characterisation. What further complicates cles in the sheath on the scatter sensitivity. matters is that lipoproteins could carry markers tradi- Funding: NWO-TTW Perspectief CANCER-ID 14195 tionally associated with EVs including PS and CD36. FT cycles did not consistently dissociate EVs and lipo- PF06.05 proteins; however, FT affected certain EV populations. Further studies are required to elucidate these findings.

Lipoprotein particles can be detected by high-resolution flow cytometry and potentially interfere with EV characterisation PF06.06 Rikke Wehner Rasmussen, Jaco Botha, Mathilde Sanden and Aase Handberg Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark, Aalborg, Denmark Analysis of fluorescent labelling efficiency of extracellular vesicles derived from different kingdoms of life with lipid-binding dyes via Introduction: Lipoproteins co-isolate with EVs and are nano-flow cytometry Ye Tiana, Chen Chenb, Qian Niua, Shaobin Zhuc and Xiaomei Yand potential confounders in EV characterisation. CD36 is a a membrane-bound scavenger receptor located on cells Department of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China, Xiamen, China (People’s and EVs capable of interacting with VLDL and LDL, Republic); bInstitute for Chemical Research, Kyoto University, Uji, Japan; which could interfere with antibody-based phenotyp- cNanoFCM Inc., Xiamen, China (People’s Republic); dDepartment of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen ing. Freezing and thawing samples was shown to University, Xiamen, China, Xiamen, China (People’s Republic) increase phosphatidylserine-positive (PS+) EVs while other common phenotype markers were unchanged. Introduction: In all domains of life – archaea, bacteria This could provide a method for disrupting lipopro- and eukarya, cells produce and release extracellular teins and EVs. Thus, we aimed to investigate the vesicles (EVs). The double-layered lipid membrane is impact of lipoproteins on EV characterisation and the most prominent feature of EVs, and fluorescent freezing/thawing samples on their dissociation from labelling with lipid-binding dyes has been frequently EVs on a high-resolution flow cytometer (hFCM). used to visualize and detect single EVs. For example, Methods: Plasma from 6 healthy individuals was sub- most conventional flow cytometers rely on fluorescence jected to either 0, 2, 4 or 6 freeze-thaw (FT) cycles and threshold triggering for single EV detection upon stained with a cocktail of lactadherin-FITC, anti-CD41- membrane labelling with lipophilic dyes. However, BV510, anti-CD36-PE and anti-ApoB-APC or lactad- the labelling efficiency of EVs with these lipid-binding herin-FITC and matched isotype controls. Samples dyes remains unknown. Here, we reported an approach were analysed on an Apogee A60 Micro-PLUS hFCM. to quantitatively analyse the labelling efficiency of Gating was performed as follows: size gates established lipid-binding dyes toward EVs by using a laboratory- on silica reference beads; phenotype gates set on 99th built nano-flow cytometer (nFCM) that enables light percentile of isotype control channel fluorescence. scattering detection of individual EVs as small as Results: hFCM was able to detect both free apolipo- 40 nm. protein B (ApoB) particles and ApoB bound to PS Methods: EVs were extracted from cultured medium of +CD41+, PS+CD36+ and PS+CD41+ CD36+ EV HCT15 cells (colorectal cancer cell line), E. coli O157: 206 ISEV2019 ABSTRACT BOOK

H7 (gram-negative), S. aureus (gram-positive) and (NTA), transmission electron microscopy (TEM) and Prochlorococcus (Pro., marine cyanobacteria) by differ- the Coomassie protein assay data collectively confirm ential ultracentrifugation. EVs isolated from E. coli the presence of EVs. To evaluate the surface charge of O157:H7 and S. aureus were further purified by floata- EVs, electrophoretic mobility was measured (Malvern tion in iodixanol density gradient. The purity of these Zetasizer Nano ZS) at varied pHs, ionic strengths and EV isolates was assessed by enumerating the particles organic contents to simulate environmental solution before and after the treatment with Triton X-100. chemistry; values were then converted to zeta potential Subsequently, the labelling efficiency of several lipophi- estimates via the Smoluchowski approximation. lic fluorescent dyes, such as PKH26, PKH67, DiI and Results: Initial tests reveal EVs to have a predomi- Di-8-Ane for EVs were evaluated by comparing with nantly negative charge, with a zeta potential of −5.4 their light scattering signals. mV in phosphate buffer. Higher ionic strengths desta- Results: The purity of EVs isolated from HCT15 cells, bilize vesicles, causing aggregation by neutralizing the E. coli O157:H7, S. aureus and Pro. were around 80% surface charge. to 90%. Compared with side scattering signals, we Summary/Conclusion: We demonstrate an initial found that almost all the EVs derived from E. coli understanding of the behaviour of how EV surface O157:H7, S. aureus and Pro. could be lightened up by charge is influenced by various environmental para- PKH26, PKH67, DiI and Di-8-Anepps. However, only meters; the effects of these changes are variable. This around 40% of EVs isolated from HCT15 cells could be implies that studying these trends mechanistically in labelled by these dyes. Morphological study by cryo- complex systems may be challenging. Changes to the TEM indicates that some vesicles secreted by HCT15 EV surface chemistry induced by alterations in the cells had surface protrusions (electron-dense spikes surrounding environment often also causes aggrega- protruding from the membrane). We suspect this tion, which has implications for fate and transport. structure may prevent these lipophilic dyes from inter- Further, work will be performed to probe the aggrega- calating with EV membrane. tion tendencies of EVs. The quantification of physico- Summary/Conclusion: The nFCM provides a straight- chemical parameters is a first step in parameterizing forward platform to analyse the labelling efficiency of future fate and transport models. EVs with different lipid-binding dyes, which will be Funding:FundedbytheNationalScienceFoundation very helpful in guiding the development of efficient (NSF) and the Environmental Protection Agency (EPA) vesicle-labelling strategies. under NSF Cooperative Agreement EF-0830093 and DBI- 1266252, Center for the Environmental Implications of NanoTechnology. PF06.07 PF06.08 Evaluating the surface charge of yeast extracellular vesicles as a function of environmental parameters Nicholas M. Rogers, Meta Kuehn, Claudia Gunsch and Mark Wiesner Isolation and characterization of bovine milk-derived EVs. Duke University, Durham, USA Saori Fukunagaa, Yuki Yamamotob and Hidetoshi Taharaa

aHiroshima University, Hiroshima, Japan; bHiroshima university, Hiroshima, Introduction: Understanding the mechanisms of Japan extracellular vesicle (EV) fate and transport is critical to predicting their targeting capabilities and delivery Introduction: Extracellular vesicles (EVs) are secreted efficiencies. Surface chemistry has been shown to be an from various cells and known to contain DNA, RNA effective predictor of the fate of nanomaterials (which and protein. Such inclusion is taken in other cells and include EVs) in complex environments. In particular, plays functionally. Since recent studies reported that ascertaining how surface charge changes based on sur- EVs are detected in food, such as fruits, vegetables rounding conditions provides a foundation for the and bovine milk, we hypothesized that functional EVs prediction of nanomaterial behaviour. Hence, the goal in food could contribute to human health. In the study, of this study is to evaluate EV surface charge as a we investigated whether the growth environment for function of environmental parameters to predict their dairy cattle affected the contents and functions of EVs ultimate environmental fate. from bovine milk. Methods: EVs were isolated from yeast (S. cerevisiae) Methods: Milk was warmed at 37ºC water bath for cell culture via the ultracentrifugation/density gradient 10 min, then mixed with 1/100 volume of acetic acid purification method. Nanoparticle Tracking Analysis at room temperature for 5 min and centrifuged at JOURNAL OF EXTRACELLULAR VESICLES 207

10,000 x g at 4ºC for 10 min to remove milk fat and were evaluated by qRT-PCR and Western blotting. debris. The supernatant was filtered with a 0.22 um Transport activity of OATP2B1 was evaluated by membrane and defined as whey. The whey was ultra- uptake of oestrone sulphate. Apple miRNA targeting centrifuged at 200,000 x g for 70 min at 4ºC. After PBS OATP2B1 predicted by in silico analysis were detected wash was performed twice, the pellet of EVs was resus- by RT-PCR. microRNA target sites for OATP2B1 were pended in PBS, and centrifuged at 10,000 x g for 5 min evaluated by deletion assay and luciferase assay. at 4ºC. The supernatant was used as EV solution. Results: Fluorescent labelled NP and nucleic acids were Particle size and concentration of EVs were measured observed in Caco-2 cells after 6 h exposure. NP sig- by qNano. Total RNA of EVs was isolated by nificantly decreased expression and transport activity miRNeasy Mimi kitand the RNA concentration was of OATP2B1 in Caco-2 cells. When NP were heat- measured by Agilent 2100 Bioanalyzer. RNA sequence denatured or broken by sonication, their decreasing was performed by Ion S5. The sequences data was effects were attenuated. In deletion assay, decrease of analysed by CLC Genomics. OATP2B1 mRNA expression was observed in only Results: We compared two bovine milks, which were plasmid construct containing 3’ untranslated region collected from different farm. Milk A and milk B were (3’UTR). Luciferase activity of pGL-OATP2B1-3’UTR both from healthy cattle who grew up with nutrient- was reduced by NP exposure. Seven miRNAs which filled pasture without giving stress, however, B was predicted to bind to this region were detected in NP. raised under better conditions. Between milk A and Moreover, decreased luciferase activity was inhibited B, bovine milk-derived EVs were almost same particle by some miRNA inhibitors for predicted miRNAs. size and concentration. Then, amount of RNA contain- Summary/Conclusion:AppleNPreducedmRNAand ing EVs were same between milk A and B. However, protein expressions and activity of OATP2B1, suggesting NGS data was revealed that EVs from milk B contained that apple miRNA in NP is involved in drug food interac- more immune-related microRNAs than milk A. tion. Moreover, it was suggested that apple miRNA con- Summary/Conclusion: This study revealed that the tributes to drug disposition by regulation of drug better growth environment of dairy cattle increased absorption mediated by OATP2B1 through NPs, immune-related microRNAs in bovine milk-derived EVs and so might be better for health. PF06.10 PF06.09

Fluorescent retroviruses as reference particles for Nanoscale flow cytometry Regulatory effect of apple-derived nanoparticle on intestinal organic Vera Tanga, Tyler Rennera, Anna Fritzschea, Dylan Burgera, Edwin van der anion transporting polypeptide (OATP) 2B1 Polb and Marc-André Langloisa Daichi Fujitaa, Hisakazu Komoria, Yuma Shirasakia, Toshiki Araia, Yui a b Iwamotoa, Tomohiko Wakayamab, Takeo Nakanishia and Ikumi Tamaia University of Ottawa, Ottawa, Canada; Amsterdam UMC, University of Amsterdam, Department of Biomedical Engineering and Physics, a Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Amsterdam, Netherlands Health Sciences, Kanazawa University, Kanazawa, Japan; bFaculty of Life sciences, Kumamoto University., kumamoto, Japan Introduction: Nanoscale flow cytometry (NFC) is a Introduction: Several studies have shown that plant- promising tool for phenotypic analysis of individual derived nanoparticles (NPs) taken up by the intestinal small particles such as extracellular vesicles (EVs) and cells affect intestinal function. Food-derived NP is viruses that are smaller than 500 nm in diameter. known to facilitate delivery of proteins, nucleic acids However, since many small EVs are currently at the including microRNA (miRNA) and other large mole- limit of detection for commercial flow cytometers, suc- cules to intestinal tissues. Therefore, such large mole- cessful detection of EVs requires optimization of both cules may affect gastrointestinal functions through sample preparation and instrument settings. These NPs. Accordingly, we investigated the effect of apple- optimizations require reference particles reflecting derived NP to intestinal transporters through contain- size, refractive index (RI), and fluorescence emission ing cargos. intensity of the labelled EVs of interest. Murine leu- Methods: NP was prepared by ultracentrifugation. kaemia virus (MLV) is a retrovirus ~114 nm in dia- Lipid membrane of NP and apple-derived nucleic meter as measured by cryo-EM, with an estimated RI acid were labelled by fluorescents to examine uptake of 1.5. Here we showcase the monodispersed nature of in Caco-2 cells using microscope. Expressions of these viruses and demonstrate their use as fluorescence mRNA and protein of transporters in Caco-2 cells reference particles for NFC. 208 ISEV2019 ABSTRACT BOOK

Methods: We engineered MLVs to express its envelope RI of the viruses could be tuned by using different glycoprotein fused to green fluorescent proteins (eGFP fluorophores. and sfGFP) on the viral surface. MLVs were character- Summary/Conclusion:MLVsaresimilartosmallEVsin ized by NFC and by nanoparticle tracking analysis. size with equivalent surface area and comparable capacity Because MLVs are monodispersed, we combined scat- for antigen expression. Unlike synthetic beads, MLVs can ter intensities and hydrodynamic diameter to obtain be genetically engineered to express protein antigens of the effective RI by solving the inverse light scattering choice in biologically relevant and consistent levels to act as problem using Mie theory. internal positive controls for phenotypic studies of EV Results: We measured an antigen density of ~300 surface marker expression. Moreover, MLVs are mono- MESF of GFP per virion. In addition, we found that disperse and have tuneable RI. Collectively, these proper- antibody labelling of this virus-associated antigen with ties support that MLVs are strong candidates as different fluorophore conjugates (PE, BV421 and fluorescence reference particles for NFC. AF647) modulates both scatter intensities and hydro- Funding: Natural Sciences and Engineering Research dynamic diameter of the labelled virus. With regard to Council of Canada (NSERC) the hydrodynamic diameter, we show that the effective JOURNAL OF EXTRACELLULAR VESICLES 209

PF07: Biogenesis II Chairs: Mathilde Mathieu; Hang Hubert Yin Location: Level 3, Hall A 15:30–16:30

groups and found that 60 proteins are associated with PF07.01 flagella, ribosome, and modification. Moreover, the role of individual proteins on the biogenesis of OMVs using knockout strains expressing proteins was Proteomic profiling of outer membrane vesicles derived from MicA, a small RNA from Escherichia coli evaluated. So Hee Leea, Yeong-Jun Parkb and Kwang-sun Kima Summary/Conclusion: All our results enabled us to aPusan National University, Busan, Republic of Korea; bPusan National elucidate the underlying mechanism of high produc- University, Pusan, Republic of Korea tion of OMVs by MicA and the information will be utilized as a vaccine platform for infectious diseases. Introduction: Outer membrane vesicles (OMVs) produced by Gram-negative bacteria are utilized as vaccine or drug delivery platforms in terms of their efficient PF07.02 immune responses to host cells. In a previous report, we identified that ectopic expression of MicA, a small Dysfunction in an autophagy-lysosome degradation pathway noncoding RNA from E. coli, produced a high produc- promotes secretion of ubiquitinated proteins via extracellular vesicles tion of OMVs as a conserved manner in both E. coli Toshihide Takeuchi, Satoko Sakai, Harue Ando and Yoshitaka Nagai and Salmonella through both up- or down-regulation Osaka University, Suita, Japan of OmpC or OmpA level, respectively, in OMV fractions. Moreover, MicA-derived OMVs showed the pro- Introduction: Autophagy-lysosome degradation is a tective role against Salmonella challenge, suggesting cellular protective mechanism that prevents aberrant that OmpC-enrichment in OMVs is important for the accumulation of cellular proteins, and thus, maintains production and function of OMVs. However, MicA protein homeostasis. Recent studies have suggested that overexpression in the knockout strain of ompA, a target autophagy impairment leads to an increase in secretion of MicA, still strongly induced the production of of aggregation-prone proteins, such as proteins that are OMVs, indicating that another underlying mechanism associated with the neurodegenerative diseases, of high production of OMV is presented. although molecular mechanisms underlying such Methods: Analysis of total and surface proteins from secretion and its biological significance still remain control- and MicA-derived OMVs from E. coli was elucidated. performed using high-resolution mass spectrometry. Methods: The extracellular vesicle (EV) fractions were The OMVs were isolated from culture supernatants, collected from the cell culture media by ultracentrifu- followed by characterization using Nanosight. We gation, and analysed by Western blotting, electron then analysed proteins of OMVs by in-gel digestion microscopy and nanoparticle tracking analysis. from SDS-PAGE, followed by nano LC-MS/MS analy- Results: Here we show that perturbation of the autop- sis. The functional analysis of candidate proteins on the hagy/lysosome pathway activates secretion of ubiquiti- biogenesis of OMVs was performed by OMV prepara- nated proteins via EVs. We found that treatment of tion, BCA quantification, and protein analysis from cells with autophagy inhibitors leads to an increase in knockout strains of specific genes. the amounts of ubiquitinated proteins, as well as Results: We found that spherical OMVs were an aver- autophagy-related proteins including LC3 and p62, in age diameter of 84.7 ± 1.3 nm and 88.2 ± 2.4 nm for the EV fraction of the culture media. We also found MicA- or control-derived OMVs, respectively. Further, that inhibitor treatment facilitates secretion of EVs we identified 1,102 (38) or 656 (40) proteins for MicA- distinct from exosomes in size, and that these EVs are or control-derived OMVs in total (or surface) fractions involved in secretion of ubiquitinated proteins. are presented. Among them the level of 84 or 15 Interestingly, analysis of knockout cells deficient for proteins from total or surface fractions, respectively, autophagy-related proteins revealed that the factors in was decreased or absent compared to control sample. the initiation step of autophagy are needed for EV- Total 99 proteins were categorized into 19 functional mediated secretion of ubiquitinated proteins. 210 ISEV2019 ABSTRACT BOOK

Summary/Conclusion: These results indicate that miRNAs, four miRNAs altered the EV secretion in autophagy impairment promotes secretion of ubiquiti- both cell lines, HCT116 and A549. nated proteins via EVs. Our data provide the mechan- Summary/Conclusion: Some of these target genes have istic link between the autophagy/lysosome pathway and reported as endosomal pathway associated protein and vesicle secretion. We propose that cells may use the shown the up-regulation in cancer cells. These findings EV-mediated secretion as an alternative pathway to suggest that the identification of target genes of these maintain protein homeostasis when cellular proteosta- miRNAs provides the new insight into the cancer cell sis machinery is functionally impaired. communication with the microenvironmental cells, Funding:ThisworkwassupportedbyJST;byKAKENHI which leads to a promising therapeutic approach (18H02585); by The Asahi Grass Foundation and The against cancer progression. Tokyo Biochemical Research Foundation. PF07.04 PF07.03

Ras Tumour microvesicles biogenesis and signalling in drosophila Vakil Ahmad, Carson Broeker, Kayla Calandro and Yves Chiswili. Chabu Identifying the miRNAs associated with EV Secretion from cancer cell lines University of Missouri, Columbia, USA Tomofumi Yamamotoa, Nobuyoshi Kosakab, Fumihiko Urabea, Yutaka Hattoric and Takahiro Ochiyab Introduction: Tumour-derived exosomes and micro- aDivision of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan; bDepartment of Molecular and Cellular vesicles are increasingly implicated in cancers. Their Medicine, Institute of Medical Science, Tokyo Medical University, respective functional contributions to cancer progres- c Shinjyuku-ku, Japan; Clinical Physiology and Therapeutics, Keio University sion and the related mechanisms remain poorly Faculty of Pharmacy, Tokyo, Japan defined. This is partly because current techniques, cen- Introduction:Extracellularvesicles(EVs)derivedfrom tered on differential centrifugation, do not permit ade- cancer cells contribute to their surrounding microenvir- quate and specific isolation of pure exosomes or MV onmental cells for their benefit. Our group has previously for targeted functional studies. More importantly, the shown that inhibiting the EVs production attenuated the paucity of animal models to address mechanistic and angiogenesis in the tumour, resulting in the suppression functional questions in tissues has further limited our of metastasis. Thus, understanding the mechanisms of EV knowledge on the role of extracellular vesicles in cancer secretion might contribute to the regulation of EV- biology mediated cancer progression. However, the precise Methods: Using a Drosophila Ras tumour model, we mechanism of EV secretion in cancer cells remains have identified a strategy to specifically label and unclear. The purpose of this study is to elucidate the genetically manipulate tumour microvesicles in tissues unknown mechanisms of EV secretion in cancer cells. for mechanistic studies. To reveal this, microRNAs (miRNAs), which regulate Results: We will discuss some of our preliminary multiple genes, are employed. results on the dynamic of microvesicle biogenesis and Methods: To identify the EV secretion associated their role in Ras tumour-macrophage signalling miRNAs, miRNA-based screening method was estab- interaction. lished. Combined with ExoScreen, which is ultra-sen- Summary/Conclusion: Together with the power of sitive detection method of EV by measuring surface Drosophila genetics, this in vivo system will enable protein of EVs, such as CD9 and CD63, miRNA- novel insights into microvesicle biogenesis and func- based screening was performed in colorectal cancer tion during tumour progression. cell line, HCT116, and lung cancer cell line, A549. The results of the screening were confirmed by the PF07.05 nanoparticle tracking analysis. Candidate genes of these miRNAs were chosen by in silico analysis. Results: From the initial 1728 miRNAs, we identified Src in endosomal membranes promotes exosome secretion and cancer progression 13 miRNAs which are associated with EV secretion in Chitose Oneyama each cell lines. Then, the target genes of these miRNAs Cancer Cell Regulation, Aichi Cancer Center Research Institute, Nagoya, were identified and confirmed that EV secretion was Japan attenuated by siRNAs against candidate genes. From 6 miRNAs, 27 genes, which were associated with EV Introduction: c-Src is a membrane-associated tyrosine secretion, were identified. Interestingly, among six kinase that has key roles in the signalling transduction JOURNAL OF EXTRACELLULAR VESICLES 211 that controls cell growth, adhesion and migration. In enzyme, S1P lyase. In the present work, we investigated the early stage of carcinogenesis, c-Src is activated the role of S1P lyase in biogenesis of the AEVs and its under the plasma membrane and transduces oncogenic molecular modulation in the apoptotic processes. signals. Previous reports demonstrate that c-Src is loca- Methods: Preparation of AEVs: The conditioned med- lized to intracellular membranes, such as those of ium was centrifuged for 10 min at 200 ×g and twice endosomes. However, the functional significance of for 20 min at 2,000 ×g to remove cellular debris and endosomal c-Src in cancer is not well understood. apoptotic bodies. The pellets were collected by over- Methods: We examined intracellular localization of night incubation in 8% PEG6000 and 0.5 M NaCl, and active c-Src, and in intermediate sections we found c- washed by ultracentrifugation at 100,000 ×g for Src localized in perinuclear regions. In co-localization 70 min. experiments with organelle markers in Src-transformed Results: S1P lyase was degraded caspases-dependently cells, active c-Src was present with the late endosome in HeLa cells by apoptotic stimuli. Over-expression of markers, including CD9 and CD63, which are also N-terminal 3X flag- and C-terminal HA-tagged S1P known as canonical exosome markers. We examined lyase turned out that C-terminal region of S1P lyase exosome secretion in c-Src-transformed cells. was degraded. However, S1P lyase was not a direct Results: Our results indicate that activated c-Src in the target of caspases because mutations of Asp residues endosomal membrane promoted the secretion of exo- at C-terminal regions did not block its degradation. somes, in which c-Src was encapsulated. In addition, Possibly, S1P lyase might be a substrate of calpain in the ESCRT-interacting molecule, Alix was identified as that co-treatment of a calpain inhibitor, PD150606 a c-Src–interacting protein in exosomes. We revealed with staurosporine inhibited the degradation of S1P that the interaction between the SH3 domain of c-Src lyase. In consistent with this, knock-down of an endo- and the proline-rich region of Alix activates ESCRT- genous inhibitor of calpain, calpastatin increased the mediated intra-luminal vesicle (ILV) formation, result- degradation of S1P lyase while knock-down of calpain ing in the upregulation of exosome secretion in c-Src– small subunit, CAPNS1 decreased the degradation of transformed cells. We observed also a correlation S1P lyase. Functionally, mutant form of S1P lyase between malignant phenotypes and Alix–dependent deleted in C-terminal 21 amino acids showed decreased aberrant exosome secretion in c-Src–upregulated can- enzyme activities as well as less inhibitory effect on cer cells. release of the AEVs when compared with wild type. Summary/Conclusion: Our findings indicate that c- Summary/Conclusion: C-terminal degradation of S1P Src-mediated activation of Alix promotes ILV forma- lyase during apoptotic processes contribute to enhance- tion in MVB, resulting in increased exosome secretion ment of biogenesis of the AEVs, possibly through from various human cancer cells with activated c-Src. decreasing enzymatic activities of S1P lyase and subse- These data suggest that dysfunctions of exosome secre- quent increment of S1P in ER region. Although degra- tion suppress cell transformation, offering a novel sig- dation of S1P lyase is caspases-dependent, S1P is not a nalling target and strategy for cancer therapeutics. direct substrate of caspases. It would be probable that Funding: JST, PRESTO Grant Number JP1005457, S1P lyase was degraded by calpain, activated caspase- Japan. dependently.

PF07.06 PF07.07

Modulation of Sphingosine-1-phosphate lyase and its implication in Super-repressor-IκB-loaded exosome improves survival in a mouse release of apoptotic exosome-like vesicle model of sepsis and attenuates sepsis-induced inflammation Jihyo Kim, Jaehark Hur and Yong Joon Chwae Youngeun Kima, Hojun Choib, Amin Mirzaaghasib, Eunsoo Kimc, Kyungsun Choic and Chulhee Choic Introduction: Biogenesis of apoptotic exosome-like aCellex LIfe Sciences Incorporated, Daejeon, Republic of Korea; bKorea vesicles (AEVs), which can function as damage-asso- Advanced Institute of Science and Technology (KAIST), Daejeon, Republic ciated molecular patterns, is reported to be regulated of Korea; cCellex Life Sciences Incorporated, Daejeon, Republic of Korea by sphingosine-1-phosphate (S1P)/S1P receptor 1/3 Introduction: The nanoparticles referred as exosomes signalling. Thus, cellular S1P levels could be key factors play an active role in intercellular communication. The in the biogenesis of AEVs. As is well-known, S1P is ability of exosomes to travel between cells and deliver synthesized from sphingosine by sphingosine kinase 1/ their cargo, which includes proteins and nucleic acids, 2-mediated phosphorylation and irreversibly degraded makes them an appealing cell-free therapy option to into fatty aldehydes and phosphoethanolamine by the treat multiple diseases. Super-repressor IκB (srIkB) 212 ISEV2019 ABSTRACT BOOK which is S32A and S36A mutant form of IκB can engineered exosome system named exosomes for pro- continuously inhibit NF-κB because it is not phos- tein loading via optically reversible protein–protein phorylated by IκB Kinase and degraded by proteasome. interaction (EXPLOR) that can deliver soluble proteins Therefore, it has the great potential as a treatment for via reversible protein–protein interactions. Here, we various inflammatory diseases. We have previously demonstrate the intracellular delivery of β -glucocereb- developed an opto-genetically engineered exosome sys- rosidase (GBA) as functional proteins from the exo- tem named exosomes for protein loading via optically somes to the target cells. We generated opto-genetically reversible protein–protein interaction (EXPLOR) that engineered exosome system to load GBA, which is an can deliver soluble proteins via reversible protein–pro- enzyme deficient in Gaucher disease patients, into tein interactions. Here, we generated opto-genetically newly generated exosomes. Treatment with GBA- engineered exosome system to load srIkB into newly loaded exosomes showed the significant increase of generated exosomes. Treatment with srIkB-loaded exo- intracellular levels of cargo proteins and their function somes significantly reduced tumour necrosis factor-α- in recipient cells in both time- and dose-dependent induced translocation and DNA binding of the p65, a manner. In the present study, we tested lysosomal subunit of NF-κB, in HeLa cells. Furthermore, srIkB- localization of GBA-loaded exosome in the target cells loaded exosomes administration improved survival in and compared the the efficacy with an analogue of the the cecal ligation and puncture (CLP)-induced sepsis human GBA, VPRIV, to suggest it as a potential drug mouse model and attenuated lipopolysaccharide (LPS)- candidate in Gaucher disease. induced systemic inflammation. In addition, in sepsis- Methods: ABC induced mice, exosomes accumulated in the spleen and Results: ABC liver after intraperitoneal injection. This finding may Summary/Conclusion: ABC be helpful for understanding the mechanism about how the administration of srIkB-loaded exosomes facilitates the recovery from sepsis. Taken together, these PF07.09 results show that srIkB-loaded exosomes could potentially be a novel anti-inflammatory and immunosup- Sequence-specific release of EV-associated RNAs pressive cure in the treatment of sepsis and septic Christian Preußer, Marie Mosbach, Lee-Hsueh Hungand Albrecht Bindereif shock. Institute of Biochemistry, Justus Liebig University of Giessen, Giessen, Methods: ABC Germany Results: ABC Summary/Conclusion: ABC Introduction:Extracellularvesicles(EVs)containdiffer- ent classes of RNAs, such as mRNA, miRNAs and circRNAs. As shown for miRNA and circular RNAs, PF07.08 RNAs are selectively exported into vesicles [1–2]. However, the factors or mechanisms that contribute to this specificity remain elusive. Thus, for example, a so- Efficient delivery of Glucocerebrosidase Lysosomal Enzyme via EXPLOR technology for treatment of Gaucher’s disease called Exo-motif has been described for miRNAs, which, Yonghee Songa, Hojun Choib, Youngeun Kimc, Kyungsun Choia and Chulhee however, cannot be transferred to all miRNAs classes, and a Choi for circRNAs a possible size-dependent export was sug- aCellex Life Sciences Incorporated, Daejeon, Republic of Korea; bKorea gested. In addition, only a few putative protein factors Advanced Institute of Science and Technology (KAIST), Daejeon, Republic of Korea; cCellex LIfe Sciences Incorporated, Daejeon, Republic of Korea involved in packaging have been described [2]. Methods: To determine the export signals for the Introduction: Many intracellular proteins with great selective release of certain RNA species into EVs, we potential as biopharmaceutical drugs have been identi- designed a modified in vivo SELEX approach fied. However, numerous challenges associated with (Systematic Evolution of Ligands by Exponential intracellular protein delivery have yet to be solved. Enrichment) for identifying putative RNA sequence Over the past years, extracellular vesicles including elements. We generated a random sequence pool exosome have been regarded as a new paradigm for (N40), which was transfected and expressed into soluble protein delivery into cells or tissues. Because of HEK293 and HeLa cells. Moreover, several expression their biological functions and features, exosomes are constructs were used, which consist of either an RNA expected to be a novel treatment for diverse diseases, Pol II or a Pol III promoter to analyze possible mod- such as cancer and rare genetic disorder diseases. We ification effects on the 5’-end of the RNA. Similarly, we have previously developed an opto-genetically introduced transcription terminators at the 3’-end to JOURNAL OF EXTRACELLULAR VESICLES 213 prevent possible polyadenylation. EVs were isolated, matrix (ECM) provide physiologically more relevant followed by RNA isolation, library preparation, RNA- system to mimic in vivo tumour growth and progres- seq analysis and bioinformatic identification of sion of invasion. However, there are currently no enriched RNA motifs. methods to efficiently isolate EVs from ECM-based Results: We developed a new SELEX-based approach 3D cultures. For that purpose, we established a proto- to identify enriched sequence motifs within EV-RNAs. col for isolating EVs from cancer cells growing in a 3D For this, we have generated constructs that express ECM-based hydrogel. long degenerate sequences but are still relatively small Methods: Human prostate cancer PC3 cells were in total (85 nts). In a first attempt, we analysed the grown in 3D to form spheroids in a commercially expression of the degenerated sequences and were able available ECM-based hydrogel and the growth media to recover these sequences from EV-RNAs. Detailed was collected every two days for a period of 14 days, sequence and motif enrichment analyses are now in during which the spheroids grew invasive. The respec- progress. tive media were differentially centrifuged at 2K, 10K Summary/Conclusion: Here we described a novel and 100K g and the pellets were resuspended in PBS. approach to determine specific sequence motifs The EVs were analysed by western blotting (WB) required for selective loading of RNA into EVs. This against the common EV markers CD81, CD63 unbiased method should contribute to our understand- and CD9. ing of how RNAs are specifically packaged into EVs. Results: Our preliminary data shows a step-wise References:[1]Preußeretal.2018,JExtracellVesicles.;[2] increase of the EV markers in the media as the PC3 Villarroya-Beltri et al. 2013, Nat Commun. spheroids formed, expanded and invaded to the surrounding 3D ECM. The EVs produced by non-invasive or invasive spheroids are currently being characterized PF07.10=OWP2.14 with nano tracking analysis, electron microscopy and WB. Summary/conclusion: This study demonstrates that Isolation of extracellular vesicles from extracellular matrix based hydrogel 3D cell cultures EVs can be isolated from 3D ECM-based hydrogel Jens Luotoa, Lea Sistonenb and Eva Henrikssona cell cultures, which recapitulate the tissue architecture a1Cell Biology, Biosciences, Faculty of Science and Engineering, Åbo of solid tumours. Our results suggest that 3D cancer Akademi University, FI-20520, Turku, Finland; 2Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FI-20521, cell cultures have dynamic EV secretion determined by Turku, Finland;, Turku, Finland; b1Cell Biology, Biosciences, Faculty of the phenotype of the spheroids. Taken together, we Science and Engineering, Åbo Akademi University, FI-20520, Turku, present a novel protocol for EV isolation from a 3D Finland; 2 Turku Centre for Biotechnology, University of Turku and Åbo Akademi University, FI-20521, Turku, Finland;, Turku, Finland culture system and provide a platform to investigate EVs from in vivo mimicking conditions. Introduction: Cancer-derived extracellular vesicles Funding: This project is funded by Magnus Ehrnrooth (EVs) are commonly studied and isolated from two- Foundation, K. Albin Johansson Foundation and Åbo dimensional (2D) cell cultures. Nevertheless, three- Akademi University. dimensional (3D) culture systems with extracellular 214 ISEV2019 ABSTRACT BOOK

PF08: EVs in Tissue Injury and Repair Chairs: Johannes Grillari; Bas van Balkom Location: Level 3, Hall A 15:30–16:30

PF08.01 PF08.02

The role of adipocyte-derived extracellular vesicles in vimentin Effect of exosomes from human adipose-derived stem cells on hair mediated fibrosis a,b c,d,e f,g h,i growth Sepideh Parvanian , Fang Cheng , John Eriksson , Jens Luoto and Hye-In Choia, Eun Wook Choib, Sang Won Yoonb and Byung-Soon Parkb Peiru Yangf,g a b a R&D Center, PROSTEMICS, Seoul, Republic of Korea; R&D Center, Cell Biology, Biosciences, Faculty of Science and Engineering, Åbo Akademi PROSTEMICS, seoul, Republic of Korea University, Turku, Finland; bTurku Centre for Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland; Turku, Finland; cCell Biology, Biosciences, Faculty of Science and Engineering, Åbo Akademi Introduction:Hairloss(alopecia)isacommon d University, Turku, Finland; Turku Centre for Biotechnology, University of medical problem affecting both men and women, Turku and Åbo Akademi University, Turku, Finland; eSchool of Pharmaceuticals, Turku, Finland; fCell Biology, Biosciences, Faculty of Science and is caused by genetics, hormonal changes, med- and Engineering, Åbo Akademi University, Turku, Finland; gTurku Centre for ical condition or medications. Adipose-derived stem Biotechnology, University of Turku and Åbo Akademi University, Turku, Finland; hCell Biology, Biosciences, Faculty of Science and Engineering, Åbo cells (ASCs) have been reported as an important Akademi University, Turku, Finland; iTurku Centre for Biotechnology, component of regenerative medicine and cell ther- University of Turku and Åbo Akademi University, Turku, Finland apy for hair loss. Recent studies have demonstrated Introduction: Vimentin is involved in wound healing that exosomes from mesenchymal stem cells and by mediating fibroblast proliferation, epithelial- DPCs may regulate the hair follicle development mesenchymal transition processing, and collagen accu- and hair growth. In this study, we investigated the mulation, but its functional contribution to this process function of ASC secreted exosomes (ASC-exos) on is not clear. Adipocyte-derived extracellular vesicles hair growth. (EVs) have the potential to promote wound healing Methods: Exosomes were isolated from the condi- by controlling molecular processes on recipient cells. tioned medium of human ASCs. The effects of ASC- Methods: Differential centrifugation was used for the exos on hDPC proliferation were evaluated using CCK- isolation of EVs from wilde-type (WT) and vimentin 8 assays. The mRNA expression of growth factors was knockout 3T3lt1 (VIM-/-) cells. Electron microscopy investigated using real-time PCR. Additionally, anagen and Western blot were used to characterize EVs from induction was evaluated using an in vivo mice model. the cell culture media. In vitro analysis of cell migration Furthermore, we analyzed the profile of exosomal and proliferation were performed using wound scratch microRNAs (exo-miRNAs) by microarray analysis, assay and cell-derived matrices (CDM) in response to which was isolated from ASC-exos. WT and VIM-/- EVs. Results: We found that the ASC-exo enhanced the cell Results:OurpreliminarydatashowsthatHumanDermal proliferation of DPCs. Also, quantitative real-time PCR Fibroblasts (HDF) show different significant migration showed that the expression of genes related with hair responses in the presence of Adipose-derived Stem Cells growth, such as transforming growth factor β-2, nog- (ASCs) EVs. Especially, exosomes treatment induced more gin, was increased after being treated with ASC-exos. migration compared to other EVs. Also, vimentin affected Additionally, ASC-exos treatment accelerated the ana- the results of the exosome treatment in fibroblast prolif- gen hair induction when topically applied to C57BL/6 eration and migration assays. mice. Based on this finding, we conducted microRNA Summary/Conclusion: This study is expected to reveal analysis and selected 12 miRNAs that contribute to novel mechanisms of ASCs-derived exosomes regulat- regulation of hair growth. ing fibroblast activities in physiological wound healing Summary/Conclusion: Our results show that the exo- and fibrosis. somes from ASCs have a potential to activate DPCs Funding: This project was funded by Sigrid Juselius and promotes hair growth in vivo and may use in Stiftelse and Åbo Akademi University. treatment of hair loss. JOURNAL OF EXTRACELLULAR VESICLES 215

PF08.03 PF08.04

Paracrine regenerative function of mesenchymal stem cells is not Cell to cell interactions orchestrated by exosomal MiRNAs between affected by chronic kidney disease pathogenic- and Non-pathogenic corneal endothelial cells a b b Bas WM. van Balkom , Femke van Rhijn-Brouwer , Diana Papazova , Dienty Kazuko Asadaa, Junji Hamuroa, Morio Uenoa, Atsushi Mukaia, Munetoyo b b a a Hazenbrink , Anke Meijer , Arjan van Zuilen , Raechel Toorop , Joost Todab, Shigeru Kinoshitab and Chie Sotozonoa Fledderusa, Hendrik Gremmelsa and Marianne Verhaara aDepartment of Ophthalmology, Kyoto Prefectural University of Medicine, a b UMC Utrecht, Utrecht, Netherlands; UMC Utrecht, Utrecht, USA Kyoto, Japan; bDepartment of Frontier Medical Science and Technology for Ophthalmology, Kyoto Prefectural University of Medicine, Kyoto, Japan Introduction: Cell-based therapies have been developed to meet the need for curative therapy in chronic Introduction: Corneal endothelial dysfunction such as kidney disease (CKD). Mesenchymal stromal cells bullous keratopathy (BK), Fuchs’ endothelial corneal (MSCs) enhance tissue repair and induce neoangiogen- dystrophy (FECD) can be restored only with corneal esis through paracrine action of secreted proteins and transplantation. We have recently developed a cell- extracellular vesicles (EVs). Administration of allo- injection therapy using cultured human corneal geneic MSCs is less desirable in a patient population endothelial cells (cHCECs) (New Eng J Med.2018). likely to require a kidney transplant, but potency of Cultured HCECs have an inclination towards cell- autologous MSCs should be confirmed, given previous state transition (CST). The expression of miRNAs is indications that CKD-included dysfunction is present. essential in the regulation of many cellular processes While the immunomodulatory capacity of CKD MSCs closely linked to CST in cHCECs. Here, we studied the has been established, it is unknown whether CKD role of exosomal miRs in pathogenesis of BK and affects wound healing and angiogenic potential of FECD. MSC-derived CM and EVs. Methods: The composition of heterogeneous cHCEC Methods: MSCs were cultured from BM obtained from subpopulations (SPs) were verified in regard to their kidney transplant recipients (N = 15) or kidney donors surface cluster determinant (CD) markers. The profiles (N = 17). Passage 3 MSCs were used for experiments of miRs in cells, culture supernatants (CS) and in fresh and collection of conditioned medium (CM). EVs were corneal tissues were detected by 3D-Gene® Human isolated from passage 8 MSCs from 13 male partici- miRNA Oligo chip (Toray). Exosome surface markers pants. In vitro pro-migratory and pro-angiogenic capa- were measured either directly by Exo Screen or by WB city of bone marrow (BM) MSC-derived CM and EVs after ultracentrifugation. PKH-labelled exosome was was assessed using an in vitro scratch wound assay and applied for the evaluation of the incorporated exo- Matrigel angiogenesis assay. Our methods are in agree- somes in cHCECs with distinct CD44 expression levels. ment with the declaration of Helsinki and we obtained Results: MiR34a-5p and miR-378 family were detected written consent from bone marrow donors. only intracellularly and were strikingly lowered in Results: Healthy and CKD MSCs exhibited similar pathogenic corneal endothelium. Candidate miRs in differentiation capacity, proliferation and senescence- CS to discriminate CD44- SPs from those with CD44 associated β-galactosidase activity. Scratch wound ++~+++ phenotypes were miRs 23a-3p, 24-3p, 184, migration was not significantly different between 1246, 1273 and 1285-3p. Among these miRs 23a-3p, healthy and CKD MSCs (p = 0.18). Healthy and CKD 24-3p and 184 have a tendency to decrease in senes- CM induced similar tubule formation (p = 0.21). There cence-disposed cHCECs, the inversely correlated was also no difference in paracrine regenerative func- decrease with upregulated CD44. It is of note that tion of EVs (tubulogenesis: P = 0.46; scratch lowered expression of cellular miR-378 induced the wound: P = 0.6). elevated gene expression of IL-8, MCP-1 and VEGF, Summary/Conclusion: Our results indicate that CKD and the increased secretion of exosomal miRs 23a-3p / does not affect the regenerative potential of CM and 24-3p / 184 / 1273e / 1285-3p. CD9+ exosomes were EVs derived from CKD BM MSCs. This suggests that more elevated in cHCEC CS with senescence-like CST autologous MSC-based therapy is a viable option than those without CST, indicating the possible import in CKD. of these extracellular vesicles into cHCECs without Funding: Netherlands Organisation for Scientific CST. Compared with non-CST, CST cHCECs have a Research (NWO) tendency to incorporate more exosomes. 216 ISEV2019 ABSTRACT BOOK

Summary/Conclusion: MiRNAs in exosomes serve as in the vesicle fraction isolated, we hypothesize, that an alternative tool to qualify cHCEC SPs. In this cur- these are not only shed upon apoptosis, hence would rent study, we present the first finding that the lowered not call the isolated fraction urinary ACPSVs. Ongoing miRs in pathogenic tissues may induce the groups of studies aim to validate the potential to initiate prolif- exosomal miRs reliant on the depolarized CD44++~++ eration on different renal cell types, to further identify + HCECs. the cellular origin as well as to determine differences in their function and content in the state of renal diseases. As these vesicles can be easily isolated in a high purity, PF08.05 they also represent a valuable source for biomarker research in various nephropathies.

Urinary CRK1 positive vesicles yield novel insight into microvesicular signaling of the kidney Fabian Brauna, Inka Homeyera, Valerie Oberübera, Victor Puelles Rodriguezb, PF08.06 Sasha Shafikhanic and Tobias B. Hubera aIII. Department of Medicine, University Medical Center Hamburg- Eppendorf, Hamburg, Germany; bIII. Department of Medicine, University Human adipose stem cells-derived vesicles improve pain and reduce Medical Center Hamburg-Eppendorf, Hamburg, Germany, Hamburg, USA; cartilage destruction in an osteoarthritis rat model a b b b b cDepartment of Medicine, Division of Hematology/Oncology, Department of Sehee Kim , Jihye Lee , Jinhee Park , Jieun Lee , Soyeon Kim , Hanlim b c Immunology and Microbiology, Rush University Medical Center, Moon and Shingyu Bae Chicago, USA aMDimune, Seoul, Republic of Korea; bStem cell team, Seoul, Republic of Korea; cMdimune corp., Seoul, Republic of Korea Introduction: While specific functions of microvesicles have been uncovered in many fields of biology and Introduction: Human mesenchymal stem cells (hMSC) medicine, very little is known about their role in kidney release extracellular vesicles (EV) containing various health and disease. Recently, a new subgroup of micro- proteins and RNAs, which can act as regulatory signals vesicles was discovered in human and murine cell between cells. hMSC-EVs also have provided signifi- culture as well as a model of glomerulonephritis. cant beneficial effects in various disease models. These vesicles are shed upon apoptosis and trigger However, most mammalian cells secret small amount proliferation in neighbouring cells, hence named apop- of EV, which is a limitation for development of ther- totic compensatory proliferative signalling vesicles – apeutics. Therefore, the next generation of EV-mimetic ACPSVs. As these vesicles could be isolated from kid- vesicles produced by serial extrusion of cells produces ney tissue, we hypothesized that a fraction is shed into higher number of vesicles, and may be easier to scale the urine and can be isolated for further analyses. up for therapeutic developments. In this study we Methods: We established a protocol of differential cen- aimed to test the efficacy of EV-mimetic vesicles trifugation and filtration to isolate ACPSVs from urine derived from human adipose-derived stem cells samples of healthy control subjects and patients suffer- (hASCs) on rat osteoarthritis (OA) model. ing from different nephropathies. With western blot Methods:hASC-derivedEV-mimeticvesicles(CDV)were analysis and immunofluorescence microscopy, we vali- produced by serial extrusions of cells through filters. The dated the presence of ACPSVs and investigated the CDVs were characterized by transmission electron micro- cellular origin of the vesicles. Whole lipid quantifica- scopy (TEM), nanoparticle analysis system (NTA), and tion was used to determine vesicle amount and to western blot and flow cytometry. CDVs were injected normalize the protein content. To identify the potential into the joints in a MIA-induced osteoarthritis (OA) rat of initiating proliferation, HeLa cells were counted 24 h model. Improvement of pain after CDV injections was after treatment with freshly isolated urinary vesicles. assessed by paw withdrawal threshold and weight bearing, Results: The employed protocol lead to a robust isola- whereas the joint destruction was evaluated by histology. tion of spherical vesicles ranging between 0.6–1.8 µm We also estimated the effects of CDV on proliferation and containing the ACPSV marker protein CRK1. Further migration of human chondrocytes in vitro by cell-counting protein analysis revealed the presence of Podocin and and scratch assays. Nephrin, pointing to a clear podocyte origin of a frac- Results: The CDV were 50–150 nm in diameter and tion of these vesicles. Similar results could be obtained carried multiple EV-associated tetraspanins (CD63, for vesicles originating from the proximal tubulus and CD9, CD81). CDV-treated OA mice had reduced paw the collecting duct. withdrawal and was more weight bearing 17 days after Summary/Conclusion: Our study represents the first treatment than PBS-treated. Further, histology showed analysis of urinary CRK1 containing vesicles. Taken reduced joint defects at 24 days. CDV-treated OA into account the presence of podocyte marker proteins models displayed significant improvement in paw JOURNAL OF EXTRACELLULAR VESICLES 217 withdrawal behaviour and weight bearing analysis. Funding:ThisprojectissponsoredbyNIHgrant Similarly, chondrocyte migration and proliferation R01DE027404 and The Osteology Foundation Advanced were enhanced by CDV in a dose-dependent manner. Researcher award. Summary/Conclusion: This study demonstrates for the first time the efficacy of hASC EV-mimetic vesicles in OA model. Most interestingly we have confirmed PF08.08 that hASC EV-mimetic vesicles can improve pain and regenerate defected cartilage. These results support the Exosomes secreted during chondrogenic differentiation of human concept that a potential application of hASC EV- adipose-derived stem cells for osteoarthritis treatment mimetic is osteoarthritis, by giving CDV locally into Ye eun Yuna, Woo Sung Kima, Hyun-A Parkb, Su Yeon Kimb and Yong Woo c affected joints. Cho aDepartment of Chemical Engineering, Hanyang University, Ansan, Republic of Korea; bExostemtech,Inc., Ansan, Republic of Korea; cHanyang University, Ansan, Republic of Korea PF08.07 Introduction: Osteoarthritis (OA) is a chronic degenerative joint disease and the most common form of

Natural and synthetic biomaterial mediated delivery of Mesenchymal arthritis. Most of the current treatments focus on Stem Cell derived exosomes pain management and treatment options for repair Chun-Chieh Huanga, Miya Kanazawab, Praveen Gajendrareddyc and Sriram a and regeneration of damaged articular cartilage are Ravindran limited. In recent years, stem cell-derived exosomes aUniversity of Illinois at Chicago, Chicago, IL, USA; bUIC College of Dentistry, Oral Biology, Chicago, IL, USA; cUniversity of Illinois, Chicago, have been the spotlight as a therapeutic candidate due Chicago, IL, USA to their regenerative and immunomodulatory capabilities. In this study, we hypothesized that exosomes Introduction: Mesenchymal stem cell (MSC) derived (Chondro-EXOs) secreted during chondrogenic differ- exosomes are versatile agents that possess immunomo- entiation of human adipose-derived stem cells (hASCs) dulatory and regenerative properties. However, sys- may contain specific biochemical cues that promote the temic delivery of natural or engineered MSC regeneration of damaged cartilage in OA animal exosomes lacks site-specificity and can trigger ectopic model. effects. Therefore, biomaterial-mediated site-specific Methods: Chondro-EXOs were isolated from condi- delivery of exosomes is important. As exosomal mem- tioned media during chondrogenic differentiation by branes are subsets of the plasma membrane. We pre-filtration in 0.2 μm, followed by tangential flow hypothesized that MSC exosomes can bound to extra- filtration (TFF) system (300 kDa MWCO). The isolated cellular matrix proteins and the property can be used as Chondro-EXOs were characterized using transmission a delivery technique. electron microscopy (TEM), nanoparticle tracking ana- Methods: To test this hypothesis, we evaluated the lysis (NTA), flow cytometry, western blot, and cytokine binding and delivery kinetics of MSC exosomes to arrays. To evaluate the therapeutic efficacy of Chon- and from fibronectin, type I collagen and their deriva- EXO, we injected a mixture of Chondro-EXOs (1×108 tive peptides followed by in vitro and in vivo evaluation particles) and hyaluronic acid hydrogel (1%) once a of their efficiency when delivered using this approach. week for 3 weeks at intra-articular site of MIA-induced Results: Results indicated that MSC exosomes bound subacute OA models. Knee joints were harvested at dose-dependently and saturably to fibronectin, type I four weeks after MIA injection and analysed histologi- collagen and their derivative peptides in an integrin cally by safranin O-fast green and haematoxylin and mediated fashion. The presence of integrins on the eosin (H&E). exosomal membrane was verified by immuno electron Results: Chondro-EXOs were approximately 50- microscopy and immunoblotting. Finally, exosomes 120 nm in diameter and expressed exosomal markers bound to 3D hydrogels containing these motifs were such as CD9, CD63, and CD81. Various soluble factors able to promote differentiation of naive MSC in vitro related to anti-inflammatory and cartilage regeneration and bone regeneration in a valvaria defect model in were contained in Chondro-EXOs. In vivo studies vivo. demonstrated that Chondro-EXOs significant pre- Summary/Conclusion: Overall, this study shows that vented proteoglycan degradation and attenuated the MSC exosomes can be tethered to natural and synthetic cartilage destruction in the damaged articular cartilage. biomaterials for site-specific delivery to aid repair and Summary/Conclusion: Our findings suggest that regeneration of tissues. Chondro-EXOs act as a biological cue for cartilage 218 ISEV2019 ABSTRACT BOOK repair and provide a new therapeutic approach for promoted YAP ubiquitination degradation reduced osteoarthritis treatment. renal interstitial fibrosis. Funding: National Natural Science Foundation of China: (81871496) PF08.09 Zhenjiang Key Laboratory of Exosomes Foundation and Transformation Application High-tech Research, China: (ss2018003) hucMSC exosomes delayed diabetic kidney diseases by transported kinase ubiquitin system promoted YAP ubiquitination degradation a b c d Si Qi Yin , Cheng Ji , Hui Qian and Jia Hui Zhang PF08.11 aJiangsu university, Zhen jiang, China (People’s Republic); bZhengjiang, China (People’s Republic); czhen jiang, China (People’s Republic); 4Zhen jiang, China (People’s Republic) Neutrophil extracellular vesicles protect from joint breakdown in Introduction: Diabetes mellitus (DM) is a type of inflammatory arthritis Bethan Lynne. Thomasa, Lucy Norlingb, Francesco Dell’Acciob and Mauro metabolic disease. Diabetic kidney disease (DKD) is b Perretti the important microvascular complications of DM, aWilliam Harvey Research Institute, Queen Mary University London, the leading cause of end-stage renal disease (ESRD). London, UK; bWilliam Harvey Research institute, Queen Mary University Human umbilical cord mesenchymal stem cell exo- of London, London, UK somes (hucMSC-Exosomes) can participated in a variety of tissue damage repair. In this study, we Introduction: Rheumatoid arthritis (RA) is a chronic demonstrated that the mechanism which hucMSC- autoimmune, inflammatory disease. Recently our Exosomes delayed the progression of DKD. understanding of the inflammatory component has Methods: The DKD rat model established by 45% progressed tremendously, however, even after the con- high-fat diet combined with streptozotocin (STZ, trol of inflammation, joint damage, in particular carti- 35 mg/kg,iv). DKD group (n = 12) and hucMSC-exo- lage breakdown, continues to progress leading to somes group (n = 12), control group (n = 6). Blood secondary osteoarthritis and patient disability. glucose, body weight and 24 h urinary albumin clear- Extracellular vesicles (EVs), with their roles in cell-to- ance were measured at 16 and 24 weeks. HE, PAS cell communication, present a novel opportunity for staining used to observed pathological of renal tissue, treatment within difficult to target joint tissues like Sirius red staining to detected renal interstitial fibrosis. cartilage. Neutrophil EVs are remarkable in their bio- YAP protein in renal tissues with time. Confocal actions and are abundant within the joints of RA microscopy observed YAP in cytoplasm and nucleus patients. Here we report the role of Neutrophil EVs location. The CO-IP showed that the ubiquitin bound in RA and their effect on cartilage breakdown. by YAP protein was significantly increased. LC-MS/MS Methods: EVs were generated from human neutrophils and west bolt confirmed CK1δ/β-TRCP existed in the stimulated with TNF (20 ng/ml; 20 min), and tested in exospores. Used the adenovirus shRNA experiment the K/BxN murine model of inflammatory arthritis. knockdown CK1δ/β-TRCP. Results: In murine inflammatory arthritis, intra-articu- 3 Results: hucMSC-exosomes can migrated to renal lar injection of neutrophil EVs (30–300x10 per joint), injury site and regulated blood glucose in tissues. reduced knee swelling and displayed cartilage protec- hucMSC-exosomes intervention delayed the progres- tive effects, measured as reduced loss of proteoglycans sion of DKD. Maintained rat weight, reduced serum and improved structural integrity in the treated joints. urea nitrogen, the degree of interstitial fibrosis signifi- Cartilage in EV-treated joints also maintained a higher cantly weakened. Sustained high glucose stimulated content of Collagen type2, an important component of activation of YAP. The YAP increased significantly healthy cartilage, and contained fewer hypertrophic with time which increased degree of interstitial fibrosis. chondrocytes, abundant in diseased cartilage. Of great hucMSC-exosomes transported CK1δ/β-TRCP translational importance, this effect lasted at least repaired kinase ubiquitin system imbalance inhibited 28 days, suggesting that administration of these EVs YAP activity that attenuated interstitial fibrosis of enacted positive circuits of protection characterized by DKD. Our experiments confirmed that hucMSC-exo- a phenotypic change within the tissue, resulting in long somes carried CK1δ/β-TRCP promoted YAP ubiquiti- lasting protective effects even after the EVs themselves nation degradation. have been cleared. In vitro, neutrophil EVs inhibited Summary/Conclusion: hucMSC exosomes delayed IL-1-induced cartilage breakdown and restored basal diabetic kidney diseases by transported CK1δ/β-TRCP expression of cartilage specific genes. JOURNAL OF EXTRACELLULAR VESICLES 219

Summary/Conclusion: Neutrophil EVs exert powerful PF08.13 and long lasting protective bioactions in inflammatory arthritis, modulating the ongoing joint inflammation Rab27a dependent exosome secretion from tubular epithelial cell while also protecting from cartilage breakdown. promotes albumin-induced tubulointerstitial inflammation a b a Funding: Medical Research Council (MRC) Ye Feng , Linli Lv and Bi-Cheng Liu Regenerative medicine research grant aInstitute of Nephrology, Southeast University, Nanjing, China (People’s Republic); bInstitute of Nephrology, Zhongda Hospital, Southeast University, Nanjing, China (People’s Republic) PF08.12 Introduction: Tubular epithelial cells (TECs) secrete

Role of small extracellular vesicles in ageing increasing exosomes under with proteinuric toxicity. Juan Antonio Fafian Labora, Ana O’Loghlen, Paula Carpintero-Fernandez However, the mechanism through which exosomes and Olga Eleftheriadou are produced and the effect on tubular cell haemostasis Epigenetics & Cellular Senescence Group, Blizard Institute, Barts and The and tubulointerstitial inflammation are unknown. London School of Medicine and Dentistry, Queen Mary University of London, London, UK Methods:Proteinuricrenaldiseasemodelwasinducedby adriamycin (ADR) administration through tail vein. Introduction: Ageing is a major risk factor for many Urinary albumin was determined at 0, 7, 14, 21 and human diseases. It is a complex process that progres- 23 days after ADR injection. For in vitro studies, TECs sively compromises most of the biological functions of were treated with albumin. Exosomes were purified from the organisms, resulting in an increased susceptibility isolated tubules of kidney and cell culture supernatant for to disease and death. Hutchinson-Gilford progeria syn- characterization and functional study. drome (HGPS) and normal aging share many cellular Results:Urinaryalbuminwassignificantlyincreasedin phenotypes: abnormal nuclear shape, dysregulated of ADR-treated mice 2 weeks after injection compared with epigenetic markers, increased DNA damage. controls. Exosome production was increased significantly in Remarkably, partial reprogramming extended the life- kidneys and tubules of ADR mice and in TECs with albumin span of the progeric mice with remodelling of the exposure, confirmed by electron microscopy, western blot- epigenetic markers. The alteration in intercellular com- ting analysis of exosome markers and EXOCET. munication with age has been demonstrated to be due Interestingly, we showed increasing levels of Rab27a to senescent cells developing a senescence-associated mRNA and protein both in the tubules of ADR-injected secretory phenotype (SASP). mice and in BSA-treated TECs in a dose dependent manner. Methods: In this study, we have a characterization of Furthermore, the increased exosome production was depen- small extracellular vesicles (sEVs) using in vitro normal dent on Rab27a up-regulation since silencing of Rab27a and premature ageing models and the rejuvenation reversed the exosomes secretion. Importantly, albumin was capacity of sEVs from young donors and iPSCs in old present in TEC-derived exosomes after BSA exposure. and progeria recipients. Impressively, lysosomal degradation of albumin was Results: Firstly, we performed the evaluation of pro- increased while the mRNA expression of inflammatory duction of sEVs using Nanoparticle Tracking Analysis cytokines was reduced after inhibition of exosome secretion (NTA) and characterization of positive CD63/CD81 by Rab27a silencing in TECs treated with BSA. To explore sEVs by flow cytometry. Then, we evaluated the reju- the effect of TEC exosome production under albumin expo- venation potential of sEVs from young and iPSCs sure, TEC-exosomes were purified and added to naïve TEC. donors on old and progeria fibroblasts. We found an Up-regulation of inflammatory cytokines were found in increment of sEVs production with the age and the receipt TECs. Lentivirus Rab27a-inhibitor intrarenal injec- capacity of sEVs from young and iPSCs donors to tion reversed tubulointerstitial inflammation and increased recover the proliferation capacity (BrdU) and epige- survival of ADR-induced mice through stably inhibiting netic marker (H3K9me3) in fibroblasts from progeria Rab27a expression. Clinically, high levels of Rab27a were and old donors. found in tubules and correlated with the magnitude of Summary/Conclusion: These findings are important urinary exosomes in patients with chronic kidney disease. to the understanding about influence of the ageing on Summary/Conclusion:TheseresultssuggestthatRab27a- sEVs and the development sEV-based therapies in age- dependent exosomes secretion drive albumin escaping related diseases. degradation and secreting into extracellular fluid may Funding: BBSRC (BB/P000223/1) and The Royal exacerbate TECs injury by enhancing inflammatory Society (RG170399) awarded to AOL. JFL and PCF response and consequently leading to tubulointerstitial are funded by the Xunta de Galicia Fellowships (Spain). inflammation. 220 ISEV2019 ABSTRACT BOOK

PF09: Detection of EV-based Biomarkers Chairs: Fabia Fricke; Shinichi Kano Location: Level 3, Hall A 15:30–16:30

PF09.01 Summary/Conclusion: The characterisation of the AF- EVs with NTA and FACS demonstrates the composition and size as well as presence of markers of different Extracellular vesicle (EV) extraction and characterisation in amniotic origin including kidney, immune system and endothe- fluid (AF) Natalia Gebaraa, Corinne Lampietrob, Benedetta Bussolatic, Chiara lium. The investigation of EV properties in healthy and Benedettod and Luca Marozioe diseased placenta could prove useful in the future as a aUniversity of Torino, Torino, Italy; bDepartment of Molecular diagnostic tool to understand and diagnose pregnancy- Biotechnology and Health Sciences, University of Torino, Torino, Italy; associated diseases. cDepartment of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy, Turin, Italy; dDepartment of Surgical Sciences, Obstetrics Funding: This work was supported by the iPlacenta and Gynecology, Torino, IL, USA; eDepartment of Surgical Sciences, project founded by the European Union’s Horizon Obstetrics and Gynecology, Torino, Italy 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 765274 Introduction: During pregnancy, placental-derived EVs have been identified in maternal blood and AF thus are implicated in cell-to-cell communication. We PF09.02 hypothesize that placental-derived EVs released in amniotic fluid may possess angio-modulating properties that could be relevant in placental angiogenesis and Evaluation of non-invasive biomarkers for monitoring functional that these characteristics may be altered in pre-eclamp- status of endometrium Mattia Criscuolia, Gaia Papinia, Davide Zoccob, Alice Luddic, Valentina sia (PE), a pregnancy complication characterised by Pavonec, Paola Piombonic and Nataša Zarovnid hypertension and proteinuria causing neonatal mor- aExosomics Siena – University of Siena, Siena, Italy; bExosomics Siena, Siena, bidity and perinatal mortality. USA; cUniversity of Siena, Siena, Italy; dExosomics, Siena, Italy Methods: The amniotic fluid was obtained from normal pregnancies during caesarean sections. The physio- Introduction: Endometrium is a complex tissue with chemical characteristics were tested by Nanosight tech- self-renewing properties, normally undergoing cyclic nology (NTA) and characterization of exosomal mar- modifications regulated by ovarian steroids divided kers was carried through FACS using microspheres and into proliferative and secretory phase. The transcrip- MASPlex exosome kit. MASPlex kit simultaneously tomic profile of the endometrium is influenced by detects 37 exosome surface epitopes. other endometrial cell types (glandular epithelial and Results: We set up a method for EV isolation from AF stromal) in both physiological and pathological condi- based on subsequent dilution with PBS; first centrifu- tions. These cells have mutual paracrine effects par- gation at 10,000 g for 30 min at 4°C, filtration through tially mediated by EVs, and they grow in a cycle- a 0.45 µM filter and ultracentrifugation at 100,000 g for dependent manner. To assess the endometrium status, 2 h in 4°C. The averages EV concentration was several invasive or expensive techniques are currently 4.34×1011 particles/ml with a mean peak of employed, including immunohistochemistry (IHC) on 240.45 nm, measured by NTA. FACS analysis showed tissue biopsy, cytology and imaging. Development of presence of angiogenic markers VEGFR 1,2,3 and protocols for the isolation of EVs from novel biological CD105, immunological markers HLA ABC, HLA DR, sources is an extremely attractive means to surrogate exosome specific markers CD81 and CD63 also CD133, endometrial biopsies. These novel protocols may which indicates kidney origin. By using the MASPlex enable the identification and sensitive detection of spe- kit, we set up a semiquantitative method for detection cific endometrial EV biomarkers for diagnostic solu- of 37 different potential AF-EV surface markers in one tions in reproductive medicine, endometriosis or sample simultaneously. We confirmed the heterogenic cancer. characteristics of AF-EVs, including expression of Methods: Samples: primary endometrial cultures, urine immune system markers CD209, CD62P, CD11c, from healthy donors in secretory phase; Differential CD20 and endothelial markers CD146 and CD41b. centrifugation, size exclusion chromatography (SEC), JOURNAL OF EXTRACELLULAR VESICLES 221 immunobeads for EV isolation; Nanoparticle Tracking five different isolation strategies were evaluated: ultra- Analysis (NTA), BCA assay, ELISA, HS Qubit, ddPCR, centrifugation, membrane affinity, spin column chro- SPR, FACS for EVs and EV markers quantification and matography, immunoaffinity and precipitation. After characterization. EV characterization by nanoparticle tracking analysis, Results: We provide new evidence that urine is a western blotting, and transmission electron micro- surrogate biofluid suitable for the detection of endo- scopy, total RNA was isolated and a library for small metrial EV biomarkers. Using pre-selected antibody RNA sequencing was prepared. The resultant success- panels, we identify specific endometrium EV binding ful strategy was then applied to all the collected sam- antibodies in relevant in vitro models. Coupling ples which were equally analysed regarding their EV immune-isolation to pre-analytical protocols for urine distribution and miRNA content. processing and sample quality testing enables detection Results: The comparative analysis disclosed huge dis- of a panel of endometrial genes in urine-recov- crepancies with respect to EV yield, population, and ered EVs. purity, as well as RNA yield and detected miRNAs. By Summary/Conclusion: Overall, the study provides a applying the best performing strategy, which was based tool for non-invasive monitoring of the functional sta- on immunoaffinity, significantly higher amounts of tus of the endometrium, supporting biomedical niches urinary EVs and several significantly differentially such as assisted fertilization and diagnosis of regulated miRNAs were observed after ABD. endometriosis. Summary/Conclusion: Urinary EVs and their miRNA Funding: ENDEvor POR Region Tuscany (identifica- profile hold indeed promising attempts for the clear tion of the project) and Exosomics R&D Programme separation of ABD and non-doped athletes. Moreover, the included complex comparative methodological analysis contributes enormously to future standardization PF09.03 and comparability of urinary EV research. Funding: The current project has been financially supported by the WADA. Unveiling autologous blood doping: comparative analysis of different purification strategies for urinary extracellular vesicles pioneering miRNA biomarker research Veronika Mussacka, Georg Wittmannb and Michael Pfafflc PF09.04 aTUM School of Life Sciences Weihenstephan, Division of Animal Physiology and Immunology, Freising, Germany, Freising, Germany; bDepartment for Transfusion Medicine, Cell therapeutics and Haemostaseology, University Extracellular vesicles as graft biomarkers to address lung Hospital LMU, Munich, Germany, München, Germany; cAnimal Physiology transplantation outcome and Immunology, School of Life Sciences Weihenstephan, Technical Mario Barilania, Ilaria Righib, Giuseppe Buonoc, Lorenzo Rossod, Mario University of Munich, Freising, Germany, Freising, Germany e c Nosotti and Lorenza Lazzari

aUnit of Regenerative Medicine – Cell Factory, Department of Transfusional Introduction: Autologous blood doping (ABD) Medicine and Hematology, Fondazione IRCCS Ca’ Granda Ospedale increases the oxygen capacity via re-infusion of a per- Maggiore Policlinico, Milano (MI), Italy; Università degli Studi di Milano, b son’s autologous red blood cells. It is therefore used by Milan, Italy; Thoracic Surgery and Lung Transplant Unit, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano (MI), Italy, endurance athletes with a high level of unreported Milano, Italy; cUnit of Regenerative Medicine – Cell Factory, Department cases, especially since reliable methods for unequivocal of Transfusional Medicine and Hematology, Fondazione IRCCS Ca’ Granda Ospedale Maggiore Policlinico, Milano (MI), Italy, Milano, Italy; dUniversità detection are still lacking. To support the Worldwide degli Studi di Milano, Milano (MI), Italy, Milano, Italy Anti-Doping Agency (WADA) in protecting the athletes’ healthiness and ensuring harmonization, herein- Introduction: In the medical practice, lung transplan- after extracellular vesicles (EVs) and their conveyed tation is the last therapeutic option for end-stage pul- cargo were used as potential biomarkers. Since monary failure, when other treatments are no longer degraded red blood cells and their content are elimi- effective. Yet, only 15-20% of the multi-organ donors nated via the kidney and urine, the urinary EV popula- have suitable lungs. Furthermore, clinical complication and their microRNA (miRNA) profile were tions may rise after organ retrieval following ische- particularly focused. mia–reperfusion lung injury, such as primary grafts Methods: After study approval by the local ethics com- dysfunction or chronic lung allograft dysfunction. mittee, written informed consent was obtained of 30 Currently, clinical parameters implemented to assess healthy men undergoing different ABD intensities and the quality of the graft have failed to evaluate tissue several sampling time points. Consistent compliance damage at the cellular level and to predict transplanta- with the “Declaration of Helsinki” was assured. Due tion outcome. Therefore, we focused our attention on to a lack of standardization in urinary EV purification, extracellular vesicles (EV) as innovative, non-invasive 222 ISEV2019 ABSTRACT BOOK biomarkers urgently needed to assess lung quality and tendency of miRNAs before and after the transplanta- monitor organ engraftment. tion. Compared with the control group, for the Methods: Research activities involving human subjects miRNAs whose signal fold multiples greater than 10 complied the Declaration of Helsinki. Informed con- folds, there were 11 miRNAs increased and 26 sent and local ethics committee approval were decreased in the aGVHD group. The expression of obtained. Size and concentration analysis were per- hsa-miR-3976, hsa-miR-122-5p, hsa-miR-3125 were formed by nanoparticle tracking analysis (Nanosight significantly up-regulated and the expression of hsa- NS300, Malvern). miR-4687-5p, hsa-miR-941, hsa-miR-4769-5p were Results: Preliminary results showed the presence of EV down-regulated; these six miRNAs were listed as can- of different sizes in bronchoalveolar lavage (BAL) and didate miRNA gene sensitive biomarkers in peripheral plasma of both donors and recipients. EV presented serum. highly polydispersed size distributions in a 50– Summary/Conclusion: Through Go, pathway and tar- 1000 nm range. Different EV production kinetics get gene analysis, candidate genes participate in regulat- were observed in the recipients (10E08-10E10 parti- ing water-soluble vitamin metabolism, mitochondrial cles/mL range): BAL samples showed concentration apoptosis and other biological processes, regulating cell peaks within 72 h post-transplant and a subsequent membrane and organelle synthesis. The specific decreasing trend, whereas plasma samples showed a mechanism will be further studied. slightly increasing trend. EV samples will be analysed Funding: Natural Science Foundation of Guangdong for RNA content and antigen expression, and correla- Province (2015–2018) tion with lung transplantation outcome will be evalu- Free application project of Shenzhen Science and ated at the conclusion of the follow-up. Technology Innovation Committee (2017–2020) Summary/Conclusion: The identification of specific EV kinetics patterns and RNA signatures represents a promising approach to define biomarkers useful for PF09.07 thoracic surgeons who want to manage in advance complications associated with lung transplantation. Circulating cancer-associated extracellular vesicles as early detection and recurrence biomarkers for pancreatic ductal adenocarcinoma PF09.05 Yusuke Yoshiokaa, Tetsuya Nakatsurab and Takahiro Ochiyac aTokyo Medical University, Shinjyuku-ku, Japan; bDivision of Cancer Immunotherapy, Exploratory Oncology Research and Clinical Trial Center, Expression profile analysis of miRNAs in serumal exosomes as National Cancer Center, Kashiwa, Japan; cDepartment of Molecular and sensitive biomarkers before and after hematopoietic stem cell Cellular Medicine, Institute of Medical Science, Tokyo Medical University, transplantation Shinjyuku-ku, Japan Daming Wanga, Lei Zhengb and Taixue Anc aShenzhen, China (People’s Republic); bClinical Laboratory Department, Introduction: Pancreatic ductal adenocarcinoma Nanfang Hospital, Southern Medical University, Guangzhou, China (PDAC) is usually found at an advanced stage, (People’s Republic); cNan Fang Hospital, Southern Medical University, Guangzhou, China (People’s Republic) although diagnosis at an early stage is unequivocally associated with better long-term survival. Therefore, Introduction: To investigate the differential expression there is an urgent need to develop early detection of miRNAs of candidate genes in peripheral blood methods to improve these outcomes. Extracellular vesi- serum of patients with acute graft-versus-host disease cles (EVs) attract much attention as potential biomar- (aGVHD) after allogeneic hematopoietic stem cell ker because tumour cells have been shown to release transplantation (allo-HSCT). EVs into circulation which mirror their cellular origin. Methods: There were a total of 30 patients with Detection of cancer-associated EVs in body fluids from aGVHD and without aGVHD within 100 days after patients could serve as a non-invasive liquid biopsy for allogeneic hematopoietic stem cell transplantation in diagnosis and monitoring of cancer. The main objec- leukaemia; serumal exosome and RNA were extracted tive of this study is the identification and detection of before and after the transplantation; image acquisition PDAC-specific EVs in patient-derived serum. and data analysis were performed after hybridization of Methods: Total EV proteins were purified from three gene chips; candidate genes associated with the occur- different stages of PDAC (stage II, III and IV, each rence of aGVHD were screened out based on the group: six patient sera pooled) and healthy donors. differential expression of miRNAs. Mass spectrometry was performed with purified EV Results: The expression profile of miRNAs after hybri- proteins, and biomarker candidates were validated by dization of gene chips showed increasing or decreasing immunoblotting in PDAC patient blood samples. JOURNAL OF EXTRACELLULAR VESICLES 223

Results: Proteomics analysis identified over 500 pro- evaluate the diagnostic and prognostic value of urinary teins in each pooled samples, and we identified nine exosomal miRNA in PCa. membrane proteins, which were detected in only Results: Five candidate miRNAs were found by NGS. PDAC patients, not in healthy donors. We focused on Significant downregulation of urinary exosomal miR- two proteins and performed immunoblotting for the 375 was observed in PCa patients comparing with validation of potential biomarkers in three stages of healthy controls, while miR-451a, miR-486-3p and PDAC blood samples. As a result, these proteins were miR-486-5p were found significantly upregulated. detected exclusively in EVs of PDAC patient sera However, no significant difference was found for including stage II. Moreover, we analysed a total of miR-16-2-3p. The expression level of urinary exosomal 33 samples from 11 PDAC patients who performed miR-375 showed significant correlation with clinical surgery at three time points; before surgery, after sur- stage and bone metastasis of the patients with PCa gery and recurrence as an early stage model. As a (p < 0.05). ROC analysis demonstrated that the urinary result, these proteins were detected in EVs derived exosomal miR-375, miR-451a, miR-486-3p and miR- from preoperative samples and recurrence samples. 486-5p are able to differentiate PCa patients from Summary/Conclusion: This study using unique recur- healthy controls, with the AUC of 0.788, 0.757, 0.704 rence samples as an early stage model shows that the and 0.796, respectively. The urinary exosomal miR-375 identified EV-associated proteins have potential as was found superior in discriminating localized PCa early detection makers and warrant further from metastatic PCa, with an AUC of 0.806. investigation. Additionally, PCa patients can be distinguished from Funding: This work was supported in part by a Grant- BPH patients by using a panel combining urinary exo- in-Aid from the Japan Science and Technology Agency somal miR-375 and miR-451a, with an AUC of 0.726. (JST) through the Center of Open Innovation Network Summary/Conclusion: These findings demonstrate for Smart Health (COINS) and a Grant-in-Aid from that the urinary exosomal miRNA can serve as a the Japan Agency for Medical Research and novel and non-invasive biomarker for diagnosing and Development (AMED) through Project for Cancer predicting the progression of PCa. Research and Therapeutic Evolution (P-CREATE: Funding: Shaanxi Health and Family Planning JP18cm0106402). Commission Foundation Project (2016D020), Xi’an Science and Technology Bureau Foundation Project (2017121SF/YX015) and Shaanxi Natural Science PF09.08 Foundation Project (2018JQ8010).

Exosome-encapsulated miRNA in urine as a non-invasive biomarker for prostate cancer PF09.09 Zhuo Li, La-Xiu Li, Yanjun Diao, Yue-yan Ma and Xiaoke Hao

Department of Clinical Laboratory Medicine, Xijing Hospital, Air Force Medical University, Xi’an, China (People’s Republic) Unlocking the secret of salivary exosomes derived from HPV-driven oropharyngeal cancer Kai D Tanga, Yunxia Wana, Natalie Bozyka, Xi Zhanga, Liz Kennyb and Introduction: Prostate cancer (PCa) is the most com- a Chamindie Punyadeera mon malignant tumours in male urinary system. Novel aThe School of Biomedical Sciences, Institute of Health and Biomedical and non-invasive biomarker with higher sensitivity and Innovation, Queensland University of Technology and The Translational specificity for the diagnosis of PCa are urgently needed. Research Institute, Queensland, Australia., Brisbane, Australia; bSchool of Medicine, University of Queensland; Royal Brisbane and Women’s Exosomal microRNAs in circulating fluids have Hospital, Brisbane; Central Integrated Regional Cancer Service, Queensland recently been reported to augment diagnosis and man- Health, Queensland, Australia., Brisbane, Australia agement of certain diseases, including cancer. The purpose of this study is to explore the diagnostic value of Introduction: There has been a significant rise in the urinary exosomal miRNAs for PCa. incidence of oropharyngeal cancer (OPC) associated Methods: A urinary exosomal microRNA expression with high-risk human papillomavirus (HPV), predomi- profiling was performed by next-generation sequencing nantly HPV-16 infections in high-income countries, using urine samples. Then, candidate miRNAs were especially when compared to HPV-negative head and selected and validated by qRT-PCR in 3 cohorts con- neck cancer (HNC). Growing evidence supports the sisting of PCa patients, healthy controls and patients concept that exosomes (30–150 nm) loaded with with benign prostatic hyperplasia (BPH). Receiver unique bio-components (DNA, RNA and protein) operator characteristic (ROC) analysis was used to play a salient role in cancer development and 224 ISEV2019 ABSTRACT BOOK progression. However, the role of exosomes in saliva more sensitive biomarkers need to be developed. obtained from HPV-driven OPC is still far from clear. Extracellular vesicles (EVs) have in the last decade Methods: Morphology and molecular features of exo- been recognized as major players in cancer biology somes derived from three different saliva sampling meth- but it’s only in recent years that isolation protocols ods: unstimulated saliva, acid-stimulated saliva and have reached enough sophistication for a truly mean- salivary oral rinses were examined using transmission ingful proteomic analysis. electron microscopy (TEM), nanoparticle tracking Methods: Tumour and non-tumour tissue (approx. (NTA) and western blot analysis. HPV-16 DNA detection 10 cm from tumour) were excised from 10 CC patients. in salivary exosome was determined using qPCR method. The tissue was sliced into approximately 1 mm 3 pieces Proteome profile of salivary exosomes derived from both and partially digested with DNase and Collagenase in cancer-free controls and HPV-driven OPC patients was cell culture medium for 30 min at 37°C. Digested tissue characterized using mass spectrometry analysis. was filtered through a 70 µm filter to remove tissue Results:Here,weshowedthatunstimulatedsalivahadthe pieces and large fragments. Vesicles were isolated from greater abundance of exosomes when compared to the the media with an isolation process consisting of dif- other saliva sampling methods. In fact, the three common ferential ultracentrifugation and density gradient floa- exosome markers (CD9, CD63 and CD81) were higher in tation aimed at isolating EVs. Isolates were then lysed, unstimulated saliva method. Nevertheless, no appreciable tryptically digested and Tandem Mass Tag labelled difference in exosome morphology was found among the before analysis by mass spectrometry. The samples, 20 three different sampling methods. Furthermore, only sali- in total, were analysed in two rounds as “Set 1” and vary exosome derived from HPV-driven OPC had a “Set 2”, with each set containing EVs derived from five detectable level of HPV-16 DNA. Intriguingly, the proteo- tumour tissues and five non-tumour tissues. mic signature of salivary exosome was significantly differ- Results: In total, approximately 4000 proteins were ent between cancer-free controls and HPV-driven OPC. identified and quantified, with 2567 and 3742 proteins Summary/Conclusion: Taken together, our results identified in Set 1 and Set 2 respectively with an over- showed that unstimulated saliva is an optimum sam- lap of 2271 proteins between the sets. Proteins which pling method for exosome characterization. More after a t-test had a p-value lower than 0.05 and a fold importantly, the development of a low cost non-inva- change of at least two were considered as being differ- sive saliva-based test (salivary exosomal DNA and pro- ently expressed in tumour tissue EVs as compared to tein) will offer an opportunity to detect HPV-driven normal tissue EVs. 299 and 592 such proteins were OPC, thereby opening new avenues in the future for identified in Set 1 and Set 2 respectively, with 125 clinical and commercial translation. meeting these requirements in both Sets. Funding: N/A Summary/Conclusion: EVs isolated directly from the tumour tissue microenvironment differ in their protein PF09.12 cargo from that of EVs resident in equivalent non- tumour tissue. Proteins carried by tumour derived EVs could potentially play a role in tumour biology Determination of the protein cargo of colon cancer tissue-derived by mediating signalling to neighbouring cells. extracellular vesicles Aleksander Cvjetkovica, Rossella Crescitellib, Helena Taflinc, Cecilia Lassera Furthermore, these differentially expressed proteins and Jan Lötvalld could potentially function as biomarker candidates. aKrefting Research Centre/University of Gothenburg1 Krefting Research Centre, Department of Internal medicine and clinical nutrition, Institute of Medicine, University of Gothenburg, Gothenburg, Sweden; bKrefting PF09.13 Research Centre, Department of Internal medicine and clinical nutrition, Institute of Medicine, University of Gothenburg, Gothenburg, Sweden; cDepartment of Surgery, Institute of Clinical Sciences, Sahlgrenska Academy at the University of Gothenburg, Sahlgrenska University Hospital, Characterization of small extracellular vesicles secreted by dermal Gothenburg, Sweden; d1 Krefting Research Centre, Dept of Internal medicine fibroblasts a b and clinical nutrition, Institute of Medicine, University of Gothenburg, Marijana Jevtić and Sarah Hetrich Gothenburg, Sweden aMaster of Pharmacy – Medical Biochemist, Berlin, Germany; bProf. Dr, Berlin, Germany Introduction: Colorectal cancer (CC) is the third most common cancer to affect both men and women, and Introduction:Dermalfibroblastsplayakeyroleinepider- the third-leading cause of cancer-related mortality. In mal proliferation and differentiation. They communicate order to be curable, CC has to be diagnosed and treated with other cell types, playing a crucial role in the regulation by surgery before the tumour cells have started to of the skin (patho)physiology.Extracellularvesicles(EVs) metastasize. In order to find CC at an early stage, are small membrane-enclosed vesicles (30-150 nm) that are JOURNAL OF EXTRACELLULAR VESICLES 225 released from all cell types into the extracellular space and culture, what provide that we isolated pure EVs represent an important mode of cell-to-cell communica- released by live cells. NTA and TEM analyses proved tion. Emerging data indicate that they play key roles in the presence of EVs, cup-shaped structure and size many (patho)physiological processes. However, there is smaller than 150 nm. With FACS analysis of EVs, we currently very little information about the content and proved that EVs are enriched with cytosolic protein the function of EVs from dermal fibroblasts. Therefore, present in EVs, Tsg101. we aimed to isolate and characterize EVs secreted by der- Summary/Conclusion: Here we present characteriza- mal fibroblasts. tion of EVs secreted by dermal fibroblasts in terms of Methods:Dermalfibroblastswereisolatedfromjuvenile size, shape and cytosolic proteins present in EVs. In foreskin and cultivated in DMEM supplemented with 7.5% next steps, we plan mass spectrometry of the proteome FBS and 2 mM L-glutamine. On day 5 of cultivation, of dermal fibroblasts and EVs secreted by dermal fibro- dermal fibroblasts were washed with PBS and further cul- blasts. EVs are able to interact with cells located nearby tured in EV-depleted medium for 24h before collecting the or distantly and EVs can be a way for carrying infor- medium. To elucidate the characteristics of EVs, EVs were mation from cell to cell. These findings may lead to isolated from conditioned medium by several ultracentri- identification of new signalling pathways in between fugation and filtration steps.ToverifythepresenceofEVs, dermal fibroblasts and other cells present in the skin, nanoparticle tracking analysis (NTA), transmission elec- what could help us to understand the regulation of the tron microscopy (TEM) and flow cytometry (FACS) were skin physiology. performed on the dermal fibroblasts-derived EVs. Funding: S.H. acknowledges financial support by the Results: With FACS analysis of dermal fibroblasts, we German Research Foundation (DFG HE 7440/4–1). proved that more than 95% of the cells were alive in the 226 ISEV2019 ABSTRACT BOOK

PF10: Advances in EV separation and concentration Chairs: Stacey Gifford; Fuquan Yang Location: Level 3, Hall A 15:30–16:30

PF10.01 as well as ApoB-100). These findings are also supported by Western Blot analysis. Summary/Conclusion: EV preparations are commonly Efficient clearance of lipoproteins from anti-coagulated and native blood-derived products to yield pure extracellular vesicle contaminated with lipoproteins due to their similar size preparations and density. The coupling of UC to separate EVs from a b b c Alexander Otahal , Olga Kuten , Andrea De Luna , Zsombor Lacza and lipoproteins by density and SEC to yield separation by Stefan Nehrerb size enabled efficient clearance of lipoproteins from aDanube University Krems, Krems An Der Donau, Austria; bDanube University Krems, Vienna, Austria; cOrthosera, Vienna, Austria CPRP or hypACT(TM) serum and obtaining pure EV preparations. Introduction: Extracellular vesicles (EVs) increasingly Funding:Theworkwasfundedbythe gain focus in regenerative medicine for promoting Wissenschaftsfonds of Lower Austria (NÖ) together with tissue repair and alleviating inflammation. However, the European Fund for Regional Development (EFRE). there are no standards for EV isolation from patient blood nor for quality assessment owing to lack of PF10.02 knowledge about active components or mechanisms of action. It is known that high, low and very low density lipoproteins (HDL, LDL, VLDL) as well as Proteomic and Lipidomic Analysis of Extracellular Vesicles from chylomicrons copurify with EVs during isolation Human Plasma and Urine Purified by Asymmetrical Flow Field-Flow Fractionation from various body fluids including blood via ultracen- Fuquan Yang trifugation (UC) or size exclusion chromatography Institute of Biophysics, Chinese Academy of Sciences, Beijing, China (SEC). The aim of our study was to develop an isola- (People’s Republic) tion strategy to purify EVs from blood derived products which are already in clinical use. Therefore, we Introduction: Extracellular vesicles (EVs) are com- analysed EV preparations from citrate-anticoagulated posed of lipid bilayer membranes and they are a platelet-rich plasma (CPRP) and hypACT™ serum. group of heterogeneous, nano-sized structures vesicles Methods: Particle concentrations after UC, SEC or a enriched with nucleic acids, proteins and lipids. EVs combination of both were assessed via nanoparticle can be released by normal and cancer cells to their tracking analysis (NTA). EVs were labelled with surrounding environments and they are also found in annexin V (AnnV), CD63 as well as CD41 and ana- diverse body fluids, including blood, urine, saliva, cer- lysed by flow cytometry (FC). LDL and HDL content ebrospinal fluid, breast milk, seminal fluid. EVs play was determined in EV preparations by labelling of many important roles in numerous physiological and Apolipoprotein A1 (ApoA1) and Apolipoprotein pathological processes. B100/48 (ApoB-100) by FC as well as detection via In recent years, various studies on EVs have been Western Blot. Presence of EVs was confirmed by cryo conducted in the clinical research. EVs are rich in electron microscopy. disease related biomarkers, and can protect the Results: NTA revealed 100-fold higher particle concen- wrapped parent cells derived materials due to their trations after SEC than after UC or UC+SEC in both, double layer membrane structures and target the spe- CPRP and hypACT(TM) serum. AnnV, CD63 as well cific cells or tissues. EVs have promising potential for as CD41 were detected on EVs via FC. It also revealed diagnostic and therapeutic applications, and can serve efficient clearance of ApoB-100 bearing particles by as biomarkers and targeting drug delivery systems. UC, while ApoA1-positive particles persisted. SEC Omics studies of EVs have been used for the dis- alone removed ApoA1-positive particles, but failed to covery of biomarkers. The isolation of EVs are the key remove ApoB-100 bearing particles. The combination step for the omics studies on EVs. of enrichment via UC and purification via SEC enabled Methods: Field-flow fractionation (FFF) technique was efficient clearance of both lipoprotein species (ApoA1 first invented in 1966 by J. Calvin Giddings. FFF has JOURNAL OF EXTRACELLULAR VESICLES 227 unique properties enabling separation and characteri- tdEVs in three size ranges (1–8 µm, 0.2-1 µm, and zation of macromolecules, polymers, proteins, colloids, 0.05–0.2 µm), EV-miRNA and ccfDNA. cells and vesicles from 1 nm to 100 m at high resolu- Results: Bead recovery was predicted with errors <18%. tion. AF4 has been reported to purify EVs from the Most notable cofounders are the 22% contamination of supernatant of cell culture. In this study, we have 1–8 µm tdEVs for TEPs, and 50–82% of tdEVs developed AF4 based mothed for isolation of EVs <200 nm for ccfDNA. Based on our model, none of from human plasma and urine. The proteomic and the evaluated protocols produces a pure biomarker. lipidomic analysis was performed using LC-MS/MS. Thus, care should be taken in interpretation of Results: EVs in human plasma were isolated from obtained results, as, for example, results from TEPs HDL and LDL with good resolution by an optimized may originate from co-isolated large tdEVs, and AF4 conditions. EVs in human urine were also isolated ccfDNA may originate from DNA enclosed in from the high abundant protein uromodulin by opti- tdEVs <1 µm. mized AF4 conditions after treatment with DTT reduc- Summary/Conclusion: The Stokes model can be tion. Transmission electron microscopy (TEM), SDS- applied to predict the behaviour of biomarkers – PAGE, Western Blot, proteomics and lipidomics are including EVs- during isolation or concentration to further applied for the studies on purified EVs from other body fluids, which may facilitate the comparison human plasma and urine. of such protocols in e.g. EV-TRACK, further standar- Summary/Conclusion: The results reveal that AF4- dization of protocols, and develop optimal bioreposi- based separation method for EVs is of high reproduci- tory conditions. bility, purity, recovery and continuous preparation and Funding: This work is supported by the Netherlands separation ability. The specific proteins and lipids have Organisation for Scientific Research – Domain Applied been identified from human plasma and urine EVs and Engineering Sciences (NOW-TTW), research pro- compared with the whole components in human grams VENI 13681 (Frank Coumans), Perspectief plasma and urine CANCER-ID 14198 (Linda Rikkert), and VENI 15924 (Edwin van der Pol). PF10.03 PF10.04 A centrifugation model to predict the behaviour of tumour biomarkers in liquid biopsies Linda Rikkerta, Edwin van der Polb, Ton van Leeuwenc, Rienk Nieuwlandd, Effects of lipoprotein destabilization on isolation and analysis of Leon Terstappene and Frank Coumansd plasma-derived extracellular vesicles Danilo Mladenovića, Paolo Guazzib, Elina Aleksejevab, Antonio Chiesib, Kairi aAmsterdam UMC, location AMC, Amsterdam, Netherlands; bAmsterdam Koorta, Davide Zoccoc, Triin Ojab and Nataša Zarovnid UMC, University of Amsterdam, Department of Biomedical Engineering and c Physics, Amsterdam, Netherlands, Amsterdam, Netherlands; dAmsterdam aTallinn University, School of Natural Sciences and Health, Tallinn, Estonia; UMC, University of Amsterdam, Department of Biomedical Engineering and bHansaBioMed Life Sciences, Tallinn, Estonia; cExosomics Siena, Siena, USA; Physics, Amsterdam, Netherlands, Amsterdam, Netherlands; dAmsterdam dExosomics, Siena, Italy UMC, University of Amsterdam, Laboratory of Experimental Clinical Chemistry, Amsterdam, Netherlands, Amsterdam, Netherlands; eMedical Cell Biophysics, University of Twente, Enschede, Netherlands Introduction: Plasma is one of the most commonly used sources of EVs since it is easy to access and is Introduction: Biomarkers in blood of cancer patients extensively used in clinical research and diagnostics. include circulating tumour cells (CTCs), tumour-edu- Isolation of pure EVs from such a complex biofluid is cated platelets (TEPs), tumour-derived extracellular hard to accomplish due to presence of many contami- vesicles (tdEVs), EV-associated miRNA (EV-miRNA), nants (lipoproteins, soluble proteins and protein aggre- and circulating cell-free DNA (ccfDNA). Because the gates) that affect downstream application. Here, we are size and density of biomarkers differ, blood is centri- exploring effects of plasma acidification on isolation, fuged to isolate or concentrate the biomarker of inter- purification and detection of EVs, as stand-alone or est. Here, we applied a model to predict the effect of combined with other pre-analytical steps: lipoprotein centrifugation on the purity of a biomarker according lipase (LPL) and low-density lipoprotein receptor to published protocols. (LDLR) treatment, in line with further purification Methods: The model is based on the Stokes equation and analytical methods. and was validated using polystyrene beads in buffer Methods: Plasma preclearing and EV isolation: differ- and plasma. Next, the model was applied to predict ential centrifugation, tangential flow filtration (TFF), the biomarker behaviour during centrifugation. The size exclusion chromatography (SEC), enzyme-coupled result was expressed as recovery of CTCs, TEPs, and affinity magnetic beads. 228 ISEV2019 ABSTRACT BOOK

Quantification and characterization of EVs: ELISA, type of EVs were measured by Nanoparticle Tracking NTA (Nanoparticle Tracking Analysis), BCA assay, Analysis at day 0, day 3, day 7 and day 14. Western Blot, total RNA extraction and quantification. Results: The concentration of micro-EVs or nano-EVs Results: Preliminary results reveal 3–5 fold increase of which were stored at 4oC or room temperature was not EV protein signal in EV-enriched SEC fractions after significantly different between days 0, 3, 7 or 14. In plasma acidification, although lipoprotein profile in contrast, the concentration of micro-EVs which were same fractions, as well as NTA counts and protein stored at −20°C was significantly reduced at both days content, stay mostly unchanged compared to normal 7(p = 0.001) and 14, compared with the concentration pH (control) samples. Additional steps aimed at of micro-EVs at day 0. The concentration of nano-EVs separation of lipoproteins from vesicles, after lipopro- stored at −20°C was significantly reduced at day 14 tein destabilization through combination of size focus- (p = 0.04), compared with the concentration of nano- ing, enzymatic digestion and ligand specific-depletion/ EVs at day 0. In addition, there was no difference in selection, are described. the modal (or mean) size of either micro- or nano-EVs Summary/Conclusion: Our experiments are addres- regardless of the storage conditions at any time point. sing the issue of plasma EV purification in attempt to Summary/Conclusion: we found that, at least in terms deplete lipoprotein particles using different preanalyti- of concentration and size, short/medium-term storage cal approaches. Acidification, along with LPL and of placental EVs at 4°C or room temperature was LDLR incubation, hold potential for lipoprotein preferable to freezing. Further work is required to removal. determine optimal storage conditions to maintain EV Funding: This research is part of TRAIN-EV project, function. funded by EU grant under the Horizon2020 Marie Sklodowska Curie Innovative Training Network (MSCA-ITN) programme. PF10.06

Only a portion of the T cell-released exosomes has a capacity to PF10.05 destruct mesenchymal tumour stroma Naohiro Seoa, Tsuguhiro Kanedaa, Junko Nakamuraa, Fumiyasu Momosea, Kazunari Akiyoshib and Hiroshi Shikua

aMie University Graduate School of Medicine, Mie, Japan; bKyoto University, The stability of placental extracellular vesicles in different short-term Kyoto, Japan storage conditions Qi Chena, Yunhui Tangb, Chunlin Sub, Michelle Wisea and Larry Chamleya Introduction: Exosomes (Exo) released from single aThe University of Auckland, Auckland, New Zealand; bFudan University of China, Shanghai, China (People’s Republic) cells have been thought to be diverse populations in membrane structures, membrane charges and bioactive Introduction: Extracellular vesicles (EVs) are attract- substances. We have reported that CD8 + T cell Exo ing considerable attention from a wide range of can deplete mesenchymal tumour stromal cells and researchers because of their signalling capacity of rele- suppress tumour invasion and metastasis (Nat. vance to health and many diseases. EVs are classified to Commun. 9: 435, 2018). In this study, we examined macro-, micro-, and nano-EVs based on their size and the diversity of CD8 + T cell Exo and destruction of carry complex cargos of RNAs, protein, DNA and mesenchymal tumour stroma. lipids that can change the behaviour of target cells. Methods: H-2Kd-restricted and mutated (m) ERK2 Given the unique characteristics of EVs and that they 136–144 peptide-specific TCR gene-transfected are challenging to isolate in large quantities for use in DUC18 mice were used in this study. DUC18 spleno- experiments especially in vivo experiments it is impor- cytes were cultured for 7 days with mERK2 peptide, tant to be able to store EVs and maintain their quality. and obtained culture supernatant (sup) was used as a In this study we began to investigate the stability of source of CD8 + T cell (CTL) Exo. Ultrafiltration (UF) human placental EVs which were extruded from first of DUC18 culture sup was performed by tangential trimester placentae. flow filtration system (KrosFlo TIFF system) using Methods: EVs were isolated from first trimester pla- mPES MidiKros Filter Modules (MWCO 500 kDa or cental explants (range from 8 to 12 weeks of gestation, 750 kDa: Spectrum) at the entrance flow rate of n = 8) and separated into micro- and nano-EVs by approximately 50 mL/min. DEAE-sepharose Fast differential centrifugation. EVs were then individually Flow (GE) was used as a carrier of cationic ion- stored in PBS at room temperature, 4°C or −20oC for exchange chromatography. DEAE-sepharose column up to 2 weeks. The concentration and the size of each (bed volume 8 cm3) was equilibrated with 10 mM JOURNAL OF EXTRACELLULAR VESICLES 229

Tris-HCl (pH7.5) containing 0.15 M NaCl. DUC18 Exo antibodies anti-CD9, anti-CD63, anti-CD81 and anti- concentrated with UF was loaded on the column, and MFG-E8. washed with TBS at over three column volumes. Exo Results: The UC method yielded a higher concentra- bound with DEAE-sepharose were eluted by linear tion of proteins in the whey than did acidification. gradient of NaCl. However, both acidification treatments yielded higher Results: By UF with 750 kDa MWCO mPES filter, amounts of EVs than UC. WB analysis revealed that CD8 + T cell Exo can be effectively concentrated acidification had partially degraded the surface marker more than 20 times without leaking. The concentrated proteins CD9 and CD81 but not CD63 or MFG-E8. CD8 + T cell Exo was adsorbed on a DEAE column Summary/Conclusion: Acidification was likely favour- and eluted with NaCl gradient of 0.15 M to 0.8 M. As a able to the removal of casein and the rapid, efficient result, the various Exo fractions could be obtained isolation of milk EVs. A higher amount of EVs were from the difference of the levels of CD9 expression, purified by acidification, but this treatment degraded CD90 expression, Granzyme B content, the Tsg101 partially some of the surface marker proteins of the content, and engulfment by mesenchymal stem cells. EVs. Our results suggest that appropriate surface mar- Interestingly, capacity of destruction of mesenchymal ker antigens should be used for evaluation of EVs from stroma was found only in Exo fraction eluted about bovine milk after acidification in the following EVs 0.25 M NaCl, indicating that a part of CD8 + T cell Exo experiments. exerts a biological function. Funding:Thisstudywaspartlysupportedbyaresearch Summary/Conclusion: We establish a novel method project for Improving Animal Disease Prevention for Exo preparation according to the negative charge. Technologies to Combat Antimicrobial Resistance 2017– Exo released from single cells are diverse populations 2021 FY of the Ministry of Agriculture, Forestry and with different physical properties, some of which exhi- Fisheries of Japan. This study was also supported in part bit biological significance. by the OGAWA Science and Technology Foundation and Funding: This work was supported by a grant from the the Morinaga Foundation for Health and Nutrition. JST CREST (JPMJCR17H2). PF10.08 PF10.07

Comparison of isolating method for obtaining extracellular vesicles from cow’s milk Mai Morozumia, Hirohisa Izumib, Muneya Tsudac, Takashi Shimizua and Evaluation of the effects of acidification on isolation of extracellular Yasuhiro Takedaa vesicles from bovine milk a b c b Md. Matiur Rahman , Kaori Shimizu , Marika Yamauchi , Ayaka Okada aMorinaga Milk Industry Co., Ltd., Zama-City, Japan; bMorinaga Milk b and Yasuo Inoshima Industry Co., Ltd., Zama-city, Japan; cMorinaga Milk Industry Co., Ltd., Zama, Japan aThe United Graduate School of Veterinary Sciences, Gifu University, Gifu, Japan; bGifu University, Gifu, Japan; cGifu University, Gifu, USA Introduction: MicroRNAs (miRNAs) are present in Introduction: Acidification has shown potential for many foods including milk, which could be involved separating casein from raw bovine milk to facilitate in various bioactivities when taken orally. Milk consists isolation and purification of extracellular vesicles mainly of two fractions, i.e. casein and whey, and most (EVs). The purpose of this study was to evaluate the of the milk miRNAs are thought to be included in effects of different acidification treatments on the yield extracellular vesicles (EVs) in whey fraction. and surface marker proteins of EVs from raw bovine Biological roles of milk miRNAs are not fully eluci- milk. dated and thus require further investigation. However, Methods: Fresh raw bulk milk was collected from procedures for isolating milk-derived EVs (M-EVs) healthy dairy cows. Casein was separated from the have not fully established. The aim of this study was raw milk by ultracentrifugation (UC), treatment with to compare methods for isolating M-EVs. hydrochloric acid, or treatment with acetic acid, fol- Methods: Aiming to minimize the contamination of lowed by filtration and preparation of the whey. The casein in whey fraction, which is the great obstacle to protein concentration of the whey was determined by determining M-EVs purity, whey fraction was sepa- spectrophotometry, and the size and concentration of rated from milk (defatted) by centrifugation only, EVs were measured by tunable resistive pulse sensing acetic acid precipitation, or EDTA precipitation analysis. Surface marker proteins of EVs were detected (n = 3). M-EVs were then isolated from each whey by western blot (WB) analysis using the primary fraction by ultracentrifugation, an exoEasy Maxi kit 230 ISEV2019 ABSTRACT BOOK

(Qiagen), a qEV kit (Izon Science) or an EVSecondL70 PF10.10 kit (GL Sciences). The number of M-EVs particles was measured using NanoSight (Malvern Instruments). Results: Acetic acid precipitation prevented casein con- ExtraSome: method for exosome isolation based on polyethylene glycol tamination to greater extents. Three combinations, Jihye Kima and Jihye Choib such as “acetic acid precipitation and qEV”, “acetic aYonsei University, Seoul, Republic of Korea; bYonsei University College of acid precipitation and EVSeocondL70” and “EDTA Medicine, Seoul, Republic of Korea precipitation and qEV” were able to collect larger numbers of total M-EVs particles than the other com- Introduction:Exosomesized30-120nmsecretedfrom binations. Among the three combinations, “EDTA pre- cells and present in blood, urine and cell media. It con- cipitation and qEV” achieved collecting the largest tains biomarkers that play important roles cell–cell com- number of M-EVs but “acetic acid precipitation and munication. Therefore, it is important to isolate exosome EVSeocondL70” was able to obtain M-EVs fractions in stable and effectively eliminate these contaminants. with high concentration. Extant method to isolate exosome include ultracentrifu- Summary/Conclusion: The combination of “EDTA gation, immunoisolation and precipitation in polymeric precipitation and qEV” is suited to collect the largest solution. Ultracentrifugation is the most conventional amount of M-EVs. The combination of “acetic acid method due to its reliability but it has the demerits of precipitation and EVSeocondL70” is capable of obtain- lengthy and laborious centrifugation, requirement for ing M-EVs fractions with high concentration. expensive equipment and low yield. Immunoisolation which uses beads conjugated with an antibody to isolate PF10.09 EVs; this method has high specificity but the EVs are hard to detach from beads, and detachment methods may reduce the functionality of the surface protein. Generating, characterizing and testing recombinant extracellular Methods: Exosomes were isolate from Fetal Bovine vesicles as biological reference material serum (FBS): After centrifugation at 2000g for An Hendrix; Edward Geeurickx; Olivier De Wever 30 min, 5 mL of FBS were combined with PEG buffer Laboratory of Experimental Cancer Research, Department of Human Structure and Repair, Ghent University, Ghent, Belgium solution, resulting in 2–30% final PEG concentration. The sample were carefully mixed and incubated at 4 C Introduction: Recent years have seen a tremendous overnight. Then the samples were pun down at 11,000 increase in the study of extracellular vesicles (EV) rpm for 1 h. The supernatant was discarded, and the geared towards biological understanding, diagnostics exosome pellet was resuspended in PBS and the num- and therapy. Concurrently EV data interpretation ber of exosomes was quantified on a Nanosight LM10 remains challenging owing to the complexity of bio- instrument. fluids and the technical variation introduced during EV Results:WeisolateexosomefromFBSusingPEGbuffer sample preparation and analysis. solution varied molecular weight (1000, 6000, 8000, Methods: To understand and mitigate these limitations 10,000, 20,000) at various concentration (2–30 w%). We we have developed a standard operating procedure to confirm the size, morphology, chemical structure and generate trackable recombinant EV (rEV). biological marker (CD63, CD81) of the exosome. As a Results: Employing complementary characterization result, we verify the optimal isolation condition of exo- methods we demonstrate that rEV are stable, commu- some for efficient system. table and share both physical and biochemical charac- Summary/Conclusion:Insummary,wehavedevel- teristics with sample EV. rEV can be accurately oped a new method for the determination of the measured using fluorescence-, RNA and protein- critical PEG value of exosome, which was proven to based technologies. Implementation of rEV reduces be very convenient and reliable. We believe that this intra-method and inter-user variability of EV sample method is highly effectiveandeconomicalandhas preparation and analysis, and improves the sensitivity great potential to be further used for the selective of EV enumeration in biofluids. separation of exosome. Summary/Conclusion: The informed use of rEV will Funding: This work was supported by a National aid method development, instrument calibration, data Research Foundation (NRF) grant funded by the normalization and routine evaluation of EV sample Korean government, Ministry of Education and preparation and analysis in various research and bio- Science Technology (NRF-2017M2A2A6A01071157, medical applications. NRF-2018R1C1B6008799). JOURNAL OF EXTRACELLULAR VESICLES 231

PF10.11=OWP3.05 (ATPS) and for comparing diagnostic ability of ATPS with conventional diagnosis. Results: With an optimized ATPS, EVs were isolated Aqueous two-phase system to isolate extracellular vesicles for with an efficiency of approximately 90%. In addition, prostate cancer diagnosis the EV-isolation time was within approximately 30 Hyunwoo Shina, Jiyoon Kima, Mee Young Kimb, Yong Hyun Parkb, Yong Goo Kimc, Ji Youl Leeb and Jaesung Parkd min, and the purity of EVs in ATPS was approximately two times better than achieved with a conventional aPOSTECH, Pohang, Republic of Korea; bDepartment of Urology, Seoul St. Mary’s Hospital, The Catholic University of Korea, Seoul, Republic of Korea, methods, ultracentrifugation and polymeric precipita- Seoul, Republic of Korea; cDepartment of Laboratory Medicine, Mary’s tion. After the ATPS isolated EVs from patients’ body Hospital, The Catholic University of Korea, Seoul, Republic of Korea, Seoul, Republic of Korea; dDepartment of Mechanical Engineering, fluid, PCR and ELISA were utilized to detect EVs POSTECH, Pohang, Republic of Korea, Pohang, Republic of Korea derived from prostate cancer cells. The expression levels of RNA and protein markers of prostate cancer Introduction: Analyzing extracellular vesicles (EVs) is were compared, and the relationship between expres- an attractive means in prostate cancer diagnosis. sion levels and clinical data was analysed. The results However, existing methods of EVs isolation have low demonstrated that diagnostic ability based on ATPS efficiency, purity and long process time, which induce was better than other conventional methods (serum low diagnostic ability. To approach the problems, we PSA and sediments). Moreover, sensitivity increased adapt a two-phase system to diagnose prostate cancer by at least 10%, and specificity was improved by at by isolating EVs from patients’ urine. Using the two- least 20% compared to conventional methods. phase system, prostate hyperplasia (BPH) patients and Summary/conclusion: High quality and quantity of prostate cancer (PCA) patients were diagnosed, and the EVs can be obtained from patients’ body fluid using diagnostic ability was compared with conventional ATPS. Using the abundant sources, which contain diagnostic methods. cancer-related proteins and genes, we can perform a Methods: Forty-two prostate cancer (PCA) patients diagnosis with high specificity and sensitivity. and 20 benign prostate hyperplasia (BPH) patients’ Therefore, ATPS offers a powerful tool for more spe- urine, plasma, saliva was collected and used for identi- cific and sensitive diagnosis. fying EVs isolation ability of aqueous two-phase system 232 ISEV2019 ABSTRACT BOOK

PF11: EV-Based Therapeutics II Chairs: Yasnouri Fujita; Xue Zou Location: Level 3, Hall A 15:30–16:30

PF11.01 ENVs can be a new source for specific cancer therapeutics. Funding: This work was supported by the Basic Therapeutic effect of plant sap-derived nanovesicles on cancer cells Science Research Program through the National a b a a Kim Kimin , Yeon Ju Hun , Yoo Hye Ju and Ruri Lee Research Foundation of Korea (NRF) funded by the aUniversity of brain education, Cheon-an, Republic of Korea; bUniversity of ministry of Education, Science and Technology (NRF- British Columbia, cheonan, Republic of Korea 2016R1C1B2013345) and Samsung Research Funding Introduction: Most of the chemical agents are toxic to Center of Samsung Electronics under Project Number both malignant and normal cells. The new anticancer SRFC-IT1701-00 agents with debilitating side effects are highly demand. Various plant sap have known to possess therapeutic PF11.02 effects like anticancer traditionally. Plant-derived nanovesicles play critical roles in intercellular and inter-species communications to transfer plant compo- Amniotic fluid stem cell extracellular vesicles derived from different species contain evolutionarily conserved microRNAs: valuable nents to mammalian cells. Plant sap-derived nanovesi- resources for regenerative medicine. cles successfully delivered contained components into Lina Antounians and Augusto Zani cells with high efficiency. The Hospital for Sick Children, Toronto, Canada Methods: We extracted plant sap-derived nanovesicles from four endemic plants: Dendropanax morbifera Introduction:Amnioticfluidstemcells(AFSCs)area (DM), Pinus densiflora (PD), Chamaecyparis obtusa population of multipotent cells that have been reported (CO) and Thuja occidentalis (TO), and investigated to hold broad regenerative potential. This regenerative endocytosis pathway of nanovesicles to malignant and capacity has been linked to a paracrine mechanism benign cells. We assessed their anti-cancer effects on mediated by microRNAs (miRNAs) contained in AFSC breast, skin, colon and melanoma cancer cells of nor- extracellular vesicles (EVs).Herein,weinvestigatedthe mal, benign and malignant origins. miRNA content of AFSC-EVs from multiple species to Results: We found that different endocytosis pathway identify commonly shared and evolutionarily conserved between malignant and benign cells, DM-derived exo- miRNAs that may be responsible for AFSC beneficial some-like nanovesicles (DM-ENVs) showed anticancer effects. effect especially on malignant breast cancer cells, while Methods: In this study, we combined data from the no cytotoxic effects were exhibited against benign cells. literature and from our laboratory. PD-ENVs showed the cytotoxic effect on malignant Literature review: Using a defined strategy, we con- skin cancer cells but not on Fibroblasts. TO-ENVs ducted a systematic review searching for studies report- and CO-ENVs showed no cytotoxic effect on most ing on AFSC-EVs and we extracted available miRNA malignant cancer cells. We also found the synergistic sequencing data. effect of the DMNVs and PDNVs on malignant breast Our study: Rat AFSCs were subjected to exosome- and skin cancer cells. We identified that combination depleted FBS in minimal essential media for 18 h. of DM-ENVs and PD-ENVs make enhancement in the Conditioned medium was collected, cleared of cells cytotoxicity against malignant cells than normal and and debris, filtered through a 0.22 µm syringe filter, benign cells. and ultracentrifuged for 14 h at 100,000g. EVs were Summary/Conclusion: We confirm that DM-ENVs assessed for size (nanoparticle tracking analysis), mor- have anticancer effects against malignant breast and phology (transmission electron microscopy) and skin cancer cells than benign breast and skin cancer expression of canonical protein markers CD63, cells. We also found synergistic effects according to the Hsp70, Flo-1 and TSG101 (Western). AFSC-EV RNA combination of DM-ENVs and PD-ENVs on malig- was isolated using SeraMir, constructed into libraries nant cells. These results provide that plant sap-derived (CleanTag Small RNA) and sequenced on NextSeq JOURNAL OF EXTRACELLULAR VESICLES 233

High Output single-end sequencing run. TargetScan and the size distribution were determined by dynamic light was used to identify species-specific and evolutionarily scattering (DLS), nanoparticle tracking analysis (NTA) conserved miRNA using seed sequences across all three and transmission electron microscopy (TEM). EVs func- species. Pathway enrichment analysis was conducted tional activity was assessed for the expression of tissue using miR-path. factor and phosphatidylserine (PS) activity. In addition, Results: Overall, data on AFSC-EVs from three species the HPLs were tested for their thrombin and plasmin (n = 2 human, n = 2 mouse, n = 1 rat) were included. activity, anti-oxidative property and thrombin generation Four miRNAs (miR-21, miR-24, miR-100 and miR- capacity 145) were found in AFSC-EVs from all three species Results: Abundant number of EVs (1010 ~1012/mL) and were reported to exert beneficial effects on lung, was found in all HPLs fractions. DLS analysis showed muscle and kidney regeneration. These miRNAs were that isolated EVs in PPL, HPPL, SCPL and HSCPL enriched in signalling pathways that involve TGF-β have size distribution approximately ranging as follows: (p = 0.004) and TNF-α (p = 0.03) and the maintenance 100 ~ 250 nm; 45 ~ 210 nm; 145 ~ 230 nm and of stem cell pluripotency (p = 0.0001). We also 55 ~ 180 nm, respectively, these data being confirmed observed species-specific miRNAs (n = 15 human, by NTA and TEM. None of the HPLs were found to n = 6 mouse, n = 6 rat) contained in AFSC-EVs. have detectable TF-expressing EVs but some significant Summary/Conclusion: AFSC-EVs isolated from differ- differences in PS-expressing EVs, as well as thrombin, ent species have some miRNAs that are shared and plasmin and anti-oxidative activity were found, possi- evolutionarily conserved. These miRNAs may have a bly linked to their EVs composition specific role in the regenerative effects that AFSC-EVs Summary/Conclusion: This study establishes that all exert in different diseases. HPLs evaluated have a high content of EVs. Differences Funding: CIHR-SickKids Foundation in functional activity were also unveiled supporting the need for further studies of the physiological functions of HPL-derived EVs in cell-based and preclinical ani- PF11.03 mal models

Extra-cellular vesicles in human platelet lysates for clinical use and human cell in vitro propagation PF11.04 Liling Delilaa, Yu-Wen Wua, Ming-Li Choub, David Devosc and Thierry Burnoufd a College of Biomedical Engineering, Taipei Medical University, Taipei, EV-mediated delivery of enzymatically fabricated size-controllable Taiwan (Republic of China); bCentre de Recherche Saint-Antoine (CRSA) – c functional DNA/RNA microstructures for therapeutic applications INSERM UMRS 938, Taoyuan, Taiwan (Republic of China); Pharmacologie Hyejin Kim, Dajeong Kim and Jong Bum Lee Médicale & Neurologie, University of Lille, University hospital center, INSERM U-1171, Lille, France; dCollege of Biomedical Engineering, Taipei Department of Chemical Engineering, University of Seoul, Seoul, Republic of Medical University, Taipei City, Taiwan (Republic of China) Korea

Introduction: Human platelet lysates (HPLs) are Introduction: Nucleic acids have been widely investi- increasingly used in regenerative medicine and cell ther- gated as a generic material with the advantage of their apy. The functional activity of HPLs to regenerate tissues wide range of biological functions, predictable molecu- in vivo and to expand cells in vitro is believed to be due to lar structure, programmability and reversibility of base- their richness in a plethora of growth factors. However, paring. Here, the following enzymatically fabricated little is known about the presence and content of extra- multiscale functional nucleic acid-based structures are cellular vesicles (EVs) in HPLs, as well as their potential introduced: bubbled RNA-based cargoes (BRCs), mes- functional activity, biological impact and involvement in senger RNA nanoparticles (mRNA-NPs) and CpG tissue repair. We aim to characterize the number, the size incorporated DNA microparticles (CpG DNA-MPs). and the biological functions of EVs present in HPLs in Methods:PrimerDNAandlonglinearDNAbearing order to develop dedicated preparations best suitable for complementary sequences to functional DNA or RNA specific clinical applications were prepared for the synthesis of circular DNA for Methods:Clinicalgradeofplateletconcentrateswerepro- the BRCs and CpG DNA-MPs. For mRNA-NPs, plas- cessed into different fractions of HPLs: platelet pellet lysate mid DNA bearing T7 promoter region, ribosome (PPL); heat-treated PPL ; serum-converted platelet lysate binding site and ORF was prepared. T7 RNA poly- (SCPL) using calcium chloride/glass bead treatment; and merase or Phi29 DNA polymerase were used to heat-treated SCPL (HSCPL). EVs in HPLs were isolated induce self-assembly of DNA or RNA-based struc- using size exclusion chromatography column. The number tures, respectively. 234 ISEV2019 ABSTRACT BOOK

Results: Adaptation and modification of rolling circle an innovative platform for the efficient production of replication (RCR) approach has enabled generating extracellular vesicles (EVs) from a renewable biosource, these functional structures with desired sizes for enabling their exploitation as tailor-made products in demonstration of regulation of biological processes. the fields of nanomedicine, cosmetics and nutraceutics The BRCs were designed to down-regulate the target (https://ves4us.eu). A core aspect of the project is to gene upon introducing to cells by bearing specific focus on an identified natural source to constitute a cleavage sites for Dicer to generate functional siRNAs. cost-effective and sustainable source of EVs. The pro- Furthermore, manipulating template DNA to polymer- ject will run for the next three years with six organiza- ase ratio in the modified RCR reaction enabled size- tions from six European countries. modulable synthesis of BRCs. We also adapted plasmid Methods: In the first phase, the focus will be the selec- DNA as template DNA in RCR to fabricate mRNA- tion of the natural source and the optimization of NPs for up-regulation of target gene expression. While culture condition at pre-industrial scale. Then, the iso- the two studies were geared towards regulation of gene lation and physiochemical characterization of the EVs expression, we also demonstrated the fabrication of will be realized. In the second phase, the functionaliza- CpG DMA-MPs for boosting immune response by tion and load of the EVs will be approached. Finally, improving efficiency of antigen presentation in the biological activity of the EVs will be explored both macrophages. in vitro and in vivo. Summary/Conclusion:Sincethepre-assembledstruc- Results: The selection of the best EV-producing nat- tures have high cargo capacity without any condensation, ural source strain/s is ongoing and it will get to the loading extracellular vesicles (EVs) with the DNA/RNA- production of the EVs needed to develop the natural based nanostructures would greatly enhance therapeutic nanocarriers. Before that happens, it exists the need of potential of nucleic acid-based therapeutics. Together with applying good research practices that include personnel the nature of EVs being safe and effective delivery agent, training on Standard Operating Procedures to control we envision that the exploitation of EVs in the field of major experimental activities for harvesting, manipu- nucleic acid engineering would provide a way of next- lating, storing, characterizing and treating EVs, as well generation gene therapy. as for key related activities. Funding:ThisresearchwassupportedbytheBio& Summary/Conclusion: Safe, efficient and specific Medical Technology Development Program of the nano-delivery systems are essential to current thera- National Research Foundation of Korea (NRF) funded by peutic medicine, cosmetic and nutraceutics sectors. the Korean Government (NRF-2016M3A9C6917402). The ability to optimize the bioavailability, stability and targeted cellular uptake of a bioactive molecule while mitigating toxicity, immunogenicity and off-tar- PF11.05 get/side effects is of the utmost priority. VES4US aims at creating a fundamentally new bioprocessing approach to generate and functionalize EVs from a VES4US: Extracellular vesicles from a natural source for tailor-made nanomaterials renewable biological source. Daniele Romancinoa, Mauro Mannob, Antonella Cusimanoa, Sabrina Funding: This project has received funding from the Picciottoa, Samuele Raccostab, Vincenzo Martoranab, Rosina Notob, Rita b c d e European Union’s Horizon 2020 research and innova- Carrotta , Elia Di Schiavi , Giovanna L. Liguori , Annamaria Kisslinger , Katharina Landfesterf, Blanca Rodriguezg, Svenja Morsbachh, Ales Iglici, tion programme under grant agreement No 801338. Veronika Iglici, Laura Corcuerah, Paolo Arosioj, Gabriella Pocsfalvik, Nicolas Touzetl and Antonella Bongiovannia aNational Research Council (CNR), Institute of Biomedicine and Molecular PF11.07 Immunology (IBIM), Palermo, Italy; bInstitute of Biophysics, Palermo, Italy; cNational Research Council of Italy (CNR) – Institute of Biosciences and BioResources, IBBR, Napoli, Italy; dNational Research Council of Italy (CNR) – e Therapeutic effects of mesenchymal stem cell exosomes in Institute of Genetics and Biophysics, Palermo, Italy; National Research Council – myocardial ischemia/reperfusion injury in a porcine model Institute of Experimental Endocrinology and Oncology (IEOS), Napoli, Italy; a b c d f g Max Silvis , Vince de Hoog , Sai Kiang Lim , Dominique de Kleijn and Leo Max-Planck Institute for PolymerResearch,Mainz,Germany; Zabala Timmerse Innovation Consulting, Navarra, Spain; hFaculty of Health Sciences, University of Ljubljana, Ljubljana, Slovenia; iETH Zurich Institute for Chemical and aUMC Utrecht, Cardiology, Nijmegen, Netherlands; bUMC Utrecht, Utrecht, Bioengineering, Zurich, Switzerland; jExtracellular Vesicles and Mass USA; cInstitute of Medical Biology, Agency for Science, Technology and Spetrometry Group, Institute of Bioscience and BioResosrces (IBBR) – CNR, Research, Singapore, Singapore, Singapore; dUMCU, Utrecht, Netherlands; Naples, Italy; kInstitute of Technology Sligo – ITSligo, Sligo, Ireland eSt. Antonius Nieuwegein, Utrecht, USA

Introduction: VES4US is a new European project Introduction:Afteranacutemyocardialinfarction, funded by the Horizon 2020-Future and Emerging restoration of the blood flow to the ischemic myocardium Technology Open programme, which aims to develop (reperfusion) is the standard and most effective treatment JOURNAL OF EXTRACELLULAR VESICLES 235 for reducing the infarct size and improving clinical out- Introduction: Adult stem cell therapy is an excellent come. The process of reperfusion, however, can induce treatment for a variety of ischemic diseases including injury as well. Studies in animal models suggest that this ischemic heart disease, limb ischemia and stroke. “reperfusion injury” accounts for up to 50% of the final size Mesenchymal stem cells (MSCs) exert their therapeutic of a myocardial infarct and in these models a number of capability via extracellular vesicles (EVs) in infarcted strategies have been shown to ameliorate lethal reperfusion tissue. Therefore, we hypothesized that MSC-derived injury. EVs (MSC-EVs) possess the equivalent sets of thera- Our laboratory previously showed that human ESC- peutic molecules to MSCs. derived mesenchymal stem cell-derived exosomes med- Methods: EVs were isolated from 3D cultures of iate cardio protection during myocardial ischemia/ Wharton’s Jelly MSC (WJ-MSC) using tangential flow reperfusion injury in a mouse model. filtration system and characterized by nanoparticle However, the therapeutic efficacy of these exosomes tracking analysis. We investigated the therapeutic effi- in a large-animal model remain to be addressed. cacy of WJ-MSC derived EVs (does tested: 0.3 ×1010, In the current study, we therefore hypothesized that 1.5 ×1010, 3 ×1010 EVs/rat) in a rat stroke model. infusion of MSC exosomes reduces infarct size and Results: The infarct size did not decrease, but the preserves cardiac function in a large-animal model. ladder walking test showed a greater improvement in Methods: Thirty female landrace pigs were subjected to behaviour in all EV groups than in the control group. 60 min transluminal balloon occlusion and treated with Immunohistochemical analysis was performed using a combination of intravenous (1 mg) and intracoronary ki-67 (proliferating cells), DCX (immature progenitor (1 mg) MSC exosomes (n = 15) or placebo (n = 15) in a neurons) and collagen IV (angiogenesis) of ipsilateral. randomized, blinded fashion. At baseline and prior to EVs groups showed significantly increased co-expres- termination 3D transesophageal echocardiography was sion of ki-67 and DCX in the subventricular zone. performed to assess cardiac function. Infarct size will Enhanced expression of collagen IV in the ischemic be calculated as the percentage of the area at risk using boarder zone was more found in the EV groups than Evans blue/TTC double staining. in the control group. Diffusion tensor imaging was Results: This is an ongoing study and no major results used to examine the regeneration of nerve fibre bun- are available yet. However, pilot studies in an open dles, and nerve fibre bundles were also increased in the chest LCx ligation model revealed that 1 mg of MSC EV groups. exosomes (200 μg i.v. 5 min before reperfusion and 800 Summary/Conclusion: Our study shows that WJ-MSC μg intracoronary immediately after reperfusion) derived EVs from 3D culture system promotes the resulted in a significant reduction of infarct size com- neurogenesis, angiogenesis and recovery of nerve fibre pared to saline injection (infarct size as a percentage of bundles in rat stroke models. These results suggest that the area at risk 22% vs 46%, p = 0.01). an effective MSC-derived EV can be used ideally for Summary/Conclusion: In this study, we will investi- the treatment of stroke. gate whether MSC exosomes are capable of reducing Funding: This study was supported by a grant from the infarct size and preserving cardiac function in a highly Korean Healthcare Technology R&D Project, Ministry translational large-animal model. of Health & Welfare (HI17C1256) and Basic Science The main goal is to establish a clinically applicable Research Program, the Ministry of Science, ICT and protocol for MSC exosomes to be used as a therapeutic Future Planning (2018M3A9H1023675). tool for the treatment of acute MI. Funding: Dutch Heart Foundation PF11.10

PF11.08 Exosomes from human urine-derived stem cells promote neurogenesis via histone deacetylase6 regulation in ischemic stroke Xiaozheng Linga, Zhang Guoweia, Qingwei Zhua, Hu Guowena, Yang Wangb a The therapeutic potential of MSC-derived extracellular vesicles for and Zhifeng Deng stroke a ’ a b c a Shanghai Jiao Tong University Affliated Sixth People s Hospital, Shanghai, Ji Hee Sung , Eun Kyoung Shin , Jeong Pyo Son , Yeon Hee Cho , Dong Hee ’ b d b b e f China (People s Republic); Shanghai Jiao Tong University Affiliated Sixth Kim , Mi Jeong Oh , Eun Hee Kim , Jae Min Cha , Oh Young Bang People’s Hospital, Shanghai, China (People’s Republic) aSamsung medical center, seoul, Republic of Korea; bSamsung medical center, Seoul, Republic of Korea; cSungkyunkwan University, Seoul, Republic of Korea; Introduction: Human urine-derived stem cells (USCs) dSungkyunkwan University, seoul, Republic of Korea; eDepartment of Mechatronics, College of Engineering, Incheon National University, Incheon, are receiving much more attention in tissue regenera- Republic of Korea; fSamsung medical center, Seoul, Republic of Korea tion and injury repair. Exosomes derived from USCs 236 ISEV2019 ABSTRACT BOOK

(USCs-Exo) have recently been suggested to mediate potential therapeutic resources for PD, NSC- the restorative effects of USCs. However, whether secreted extracellular vesicles (EVs) including exo- USCs-Exo play a protective role in stroke remains somes are key mediators of positive paracrine unknown. effects. Direct evidence for neuronal protective Methods: Therapeutic effect of USCs-Exo in vivo was effects of EVs is essential for developing new PD evaluated by cerebral infarction and neurological beha- therapeutics. viour. The proliferation and differentiation of neural Methods: To trace EV movement, a lentivirus contain- stem cells (NSCs) was determined by double immu- ing Palm-tandem dimer tdTomato (Palm-td) was noﬂuorescent staining. The viability, apoptosis, prolif- transduced into F3 NSCs. EVs isolated from Palm-td- eration, and differentiation of NSCs subjected to infected F3 cells showed high tdTomato fluorescence oxygen glucose deprivation/reoxygenation (OGD/R) intensity enough to visualize their functional actions were assessed, respectively. The protein expression such as secretion, migration and engulfment between and activity of HDAC6, and the expression levels of cells. We found that pretreatment with EVs dramati- miRNAs both in vivo and in vitro were assessed to cally prevented 6-OHDA-induced toxicity by reducing detect the possible mechanism. intracellular reactive oxygen species (ROS), percentage Results: We found that intravenous injection of USCs- of apoptotic cells and caspase-3/7 activity. Exo reduced brain infarct volume and improved func- Results: These results indicate that NSC-derived EVs tional recovery by enhancing the proliferation and have neuroprotective effects against the cell damage, differentiation of NSCs in ischemic rats. The in vitro possibly through antioxidant and anti-apoptotic action. results suggested that USCs-Exo increase viability, Specifically, F3-derived EVs effectively prevents 6- reduce apoptosis, and promote the proliferation and OHDA-induced the production of ROS, NSC-EVs neuronal differentiation of NSCs after OGD/R. We that inhibit ROS production might be applied as an further found that miR-206 contained in USCs-Exo is important therapeutic agent for neuroprotection. EV- associated with the therapeutic efficacy for neurogen- mediated neuroprotection likely acts by inhibiting the esis through the inhibition of histone deacetylase6 generation of caspase-3/7, leading to reduce apoptosis (HDAC6). induction caused by 6-OHDA neurotoxicity. Summary/Conclusion: Our study demonstrates that Summary/Conclusion: In summary, this study demon- USCs-Exo can improve neurological function recovery strates that NSC-derived EVs protected dopaminergic by promoting neurogenesis, which is attributed to cells from oxidative insults. While 6-OHDA-induced miR-206-mediated HDAC6 inhibition. Our research cell death in SH-SY5Y cells occurs by the generation of suggests that the use of USCs-Exo represents a novel ROS, the neurotoxin-mediated cell death was sup- therapeutic strategy for stroke recovery. pressed by F3-derived EVs via their protective effects Funding: This work was funded by National Natural on ROS-induced cell damage. We therefore expect that Science Foundation of China (Numbers 81671209, further investigations into the therapeutic applications 81471243 and 81472152). of NSC-derived EVs will reveal additional advantages for EV-based PD therapies in comparison to cell transplantation. PF11.11 PF11.12 Protective effect of extracellular vesicles released from the neural stem cells on 6-hydroxydopamine induced pathological condition of Parkinson’s disease Eun Ji Leea, Dowon Hwangb and Dong Soo Leea The function of extracellular vesicles secreted by human-induced pluripotent stem cell-derived mesenchymal stem cells on a cellular a Department of Nuclear Medicine, Seoul National University Hospital, Seoul, ischemic stroke model b Republic of Korea; Seoul National University Hospital, Seoul, Republic of Gang Lu, Man Sze Wong, Xian Wei Su, Rui Can Cao, Hoi Hung Cheung and Korea Wai Yee Chan CUHK-SDU joint laboratory on reproductive genetics, School of Biomedical Introduction:Parkinson’sdisease(PD)isaneuro- Sciences, The Chinese University of Hong Kong, Hong Kong, Hong Kong degenerative disease characterized by bradykinesia, resting tremors and postural instability. A key Introduction:Strokeisthesecondleadingcauseof symptom of PD is the loss of the nigral dopaminer- death and the primary cause of long-term disability, gic neurons and subsequent dopamine deficit in the but yet lack of effective treatment. Studies suggested brain. However, the precise mechanism is still the transplantation of mesenchymal stem cells unknown. While neural stem cells (NSCs) are (MSCs) improved recovery from stroke in animal JOURNAL OF EXTRACELLULAR VESICLES 237 models, similar therapeutic effect was found with aimed to load EVs with Cre recombinase (Cre) as a model the injection of MSCs medium. MSCs medium was protein cargo and determine whether functional delivery found enriched with extracellular vesicles, hence to cells could be improved by using uptake-enhancing leads to the focus on utilizing extracellular vesicles compounds. to treat neurological diseases, due to the evidence Methods:Expi293FcelllinewasusedforisolatingCre that extracellular vesicles are able to penetrate the loaded EVs by differential centrifugation after trans- blood–brain barrier. This project aims to develop a fecting releasing cells with constructs for protein product with enriched extracellular vesicles and to expression. EVs were then analysed by nanoparticle evaluate its therapeutic efficacy in ischemic stroke. tracking analysis, western blotting, RT-qPCR and Methods:MSCs,withsamepassagenumber,werederived cryo-electron microscopy including detergent and from human-induced pluripotent stem cells-MSCs for the nuclease digestion controls. Uptake of Cre loaded isolation of extracellular vesicles. The derived MSCs were EVs was assessed using modified Hek293T cells then confirmed by the adherence to plastic, multipotent expressing a fluorescent reporter cassette consisting differentiation potential and surface antigen expressions. of LoxP – GFP – LoxP – RFP. Three methods (ultracentrifugation, ultrafiltration and Results: Endosomal escape enhancers chloroquine and polyethylene glycol) were used to extract extracellular Unc10217939 increased TATcre functional delivery by vesicles, which were further analysed by the expression of ≥50%. CreFRB protein was loaded into EVs by rapalog- surface proteins, electron microscopy, ribosomal RNA induced dimerisation to CD81FKBP. Cells treated with detection and oxygen–glucose deprivation (OGD) in 20 µg/mL CreFRB loaded EVs showed functional Cre vitro stroke model. activity only in the presence of 25 µM chloroquine or Results:DifferentiatedMSCsexhibitedadherenceto 2 µM unc10217939. plastic, ability to differentiate into osteoblasts, adipocytes Summary/Conclusion: Passively loaded protein and and chondroblasts, and ≥95% population expressed mRNA was effectively delivered to recipient Hek293T CD105, CD73 and CD90, and lack of CD45, CD34 fluorescent Cre reporter cells in the presence of endo- and HLA class II. The isolated extracellular vesicles somal escape enhancing compounds. This finding expressed CD9, CD63 and CD81, with the size between shows that endosomal escape enhancing compounds 30 and 200 nm and contained RNA with a peak may have a place in the clinic to improve delivery between 25 and 200 nucleotides. Products from ultrafil- efficiency of nanoparticle-based therapies. tration were found to increase cell viability in vitro stroke model most significantly. Summary/Conclusion:Extracellularvesicleswere PF11.14=OWP1.02 able to increase the viability of neuronal cell (HT22) in oxygen–glucose deprivation in vitro stroke model, indicating the potential use of extra- MSC exosome works through a multifaceted mechanism of action in joint repair cellular vesicles injection as an alternative therapy Shipin Zhanga, Yedan Wangb, Francis Keng Lin Wongc, Ming Wangb, Ruenn for ischemic stroke. Chai Laid, James Hoi Po Huib, Sai Kiang Limd and Wei Seong Toha Funding: Innovation and Technology Fund ITS-053– aFaculty of Dentistry, National University of Singapore, Singapore, Singapore, b 17FX, the Government of the Hong Kong Special Singapore; Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore; Administrative Region. cDepartment of Orthopaedic Surgery, Sengkang General Hospital, Singhealth, Singapore, Singapore, Singapore; dInstitute of Medical Biology, Agency for Science, Technology and Research, Singapore, Singapore, PF11.13 Singapore Introduction: Mesenchymal stem cell (MSC) exosome Endosomal escape enhancing compounds facilitate functional is increasingly accepted as the principal agent that delivery of EV cargo underpins the therapeutic efficacy of MSC in tissue Nikki Heatha, Xabier Osteikoetxeab, Taiana Mia de Oliveirac, Elisa Lázaro- Ibáñezd, Olga Shatnyevae, Christina Schindlerf, Natalie Tiguef, Lorenz Mayrg, repair. Here, we aim to elucidate the mechanism of Niek Dekkerh, Ross Overmanb and Rick Daviesb action of MSC exosome in immunocompetent rat aAstraZeneca, Vancouver, Canada; bAstraZeneca, Macclesfield, UK; cAstraZeneca, models of osteochondral defect and osteoarthri- Cambridge, UK; dAstraZeneca, Molndal, Sweden; eAstraZeneca, Molndal, tis (OA). Sweden; fMedImmune, Cambridge, UK; gAstraZeneca, Cambridge, USA; hAstraZeneca, Mölndal, Sweden Methods: Exosomes were purified from conditioned medium of human MSCs by size fractionation. Introduction:Extracellularvesicles(EVs)aredesirable Osteochondral defect creation or anterior cruciate liga- carriers and delivery vehicles for therapeutic cargoes. We ment transection to induce OA were performed in 72 238 ISEV2019 ABSTRACT BOOK adult rats. Thereafter, weekly 100 µl intra-articular versus host disease. There is novel work exploring the injections of 100 µg exosome or PBS vehicle were utility of haploidentical cellular therapy infusion in given. Analysis included weight distribution, histology, order to incite purposeful recipient immune response immunohistochemistry and cytokine assay. Cellular and subsequent cytokine storm to treat refractory assays using chondrocytes were performed to deter- AML. Our group has demonstrated the healing poten- mine the exosome-activated cellular processes and sig- tial of bone marrow-derived mesenchymal stem cell nalling pathways. extracellular vesicles (MSC-EVs) across multiple dis- Results: We observed that exosome-mediated repair of ease states, most recently demonstrating the pro-apop- osteochondral defects was characterized by increased toic signalling imparted by these nanoparticles on cellular infiltration and proliferation, enhanced matrix nascent leukemic cells in vivo; as well as the potentiat- synthesis, together with a regenerative M2 macrophage ing effects of MSC-EVs when used as an adjunct to phenotype and a reduction in pro-inflammatory cyto- standard cytarabine chemotherapy. We have also kines IL-1β and TNF-α. In OA joints, MSC exosome shown the protective role of HMSC EV on radiated mediated an early suppression of pain and degenera- BM and stem cell recovery. tion with reduced inflammation, followed by sustained Methods: Kasumi AML cells lines were seeded with proliferation and matrix restoration that led to cartilage MSC-derived EVs. Vesicles were isolated using an and subchondral bone regeneration. Using chondro- established differential centrifugation technique, and cyte cultures, we could attribute some of these cellular were co-cultured with Kasumi cells for various time activities during exosome-mediated joint repair to exo- points. To study cellular viability, we used a fluores- somal CD73-mediated adenosine activation of AKT cence-based method for quantifying viable cells. and ERK signalling. These effects were partially abro- We also explored various modes of death EVs may gated by wortmannin or U0126, which inhibited AKT illicit via a tri-dye Abcam assay designed to simulta- and ERK phosphorylation, respectively. The role of neously monitor apoptotic, necrotic and healthy cells. exosomal CD73 was confirmed using CD73 inhibitor Both assays were used to measure viability and apop- and theophylline that showed inhibition of exosome- tosis in similar experiments employing cytarabine induced AKT and ERK phosphorylation. Results: AML cell proliferation decreased after 1 Summary/conclusion: Our observations suggest that −6 days of co-culture with hMSC-derived EVs. MSC exosome works through a multifaceted MoA Apoptosis is the primary mode of death induced. that involved multiple cellular processes to restore AML cell Proliferation Decreased synergistic after 1– joint homeostasis and promote regeneration. 6 days of co-culture with hMSC-derived EVs ± Funding: National Medical Research Council Cytarabine. Singapore (NMRC/CNIG/1168/2017 and NMRC/ Summary/conclusion: MSCs inhibits the proliferation CIRG/1480/2017). of the AML cell line in vitro and work synergistically with cytarabine chemotherapy to promote apoptotic PF11.15=OWP1.04 death in AML cell lines. Our prior work has shown that MSC-EVs can abate the effects of toxic chemo/ radiation and serve to protect stem cell allowing for Exosome-mediated enhancement of cellular therapy in acute quicker recover in cell blood counts. myelogenous leukemia (AML) Based on the innate ability of MSC-EV to directly alter Theo Borgovana, Peter Quesenberryb, Mike Deltatto; Sicheng Wenb, Mark Doonerb the cellular machinery of abnormal leukemic cell and aBrown University Department of Hematology Oncology; Rhode Island of nascent immune cells our corollary hypothesis is Hospital, Pawtucket, USA; bBrown University Department of Hematology that BM-derived MSC-EVs may serve as suitable alter- Oncology; Rhode Island Hospital, Providence, USA native to conditioning chemo/radiation in the AML setting and will enhance the effects seen by cellular Introduction: Of the acute myelogenous leukemia therapy infusion. (AML) patients able to tolerate curative therapy with Funding: t32 chemotherapy and stem cell transplant, many are challenged by treatment related toxicities as well as graft JOURNAL OF EXTRACELLULAR VESICLES 239

PF12: Advances in EV Cargo Profiling Chairs: Leonid Margolis; Yutaka Naito Location: Level 3, Hall A 15:30–16:30

PF12.01 candidates (miR-381-3p, −889-3p, −323a-3p) were found to be upregulated in both TGFBR2-proficient EVs and parental cells. Tumor driver TGFBR2-dependent microRNA profiles in colorectal cancer cells and their EVs Summary/Conclusion: Our results emphasize a broad Fabia Frickea, Veronika Mussackb, Dominik Buschmannb, Michael Pfafflc, overlap of miRNAs between EVs and their parental Jürgen Kopitzd and Johannes Gebertd cells but also highlight the impact of the recurrent a Department Applied Tumor Biology, University Hospital Heidelberg, MSI tumour driver TGFBR2 on aberrant miRNA sig- German Cancer Research Center (DKFZ), Heidelberg, Germany; bTUM School of Life Sciences Weihenstephan, Division of Animal Physiology and natures in MSI cancer cells and their small EVs. Immunology, Freising, Germany; cAnimal Physiology and Immunology, Funding: This work was supported by intramural School of Life Sciences Weihenstephan, Technical University of Munich, Freising, Germany; dApplied Tumor Biology, University Hospital funding from the Technical University Munich (MP) Heidelberg, Heidelberg, Germany and the University Hospital Heidelberg (JG, JK).

Introduction: Microsatellite unstable (MSI) colorectal cancers accumulate frameshift mutations at short repe- PF12.02 titive DNA sequences (microsatellites). MSI-specific mutation patterns in tumour driver genes such as Transforming Beta Receptor Type 2 (TGFBR2) were Orthologous grouping and comparison of prokaryotic and eukaryotic EV proteomes found to be reflected in the cargo of MSI cell line- Tae-Young Roha, Seokjin Hamb, Dae-Kyum Kimc, Jaewook Leec and Yong derived extracellular vesicles (EVs). In previous work, Song Ghod we have shown that TGFBR2 reprograms the protein aDiv. of IBB, Department of Life Sciences, Pohang University of Science and b content of MSI tumour cells and small EVs derived Technology (POSTECH), Pohang, Republic of Korea; Department of Life Sciences, Pohang University of Science and Technology (POSTECH), thereof. Here, we report on TGFBR2-dependent altera- Pohang, Republic of Korea; cDepartment of Life Sciences, Pohang tions of miRNA expression in small EVs and their University of Science and Technology (POSTECH), Pohang, Republic of Korea; dDepartment of Life Sciences, Pohang University of Science and corresponding parental MSI tumour cells. Technology, Pohang, Republic of Korea Methods: To identify TGFBR2-regulated miRNAs in an isogenic background, the established doxycycline Introduction: Most prokaryotic and eukaryotic cells (dox)-inducible MSI model HCT116-TGFBR2 was secrete extracellular vesicles (EVs) with bioactive mole- used. RNA was isolated from four biological replicates cules, including proteins and nucleic acid. Protein car- of TGFBR2-proficient (+dox) and TGFBR2-deficient gos important for EV biogenesis and/or biological (-dox) cells and their EVs. EVs were isolated by differ- functions can be found using proteomic analyses. ential centrifugation, ultrafiltration, and precipitation Methods:Toanalysethesimilarityanddifference and characterized by electron microscopy, Western between prokaryotic and eukaryotic EVs, EV pro- blot, and nanoparticle tracking. RNA quality and con- tein databases was obtained from EVPedia (http:// centration were determined by capillary electrophor- evpedia.info), regardless of EV sources and analys- esis. cDNA libraries for small RNA fractions were ing platforms. EV proteins were catalogued into generated and RNA sequencing was performed. orthologous groups and annotated these groups TGFBR2-regulated miRNA expression was assessed using eggNOG database. Gene set enrichment ana- by DESeq2 and validated by RT-qPCR. lysis (GSEA) was employed to determine how much Results: From 471 identified miRNAs, the majority the orthologous groups are enriched in EVs of pro- (n = 263) was unaffected by TGFBR2 expression and karyotic or eukaryotic species. The core network of shared by small EVs and parental MSI cells. In addi- prokaryotic and eukaryotic EV orthologous groups tion, we detected specific miRNAs exclusively present were explored by Generalized HotNet analysis. Only in EVs from TGFBR2-deficient (n = 4) or TGFBR2- hot clusters with more than four orthologous proficient (n = 14) MSI cells. Differential expression groups were visualized by Cytoscape. analysis revealed TGFBR2-regulated miRNAs in EVs Results: A total of 6634 proteomic orthologous groups (n = 10) and MSI donor cells (n = 15). Three were identified from 33 prokaryotes and 22 and 240 ISEV2019 ABSTRACT BOOK separated into two distinct groups. Each orthologous trypsin treatment, we classified the vesicular proteins group was annotated with gene symbols, GO terms, as into 363 candidate real-vesicular proteins and 151 con- well as functional interactions. Frequently detected taminated extravesicular proteins. Protein interaction orthologous groups were related with mainly mem- network analyses showed that candidate real-vesicular brane-associated compartments. The GSEA analysis proteome is composed of proteins derived from plasma showed some common and specific proteins to prokar- membrane (46.8%), cytosol (36.6%), cytoskeleton yote or eukaryote in the categories of biological process (8.0%) and extracellular region (2.5%). On the other and cellular component. The correlation network ana- hand, most of the identified proteins derived from lysis clearly provided a domain-specific terms such as other cellular organelles including nucleus, Golgi appa- intracellular organelle cilium, cytoplasm ribosome, and ratus, endoplasmic reticulum and mitochondria were ribosome proteasome complex for eukaryotes, and considered as the contaminated extravesicular proteins. cytoplasm envelope, extracellular exosome and cell In addition, protein complexes, including ribosome outer membrane for prokayrotes. and T-complex proteins, were classified as the con- Summary/Conclusion: Our comprehensive EV pro- taminated extravesicular proteins. teome analysis could provide a functional modules Summary/Conclusion: Taken together, this trypsin related with characteristic biological mechanisms in treatment to EVs with large-scale quantitative proteo- prokayrotes and eukaryotes. This analytical strategy mics allows the evaluation of the real-vesicular proteins will also provide a new integrative method to investi- in isolated EVs as well as the sub-vesicular localization gate EV proteins and propose an evolutionary protein of identified proteins. Therefore, our results provide repertoire of EV. the applicable approach to identify the reliable diagnostic markers of EVs. PF12.03 PF12.04

Quantitative proteomic analysis of trypsin-treated extracellular vesicles to evaluate the real-vesicular proteins a a a a Characterization of sweat extracellular vesicles Gyeongyun Go , Dong-Sic Choi , Dae-Kyum Kim , Jaewook Lee and Yong a a b c b Genevieve Bart , Anatoliy Samoylenko , Daniel Fischer , Anna Kaisanlahti , Song Gho Artem Zhyvolozhnyia, Marko Suokasd, Prateek Singha, Justus Reunanenc d aDepartment of Life Sciences, Pohang University of Science and Technology and Seppo Vainio b (POSTECH), Pohang, Republic of Korea; Department of Life Sciences, aUniversity of Oulu, Biocenter Oulu, Laboratory of developmental Biology, Pohang University of Science and Technology, Pohang, Republic of Korea Oulu, Finland; bNatural Resources Institute Finland (Luke), Animal Genomics, Jokioinen, Finland; cUniversity of Oulu, Biocenter Oulu, Cancer Introduction: Extracellular vesicles (EVs) are nano- and Translational Medicine Research Unit, Oulu, Finland; dUniversity of Oulu, Biocenter Oulu, Department of Biology, Oulu, Finland; eUniversity of sized vesicles surrounded by a lipid bilayer and released Oulu, Biocenter Oulu, Laboratory of Developmental Biology, Oulu, Finland into the extracellular milieu by most of cells. Up to date, various isolation methods of EVs have been estab- Introduction:Theviewthathumanbeingsaremore lished. However, most of the current methods isolate complex than originally thought and could be described EVs with the contaminated extravesicular proteins, as a mixture of human and microorganism is gaining which are co-isolated proteins or non-specifically momentum and even biofluids which had always been bound proteins. Since it is difficult to isolate EVs with- considered sterile have now been shown to contain bac- out any contaminations, the evaluation of the real- teria originating molecules and, in some cases, bacteria. vesicular proteins could be valuable for the quality Healthy human skin is populated by many species of control of EVs. unicellular organisms, a number of which are known to Methods: SW480 EVs were isolated from the condi- secrete extracellular vesicles (EVs). Our study of sweat EV tioned medium by sucrose cushion and iodixanol cargo using omics is aiming to shed some light on these buoyant density gradient ultracentrifugation. The iso- complex interactions. lated EVs were treated with trypsin or control for 6 h Methods:Wehavecollectedsweatfromtheupperbody and then pelleted by ultracentrifugation, before under- of exercising individuals (men and women) and isolated going LC-MS/MS. EVs and EV RNA using concentration and filtration. EVs Results: Trypsin treatment could digest the contami- were checked by TEM and NTA then subjected to pro- nated extravesicular proteins without influencing the teomics analysis. For RNA extraction EVs were directly intravesicular (luminal) proteins, as well as size and lyzed on filter. 1–10 ng of RNA was used to make morphology of EVs. By the quantitative proteomic libraries for sequencing. Filtered and trimmed reads analyses between vesicular proteins with and without were aligned to human genome using Bowtie. JOURNAL OF EXTRACELLULAR VESICLES 241

Unmapped reads were blasted against the EMBL database be coupled to the MSC-EVs’ common therapeutic to identify and classify metagenomics reads. potential. Results:Afewhundredhumanproteinswereiden- Summary/Conclusion:Thisproteinsignaturemay tified but also a number of bacterial proteins. In the be helpful in developing MSC-EV quality control case of RNA the number of unmapped reads was platforms required to confirm the identity and test larger than is usually observed with extracellular for the purity of potential therapeutic MSC-EVs. small RNA sequencing. Metagenomic analysis provided information about species but only a certain PF12.06 number of reads could be assigned, probably due to the lack of available genome data. There is also an uncertainty about the precise species as we can only Comparative analysis of stool extracellular vesicles between germ- free, bifidobacteria-di-associated and SPF mice identify with any precision taxonomy at the level of Hirohisa Izumia, Tatsuya Eharab, Mai Morozumib, Fuuka Tabatab, Yosuke order. Komatsub, Takashi Shimizub and Yasuhiro Takedab Summary/Conclusion:SweatEVsareamixtureofhuman aMorinaga Milk Industry Co., Ltd., Zama-city, Japan; bMorinaga Milk and microbe-derived EVs and their complete characteriza- Industry Co., Ltd., Zama-City, Japan tion will depend on the availability of genomic information Introduction:Gutmicrobiotaiscloselyrelatedto including for difficult to cultivate strains. host immune and metabolic functions. Recently, Funding: Academy of Finland Biofuture2025 microRNA (miRNA) in extracellular vesicles (EVs) produced by host intestinal epithelial cells participate in shaping the gut microbiota. However, effects PF12.05 of microbiota on host gut EVs and miRNAs are not fully elucidated. In this study, we investigated the effects of microbiota on host stool EVs and miRNAs Proteomic signature of mesenchymal stromal cell-derived small using germ-free (GF), bifidobacteria-di-associated, extracellular vesicles. Bas WM. van Balkoma, Hendrik Gremmelsa, Bernd Giebelb and Sai Kiang and specific pathogen free (SPF) mice. Limc Methods: GF mice and SPF mice (11 week, male) were aUMC Utrecht, Utrecht, Netherlands; bUniversitatsklinikum Essen, Essen, obtained. At 12-week-old age, part of GF mice were c Germany; Institute of Medical Biology, Agency for Science, Technology inoculated with Bifidobacterium longum BB536 and B. and Research, Singapore, Singapore, Singapore breve M-16V (1109 each) once, and maintained with 5% Introduction:Smallextracellularvesicles(EVs)are50– fructooligosaccharides (bifidobacteria-di-associated 200 nm vesicles secreted by most cells. They are considered mice). Mice were sacrificed at 15-week-old age by deep as mediators of intercellular communication, and EVs anaesthesia with sevoflurane, and tissue samples and from specific cell types, in particular mesenchymal stem/ stool samples were harvested. Stool EVs were purified stromal cells (MSCs), offer powerful therapeutic potential, using exoEasy Maxi Kit, and were analysed by and could provide a novel therapeutic strategy. They NanoSight LM10. Total RNAs were purified from stool appear promising and safe (as EVs are non-self-replicat- EVs using miRNeasy, and were analysed by microarray. ing), and eventually MSC-derived EVs (MSC-EVs) may be Results: NanoSight analyses revealed that mean EV developed to standardized, off-the-shelf allogeneic regen- size of GF stool was significantly smaller than that of erative and immunomodulatory therapeutics. Promising SPF stool, and EV quanity of bifidobacteria-di-asso- preclinical data have been achieved using MSCs from ciated stool was tend to be increased than that of GF different sources as EV-producing cells. Similarly, a variety stool. Moreover, microarray analysis revealed that EV isolation and characterization methods have been microbiota could change miRNA expression profiles applied. Interestingly, MSC-EVs obtained from different of stool EVs. sources and prepared with different methods show in vitro Summary/Conclusion: Microbiota might affect host and in vivo therapeutic effects, indicating that isolated EVs gut function via alterations of EVs’ characteristics. share a common potential. Methods:WeanalysedpublishedMSC-EVproteomics PF12.07=OWP3.07 datasets to identify a unique and robust proteomics signature. Shed microvesicles released from human primary and metastatic Results:Here,wecomparewell-characterizedandcon- colorectal cancer cell lines contain key cancer progression proteins trolled publicly available proteome profiles of MSC-EVs to and RNA species Wittaya Suwakulsiria, Alin Raia, Rong Xua, Maoshan Chena, David Greeningb identify a common MSC-EV protein signature that might and Richard Simpsona 242 ISEV2019 ABSTRACT BOOK aDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular PF12.08=OWP3.08 Science, La Trobe University, Melbourne, Victoria 3086, Australia, Melbourne, Australia; bDepartment of Biochemistry and Genetics, La Trobe Institute for Molecular Science, La Trobe University, VIC, Australia, Melbourne, Australia Mass spectrometry analysis of small extracellular vesicles isolated from ovarian cancer ascites Introduction: Extracellular vesicles (EVs) function in Anna Kotrbováa, Kristina Gomoryovaa, David Potesilb, Vit Weinbergerc, Igor c d c b a – Crha , Eva Jandakova , Lubos Minar , Zbynek Zdrahal , Vítězslav Bryja and bidirectional cell cell communication and contribute Vendula Pospíchalovaa to the sustained growth, invasion, and metastasis of aDepartment of Experimental Biology, Faculty of Science, Masaryk cancer cells within the tumour microenvironment University, Brno, Czech Republic; bCore Facility Proteomics, Central (TME). EVs comprise two main classes – exosomes European Institute of Technology, Masaryk University, Brno, Czech Republic; cDepartment of Obstetrics and Gynecology, Faculty Hospital and shed microvesicles (sMVs, also termed micropar- Brno, Brno, Czech Republic; dDepartment of Pathology, University Hospital ticles and ectosomes) – with distinct modes of biogen- Brno, Brno, Czech Republic esis. Within each EV class, subtypes exist that can be distinguished by their distinct protein/RNA signatures. Introduction: High-grade serous carcinoma (HGSC) Whilst much is known about exosome cargo content of the ovaries, fallopian tube and peritoneum is the and functionality, sMVs are poorly understood. deadliest gynaecological malignancy with 5-year survi- Methods: Here, we compare protein/RNA profiles and val rate below 30%. HGSC is frequently accompanied functionality of sMVs and exosomes secreted from by ascites, a pathological accumulation of fluid in the human primary (SW480) and metastatic (SW620) col- peritoneum, which can be exploited as a liquid biopsy orectal cancer cell lines. Milligram amounts of EVs containing not only cancer cells but also the tumour were purified from cell culture media using a combina- microenvironment including extracellular vesicles tion of differential ultracentrifugation/isopycnic iodix- (EVs). Tumour cells produce substantially more EVs anol density centrifugation. Label-free quantitative than healthy cells, thus malignant ascites is the source mass spectrometry was performed to obtain protein of enriched pool of EVs of HGSC origin. profiles for SW480-derived and SW620-derived sMVs. Methods: Ascitic fluids depleted of cells were frac- Results: We show that sMVs, unlike exosomes, are tioned using size-exclusion chromatography and two ALIX-, TSG101-, CD63- and CD9- and contain a dif- fractions – containing and not containing EVs – were ferent suite of key cancer progression modulators. further analysed. In parallel, small EVs were also iso- Protein/RNA signatures for SW480-derived sMVs and lated from ascitic fluids using differential ultracentrifu- exosomes differ from each other and also from their gation followed by purification step in sucrose/D2O SW620-derived counterparts. SW480-derived sMVs are cushion. In total, 24 malignant ascites and 5 non- enriched in ITGA/B, ANXA1, CLDN7, CD44 and malignant ascites were used for EV isolation and EGFR/NOTCH signalling networks, while SW620- further analysed using high resolution hybrid mass derived sMVs are enriched in PRKCA, MACC1, spectrometer Orbitrap Fusion Lumos Tribrid. The sub- FGFR4 and MTOR/MARCKS signalling networks. sequent data visualization and statistical analyses were Fibroblast invasion capabilities of SW480-derived and performed using in-house developed pipelines in SW620-derived sMVs are comparable. KNIME environment. Summary/conclusion: Furthermore, we report for the Results: We identified 2441 proteins in total in the EVs first time a comprehensive biochemical/functional ana- from the ascites among which 21 were present in all 29 lysis of a hitherto undescribed subpopulation of sMVs. EV samples and not in non-vesicular fractions. Several We anticipate our in-vitro findings will be a starting of these proteins were specifically enriched in small point for more sophisticated studies aimed at elucidat- EVs in malignant ascites in comparison with non- ing the biochemical and functional properties of EV malignant ascites. These proteins are now being eval- subtypes in vivo. The emerging roles of specific EV uated as biomarkers. subtypes in the TME we believe will alter our view of Summary/conclusion: Using advanced mass spectro- cancer biology and might present new targets for ther- metry, we identified candidate proteins which are spe- apeutic intervention. cifically enriched in small EVs of HGSC. These Funding: Funding support from La Trobe University, proteins warrant further investigation as they may act Melbourne, Australia. as important players in HGSC progression as well as JOURNAL OF EXTRACELLULAR VESICLES 243 serve as potential prognostic/diagnostic/screening bio- CC10 expression). AFSC-EVs contain 901 microRNAs, markers of HGSC. some of which are crucial for foetal lung development, Funding: Czech Science Foundation, Grant No. GJ17- such as miR17 ~ 92 cluster. 11776Y Summary/conclusion: Administration of AFSC-EVs rescues impaired foetal lung development in experi- PF12.09=OWP1.05 mental models of PH. AFSC-EV regenerative ability is exerted via the release of miRNAs some of which regulate genes involved in foetal lung development. Extracellular vesicles derived from amniotic fluid stem cells rescue AFSC-EVs represent a promising therapeutic strategy impaired foetal lung development via the release of microRNAs Lina Antounians, Vincenzo Catania, Benjamin Liu, Areti Tzanetakis, Louise for PH in foetuses. Montalva and Augusto Zani Funding: CIHR-SickKids Foundation The Hospital for Sick Children, Toronto, Canada

Introduction: Incomplete lung development, also PF12.10=OWP2.10 known as pulmonary hypoplasia (PH), is a recognized cause of neonatal death. To date, there is no effective HIV-specific antibody-mediated targeting of ENV+ tissues by treatment that promotes foetal lung growth and exosomes maturation. Herein, we describe a stem cell based Zou Xue, Yuan M’eng, Zheng Nan and Wu Zhiwei approach that enhances foetal lung development via Nanjing University, Nanjing, China (People’s Republic) the administration of extracellular vesicles (EVs) derived from amniotic fluid stem cells (AFSCs) in rat Introduction: ART (Antiretroviral Therapy) can effec- models of PH. Moreover, we report the microRNAs tively suppress HIV replication in the peripheral blood present in AFSC-EVs that are responsible for these to an undetectable level. However, efforts to eradicate beneficial effects. the latent virus in reservoirs remain a challenge and are Methods: AFSC-EVs were isolated by ultracentrifuga- a major obstacle in the treatment of HIV patients. tion from conditioned medium (CM) of c-Kit+ rat Exosomes exhibit huge promise as an endogenous AFSC that were grown in exosome-depleted FBS for drug delivery nanosystem for delivering drugs to reser- 18 h. AFSC-EVs were assessed for size (nanoparticle voir tissues given their unique properties, including tracking analysis), morphology (TEM), and expression low immunogenicity, innate stability, high delivery effi- of CD63, Hsp70, Flo-1 and TSG101 (Western). ciency and mostly importantly the ability to penetrate Ex vivo: Pregnant dams were gavaged nitrogen at E9.5 solid tissues due to their lipophilic properties. to induce foetal PH. At E14.5, foetal lungs were har- Methods: In this study, we engineered and expressed vested, and incubated with culture medium alone, the ScFv of a high affinity HIV-specific monoclonal AFSC-CM, or AFSC-EVs. Foetal lungs from untreated antibody, 10E8, on exosome surface. Exosomes from dams served as control. Lungs were compared for 293T cells were loaded with curcumin via saponin, terminal bud density and surface area at 72 h, by two with efficient up to 34%. 10E8ScFv-expressing exo- independent investigators. somes (10E8-Exo) showed highly efficient targeting of In vitro: Foetal rat lung organoids were generated with and curcumin delivery to CHO cell that expresses a epithelial cells from normal and hypoplastic lungs. trimeric gp140 on its surface (ENV+ cells) in vitro as Organoids were cultured for 10 days in either medium demonstrated by confocal imaging and flow cytometry. alone or medium supplemented with AFSC-EVs. Lung We showed that 10E8-Exo could effectively bind to organoids from untreated normal pups served as con- CHO cell that expresses a trimeric gp140 on its surface. trol. Organoids were assessed for proliferation (Ki67) The exosomes loaded with curcumin, a chemical that and markers of epithelial cell differentiation via was shown to kill HIV-infected cells, showed specific immunofluorescence. killing of the trimeric gp140-expressing CHO cells. In RNA-sequencing: RNA was isolated using SeraMir, an NCG mouse model that was grafted with the constructed into libraries (CleanTag Small RNA), and tumourigenic gp140-CHO cells and developed solid sequenced on NextSeq High Output single-end sequen- tissue tumours intravenously injected 10E8-Exo tar- cing run. geted the ENV-expressing tissues and delivered curcu- Results: Administration of AFSC-EVs increased term- min to induce a strong suppression of the ENV+ inal bud density and surface area of lung explants back tumour growth with a low toxicity. to control levels and promoted lung epithelial cell Results: Our results demonstrated that engineered exo- differentiation in lung organoids (increased SPC and somes can deliver anti-HIV agents to solid tissues by 244 ISEV2019 ABSTRACT BOOK specifically targeting cells expressing viral ENV and (2016YFC1201000), Nature Science Foundation of induce cell killings. Jiangsu Province (BY2015069-02) and National Summary/conclusion: It suggesting that such an Nature Science Foundation of China (81672020). The approach can be developed for eradicating virus- funders had no role in study design, data collection and infected cells in tissue reservoir analysis, decision to publish, or preparation of the Funding: This study was supported by The National manuscript. Key Research and Development Program of China JOURNAL OF EXTRACELLULAR VESICLES 245

Late breaking- EVs and cancer Chairs: Sonia Melo; Golnaz Morad Location: Level 3, Hall A 15:30–16:30

for Cancer Research, Tokyo, Japan; cCancer Proteomics Group, Cancer LBF01.01 Precision Medicine Center, Japanese Foundation for Cancer Research, Tokyo, Japan

Exosomes from LNCaP cells promote the activity of osteoblasts through the transfer of mir-375 Introduction: For improvement of prognosis of color- Yun Yea and Su-liang Lib ectal cancer (CRC), detection at an earlier stage of CRC aProstate Cancer, Xi’an, China (People’s Republic); bCancer, Xi’an, China is essential. Exosomes are nanovesicles secreted from (People’s Republic) plasma membrane, and have potential to be served as biomarker carriers. In this study, we performed pro- Introduction: Studies have shown that exosomes influ- teomic profiling of exosomes secreted from viable CRC ence tumour metastasis, diagnosis and treatment. It has tissues. been found that exosomal miRNAs are closely linked Methods: To identify early detection biomarkers for to the metastatic microenvironment. However, the reg- CRC, we performed comprehensive proteome analysis ulatory role of exosomes from prostate cancer (PCa) of tissue-exudative extracellular vesicles (Te-EVs), cells in bone metastasis remains poorly understood. which were obtained from culture media of freshly Methods:Weisolatedandpurifiedexosomesbyultracen- resected viable CRC tissue or adjacent normal mucosa trifugation, isolated total RNA from cells and total miRNA (n = 17). Among the identified Te-EV proteins, we from exosomes, and analysed the level of miR-375 by RT- narrowed down the biomarker candidate by selecting PCR. Exosome libraries from LNCaP cells and RWPE-1 proteins which are statistically upregulated (p < .05, cells were sequenced and filtered with an Illumina fold change > 5.0) in Te-EVs from CRC tissues than HiSeqTM 2500 system. The activity of alkaline phospha- those from adjacent normal tissues. Then we per- tase, the extent of extracellular matrix mineralization and formed functional analysis of the biomarker candidate the expression of osteoblast activity-related marker genes specifically. were measured to evaluate osteoblast activity. Results: Comprehensive LC/MS analysis identified Results:Morphologicalobservation,particlesizeanalysis 6149 Te-EV proteins, in which 641 proteins showed and molecular phenotyping confirmed that the isolated significant upregulation in Te-EVs from CRC tissues extracts contained exosomes. Differential expression ana- (p < . 05, fold change > 5. 0) compared to those from lysis confirmed the high expression of miR-375 in LNCaP adjacent normal mucosa. We focused especially on cell-derived exosomes. We further determined which exo- GAM (p = 7.0 ×10–5, fold change = 7.4) as a novel somes could enter osteoblasts and increase their miR-375 biomarker candidate. GAM protein was significantly level. In addition, exosomal miR-375 could significantly overexpressed in CRC tissues compared with adjacent promote the activity of osteoblasts. normal mucosa. In EV-sandwich ELISA assay, the Summary/conclusion: This study lays the foundation expression level of GAM on plasma EVs from CRC for further investigations on the function of exosomal patients was significantly higher than that from miR-375 in the activation and differentiation of osteo- healthy donors in EV-sandwich ELISA assay blasts and the mechanism of bone metastasis in PCa. (n = 133, p = 4.0 ×10–7). In addition, the uptake of Funding: none GAM-overexpressing EVs enhanced vascular endothelial cell growth and angiogenesis through modulation LBF01.02=OWP1.13 of nitric oxide metabolism. Summary/conclusion: EV-GAM might have great potential as a target for both CRC diagnosis and ther- Colorectal cancer cell-derived exosome enhances microenvironmental angiogenesis through modulation of intracellular metabolism apy. Our strategy for identification of exosomal bio- Atsushi Ikedaa, Satoshi Nagayamab and Koji Uedac marker by proteomic profiling of Te-EV proteins can aCancer proteomics group, Cancer Precision Medicine Center, Japanese be applied to other cancers. Foundation for Cancer Research, Tokyo, Japan; bDepartment of Gastroenterological Surgery, Cancer Institute Hospital, Japanese Foundation 246 ISEV2019 ABSTRACT BOOK

LBF01.03 LBF01.04

Augmentation of Exosome secretion in Ha-RasV12-induced cell softening and multilayer cellular aggregates in Madin-Darby canine Longitudinal analysis of serum-derived extracellular RNA for kidney cells monitoring of treatment response to targeted therapy in Ming Jer Tang and Hsi Hui Lin glioblastomas Leonora Balaja, Rob Kitchenb, Bob Carterc, Xandra Breakefieldc and Johan Deaprtment of Physiology, National Cheng Kung University Medical College, Skogd Tainan, Taiwan (Republic of China) aMassachusetts General Hospital, Boston, USA; bExosome Diagnostics, c d V12 Waltham, USA; MGH, Boston, USA; Exosome Diagnostics, Inc., Introduction: Ha-Ras overexpression-induced cell Waltham, Massachusetts, USA, Waltham, USA softening and multilayer cellular aggregates (cell clumping) during confluence in MK4 cells (inducible Introduction: We performed longitudinal whole-tran- Ras harbouring MDCK cells). We have demonstrated scriptome profiling of serum exosomes from patients that Ha-RasV12-induced cell softening and transforma- suffering from recurrent glioblastoma (GBM) enrolled tion required Cav-1 downregulation and augmented in a clinical trial to assess response to Dacomitinib, a secretion of Wnt-5a containing exosome. Here, we second-generation irreversible EGFR tyrosine kinase explored the role of exosome secretion and cell soft- inhibitor. All patients had failed standard of care ther- ening in Ha-RasV12-induced cell clumping. apy and had tumours amplified for EGFR. They under- Methods: We employed MK4 cells (inducible Ha-Ras- went daily oral administration of Dacomitinib and harbouring MDCK cells). Cell stiffness was measured blood serum samples were collected immediately by atomic force microscope (Biowizard II, JPK) and prior to first treatment and monthly thereafter. cell aggregates were examined under confocal micro- Methods: Deep sequencing of exosomal RNA scope. Quantitation of exosome secretion in culture (exRNA), derived from just 2 ml of patient serum, media was assessed by Nanoparticle analysis. revealed robust signatures of treatment response, as Results: We found that Induction of Ha-RasV12-trig- defined by >6-month progression-free survival. gered YAP nuclear translocation and subsequently Results: Non-responders were found to have signifi- YAP-targeted gene expression. However, verteporfin, cantly elevated levels of a number of transcripts includ- a YAP/TEAD binding inhibitor, failed to prevent Ha- ing colony stimulating factor 1, which regulates the RasV12-induced multilayer cellular aggregates. proliferation, differentiation, and survival of macro- Overexpression of Cav1 inhibited Ha-RasV12-induced phages and microglia. We further identified robust YAP activation and multicellular cell aggregates, signatures of treatment response in post-treatment whereas knockdown of Cav1 in MDCK cells resulted serum samples, including the suppression of DNA in activation of YAP, but not cellular aggregates and methyltransferase 3 alpha, an important player in cellular transformation. Activities of Rac and RhoA, DNA methyl transfer for de-novo methylation, as well both associated with cell extrusion, were increased in as Adenosine A2B receptor, a member of the G pro- Ha-RasV12-overexpressed MK4 cells. EHT1864 (Rac tein-coupled receptor superfamily which is overex- inhibitor) abolished multilayer cellular aggregates in pressed in a variety of cancers and has been shown to Ha-RasV12-overexpressed MK4 cells, whereas Y27632 play a role in tumour progression via increased angio- (ROCK inhibitor) induced multilayer cellular aggre- genesis and metastasis. Furthermore, we identified gen- gates in MK4 cells. However, neither EHT1864 nor eral decreases in oncogene abundance following Y27632 inhibited Ha-RasV12-induced YAP nuclear Dacomitinib treatment, including tumour protein p53 localization. Finally, Ha-RasV12-overexpression and Ovo-like transcriptional repressor 1, a zinc finger- induced a marked increase in exosome secretion and containing transcription factor, shown to be a critical cell softening, which preceded cell clumping. inducer of epithelial-to-mesenchymal transition in can- Summary/conclusion: Taken these data together, our cer. Finally, in comparison to healthy control serum, results indicate that Cav-1 downregulation is required we find hundreds of transcripts exhibiting differential for Ha-RasV12-induced YAP activation and cellular abundance in pretreatment GBM patients that may transformation. However, only Cav1 downregulation serve as general non-invasive biomarkers for this and Rac activation, but not YAP activation, are devastating disease. required for Ha-RasV12-induced multilayer cellular Summary/conclusion: This study is unique because it aggregate. represents the first longitudinal profiling of the exoso- Funding: This work was partially funded by MOST mal transcriptome in a cohort of genomically selected 107-3017-F-006-002 to MJ Tang. GBM patients. These findings are a tantalizing step JOURNAL OF EXTRACELLULAR VESICLES 247 towards exosome-based biomarkers for the detection of in NSCLC patients, with band intensity increasing with GBM, as well as patient stratification and monitoring. the grade of lung cancer, compared to healthy controls. Funding: CA069246 CA230697 TR000931 Summary/conclusion: LESP-1 in exosomes was highly expressed in the blood plasma of lung cancer patients, which suggests that LESP-1 could serve as a potential LBF01.05 biomarker for diagnosis of NSCLC. Funding: This research was supported by the Korea Health Technology R&D Project (No. HR14C0007)

Lung cancer exosome specific protein 1 (LESP-1) is increased in the funded by the Ministry of Health & Welfare, Republic plasma of non-small cell lung cancer patients of Korea. Byeonghyeon Choia, Yu Hua Quanb, Jiyun Rhob, Hyesun Jeongc, Jik Han Jungd, Sunghoi Hongc, Ji-Ho Parkd, Yeonho Choie, Yong Parkf, Kook Nam g g g Han , Young Ho Choi and Hyun Koo Kim LBF01.06 aKorea university, Seoul, Republic of Korea; bDepartment of Biomedical Sciences, College of Medicine, Korea University, Gurogu, Republic of Korea; cSchool of Biosystem and Biomedical Science, Korea University, seongbuk-gu, Republic of Korea; dDepartment of Bio and Brain Chloride intracellular channel protein 4 (CLIC4) is a serological cancer Bioengineering, Korea Advanced Institute of Science and Technology biomarker released from tumour epithelial cells via extracellular (KAIST), Daejeon, Republic of Korea; eDepartment of Bioconvergence vesicles and required for metastasis Engineering, Korea University, seongbuk-gu, Republic of Korea; fDivision Vanesa C. Sanchez, Alayna Craig-Lucas, Ji Lou; Kent Hunter and Stuart of Hematology-Oncology, Department of Internal medicine, Korea Yuspa University Anam Hospital, seongbuk-gu, Republic of Korea; gDepartment of Thoracic and Cardiovascular Surgery, Korea University Guro Hospital, National Institutes of Health (NIH), Bethesda, USA Korea University College of Medicine, Gurogu, Republic of Korea Introduction: Chloride intracellular channel protein 4 Introduction: Lung cancer remains to be the leading (CLIC4) is a highly conserved metamorphic protein cause of cancer-related mortality worldwide. Finding originally described as an ion channel. It translocates new non-invasive biomarkers for lung cancer is still a to the nucleus serving as an integral component of significant challenge. Exosomes are endosome-derived, TGF-β signalling. In multiple cancers, CLIC4 is a nano-sized (30–150 nm), extracellular microvesicles tumour suppressor, excluded from the nucleus and released from many cell types, and that play a key lost from the cytoplasm of progressing cancer cells. In role for in cell-to-cell communication. Use of exosomes contrast, CLIC4 is upregulated in the tumour stroma as biomarkers in of lung cancer, in a liquid biopsy, is a acting as a tumour promoter. Recent reports indicate rising emerging field in nanotechnology in a liquid that CLIC4 is detected in the circulation of cancer biopsy. This research work focused on identifying exo- patients serving as possible biomarker and has been some-specific proteins (LESP) of in non-small cell lung detected in extracellular vesicles (EVs). cancer (NSCLC) by using proteomics and assessed Methods: EVs from multiple sources were isolated by their concentration efficacy within exosomes derived differential centrifugation, following ultracentrifuga- from the plasma of normal and NSCLC patients. tion and Optiprep density gradients. EV size distribu- Methods: Proteomics analysis was performed to inves- tion and concentration were analysed by NTA and tigate lung cancer-specific proteins within exosomes TEM. The presence of prototypical markers and isolated from five NSCLC (H522, A549, H1299, CLIC4 were analysed by immunoblot and by tissue H1650, PC9) and one normal lung alveolar cell lines staining. (Human pulmonary alveolar epithelial cell), using size Results: CLIC4 was present in EVs released from pri- exclusion chromatography. We then isolated plasma mary normal and multiple breast tumour cell lines and exosomes from healthy controls and NSCLC patients increased in EVs from TGF-β-induced myofibroblasts. (17 controls and 54 patients) using dual size exclusion In vivo, in two different orthotopic syngeneic mouse chromatography. ELISA and Western blot were utilized breast cancer models, CLIC4 levels in EVs isolated to validate the proteomic results in NSCLC patients from plasma increased with tumour burden and lung and compare with healthy controls. metastatic load. Moreover, CLIC4 levels in EVs isolated Results: Using proteomics analysis, we identified from plasma of breast cancer patient was elevated when LESP-1 in the exosomes from NSCLC cells, but not compared to healthy age and race matched controls. To in those from normal cells. LESP-1 concentration was dissect the contribution of stromal vs tumour epithelial higher in lung cancer patients compared to the healthy compartments as the source of the CLIC4-high EVs, controls (p < .01), and increased according to the grade CLIC4 was either deleted in tumour cells lines by of lung cancer, in peripheral blood (p < .01). Moreover, CRISPR/Cas9 or CLIC4 KO females were implanted Western blot results confirmed the increase in LESP-1 CLIC4 WT tumour cells. CLIC4 is reduced in 248 ISEV2019 ABSTRACT BOOK circulating EVs from CLIC4 KO tumour bearing mice similarities and differences between gefitinib-resistance when compared to WT and it is present in circulating of exosomes and whole cells, through pathway analysis EVs from CLIC4 KO females bearing WT tumours, of the core functional proteins. indicating that the major contribution of CLIC4 into Summary/conclusion: The results might suggest that circulation is from tumour epithelium. Additionally, functional exosomal proteins secreted from gefitinib CLIC4 KO females display no difference in primary resistant lung cancer cells contain specific signatures tumour size and a significant reduction in both size via horizontal transfer from whole cells of NSCLC and number of lung metastases. Funding: This work was supported by the Industrial Summary/conclusion: CLIC4 levels in EVs from bio- Strategic Technology Development Program logical fluids may have value as a cancer biomarker, in (10077559) funded by the Ministry of Trade, Industry conjunction with other markers, to detect or analyse & Energy (MOTIE, Korea). tumour progression or recurrence. The low lung metastasis frequency in CLIC4 KO females may due to a defect in lung tissue to recruit neutrophils and to LBF01.08 induce neovasculature. Funding: National Institutes of Health Extracellular vesicles derived from bone marrow stromal cells promote evasion of multiple myeloma cells from natural killer cell antitumour activity LBF01.07 Tomohiro Umezua, Chiaki Kawanaa, Satoshi Imanishib, Junko Ohyashikia and Kazuma Ohyashikia aTokyo Medical University, Tokyo, Japan; bTokyo University of Science, Tokyo, Japan Comparative proteomic analysis of exosomes and whole cells from NSCLC cell lines: focus on gefitinib resistance Mi young Lee, Ye-Eun Jeong and A-Reum Ryu Introduction: Natural killer (NK) cells are a major Soonchunhyang University, Asan, Republic of Korea component of the antitumour immune response. NK cell dysfunctions have been reported in various haema- Introduction: Overexpression of epidermal growth tologic malignancies, including multiple myeloma factor receptor (EGFR) is a typical feature of approxi- (MM). In the bone marrow of MM patients, bone mately 90% of NSCLC patients. EGFR mutations marrow stromal cells (BMSCs) interact with MM induce excessive activation of tyrosine kinase domain cells, and also create a permissive microenvironment of EGFR, eventually inducing oncogenic alterations. for MM cell survival and immunosuppression. In this Thus, EGFR has become a therapeutic target for study, we investigated the biological property of the NSCLC patients harbouring activating EGFR muta- extracellular vesicles (EVs) and EV-miRNAs of tions with tyrosine kinase inhibitor (TKI) such as gefi- BMSCs derived from MM patients (MM-BMSCs), aim- tinib. However, more than 50% of patients with ing to clarify the involvement of EVs derived from NSCLC receiving gefitinib showed resistance to gefiti- MM-BMSCs in tumour immune microenvironment. nib. Thus, acquired resistance to EGFR TKI is a major Methods: BM samples were obtained from MM challenge in the lung cancer treatment. Although sev- patients (age 68–79, n = 6) in accordance with the eral mechanisms have been attributable to acquired Declaration of Helsinki and using protocols approved resistance, the information on exosomal studies on by the research Ethics Committee of Tokyo Medical EGFR-TKIs resistance of NSCLC is limited. University (IRB No. 2648), and BMSCs derived from Methods: In this study, comparative proteomic analy- MM patients (MM-BMSCs) were isolated by the clas- sis of exosomes and whole cells from EGFR mutant sical adhesion method. BMSCs from healthy donors gefitinib-sensitive NSCLC cell lines (PC9) and gefiti- (Normal-BMSCs) were purchased from Lonza Inc. nib-resistant cell line (PC9/GR) were conducted by The EVs were isolated from conditioned medium of quantitative proteomics. The significant protein BMSCs using Exoquick-TC Reagent (System expression changes observed in each analysis, and the Biosciences). EV-miRNA profiling was done using a differences of gefitinib resistance-related proteins from TaqMan low-density array (Applied Biosystems). A exosomes and whole cells were examined. rapid flow cytometry assay for quantifying target cell Results: Biological processes, molecular functions and killing after CellVue (Sigma)-labelled MM cell lines cellular components associated with gefitinib resistance (RPMI8226, U266) prior to co-incubation with NK and key pathways related with gefitinib resistance have cells (Biotherapy Institute of Japan, Inc.). been identified in exosomes and whole cell lysates from Results: There were no significant differences in size PC9 and PC9/GR cells. The results also revealed the and amount of EVs among Normal-BMSCs and MM- JOURNAL OF EXTRACELLULAR VESICLES 249

BMSCs. We found that the expression of some cells to MDSCs. Besides that, lncRNA NBR2 conveyed miRNAs, including miR-146a, in the EVs derived by TEXs induced the development and suppressive from MM-BMSCs were higher than that of Normal- function of MDSCs by interacting with STAT3 and BMSCs. Furthermore, miR-146a in MM-BMSC-EVs enhancing its phosphorylation. was significantly up-regulated by co-culturing with Summary/conclusion: Thus, this present study indi- MM cell lines (RPMI8226, U266). Functionally, when cates that exosomal lncRNA NBR2 can increase the the miR-146a-enriched MM-BMSC-EVs was added to immunosuppression of MDSCs by regulating the phos- the NK cell-MM cell co-culture system, RPMI8226 phorylation of STAT3, which will provide sufficient become less sensitive to NK cell killing compared scientific basis for the immunotherapy of cancer. with adding the Normal-BMSC-EVs. Summary/conclusion: These results indicated that MM-BMSC-EVs, including miR-146a, may function LBF01.10 as immune regulator to inhibit NK cell function against tumours, therefore providing a new therapeutic target In vivo imaging of natural killer cells labelled with fluorophore- for tumour treatment. loaded extracellular vesicle mimetics Hye Sun Parka, Gyeongyun Gob, Yong Song Ghob and Kwan Soo Hongc aKorea Basic Science Institute, Cheongju-si, Republic of Korea; bDepartment LBF01.09 of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea; cBioimaging Research Team, Korea Basic Science Institute, Cheongju, Republic of Korea

Exosomal long noncoding RNA NBR2 facilitates the immunosuppression of MDSCs by enhancing the phosphorylation of Introduction: In the field of cancer immunotherapy, signal transducers and activators of transcription 3 in-vivo biodistribution of immunotherapeutic moiety a b Xinyu Tian and lI Zhiyang has emerged as important issue as well as its therapeu- aNanjing Drum Tower Hospital & the affiliated Hospital of Nanjing tic efficacy. This is because it plays an important role in University Medical School, Nanjing, China (People’s Republic); bNanjing Drum Tower Hospital Clinical College, Nanjing, China (People’s Republic) assessing the pharmacokinetic aspects associated with the bio-toxicity of the immunotherapeutic moieties Introduction: The immunosuppression induced by injected in vivo and evaluating the therapeutic effects myeloid-derived suppressor cells (MDSCs) plays a cru- associated with homing to lesion sites. Natural killer cial role in tumour escape. And long noncoding RNA (NK) cells have non-specific antitumour activity, and (lncRNA) has been indicated as a core regulator of have been employed to treat tumours. Unlike other tumour immunity. However, the effect and regulatory immune cells, NK cells cannot perform phagocytosis mechanism of lncRNA on the immunosuppressive sufficiently, so it is difficult to label NK cells with function of MDSCs remain unclear. Exosomes are imaging materials such as nanoparticles. Difficulty in cell-derived vesicles which are present in high abun- labelling NK cells makes it difficult to validate the dance in the tumour microenvironment where they distribution and antitumour activity of NK cells in transfer information between cells. vivo. Methods: Ultracentrifugation was used to isolate exo- Methods: In this study, we tried to develop NK cell somes from cancer cells. MDSCs and T cells were labelling technology using exosome mimetics, based on sorted from the spleen of tumour-bearing mice and the fact that exosome mimetics can deliver their cargos wild type mice, respectively, with immunomagnetic to target cells via receptor-mediated endocytosis. We beads. CFSE was performed to estimate the influence analysed cell adhesion molecules that were overex- of MDSCs on the proliferation of T cells. And real-time pressed in NK cells and produced the cell line that fluorescence quantification PCR (qRT-PCR) was used overexpress them using cell transformation techniques. to detect the expression of lncRNA NBR2, while wes- We also labelled NK cells with exosome-mimetic nano- tern-blot was used to confirm the phosphorylation of vesicles loaded with magnetic nanoparticles and fluor- signal transducers and activators of transcription 3 ophores, and evaluated biomedical imaging and (STAT3). therapeutic effects of the NK cells using mouse tumour Results: Herein, we found that tumour-derived exo- models. somes (TEXs) could enhance the development and Results: We analysed cell adhesion molecules immunosuppression of MDSCs. Furthermore, it was expressed in NK cells and constructed cell lines that indicated that the regulation of TEXs to the develop- overexpress counter receptors. We succeeded in label- ment and immunosuppression of MDSCs depending ling NK cells with a fluorophore-loaded exosome on the transportation of lncRNA NBR2 from cancer mimetics and also quantitatively evaluated the 250 ISEV2019 ABSTRACT BOOK biomedical imaging and therapeutic effects of the Summary/conclusion: These data suggest that the labelled NK cells. number of secreted EVs and/or the concentration of Summary/conclusion: We produced and characterized MMP-13 in EVs play an important role in the meta- NK cell-targeting exosome-mimetic nanovesicles. static ability of human osteosarcoma cells. Exosome mimetics-based cell labelling technology developed in this study will overcome the limitations of current technology and can be widely applied to in LBF01.13 vivo image-tracking of immune cells in cancer immunotherapy. Exosomal long noncoding RNA NBR2 induces the autophagy of lung cancer cells by interacting with high-mobility group box 1 Ting Wanga and Xinyu Tianb LBF01.11 aJiangsu Cancer Hospital & Jiangsu Institute of Cancer Research & The Affiliated Cancer Hospital of Nanjing Medical University, Nanjing, China (People’s Republic); bNanjing Drum Tower Hospital, Nanjing, China (People’s Republic) Comparison of MMP-13-containing extracellular vesicles with metastatic ability in human osteosarcoma cells Introduction a b b : Lung cancer has become the leading Ryo Sasaki , Mitsuhiko Osaki and Futoshi Okada cause of disease-related death worldwide. It has been a Division of Pathological Biochemistry, Department of Biomedical Sciences, confirmed that high-mobility group box 1 (HMGB1) is Faculty of Medicine, Tottori University, Yonago, Japan; bDivision of Pathological Biochemistry, Department of Biomedical Sciences, Faculty of closely correlated with the progression of lung cancer. Medicine, Tottori University, Yonago, Japan However, it still remains unclear about the accurate mechanisms of regulating the expression and secretion Introduction: Osteosarcoma commonly develops from of HMGB1 in lung cancer cells. Exosomes are cell- bone and mainly affects children and adolescents. derived vesicles which are present in high abundance Although therapy for primary osteosarcoma, such as in the tumour microenvironment where they transfer adjuvant chemotherapy combined with surgical wide information between cells. – resection, is being improved, 30 40% of osteosarcoma Methods: Exosomes from cultivate supernatant of lung patients die of lung metastasis. Therefore, it is impor- cancer cells were isolated with ultracentrifugation. tant to elucidate the mechanism of lung metastasis to Western-blot and immunofluorescence were per- establish specific new therapies based on the mechan- formed to confirm the expression of HMGB1 in lung ism. We previously reported that the down-regulation cancer cells, and ELISA was used to detect the secreted of miR-143 promotes cellular invasion of 143B cells, a HMGB1. The expression of long noncoding RNA human osteosarcoma cell line, and that intravenous (lncRNA) NBR2 was detected with real-time fluores- injection of miR-143 significantly suppresses lung cence quantitative fluorescence (qRT-PCR). Western- metastasis of osteosarcoma cells in mice. In addition, blot and transmission electron microscope were used matrix metalloproteinase-13 (MMP-13) was identified to make sure the autophagy of lung cancer cells. as one of the miR-143 target genes, and knockdown of Results: Herein, we demonstrated that exosomes from MMP-13 was able to suppress the invasion of 143B lung cancer cells could promote the both the expres- (metastatic osteosarcoma cell line) cells in vitro. sion and secretion of HMGB1, and therefore induce Methods: These data motivated us to examine whether the autophagy of lung cancer cells. Besides that, it was MMP-13 concentration in extracellular vesicles (EVs) also demonstrated that exosomes from lung cancer secreted by 143B was higher than in that secreted by cells promoted the expression and release of HMGB1 HOS (non-metastatic cell line). In this study, we exam- by conveying lncRNA NBR2 which could interact with ined the number of secreted EVs and MMP-13 con- HMGB1 protein and enhance its stability. Moreover, centration in the EVs of two human osteosarcoma cell high level of HMGB1 facilitated the autophagy of lung lines-143B and HOS. cancer cells via activating RAGE-Erk1/2 pathway, and Results: The number of EVs secreted by 143B was four accelerated the progression of lung cancer. times higher than those secreted by HOS. Moreover, Summary/conclusion: Taken together, our study indi- Western blot analysis revealed that MMP-13 concen- cates that exosomal lncRNA NBR2 induces the autop- tration per 3 µg of EVs was increased 2.5 times in EVs hagy of lung cancer cells and accelerates the tumour derived from 143B in comparison to those derived progression by interacting with HMGB1. from HOS. JOURNAL OF EXTRACELLULAR VESICLES 251

LBF01.14 studied based on the H&E-stained slides and on their diagnosis available for each case. Each organ and tissue specific tumours were selected after systematically Morphology of tissue disruption at the sites of high-grade tumour browsing all seminars. Control cases were chosen Mousumi Chaudhury based on their diagnosis notes as non-invasive benign GIM foundation, Little Rock, USA tumour condition. In this study, image J was used for its different potentials. It was used to prepare the Introduction:Invasivecancersoriginatingfromdiverse figures to show boundary, measure the perimeter, mak- organs like breast, ovary and lung metastasize to distant ing efficient colour contrast figures. sites. However, the structural changes at the primary site of Results: The preliminary observations reported in this these high-grade tumours have not been well character- study suggest that there may be excessive force acting ized. In this observational study using images of hematox- in the periphery of the cell, which likely leads to this ylin-eosin stained human tumour tissues, it is reported that cytosol enclosed membrane protrusions, likely to con- intriguingly, the morphology of the tissues at the meta- tain the nuclei. Some of these protrusions have nuclei static front or at the primary site appears similar, irrespec- and others do not. Also, there is clear evidence of tive of the organ of origin of the primary tumour. These enhanced membrane synthesis in high-grade tumour structures appear as a bulb-like projection, emanating tissues. from the tumour cell membrane. Mostly, they appear Summary/conclusion: This study suggests that there is similar as a ball and stick bulge. Although structures are a possible final common pathway of mechanical force not exactly similar in their dimension, there is a conserved generation, likely via cytoskeleton remodelling, which bulb like morphological protrusion in all tumours. Earlier may arise irrespective of organ-specific genomic mod- studies have described in in vitro or mouse models of ulation related to the primary disease process. These similar structures as invadopodia, invadosome or podo- observations provide visual evidence of epithelial- some, but their morphology has not been well character- mesenchymal transition during tumour metastasis. ized and examined in detail. Funding: none Methods: Web pathology website of H&E-stained human tumour slides were used. Cases have been 252 ISEV2019 ABSTRACT BOOK

Late breaking- EVs and physiology Chairs: Elie Beit-Yannai; Benedikt Kirchner Location: Level 3, Hall A 15:30–16:30

LBF02.01=OWP1.10 ectosomes) only upon LPS stimulus, and not in the EVs-free medium, suggesting that TG2 is a cargo of exosomes during neuroinflammation. Type-2 transglutaminase affects calcium homeostasis in neurons and is released in association with astrocytes-derived exosomes Summary/conclusion: TG2 is externalized through Elisa Tonolia, Ilaria Pradab, Claudia Verderioc and Elisabetta Verderioa astrocyte-derived exosomes upon neuroinflammatory aNottingham Trent University, Nottingham, United Kingdom; bCNR stimuli. Extracellular TG2 mediates the opening of L- c Institute of Neuroscience, Milan, Italy., Milan, Italy; CNR Institute of type VOCCs in neurons and sets basal [Ca2+]i at Neuroscience, Milan, Italy, Milan, Italy higher levels, which could have a significant impact Introduction: Type-2 transglutaminase (TG2) has been on neuronal activity in neuroinflammation. linked to calcium (Ca2+) dysregulation in conditions Funding: John Turland PhD bursary (NTU) and IBRO such as neurodegeneration. Recent evidences suggest travel fund. that extracellular vesicles (EVs) contribute to the onset and progression of neurological diseases, and we have recently shown that TG2 is a cargo of EVs in biological LBF02.02=OWP1.12 fluids. Here, we hypothesise that TG2 could be released by EVs, interact with neurons and affect neuronal Ca2 Plasma exosomes regulate proliferation and migration of vascular + homeostasis. smooth muscle cells Methods: Primary hippocampal neurons were estab- Kosuke Otania, Mai Yokoyab, Muneyoshi Okadac and Hideyuki Yamawakib lished from E18 rat embryos. Extracellular TG2 was aLaboratory of Veterinary Pharmacology, School of Veterinary Medicine, modulated in neurons either by lipofectamine transfec- Kitasato University, Towada, Japan; bKitasato University, Towada, Japan; cKitasato University, School of Veterinary Medicine, Laboratory of tion of a TG2-EGFP construct or by addition of pur- Veterinary Pharmacology, Towada, Japan ified TG2. Intracellular Ca2+ concentration ([Ca2+]i) was assessed by live imaging in fura-2/AM-loaded neu- Introduction: We previously reported that systolic rons. EVs were isolated from primary astrocytes (60 blood pressure in spontaneously hypertensive rats, an DIV) by serial centrifugation, characterized by Western animal model of essential hypertension, was partly blotting (flotillin-2 and alix) and nanoparticle tracking modulated by circulating exosomes (BBRC 2018). analysis (ZetaView). Experiments to assess TG2 influ- Vascular wall remodelling regulated by proliferation ence on exosomes-to-neural cells interactions, using a and migration of vascular smooth muscle cells Renilla sensor based on miR-146a-5p-transfer, are still (VSMCs) mediates development of hypertension. We ongoing. aimed to clarify the effects of plasma exosomes derived Results: Increase of extracellular TG2 levels in neurons from SHR and control Wistar Kyoto Rats (WKY) on induced an influx of extracellular Ca2+ ions, leading to proliferation and migration of VSMCs. a significant raise in basal [Ca2+]i both in normal Methods: Exosomes were isolated from rat plasma by conditions (ΔF340/380 = 0.126 ± 0.014; N = 23; an ultracentrifuge method, and identified through p <10–5) and with inhibited synaptic transmission measurement of particle size distribution by a tunable (tetrodotoxin) (ΔF340/380 = 0.058 ± 0.005; N = 33; resistance pulse sensing. For exploring exosome inter- p <10–5). Nifedipine, a blocker of L-type voltage-oper- nalization in VSMCs, the isolated exosomes were ated Ca2+ channels (VOCCs), partially prevented TG2- labelled with PKH67 dye and observed by a fluores- dependent Ca2+ response (average inhibition 36%; cence microscopy. Proliferation and migration of N = 21; p <10–5), suggesting that Ca2+ influx may SMCs were determined by a bromodeoxyuridine incor- occur through L-type VOCCs. To identify the source of poration and Boyden chamber assay, respectively. extracellular TG2, we analysed EVs isolated from rat Actin cytoskeleton was visualized by a rhodamine- primary astrocytes, previously reported to release TG2 phalloidin staining. Expression of protein and into the matrix especially in inflammatory conditions. microRNA in exosomes was determined by Western TG2 was detected in astrocytic exosomes (but not in blotting and microarray, respectively. JOURNAL OF EXTRACELLULAR VESICLES 253

Results: There was no difference in size and concen- GTPase Cdc42 and decreased actin polymerization. tration of plasma exosomes between WKY and SHR. ExNef increased susceptibility of MDM to fusion with Exosomes were incorporated into VSMCs, while the HIV. These changes resulted in activation of NLRP3 internalization of SHR exosomes was significantly inflammasome and increased secretion of pro-inflam- lower than WKY exosomes. Both WKY and SHR exo- matory cytokines. The effects of exNef were reversed somes similarly stimulated proliferation, migration and by overexpression of a constitutively active mutant of cytoskeletal changes such as formation of filopodia and Cdc42. The findings were validated with exosomes lamellipodia in VSMCs. Heparin, an inhibitor of exo- produced by HIV-infected cells and with exosomes some internalization, completely blocked the migration isolated from plasma of HIV-infected subjects, but and proliferation. Protein expression of CD9 and not from ΔNef-HIV-infected subjects. Mice injected CD63, an exosomal marker, was significantly higher with exNef displayed altered cholesterol metabolism, in exosomes from WKY than SHR. The expression of monocytosis, increased raft abundance and augmented several microRNAs in SHR exosomes changed com- inflammation. pared with WKY exosomes. Summary/Conclusion: Nef containing exosomes Summary/conclusion: These results suggest that potentiate pro-inflammatory responses by impairing plasma exosomes play physiological, but not patholo- cholesterol metabolism and reorganizing lipid rafts, gical, role on VSMCs irrespective of their origin (nor- increasing cell susceptibility to HIV infection and con- motensive or hypertensive rats). Further research is tributing to co-morbidities of HIV disease. required for determining whether the changes in mole- Funding: Grants from NIH (HL131473, AI055019), cular profiles of circulating exosomes mediate the American Heart Association (17GRNT33630163), and development of high blood pressure in SHR. NHMRC (GNT1036352).

LBF02.03 LBF02.04

Exosomes derived from human mesenchymal stem cells ameliorates Exosomes containing HIV protein Nef reorganize lipid rafts inducing autistic-like behaviours in BTBR and Shank3 mice models of autism inflammatory responses of immune cells and increasing their Nisim Peretsa, Ehud Maromb, Yona Geffenc, Uri Danond, Oded Orone, Evan susceptibility to HIV infection Eliote and Daniel Offenf Dmitri Sviridova and Michael Bukrinskyb a a b Sagol School of neuroscience, Tel Aviv University, Israel, Tel aviv, Israel; Baker Heart and Diabetes Institute, Melbourne, Australia; George bStem Cell Medicine LTD, Jerusalem, Israel., Jerusalem, Israel; cStem Cell- Washington University School of Medicine, Washington, USA Medicine LTD, Jerusalem, Israel., Jerusalem, USA; dStem Cell Medicine LTD, Jerusalem, Israel, Jerusalem, Israel; eFaculty medicine, Bar-Ilan University, Introduction: HIV disease is associated with co-mor- Israel, Ramat Gan, Israel; fSagol School of neuroscience, Tel Aviv University, Israel, Sacklar school of medicine, department of human genetics and bio- bidities, which persist even after successful application chemistry Tel Aviv University, Israel., Tel Aviv, USA of antiretroviral therapy. One important factor responsible for these effects is exosomes containing the main Introduction: Autism spectrum disorders (ASD) are HIV pathogenic factor, Nef, which can be found in neurodevelopmental disorders characterized by three blood of HIV subjects with undetectable HIV load. In core symptoms that include severe impairment of this study, we investigated the effect of Nef-containing social interaction and communication skills, increased exosomes on uninfected macrophages and T cells. repetitive behaviours and cognitive inflexibility. Rate of Methods: Exosomes were purified by differential cen- ASD is steadily increasing in children over the past trifugation from HEK293 cells transfected with Nef- several years, with no effective treatment. expressing or empty vector, monocyte-derived human Methods: BTBR and Shank3 are accepted mouse mod- macrophages infected with Nef-positive or Nef-defi- els used for evaluating autistic-like behaviours as it cient HIV-1, or plasma of uninfected subjects or presents all core symptoms and genetic human muta- patients infected with wild-type or Nef-deficient HIV- tion of ASD. We have previously shown that transplan- 1. Exosomes were adjusted by protein content and tation of human bone marrow mesenchymal stem cells added to cells at 0.2 ng/ml of Nef protein. Mice were (MSCs) to the lateral ventricles of BTBR mice results in injected with Nef exosomes IP (2 μg per injection) 3 long lasting improvement in their autistic behavioural times a week for 2 weeks. phenotypes. Recent studies point of exosomes as the Results: Exosomes containing HIV protein Nef (exNef) main mediators of the therapeutic effect of MSCs. are internalized by macrophages altering cholesterol Results: Here we show that intranasal administration metabolism and causing sharp increase in the abun- of exosomes derived from bone marrow or adipose dance of lipid rafts through reduced activation of small tissue MSCs, ameliorate autistic-like behaviour in 254 ISEV2019 ABSTRACT BOOK

BTBR and Shank3 mice. Including significant increase almost completely removed. Especially, aOMVs were of social interaction and ultrasonic vocalizations, observed to induce less inflammation in macrophages reduced repetitive behaviours and improve maternal compared to OMVs. In addition, immunization with behaviours of pup retrieval. No negative safety symp- aOMVs induced strong IgG antibody response to the toms were detected following exosomes intranasal bacterial proteins, and a greater increase in IFN- treatments in mice. gamma from CD4+ T cells compared to OMVs. Summary/conclusion: The beneficial effects of the Moreover, aOMV-immunized mice showed dramati- exosomes treatment in mice models may be translated cally reduced lung inflammation caused by bacterial to a novel, easy to administer, therapeutic strategy to challenge. reduce the symptoms of ASD. Summary/conclusion: This study shows that aOMVs Funding: Partially by Stem Cell Medicine LTD and can be produced in a large amount with high purity, Kamin and have protective effect against P. aeruginosa- induced pneumonia through a balanced humoral and cellular immune response, suggesting that aOMVs may LBF02.05 be novel bacterial vesicle-mimetics to clinically applicable to infectious diseases. Funding: This work was supported by Swedish Heart- The use of artificially produced bacterial vesicles as an immunotherapeutic vaccine against Pseudomonas aeruginosa Lung Foundation (20150588). pneumonia Kyong-su Parka and Jan Lötvallb aKrefting Research Centre, University of Gothenburg, Gothenburg, Sweden; LBF02.06 bKrefting Research Centre, Institute of Medicine at the Sahlgrenska Academy, University of Gothenburg, Göteborg, Sweden, Gothenburg, Sweden Herpesvirus infection of infant tonsil mucosal epithelia containing intravesicular Human Immunodeficiency Virus induces release of HIV Introduction: Pseudomonas aeruginosa is an opportu- from epithelial cells nistic pathogen which is involved in pneumonia and Rossana Herrera, Kristina Rose and Sharof M. Tugizov cystic fibrosis. Immunization with outer membrane University of California San Francisco, San Francisco, USA vesicles (OMVs), which has naturally budded off from bacteria, is an evolving field in infectious diseases Introduction: Mother-to-child transmission (MTCT) due to their highly immunogenic properties. However, of HIV is an important pathway for the spread of the OMVs are difficult to produce naturally in large quan- virus from mother to child; however, the molecular tities with high purity. This study aims to develop an mechanisms of HIV MTCT are poorly understood. artificial OMVs (aOMVs) isolation method, and to Our recent work showed that >90% of virions inter- investigate the protective effects of aOMVs against P. nalized into polarized infant tonsil epithelium were aeruginosa-induced pneumonia. sequestered, that is, trapped in the endosomes, includ- Methods: Outer membranes were obtained from P. ing multivesicular bodies (MVBs) and vacuoles of aeruginosa by lysozyme and detergent treatment, fol- epithelial cells, for up to 9 days. The primary goal of lowed by ionic stress and applied mild energy to gen- this work was to investigate the role of common oral erate aOMVs. The yield and purity of aOMVs were viral pathogens herpes simplex virus-1 (HSV-1), analysed by nanoparticle tracking analysis and trans- human cytomegalovirus (HCMV), and Epstein-Barr mission electron microscopy. The protein and RNA virus (EBV) in the release of endosomal HIV, which contents were examined by label-free quantitative may play a role in HIV MTCT. mass spectrometry and bioanalyzer. Inflammation was Methods: Polarized tonsil epithelial cells were incu- evaluated in lung macrophages by measuring cytokine bated with HIV-1. After 4 h, the extracellular virus release. Naturally produced OMVs or aOMVs were was removed, and cells were maintained for 3 days. weekly injected in mice for 3 weeks, and then blood Cells were then infected with HSV-1, HCMV, and and spleen were obtained for antibody titer and sple- EBV. AP and BL medium was independently collected nocyte cytokine study. Lung inflammation by P. aeru- after herpesvirus infection and HIV release was exam- ginosa challenge was assessed in H&E stained lungs. ined by p24 ELISA assay. Results: The aOMVs were isolated in higher yield Results: Our data showed that the infection of HSV-1, (fivefold) compared to OMVs. They had similar sphe- HCMV and EBV in tonsil epithelial cells containing rical shape and size as OMVs, but had better purity. intravesicular HIV-1 led to the release of HIV virions, Outer membrane components were more enriched in which were infectious for peripheral blood mononuc- aOMVs, and cytosolic protein and RNA contents were lear cells. HIV release was correlated with the reduction JOURNAL OF EXTRACELLULAR VESICLES 255 of TER in HSV-1-, HCMV- and EBV-infected polar- infectivity of HCV released from syntenin expressing ized epithelial cells; that is, herpesvirus-induced depo- hepatoma cell and PHHs was more resistant to neu- larization of epithelial cells was critical for HIV release. tralization by E2-specific antibodies and chronic-phase HSV-1 and HCMV infection in tonsil epithelial cells patient serum. Last, high E2/syntenin levels in sera substantially increased the expression of GTPases correlates to lower serum neutralization capability. Rab27a and Rab27b, which may regulate the movement Summary/conclusion: E2- and syntenin-containing of MVBs and vacuoles to the plasma membrane and exosomes is a major type of particles released from their fusion with the epithelial cell membrane. cells high expressing syntenin. Efficient production of Summary/conclusion: HSV-1- and HCMV-induced E2-coated exosomes in hepatoma cells and PHHs ren- activation of Rab27a and Rab27b in epithelial cells ders HCV infectivity less susceptible to antibody containing intravesicular HIV may promote virus neutralization. release, that is, exocytosis of virions. HSV-1-, HCMV- Funding: This work was supported by from the strate- and EBV-induced depolarization of tonsil epithelial gic priority research program of the Chinese Academy cells also may play critical in the release of endosomal of Sciences (XDB29010000), the National Science and HIV. Herpesvirus interaction with infant tonsil epithe- Technology Major Project of the Ministry of Science lial cells containing HIV may lead to the release and and Technology of China (2015CB554300 and spread of HIV into CD4+T lymphocytes, macrophages 2016YFC1200400) and the National Nature Science and Langerhans/dendritic cells, leading to HIV MTCT. Foundation of China (81761138046). Work by R.B. Funding: R01DE028129, NATIONAL INSTITUTE OF was supported by the Deutsche DENTAL & CRANIOFACIAL RESEARCH Forschungsgemeinschaft, collaborative research center (TRR) 179, TP9. LBF02.07 LBF02.08

Syntenin regulates Hepatitis C virus sensitivity to neutralizing antibody by promoting E2 secretion through exosomes Lipidomics profiles of plasma microvesicles differ in experimental Libin Deng and Gang Long cerebral malaria, compared to malaria without neurological complications Institut Pasteur of Shanghai, Shanghai, China (People’s Republic) Amani M. Batarseha, Elham Hosseini-Beheshtib, Alex Chenc, Amy Cohenb, Annette Juillardd, Michael Marianie and Georges Graub Introduction: Hepatitis C virus (HCV) is a major aBCAL Dx, Eveleigh, NSW, Australia 2015, Eveleigh, Australia; bVascular cause of chronic liver disease, infecting approximately Immunology Unit, Faculty of Medicine & Health, University of Sydney, Camperdown, NSW, Australia 2050, Camperdown, Australia; cThermo 71 million people worldwide. Assembly of infectious Fisher Scientific, Scoresby, VIC, Australia 3179, Scoresby, Australia; HCV particles involves host lipoproteins, giving rise to dVascular Immunology Unit, Faculty of Medicine & Health, University of Sydney, Camperdown, NSW, Australia 2050, Sydney, Australia; eThermo unique lipo-viro-particles (LVPs), but proteome studies Fisher Scientific, North Ryde, NSW, Australia 2113, North Ryde, Australia suggest that additional cellular proteins are associated with HCV virions or other particles containing the Introduction: Cerebral malaria (CM), a fatal complica- viral envelope glycoprotein E2. Many of these host tion of Plasmodium infection affecting children in sub- cell proteins are common markers of exosomes, most Saharan Africa and adults in South-East Asia, results notably the intracellular adaptor protein syntenin from incompletely understood pathogenetic mechan- required exosome biogenesis. These observations sug- isms, which include sequestration of infected erythro- gest that E2 might be a component of both LVPs and cytes, cytokine overproduction, accumulation of exosomes produced from HCV infected cells. inflammatory cells, and excessive release of microvesi- Methods: Using HCVcc in both hepatoma cells and cles (MV). Plasma MV levels are elevated in CM primary human hepatocytes (PHHs), we studied bio- patients and in the experimental mouse model. Here, genesis and function of E2-coated exosomes. MV lipidomics profile was studied in relation to the Results: Knockout of syntenin had negligible impact development of cerebral complications. on HCV replication and virus production whereas Methods: Plasma MV was enriched using differential ectopic expression of syntenin at physiological level centrifugation (El-Assaad 2014). Lipids were extracted reduced intracellular E2 abundance concomitant with according to Matyash et al. (2008), loaded on a C30 increased secretion of E2-coated exosomes. Acclaim column using a Vanquish liquid chromatogra- Importantly, cells expressing syntenin and HCV struc- phy (LC) system and analysed using a Fusion mass tural proteins efficiently released exosomes containing spectrometer (MS). LipidSearch software was used for E2 but lacking the core protein. Furthermore, lipid species annotation and quantification. 256 ISEV2019 ABSTRACT BOOK

Results: We compared lipid profiles in circulating MV significant up- or down-regulation in both biological purified from CBA mice with P. berghei ANKA (PbA), samples. which causes CM, to those from P. yoelii (Py), which Results: We were able to quantitate 13,013 peptides, does not. Plasma MV produced at the time of CM which corresponds to 1264 proteins from two biologi- dramatically differed from those from non-CM mice, cal replicates. Thirty-two differentially expressed pro- in spite of identical levels of parasitaemia: using high- teins were shortlisted, among them some are nuclear resolution LCMS, we identified over 200 lipid species protein and protein relevant to lipid metabolism. within 12 lipid classes. Total phosphatidylethanolamine Further pursuing this, we treat hepG2 with ABL006, (PE) levels were significantly higher in MV from PbA and study the differential protein expression in the mice compared to those from uninfected control and conditioned medium, hoping to understand further Py. Using fragmentation MS, we identified that this PE the lipid regulating action of ABL006. The differentially increase is due at least in part to PE (16:0_22:6), PE expressed proteins between treated and non-treated (18:0_22:6) and PE (18:1_22:6) species identified in were short-listed to 33 proteins. These proteins were PbA vs Py and uninfected control. Total phosphatidyl- checked against the 100 top expressing proteins serine (PS) was significantly higher in both PbA and Py secreted by the exosomes (Exocarta, http://exocarta. compared to uninfected control. Conversely total lyso- org/index.html). Out of 33 most significantly regulated phosphatidylcholine (LPC) and lysophosphatidyletha- proteins, 8 were exosomal markers, and almost all were nolamine (LPE) were significantly lower in PbA down-regulated upon ABL006 treatment. compared to uninfected mice, while they were Summary/conclusion: This suggested that exosomes unchanged in Py MV. release from hepG2 is reduced upon ABL006 Summary/conclusion: These results suggest, for the treatment. time, that experimental CM is characterized by specific Funding: MOST 107-2632-B-324-001. changes in lipid composition of circulating MV, pointing towards PE subsets, LPC and LPE as potential important players in CM pathogenesis. LBF02.10 Funding: NHMRC Project grant APP1099920 to GG.

Placental cells function as environmental sensors that respond to changes in the extracellular milieu via extracellular vesicles LBF02.09 Carlos Palmaa and Carlos Salomonb aExosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane QLD 4029, Australia, b Compound extracted from cinnamomum osmophloeum leaves Brisbane, Australia; Exosome Biology Laboratory, Centre for Clinical reduced exosomes release from hepG2 cells Diagnostics, University of Queensland Centre for Clinical Research, Royal Wei-chi Kua, Shu-yu Yangb, Jen Ying Lib and Meng-Jen Leec Brisbane and Women’s Hospital, The University of Queensland, Brisbane QLD 4029, Australia., Brisbane, Australia aFu Jen Catholic University, New Taipei, USA; bTsu-chi Hospital, Taichung, Taiwan (Republic of China); cDepartment of applied chemistry, Taichung, USA Introduction: Placenta-derived extracellular vesicles (PdEVs) are present in maternal circulation as early Introduction: Cinnamomum osmophloeum belongs to as 6 weeks of gestation. Changes in the concentration the genus of Cinnamon, the same genus as the species of PdEVs are found in gestational diabetes, preeclamp- used for commercially sold cinnamon. Compounds of sia and preterm birth. The aim of this study was to the extracted Cinnamomum osmophloeum leaves have characterize the release and biogenesis of EVs from good potential to be developed into new drugs. placental cells in response to extracellular glucose, Further, usage of the leaves of the tree is much more insulin, lipopolysaccharide (LPS) and tumour necrosis sustainable and cost effective than the bark. ABL006 is factor a (TNF-a) in vitro. a major compound isolated from Cinnamomum Methods: Bewo cells were used as a placental model. osmophloeum that previously known for insulin Cells were incubated with forskolin for 24 h to stimu- mimetick effect. For fear of side effect of pro-inflam- late syncytium formation in vitro. After syncytializa- matory effect to the central nervous system, we tested tion, cells were incubated in the presence of forskolin using proteomic approach to study differential protein with D-glucose (5 mM or 25 mM), insulin (1 nM), LPS expression after ABL006 treatment in astrocytic cells. (0–10 μg/ml) and TNF-a (0–20 ng/ml) for 48 h. EVs Methods: We used dimethyl labelling on the peptide were isolated from cell-conditioned media by differen- level and LC-MS/MS to select differentially expressed tial centrifugation and characterized by their size dis- proteins. The selection criterion was based on tribution, protein abundance and morphology using JOURNAL OF EXTRACELLULAR VESICLES 257 nanoparticle tracking analysis, Western blot and elec- Methods: Plasma samples were collected from preg- tron microscopy, respectively. The effect of the extra- nant women during the first trimester of pregnancy cellular milieu on the release of PdEVs was evaluated in (n = 10). EVs were isolated through differential centri- four different subpopulations according to size; <50, fugation, at 2000g for 30 min (pellet 1); 12,000g for 50–150, 150–200 and >200 nm. 45 min (pellet 2) and at 100,000g for 120 min (pellet 3). Results: Differential changes in the release of PdEVs The supernatant after the last centrifugation was subpopulations in response to glucose, insulin, LPS and termed “soluble fractions”. EVs were characterized by TNF-a were observed. High glucose induced the release size distribution, abundance of proteins associated with of EVs <50 nm, and >200 nm although this effect was EVs (i.e. CD63, Flotilin-1 and TSG101), negative con- abolished by insulin. High glucose and insulin trol for Grp94, and morphology, according to the decreased the release of EVs 150–200 nm and EVs recommendations of the International Society of 50–150 nm, respectively. The effect of LPS on the Extracellular Vesicles, using Nanoparticle Tracking release of PdEVs was size-dependent with the greatest Analysis (NTA), Western blot analysis and electron effect on EVs of >200 nm. Finally, TNF-a increased the microscopy, respectively. The concentration of IL-10, release of EVs in size and concentration-dependent IL-6, IFN-γ and TNF-a in the EVs and the soluble manner with a maximum effect on EVs >200 nm and fractions were established by cytokine array analysis 2 ng/ml. Changes in the release of exosomes were (Bioplex-200). associated with a differential abundance of proteins Results: Specific changes in the levels of cytokines, in associated with ESCRT machinery. the different population of vesicles, and in the soluble Summary/conclusion: The effect of the extracellular fractions were identified. The levels of IL-10, IL-6, IFN- milieu on PdEVs release may be recapitulated and is γ and TNF-a were significantly higher (p < .05) in the of clinical relevance in vivo in association with hyper- exosome fraction (pellet 3) compared to the values glycaemia (glucose and insulin), infection (LPS) and observed in pellet 1 and pellet 2 (macro and micro- inflammatory (TNF-a) conditions. vesicles fractions). The levels of IL-10, IFN-γ and TNF- Funding: Lions Medical Research Foundation, a were significantly higher (p < .05) in the soluble National Health and Medical Research Council fractions compared with the exosomal fraction. No (NHMRC; 1114013), Fondo Nacional de Desarrollo significant difference in the level of IL-6 in the exoso- Científico y Tecnológico (FONDECYT 1170809), and mal and soluble fraction was observed. CONICYT PFCHA/DOCTORADO BECAS CHILE/ Summary/conclusion: This study established that cyto- 2018-72190513 kines are packaged within EVs (in which these molecules are protected), suggesting a novel mechanism of LBF02.11 action through which cytokines via EVs can lead to distal interactions. Funding: Lions Medical Research Foundation, Association of cytokines with circulating populations of extracellular National Health and Medical Research Council vesicles at early gestation Katherin Scholz-Romeroa, Andrew Laia, Carlos Palmaa, Gregory Duncombea, (NHMRC; 1114013), and Fondo Nacional de Gregory Ricea and Carlos Salomonb Desarrollo Científico y Tecnológico (FONDECYT aExosome Biology Laboratory, Centre for Clinical Diagnostics, University of 1170809). Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane QLD 4029, Australia, Brisbane, Australia; bExosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal LBF02.12 Brisbane and Women’s Hospital, The University of Queensland, Brisbane QLD 4029, Australia., Brisbane, Australia Mesenchymal stem cell-derived exosomes attenuate inflammation Introduction: Cytokines have several roles across and protect ischemic neuronal damage a b b gestation, including implantation, placentation and May-Jywan Tsai , Dann-Ying Liou and Henrich Cheng immune response, which are all essential for the con- aNeurological Institute, Taipei Veterans General Hospital, Taipei, Taiwan (Republic of China); bNeurological Institute, Taipei Veterans General tinuation of pregnancy. The aim of this study was to Hospital, Taiwan, Taipei, Taiwan (Republic of China) isolate and characterize different populations of extracellular vesicles (EVs) from maternal plasma at the first Introduction: Bone marrow mesenchymal stem cells trimester of pregnancy, and quantify the levels of inter- (BM-MSC) are the most extensively used stem cells in leukin 10 (IL-10), 6 (IL-6), Interferon gamma (IFN-γ), tissue engineering due to their easy access, rapid ex and tumour necrosis factor a (TNF-a), associated vivo expansion and poor immunogenicity. MSCs with EVs. secrete various factors that can modulate a hostile 258 ISEV2019 ABSTRACT BOOK environment, which is called the paracrine effect. in neuron-glial cultures. Further comparison of the MSCs have a strong capacity for secretion of exosomes beneficial effects of MSCcm and MSCexo will be con- which are suspected to participate in paracrine cellular ducted in in vivo SCI rats via vascular administration. communication. Summary/conclusion: This cell-free therapy, avoiding Methods: We compare the effects of BM-MSC condi- the risks associated with direct MSC transplantation, tioned medium (MSCcm) and MSC-derived exosomes may improve nerve regrowth and functional recovery (MSCexo) in neuron-glial cultures as well as in spinal after SCI. cord injured (SCI) rat model. Funding: Research grant (V107C-087) from the Taipei Results: We found that both MSCcm and MSCexo Veterans General Hospital in Taiwan, and grants (106- were effective in reducing LPS stimulation and oxygen 2314-B-075-023 & 107-2314-B-075-021) from the glucose deprivation, an in vitro stroke model, damage Ministry of Science and Technology in Taiwan. JOURNAL OF EXTRACELLULAR VESICLES 259

Symposium Session 19: EV Cargo Profiling Chairs: Tang-Long Shen, Lei Zheng Location: Level B1, Lecture Room 16:30–18:00

Summary/Conclusion: The heterogeneity of exRNA OF19.01 cargo types exceeds the capabilities of current experimental methods to reproducibly isolate defined carrier subpopulations from human biofluids. While this pro- Distinct extracellular RNA cargo types associate with specific vesicular and non-vesicular RNA carriers across human biofluids blem calls for the development of new carrier isolation Aleksandar Milosavljevic methods, we have now demonstrated the power of Department of Molecular and Human Genetics, Baylor College of Medicine, computational deconvolution to complement and Houston, USA enhance current isolation methods and have created the first comprehensive survey of exRNA cargo types Introduction: The Extracellular RNA Communication and their carriers in human biofluids. Consortium (ERCC) has developed the ExRNA Atlas, a Funding: Common Fund of the NIH (5U54 reference catalogue of exRNAs present in human bio- DA036134). fluids. A computational deconvolution analysis identified six RNA cargo types (CT1, CT2, CT3A, CT3B, CT3C, CT4) present across human biofluids repre- OF19.02 sented within the Atlas. Extensive experimental analyses by the ERCC show association of these cargo types with specific vesicular and non-vesicular (lipo- Heparan sulphate glycosaminoglycans on the extracellular vesicle surface bind a variety of proteins protein, RNP particle) carriers. Sara Veigaa, Alex Shepharda, Alex Cocksa, Aled Claytona and Jason Webberb Methods: To identify carriers for the six CTs, we aTissue Microenvironment Group, Division of Cancer and Genetics, School performed: cushioned density gradient ultracentrifuga- of Medicine, Cardiff University, Cardiff, UK; bCardiff University, Cardiff, UK tion of serum and plasma using the OptiPrep density gradient, RNA-seq, western blot and mass spectrome- Introduction: Cancers develop in complex tissue try analysis of the density fractions; RNA-seq profiling environments, comprising multiple cell types that con- of ultracentrifugation products, of size fractionation tribute to tumour growth, invasion and metastasis. Our using nanoscale deterministic lateral displacement group has previously demonstrated that prostate cancer (nano-DLD) arrays; of lipoprotein fractions; and of derived EVs mediate the delivery of TGFβ, via heparan AGO-1 immuno-pulldowns. We also carried out sulphate (HS) glycosaminoglycans on the EV surface RNA-seq of a shared pool of human plasma and and stimulate fibroblast to myofibroblast differentia- serum samples processed using 10 widely used RNA tion. Given the potential capacity for HS to bind isolation methods. other “soluble” factors we have herein explored the Results: CT1 correlates with RNA-seq profiles of car- repertoire of proteins associated vesicular HS. riers of exosomal size and density (OptiPrep fractions Methods: EVs were isolated from DU145 prostate can- 4–7 that are CD9 and Flotillin positive). CT2 correlates cer cells by differential centrifugation followed by with the exRNA profiles of lipoprotein carriers (HDL, ultra-centrifugation on a sucrose cushion and washed LDL, VLDL, Chylomicron); the carrier shows HDL with PBS. Specific removal of Heparan sulphate side density (OptiPrep fractions 9–12) and is APOA1 posi- chains from the vesicle was performed by enzymatic tive. CT3A and B show high miRNA content and digestion using heparinase III (HEPIII). Differences in correlate with RNA-seq profiles of AGO2 immuno- proteins with vs. without digestion were identified by a pulldowns. CT4 correlates with the RNA-seq profiles sensitive multiplex proximity extension assay and select of both low-density vesicles (OptiPrep fractions 1-3) targets validated by ELISA. and HMC-1 cell-line derived vesicles of higher-density. Results: Protein profiles identified approximately 60 The 10 widely used commercial RNA isolation kits factors that were significantly differentially expressed show distinct preferences for specific CT subsets. On on control versus HS-deficient EV’s. Some but not all average, across all kits, CT4 was captured at highest of these have been previously identified as HS-asso- and CT3B at second-highest relative abundance. ciated factors. Gene ontology analysis points to 260 ISEV2019 ABSTRACT BOOK functional relationships with angiogenesis, invasion by differential centrifugation and characterized by SEM and immune regulation. Using ELISA, we have been and immunoblotting. Total lipids from EVs and their able to quantify six selected candidates on wild type parental cells were analysed by LC/MS-MS. vesicles, some of these are lost following HS-digestion. Results: EVs released by HuH7 cells expressed positive We went on to examine functional consequences of markers (Alix and CD63), whose levels were higher in HS-deficiency in relation to cell-uptake, and angiogenic PA-EVs. SEM images showed vesicles with the typical responses. cup-shaped morphology and size, not altered by the Summary/Conclusion: These data demonstrate a various treatments. Lipid composition comparison of diverse repertoire of proteins that are bound to the EVs released by treated- and control cells showed a surface of exosomes via HS glycosaminoglycans. We higher content of all phospholipid subclasses, in PA- anticipate that removal of EV-associated HS will result EVs, except for phosphatidic acid and phosphatidyli- in attenuated delivery of multiple factors to recipient nositol, whereas no differences were observed in EVs cells, and this will have major implications on EV released by OA-, Tg- or SCD1i-treated cells. Specific functions and their ability to modulate tissue differences have been observed in the levels of numer- environments. ous phospholipid molecular species in PA-EVs as com- Funding: Cancer Research Wales. pared to other treatments. Summary/Conclusion: In HuH7 cells, the most relevant changes in lipid composition were observed in OF19.03 PA-EVs, even if all treatments, except OA, induced ER stress. These results suggest that the increase of membrane phospholipid saturation is crucial to deter- Membrane lipid saturation modifies the lipid signature of extracellular vesicles released by HuH7 hepatocarcinoma cells mine which lipids discarded via EVs. Eva Costanzia, Yuta Shimanakab, Lorena Urbanellic, Krizia Saginic, Stefano Giovagnolid, Hiroyuki Araie, Carla Emilianic and Sandra Burattac aDepartment of Chemistry, Biology and Biotechnology (University of OF19.04 Perugia), Foligno, Italy; bGraduate School of Pharmaceutical Sciences (University of Tokyo), Tokyo, Japan; cDepartment of Chemistry, Biology and Biotechnology (University of Perugia), Perugia, Italy; dDepartment of Deconvolution analysis of small RNAseq data from exRNAs isolated Pharmaceutical Sciences (University of Perugia), Perugia, Italy; eGraduate using different methods reveals multiple carrier subclasses and School of Pharmaceutical Sciences (University of Tokyo), Perugia, Italy identifies optimal methods for isolation of total and EV-specific exRNA Introduction: Extracellular vesicles (EVs) are mem- Srimeenakshi Srinivasana, Ashish Yerib, Pike See Cheahc, Kirsty M. Danielsond, Peter DeHoffa, Justyna Filante, Anu Paulf, Ravi Shahb, Bridget brane-limited particles released by cells during physio- Simonsonb, Irene Yang, Cuong Trana, Xuan Zhangc, Leonora Balajh, Xandra logical and pathological conditions involved in cell Breakefieldi, Roopali Gandhif, Jodi Lapidusj, Eric Londink, Tushar Patelg, l e b m communication. An increased ceramide-enriched pro- Robert Raffai , Anil Sood , Saumya Das and Louise Laurent inflammatory EV release has been demonstrated in in aUCSD, San Diego, USA; bCardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, USA; cNeuology and vitro lipotoxic conditions and in non-alcoholic steatohe- Radiology Services and program in Neuroscience, Harvard Medical School, patitis mouse models and patients. So far, lipid profile Massachusetts General Hospital, Boston, USA; dUniversity of Otago, Wellington, Wellington, New Zealand; eThe University of Texas MD changes in EVs released under lipotoxic conditions have Anderson Cancer Center (MDACC), Houston, USA; fCenter for Neurologic not been investigated, despite the evidence that EVs Diseases, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA; gMayo Clinic Florida, Jacksonville, USA; hMassachusetts General shuttle many membrane-derived bioactive lipids playing Hospital, Boston, USA; iNeurology and Radiology Services and program in crucial role in several processes, including inflammation. Neuroscience, Harvard Medical School, Massachusetts General Hospital, j In this study, we carried out a comprehensive lipidomic Charlestown, USA; Biostatistics & Design Program, Oregon Health and Science University, Oregon Health & Science University – Portland State analysis of EVs released by HuH7 cells under membrane University School of Public Health, Portland, USA; kSidney Kimmel College lipid saturation conditions induced by lipotoxic palmi- of Medicine, Thomas Jefferson University, Philadelphia, USA; lUniversity of California & VA Medical Center, San Francisco, San Fransisco, USA; tate (PA) or Δ9 desaturase inhibition (SCD1i). Since mUCSD, La Jolla, USA membrane lipid saturation induces ER stress, HuH7 cells were also treated with Thapsigargin (Tg), a con- Introduction: Reproducibility has been a major chal- ventional ER stress inducer, and with oleate (OA), a lenge in extracellular RNA (exRNA) research both due nontoxic monounsaturated fatty acid. to low concentration and heterogeneity of exRNA car- Methods: EVs were isolated from culture media of riers in biofluids, such as EVs, RNPs and LPPs. Lack of HuH7 cells treated for 16 h with fatty acids (400 μM), knowledge regarding the efficiency/reproducibility of or Tg (2.5 nM), or SCD1i (CAY 10566, 5 μM). All different isolation methods in accessing the exRNAs treatments were performed in serum-free medium con- in different carriers has hindered rational selection of taining 0.1% free fatty acids-BSA. EVs were recovered standardized methods. JOURNAL OF EXTRACELLULAR VESICLES 261

Methods: Using small RNAseq, we compared the per- production. However, the direct effect of SR1 on EC formance of 10 exRNA isolation methods on standar- biology and EV production is largely unknown. dized samples of five biofluids across multiple Methods: Human umbilical vein EC (HUVEC) and laboratories. We found that the read depth required HSPC were obtained per approved IRB protocol. EC to maximize miRNA complexity in each biofluid was culture and EC-HSPC in vitro co-culture was performed different: 1 million in Bile (~200 detected miRNAs), 0.5 as described previously. EC-EV harvest was collected in million in Cell culture supernatant (~300), 2 million in serum free medium. The effect of SR1 on HUVEC Plasma/Serum (~450), and 50,000 in Urine (~100). growth was evaluated by EC-colony-forming cell (EC- While the miRNA profiles varied greatly among CFC) assay. HSPC population analysis was done by exRNA isolation methods in Plasma, Serum, and Bile, FACS. EV was purified by ultracentrifugation. EV pro- Cell culture supernatant and Urine showed similar teins were identified by LC-MS/MS. Student t-test was profiles for all tested methods. performed with p < 0.05 for statistically significance. Results: We performed small RNAseq on purified Results: HSPC co-cultured with HUVEC in the pre- exRNA carriers from Plasma and Serum; and used sence of SR1 results in a ~ 2-fold increase of more the resulting carrier-specific miRNA signatures to primitive HSPC subpopulation compared to the con- computationally deconvolute the miRNA profiles trol group. SR1 treated HUVEC leads to a significant from each of the isolation methods. We found that >2-fold increase in EC-CFC numbers and >67% ExoRNeasy, ME, and Ultracentrifugation purified increases in the colony diameter. A total of 327 pro- miRNAs that were predominantly carried in EVs, teins were detected in ECs derived from HUVEC. As a while Exiqon, ExoQuick, and Norgen isolated both quality control, many commonly reported “EV- EV- and AGO2+ RNP-associated miRNAs. enriched” proteins per “ISEV position statement” Summary/Conclusion: Our studies identified several were identified. A small fraction of proteins recovered factors that contribute to difficulties with reproducibil- from the EV fraction (<2%) is found on EC membrane. ity in exRNA studies, like inefficient and variable Among the 327 proteins, 46 of them showed a signifi- exRNA isolation for many of the available methods, cant change with SR1 treatment. Functional annotation differences in accessibility of miRNA cargo associated by DAVID bioinformatics enrichment tools classified with different carriers among methods, and insufficient three EV proteins associated with enhanced angiogen- sequencing depth. To help investigators select an opti- esis signalling pathways. mal method, we developed an interactive web-based Summary/Conclusion: SR1 differentially regulates application, miRDaR, that will provide a ranked list angiogenic EV production that associates with of tested exRNA isolation methods by complexity/ increased robustness in endothelial growth and expression level and reproducibility, specific to their enhanced haematopoiesis. Future investigations on biofluid and miRNA of interest. the biological effects of HUVEC EV differentially pro- Funding: This study was supported by the Extracellular duced by SR1 are in progress and needed. RNA Communication Consortium funded by the NIH Common Fund. OF19.06 OF19.05 Evaluation of circulating extracellular vesicles derived miRNAs as biomarkers of early colon cancer: a comparison with plasma total miRNAs Novel angiogenic extracellular vesicles induced by StemRegenin1 Li Mina, Shengtao Zhua, Lei Chena, Xiang Liub, Rui Weia, Libo Zhaob, Yuqing Yen-Michael Sheng Hsua, Jae-Hung Shiehb, Taojunfeng Suc, Zhen Zhaoa Yangb, Guanyi Kongb, Peng Lic and Shutian Zhangc aDepartment of Pathology and Laboratory Medicine, Weill Cornell Medicine, aDepartment of Gastroenterology, Beijing Friendship Hospital, Capital New York, USA; bDepartment of Medicine, Weill Cornell Medicine, New Medical University, Beijing, China (People’s Republic); bEcho Biotech Co., York, NY, USA; cProteomics&Metabolomics Core Facility, Weill Cornell Ltd, Beijing, China (People’s Republic); cDepartment of Gastroenterology, Medicine, New York, NY, USA Beijing Friendship Hospital, Capital Medical University, Beijing, P. R. China

Introduction: Aryl hydrocarbon receptor antagonists, Introduction: Early diagnosis of colon cancer (CC) is such as StemRegenin1 (SR1), have been recently shown clinically important, as it can significantly improve to enhance expansion of hematopoietic stem progeni- patients’ survival rate and quality of life. Although the tor cells (HSPCs). Our preliminary data showed that potential role for small extracellular vesicles (sEVs) in SR1 enhances endothelial cells (EC) to promote HSPC early detection of many diseases has been repeatedly expansion possibly via direct and indirect intercellular mentioned, systematic screening of plasma sEVs interactions, including extracellular vesicle (EV) derived early CC biomarkers has not yet been reported. 262 ISEV2019 ABSTRACT BOOK

Methods:PlasmasEVswerederivedfrom15early consisting of 134 participants (58 CC and 76 NC). In stage CC patients and 10 normal controls (NC) and the validation cohort, the AUC of 4 individual miRNAs characterized according to MISV2014 standard. The ranged from 0.680 to 0.792. A logistic model combin- total circulating sEVs derived microRNA (miRNA) ing two miRNA (i.e. let-7b-3p and miR-145-3p) expression profile of all participants was investigated achieved an AUC of 0.901. Adding the 3rd miRNA by next-generation sequencing (NGS). Selected (miR-139-3p) into this model can further increase the miRNA candidates were further verified in both AUC to 0.927. Side by side comparison revealed that plasma-derived sEV miRNA and plasma total sEVs miRNA profile outperformed cell-free plasma miRNA of an independent cohort consisting of 134 miRNA in the diagnosis of early CC. participants. Summary/Conclusion: Circulating sEVs have a dis- Results: RNA sequencing identified a total number of tinct miRNA profile in CC patients, and sEVs derived 95 sEVs miRNA with differential expression between miRNA could be used as a promising biomarker to CC and NC, most of which (60/95) was in well accor- detect CC at an early stage. dance with tissue results in the Cancer Genome Atlas Funding: This work was supported by grants from the (TCGA) dataset. Among those miRNAs, we selected National Natural Science Foundation of China let-7b-3p, miR-139-3p, miR-145-3p, and miR-150-3p (81702314). for further validation in an independent cohort JOURNAL OF EXTRACELLULAR VESICLES 263

Symposium Session 20: EV Therapeutics II Chairs: Minh Le; Lucia Languino Location: Level B1, Hall B 16:30–18:00

OF20.01 standalone biological or as a carrier for the targeted delivery of therapeutics, such as anti-inflammatory agents and growth factors. Ongoing in vivo studies Nano-Ghosts: mesenchymal stem cells derived nanoparticles as a are focusing on confirming the NGs’ targeting and novel approach for cartilage regeneration. anti-inflammatory capacity. ’ a b c d Domenico D Atri , Joao Garcia , Laura Creemers and Marcelle Machluf Funding: This project has received funding from the a b Technion Israel Institute of Technology, Haifa, Israel; UMC Utrecht, European Union’s Horizon 2020 research and innova- Utrecht, Netherlands; cDept Orthopaedics, University Medical Centre Utrecht, Utrecht, Netherlands; dTechnion – Israel Institute of Technology, tion programme under Marie Sklodowska-Curie grant Haifa, Israel agreement No 642414

Introduction: Osteoarthritis is the most common inflammatory disease of the joints which is character- OF20.02 ized by cartilage degeneration and bony overgrowth. Mesenchymal stem cells (MSCs) play an essential role Combining virus-based therapeutics and EV therapy for the in inflammation, due to their aptitude to home to treatment of pancreatic cancer inflamed tissues and modulate the process. We Marie-Ève Wedge and Carolina Ilkow designed a new kind of particles termed Nano-Ghosts Ottawa Hospital Research Institute, Ottawa, Canada (NGs), derived from the cytoplasmic membrane of the MSCs. Retaining MSCs’ surface properties, NGs are Introduction:Pancreaticcancer(PC)isahighly expected to target inflamed tissue and modulate aggressive disease with unmet therapeutic needs. inflammation. In this study, we demonstrate NGs’ abil- Recent advances in the use of cancer killing oncolytic ity to target human articular chondrocytes (hACs) and viruses (OVs) as cancer therapeutic agents bring new cartilage explants while reducing inflammation. hope to fight the notorious disease that is PC. Methods: Targeting was evaluated by flow cytometry Although OVs have shown promising results in cer- and confocal microscopy. NGs’ anti-inflammatory tain cancers, some tumours remain resistant to OV properties were studied in vitro on TNF-stimulated therapy due to their inherent residual antiviral and non-stimulated hACs and, ex vivo, on cartilage mechanisms. We hypothesized that the use of OV- explants. qPCR and ELISA of various markers assessed encoded artificial microRNAs (amiRs) could help anti-inflammatory effect. Smooth muscle cell (SMC)- target the cellular antiviral components associated NGs were used as a non-MSC control. with the observed OV resistance and could also Results: Flow cytometry showed that NGs can target sensitize neighbouring tumour cells to OV therapy hACs’ 2 times more efficiently compared to SMC-NGs. and small molecule inhibitors through the secretion Moreover, NGs showed 4 times higher targeting to of amiR-containing extracellular vesicles (EVs) from TNF-stimulated hACs. Targeting was confirmed by infected cells. confocal microscopy and imaging flow cytometry Methods: To find such amiRs, we passaged a viral which showed that NGs bound the membrane and library encoding ~16,000 unique amiRs in several PC were taken up by the cells. Similar results were cell lines and patient-derived xenograft samples to achieved in human explants where the particles showed enrich for sequences that could enhance OV 4 times higher binding to TNF-stimulated explants. To replication. test the anti-inflammatory effect, different markers Results:WeidentifiedanamiRthatimprovesPCcell such as NO2, IL6, PGE2 and MMP13 were analysed. killing (amiR-PC) when expressed from an OV. Our data showed that NGs reduce inflammation by Target identification of amiR-PC revealed ARID1A more than 50% both at the protein and RNA level. as a key player in resistance to OV therapy in PCs. Summary/Conclusion: Here we provide a proof-of- This target is of particular interest since its down- concept for the utility of NGs with intrinsic capabilities regulation acts in a synthetic lethal fashion with inhi- for targeted cartilage regeneration, either as a bition of the EZH2 methyltransferase. Combining an 264 ISEV2019 ABSTRACT BOOK amiR-PC-expressing OV with a small molecule inhi- inhibition of exosome secretion and uptake by GW4869 bitor of EZH2 enhances PC cell death. Moreover, we and E1PA inhibited CD47 expression in ovarian cancer have shown that amiR-PC is packaged in cancer cell- cells, suggesting that CD47 is released from cells via exo- secreted EVs which have theabilitytoreachneigh- somes and thereafter recycled via pinocytosis. The co- bouring naïve cells to sensitize them to EZH2 inhibi- culture assay revealed that the inhibition of exosomal tion-mediated cell death and to spread the OV- CD47 enhanced the phagocytosis of macrophage-like mediated tumour killing effect throughout the cells against cancer cells, which may lead to cancer cell tumour. These results translate into an impressive survival in vivo. improvement in tumour debulking and survival in Summary/Conclusion:CD47expressionwascorre- animal models of highly aggressive PC. lated with poor OS in HGSOC patients, suggesting Summary/Conclusion: This work not only broadens the importance of immune evasion. CD47 was our knowledge on the resistance of select tumours to expressed on exosomes and the inhibition of exosome oncolytic virotherapy and the EV-mediated bystander recycling enhanced the phagocytosis of macrophage- killing effect in OV-infected tumours, but it also pro- like cells against cancer cell through the down-regula- vides new hope for a cure to the grim disease that tion of CD47 expression in cancer cells. Our data is PC. indicates that cancer derived exosomes can be considered as a therapeutic target of HGSOCs. OF20.03 OF20.04

CD47, a “don’t eat me signal” expression in ovarian cancer cells were regulated by circulating exosomes – the therapeutic potential of The impact of in vitro ageing on the release of extracellular vesicles targeting exosomes by inhibiting immune evasion from human mesenchymal stem cells Aasa Shimizua, Kenjiro Sawadab, Masaki Kobayashib, Mayuko Miyamotob Xiaoqin Wanga, Jincy Philipa, Forugh Vazirisanib, Chrysoula Tsirigotia, Peter b a and Tadashi Kimurab Thomsen and Karin Ekström aOsaka University, Sjuita, Japan; bOsaka University, Suita, Japan aDepartment of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, University of Gothenburg, Gothenburg, USA; bDepartment of Biomaterials, Institute of Clinical Sciences, Sahlgrenska Academy, Introduction: CD47, a “don’t eat me” signal, is over- University of Gothenburg, Gothenburg, Sweden expressed on the surface of various tumours to allows tumour immune evasion. However, the role and reg- Introduction: Ageing increases the risk of bone degen- ulation of CD47 in high grade serous ovarian carci- erative diseases, which are partially caused by ageing- noma (HGSOC) remains undetermined. CD47 is related changes in mesenchymal stem cells (MSCs). known to be present on exosomes. Herein, we tested Both in vitro ageing (reflected by the passage number the following hypothesis that CD47 present on exo- in culture) and ageing (donor age) reflect a loss of somes mediates immune evasion of cancer cells from regenerative capacity in terms of the proliferation and macrophages. osteogenic differentiation of MSCs. Extracellular vesi- Methods:PrognosticsignificancesofCD47expressionin cles (EVs) secreted from MSCs have been shown to HGSOCs were examined using a public database includ- exert therapeutic effects that contribute to the regen- ing 1656 HGSOC patients (Kaplan-Meier Plotter; http:// eration of various tissues, but there is scarce informa- kmplot.com/analysis) and validated with 80 HGSCOs tion on whether ageing, particularly in vitro ageing, treated at Osaka University Hospital between 2013 and influences the release of EVs by MSCs. 2017. CD47 expression in exosomes derived from several Methods:Anin vitro ageing model in which low- and HGSOC cell lines were examined by western blot. The high-passage cells (LP and HP MSCs, respectively) effect of exosomal CD47 in HGSOCs was examined by represent “young” and “aged” cells, respectively, was inhibiting exosome secretion with GW4869, or by inhi- utilized. Both LP and HP EVs were characterized by biting exosome uptake with E1PA. Further, the co-culture NTA and WB. The EV protein contents were further experiments of HGSOCs with THP-1-derived macro- explored and the functions of LP and HP EVs on the phage were performed and the effect of exosomal CD47 survival and proliferation of MSCs were investigated. on phagocytosis was analysed. Results: The results showed that in vitro ageing Results:HighCD-47expressionwascorrelatedwithpoor retained the phenotypic signature of MSCs but resulted prognoses of HGSOC patients compared with low CD-47 in morphological changes and decreases in the prolif- expression (19.0 months vs. 23.6 months in overall survival eration and osteogenic differentiation capacity of the (OS)). Exosomes derived from SKOV3ip1, OVCAR3 and cells. Both LP and HP MSCs secreted EVs with similar A2780 cell lines showed strong CD47 expression. The characteristics in terms of size and typical exosomal JOURNAL OF EXTRACELLULAR VESICLES 265 protein markers, but HP MSCs secreted more EVs than express Foxp3 and EGFP. Treg in blood of CML LP MSCs. The global proteome of the LP and HP EVs patients were analysed using 13-colour flow cytometry. revealed that the vast majority of the identified proteins Results: Leukemic EVs potentiate suppressive function were in fact associated with EVs. The most abundant of regulatory T cells. This effect is driven by EV- proteins in LP and HP EVs shared similar but not mediated upregulation of Foxp3 – a transcription fac- identical functional characteristics, and the proteins tor responsible for Treg suppressive phenotype. showing significant differential expression between Proteomic analyses revealed that CML-derived EVs HP and LP EVs were predicted to be enriched in contain BCR-ABL oncoprotein. Interestingly, further Gene Ontology biological process terms mainly related functional studies revealed that inhibition of kinase to transport and secretion and to pathways regulating activity of BCR-ABL in EVs has abolished the increase cellular morphology, growth/proliferation and develop- in Foxp3 level in EVs-treated Treg. Similarly to our in ment. Both LP and HP EVs promoted MSC survival vitro findings, also Treg in CML patients seem to have and proliferation in autocrine and paracrine manners, more suppressive phenotype, as demonstrated by e.g. and the degree of proliferation was dependent on the larger amount of highly suppressive CD39+ Tregs. applied EV dose and associated with the characteristics Summary/conclusion: CML-derived EVs seem to of the recipient cells. modulate immunosuppression in leukemia, by increas- Summary/conclusion: The above-described results ing suppressive activity of regulatory T cells. This effect demonstrate that in vitro ageing influences the secre- is largely driven by BCR-ABL contained in leukemic tion of EVs by MSCs, particularly the number and EVs. However, precise mechanism of this regulatory protein cargoes of the EVs. pathway is yet to be dissected. Funding: Grants from National Science Centre: 2013/ 10/E/NZ3/00673 to KP, 2018/29/N/NZ3/01754 to JS OF20.05 and Foundation for Polish Science grant TEAM TECH Core Facility Plus/2017-2/2 to KP. Novel role of BCR-ABL-containing leukemic extracellular vesicles in controlling the function of regulatory T cells Julian Swatlera, Wioleta Dudka-Ruszkowskaa, Lukasz Bugajskib, Ewa OF20.06 Kozlowskac and Katarzyna Piwockaa aLaboratory of Cytometry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, Warsaw, Poland; bLaboratory of Cytometry, Nencki Immunomodulatory function of human mesenchymal stromal cells Institute of Experimental Biology, Polish Academy of Science, Warsaw, (MSC)-derived extracellular vesicles (EVs) on type-i interferon Poland; cDepartment of Immunology, Faculty of Biology, University of response in human plasmacytoid dendritic cells (pDCs) and its Warsaw, Warsaw, Poland therapeutic effect on murine lupus model Lin Kuia, Godfrey Chanb and Pamela PW Leea Introduction: BCR-ABL-positive chronic myeloid leu- aDepartment of Paediatrics and Adolescent Medicine, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong, Hong Kong; bThe kemia (CML) has only recently been recognized as a University of Hong Kong, Hong Kong, Hong Kong malignancy associated with an immunosuppressive microenvironment, which includes increased amount Introduction: Immunoregulatory effect of mesenchy- of Foxp3+ regulatory T cells (Treg). However, mechan- mal stem cell (MSC) is attributed to extracellular vesi- isms driving Treg differentiation and function in CML cles (EVs) secretion. Given its effectiveness in are mostly unknown. We hypothesize that extracellular preclinical studies of autoimmune disease, no one has vesicles (EVs) released by leukemic cells may be examined its effect on SLE pathogenesis, signify by engaged in this phenomenon and mediate immuno- excessive type-I IFN production by plasmacytoid den- suppression both inside and outside the CML bone dritic cells (pDCs) and animal models. marrow niche. Objective: To investigate the effect of MSC and MSC- Methods: We isolated EVs from murine BCR-ABL- EVs on regulating cytokines production in pDCs, and expressing progenitor cells, using differential centrifu- the immunomodulatory role of MSC-EV in NZB/ gation. EVs were characterized using TEM, Western W F1. blotting, Nanosight analysis and uptake of EVs by Methods: htMSC (immortalized human MSCs), was lymphocytes was analysed by flow cytometry. cultured in CDPF medium for 48 h. EV were isolated Influence of CML-derived EVs on Treg was analysed by ultracentrifugation at 100,000g, 3 h, 4°C and char- in different ex vivo settings, such as an in vitro suppres- acterized. Comparison of immunosuppressive function sion assay and differentiation of Treg on dendritic cells. between htMSC-EV and TSG-6 knockdown htMSC on To precisely analyse Foxp3 expression in Treg, we have TLR9-mediated cytokine production in pDC was deter- used cells from B6.Cg-Foxp3tm2Tch mice that co- mined with GEN2.2, a human pDC cell-line, following 266 ISEV2019 ABSTRACT BOOK activation by CpG-A, and analysis by qPCR and ELISA. CpG-A-primed GEN2.2 resulted in downregulation of Finally, we compared the survival, pDC IFN-α intra- IFN-α transcription and protein expression, mediated cellular expression and serum IgG autoantibodies via reduction in total and phospho-IRF7. NZB/W F1 expression of htMSC-EV-treated NZB W/F1 mice mice treated with htMSC-EV resulted in improved with those that received htMSC cellular treatment, survival, followed by reduction in splenic pDC IFN-α and PBS control-group. expression, and IgG autoantibodies in serum. Results: Upon activation of TLR9 by CpG-A, IL-1ß, Summary/conclusion: We showed that MSC downre- TNF-α and IFN-α transcription was upregulated in gulated TLR9 activation in human pDCs, in a TSG-6 GEN2.2. Such response was reduced when CpG-A- dependent manner. Furthermore, htMSC-EV contains primed GEN2.2 were co-cultured with htMSC. TSG-6 and suppress IFN-α response in CpG-A-acti- Knockdown of TSG-6 in htMSC dampened its capacity vated pDCs through reducing total and phospho-IRF7. to suppress IL-1ß, TNF-α, IFN-α and IRF7 transcrip- Finally, htMSC-EV treatment improved survival, and tion in GEN2.2. To find out whether MSC exert its modulated pDCs and serum IgG autoantibodies immunosuppressive effect by means of EV, we isolated expression in NZB/W F1 mice. EVs from hTERT MSCs and found htMSC-EV contained TSG-6 protein. Co-culture of htMSC-EV with JOURNAL OF EXTRACELLULAR VESICLES 267

Symposium Session 21: EVs in Migration and Cancer Chairs: Akiko Takahashi; Yoshinobu Takakura Location: Level 3, Hall B 16:30–18:00

OF21.01 expression is dynamic during the different stages of disease progression, but only Rab27a shows an increased expression in metastatic lesions. Using a Targeting Rab27a in pancreatic cancer Nuno Bastosa, Carolina Ruivoa, Bárbara Adema, Christoph Kahlertb, Bruno Rab27a small molecule inhibitor in KPC mice we see Gonçalvesc, Nuno Diasc, Raghu Kallurid, Fátima Carneiroe, José Machadof a decrease in the number of liver macro-metastasis and f and Sónia Melo increase in overall survival. In addition, we developed a ai3S – Instituto de Investigação e Inovação em Saúde, Porto University, conditional and inducible Rab27aKO mouse and show Portugal; IPATIMUP – Instituto de Patologia e Imunologia Molecular da Universidade do Porto; ICBAS – Instituto de Ciências Biomédicas Abel that pancreas conditional KO of Rab27a does not affect Salazar da Universidade do Porto, Porto, Portugal; bUniversitätsklinikum the normal development and physiology of the Carl Gustav Carus Dresden, Dresden, Germany, Dresden, Germany; cHospital de Viseu, Viseu, Portugal, Viseu, Portugal; dMD Anderson pancreas. Cancer Center, University of Texas, Texas, USA, Texas, USA; ei3S – Summary/Conclusion: We are currently assessing the Instituto de Investigação e Inovação em Saúde, Porto University, Portugal; IPATIMUP – Instituto de Patologia e Imunologia Molecular da Universidade effects of Rab27a conditional KO in PDAC GEMMs. do Porto; Department of Pathology, Centro Hospitalar São João, Porto, Funding:ProjectNORTE-01–0145-FEDER-000029 from 6 Portugal, Porto, Portugal; i3S – Instituto de Investigação e Inovação em NORTE 2020. IF/00543/2013/CP1184/CT0004, PTDC/ Saúde, Porto University, Portugal; IPATIMUP – Instituto de Patologia e Imunologia Molecular da Universidade do Porto; FMUP – Faculdade de BIM-ONC/2754/201 and, POCI01-0145-FEDER-32189 Medicina da Universidade do Porto, Porto, Portugal, Porto, Portugal from FCT – Foundation for Science and Technology. FAZ Ciencia Award from Astrazeneca Foundation. Introduction: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with very limited therapeutic options. PDAC lesions are unique OF21.02 due to their extensive desmoplastic reaction and sparse cancer cells, highlighting the potential role of cell communication in PDAC progression. Despite cell com- Roles of lysyl oxidase like 2 (LOXL2) in exosomal fraction on lymph node metastasis of head and neck squamous cell carcinoma munication being intrinsically involved in tumour Hajime Yano, Afsana Islam, Teppei Kaminota, Reina Tanimoto, Naohito progression, this process of tumorigenesis is still off Hato and Junya Tanaka the cancer therapy landscape. Exosomes have emerged Graduate School of Ehime University Medical School, To-on, Japan as crucial mediators of intercellular communication in cancer. Rab27a and −27b have been described as key Introduction: The secretory enzyme lysyl oxidase like players in cancer exosomes release. 2 (LOXL2) is assumed to contribute to tumour pro- Methods: Therefore we decided to evaluate if inhibi- gression through participation in cellular events tion of cancer exosomes communication could repre- including remodelling extracellular matrix and epithe- sent a novel therapeutic strategy in PDAC. lial-mesenchymal transition. In a previous study, we Results: We demonstrate that Rab27a, but not Rab27b, identified elevated expression of LOXL2 in human expression correlates with poor survival in patients head and neck squamous cell carcinoma (HNSCC) with metastatic PDAC, but the same is not true for lymph node metastases, and localization of LOXL2 in early stage PDAC. We further demonstrate that Rab27a exosomal fraction of a metastatic HNSCC cell. Here we knockout in pancreatic cancer cells is lethal, further assessed the significance of LOXL2 in the metastasis stressing the crucial role of Rab27a for cancer cells and of liquid biopsies for detecting HNSCCs and their survival. When using an inducible TetON knockdown risk of the metastasis. system for Rab27a, downregulation of this protein Methods: A mouse model of lymph node metastasis of impairs tumour growth in orthotopic models and, HNSCC was established to assess metastatic activity most strikingly, inhibits liver metastatic colonization. and responsible factors for the metastasis. LOXL2 pro- Next we evaluated Rab27a, −27b, −5 and −7 expression tein expression as well as lysyl oxidase enzyme activity during disease progression in a genetically engineered were assessed in exosomal fraction of human meta- mouse model (GEMM) that spontaneously develops static HNSCC cell. Effects of LOXL2 knockdown on PDAC (KPC) and reflects the human disease. Rabs the metastasis was assessed in the mouse model. 268 ISEV2019 ABSTRACT BOOK

LOXL2 levels were assessed in immunohistochemistry confirm senescence induction. Receptor activator of and immunoblotting in HNSCC tissues as well as nuclear factor kappa-Β ligand (RANKL), a key pro- serum from patients. Exosomal LOXL2 levels from osteoclastogenic factor, and p16INK4a transcript abun- further 38 serum samples from HNSCC patients were dance was assessed by qPCR. Conditioned media was subjected to statistical analysis. collected and an enzyme-linked immunosorbent assay Results: LOXL2 localization was identified in exosome used to detect soluble RANKL protein secretion. from metastatic HNSCC cell but not in non-metastatic Furthermore, osteoclastogenesis and pit formation on cancer cell. Knockdown of LOXL2 attenuated the a synthetic bone substrate was assessed in response to lymph node metastasis. Immunoblot analyses revealed proliferating and senescent NOF-derived EV condi- that LOXL2 was present in human serum exosomal tioned media. fractions, and the mean LOXL2 level was approxi- Results: Readily detectable p16INK4a and DPP4 mately three-fold higher in patients than healthy expression was observed in fibroblastic stroma adjacent volunteers. Immunohistochemical LOXL2 staining to bone, indicative of the presence of senescent fibro- was detected in HNSCC cells in a human tongue blasts. A significant increase in RANKL and p16INK4a HNSCC sample. Further measurements of exosomal expression was observed following 15 days of senes- LOXL2 showed higher LOXL2 levels in patients. cence induction. RANKL secretion by NOFs was sig- Summary/Conclusion: Elevated serum exosomal nificantly upregulated following in vitro induction of LOXL2 levels can be an indicator of HNSCC. A fol- senescence. Conditioned media from senescent oral low-up clinical study will be required to determine the fibroblasts provoked a marked increase in osteoclasto- clinical utility of using LOXL2 to diagnose HNSCC genesis, shown by an increase in tartrate resistant acid and/or determine the risk of metastasis. phosphatase activity, multinucleation and osteoclastic Funding: This study was supported in part by grants resorption/pit formation. from Ehime University (#0101010010, #0101050003, Summary/Conclusion: Here we provide novel evi- and #0101390003), Daiwa Securities Health Care dence that senescent oral fibroblasts and derived EVs Foundation (#14–23), and the Ministry of Education, play an active role in OSCC bone invasion. The ability Culture, Sports, Science, and Technology of Japan of senescent fibroblast-derived factors to promote (15K10809 and 18K09379). osteoclastogenesis may have implications more broadly for age-related bone pathologies, and this is the focus of our ongoing investigations. OF21.03 OF21.04 Growing old disgracefully – a novel role of extracellular vesicles in bone invasion Areeg A. Elmusrati, Daniel Lambert and Syed Ali Khurram The multifaceted role of breast cancer-derived extracellular vesicles in brain metastasis The University of Sheffield, Sheffield, UK Golnaz Morada, Christopher Carmanb and Marsha Mosesc

aHarvard Graduate School of Arts and Sciences, Boston Children’s Hospital, Introduction: Bone invasion is a common feature of Boston, USA; bMolecular and Integrative Physiological Sciences Program, oral squamous cell carcinoma (OSCC) and is associated Harvard T.H. Chan School of Public Health, Boston, USA; cVascular Biology Program, Boston Children’s Hospital; Department of Surgery, with poor prognosis. The mechanism of OSCC bone Harvard Medical School and Boston Children’s Hospital, Boston, USA invasion remains unclear, but our recent work indicated a key role for cancer-associated fibroblasts (CAF) Introduction: Breast cancer brain metastasis is often Methods: In this study we sought to investigate associated with a dismal prognosis. Elucidation of the whether senescent fibroblasts and derived extracellular early events that lead to brain metastasis will pave the vesicles (EV) play a role in bone invasion in OSCC. way to identifying potential diagnostic and therapeutic Immunohistochemistry (IHC) for senescence markers targets for early intervention. We have previously p16INK4a and dipeptidyl peptidase 4 (DPP4) was car- shown that extracellular vesicles (EVs) derived from ried out on bone resection cases with cortical and the brain-seeking MDA-MB-231 breast cancer cell medullary OSCC invasion. Senescence in normal oral line can increase brain metastasis growth. To investi- fibroblasts (NOF) was experimentally induced through gate the mechanisms underlying the EV-induced facil- replicative mitotic exhaustion, as well as exposure of itation of brain metastasis, we studied the mechanisms NOF at low passage to hydrogen peroxide, and the with which EVs interact with and modulate the blood chemotherapeutic drug cisplatin. Senescence-associated brain barrier (BBB), as an initial niche for tumour cell beta-galactosidase (SA-βgal) activity was monitored to growth. JOURNAL OF EXTRACELLULAR VESICLES 269

Methods: EVs were isolated from the parental MDA- with exposed PS; this subtype has lower density, larger MB-231 breast cancer cell line (P-EVs) and its brain- size, more negative zeta potentials and lower abun- seeking variant (Br-EVs). Through retro-orbital and dance of exosomal proteins. Because PS and PE have intracardiac injection of EVs in mouse and zebrafish often been reported to change their membrane locali- models, we studied the distribution of EVs to the brain. zation in a closely associated manner, in this study, we A combination of in vitro and in vivo BBB models was aimed to examine if PE is also present in this subtype used to study the mechanisms with which EVs interact of sEVs. with an intact BBB. We next conducted continuous in Methods: An sEV fraction was prepared from a con- vitro and in vivo treatment with EVs to elucidate the ditioned medium of tumour cell lines (HT-29 and HT- effects of EVs on the behaviour of the luminal and 1080) that were propagated in a serum-free medium abluminal components of the BBB. for approximately 68 h. After differential centrifuga- Results:Ourdistributionstudiesdemonstratedthat tions (10,000g for 30 min and 160,000g for 70 min) and breast cancer-derived EVs could enter the brain parench- filtration with a 0.22-µm pore filter, the sEVs were yma via an intact BBB. Using state-of-the-art models of further differentiated by continuous density-gradient the BBB and high-resolution microscopy, we have identi- centrifugation (8–40% iodixanol, 100,000g, 17 h) into fied, for the first time, the mechanisms with which Br-Ex 10 fractions. Thereafter, the fractions were washed with interact with the endocytic pathway in brain endothelial phosphate-buffered saline and analysed by Western cells to cross the endothelium. Interestingly, our mechan- blotting, silver staining, nanoparticle tracking and istic studies showed that through transferring miRNAs, atomic force microscopy (AFM). To detect PS or PE Br-EVs could modulate the endothelial endocytic path- on the surfaces of the vesicles, sEVs were labelled with way to decrease EV degradation. Moreover, we have gold nanoparticles (GNPs) using MFG-E8 or duramy- shown that following their transport across the brain cin, respectively, followed by AFM observation. endothelium, Br-EVs can exclusively alter the expression Results: Continuous density-gradient centrifugation profile of astrocytes to provide a suitable environment for showed two subtypes of sEVs. One subtype was metastatic growth. enriched with canonical exosome markers, including Summary/Conclusion: These findings indicate that EVs CD63, CD81, Alix and Tsg101, and had a density of derived from a brain-seeking subpopulation of breast 1.10 g/ml. The other subtype, however, was scarce for cancer cells can exclusively modify the physiological these markers and had a lower density of 1.04 g/ml. regulation of the BBB at multiple levels to accelerate The estimation of the amounts of exposed PS and PE metastatic growth in the brain microenvironment. by GNP in AFM showed that the second subtype of Funding: This work was supported by the Breast sEVs had exposed PE as well as PS. Cancer Research Foundation and NIH R01CA185530. Summary/conclusion: The subtype of sEVs with lower density and fewer canonical exosome markers in den- OF21.05 sity-gradient centrifugation contained not only exposed PS but also PE, which defined a new subtype of sEVs from tumour cells. Exposed aminophospholipids enriched in a subtype of small Funding: This work was supported by JSPS KAKENHI extracellular vesicles from tumour cell lines Sachiko Matsumuraa, Tamiko Minamisawab, Kanako Sugac and Kiyotaka Grant Numbers 16K07152 to SM and 17H06255 to KS. Shibad aJapaese Foundation for Cancer Research, Koto-ku, Japan; bJapanese Foundation for Cancer Research, Tokyo, Japan; cJapanese Foundation for OF21.06 Cancer Research, Tokyo, Japan; dJapaese Foundation for Cancer Research, Tokyo, Japan Mesenchymal stem cells-derived exosomes present natural migration and homing abilities to specific neuropathological areas Abstract: Aminophospholipids such as phosphatidyl- Nisim Peretsa, Oshra Betzerb, Ronit Shapirac, Shmuel Berensteind, Areil serine (PS) and phosphatidylethanolamine (PE) nor- Angele, Tamar Sadanb,Uri Asheryf, Rachela Popovtzerb and Daniel Offeng mally exist in the inner leaflet of the plasma aSagol School of neuroscience, Tel Aviv University, Israel, Tel Aviv, Israel; membrane. Tumour cells, however, expose PS on bFaculty of Engineering and the Institute of Nanotechnology & Advanced Materials, Bar-Ilan University, Israel, Ramat Gan, USA; cSchool of their surfaces and release the extracellular vesicles Neurobiology, Biochemistry and Biophysics, Life sciences faculty, Tel Aviv (EVs) enriched with the exposed PS, which have been University, Israel, Tel Aviv, Israel; dSacklar School of medicine, department of human genetics and biochemistry Tel Aviv University, Israel, Petah Tikva, proposed to play an important role in communication Israel; eSacklar School of Medicine, Department of Human Genetics and between tumour cells and other surrounding or distal Biochemistry Tel Aviv University, Israel, Petah Tikva, USA; fSagol School of neuroscience, Tel Aviv University, Israel. School of Neurobiology, cells. We have recently identified a subtype of small Biochemistry and Biophysics, Life Sciences Faculty, Tel Aviv University, EVs (sEVs) from tumour cell lines that were enriched Israel, Tel Aviv, Israel; gSagol School of Neuroscience, Tel Aviv University, 270 ISEV2019 ABSTRACT BOOK

Israel, Sacklar School of Medicine, Department of Human Genetics and given via intranasal administration to mice with differ- Biochemistry Tel Aviv University, Israel, Tel Aviv, USA ent pathologies. All mice were scanned with CT 1, 24 Introduction: Though exosoemes have been found to and 96 h post administration. Moreover, using PKH26 cross the blood–brain barrier, their migration and MSC-exo were labelled and were visualized with whole homing abilities within the brain remain unstudied. brain florescence. We have recently developed a method for longitudinal Results: Altogether, our DATA suggests that MSC-exo and quantitative in vivo neuroimaging of exosomes, present distinct neurodistribution which is pathology- based on the superior visualization abilities of CT, specific in each of the mice models visualized both in combined with gold nanoparticles as labelling agents. vivo and ex-vivo. ’ Here, we used this technique to track the migration In both the induced stroke and Parkinson s models, the and homing patterns of intranasally administrated exo- MSC-exo were visualized mainly in the damaged tissue ’ somes derived from bone marrow mesenchymal stem (Striatum). In Alzheimer s model, they were visualized cells (MSC-exo) in different brain pathologies, includ- mainly in the hippocampus, and in the Autism mice ing stroke, autism, Parkinson’s disease and Alzheimer’s model, they were visualized both in the prefrontal disease. We found that MSC-exo specifically targeted cortex and the cerebellum. Interestingly, in healthy and accumulated in pathologically-relevant murine mice the exosomes did not home to any specific loca- models brains regions up to 96 h post administration, tion and the signal was lost 24 h post administration while in healthy controls they evacuated. The neuro- both in vivo and ex vivo. In the damaged tissue, the inflammatory signal in pathological brains was highly MSC-exo were found mainly in the neurons and not in correlated with MSC-exo accumulation. In addition, other cells. MSC-exo were selectively uptaken by neuronal cells Summary/conclusion: Taken together, these findings in the pathological regions. can significantly promote the application of exosomes Methods: Exosomes were extracted from human bone for therapy and targeted drug delivery in various brain marrow mesenchymal stem cells. They were loaded pathologies via intranasal administration. with glucose-conjugated gold nanoparticles and were JOURNAL OF EXTRACELLULAR VESICLES 271

Symposium Session 22: Novel Methods of EV Analysis Chairs: An Hendrix; John Nolan Location: Level B1, Hall A 16:30–18:00

clinical EV samples can be analysed in parallel and OF22.01 total 384 samples in one run. Processing 16 EV samples required less than 2 min. To our knowledge, this is the first time BLI has been used as a diagnostic tool. We Biolayer interferometry – extracellular vesicles (BLIEV) platform for liquid biopsy of ovarian cancer strongly believe that the remarkable sensitivity and Tatu Rojalina, Randy Carneya and Kit Lamb specificity of our BLIEV platform exhibit clinical sig- aUC Davis, Davis, USA; bUniversity of California, Davis, Sacramento, USA nificance for the next-generation cancer diagnostics. Funding: Sigrid Juselius Foundation, Finland. Introduction: Rigorous efforts are being addressed to tackle the hurdles in extracellular vesicle (EV)-based cancer diagnostics – in particular liquid biopsies. We OF22.02 introduce a promising optical platform, biolayer interferometry – EV (BLIEV) for EV-based liquid biopsies. Enhanced detection and visualization of exosomes with BLIEV utilizes “dip-and-read” optical fibre biosensors interferometric reflectance imaging without any microfluidics that can be clogged by crude M. Selim Ünlüa, Ayca Yalcin Ozkumura, Celalettin Yurdakula, Marcella b a a a c and complex sample matrices. Chiari , Lei Tian , Fulya Ekiz Kanik , Nese Lortlar Unlu and Elisa Chiodi Methods: A ForteBio Octet RED384 System and SSA aBoston University, Boston, USA; bConsiglio Nazionale delle Ricerche (CNR), Istituto di Chimica del Riconoscimento Molecolare (ICRM), Milano, Italy; Streptavidin sensor tips were used for the BLI. EVs cCNR, Milano, Milano, Italy were collected from the serum of 16 ovarian cancer (OvCan) patients and 16 healthy (H) individuals. A Introduction: Biological sensors that detect whole group of 16 test samples was also prepared by spik- viruses and exosomes with high specificity, yet without ing-in several concentrations of OvCan EVs into chemical labelling, are promising because they generally healthy human plasma. The sensor tips were functio- reduce the amount and complexity of sample prepara- nalized with biotinylated CD9 or CD63. Bulk EVs from tion required by molecular amplification methods and OvCan and H preparations were captured by the may improve measurement quality by retaining infor- BLIEV-CD9 or BLIEV-CD63 biosensors. The same mation about nanoscale biological structure. We devel- procedure was repeated for the spiked-in clinical test oped an optical sensing technology, Interferometric samples. Cancer-specific signals were acquired by dip- Reflectance Imaging Sensor (IRIS), a multifunctional ping the EV-containing BLIEV biosensors to the solu- platform for quantitative, label-free and dynamic detection of an in-house cyclic reporter peptide, LXY30, tion. In high-magnification modality, Single-Particle which has been demonstrated to bind the α3β1 integrin Interferometric Reflactance Imaging (SPIRI) has the overexpressed on OvCan cells and EVs. ability to detect and characterize individual biological Results:TheEVcapturingefficiencyofBLIEV-CD9and nanoparticles. We have recently improved the contrast BLIEV-CD63 sensors was very high, enabling the sub- and spatial resolution of SPIRI by pupil function engi- sequent detection with LXY30. Regardless the used bio- neering and computational imaging. sensor type, high signals were recorded in the bulk- Methods:InSPIRI,theinterferenceoflightreflectedfrom capturing phase for OvCan EV preparations while the the sensor surface is modified by the presence of particles healthy EV samples gave negligible signals. Titration of producing a distinct signal that reveals the size of the the prepared spiked-in clinical test samples yielded particle that is not otherwise visible under a conventional remarkably high OvCan detection specificity (>90%), microscope. Using this instrument platform, we have and the sensitivity reaching as low as < 1500 EVs/µL. demonstrated label-free identification and visualization of Our integrin-binding reporter peptide LXY30 enabled various viruses in multiplexed format in complex samples significant detection specificity towards the OvCan EVs. in a disposable cartridge. Recently, our technology was Summary/Conclusion: We have developed a robust, applied to detection of exosomes and commercialized by sensitive, label-free, cost-effective, and high-throughput Nanoview Biosciences for quantified measurement of exo- BLIEV platform for EV-based liquid biopsies. Up to 16 somes on dry sensor chips. We are currently focusing on 272 ISEV2019 ABSTRACT BOOK various in-liquid detection as well as further improvement use of multiple recognition events in combination with of the technique using pupil function engineering. signal amplification allows detection of exosomes with Results: By acquiring multiple images with a parti- high specificity and sensitivity. tioned pupil (resulting in structured illumination) and Summary/Conclusion: Here, we discuss the applica- computational imaging, we have demonstrated signifi- tion of proximity assays for sensitive detection of exo- cant improvement in visibility of low-index nanoparti- somes in body fluids, to visualize the uptake of cles in liquid. Furthermore, spatial resolution has been exosomes by cells, and the potential of such approach improved beyond the diffraction limit approaching to be used to better understand the biology of the 100 nm in the visible microscopy. We have developed exosomes and to identify exosomes as disease compact and inexpensive sensor chips and microfluidic biomarkers. cartridges allowing for study of biological particles (exosomes and other extracellular vesicles) directly in the bodily fluids without labels. OF22.04 Summary/Conclusion: In summary, we have demonstrated improved visibility of exosomes in SPIRI using A 96 well plate format lipid quantification assay with improved pupil function engineering. sensitivity for standardization of experiments with extracellular Funding: EU-INDEX vesicles Tamas Visnovitza, Xabier Osteikoetxeab, Barbara W. Sódarc, Judith Mihalyd, Péter Lőrincze, Krisztina V. Vukmana, Eszter Ágnes Tótha, Anna Koncza, Inna Székácsf, Robert Horváthf, Zoltan Vargag and Edit I Buzásc

OF22.03 aSemmelweis University, Dept. of Genetics, Cell- and Immunobiology, Budapest, Hungary; bAstraZeneca, Macclesfield, UK; cSemmelweis University, Budapest, Hungary; dRCNS HAS, Budapest, Hungary; eDepartment of Anatomy, Cell and Developmental Biology, Eötvös Loránd Proximity assays for detection and characterization of exosomes University, Budapest, Hungary; fNanobiosensorics Laboratory MTA-EK- Masood Kamali-Moghaddam, Ehsan Manouchehri, Alireza Azimi, Qiujin MFA, Budapest, Hungary; gResearch Centre for Natural Sciences, Shen, Radiosa Gallini and Claudia Fredolini Hungarian Academy of Sciences, Budapest, Hungary Uppsala University, Uppsala, Sweden Introduction: Current EV studies usually standardize Introduction: Exosomes receive an increased attention EV samples on the basis of their protein content, in basic biology as well as in medicine. They are shown particle number or both. Even with this latter approach to be involved in many biological processes, and are may lead to inaccuracy and overestimation of the EV proven to hold great potentials as diagnostic and ther- concentration. Lipid bilayers are defining components apeutic tools. However, there is an unmet need for new of EVs. Therefore, a lipid-based quantification, espe- and improved technologies for quantitative and quali- cially in combination with protein content and/or par- tative characterization of exosomes to meet challenges ticle count determination, appears to be a related to these vesicles, such as low concentrations in straightforward approach for quantification of EVs. body fluids, the small size of the exosomes or the low Here we set the goal to improve the sensitivity of the copy numbers of antigens present on the surface of the previously reported sulfo-phospho-vanillin (SPV) lipid exosomes. assay. Methods: We have developed a large number of affi- Methods: We to replace the traditional purified lipid nity-based proximity assays for single- and multiplex standards (diluted in organic solvents) with an aqueous detection of proteins and large complexes with high phase liposome standard (DOPC), and we optimized specificity and sensitivity. Several of these technologies, the concentration of the vanillin reagent of the assay. such as proximity ligation assay combined with flow Results of the lipid assay were compared with the cytometry readout, multiplex proximity extension previously described ATR-FTIR spectroscopy-based assays and proximity barcoding assays, are used for lipid quantification approach. The assay was validated sensitive detection and characterization of individual with EPIC biosensor system, qNano, commercially exosomes. available lipid assay and commercial LDL. Using the Results: Commonly, in these assays the exosomes are optimized lipid assay, we tested liposomes of known recognized by several affinity binders, each equipped composition as well as EVs secreted by four different with a DNA oligonucleotide. Upon binding of the cell lines. EV markers were documented by immune target exosomes by the affinity probes, the DNA oligo- electron microscopy. nucleotides are brought in proximity, subjected to Results: Elimination of organic solvents from the reac- enzymatic ligation or polymerization, which results in tion mixture abolished the background colour that formation of an amplifiable reporting molecule. The previously interfered with the assay. Comparison of JOURNAL OF EXTRACELLULAR VESICLES 273 the optimized assay with a commercial lipid kit (also frequency (>1 MHz) to the low frequency (e.g. based on the original SPV lipid assay) showed an 500 kHz), which provided a parameter independent of increase of sensitivity by approximately one order of the number of vesicles, reflecting the changes in dielectric magnitude, and the lipid-based quantification of EV properties including their membrane capacitance and samples have clearly increased the reliability of the cytosolic conductance. Extracted exosomes from different experiments. cell of origins were measured five times and the result Summary/Conclusion: The optimized lipid assay with showed the changes in opacity measurements at the improved sensitivity provides a fast, reliable and sensi- intermediate and high frequency ranges which represents tive test that addresses an existing need in EV standar- the difference between the membrane composition and dization. This optimized lipid assay for EV lipid cytosolic conductance of the exosomes. measurements can be as easy as a simple BCA test for Summary/Conclusion: A new class of electrical impe- protein determination. dance measurement system was developed with the Funding:NVKP_16-1-2016-0017,OTKA11958,OTKA1 capability to characterize and distinguish exosomes 20237, OTKA PD112085, VEKOP-2.3.2-16-2016-00002 based on their unique dielectric properties as their and VEKOP-2.3.3-15-2016-00016, KH_17 grant, ERC hu biogenesis was subjected to systematic changes under and Lendület, Institutional Higher Education Excellence different culture conditions. This technique can be Program of the Ministry of Human Resources in the theme further utilized for classification of exosomes based “Therapeutic development”.JánosBolyaiResearch on their cell of origin and can be evolved as a diag- Fellowship of HAS. nostic tool for characterizing the pathogenic exosomes. Funding: UC Faculty Development Fund. OF22.05 OF22.06

Characterization of exosomes-based on their unique dielectric properties by a novel electrical impedance measurement system A snorkel-tag based method for in vivo isolation of recombinant Yuqian Zhanga, Esam Salemb, Takahisa Nakamurac and Leyla Esfandiard extracellular vesicles Madhusudhan Reddy Bobbilia, Stefan Vogtb, Severin Muehlederc, Carolina aUniversity of Cincinnati, Non-hispanic or Latino, USA; bCincinnati Patriolid, Samir Barbariab, Markus Schossererb, Wolfgang Holnthonee, Heinz Children’s Hospital Medical Center, Cincinnati, USA; cCincinnati Redle, André Görgensf, Samir El Andaloussig and Johannes Grillarih Children’s Hospiltal Medical Center, Cincinnati, USA; dUniversity of Cincinnati, Cincinnati, USA aUniversity of Natural Resources and Life Sciences Vienna (BOKU), Vienna, Austria; bUniversity of Natural Resources and Life Sciences, Vienna (BOKU), Vienna, Austria; cLudwig Boltzmann Institute for Experimental and Clinical Introduction: Exosomes are composed of a lipid Traumatology, vienna, Austria; dUniversity of Natural Resources and Life bilayer membrane containing nucleic acids, proteins Sciences, Vienna (BOKU), vienna, Austria; eLudwig Boltzmann Institute for f and lipids in the lumen and their compositions reflect Experimental and Clinical Traumatology, Vienna, Austria; Karolinska Institutet, Department of Laboratory Medicine, Stockholm, Sweden; their cell of origin. Thus, when the secreting cells are in gDepartment of Laboratory Medicine, Clinical Research Center, Karolinska abnormal microenvironments, the exosomes undergo Institutet, Sweden., Stockholm, Sweden; hChristian Doppler Laboratory on Biotechnology of Skin Aging, University of Natural Resources and Life the compositional changes. We have developed a new Sciences, Vienna (BOKU), Vienna, Austria class of electrical impedance measurement system to non-invasively characterize exosomes based on their Introduction: Extracellular vesicles (EVs) emerged as unique dielectric properties. Although, the biophysical an important mode of cell-to-cell communication in properties of exosomes such as size, density and shape both normal and pathological conditions by transfer- have been characterized before, their dielectric proper- ring the cargo from donor cell to recipient cell. It is ties have not been investigated. their apparent natural ability to transfer cargo from Methods: An electrokinetic-based system has been donor cell to recipient cell and thus regulating via developed to characterize the dielectric properties of paracrine or endocrine mode. Over a decade, lot of exosomes extracted from human hepatocellular carci- research has been done to understand the omics, noma (HuH-7) cells under different culture conditions. mode of secretion and uptake mechanisms. However, Extracted exosomes were initially trapped with dielec- trafficking of EVs in vivo is still poorly understood. trophoresis and further characterized by their dielectric Methods: We used recombinant tetraspanin (tetraspa- properties as 0.2Vpp was swept from 1 kHz to 50 MHz. nin with C-terminus snorkel tag (1)) as a tool to under- Results:Theprincipleoftheimpedancemeasurement stand trafficking of EVs in vivo. As a first step we was adapted from the Maxwell’smixingtheoryapplied established a method for isolating functional EVs car- to analyse the dielectric behaviour of cells. Opacity was rying recombinant tetraspanins from stably expressing defined as the ratio of impedance magnitude at high cells in vitro. The presence of snorkel-tagged 274 ISEV2019 ABSTRACT BOOK tetraspanins on EVs are not affecting the surface plasma and isolate recombinant EVs from this complex protein signature (2). This method uses a combination matrix using this method and confirm by multiplex of anti-HA (hemagglutinin) affinity matrix and bead-based assay. In addition, to determine the func- Prescission protease to isolate EVs from cell culture tionality of recombinant EVs, we used CRE-LoxP supernatants without damaging the integrity of the method (3) to confirm the recombinant EV uptake in EV membrane. recipient cells. Results: EVs isolated by this method are further char- Summary/Conclusion: Ultimately, we are comparing acterized by using multiplex bead-based flow cytometry the RNA content of recombinant EVs isolated by snor- assay and electron microscopy. The multiplex bead- kel-tag to CD81+ affinity purified EVs with the total based assay results showed us that we are able to pull EV population in order to investigate the specific RNA out EVs carrying only snorkel tag from a mixture of loading by RNA seq. different EVs from different sources. Furthermore, we Funding: This work supported by the FWF Doctoral plan to spike in human recombinant EVs into mouse Program BioToP [W1224] JOURNAL OF EXTRACELLULAR VESICLES 275

Plenary Session 3: RNA Saturday 27 April Chairs: Jan Lötvall; Marca Wauben Location: Level 3, Hall B 10:00–11:20 piRNA biogenesis and functions in drosophila functional in repressing transposons. The details of Mikiko C. SIOMI our new findings will be presented at the meeting. University of Tokyo, Tokyo, Japan EV as a novel therapeutic target for cancer metastasis PIWI-interacting RNAs (piRNAs) are small non-cod- Takahiro Ochiya, Ph.D., Chief and professor ing RNAs enriched in animal gonads where they arm National Cancer Center, Tokyo and Tokyo Medical University race with transposons to maintain germline genome integrity. Although transposons are powerful agents Extracellular vesicles, known as exosomes and micro- contributing to evolution, they are also regarded as vesicles, serve as versatile intercellular communication selfish DNA parasites. Indeed, loss of piRNAs causes tools. Increasing evidence has suggested that cancer derepression of transposons, leading to DNA damage cell-derived exosomes carry pathogenic components. and failure in gonadal development and fertility. Thus, Exosomal transfer of cancer pathogenic components piRNA-mediated transposon silencing is indispensable enable long-distance-crosstalk between cancer cells for animals that undergo obligate sexual production, and target organs and tissues, resulting in the promo- including humans. Since the discovery of piRNAs, stu- tion of the initial steps for pre-metastatic niche forma- dies have intensively been conducted worldwide and tion. Furthermore, the circulating exosome have also fundamental features of the pathway have emerged. We been of interest as a source for liquid biopsies. now know that piRNAs are primarily produced from Circulating exosome in body fluids provides a reliable piRNA clusters, discrete intergenic elements composed source of miRNAs, mRNAs, DNAs, proteins and onco- of transposon remnants, and loaded onto PIWI pro- metabolites for cancer biomarkers. We also suggest our teins to form piRISCs. Cytoplasmic piRISCs silence current knowledge on the tumour-specific DNA transposons post-transcriptionally while piRISCs in methylome in exosomes effectively provide various the nucleus repress target genes co-transcriptionally. messages on the physiological and pathological status However, the molecular mechanism is not yet fully of cancer patients. In this talk, we provide an overview understood. To solve the problem, we have use of current research on exosomes in cancer. We also Drosophila and Bombyx as model systems, particularly propose new therapeutic strategies by targeting cancer- their cultured cell lines where piRNAs are fully specific exosomes to inhibit tumour metastasis. 276 ISEV2019 ABSTRACT BOOK

Featured Abstracts- Session 2 Chairs: Location: Level 3, Hall B 11:20–12:00

FA2.01 Knockdown of multiple targets in endocytosis and/or intracellular membrane trafficking in reporter cells significantly decreased reporter activation, suggesting vital A novel CRISPR/Cas9-based reporter system enables detection of EV- mediated functional transfer of RNAs on a single-cell level roles for these processes in EV-mediated RNA transfer. Olivier G. de Jonga, Dan E. Murphyb, Imre Mägerc, Eduard Willmsc, Sander Summary/Conclusion: Here we demonstrate a b b d A.A. Kooijmans , Raymond Schiffelers , Samir El Andaloussi , Matthew J. A. CRISPR/Cas9-based reporter system that for the first Woodc and Pieter Vaderb time allows the study of functional delivery of small aDepartment of Physiology, Anatomy and Genetics, University of Oxford, UK, Utrecht, Netherlands; bLaboratory of Clinical Chemistry and non-coding RNAs with single-cell resolution. This Hematology, University Medical Center Utrecht, The Netherlands, Utrecht, novel approach allows the study of EV cargo proces- Netherlands; cDepartment of Physiology, Anatomy and Genetics, University of Oxford, UK, Oxford, UK; dDepartment of Laboratory Medicine, Clinical sing in the context of functional RNA delivery, and Research Center, Karolinska Institutet, Sweden., Stockholm, Sweden may help to increase our understanding of the regulatory pathways that dictate the underlying processes. Introduction: In recent years, multiple studies have shown that extracellular vesicles (EVs) play a role in intercellular communication through transfer of RNAs. FA2.02 Unfortunately, our understanding of the mechanisms regulating EV-mediated RNA delivery and processing is lacking, due to the absence of suitable readout sys- From nanoscale to organisms: a multi-resolution imaging system of tems for functional RNA transfer. Here, we describe a endogenously released extracellular vesicles with bioluminescence novel highly-sensitive CRISPR/Cas9-based reporter resonance energy transfer Anthony Yan-Tang Wua, Yun-Chieh Sungb, John Jun-Sheng Koc, Alan Ling system that, for the first time, allows direct functional Yangd, Vanessa Guoe, Yunching Chenf and Charles P. Laie study of EV-mediated transfer of small non-coding aDepartment and Graduate Institute of Pharmacology, National Taiwan RNA molecules on a single-cell level. University, Taipei, Taiwan (Republic of China); bDepartment of Chemical Methods: We generated a CRISPR/Cas9-based stop- Engineering, National Tsing Hua University, Hsinchu, Taiwan (Republic of China); cInstitute of Biomedical Engineering, National Tsing Hua University, light reporter system, in which eGFP expression is Zuoying, Taiwan (Republic of China); dInstitute of Atomic and Molecular activated upon functional delivery of targeting single- Sciences, Taoyuan, Taiwan (Republic of China); eInstitute of Atomic and Molecular Sciences, Academia Sinica, Taipei, Taiwan (Republic of China); guide RNAs (T-sgRNAs). Donor cell lines were gener- fNational Tsing-Hua University, Hsinchu, Taiwan (Republic of China) ated stably expressing either T-sgRNAs or non-targeting sgRNAs (NT-sgRNAs). Intercellular functional Introduction:Accuratevisualizationandmonitor- RNA transfer was assessed by measuring eGFP expres- ing of EVs is paramount to the understanding of its sion in reporter cells after direct co-culture, transwell biological mechanism. However, EV labelling with co-culture, and upon addition of isolated EVs, using organic dyes such as DiI and PKH have an extended fluorescence microscopy and flow cytometry. The role half-life (e.g. PKH26: >100 days) and self-assemble of potential regulators of EV-mediated RNA transfer into micelles, which may lead to inaccurate inter- was assessed after RNAi-mediated target knockdown in pretation of EV spatiotemporal properties. reporter cells, prior to co-culture experiments. Furthermore, reporters which fuse EV and fluores- Results: Expression of sgRNAs in donor cells and EVs cent proteins (e.g. CD63-GFP) may only detect spe- was confirmed by RT-PCR. A significant activation of cific EV subpopulations. eGFP expression was observed in reporter cells after Methods: To overcome these limitations, here we direct co-culture and transwell co-culture with donor developed a next-generation reporter to enable EV cells expressing T-sgRNAs, but not NT-sgRNAs. imaging from organismal to super-resolutions by engi- Addition of EVs from cells expressing T-sgRNAs, and neering enhanced green fluorescent protein fused to a not NT-sgRNAs, also resulted in significant reporter palmitoylation signal and nanoluciferase, termed activation. Reporter activation was substantially PalmGp. HEK293T and HCA-1 cells were lentivirally decreased after blocking EV production through addi- transduced to stably expressed PalmGp as EV donor tion of GW4869 or Rab27A knockdown in donor cells. cells. In vivo EV and nanoscopic imaging were JOURNAL OF EXTRACELLULAR VESICLES 277 achieved by in vivo imaging system (IVIS) and super- Summary/Conclusion: To our knowledge, this is the resolution radial fluctuations nanoscopy, respectively. first report of a multi-resolution reporter strategy that Results: PalmGp predominantly labels EV inner mem- enables EV imaging. Efforts are currently underway in brane, and exhibited a robust bioluminescent and employing the PalmGp EV reporter to elucidate the BRET-GFP signals for EV imaging upon addition of spatiotemporal property and mechanism(s) of cancer its substrate, furimazine. IVIS imaging of liver cancer EVs during disease progression. HCA1-PalmGp bearing mice showed sustained biolu- Funding:MinistryofScienceandTechnology(MOST) minescent and fluorescent signals at the primary and grants104-2320-B-007–005-MY2 (C.P.L.), 106–2320-B- metastatic sites, indicating the high sensitivity of 007–004-MY3 (C.P.L.), and Academia Sinica Innovative PalmGp to enable biodistribution and clearance of Materials and Analysis Technology Exploration (i-MATE) endogenously released EVs. PalmGp-EVs could further Program AS-iMATE-107–33 (C.P.L.) be visualized at super-resolution (50–150 nm) to monitor EV subcellular trafficking. 278 ISEV2019 ABSTRACT BOOK

Symposium Session 23: EV Engineering II Chairs: Cherie Blenkiron; Thomas Kislinger Location: Level 3, Hall B 13:00–14:00

PBMC assays with similar EC50 values as free recombinant OS23.01 CD40L. Summary/Conclusion: We have identified and refined a short peptide, exoTOPE, that can be used to load exoTOPE: loading bioactive molecules into exosomes using a short- exosomes with diverse classes of cargos, including pro- peptide fusion Russell McConnell, Madeleine Youniss, Ke Xu, Kevin Dooley, Bryan Choi, teins and nucleic acids. The small size of this peptide Rane Harrison, Sonya Haupt, Damian Houde, Nuruddeen Lewis, Shelly tag makes this system readily adaptable to a wide Martin, Chang Ling Sia and Sriram Sathyanarayanan variety of applications and represents a significant Codiak BioSciences, Cambridge, USA advance in our ability to engineer exosomes with biologically active cargos. Introduction: Exosomes represent a promising thera- Funding: Funded by Codiak Biosciences. peutic platform for the selective delivery of diverse classes of payloads; however, loading exosomes with non-native cargo molecules has historically been a sig- OS23.02 nificant barrier to unlocking this potential. We reasoned that it would be possible to load therapeutically Retrograde dicer-independent AGO-loading of extracellular single relevant proteins into exosomes by identifying and co- stranded miRNA in recipient human cells a b opting peptide sequences that natively enrich proteins Bartika Ghoshal and Suvendra N. Bhattacharyya in exosomes. aCSIR_Indian Institute of Chemical Biology, Kolkata, India, Kolkata, India; bMolecular Genetics Division, CSIR-Indian Institute of Chemical Biology, Methods: Differential and density gradient ultracentri- Kolkata, India fugation were used to purify exosomes from cell culture supernatant. LC-MS/MS was used to identify Introduction: microRNAs are tiny regulator of gene proteins present in purified exosomes, the amino acid expression that can be transferred between neighbour- sequences of highly abundant proteins were analysed ing cells in mammalian tissue to control the expression for common sequence features, and plasmids encoding of genes in both donor and recipient cells. How the candidate peptide sequences fused to cargo proteins extracellular vesicle (EV)-derived miRNAs are getting were expressed in stably selected cells. The enrichment internalized and become functional in target cells is an of fusion proteins in purified exosomes was assessed unresolved question. using biochemical, flow cytometric and functional Methods: We used mammalian cells in culture to study analyses. the EV-mediated miRNA delivery to target cells. Using Results:Amongthemostabundantnativeexosomalpro- miR-122 negative HeLa cells as recipient cells and miR- teins identified by LC/MS-MS were three members of the 122 containing exososmes isolated from miR-122 posi- MARCKS family. All three MARCKS family members tive cells, we have delineated the mechanistic detail of were found to strongly localize to purified exosomes the import process. when overexpressed as GFP fusions. Using truncated and Results: We have identified that, through a unique point mutant versions of sequences derived from these mechanism, the EV-associated miRNAs that are primar- proteins, we identified a seven amino acid consensus pep- ily single stranded can get loaded with the Ago proteins tide sequence that is able to load non-native cargo proteins present in the target cells to become functional there. The into the exosome lumen at extremely high levels, compris- loading of EV-derived miRNAs to host cells Ago proteins ing up to ~10% of the total exosomal protein. Sequences is not dependent on the Dicer1 that otherwise required containing this seven amino acid “exoTOPE” tag were for the loading of the Ago proteins with double stranded used to load exosomes with cytosolic cargos such as fluor- miRNAs before one strand get cleaved and dislodged escent proteins, RNA-binding proteins and mRNA, Cas9, from Ago2. The EV-derived miRNA loading of Ago2 antigenic peptides and proteins, and the type 2 transmem- happens on the endosomal membrane where the pH brane protein CD40 ligand (CD40L). Exosomes carrying dependent fusion of the internalized EV membrane exoTOPE-CD40L activated antigen presenting cells in with endosomal membrane releases the miRNAs that JOURNAL OF EXTRACELLULAR VESICLES 279 get loaded with unloaded Ago2 present on the endosomal single particle level, using monomeric EGFP as a membrane. This process is depenent on memebrane reference. dynamics and restriction of memebrane dynamics either Results: The screening of EGFP fused to the N- or C- due to mitochondrial depolarization or other ways affects terminal of EV proteins served as a quantitative the loading of EV-derived miRNAs with Ago2. method to identify protein candidates for the surface Leishmania donovani, a protozoan parasite affect mem- display of EV-associated cargo. Fusions to CD47 and brane dynamics in infected macrophage cells and thus it luminal EV proteins with a snorkel domain allowed the restrict the internalization of miR-122 containing EVs display of EGFP at the surface of EVs, with CD47 as that otherwise cause an inflammatory response in mam- good candidate for surface display. Alternatively, malian macrophage-a process detrimental for the fusions of EGFP to EV proteins with either C- or N- pathogen. in topology like Tspan14 and CD63 allowed for loading Summary/Conclusion: therefore we conclude that of EGFP within the EV lumen. Single EV analysis using Leishmania donovani Restricts Retrograde Dicer- TIRF microscopy enabled the quantification of the Independent Loading of Extracellular Single Stranded average number of EGFP molecules per single engi- miR-122 in Host Cell Agos to Prevent Inflammatory neered vesicle, which was between 15 and 136 EGFP/ Response. EV depending on the fusion protein. Funding: SERB, Dept of Science and Technology, Summary/Conclusion: The screening of EGFP-fusions Govt. of India and Swarnajayanti Fellowship Fund, to EV proteins revealed several protein candidates for Dept of Science and Technology, Govt. of India. both surface display and intra-luminal cargo loading in EVs. These results contribute to the understanding of EV biogenesis and are relevant for exploiting the OS23.03 potential of engineered EVs as drug delivery systems. OS23.04 Engineering of extracellular vesicles for surface display of targeting ligands Elisa Lázaro-Ibáñeza, Anders Gunnarssonb, Gwen O´Driscollb, Olga c d b Endogenous drug loading of extracellular vesicles using microbubble- Shatnyeva , Xabier Osteikoetxea and Niek Dekker assisted ultrasound a a a a b Yuana Yuana , Kim van der Wurff-Jacobs , Banuja Balachandran , Linglei AstraZeneca, molndal, Sweden; AstraZeneca, Mölndal, Sweden; b c cAstraZeneca, Molndal, Sweden; dAstraZeneca, Macclesfield, UK Jiang and Raymond Schiffelers aDivision Imaging, UMC Utrecht, The Netherlands, Utrecht, Netherlands; Introduction:Cellengineeringisoneofthemostcom- bDepartment of Clinical Chemistry and Haematology, UMC Utrecht, The Netherlands; cLaboratory of Clinical Chemistry and Hematology, University mon strategies to modify extracellular vesicles (EVs) for Medical Center Utrecht, Utrecht, Netherlands therapeutic drug delivery. Engineering can be applied to optimize cell tropism, targeting, and cargo loading. In this Introduction:Developmentofextracellular vesicles (EVs) study, we screened several EV proteins fused with EGFP to as nanocarriers for drug delivery relies on loading a evaluate the surface display oftheEV-associatedcargo.In substantial amount of drug into EVs. Loading has been addition, we screened for EV proteins that could efficiently done from the simplest way by co-incubating the drug traffic cargo proteins into the lumen of EVs. We also with EVs or producer cells until using physical/chemical developed a novel technology to quantify the number of methods (e.g. electroporation, extrusion, and EV surface EGFP molecules per vesicle using total internal reflection functionalization). We use physical method combining (TIRF) microscopy for single-molecule investigation. gas-filled microbubbles with ultrasound known as sono- Methods: Human Expi293F cells were transiently poration (USMB) to pre-load drug in the producer cells, transfected with DNA constructs coding for EGFP which are eventually loaded into EVs. fused to the N- or C-terminal of EV proteins (e.g., Methods: Cells were grown overnight in 0.01% poly-L- CD63, CD47, Syntenin-1, Lamp2b, Tspan14). 48 h lysine coated cell culture cassette. Prior to USMB, cells after transfection, cells were analysed by flow cytome- were starved for 4 h. Treatment medium containing try and confocal microscopy for EGFP expression and microbubbles and 250 µg BSA-Alexa Fluor 488 as a EVs were isolated by differential centrifugation fol- model drug was added to the cells grown in the cas- lowed by separation using iodixanol density gradients. sette. Cells were exposed directly to pulsed ultrasound EVs were characterized by nanoparticle tracking ana- (10% duty cycle, 1 kHz pulse repetition frequency, and lysis, western blotting, and transmission electron 100 μs pulse duration) with up to 845 kPa acoustic microscopy. Single-molecule TIRF microscopy was pressure. After USMB, cells were incubated for used to determine the protein number per vesicle at a 30 min and then treatment medium was removed. 280 ISEV2019 ABSTRACT BOOK

Cells were washed and incubated in the culture med- Methods:Theinvestigatedredbloodcell-derivedvesicles ium for 2 h. Afterward, EVs in the conditioned med- (REVs) were isolated from blood using a standard protocol ium were collected and measured. and purified using size-exclusion chromatography. REVs Results: Cells took up BSA-Alexa Fluor 488 after were subjected to IR, CD and flow-Linear Dichroism USMB treatment as measured by flow cytometry. spectroscopy, freeze-fracture Transmission Electron These cells released EVs in the conditioned medium Microscopy as well as Dynamic Light Scattering. which were captured by anti-CD9 magnetic beads. Results:Herewedemonstratethatpolarizedlightspectro- About 5% of the CD9-positive EVs contained BSA- scopy techniques can provide important information on Alexa Fluor 488. The presence of CD9-positive EVs REVs and molecules inserting into their bilayer. Flow- containing BSA also were confirmed by immunogold linear dichroism (flow-LD) measurements show that EVs electron microscopy. can be oriented by shear force, insight into properties of Summary/Conclusion: USMB serves as a tool to pre- oriented macromolecules in the vesicles. The Soret-band of load the model drug, BSA-Alexa Fluor 488, endogen- the LD spectra demonstrates that hemoglobin molecules ously and to produce EVs loaded with this model drug. are oriented and associated to the lipid bilayer in freshly USMB setup, incubation time, and type of drugs will be released REVs [1]. investigated to further optimize the production of Further on, we selected three different antimicrobial drug-loaded EVs and to explore possible application peptides (AMPs), CM15, melittin and gramicidin and for in situ drug delivery system. investigated their interactions with REVs using a Funding: This research is funded by Focused diverse set of techniques. The peptide-membrane inter- Ultrasound Foundation. actions reveal several novel function of AMPs, including their ability to remove associated proteins from the OS23.05 surface of REVs (Figure 1). [1] I. Cs. Szigyártó, R. Deák, J. Mihály, S. Rocha, F. Zsila, Z. Varga, T. Beke-Somfai. Flow-alignment of Extracellular Vesicles for new Molecular Insight to Biomolecular extracellular vesicles: structure and orientation of Interactions Tamas Beke-Somfaia, Priyanka Singhv, Imola Szigyarto and Zoltan Vargac membrane associated biomacromolecules studied with aPI, Budapest, Hungary; bMs, Budapest, Hungary; cResearch Centre for polarized light. ChemBioChem. 2018;19:545–551 Natural Sciences, Hungarian Academy of Sciences, Budapest, Hungary Summary/Conclusion:Inconclusion,EVsprovideexcel- lent opportunities to better understand the function and Introduction: The potential of extracellular vesicles mechanism of natural membrane active biomolecues. (EVs) to revolutionize the diagnosis and therapy of Funding: This work was funded by the Momentum various diseases has been realized and thus it is an programme (LP2016-2), by the National Competiti- extensively studied direction. However, EVs are also veness and Excellence Program (NVKP_16-1-2016- in the size range suitable for membrane biophysics, 0007) and BIONANO_GINOP-2.3.2-15-2016-00017. while they preserve the complex composition of a The János Bolyai Research Scholarship (Z.V.) is greatly biological bilayer. Consequently, they are optimal for acknowledged. monitoring the structure, orientation and function of biomolecules associated to EVs. JOURNAL OF EXTRACELLULAR VESICLES 281

Symposium Session 24: Mechanisms of EV Delivery Chairs: Pieter Vader; Hang Hubert Yin Location: Level B1, Hall B 13:00–14:30

Summary/Conclusion: This research offers new real- OS24.01 time methods to investigate EV kinetics with living cells and complements the existing techniques. The findings of the study improve the current knowledge State of the art microscopy for live cell study of the extracellular vesicle-mediated drug delivery in exploiting EVs as drug delivery systems. Ekaterina Lisitsynaa, Kaisa Rautaniemia, Heikki Saarib, Timo Laaksonena, Funding: The research is funded by Academy of Marjo Yliperttulab and Elina Vuorimaa-Laukkanena Finland projects 311362 and 258114. aLaboratory of Chemistry and Bioengineering, Tampere University of Technology, Tampere, Finland; bDivision of Pharmaceutical Biosciences and Drug Research Program, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland OS24.02

Introduction:Extracellularvesicles(EVs)providea Fusion of extracellular vesicles (EVs) and delivery of internal EV compelling alternative for targeted drug delivery due cargos to host cells is dependent upon circulating or endogenous viral envelope proteins to the unique set of their properties: (1) natural Zach A. Troyera, Aiman Haqqanib and John Tiltonb protection of EV content from degradation in the aCase Western Reserve University, Shaker Heights, USA; bCase Western circulation; (2) EVs’ intrinsic cell targeting proper- Reserve University, Cleveland, USA ties and (3) innate biocompatibility. However, their mechanisms of interacting with living cells are Introduction: Extracellular vesicles (EVs) contain pro- poorly understood. teins and small RNAs that are posited to mediate cell- Methods: Microvesicles (MVs) and exosomes (EXOs) to-cell communication; however, the precise molecular derived from prostate cancer cells were studied. The mechanisms of EV fusion to host cells and delivery of EVs were passively loaded with the conjugate of cancer internal cargos remains poorly defined. Delivery of drug Paclitaxel (Ptx) and fluorescent probe Oregon internal EV cargos to target cells requires fusion Green (OG). Ptx-OG EVs were applied to the cells between the EV and cell membranes; otherwise, the autologously and imaged by fluorescence lifetime EV and its contents are degraded by lysosomal microscopy (FLIM). Simultaneous labelling of cell enzymes. In this study, we probed the molecular organelles with the FRET pairs to OG was done to mechanisms of EV fusion by adapting and employing utilize FLIM in combination with Foerster resonance a validated and powerful viral fusion assay. energy transfer (FLIM-FRET). Time-resolved fluores- Methods: EVs were produced in HEK 293T cells and cence anisotropy imaging (TR-FAIM) was applied for labelled with beta-lactamase (BlaM) by overexpression the first time to study the EV-based drug delivery. or with BlaM-CD9/CD63/CD81 chimeric proteins. In Confocal microscopy was used as a standard method some conditions, the HEK 293T cells were also trans- of live cell imaging. fected with plasmids encoding viral envelope glycopro- Results:ByFLIM,weshowdistinctcellularuptake tein (Env) proteins. EVs were isolated by mechanisms for EXOs and MVs loaded with the ultracentrifugation and size exclusion chromatography, drug-dye conjugate Ptx-OG. We demonstrate differ- characterized by TEM imaging, and titered with ences in intracellular behaviour and drug release microBCA assay. To test EV fusion, EVs were added profiles of Ptx-containing EVs in correlation with to target cells containing CCF2-AM FRET dye. Fusion the intracellular position. Based on FLIM and con- was measured by flow-cytometric evaluation of CCF2- focal data we suggest that EXOs deliver the drug AM dye cleavage by BlaM. mostly by endocytosis while MVs enter the cells by Results: EVs produced in the absence of viral Env both endocytosis and fusion with the cell mem- showed no evidence of fusion with target cells. In brane. TR-FAIM shows that Ptx-OG binds some contrast, EVs produced in cells co-transfected with intracellular target inside the cell that is in accor- vesicular stomatitis virus Env (VSV-G) were highly dance with the known fact that Ptx interacts with fusogenic even at low doses. EV fusion was dependent microtubules network. on the presence of functional viral Env machinery, 282 ISEV2019 ABSTRACT BOOK either from actively circulating viruses – including Results: Cellular uptake of Exo was time- and dose- VSV-G, rabies, influenza, and mokola viruses – or dependent profiles. PC derived PANC-1 Exo showed from human endogenous retroviruses (HERVs) Env significantly higher and not saturable uptake in PANC- proteins – such as syncytin-1. 1 cells compared to B16-F10 Exo (cancer-derived) and Summary/Conclusion: EVs produced in the HEK-293 Exo (non-cancer derived) which showed absence of viral Env machinery are poorly fusogenic lower and saturable uptake profile at 24 h. In vivo and are unlikely to be efficient mediators of cell-to- biodistribution studies of PANC-1 Exo in subcuta- cell communication via the delivery of EV contents neous PC xenograft further confirmed that PANC-1 to the cytoplasm. In contrast, viral Env proteins Exo favoured accumulation in PC tumours over mela- significantly enhance EV fusogenicity, suggesting noma (B16-F10) tumours. that EV fusion and communication may occur and Summary/Conclusion: A simple and highly efficient play a significant role during viral infections. surface modification approach via click chemistry was Furthermore, cells expressing the HERV Env syncy- developed enabling both in vitro and in vivo tracking of tin-1 – including many human cancers – also give Exo. DoE modelling predicted PC cells’ preference to rise to fusogenic EVs that may contribute to tumour PC-derived Exo which was confirmed also in vivo. This establishment, growth, and metastasis. These find- Exo dosimetry study could facilitate a rationalized ings suggest that blocking syncytin-mediated EV approach in Exo-based therapeutics for treatment of fusion may be an effective strategy to block EV cancer in pre-clinical studies. communication in human cancers. Funding: The K. C. Wong Education Foundation and The Marie Skłodowska-Curie actions, European Commission “Horizon 2020”, EU (H2020-MSCA-IF- OS24.03 2016)

Preferential accumulation of copper-free click chemistry-modified exosomes to own pancreatic xenograft in vivo OS24.04 Lizhou Xua, Revadee Liam-Orb, Farid N. Faruqub, Omar Abedc, Danyang Lib, Julie Wangb and Khuloud Al-Jamalb aSchool of Cancer and Pharmaceutical Sciences, King’s College London, Specific transfer of hollow gold nanoparticles within exosomes is London, UK; bKing’s College London, London, UK; cKing’s College determined by the exosome origin London, London, UK Maria Sancho-Alberoa, Nuria Navascuésb, Gracia Mendozab, Victor Sebastiana, Manuel Arrueboa, Pilar Martin-Duquec and Jesus Santamariaa Introduction: Pancreatic cancer (PC) is one of the aDepartment of Chemical Engineering, Aragon Institute of Nanoscience deadliest malignancy with few effective approaches (INA), University of Zaragoza, Campus Río Ebro- Edificio I+D, C/ available for early diagnosis or therapy. Exosomes Mariano Esquillor S/N, 5018- Zaragoza, Spain.///Networking Research (Exo) as one type of extracellular vesicles are cur- Center on Bioengineering, biomaterials and Nano, Zaragoza, Spain; bDepartment of Chemical Engineering, Aragon Institute of Nanoscience rently being investigated aspotentialtheragnostic (INA), University of Zaragoza, Campus Río Ebro- Edificio I+D, C/ tools in cancer. However, it is not yet well-under- Mariano Esquillor S/N, 5018- Zaragoza, Spain, Zaragoza, Spain; cInstituto Aragonés de Ciencias de la Salud/ IIS Aragón// Fundación Araid, Zaragoza, stood how Exo are taken up by PC cells. This work Spain aims to study the Exo dosimetry and preferential Exo-cell affinity in PC cells in vitro and in vivo for Introduction: Exosomes are considered key elements exploitation of Exo-based delivery of therapeutics. for communication between cells but very little is Methods: Exo are isolated by sucrose cushion ultra- known about the mechanisms and selectivity of the centrifugation and characterized for exosomal marker transference processes involving exosomes released expression, number, purity and shape. Exo were fluor- from different cells. escently labelled by copper-free click chemistry to Methods: In this study we have investigated the trans- enable uptake quantification in cells using the Design fer of hollow gold nanoparticles (HGNs) between dif- of Experiments (DoE) approach. Cellular uptake of Exo ferent cells when these HGNs were loaded within was investigated using flow cytometry and confocal exosomes secreted by human placental mesenchymal microscopy. Factors studied are donor Exo source, stem cells (MSCs). These HGNs were successfully dose, receipt cell type, and incubation time. incorporated in the MSCs exosome biogenesis pathway Responses identified are Exo “Taken up numbers” and released as HGNs-loaded exosomes, by using time- and “Percentage uptake” per cell. Candidate PC Exo lapse microscopy and atomic emission spectroscopy uptake was then assessed in vivo and compared Results: Those studies allowed us to demonstrate the between PC and melanoma xenograft models in NSG selective transfer of the secreted exosomes only to the mice following intravenous administration. cell type of origin when studying different cell types JOURNAL OF EXTRACELLULAR VESICLES 283 including cancer, metastatic, stem or immunological diseases. We have developed a mouse neuroblastoma cells. cell-based assay to identify compounds that modulate Summary/Conclusion: In this study we demonstrate exosome uptake and subsequent protein aggregate for- the selectivity of in vitro exosomal transfer between mation in recipient cells. In this novel cell-based assay, certain cell types and how this phenomenon can be we take advantage of the non-toxic Saccharomyces exploited to develop new specific vectors for advanced cerevisiae prion domain Sup35NM that forms self-tem- therapies. We show how this preferential uptake can be plating protein aggregates in mammalian cells capable leveraged to selectively induce cell death by light- of spreading through cell cultures. The addition of induced hyperthermia only in cells of the same type fibrils produced from bacterially expressed Sup35NM as those producing the corresponding loaded exo- to cells expressing soluble NM efficiently induces somes. We describe how the exosomes are preferen- appearance of NM aggregates which are faithfully tially transferred to some cell types but not to others, inherited by daughter cells. Importantly, EVs released thus providing a better understanding to design selec- from donor cells containing NM aggregates are infective therapies for different diseases. tious and induce the aggregation of soluble NM-GFP in Funding: We thank the ERC Consolidator Grant pro- recipient cells after 12 h incubation time. We here gram (ERC-2013- CoG-614715, NANOHEDONISM) introduce a high throughput assay to screen for func- for the financial support, and CIBER-BBN, financed tional EVs that trigger NM reporter protein aggrega- by the Instituto de Salud Carlos III. tion in target cells. Methods: We have developed a quantitative high- throughput screen assay to identify modulators (inhi- OS24.05 bitors and activators) on exosome uptake. The read-out of this functional EV assay is the percentage of recipi-

A high-throughput screen for functional extracellular vesicles ent cells with induced NM-GFP aggregates. Shu Liua, André Hossingera, Philip Dennera and Ina Vorbergb Results: A total of 4135 small molecules were screened aGerman Center for Neurodegenerative Diseases Bonn (DZNE e.V.), Bonn, from three well-defined compound libraries (LOPAC, Germany, Bonn, Germany; bGerman Center for Neurodegenerative Diseases TOCRIS and SELLECKCHEM). Thirty-three inhibitors Bonn (DZNE e.V.), Bonn, Germany / Rheinische Friedrich-Wilhelms- Universität Bonn, Bonn, Germany, Bonn, Germany and 35 activators were found to decrease or increase the EV-mediated aggregate induction in recipient cells, Introduction: Prions are infectious protein aggregates respectively. Lead compounds identified in this screen that self-propagate and infect naïve cells by direct cell affect active and selective EV uptake in recipient cells. contact or via secreted vesicles. Several lines of evi- Summary/Conclusion: We successively developed a dence argue that also protein aggregates associated cell-based assay for functional extracellular vesicles with common neurodegenerative diseases can intercel- and performed high-throughput screening to identify lularly propagate their aggregated states in a prion-like the mechanisms of active extracellular vesicle uptake. I manner. Thus, targeting extracellular vesicles (EVs) has will present some interesting findings out of the screen. potential clinical implications for neurodegenerative 284 ISEV2019 ABSTRACT BOOK

Symposium Session 25: EVs in Neurological Diseases Chairs: Andrew Hill; Yiyao Huang Location: Level B1, Hall A 13:00–14:30

OS25.01 participants. Blocking the formation of the complement Membrane Attack complex with CD59 rescues the toxicity.

Circulating extracellular vesicles of astrocytic origin carry neurotoxic Summary/Conclusion: This is the first demonstration complement in Alzheimer’s disease that blood-borne EVs from AD patients are neurotoxic Carlos Nogueras-Ortiza, Francheska Delgrado-Perezab, Vasiliki Machairakib and Dimitrios Kapogiannisa through a complement-mediated mechanism. These findings indicate a novel mechanism for induction aNational Institute on Aging, Baltimore, USA; bJohns Hopkins University, Baltimore, USA and perhaps propagation of neurodegeneration in AD through circulating EVs with important therapeutic Introduction:Recentresearchhasdocumentedthe implications. role of reactive astrocytes in neuroinflammation in Funding: This research was supported entirely by the Alzheimer’sdisease(AD),andofExtracellularvesi- Intramural Research Program of the National Institute cles (EVs) in the transneuronal propagation and on Aging, NIH. seeding of Aβ,tauandotherpathogenicprotein mediators. However, the mechanisms underlying the initial induction and propagation of neurodegen- OS25.02 eration in AD remain elusive. In our Lab, we have pioneered the isolation of neuronal- and astrocytic- Platelet extracellular vesicles as first liquid biopsy biomarkers to derived EVs (NDEVs, ADEVs) from peripheral diagnose acute ischaemic stroke blood and have found that, in AD patients, NDEVs Aleksandra Gaseckaa, Ceren Eyiletenb, Edwin van der Polc, Rienk Nieuwlandd, Krzysztof J. Filipiake and Marek Postulab contain pathogenic Aβ and tau, whereas ADEVs a1st Chair and Department of Cardiology, Medical University of Warsaw, contain high levels of potentially toxic complement. Warsaw, Poland; bDepartment of Experimental and Clinical Pharmacology, Based on these observations we hypothesized that Centre for Preclinical Research and Technology, Warsaw Poland, Warsaw, c ADEVs and/or NDEVs circulating in the plasma of USA; Amsterdam UMC, University of Amsterdam, Department of Biomedical Engineering and Physics, Amsterdam, Netherlands, Amsterdam, AD patients are neurotoxic. Netherlands; dAmsterdam UMC, University of Amsterdam, Laboratory of Methods:WeisolatedplasmaADEVs,NDEVsand Experimental Clinical Chemistry, Amsterdam, Netherlands, Amsterdam, Netherlands; e1st Chair and Department of Cardiology, Medical University CD81+ EVs from patients with sporadic AD and age- of Warsaw, Poland, Warsaw, USA matched controls. To assess their ability to induce neurotoxicity, we used them to incubate cultures of Introduction: Acute ischemic stroke is the second rat cortical neurons and human iPSC-derived neu- most common cause of death in Europe, accounting rons. We studied neuronal viability using the MTT for almost 1.1 million deaths annually. Diagnosis of assay and neurite density quantification; necrosis stroke relies on neurologic deficits and brain imaging. using fluorescent detection of EthD-1; and apoptosis Because time is brain, stroke is preferably already diag- using caspase 3/7 assays in vitro.Weusedthephy- nosed in the ambulance, which requires a liquid biopsy siologic inhibitor of the terminal complement path- biomarker. Our aim is to determine whether EVs from way CD59 in rescue experiments. In evolving vivo platelets, leukocytes and endothelial cells can be used as experiments, we perform hippocampal injections in biomarker to diagnose stroke. rats and study neurodegeneration and induction of Methods: The study was approved by the medical Aβ and tau pathology. ethics committee. Venous blood was collected at days Results: Neurons incubated with NDEVs and ADEVs 1 (acute phase) and 7 (late phase) after the onset of from AD patients exhibited significantly decreased stroke from fasting patients (n = 19, mean age neurite density, cell viability, and increased necrotic 53.8 ± 5.4 years, 55% male) and controls (patients and apoptotic cell death, compared to neurons treated with Parkinson or Alzheimer disease, n = 9, mean age with control EV subpopulations (CD81+, total EVs) 57.1 ± 3.2 years, 53% male). Flow cytometry (Apogee from patients or ADEVs or NDEVs from control A60 Micro) was used to determine plasma JOURNAL OF EXTRACELLULAR VESICLES 285 concentrations of EVs labelled with antibodies for acti- techniques as Surface Plasmon Resonance imaging vated platelets (CD61, CD62p; PEVs), leukocytes (SPRi) and Raman Spectroscopy (RS). (CD45; LEVs) and endothelial cells (CD146; EEVs). Methods: EVs were isolated by size exclusion chro- Flow cytometry data files were processed using in- matography and ultracentrifugation from plasma house developed, automated software (MATLAB and serum of Stroke, AD and PD patients and R2018a), enabling flow rate stabilization, diameter healthy controls recruited according to a protocol and refractive index determination, MESF calibration, approved by the medical Ethics Committee. We fluorescent gate determination and application, and developed an SPRi antibody array to separate and statistics reporting. To standardize and differentiate characterize plasma EVs of different neural origins. EVs from small platelets and lipoproteins, only events In parallel, RS was applied to serum EVs in order to between 200 and 700 nm and with a refractive index obtain a snapshot of their biochemical profiling. <1.42 were included. Statistical analysis was applied for the comparison Results: Concentrations of PEV were elevated in stroke of SPRi and Raman data from healthy subjects and patients compared to controls, both at day 1 and day 7 neurological patients. (p = 0.035, p = 0.059, respectively). Concentrations of Results: After the successful SPRi detection of EVs LEVs were comparable at day 1 (p = 0.83) and from neurons, oligodendrocytes, astrocytes and decreased at day 7 (p = 0.059), whereas concentrations microglia, the quantification of specific surface of EEVs decreased at day 1 (p = 0.048) and normalized molecules related to pathological or recovery pro- to control levels at day 7 (p = 0.91). cesses has revealed variations of EVs specific con- Summary/Conclusion: Concentrations of platelet EVs tent during a disease. Moreover, the bulk are elevated in patients with stroke both at day 1 and characterization of EVs by RS demonstrated the day 7, compared to controls. In follow-up studies, we presence of EVs loaded with atypical cargoes when are going to validate platelet EVs as the first liquid compared to healthy controls. biopsy biomarker to diagnose ischemic stroke. Summary/Conclusion: Our results provide support for Concentrations of LEVs and EEVs fluctuate between using EVs as biomarkers for monitoring the progres- day 1 and day 7 after stroke, likely reflecting activation sion of Stroke, AD and PD, suggesting the possibility to of immune system and endothelium following brain use the SPRi-biosensor and Raman fingerprint to iden- damage. tify and verify the neuro-pathological or recovery pro- Funding: Polish National Science Centre (2017/25/N/ cesses ongoing in neurological patients. NZ5/00545) Funding: This study was supported by the Italian Ministry of Health.

OS25.03 OS25.04

Circulating extracellular vesicles as a novel source of biomarkers for Extracellular vesicles of Alzheimer’s disease patients as a biomarker diagnosis and monitoring of neurological diseases for disease progression a b c d Silvia Picciolinia, Alice Gualerzia, Cristiano Carlomagnoa, Andrea Sguasseroa, Anat Aharon , Polina Spector , Rwan Sayed Ahmed , Nizar Horrany , Anni e f g Massimo Masserinib and Marzia Bedonia Sabach , Benjamin Brenner and Judith Aharon-Peretz a b aIRCCS Fondazione Don Carlo Gnocchi, Milan, Italy; bNanomedicine Center Tel Aviv Sourasky Medical Center, Tel Aviv, Israel; Cognitive NANOMIB, Monza, Italy Neurology Unit, Rambam Health Care Campus, Haifa, Israel; cDepartment of Hematology, Tel Aviv Sourasky Medical Center, Tel- Aviv, Israel; dCognitive Neurology Unit, Rambam Health Care Campus, Introduction: One of the main hurdle in clinical Haifa, Israel; eDepartment of Hematology and Bone Marrow approach to neurological diseases is the lack of Transplantation, Rambam Health Care Campus, Haifa, Israel; fBruce Rappaport Faculty of Medicine, Technion-Israel Institute of easily accessible and sensitive biomarkers. Brain- Technology, Haifa, Israel; gBruce Rappaport Faculty of Medicine, derived extracellular vesicles (EVs) circulate in Technion-Israel Institute of Technology, Haifa, Israel blood, cross the blood–brain barrier and are Introduction: Introduction: Alzheimer’sdisease involved in the onset and progression of neurologi- (AD) is progressive irreversible neurodegenerative cal disorders, but reliable, sensitive and reproduci- pathology and the most common cause of degenera- ble methods that can cope with their nanoscale tive dementia. AD becomes symptomatic only after dimensions are still lacking. brain changes occur over years.Accumulating evi- We propose to validate brain-derived EVs as new dence suggests that extracellular vesicles (EVs) that biomarkers of Stroke, Alzheimer’s disease (AD) and contain cytokines and microRNA are involved in the Parkinson’s disease (PD) by using biophotonics-based regulation of inflammation. The current study aimed 286 ISEV2019 ABSTRACT BOOK to characterize the EVs of AD patients as a biomar- especially in the early stages of Alzheimer’sdisease(AD), ker for disease progression. are lacking. Such biomarkers could be present in easily Methods: Blood samples were collected after available fluids, such as blood, due to the breakdown of obtaining signed informed consent (No. 0462-14- the blood–brain barrier (BBB) early in AD. However, the RMB) from 39 AD patients at three stages of disease identification of specific and sensitive blood-based bio- severity and from 14 healthy controls (HC). markers is a challenging task. Therefore, extracellular Cerebrospinal fluid was collected from five patients vesicles (EVs) may provide a window into AD etiology and three HC. EV size and concentration were and therapeutic targets, as brain-derived EVs have been studied by Nano-tracking analysis. Membrane anti- shown to cross the BBB and are present in blood. As gens were characterized by their cell origin as biomarkers, proteins are a potential source of relevant defined by flow cytometry. EV protein contents information relating to biological function. Thus, we were screened by protein array, and miRNA content investigated a subset of proteins hypothesized to be was screened by Nano-string technology and vali- involved in neurological processes in plasma and EV dated by RT-PCR. samples using the Proximity Extension Assay (PEA). Results: The AD patients’ EVs were significantly smal- Methods: EVs were isolated from platelet poor plasma ler and the levels of neural cell markers were higher from 10 healthy controls (HC), 10 patients with Mild than EVs obtained from HC. Moderate or severe AD Cognitive Impairment (MCI) and 10 patients with patients’ EVs had a significantly higher level of the mild/moderate AD. Isolation was performed using cen- Myelin oligodendrocyte glycoprotein (MOG), com- trifugation at 20.000 xg, 1 h, 4°C with a subsequent pared to the EVs obtained from patients with mild washing of the pellet at the same g-force. For the AD (P = 0.0002 and P = 0.036). Levels of the EVs characterization of the EV isolates, Nanosight and wes- that expressed the axonal glycoprotein CD171 were tern blotting (CD9) are performed. A neurology panel significantly higher in the patients with severe AD of 92 biomarkers were assessed in plasma and EVs compared to HC (P = 0.0066), possibly indicating using the PEA. Written informed consent was obtained injured apoptotic neural cells. There was also a signifi- from all study participants and the study was approved cant increase in EVs originating from endothelial cells by The North Denmark Region Committee on Health (labelled with CD31+ CD41-, P = 0.0115 and with Research Ethics (N-20150010). CD144, P = 0.0276) in patients with moderate AD Results: PEA showed no significant difference of pro- compared EVs obtained from the HC. A >2-fold tein levels comparing the three groups for the plasma increase was measured in the content of inflammatory samples. Interestingly, EV samples showed four statis- cytokines (TNFα, IL8, IL-2, IFNγ) as was a >50% tically significant proteins; Siglec-9, CLM-1, CLM-6 reduction in growth factors (FGF, EGF VEGF) and and CD38, which were less expressed in the MCI and their receptors in the EVs of moderate AD patients. AD groups compared with the HC group with a false miR-146a-5p and several other miRNAs obtained from discovery rate adjusted p-values of 0.014, 0.024, 0.035 the EVs of severe AD patients had significantly low and 0.031, respectively. These proteins have been docu- levels compared to HC. mented to be involved in neurotoxicity protection and Summary/Conclusion: The neural and endothelial inflammatory regulation. damage severity as reflected by AD patients’ EVs (anti- Summary/Conclusion: Our preliminary results gen profiles cytokine and miRNA) may serve as a demonstrate that EVs, compared to plasma, hold biomarker for disease dynamics. potential as candidate diagnostic biomarkers in AD.

OS25.05 OS25.06

Proteomic and transcriptomic profiting of extracellular vesicles Novel Blood-derived Extracellular Vesicle-based Biomarkers in isolated from immune-stimulated human primary astrocytes Alzheimer’s Disease by the Proximity Extension Assay a a b b a b c a Tsuneya Ikezu , Yang You , Kathleen Borgmann , Satomi Stacy and Anuja Jonas E. Nielsen , Kamilla Pedersen , Karsten Vestergård , Søren Kristensen Ghorpadeb and Shona Pedersena aBoston University School of Medicine, Boston, USA; bUniversity of North aDepartment of Clinical Biochemistry, Aalborg University Hospital, Aalborg, b c Texas Health Science Center, Fortworth, USA Denmark; BioXpedia A/S, Aarhus, Denmark; Department of Neurology, Aalborg University Hospital, Aalborg, Denmark Introduction: Astrocytes are abundant glial cells in the Introduction: Biomarkers capable of identifying complex central nervous system that provide supportive neuro- pathways contributing to neuropathological development, nal functions. They have critical roles in regulating JOURNAL OF EXTRACELLULAR VESICLES 287 neuronal activities in response to pro-inflammatory Results: Atotalof539commonproteinswereiden- factors in neurodegenerative diseases. Exosomes, typi- tified. IL1-β-stimulated astrocytes enhanced the cally 50–150 nm in size extracellular microvesicles, are cargo load of proteins in the EVs. IL-1b stimulation known to carry a large diversity of molecules such as induced activation of immune response and modula- proteins and RNA species that can modify the physiol- tion of cell adhesions. The EVs from resting astro- ogy of recipient cells. Here, we hypothesized that astrocytes play a role in protein metabolism, cell growth cyte-derived exosomal proteins are regulated when and maintenance. Finally, similar proteomic results exposure to pro-inflammatory factors, thus transported were also obtained from exosomes derived from to control neuronal function and plasticity. astrocytes cultured in serum-free media with IL1-β Methods: We performed a quantitative proteomic stimulation, further validating the alteration of exo- and transcriptomic analysis of exosomes purified somal proteins in activated astrocytes which can be from human primary astrocytes with or without transferred to control neuronal function and interleukin 1-β (IL1-β)stimulationin vitro. plasticity. Exosome-enriched fractions were purified by size- Summary/Conclusion: Our finding will be helpful to exclusion columns. The total proteins isolated from elucidate the pathophysiological functions of astrocyte- the EVs were run on 1D SDS-PAGE and mass derived exosomes in regulating neuronal networks and spectrometry. miRNA was isolated from EVs and provide new insights into the diagnostics and thera- subjected to Affymetrix miRNA 4.0 Array. The peutics of inflammatory diseases. data are subjected to bioinformatic analysis and Funding: NIH 1R01AG054672, 1R56AG057469 and validation for select molecules. 1RF1AG054199 (TI), 5R24HD0008836 288 ISEV2019 ABSTRACT BOOK

Saturday Poster Session PS01: Engineering and Loading EVs Chairs: Hang Hubert Yin; Antonella Bongiovanni Location: Level 3, Hall A 15:00–16:00

PS01.01 expression. The expression of PSMA targeting peptide and its binding to PSMA receptors was confirmed in vitro. Finally, successful tumour accumulation of Targeting prostate cancer via PSMA-peptide decorated exosome- PSMA-targeted EMs was achieved in vivo with the mimetics Maja Severic, Guanglong Ma, Hatem Hassan, Sara Pereira, Calvin Cheung absence of in vivo toxicity. and Wafa AL-Jamal Summary/Conclusion: Our engineered PSMA-tar- Queen’s University Belfast, Belfast, UK geted EMs, could offer a promising drug delivery system for PC, based on its drug loading capacity, tumour Introduction:Prostatecancer(PC)isthemost targeting and safety in vivo. common type of cancer and the second cause of Funding:RosetreesTruststudentship(A1108),PCUK death in men worldwide. A range of effective antic- (CDF-12-002 Fellowship) and EPSRC (EP/M008657/1). ancer drugs have been used to treat advanced PC, however, their systemic toxicity has limited their clinical use. Therefore, there is an unmet need to PS01.02 develop novel strategies to deliver cancer therapeutics to PC tissues. Exosomes are nanosized, cell- derived vesicles that carry proteins and RNAs for Improved loading of plasma-derived extracellular vesicles to encapsulate antitumour miRNAs intercellular communication. They could also deli- Margherita A. C. Pomattoa, Benedetta Bussolatib, Sergio D’Anticoc, Sara ver their cargo across the plasma membrane and Ghiottoc, Ciro Tettad, Maria Felice Brizzia and Giovanni Camussia delay premature drug transformation and elimina- aDepartment of Medical Sciences, University of Turin, Turin, Italy; tion. Exosomes have shown an intrinsic homing bDepartment of Molecular Biotechnology and Health Sciences, University of Turin, Turin, Italy; cBlood Bank, A.O.U. Città della Salute e della ability to a wide range of cells. Furthermore, a Scienza, Turin, Italy; dUnicyte srl, Turin, Italy new approach has been proposed to combine the intrinsic homing ability of exosomes with active Introduction: Extracellular vesicles (EVs) are particles targeting to enhance their tumour accumulation. released by cells that carry a complex cargo of mole- In the present work, we report the development of cules and mediate intercellular communication. novel prostate-specific membrane antigen (PSMA)- Recently, they have raised great interest as drug deliv- targeted exosome-mimetics (EMs) for advanced PC. ery systems and several engineering methods are cur- Methods: Stably transfected PSMA-peptide expressing rently under investigation. Numerous factors, however, monocytes U937 cell line was established. PSMA-tar- influence the transfection yield, including protocol geted EMs were prepared by serial extrusion of the variability and EV damage. transfected U937 monocytes. The PSMA-targeted Methods:Theelectroporationwasinvestigatedas EMs were characterized by dynamic light scattering, method to directly load miRNAs in plasma-derived nanoparticle tracking analysis, transmission electron EVs. Different parameters (voltage and number of microscopy, bicinchoninic acid assay and western blot- pulses) were compared for their effect on EV mor- ting. Furthermore, the binding of the PSMA-targeted phology and loading capacity of a synthetic miRNA, EMs to the recombinant human PSMA protein was cel-39, including miRNA enrichment in EVs and its confirmed by ELISA. Their drug loading capability transfer to target cells. Next, analyses were per- was assessed by loading doxorubicin and its derivatives. formed to evaluated the transfection effect on EV Next, in vivo biodistribution and safety studies of tar- endogenous cargo and the exogenous miRNA pro- geted EMs were carried out in C4-2B and PC3- tection from RNAse degradation. Then, EVs were tumour-bearing mice. loaded with antitumour miRNAs and their pro- Results: The engineered EMs exhibited high protein apoptotic effect was evaluated on a cell line of yield, good drug loading and exosome markers hepatocellular carcinoma, HepG2 cells. JOURNAL OF EXTRACELLULAR VESICLES 289

Results:Thecomparisonofdifferentelectropora- fragments thereof were expressed in a cell line and the tion settings demonstrated the importance of choos- minimum PrX domain requirements for exosomal ing the more appropriate protocol parameters to enrichment were determined. Leveraging PrX as a scaf- obtain an efficient EV transfection yield, under- fold for exosome surface display, we developed our stood as both molecules loading and EV damage. engEx platform to generate engineered exosomes func- In particular, we observed the superiority of one tionalized with a variety of pharmacologic payloads electroporation protocol (using 750 Volt and 10 including enzymes, antibodies, type I cytokines and pulses) that allowed the most efficient miRNA TNF superfamily members. Biological activity of these packaging and transfer to target cells, without struc- engineered exosomes was assessed in an array of in turally damaging EVs. The most efficient electro- vitro assays and compared to previously described poration protocol was also proven to allow a more scaffolds. efficient miRNA loading in respect to incubation, Results:StableexpressionofPrXinanexosome better protecting miRNA from enzymatic digestion. producing cell line resulted in 200-fold enrichment In addition, our findings suggested that electropora- of PrX on secreted exosomes. Interestingly, over- tion preserved the naïve EV cargo, including RNAs expression of PrX structural paralogs did not result and proteins, and did not alter their uptake in cells. in similar levels of enrichment, suggesting PrX is EVs engineered with antitumor miRNAs (miR-31 unique. Exosomes expressing PrX-GFP exhibited and miR-451a) successfully promoted the apoptosis 100-fold increase in relative fluorescence compared of HepG2 cells, downregulating their target genes to LAMP2B and pDisplay GFP fusions. Similar related to apoptotic pathways. levels of high-density expression were achieved Summary/Conclusion: In conclusion, our findings with a variety of topologically diverse therapeutic indicate an efficient and functional miRNA encapsula- proteins fused to full-length or truncated forms of tion in plasma-derived EVs following an electropora- PrX. Exosomes engineered to display IL7, CD40 tion protocol that preserves EV integrity. ligand, IL12 and antibody fragments via PrX fusion Funding: Associazione Italiana per la Ricerca sul exhibited up to 1500-fold improvement in potency Cancro (A.I.R.C.), Unicyte AG (Switzerland) compared to previously described scaffolds. Summary/Conclusion: This work demonstrates the potential of our engEx platform to generate novel exo- PS01.04 some therapeutics, specifically through high density surface display mediated by PrX.

Development of a platform for exosome engineering using a novel and selective scaffold protein for surface display Kevin Dooley, Ke Xu, Sonya Haupt, Shelly Martin, Russell McConnell, Nuruddeen Lewis, Christine McCoy, Chang Ling Sia, Jorge Sanchez-Salazar, PS01.05 Nikki Ross, Rane Harrison, Bryan Choi, Damian Houde, John Kulman and Sriram Sathyanarayanan Codiak BioSciences, Cambridge, USA Leptin-loaded macrophage-derived exosome: high-efficiency loading Introduction: Membrane proteins preferentially parti- method and its properties tioned into exosomes can be co-opted to display phar- Ryo Kojima, Elena Batrakova and Alexander Kabanov macologically active molecules on the exosome surface, University of North Carolina at Chapel Hill, Chapel Hill, USA which is an important strategy for maximizing the potential of therapeutic exosomes. Previously published Introduction:Exosome,oneofextracellularvesi- approaches have relied on “canonical” scaffolds includ- cles, is considered to be an important player in ing multi-pass transmembrane tetraspanins (CD9/ intercellular communication. Application of exo- CD63/CD81), LAMP2B, or non-exosomal domains some to drug delivery system is expected to target such as pDisplay or GPI anchors. We sought to identify specific cells. Especially macrophage-derived exo- novel scaffolds that enable more uniform, higher den- some is known to cross blood–brain barrier (BBB) sity surface display of structurally and biologically and deliver its cargo after intravenous administra- diverse molecules. tion. Leptin is hormone to regulate energy balance Methods: Proteomic analysis of stringently purified by inhibiting hunger, and leptin receptor is located exosomes led to the identification of highly abundant on neurons of hypothalamus. Drug delivery system and unique exosomal proteins, including a single-pass of leptin to brain is anticipated because leptin trans- transmembrane glycoprotein (Protein X, PrX) belong- porter at BBB is known to be impaired in obesity ing to the immunoglobulin superfamily. Protein X and models. However, it has been challenging to load 290 ISEV2019 ABSTRACT BOOK enough amount of protein drugs into exosome therapeutic and paracrine effects of MSCs. With the without changing its original properties. rapid increase of attention and being of great potential Purposes of this research are to develop leptin- as a future medical regimen for human disease, the loading method into exosome with high efficiency information of fate and behaviour of EVs in the living and to evaluate its physicochemical and biological subject should be urgently gathered. However, investi- characteristics. gators still have not developed an effective method to Methods: Exosome was isolated from IC-21 (mouse monitor the in vivo behaviour of EVs. Therefore, here macrophage) cells by an ultracentrifuge method. in our study, EVs derived from Wharton’s jelly MSCs Particle-size distribution of the exosome was measured were isolated, characterized and radiolabeled with by Nanoparticle Tracking Analysis. Expression of exo- 111In-oxine followed by biodistribution study and in some-marker protein was confirmed by Simple vivo SPECT/CT imaging. Western. Leptin was loaded into the exosome by Methods:Conditionedmediumwascollectedfol- using a probe sonicator, and free leptin was removed lowed by exosome isolation using Exo-Prep kit by gel filtration chromatography. Loaded amount of (Hansa BioMed) followed by purification with PD- leptin was measured by ELISA. Release profile of leptin 10 columns and 100 kDa concentration. Expression from the exosome was evaluated in mouse serum at 37° of EVs specific proteins CD63 and HSP70 was ver- C. In order to evaluate protection ability of exosome ified by western blot. Morphology and size were formulation against protease, the leptin-loaded exo- characterized by transmission electron microscopy some was treated with pronase and remained leptin nanoparticle tracking analysis (NTA). For radiola- was quantified. Stability of the exosome was also beling, EVs were incubated with 111In-oxine in PBS investigated. at 37oc for 1 h followed by purification and further Results:IC-21derivedexosomehad100–110 nm of characterization. Biodistribution and in vivo mean size and contained exosomal markers, such as SPECT/CT imaging of 111In-oxine- labelled EVs Alix and Rab11A. Size distribution and exosomal were performed at 1, 3, 6 and 24 h after intravenous marker level of the leptin-loaded exosome prepared injection into C57BL/6 mice. under optimized condition were similar to those of Results: CD63 and HSP70 expression were observed bare exosome. Drug-loading efficiency was 7% in on EVs as well as 111In-oxine-EVs. Radiochemical this condition. Although ~50% of leptin burst purity of 111In-oxine-EVs as higher than 90% and from the exosome after release study and ~70% of remained stable for at least 48 h. Result of biodistribu- leptin was degraded by protease challenge test, the tion showed that 111In-oxine-labelled EVs accumu- other leptin was considered to be retained in the lated in liver, spleen, bone marrow and cleared exosome. Particle-size distribution and leptin con- rapidly from the circulation. In vivo SPECT/CT ima- centration of the exosome were stable at 4°C for ging of 111In-oxine-labelled EVs showed high accumu- 1month. lation in liver, bone, spleen and liver, but not in brain Summary/Conclusion: This methodology to load pro- and circulation. tein drugs into exosome is promising strategy for its Summary/Conclusion:Inthisstudy,wehavepre- drug delivery application. liminarily demonstrated the feasibility of in vivo tracking of MSC- derived EVs labelled with 111In- oxine. Further investigation is still needed and PS01.06 underway to monitor the in vivo fate and behaviour of EVs.

Characterization and in vivo imaging of mesenchymal stem cells derived extracellular vesicle Cheng-Hsiu Lu, Yi-An Chen, Chien-Chih Ke and Ren-Shyan Liu PS01.07 National Yang-Ming university, Taipei, Taiwan (Republic of China) EVs as siRNA delivery vehicles for functional knockdown in cells Introduction: Mesenchymal stem cells (MSCs) are Senny Nordmeier, Victoria Portnoy and Frank Hsiung multipotent stromal cells which show the great poten- System Biosciences, Palo Alto, USA tial in tissue engineering, regenerative medicine and the treatment of various diseases. Deep into mechan- Introduction: Extracellular vesicles (EVs) mediate cell- isms, paracrine effect has been reported to be the major to-cell communication by delivering cargo, composed role in MSC therapy. Further, extracellular vesicles of nucleic acids, proteins and various other molecules, (EVs) are reportedly the major player mediating the from secreting cells to specific tissues and recipient JOURNAL OF EXTRACELLULAR VESICLES 291 cells. This method of cellular delivery has generated cells and tissues. However, the biocompatible system great interest in targeted delivery of therapeutics, such for screening combinational libraries have not been as chemical probes, proteins and RNA. In particular, described. The goal of this project is to develop a EV RNA has gained increasing attention not only in biocompatible system for peptide screening using EV- biomarker development but also in the regulation of peptide display library. gene expression in cells. The delivery of siRNA by EVs Methods: Homologous recombination cloning using is one method to induce gene silencing. Here we devel- regenerative oligos are used to create peptide library oped and optimized a method of loading EVs with plasmids where peptide sequences are fused to the siRNA using a chemical transfection reagent. phosphatidylserine binding domain (C1C2) of human Methods:IsolatedEVsfromtissueculturemedia lactadherin for peptide display to the vesicle surface. were loaded with siRNA using a chemical transfec- Fluorescent reporter gene was cloned with C1C2 tion reagent. Various compositions of the chemical, domain for imaging purposes. Plasmid DNA was trans- as well as molar ratio between the chemical and fected to HEK293FT cells and EVs were harvested by siRNA, temperatures and EV concentrations were ultracentrifugation followed by differential centrifuga- examined to determine optimal conditions. The tion. DNA sequence was recovered from EV by direct transfected EVs were added onto cells and incu- PCR amplification. bated for 24–48 hrs. After the incubation period, Results: Homologous recombination cloning was suc- the cells were imaged for fluorescence and cell cessfully used for EV library construction. Chimeric morphology changes. Then, the cells were har- protein expression on EVs was determined by vested, lysed and analysed by Western blot (protein Western blot analysis and that of reporters was verified level). Additionally, cell lysates RNA was isolated fluorescent microscopy. Direct detection of plasmid and analysed by RT-qPCR. DNA was verified from isolated EVs and the targeting Results:Fluorescentimagingshowedadose-depen- with known targeting peptide is in progress. dent incorporation of control Cy3-labelled siRNA Summary/Conclusion: In this study, our current pro- into cells. The cell nuclear morphology was gress on developing in vivo peptide screening strategy examined after delivery of ECT2 and TOR1AIP1 using degenerative oligonucleotide library EVs will be siRNA. In addition, RT-qPCR and Western blotting discussed. The success of this approach may provide a analyses were used to measure the level of knock- novel biocompatible system for peptide screening both down of the housekeeping gene, HPRT. CCK8 in vitro and in vivo. assay was used to determine cell viability after Funding: Michigan State University Startup funding delivery of EVs loaded with control siRNA and other siRNAs. Summary/Conclusion:Ourresultsprovideaspecific PS01.09 and efficient approach for loading siRNA directly into EVs for the implementation of targeted gene The construction of nanogel/exosome hybrid by exosome surface silencing. polymer engineering Shin-ichi Sawadaa, Yuko T. Satob, Riku Kawasakib, Yoshihiro Sasakia and Kazunari Akiyoshia PS01.08 aKyoto University, Kyoto, Japan; bKyoto University, Kyoto-shi, Japan Introduction:Extracellularvesiclessecretedbyvar- ious cells have attracted attention as a new system Development of an in vivo extracellular vesicle-based peptide library screening tool in cell-to-cell communication. We focus on the uti- Masako Harada lization of exosomes as biological molecule delivery Institute for Quantitative Health Science and Engineering (IQ), Michigan systems. However, it is notalwayseasytocontrol State University, East Lansing, USA the delivery and internalization of exosomes to var- Introduction: Extracellular vesicles (EVs) including ious cells. We propose here a new strategy for the exosomes and microvesicles are heterogenous popula- effective delivery of exosomes into cells using function of membrane-bound vesicles with cargos includ- tional macromolecular carriers such as amphiphilic ing protein, lipids and nucleic acids of DNA and RNA nanogels. Surface polymer engineering was applied species. Recently, EVs have gained attention as a deliv- with a carrier of exosomes,namely,amphiphilic ery vehicle for targeted delivery of oligo nucleotide cationic CHP (cCHP) nanogel, to improve the drugs. Previous reports suggest that particles coated delivery of exosome content by forming complexes with targeting peptide can be delivered to selected with the exosomes. In this study, we developed the 292 ISEV2019 ABSTRACT BOOK preparation method of exosome hybrids with nano- Methods: Human telomerase overexpression immorta- gel, and the hybrids were evaluated the character- lizes cells while keeping primary like characteristics istics and the biological functions. intact. Ectopic overexpression and charcterization of Methods: Mouse macrophage cells were used to pro- mesenchymal stem cells was used to establish produc- duce the exosomes, which were then mixed with cCHP tion cell lines. nanogel to form a hybrid. Various characteristics of Results:Herewedescribethedevelopmentofhuman these hybrid particles were examined by TEM observa- continuously growing cell lines from various tissues tion, nanoparticle tracking analysis to determine their that show a high potential as innovative production size, measurements of their ζ-potential. The interac- systems for extracellular vesicles with use for clinical tions between the hybrids and cells were evaluated by applications. confocal scanning laser microscopy and flow Summary/Conclusion: These cell lines will be used for cytometry. the production of standardized EV preparation. Results:TEMrevealedthatthesurfaceofeach exosome was coated by cCHP nanogel particles. Flow cytometry also showed significant uptake of PS01.11=OWP1.06 this exosome/nanogel hybrid by cells, with the main mechanism behind this internalization being endo- Extracellular vesicles from Fat-laden hypoxic hepatocytes activates cytosis. A range of different molecules that inhibit pro-fibrogenic signals in Hepatic Stellate Cells Alejandra Hernandeza, Yana Gengb, Daniel Cabrerac, Nancy Solisd, Han different types of endocytosis were also applied to Moshagee and Marco Arresed determine the particular pathway involved, with a aPontificia Universidad Católica de Chile; University Medical Center of caveola-mediated endocytosis inhibitor being Groningen, Groningen, Netherlands; bUMCG, Groningen, Netherlands; revealed to markedly affect hybrid uptake. Next, cPontificia Universidad Católica de Chile/Universidad Bernardo O´Higgins, SANTIAGO, Chile; dPontificia Universidad Católica de Chile, Santiago, we evaluated revealing the functional efficacy of Chile; eUniversity Medical Center Groningen, Groningen, Netherlands this approach, we showed that the nanogel system could successfully deliver functional exosome into Introduction: Background: Transition from isolated cells as indicated by its ability to induce neuron-like steatosis (IS) to non-alcoholic steatohepatitis (NASH) cell differentiation in the recipient cells. is a key issue in non-alcoholic fatty liver disease Summary/Conclusion: These results indicate that the (NAFLD). Recent observations in patients with newly developed cationic nanogel systems for exosome obstructive sleep apnea syndrome (OSAS), suggest delivery are powerful tools to investigate the biological that hypoxia may contribute to disease progression functions of exosomes. mainly through activation of hypoxia inducible factor 1α (HIF-1α)-related pathways. Release of extracellular PS01.10 vesicles (EV) by injured hepatocytes may be involved in NAFLD progression. Aim: To explore whether hypoxia modulates the Human telomerized cells for production of extracellular vesicles release of EV from free fatty acid (FFA)-exposed hepa- a b a b Regina Grillari , Susanne Neubert , Matthias Wieser and Johannes Grillari tocytes and assess cellular crosstalk between hepato- aEvercyte GmbH, Vienna, Austria; bChristian Doppler Laboratory on cytes and LX-2 cells (human hepatic stellate cell line). Biotechnology of Skin Aging, University of Natural Resources and Life Sciences, Vienna (BOKU), Vienna, Austria Methods: HepG2 cells were treated with FFAs (250 μM palmitic acid + 500 μM oleic acid) and chemical Introduction:Humancellsareofeverincreasing hypoxia (CH) was induced with Cobalt (II) Chloride, importance as in vitro test system to represent the which is an inducer of HIF-1α. Induction of CH was in vivo situation. Additionally,highlydifferentiated confirmed by Western blot (WB) of HIF-1α. EV isola- cells are also essential production systems for com- tion and quantification was performed by ultracentri- plex biopharmaceuticals. However, the use of such fugation and nanoparticle tracking analysis cell systems are limited due to the fact that the cells respectively. EV characterization was performed by enter replicative life span and therefore can only be electron microscopy and WB of CD-81 marker. LX-2 propagated for a limited number of population dou- cells were treated with 15 μg/ml of EV from hepato- blings in vitro,whichlimitedstandardizationof cytes obtained from different groups and markers of experiments as well as production processes. pro-fibrogenic signalling were determined by quantita- Moreover, reports have shown that the number of tive PCR (qPCR), WB and immunofluorescence (IF). secreted vesicles significantly reduced with increas- Results: FFA and CH-treatment of HepG2 cells ing age of normal cells. increased gene expression of IL-1β and TGF-β1in JOURNAL OF EXTRACELLULAR VESICLES 293

HepG2 cells and increased the release of EV compared Summary/conclusion: CH promotes EV release from to non-treated HepG2 cells. Treatment of LX-2 cells HepG2 cells. EV from hypoxic FFA-treated HepG2 with EV from FFA-treated hypoxic HepG2 cells cells evoke pro-fibrotic responses in LX-2 cells. increased gene expression of TGF-β1, CTGF, α-SMA Further genomic and proteomic characterization of and Collagen1A1 compared to LX-2 cells treated with EV released by steatotic cells under hypoxia are neces- EV from non-treated hepatocytes or LX-2 cells exposed sary to further delineate their role in the crosstalk to EV-free supernatant from FFA-treated hypoxic between hepatocytes and stellate cells in the setting of HepG2 cells. Moreover, EV from FFA-treated hypoxic NAFLD and OSAS. HepG2 cells increased Collagen1A1 and α-SMA pro- Funding: FONDECYT 1150327-1150311. tein levels. 294 ISEV2019 ABSTRACT BOOK

PS02: EVs in Infectious Diseases and Vaccines II Chairs: Norman Haughey; Ryosuke Kojima Location: Level 3, Hall A 15:00–16:00

PS02.01 (DDEL), Helmholtz-Institute for Pharmaceutical Research Saarland (HIPS), Saarbrücken, Germany

Host:pathogen interactions and host cell internalization of Trichomonas vaginalis exosomes Introduction: Less and less novel anti-infectives Patricia J. Johnsona and Anand Raib against diseases caused by Gram-negative bacteria aUniversity of California, Los Angeles, Los Angeles, USA; bUCLA, Los reach the market while bacterial resistance is steadily Angeles, USA increasing. Among the many hurdles of an antibiotic on its way from development to clinical use, the Gram- Introduction:TheparasiteTrichomonas vaginalis is the negative cell envelope is one crucial factor strongly causative pathogen of the sexually transmitted infection delimiting access to inner bacterial targets and thus trichomoniasis. Depending on the parasite strain and host, decreasing efficacy. As a model to study and optimize infections can vary from asymptomatic to highly inflam- the permeation of anti-infectives, outer membrane matory. We previously reported that T. vaginalis generates vesicles (OMV) were selected to create an in vitro and secretes vesicles with physical and biochemical proper- membrane model on a 96-well filter plate. ties similar to mammalian exosomes that deliver their Methods:E.coliBL21wereculturedinLuria-Bertani contents to human host cells. T. vaginalis exosomes mod- medium until stationary phase. Bacteria were separated ulate host cell immune responses and likely assist in para- by centrifugation (15 min, 9500g)andfiltration(0.2or site colonization of the host. 0.45 µm membrane pore size). OMV’swereisolatedby Methods: In our current study, we are optimizing adding 33% (w/w) PEG 8000 solution to the filtrate (ratio methods to study the uptake of T. vaginalis exosomes 4:1), shaking and overnight incubation at 4°C. The pre- into the host cells. cipitate was centrifuged (30 min, 16,233g)andthepellet Results: The data obtained from our studies show that resuspended in 100 µl filtered PBS. This suspension was exosome uptake is a time-dependent process, regulated characterized by nanoparticle tracking analysis and coated by many factors such as temperature, etc. Our findings onto 96-well filter plates using a vacuum oven (15 min, 37° also suggest that exosome uptake is mediated by endo- C, 100 mbar). Coating morphology was imaged by scan- cytosis, with specific host cell lipids playing a critical ning electron microscopy and confocal laser scanning role in this process. We have also identified target microscopy. For permeation studies the OMV coating molecules present on the surface of T. vaginalis exo- was covered with 0.5% (w/v) agarose gel before adding somes that induce exosome uptake into the host cell. solutions of different antibiotics to the donor compartment Summary/Conclusion: This work expands our general and determining the concentration time course in the knowledge of exosome uptake by target cells and our acceptor compartment using UV-spectroscopy. understanding of the mechanisms used by exosomes to Results: The filtration through 0.2 and 0.45 µm pores mediate T. vaginalis host-pathogen interactions. led in both cases to sterile filtrates, whereas 0.45 µm Funding: National Institutes of Health pores led to larger vesicles and higher yield. The applied microscopy methods indicated that a PS02.02 complete and homogenuous OMV coating was achieved. Preliminary permeation studies revealed kinetic differences between antibiotics. Coating filter membranes with bacterial derived vesicles to study the Summary/Conclusion: The OMV isolation and purifi- permeation of anti-infectives across the Gram-negative cell envelope Robert Richtera, Adriely Goesb, Marcus Kochc, Gregor Fuhrmannd, Nicole cation protocol allowed for a yield sufficient to coat 96- Schneider-Daume and Claus-Michael Lehre well filter supports. The measured permeated amounts aDepartment of Drug Delivery (DDEL), Helmholtz-Institute for allow to distinguish the permeability of different anti- Pharmaceutical Research Saarland, Saarbrücken, Germany; bBiogenic biotics. Compared to artificial phospholipid membrane Nanotherapeutics (BION), Helmholtz Institute for Pharmaceutical Research Saarland, Saarbrücken, Germany; cLeibniz Institute for New Materials (INM), models, fluxes across OMV derived membranes were Saarbrücken, Germany; dHelmholtz-Institut for Pharmaceutical Research significantly higher, facilitating faster analytics. An Saarland (HIPS), Saarbrücken, Germany; eDepartment of Drug Delivery JOURNAL OF EXTRACELLULAR VESICLES 295 involvement of outer membrane proteins in this model isolation of microbial EVs from both laboratory cul- is subject of ongoing investigations. tures and from clinical samples. Funding: School of Medicine Performance-Based Research Fund; Maurice and Phyllis Paykel Trust PS02.03 Project Grant [8.1.17]; Lottery Health Research Grant [326702]; Health Research Council, Explorer Grant [14/805]; Ministry of Business, Innovation and

Quality markers for microbial EVs Enterprise, Smart Ideas Grant [UOAX1507]. Simon Swifta, Jiwon Honga, Zachary Devereuxa, Priscila Dauros Singorenkoa, b c Cherie Blenkiron and Anthony Phillips PS02.04 aUniversity of Auckland, Auckland, New Zealand; bThe University of Auckland, Auckland, New Zealand; cDepartment of Surgery, Faculty of Medical and Health Sciences, The University of Auckland, Auckland, New Zealand Akt and CD9 in urine exosomes as potential markers for urinary tract infection Kosuke Mizutania, Kyojiro Kawakamib, Kengo Horiea, Yasunori Fujitab, Koji Introduction: Microbial EVs have potentially impor- Kameyamac, Taku Katoa, Keita Nakanec, Tomohiro Tuchiyad, Mitsuru c e e e tant roles in interactions with cells in populations of Yasuda , Koichi Masunaga , Yutaka Kasuya , Yoshishige Masuda , Takashi Deguchif, Takuya Koiec, Masafumi Itob the same species, with other microbial species and with aDepartment of Urology, Gifu University Graduate School of Medicine, Gifu, eukaryotic cells. To investigate the effect of these inter- Japan; bResearch Team for Mechanism of Aging, Tokyo Metropolitan actions in target cells it is important to define the EVs Institute of Gerontology, Itabashi-ku, Japan; cGifu University Graduate School of Medicine, Gifu, Japan; dGifu University Graduate School of under test. Medicine, Gifu, Japan; eDepartment of Urology, Tokyo Metropolitan Methods:PathogenicEscherichia coli 536 and 2348/69 and Geriatric Hospital, Tokyo, Japan; fTokyo Metropolitan Geriatric Hospital, g probiotic Nissle 1917 were cultured in RPMI 1640 ± FeCl3. Tokyo, Japan; Department of Urology, Kizawa Memorial Hospital, Minokamo, Japan Candida albicans and C. auris were cultured in YPD broth. Microbial EVs were separated from cells by centrifugation, Introduction: Urinary tract infections (UTI) is one of filtration (0.2 μmforbacteriaor0.45μmforyeast)fol- the most common bacterial infections. UTI is treated lowed by concentration (100,000 kDa cut-off filter) and with antibacterial agents, but asymptomatic bacteriuria ultracentrifugation. EVs were further enriched by either (ABU) that is diagnosed by bacteriuria without any density gradient centrifugation (DGC, bacterial samples) urinary tract symptoms should not be treated except or size exclusion chromatography (SEC, bacterial and yeast pregnant women and patients who will undergo trau- samples). An iTRAQ proteomic approach was used to matic urologic interventions. However, there has been identify proteins from bacterial cells, crude EV pellets no clinically available biomarker to distinguish UTI and DGC and SEC fractions. Yeast proteins were fractio- from ABU. Exosomes are 40–150 nm sized membrane nated by SDS/PAGE and proteins in EV-enriched and vesicles containing proteins and nucleic acids that are non-EV fractions were identified using mass spectrometry present within cells from which they are released and techniques. thus have the potential as biomarkers for various dis- Results: A number of outer membrane proteins were eases. It is likely that urine may contain exosomes identified in E. coli EVs, but with some variation released from uroepithelial cells and white blood cells. between strains and media used. Cytoplasmic protein In the present study, we aimed to identify urinary GroEL was also common. There were no obvious pro- exosomal markers that are useful to discriminate teins removed by the purification of EVs and the major between UTI and ABU. differences in proteome were due to changes in envir- Methods:Exosomeswerecollectedbyultracentrifugation onmental growth conditions. For Candida, a clear set from the culture medium of SV-HUC-1 (immortalized of EV-associated envelope proteins were identified. In uroepithelial cell line) andTHP-1(acutemonocyticleu- addition, a series of proteins removed from the crude kaemia cell line) co-cultured with or without Escherichia EV prepartion by further enrichment were identified coli or treated with or without LPS. The protein expression for Candida species that may represent non-EV was examined by western blot analysis. Urinary exosomes contaminants. were isolated from urine by Tim4-conjugated magnetic Summary/Conclusion: A number of possible markers beads. Expression of Akt and CD9 in isolated exosomes for E. coli and Candida species have been identified, was analysed by ELISA and CLEIA, respectively. which now need verification by alternative techniques Results: Expression of Akt, ERK and NF-κB was and the screening of a range of pathogenic and non- increased in exosomes isolated from SV-HUC-1 and pathogenic isolates grown in different conditions. THP-1 cells co-cultured with E. coli or treated with LPS These findings offer promising new markers for compared to without co-culture or treatment. The 296 ISEV2019 ABSTRACT BOOK levels of Akt and CD9 in urinary exosomes from and stimulated the expression of pro-inflammatory patients with UTI were higher than those from ABU cytokine and chemokine genes in Caco-2 cells in a patients. dose-dependent manner. Moreover, EVs/LIN were Summary/Conclusion: Our results suggest that intra- more cytotoxic towards Caco-2 cells than EVs/BHI cellular signalling molecule Akt and cell surface-resi- and EVs/VAN, whereas EVs/VAN induced more pro- dent exosomal marker CD9 in urinary exosomes have inflammatory cytokine and chemokine gene expression the potential to discriminate UTI from ABU, thus in Caco-2 cells than EVs/BHI and EVs/LIN. providing novel objective markers for their differential Summary/Conclusion: The sublethal dose of antibio- diagnosis, which will allow better diagnosis and treat- tics modulates the EV biogenesis in E. faecium. EVs ment of UTI and ABU patients. produced by E. faecium under different antibiotic stress Funding: JSPS KAKENHI Grant Number JP18K09190, condition show different host cell responses, which GSK Japan Research Grant 2015 plays a role in bacterial pathogenesis. Funding: This work was supported by the National Research Foundation of Korea (NRF) grant funded by PS02.05 the Korea government (NRF-2017R1A2A2A05001014).

Different protein profile and host immune response induced by extracellular vesicles from Enterococcus faecium cultured with or PS02.06 without antibiotics Mi Hyun Kima, Se Yeon Kima, Seung Il Kimb, Joo Hee Sona and Je chul Leec aDepartment of Microbiology, School of Medicine, Kyungpook National The RNA profile of extracellular vesicles released from Trypanosoma University, Daegu, Republic of Korea; bDrug & Disease Target Team, Korea brucei a b b Basic Science Institute, Ochang, Republic of Korea; cDepartment of Christian Preußer , Lee-Hsueh Hung and Albrecht Bindereif Microbiology, School of Medicine, Kyungpook National University, Daegu, aJustus Liebig University of Giessen, Institute of Biochemistry, Giessen, Republic of Korea Germany; bJustus Liebig University of Giessen, Institute of Biochemistry, Giessen, Germany Introduction: Vancomycin-resistant Enterococcus faecium is of medical importance associated with multi- Introduction: Trypanosomes are unicellular eukar- drug resistance and opportunistic infection. E. faecium yotes, which are vector-borne ubiquitous parasites of produces extracellular vesicles (EVs), but EV produc- vertebrates and have a high impact on global health. tion in E. faecium under antibiotic stress condition and The well-known Trypanosoma brucei for instance is the their pathogenic roles have not been determined yet. causative agent of the human African trypanosomiasis, This study investigated the production of EVs in van- a lethal tropical disease, and the nagana-cattle disease comycin-resistant E. faecium strain cultured with or in domestic livestocks in sub-Saharan Africa. Besides without the sub-minimum inhibitory concentrations this, trypanosomes have become an important model (MICs) of vancomycin and linezolid, and determined organism, because of various biochemical and cellular the pathogenic roles of EVs in colon epithelial Caco-2 characteristics such as trans spliced mRNAs. As other cells. parasites, trypanosomes produce extracellular vesicles Methods: The EVs were purified from vancomycin- (EVs), which contribute to parasite-host interactions. resistant E. faecium ATCC 700221 cultured with or Here we analysed for the first time the RNA profile without the 1/2 sub-MICs of vancomycin and linezolid. from EVs produced by parasitic T. brucei. Caco-2 cells were incubated with E. faecium EVs and Methods:WeisolatedEVsreleasedfromtwodifferentlife then analysed for cytotoxicity and pro-inflammatory cycle stages (procylic and bloodstream) of T. brucei,usinga cytokine gene expression. combination of differential centrifugation, size exclusion Results: E. faecium ATCC 700221 produced EVs dur- chromatography and ultracentrifugation. Subsequently we ing in vitro culture. E. faecium cultured with 1/2 sub- performed RNA-seq analysis of long RNAs (>200 nts) and MIC of vancomycin and linezolid produced 4.5 and 2 small RNAs (<200 nts), followed by bioinformatic identi- times more EV proteins than bacteria cultured without fication; validation of trypanosome and EV-associated antibiotics, respectively. A total of 438, 461 and 513 RNAs was based on quantitative RT-PCR. proteins were identified in the EVs isolated from E. Results: Our analysis of RNAs revealed different RNA faecium cultured in brain heart infusion (BHI) broth species in trypanosome-derived vesicles. Interestingly, (EVs/BHI), EVs from E. faecium in BHI broth with 1/2 we observed specific release of fragments from certain sub-MIC of vancomycin (EVs/VAN) and EVs from E. mRNAs into the vesicles, whereas metabolically impor- faecium in BHI broth with 1/2 sub-MIC of linezolid tant mRNAs were retained in the parasite, suggesting a (EVs/LIN), respectively. EVs/BHI induced cytotoxicity role in RNA disposal. We are currently comparing the JOURNAL OF EXTRACELLULAR VESICLES 297 mammalian- and insect-specific life cycle stages of the cepacia cultured with 1/4 sub-MIC of MEM (OMVs/ parasites, which should further clarify a potential func- MEM). Intratracheal injection of OMVs/LB, OMVs/ tional role of vesicle-mediated host-parasite interac- MEM, and OMVs/CAZ induced histopathology and tions. pro-inflammatory responses in the mouse lung, but Summary/Conclusion: Trypanosome-derived extracel- OMVs/SXT did not induce pro-inflammatory lular vesicles contain several RNA species, which are responses in the mouse lung. The expression of the selectively released, representing a class of diagnostic interleukin-1β and GRO-α genes was significantly biomarkers for diseases caused by these parasites. higher in the mice treated with OMVs/CAZ than the Funding: LOEWE Center DRUID (Novel Drug Targets mice treated with other OMVs. against Poverty-Related and Neglected Tropical Summary/Conclusion: OMVs produced by B. cepacia Infectious Diseases). exposed to different antibiotics represent different host cell responses, which may modulate influence on the PS02.07 bacterial pathogenesis. Funding: This work was supported by the National Research Foundation of Korea (NRF) grant funded by Host immune response induced by outer membrane vesicles derived the Korea government (NRF-2017R1A2A2A05001014). from Burkholderia cepacia cultured with different antibiotics Se Yeon Kima, Mi Hyun Kima, Joo Hee Sona, Seung Il Kimb and Je chul Leec aDepartment of Microbiology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea; bDrug & Disease Target Team, Korea PS02.08 Basic Science Institute, Ochang, Republic of Korea; cDepartment of Microbiology, School of Medicine, Kyungpook National University, Daegu, Republic of Korea Thymol suppresses the inflammatory responses induced by Staphylococcus aureus-derived extracellular vesicles in cultured Introduction: Burkholderia cepacia is an opportunistic keratinocytes Joo Hee Sona, Se Yeon Kima, Mi Hyun Kima, Sang Hyun Kimb and Je chul pathogen that usually infects the patients with cystic Leec fibrosis or indwelling hardware. This study investigated aDepartment of Microbiology, School of Medicine, Kyungpook National the production of outer membrane vesicles (OMVs) in University, Daegu, Republic of Korea; bDepartment of Pharmacology, B. cepacia strain cultured with the sub-minimal inhibi- Kyungpook National University, School of Medicine, Daegu, Republic of Korea; cDepartment of Microbiology, School of Medicine, Kyungpook tory concentrations (MICs) of antibiotics and their National University, Daegu, Republic of Korea pathogenic roles in vitro and in vivo. Methods: OMVs were purified from the culture super- Introduction: Staphylococcus aureus-derived extracel- natants of B. cepacia ATCC 25416 cultured with the 1/ lular vesicles (EVs) deliver effector molecules to host 4 sub-MICs of ceftazidime (CAZ), trimethoprim/sulfa- cells and induce host cell pathology. This study inves- methoxazole (SXT) or meropenem (MEM). A549 cells tigated whether thymol could disrupt S. aureus EVs were incubated with B. cepacia OMVs and then ana- and suppress the pathology of the keratinocytes lysed for cytotoxicity and pro-inflammatory cytokine induced by S. aureus EVs. gene expression. Mice were treated with B. cepacia Methods: Membrane disruption of the S. aureus EVs OMVs intratracheally, and lung pathology was treated with thymol was determined using transmis- evaluated. sion electron microscopy. Human keratinocyte HaCaT Results: B. cepacia produced OMVs during in vitro cells were incubated with either intact or thymol-trea- culture. A total of 265 proteins were identified in ted S. aureus EVs and then analysed for cytotoxicity OMVs isolated from B. cepacia cultured in Luria- and pro-inflammatory cytokine gene expression. Bertani broth (OMVs/LB) using proteomic analysis. Results: Thymol inhibited the growth of S. aureus OMVs/LB induced cytotoxicity and stimulated the strains and disrupted the membranes of the S. aureus expression of pro-inflammatory cytokine genes in EVs. Thymol-treated S. aureus EVs inhibited the cyto- lung epithelial A549 cells in a dose-dependent manner. toxicity of HaCaT cells when compared to intact S. B. cepacia produced more OMVs under antibiotic aureus EVs; however, the cytoprotective activity dif- stress condition than under no antibiotic condition. fered between the EVs derived from S. aureus strains. Host cell cytotoxicity and pro-inflammatory response Intact S. aureus EVs stimulated the expression of the were significantly higher in A549 cells treated with pro-inflammatory cytokine and chemokine genes in OMVs from B. cepacia cultured with 1/4 sub-MIC of keratinocytes. The expression levels of the cytokine CAZ (OMVs/CAZ) than in the cells treated with genes differed between thymol-treated EVs from dif- OMVs/LB, OMVs from B. cepacia cultured with 1/4 ferent S. aureus strains, but thymol-treated S. aureus sub-MIC of SXT (OMVs/SXT) or OMVs from B. EVs suppressed the expression of these genes. Thymol- 298 ISEV2019 ABSTRACT BOOK treated S. aureus EVs delivered lesser amounts of the susceptible cells, even after being pretreated with EV component to host cells than intact EVs. RNase A. This indicates that the viral RNA resides Summary/Conclusion: Our results suggest that the thy- inside the IEVs. Using impedance measurements on mol-induced disruption of the S. aureus EVs inhibits the HBMEC/D3 cell monolayers, we observed that IEVs, delivery of effector molecules to host cells, resulting in as well as virus control caused similar and temporal the suppression of cytotoxicity and inflammatory disturbances on the monolayer’s integrity within responses in keratinocytes. Thymol may attenuate the 30 min post infection. No disturbances were seen host cell pathology induced by an S. aureus infection via upon addition of non-infected EVs. both the antimicrobial activity against the bacteria and Summary/conclusion: Our study demonstrates that the disruption of the secreted EVs. EVs-derived from ZIKV-infected cells are able to trans- Funding: This work was supported by the National fer proteins and viral RNA to recipient cells. Since both Research Foundation of Korea (NRF) grant funded by IEVs and viral particles can induce similar changes on the Korea government (NRF-2017R1A2A2A0500 barrier’s integrity it is possible that IEVs are involved 1014). in an alternative mechanism of ZIKV transmission.

PS02.09= OWP2.09 PS02.10=OWP2.11

Deciphering the role of extracellular vesicles on the blood–brain In vivo testing of OMV-based vaccine prototypes against barrier during Zika virus infection Gallibacterium anatis Antonios Fikatas, Sam Noppen, Peter Vervaeke, Jordi Doijen, Mohammed a b b b Fabio Antenucci , Homa Arak , Jianyang Gao , Toloe Allahghadry , Ida Benkheil, Christophe Pannecouque and Dominique Schols Thøfnerb and Anders Miki Bojesenc Laboratory of Virology and Chemotherapy, Rega Institute, KU Leuven, aUniversity of Copenhagen, København S, Denmark; bUniversity of Belgium, Leuven, Belgium Copenhagen, Copenhagen, Denmark; cUniversity of Copenhagen, Copenhagen, USA Introduction: The association of Zika virus (ZIKV) with severe neurological disorders has gained increased Introduction: Outer membrane vesicles (OMVs) are interest over the last decade. However, the mechanism produced by the majority of Gram-negative bacteria. by which ZIKV crosses the blood–brain barrier (BBB) Thanks to the antigenic similarity between OMVs and and reaches the brain remains to be elucidated. It is the bacterial outer membrane, OMVs have proven to known that viruses incorporate viral material in extra- be promising for the development of novel vaccines cellular vesicles (EVs) as a spreading strategy. These against bacterial pathogens. In this work, we describe membrane-enclosed vesicles play a vital role in inter- the testing of OMV-based vaccine prototypes against cellular communication. Currently, there is a lack of Gallibacterium anatis, a Gram-negative pathogen of knowledge on the possible involvement of EVs in great veterinary interest. ZIKV pathogenesis. Our study aims to unravel the Methods: OMVs were isolated from a G. anatis hyper- role of EVs in ZIKV RNA transmission to the brain, vesiculating mutant using a modified version of the via the BBB. Hydrostatic Filtration protocol described by Musante Methods: Human brain microvascular endothelial cells et al. (2014). 120 16-week-old Lohmann-Brown chick- (HBMEC/D3) were used in our study since they repre- ens were divided in six groups and immunized twice sent the BBB in vitro. Three different EV isolation intramuscularly with different combinations of buffer methods (precipitation kit, density gradient and size (controls), OMVs and selected recombinant immuno- exclusion chromatography combined with the density gens. Two weeks after second immunization, the effec- gradient) were performed. Western blot, Transmission tiveness of the immunization regimes adopted was electron microscopy and Nanosight tracking analysis tested by challenging the animals intraperitoneally confirmed the presence of EVs in the supernatant of with live CFUs from a heterologous G. anatis strain. HBMEC/D3 cells. The presence of ZIKV RNA in One week post-challenge, the animals were sacrificed infected-EVs (IEVs) was evaluated by immunofluores- and an established lesion score model was used during cence and qPCR. In addition, the effect of IEVs on the necropsy to evaluate the clinical outcome of infection. BBB was assessed using a label-free impedance-based Results: Statistical analysis of the recorded lesion biosensor (ECIS, Applied BioPhysics). scores showed that the group immunized with G. ana- Results: We confirmed the presence of viral compo- tis OMVs presented an average total score of 2.95, as nents in our IEVs, including the NS1 and E proteins of opposed to an average total score of 8.77 in the control ZIKV. The obtained IEVs were able to re-infect group. The approximately threefold reduction in total JOURNAL OF EXTRACELLULAR VESICLES 299 average lesion score observed demonstrates that immu- manner. Accordingly, we hypothesize that dose/ nization with G. anatis OMVs is able to effectively response optimization and the enrichment of G. anatis decrease the morbidity of G. anatis infection in the OMVs with selected immunogens should result in an immunized animals. improvement of the effectiveness of the vaccination Summary/conclusion: Our results show that G. anatis regime proposed. OMVs represent a promising candidate for the devel- Funding: This research project is being funded by a opment of cost-effective vaccination strategies for the grant from Huvepharma (https://www.huvepharma. prevention of G. anatis infections in a cross-serovar com/). 300 ISEV2019 ABSTRACT BOOK

PS03: EVs in Cardiovascular Disease Chairs: Oh Youn Kim; Caroline Reddel Location: Level 3, Hall A 15:00–16:00

PS03.01 27a, −144/Nrf2 pathway that mediates the development of CIH-associated endothelial dysfunction. Funding: This study was supported by National Serum exosome mediates chronic intermittent hypoxia-associated Natural Science Foundation of China (No.81471082, endothelial dysfunction through regulating miR-144, −27a/Nrf2 pathway 31741064 and 81470567) Huina Zhang

Beijing An Zhen Hospital, Capital Medical University, Beijing, China (People’s Republic) PS03.02

Introduction: Endothelial dysfunction plays a crucial role in the development of OSAHS-related vasculopa- Influence of cardiovascular risk markers on numbers and characterization of circulating extracellular vesicles thy, but the mechanisms are not fully understood. Ruihan Zhou, Dionne Tannetta, Plinio Ferreira; Esra Bozbas, Jon Gibbins, Exosomes, abundant in blood, deliver various mole- Chris Jones and Parveen Yaqoob cules to recipient cells. However, whether serum exo- University of Reading, Reading, UK somes (SExos) are involved in chronic intermittent hypoxia (CIH)-associated vasculopathy is largely Introduction: Extracellular vesicles (EVs) are small unknown. plasma membrane-derived vesicles released from var- Methods: SExo was purified by ultracentrifugation. ious cells, which potentially affect many pathophysio- TEM and NTS were used to evaluate the purity of logical processes involved in cardiovascular diseases exosome. Myograph was used to detect endothelial (CVDs). However, there is little information about function. Western blotting and quantitative polymerase the relationship between gender, CVD risk markers chain reaction (qPCR) were used to measure the (Body Mass Index (BMI), blood pressure (BP), trigly- expression of protein or miRNA respectively. ceride level, cholesterol level and HDL level), CVD risk Confocal microscopy was used to detect the signal of score and circulating EVs. exosome or oxygen free radical in endothelial cells. Methods: Subjects (n = 27) aged 40–70 years with Results: Endothelial dysfunction caused by CIH was moderate risk of CVDs (QRISK2 score) were recruited related with oxidative stress. Furthermore, SExos from and assessed for BMI, BP and blood lipid profile. EVs CIH mouse (CIH SExos) severely impaired endothelial were isolated from platelet-free plasma by size exclu- function and enhanced the oxygen free radical in sion chromatography and analysed by nanoparticle endothelial cells from normal C57BL/6 mice. Western tracking analysis (NTA) and flow cytometry (FCM). blotting showed that the expression of antioxidant NTA measured the concentration and size distribution transcription factor Nrf2 and its downstream target of EVs, and EVs were phenotyped by FCM via a 3- catalase were significantly decreased in CIH SExos- colour panel, including Annexin V (for the majority of treated endothelial cells ex vivo or in vitro. qPCR circulating EVs), CD41 (for platelet-derived EVs) and assay showed significant increasement of exosomal CD105 (for endothelial-derived EVs). miR-27a and miR-144 under CIH status. Results: • Subjects unexpectedly fell into two clear Correspondingly, silencing miR-144 and miR-27a groups: high EVs group (total EV numbers: 4*10^10/ with CIH SExo-packaged antagomiR-27a and mL blood ~ 8*10^10/mL blood, n = 9 or Annexin V antagomiR-144 confirmed the pivotal role of SExo + EV numbers: 2.6*10^7/mL blood ~ 5*10^7/mL miR-27a and miR-144 in CIH SExo-inhibited Nrf2 blood, n = 17) and low EVs group (total EV numbers: expression, CIH SExo-induced endothelial dysfunction 1*10^10/mL blood ~ 3.9*10^10/mL blood, n =18or and the excess oxygen free radical generation in Annexin V+ EV numbers: 9*10^6/mL blood ~ endothelial cells. 2.5*10^7/mL blood, n = 10). Summary/Conclusion: This study demonstrates the • Males accounted for 78% of the subjects in high total adverse effect of CIH SExo on endothelial cells, repre- EVs group. Overweight subjects (BMI ≥ 24.9 kg/m^2) senting a novel cellular mechanism of exosomal miR- contributed to 89% of the subjects with high total EV JOURNAL OF EXTRACELLULAR VESICLES 301 numbers, while 93% of the subjects with normal weight particle concentration. Immunoblotting and electron were classified into low EVs group. The high Annexin microscopy confirm the presence of exosomes. V+ EVs group had significantly higher diastolic BP Samples were stored for proteomic, microRNA and in levels (p = 0.02) and higher cholesterol levels vitro analysis. (p = 0.03) than those with low EV numbers. Those Results: Mean particle sizes at each time point were with higher total EV numbers had a higher average within the known size distribution of exosomes. CVD risk score (p = 0.02). Particle concentration at the completion of cooling • Overweight subjects had a significantly higher num- was decreased from baseline. Thereafter, particle con- ber of endothelial-derived EVs than subjects with nor- centration showed an increase after DHCA and a mal weight (p = 0.02). further increase during chest closure at the conclusion Summary/Conclusion: The majority of subjects with of the surgery. high total EV numbers were male. Overweight contrib- Summary/Conclusion: Our data show that cooling can uted to the elevation of total EV and endothelial- decrease exosome levels in blood, while whole body derived EV numbers. Higher BP level, cholesterol ischaemia reperfusion associated with DHCA in level and CVD risk score were associated with higher patients may be a stimulus for exosome release. As numbers of circulating EVs. more samples are collected, we will assess changes in Funding: This project is supported by Biotechnology the proteome and microRNA content of exosomes and Biological Sciences Research Council (BBSRC)– before and after DHCA as a function of age. This Diet and Health Research Industry Club in UK model also lends itself well to further detailed investigation of tissue and organ-specific responses to ischaemia reperfusion in young and aged patients. PS03.03 Funding: This work was funded by the National Institutes of Health, USA.

Changes in exosome release in ageing: a pilot study in a human model of ischemia reperfusion Ying Qiu Zhoua, Liem Nguyena, Michael Madanib, Victor Pretoriusb, Hemal PS03.04 Patelb and David Rotha aUniversity of California, San Diego, USA; bUniversity of California, San Diego, La Jolla, USA Intracardiac extracellular vesicle release in post-infarction diabetic hearts Introduction: The growing aged population necessi- Stephane Mazlana, Vincent Duvala, Cecile Devuea, Michael Robillarda, b a a tates better understanding of cellular and physiological Chantal Boulanger , Jean-Sebastien Silvestre and Xavier Loyer changes in ageing to improve future healthcare delivery aINSERM, Paris, France; bINSERM ‘ParCC’ Paris-Cariovascular Research Center, Hôpital Européen Georges Pompidou, Assistance Publique- and cost. The role of exosomes, extracellular vesicles Hôpitaux de Paris, and Université Sorbonne, Paris, France carrying biologically active cargo secreted by almost all cells, may have major impacts on perioperative care Introduction: Cardiovascular disease (CVD) is the and monitoring. Deep hypothermic circulatory arrest main cause of death in non-communicable diseases. (DHCA) is a profound perioperative stress event invol- In response to myocardial infarction (MI), extracellular ving hypothermia, arrest of circulation to major organ vesicles (EVs), including large (lEVs) and small (sEVs), systems and whole body ischaemia reperfusion. DHCA are released within and from the heart to facilitate is used during pulmonary thromboendarterectomy, for intercellular communication and maintain cardiac which the University of California, San Diego, USA, homeostasis. As diabetes increases the risk of CVD, serves as a leading centre. With a patient age range of the purpose of the study was to investigate how dia- 14–80 years old, we use DHCA as a model of whole betes influences the release of intracardiac EVs body ischaemia reperfusion to test the novel hypothesis after MI. that DHCA alters the amount of exosome release, Methods: C57BL/6J male mice were fed normal chow content and ability of exosomes to affect cellular meta- diet or high-fat diet (HFD) for 3 months. HFD-fed bolism and function in an age-dependent manner. mice were glucose intolerant as attested by the measure Methods: Plasma was obtained from patients under- of GTT above 200 mg/mL. Mice were then subjected to going DHCA: after induction of anaesthesia (baseline), MI by permanent ligation of the left anterior descend- at initiation of cardiopulmonary bypass (CPB), com- ing artery, and sham animals underwent similar surgi- pletion of cooling, after circulatory arrests and at chest cal procedure without ligation. Left ventricles from closure. Exosomes were isolated with ExoQuick. sham or MI mice were then harvested at either 15, Nanoparticle tracking analysis (NTA) measured 24, 48 or 72 h after surgery (n = 5 per group at each 302 ISEV2019 ABSTRACT BOOK time point) and processed for EV extraction by differ- distribution, according to the MISEV guidelines. ential centrifugation. lEVs and sEVs were then quanti- Exosomal LDLR and ABCA1 protein expressions fied and analysed via Tunable Resistive Pulse Sensing were analysed by flow cytometry, FACS. Furthermore, Technology (TRPS), flow cytometry and Western blot. the exosome specific markers CD9, CD63 and CD81 Results: In chow diet-fed mice, release of both lEVs were simultaneously detected in exosome-EXÖBead and sEVs was increased at 24 h post-surgery when complexes by multiple fluorescent antibody staining compared to shams. These findings were in agreement and FACS. We incorporated 10% exosome-free “foetal with previous data obtained in younger control ani- bovine serum” in PBS as the antibody staining negative mals. In diabetic mice, lEVs peaked at 24 h post-MI control. and this increase was slightly greater than that Results: The exosome size distribution and morphol- observed in chow diet-fed animals. However, there ogy were similar between the plasma sample from were no differences in sEV release between sham and healthy and CAD groups. The geometric mean fluor- MI diabetic mice. TRPS analysis revealed that diabetes escence intensity, MFI of CD9, CD63, CD81, LDLR does not change EV size (diameter) and population. and ABCA1 were not different between these two Furthermore, both control and diabetic-derived EVs groups. However, the corrected MFI ratio of LDLR/ harboured cardiomyocyte marker (Troponin T) as CD9 in healthy donors was significantly higher com- revealed by Western blot. pared to CAD patients (p = 0.044). Similar significant Summary/Conclusion: Our results thus show that dia- changes in ratio of LDLR/CD63 (p = 0.026) and LDLR/ betes modulates the release of both large and small CD81 (p = 0.027) were also observed. Besides, there is intracardiac EVs after MI. Further work will be needed no significant change in exosomal ABCA1 between to fully investigate the functional impact of cardiac EVs healthy donors and CAD patients. in the diabetic heart after MI. Summary/Conclusion: Declined expressions of LDLR/ Funding: INSERM and ANR-16-CE92-0032-02 exosome in patients with CAD were observed in our study. These results may be an essential clue for exploring the function of exosomal LDLR in lipid metabolism PS03.05 and atherosclerosis. Further approaches regarding cell- to-cell communication of exosomal LDLR will be Exosomal low-density lipoprotein receptor (LDLR) as a potential addressed in the future. biomarker in patients with coronary artery disease Dapi Meng Lin. Chianga, Liv Weichien Chenb, Michael Pfafflc and Chin- Sheng Linb PS03.06 aBiovesicle, Taipei, Taiwan (Republic of China); bDivision of Cardiology, Tri- Service General Hospital, Taiwan & National Defense Medical Center, Taipei c City, Taiwan (Republic of China); Animal Physiology and Immunology, Therapeutic EV rescue a deficient hypoxic response in pulmonary School of Life Sciences Weihenstephan, Technical University of Munich, arterial hypertension Freising, Germany David Marciano, Rebecca Harper, Vignesh Viswanathan, Marlene Rabinovitch and Michael Snyder

Introduction: Atherosclerosis is one of the key factors Stanford University, Stanford, USA contributing to cardiovascular disease. Exosomes have been documented to be associated with atherosclerosis Introduction: Complex gene-environment interactions pathogenesis. However, the potential exosome-related can determine the penetrance of a genetic mutation biomarkers in atherosclerosis patients has not been leading to disease. Pulmonary arterial hypertension analysed and characterized yet. In this study, we (PAH) is a lethal disease that is highly associated with aimed for assessing the potential biomarker in serum loss-of-function bone morphogenetic protein receptor exosome for coronary artery disease (CAD). II (BMPR2) mutations and persistent hypoxic stress. Methods: Plasma samples were collected from patients While these genetic and environmental determinants of undergoing coronary angiography. To assess exosomal PAH are clearly defined, little is known about how they low-density lipoprotein receptor (LDLR) and ATP- are interconnected to potentiate disease. binding cassette transporter A1 (ABCA1) expression, Methods: Pulmonary arterial endothelial cells (PAEC) we isolated exosome by incubating glycan recognition were exposed to hypoxia (0.5% O2) for 24 h and beads, EXÖBead (Biovesicle) with 250 µL pre-cleared culture media collected. Extracellular vesicles (EVs) plasma from healthy donors (n = 28) and CAD patients were isolated using differential ultracentrifugation and (CADs, n = 26) as manufacturer’s protocol. To confirm characterized with electron microscopy and nanoparti- the purity of our isolation, we used NTA and TEM to cle analysis. qPCR analysis after RNAse digestion was demonstrate exosome morphology and size performed to identify packaged mRNA. EV treatment JOURNAL OF EXTRACELLULAR VESICLES 303 of mice was administered via tail-vein injection. In vivo stress response. Consent was obtained by the biodistribution was visualized by Gallium-68 labelling University Health Network Research Ethics Board. coupled with positron emission tomography (PET) Results: We identified selective dysregulation in each imaging. treatment group associated with CML and as well as Results: Here we report that BMPR2 is induced by specific cardiovascular pathophysiology, e.g. miR-let- hypoxia in PAEC, and loss of BMPR2 partially blocks 7e-5p, miR-502-5p and miR-548a-5p (p < 0.05). In the release of extracellular vesicles under these condi- vitro, we identified a surprising cardioprotective action tions. EVs derived from hypoxic PAEC are enriched of ponatinib-patient EVs on cardiomyocytes, indicated with BMPR2 mRNA and can restore abnormal pheno- by a significant decrease in free BNP in the media of types in BMPR2 mutant cells in vitro. BMPR2 knock- cardiomyocytes treated with EVs compared to other out mice exposed to intermittent hypoxia develop PAH treatment arms (p < 0.01). phenotypes that are prevented by treatment with Summary/Conclusion: This study represents a novel hypoxic PAEC derived EV. approach investigating the utility of EVs and their Summary/Conclusion: These results show that PAEC- associated miRNAs as biomarkers and effectors of derived EVs are critical for the maintenance of vascular TKI-induced cardiovascular toxicity. Our results high- homeostasis, loss of this signal due to BMPR2 dysfunc- light a distinct profile of miRNAs associated with TKI tion contributes to PAH pathogenesis, and replacement treatment. Understanding the complex role of EVs in with exogenous EV has therapeutic potential in PAH. TKI therapy will elucidate the complexities of cardio- Funding: F32 HL 132452, NIH NHLBI vascular toxicity and aid in tailoring the risk management of individual patients. Funding: This project was funded by the Princess PS03.07 Margaret Cancer Centre.

Role of extracellular vesicles in cardiovascular toxicity induced by BCR-ABL tyrosine kinase inhibitors Dakota D. Gustafsona, Nazanin Aghelb, Jason Fishc, Diego Delgadob and Jeff PS03.08 Liptond aDepartment of Laboratory Medicine and Pathobiology, University of b Toronto, Toronto, ON, Canada; Peter Munk Cardiac Centre, Toronto, Extracellular vesicles derived from genetically modified human Canada; cToronto General Hospital Research Institute, University Health d induced pluripotent stem cells enhance cardiomyogenesis and Network, Toronto, ON, Canada; Princess Margaret Cancer Centre, angiogenesis in vitro and in vivo Toronto, Canada Katarzyna Kmiotek-Wasylewska, Sylwia Bobis-Wozowicz, Anna Labedz- Maslowska, Elzbieta Karnas, Zbigniew Madeja and Ewa Zuba-Surma Introduction: Despite their efficacy as an anti-cancer Department of Cell Biology, Faculty of Biochemistry, Biophysics and therapeutic against chronic myelogenous leukaemia Biotechnology, Jagiellonian University, Krakow, Poland (CML), tyrosine kinase inhibitors (TKIs) can be associated with deleterious cardiovascular effects. Introduction: Extracellular vesicles (EVs) represent Considerable progress has been made in identifying population of small circular membrane vesicles the excess risk of cardiovascular events related to TKI secreted by most cells including stem cells (SCs). It exposure; however, the data on the underlying has been reported that EVs may carry bioactive cargo mechanisms and possible predictive biomarkers are including proteins, microRNAs and mRNAs. They also currently inadequate. To this end, we sought to exam- play a crucial role in cell-to-cell communication in ine EV-associated miRNAs as a means of elucidating both physiological and pathological conditions. their potential as effectors and biomarkers of TKI- The aim of this study was to verify the impact of induced cardiovascular toxicity in CML. EVs derived from human induced pluripotent stem Methods: We obtained informed consent and recruited (iPS) cells (hiPS-EVs) overexpressing procardiomyo- 24 age- and sex-matched response stable CML patients genic miR1 or miR199a, or proangiogenic miR126, on either off-TKI (median 32.26 months, n =6)oron various properties of human cardiac and endothelial long-term treatment with imatinib, nilotinib or pona- cells. tinib (median 79.01 months, n = 6/group), and assayed Methods: hiPS-EVs were isolated from conditioned plasma-derived EV-associated miRNAs using the hiPS culture media by differential centrifugation nCounter® Analysis System. Concurrently, in vitro stu- including ultracentifugation. Cardiac cells and dies were conducted to examine the responses of iPSC- endothelial cells were used as target cells in vitro, and derived human cardiomyocytes to plasma-derived EVs their functional properties were evaluated after hiPS- using BNP as a surrogate marker of the cardiovascular EVs treatment. The regenerative capacity of hiPS-EVs 304 ISEV2019 ABSTRACT BOOK was also examined in vivo – in murine model of acute investigated (using Boyden’s Chamber assay, MTT limb ischaemia (LI). assay and western blot analysis/phosphokinase arrays, Results: Our data indicate that hiPS-EVs carrying pro- respectively). cardio- and proangiogenic miRNAs may protect car- Results: Isolated AFSC sEVs were CD9/CD63/CD81- diac cell types from apoptosis as well as enhance their positive and of high purity (up to 1.2x10^10 particles/ proliferation, metabolic activity, migration and cardio- µg protein). These vesicles were not cardioprotective in myogenic differentiation. The hiPS-EVs enhanced also models of simulated ischaemia/reperfusion injury in proangiogenic capacity, migration and metabolic activ- primary cardiomyocytes in vitro. Nevertheless, AFSC ity of HCAEC cells in vitro. The vesicles also promoted sEVs carried promigratory cytokines and angiogenic angiogenesis and increased blood flow recovery in factors (e.g. SDF-1, MIF, PTX3) and promoted murine ischaemic limb injury model in vivo. endothelial cell migration and proliferation in vitro. Summary/Conclusion: These results may indicate (i) Pharmacological inhibition of PI3K (a promigratory feasibility of genetic modifications of EVs enforcing signalling pathway) in target endothelial cells reduced their regenerative proprieties as well as (ii) enhanced sEV-stimulated migration by 54 ± 15% (p < 0.001). activity of EVs from hiPS cells overexpressing miR1, However, sEVs did not induce phosphorylation of miR199a and miR126 in regeneration of ischaemic downstream PI3K targets, indicating that sEV effects tissues. We conclude that EVs from genetically mod- may be multifactorial and may involve multiple ified hiPS cells may represent new safe tool for tissue pathways. repair alternative to whole-cell therapies in vivo. Summary/Conclusion: AFSC sEVs did not have direct Funding: This study was funded by NCN and NCBR protective effects on cardiomyocytes in vitro but pos- grants: SONATA BIS-3 (UMO-2013/10/E/NZ3/ sessed proangiogenic potential which requires, but is 007500) and STRATEGMED III (STRATEGMED3/ not solely dependent on, PI3K signalling. Ongoing 303570/7/NCBR/2017) to EZS and PRELUDIUM-11 experiments include analyses of the sEV proteome, (UMO-2016/21/N/NZ3/00363) to KKW. their cardioprotective properties in a model of rat myocardial ischaemia/reperfusion injury in vivo and their role in capillary sprouting from rat aortic PS03.09 explants. Together, these data will define the potential for using AFSC sEVs as cardioprotective and proangio-

Cardioprotective and proangiogenic potential of small extracellular genic therapy. vesicles secreted from amniotic fluid stem cells Funding: BHF Kaloyan Takova, Filipa Vlahovab, Pascale Guillotb, Derek Yellona and Sean Davidsona aThe Hatter Cardiovascular Institute, University College London, London, PS03.10 UK; bInstitute for Women’s Health, University College London, London, UK

Introduction: Mesenchymal stem cells (MSCs) exhibit CystatinC and CD14 in plasma extracellular vesicles are associated with both renal dysfunction and heart failure in patients presenting antiapoptotic and proangiogenic functions in models with dyspnoea of myocardial infarction, a common cause of death and Mirthe Dekkera, Farahnaz Waissib, Laura Verbree, Irwani Ibrahim, Shirley Ooi, Jiong-Wei Wangc, Win Kuand, Siew Chanc, Linda Peelene, Diederick disability. These effects are partially mediated by Grobbee, A. Mark Richards, Carolyn Lam, Ya-Nan Zhang, Muhammad I secreted small extracellular vesicles (sEVs). Amniotic Mazlan, Dominique de Kleijnf fluid stem cells (AFSCs) are foetal MSCs with superior aUMC Utrecht, Utrecht, Netherlands; bUMC Utrecht, Utrecht, Netherlands; functional potential to adult MSCs. We hypothesized cNational University of Singapore, Singapore; dNational University Hospital, Singapore; eJulius Center for Health Sciences and Primary Care UMC that sEVs released by AFSCs are cardioprotective and Utrecht, Utrecht, Netherlands; fUMCU, Utrecht, Netherlands proangiogenic. Methods: Human AFSC sEVs were isolated from Introduction: Heart failure and renal failure com- serum-free conditioned medium by size-exclusion monly coexist: heart failure patients have higher chance chromatography and characterized using nanoparticle of developing renal failure and vice versa.1,2 Declines in tracking, dot blots, protein and immunoassays, electron renal function are associated with the development of microscopy and protein arrays. Their cardioprotective ventricular dysfunction and worsen prognosis in heart potential was examined in models of hypoxia/reoxy- failure.3,4 However, underlying pathophysiological genation- and reactive oxygen species-induced death of mechanisms in cardiac-renal cross talk are not fully primary adult rat cardiomyocytes in vitro. AFSC sEV understood1. The role of plasma extracellular vesicles effects on human endothelial cell migration, prolifera- (EVs) in combined organ failure such as cardiorenal tion and signalling pathway activation were also syndrome has not been investigated. The primary aim JOURNAL OF EXTRACELLULAR VESICLES 305 of this study is to investigate if the extracellular vesicle Introduction: Acute myocardial infarction (AMI) is a proteins CystatinC, CD14, SerpinG1 and SerpinF2 that major cause of death. To diagnose AMI, measuring have been associated with heart failure are also asso- troponin concentration is the gold standard. Since tro- ciated with renal dysfunction in patients with acute ponin is unspecific for AMI, novel biomarkers for AMI dyspnoea. are urgently needed. After the onset of AMI, platelets, Methods: Blood samples were prospectively collected endothelial cells and blood cells release specific extra- in 404 patients presenting with breathlessness at the cellular vesicles (EVs). Our aim is to identify these EVs emergency department at National University Hospital, as biomarkers for AMI diagnosis and treatment Singapore. Renal dysfunction was defined as estimated monitoring. glomerular filtration rate below 60 mL/min/1.73m2. Methods: The study was approved by the medical The presence of heart failure was independently adju- ethics committee. Venous blood was collected 24 dicated by two clinicians. EVs were precipitated in 3 hours, 72 hours and 6 months after AMI from fasting sub-fractions from the plasma using dextrane sulphate patients (n=60, 64.5±10.8 years, 68% male) and healthy for the LDL and HDL plasma-subfractions or controls (n=30, 57.7±6.6 years, 62% male). Flow cyto- ExoQuick. After precipitation, the EVs were lysed and metry (Apogee A60 Micro) was used to determine the four selected proteins were measured quantitatively plasma concentrations of EVs labelled with antibodies using immunobead assays and tested for their associa- for activated platelets (CD61, CD62p; PEVs), endothe- tions with renal dysfunction, heart failure and the lial cells (CD146; EEVs) and red blood cells (CD235a; concurrence of both conditions using multinomial RBC-EVs). Processing of 1,224 flow cytometry data regression analysis. files was performed using in-house developed, auto- Results: CystatinC was associated with renal dysfunc- mated software (MATLAB R2018a), enabling flow tion, heart failure and their combination in all three rate stabilization, diameter and refractive index deter- EV-sub-fractions and in plasma. CD14 was associated mination, MESF calibration, fluorescent gate determi- with both renal dysfunction and the combined renal nation and statistics reporting. dysfunction and heart failure in all EV-sub-fractions, Results: Between AMI patients and controls, PEV con- and with the presence of heart failure in the HDL-sub- centrations in plasma were comparable (p=ns), EEV fraction but these associations were only seen in the EV concentrations increased (p<0.0001), and RBC-EV subfractions and not in plasma. concentrations decreased (p<0.0001). Antiplatelet Summary/Conclusion:Weprovidethefirstdatashowing drug ticagrelor decreased concentrations of PEVs that EV CystatinC and CD14 are associated with both (p=0.03), compared to less potent clopidogrel, but did renal dysfunction and heart failure in patients presenting not affect EEVs and RBC-EVs. In turn, concentrations with acute dyspnoea. These data suggest that extracellular of EEVs, but not PEVs and RBC-EVs, positively corre- vesicle proteins may be involved in the combined organ lated with the dose of atorvastatin (p<0.001). The anti- failure of the cardiorenal syndrome, and represent possi- oxidative β-blocker carvedilol increased concentrations ble targets for prevention or treatment. of RBC-EVs, compared to nebivolol (p=0.05), but did not affect PEVs and EEVs. PS03.11=OWP1.03 Summary/Conclusion: Flow cytometry and automated data processing were used to find biomarkers for AMI based on EVs in plasma. During treatment, ticagrelor Identification of extracellular vesicles as biomarkers for myocardial infraction by flow cytometry and automated data processing decreased PEV concentrations, atorvastatin increased Aleksandra Gasecka1; Edwin van der Pol2; Frank Coumans3; Kinga Pluta4; EEV concentrations, and carvedilol increased RBC-EV 4 5 3 Grzegorz Opolski ; Krzysztof J. Filipiak ; Rienk Nieuwland concentrations, suggesting that EVs might be used to 11st Chair and Department of Cardiology, Medical University of Warsaw, monitor AMI treatment. AMI patients differed from Warsaw, Poland; 2Amsterdam UMC, University of Amsterdam, Department of Biomedical Engineering and Physics, Amsterdam, Netherlands, Amsterdam, controls regarding EEV and RBC-EV concentrations, Netherlands; 3Amsterdam UMC, University of Amsterdam, Laboratory of but not PEVs, likely because blood was collected 24 Experimental Clinical Chemistry, Amsterdam, Netherlands, Amsterdam, Netherlands; 41st Chair and Department of Cardiology, Medical University of hours after the start of antiplatelet therapy. In follow- Warsaw, Poland, Warsaw, Poland; 51st Chair and Department of Cardiology, up studies, it is crucial to collect blood prior to Medical University of Warsaw, Poland, Warsaw, USA treatment. 306 ISEV2019 ABSTRACT BOOK

PS04: Affinity and Microfluidic Separation Chairs: Kazunari Akiyoshi; Yanling Cai Location: Level 3, Hall A 15:00–16:00

EVs were successfully isolated from human plasma PS04.01 with almost same recovery rate. Summary/Conclusion: The difference of diffusion velocity in laminar flow was dominant factor in separ- Isolation of extracellular vesicles from small volume of plasma by ating proteins from EVs in our microfluidic ATPS. microfluidic aqueous two phase system Bohoon Hana, Sumi Kima, Yeseong Choia, Seok Chungb and Ji Yoon Kanga Other body fluids will be tested with our modified system. We expect that our device will provide more aKorea Institute of Science and Technology, Seoul, Republic of Korea; bKorea University, Seoul, Republic of Korea useful application in isolation of EVs.

Introduction: Isolation of extracellular vesicles (EVs) from small volume of sample is a major issue of point- PS04.02 of-care testing and it leads to great attention in microfluidic device. However, previous microfluidic immu- Extracellular vesicle-associated microRNAs show stronger correlations noaffinity approach has possibility of the loss of EVs with cardiovascular disease protein biomarkers than cell-free that might have more useful information due to het- microRNAs in human plasma Shi Chena, Shu-Chu Shieshb, Gwo-Bin Leec and Chihchen Chena erogeneity of EVs. In the case of microfluidic device a applying external forces, has drawback in complicated Institution of NanoEngineering and MicroSystems, National Tsing Hua University, Hsinchu, Taiwan (Republic of China); bDepartment of Medical fabrication process and possibility in deformation of Laboratory Science and Biotechnology, National Cheng Kung University, EVs. Therefore, this paper suggests a microfluidic aqu- Tainan, Taiwan (Republic of China); cDepartment of Power Mechanical Engineering, National Tsing Hua University, Hsinchu, Taiwan (Republic of eous two phase system (ATPS) in isolation of EVs from China) stable laminar two phase flow with just simple design of chip. Introduction: This abstract presents a high-efficiency Methods: EV-protein mixture was tested to investigate method utilizing two sets of magnetic beads to isolate the partitioning behaviour. EVs were isolated by ultra- extracellular vesicles (EVs) and EV-associated centrifuge from human plasma, then bovine serum microRNAs (EV-miRNAs) from human platelet-poor albumin was added to prepare EV-protein mixture. plasma samples. Our goal is to develop a platform for Polyethylene glycol (PEG, 3.5 wt%) dissolved in phos- risk assessment of cardiovascular diseases (CVDs) and phate-buffered saline was injected to top and bottom compare the expression levels of circulating cell-free inlet. Dextran (DEX, 1.5 wt%) dissolved in sample was miRNAs and EV-miRNAs. In contrast to the rapid injected to middle inlet. Fluorescence intensities of EV peaking and falling of cardiac troponin I (cTN-I), a and albumin were imaged to investigate the partition- conventional CVD biomarker, the level of circulating ing behaviour in real time from EV-protein mixture. miR-126 remains downregulated even one week after Concentrations of collected EV and albumin were mea- the onset of acute myocardial infarction (AMI). sured to confirm the fluorescence imaging. Also, same Methods: In this study, we first used anti-CD63 anti- experiment was performed with only PEG without body-coated magnetic beads to separate CD63+ EVs. dextran to investigate the effect of ATPS. EV isolation EV-miRNAs were released after EV lysis and subse- from human plasma was also performed and charac- quently extracted by using oligonucleotide-conjugated terized by western blot and atomic force microscopy. magnetic beads. Expression levels of cell-free and EV- Results: Most of green EVs were remained in middle associated microRNAs in six clinical plasma samples phase where red BSA seems almost fully diffused out were quantified using quantitative reverse transcription for the equilibrium state in fluorescence experiment. polymerase chain reaction (RT-qPCR) with a spike-in Microfluidic ATPS could isolate the EV with 83.43% of exogenous cel-miR-238 control. recovery efficiency and protein removal of 65.46% Results: Experimental results showed the levels of from EV-protein mixture. Microfluidic without ATPS miRNAs in CD63+ EVs were ~74% of cell-free could isolate the EV with recovery rate of 67.14%. Also, miRNAs in plasma, whereas the miRNA extraction JOURNAL OF EXTRACELLULAR VESICLES 307 efficiency was >87% and exhibited no apparent depen- Results: As results of LAC evaluations, both ConA- dence on the concentration of miRNA and the medium SPM and SSA-SPM showed selective lectin affinity for evaluated. Compared with the levels of conventional the glycoproteins, only the glycoproteins associated to CVD protein biomarkers, EV-derived miR-126 levels each lectin were selectively separated from the mixture were negatively correlated with N-terminal pro-b-type samples. Additionally, an Ins-SPM allowed the effective natriuretic peptide (NTproBNP) and cTN-I levels with permeability against liposome and exosome. This R^2 = 0.70 and R^2 = 0.61, respectively. In contrast, means that the protein-immobilized SPM was suitable circulating total miR-126 levels were only weakly cor- for the separation media of nanometer sized particles related with these biomarkers (R^2 = 0.14, R^2 = 0.02, without any non-specific adsorption. Finally, we respectively). demonstrated the selective separation of exosome due Summary/Conclusion: We have developed methods to to lectin affinity. As a result, SSA-SPM provided the isolate EVs from human plasma samples, and subse- effective adsorption of exosome based on the interac- quently to extract miRNAs carried by EVs by using two tion between SSA and sialic acid on exosome. sets of magnetic beads. Our preliminary results suggest Summary/Conclusion: According to these results, the that EV-associated miR-126 may serve as a better bio- newly developed lectin-SPMs can be used for the marker than the total circulating miR-126. More clin- separation of exosomes based on the difference of the ical samples are currently being investigated. surface sugar chains. We believe that the increase of Funding: Taiwan Ministry of Science & Technology number of lectin-SPMs and other affinity-SPMs will (MOST 106–2221-E-007–003-, MOST 105–2221-E- lead to the detailed classification of exosomes due to 007–009-, and MOST 106–2221-E-007–002-) and the its surface chemistry. Taiwan Ministry of Education (Higher Education (1) Kubota, K.; Kubo, T.; Tanigawa, T; Naito, T.; Sprout Project: grant no. 107Q2713E1). Otsuka, K. Sci. Rep. 2017, 7, 178.

PS04.03 PS04.04

A microfluidic module for extracellular vesicle separation coupled to Effective separation of exosomes based on its surface sugar chains microarray-based phenotyping using a macroporous spongy monolith Marina Creticha, Dario Brambillaa, Alessandro Romanatoa, Maria Teresa a b b b Takuya Kubo, Raga Ishikawa, Seiya Kato, Toyohiro Naito, Yoshihiro Sasaki, Odinolfi , Stephanie Descroix , Lucile Alexandre , Laura Trapiella ,M. c d a Kazunari Akiyoshi and Koji Otsuka Selim Ünlü , Natasa Zarovni and Marcella Chiari Kyoto University, Kyoto, Japan aConsiglio Nazionale delle Ricerche (CNR), Istituto di Chimica del Riconoscimento Molecolare (ICRM), Milano, Italy; bCurie Institute, Paris, France; cBoston University, Boston, USA; dHansaBioMed Life Sciences Ltd, Introduction: The surface sugar chains on exosomes Tallin, Estonia contribute the communication among cells. But, in the present separation procedures, the effective separations Introduction: Standard approaches to characterize EVs of exosomes based on the differences of sugar chains are usually either low-throughput, laborious or based have never reported. We focus on a lectin affinity on sophisticated equipment not applicable to clinical chromatography (LAC) with a macroporous spongy routines. An integrated microfluidic platform that iso- monolith (1), which is suitable for a high throughput lates and characterizes EVs available in bodily fluids by and selective separations for biomolecules. In this combining capture, release and phenotyping of bio- study, we prepared a few lectin-immobilized spongy- nanoparticles would significantly accelerate the transi- monolithic columns and evaluated for typical LAC tion of EV-based research to real clinical utility. The analyses. Additionally, the columns were applied for realization of such a system is the goal of INDEX, a the separation of exomes to determine the fundamental project recently funded in the frame of Horizon 2020 adsorption/desorption conditions. FET-OPEN programs. Methods:Poly(ethylene-co-glycidylmethacrylate)(PE Methods: The platform consists of two modules, one GM)-based spongy monolith (PEGM-SPM) was packed for the extraction of EVs in complex samples and one into columns, and then concanavalin A (ConA) or for the interferometric label-free identification and Sambucus sieboldiana agglutinin (SSA) was immobilized. visualization in a disposable cartridge. In order to Additionally, bovine serum albumin or insulin (Ins) was integrate the two modules, a new approach for the further immobilized to block the hydrophobic surface of reversible capture of EVs from serum was developed PEGM-SPM. The obtained columns were simply analysed and demonstrated using the so called magnetic flui- by LAC and applied for the separation of exosomes. dized bed technology recently developed by Pereiro et 308 ISEV2019 ABSTRACT BOOK al. (Lab Chip, 2017, 17, 1603–1615). This technology is sensing protocol is established and carried out by based on the use of functionalized magnetic particles to using the two platforms and, subsequently, the proce- perform a solid phase extraction step. We report on the dure is transferred in a microfluidic environment. Gold progress of the optimization of the technology itself nanoparticles are first deposited on glass substrates and and how it can be used to recover intact EVs. annealed to form gold nano-islands, whereas the gold Results: In the optimization of the magnetic fluidized nanoparticles were in-situ synthesized in a PDMS bed system as tool for the isolation and pre-concentra- matrix by the immersion method. EVs are affinity tion of EVs in plasma/serum patient samples, several captured by a peptide (Vn96) in this protocol. The steps/issues have been evaluated including the immo- two platforms were individually bonded to a PDMS bilization chemistry of antibodies on the surface of sample having a channel to form a microfluidic device. magnetic particles for increased EV recovery, the The entities involved in the biosensing protocol are dimensions of the chip, the flow rate and sample flown through the channel, and the absorption spec- volume. Capture and release efficiency were evaluated trum is measured after each step. by direct on-chip monitoring of the fluorescence when Results: A graph showing the LSPR shift of the gold working with fluorescent samples or by ELISA test. plasmon band for different concentrations of EVs is Capturing and release steps were also monitored on plotted for the two platforms. The exosomes from anti-tetraspanins antibody microarrays by fluorescence breast cancer cell line (MCF7)-conditioned media and interferometric detection. have been grown in a small bioreactor. Comparable Summary/Conclusion: In summary, we have demon- results in terms of sensitivity have been found for the strated that intact EVs can be released from the first two platforms. module of the sensing platform and transferred to the Summary/Conclusion: Compared to the macro detec- second module devoted to nanoparticle sizing and tion method, the microfluidic detection of EVs proved phenotyping to be highly reproducible and more sensitive as very Funding: This project has received funding from the small amount of chemicals and EVs are necessary for European Union’s Horizon 2020 research and innova- the analysis. tion programme under grant agreement no. 766466. INDEX. PS04.06 PS04.05

Dielectrophoretic nanovesicle sorter Yong-Sang Ryua, Avijit Barikb, Nathan J. Wittenbergb, Daniel A. Mohrb and Comparison of extracellular vesicles detection by microfluidic Sang-Hyun Oh3 plasmonics of gold nano-island and nanocomposite platforms a b Muthukumaran Packirisamya, Srinivas Bathinia, Simona Badilescub, Sensor System Research Center, Seoul, Republic of Korea; University of c Duraichelvan Rajua, Anirban Ghoshc and Rodney J Ouellettec Minnesota, Minneapolis, USA; University of Minnesota, Minneapolis, Minneapolis, USA aConcordia University, Montreal, Canada; bConcordia University Montreal, Montreal, Canada; cAtlantic Cancer Research Institute (ACRI), Moncton, Canada Introduction: Extracellular vesicles are membrane- bound particles that play important roles in cellular Introduction: Extracellular vesicles (EVs) are groups of communications, packaging of genetic material and nanoscale extracellular communication organelles in waste management. An important category of extracel- the order of 30–100 nm, which can be used as disease lular vesicles, exosomes, are only 30-100 nm in size. To biomarkers for cancer. In this work, we have developed investigate the biological functions of these extracellu- different platforms for the detection and characteriza- lar vesicles and to use them for applications in diag- tion of EVs by using a localized surface plasmon reso- nostics and drug delivery, rapid isolation with high nance (LSPR) method based on the sensitivity of the collection efficiency and selectivity is of great impor- gold plasmon band to the environment of gold tance. Small unilamellar vesicles (SUVs), as a model nanoparticles. type of exosomes, have been extensively exploited to Methods: EVs from breast cancer cell line (MCF7) are characterize the role of extracellular vesicles during the detected and characterized by using a gold nanoparti- processes. cle-based plasmonic platforms. Here, two different Methods: 2.1. Fabrication of 10 nm-width-gap elec- platforms have been developed, a gold nano-island trode device platform on glass substrate and a gold poly(dimethyl) 2.2. SUV preparation and size characterization siloxane (Au-PDMS) nanocomposite. A plasmonic 2.3. Dielectrophoresis on nanogap electrodes JOURNAL OF EXTRACELLULAR VESICLES 309

Results: Here we demonstrated that dielectrophoresis platform to separate intact EVs based on specific sur- (DEP) can be used to collect and sort sub-100 nm face signatures and compare their properties. SUVs, a model of exosomes, based on their size and Methods: EVs were isolated from MDA-MB-231 cells the electrical properties of their cargo. The DEP plat- using size exclusion chromatography. EV subpopula- form is based on a 0.8 mm-long, 10 nm-wide gap tions expressing specific surface markers were captured between gold electrodes, capable of generating ultra- on magnetic beads and released using a novel release high electric field gradients with low voltages. We protocol. Released EVs were characterized by western determine the DEP trapping threshold voltages as a blotting, nanoparticle tracking analysis (NTA) and function of vesicle size for the selective capture. transmission electron microscopy (TEM). Uptake of Furthermore, SUVs with different internal conductiv- fluorescently labelled EV subpopulations by various ities can be sorted by varying DEP frequency. cell types was examined using flow cytometry. 3. 1. Dielectrophoretic trapping of SUV and size- Results: Isolated MDA-MB-231 EVs showed typical dependent sorting EV properties, including the presence of EV marker 3.2. SUV sorting based on internal conductivity. proteins, heterogeneous size distribution (mode size of Summary/Conclusion: Such differential DEP 120 nm) by NTA and intact, “cup-shaped” morphology responses may allow the isolation of membrane-free as visualized by TEM. When these EVs were subjected macromolecular aggregates in the presence of empty to the capture-and-release platform, EV subpopula- vesicles down to size ranges of d ≤ 100 nm without tions with different properties were obtained. labelling processes required for detection methods used Released subpopulations appeared intact as demon- with other separation techniques. Our electronic DEP strated by TEM, but differed in their size distribution. sorter can readily be applied to diverse biological mate- Furthermore, EV subpopulations showed different rials including viruses, proteoliposomes, functionalized enrichment/depletion patterns of canonical EV pro- nanobeads, DNA molecules and other biomolecules. teins as shown by western blot. Lastly, uptake of EVs Funding: This research was supported by grants from by target cells differed between EV subpopulations and the Minnesota Partnership for Biotechnology and between target cell types. Medical Genomics, MnDrive Research Initiative, NSF Summary/Conclusion: In this work we showcase a through the National Nanotechnology Coordinated novel capture-and-release platform to separate intact Infrastructure (NNCI) program, and internal project EV subpopulations based on their expression of speci- of KIST. fic surface markers. Using a small panel of antibodies against EV surface markers, we show differences between EV subpopulations in terms of protein com- PS04.07 position, size distribution and cellular uptake by target cells. We anticipate that this tool can help to clarify relationships between the surface signature of EVs and

A novel capture-and-release platform to isolate extracellular vesicle their functionality, and facilitate the enrichment of EVs subpopulations reveals functional heterogeneity among EVs with with desirable characteristics for therapeutic purposes. different surface markers Olivier G. de Jonga, Mark Tielemansb, Raymond Schiffelersc, Pieter Vaderc and Sander A. A. Kooijmansc aDepartment of Physiology, Anatomy and Genetics, University of Oxford, PS04.08 Utrecht, Netherlands; bDepartment of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, Netherlands; cLaboratory of Clinical Chemistry and Hematology, University Medical Center Utrecht, Utrecht, Netherlands Nanopillar and nanochannel fabrication via mixed lithography Sung-Wook Nama, Sun-Woong Leea and Moon-Chang Baekb Introduction: Extracellular vesicles (EVs) are hetero- aSchool of Medicine, Kyungpook National University, Daegu, Republic of Korea; bSchool of medicine, Kyungpook National University, Daegu, geneous in terms of size and molecular composition, Republic of Korea which may also reflect functional differences. For example, given that the EV surface dictates interactions Introduction: Extracellular vesicle (EV) sorting and with their environment, EVs with different surface separating by nanostructure is essential to achieve a profiles may be taken up and processed by target cells size-dependent analysis of protein and miRNA inside in different ways. Unfortunately, tools to isolate and the vesicles. In this regard, implementation of lab-on-a- functionally compare EV subpopulations based on chip devices having the EV sorting functionality has their surface marker expression are currently not avail- been pursued by utilizing the physical properties of the able. Here, we describe a novel capture-and-release particles. 310 ISEV2019 ABSTRACT BOOK

Methods: Nanopillar array is a useful template for calibration particles (polystyrene and melanin resin sorting and separating EVs. We report a method of nanoparticles) biofunctionalized with proteins and fabricating nanopillar array coupled with large-scale mimicking EVs in buffer solution. fluidic structures. To do this, we introduce mixed Results: Sample was introduced into the chip using a lithography by which both nanometer-scale functional syringe pump or a pressure generator and the filtered features and large-scale guiding structures are gener- sample was simply collected at the chip outlet and ated in the same level upon 200 mm silicon wafers. redirected towards a biodetection chamber designed Results: Upon 200 mm silicon wafer, nanometer fea- as an array of gold plots functionalized with antibodies. tures are firstly produced by electron beam lithography We demonstrated the high quality separation of 490 (EBL) in the extremely localized area which is subse- nm nanoparticles from 920 nm particles in concen- quently connected by the micrometer structures pro- trated solution (2.109 to 2.1011 particles/µL). duced by photolithography. By introducing hard- Following sorting step, biosynthetic particles were masking oxide layer, we can create the coupled geome- immunocaptured in a miniaturized module of the try in the same level structure. For the nanometer NBA platform (2, 3) for their subsequent analysis. fluidic channels, we examine wetting of a liquid solu- Summary/Conclusion: We did the proof-of-concept of tion containing fluorescent polystyrene particles. on-chip nanoparticles separation and capture demon- Summary/Conclusion: We demonstrate EV sorting strating the ability of miniaturized systems to perform devices by implementing nanostructures in lab-on-a- sample fractionation. The tunable properties of the chip structure. Our method may offer a way to produce device open the way to a versatile tool for pre-analy- biochips that have versatile functions including sorting tical steps of EVs, including sorting and concentration, and separating EVs. even in complex media. Funding: This research was supported by the Bio & Funding: ANR: Agence Nationale de la Recherche Medical Technology Development Program of the National Research Foundation (NRF) funded by the Ministry of Science & ICT (2017M3A9G8083382). PS04.10

PS04.09 Acoustophoretic-based microfluidic platform for sorting extracellular vesicles Erfan Taatizadeha, Arash Dalilib, Nishat Tasnima, Cathie Garnisc, Mads d e f Towards on-chip EVs separation: a lab-on-chip approach Daugaard , Isaac Li , Mina Hoorfar a b Lyne Pillemont, Daniel Guneysu, Celine Elie-Caille , Wilfrid Boireau and a b University of British Columbia Okanagan, Kelowna, Canada; University of Anne-Marie Guec British Columbia Okanagan, Kelowna, Canada; cAssociate Professor, Faculty aFEMTO-ST Institute, Besançon, France; bFEMTO-ST Institute, UBFC, of Medicine, Department of Surgery, Division of Otolaryngology, University CNRS, Besançon, France; cCNRS, Toulouse, France of British Columbia Senior Scientist, Genetics Unit, Integrative Oncology Department, BC Cancer Research Centre, Vancouver, Canada; dVancouver Prostate Centre Head, Molecular Pathology & Cell Imaging Core Facility, Introduction: Owing to their complexity in size, ori- Vancouver Prostate Centre Assistant Professor, Department of Urologic gin, membrane markers, there is currently no ideal Sciences, University of British Columbia, Vancouver, Canada; eDepartment of Chemistry, University of British Columbia Okanagan, Kelowna, Canada; technology available to relate cell-derived microvesicles fUniversity of British Columbia, Kelowna, Canada (EVs) structure and functions. All currently available methods (flow-cytometry, DLS, TRPS, etc.) have limits Introduction: Conventional methods used for isolation in their ability to capture the whole diversity of EVs of extracellular vesicles (EVs) are time-consuming, populations and are not amenable to automation and produce low purity samples and may change the struc- large-scale analysis of numerous samples. In that con- ture of EVs. To address these problems, microfluidics- text, the overall objective of this study is to develop a based EV isolation methods have been introduced. In miniaturized platform allowing the isolation, fractiona- particular, acoustic-based cell isolation (functioning tion and qualification of microvesicles in µL volume. based on size, density and compressibility differences Methods: Based on previous works (1), we propose a of bioparticles and medium) have shown potentials. lab-on-chip coupling a hydrodynamic separation mod- However, the geometrical and operational parameters ule enabling EVs separation according to their size to of such a platform still need to be optimized to produce an affinity-trapping chamber compatible with subse- high throughput and reproducible results. This study quent SPR and AFM characterization. We designed focuses on the optimization of an acoustophoretic- and fabricated 2.5 ×2.5cm chips enabling the separa- based microfluidic platform using first colloidal partition of vesicles at tunable cut-off (150-900nm). The cles following by EVs isolated from culture media from proof-of-concept was done using fluorescent cancer cell lines. The results are compared against the JOURNAL OF EXTRACELLULAR VESICLES 311 conventional method to show high yield and purity of In this study, we aimed to establish a method to effi- the proposed platform. ciently recover exosomes from serum, plasma and Methods: The acoustic pressure field can be generated urine using IP and UC method, considering practical inside a microchannel by applying a voltage to pat- use at the clinical site. terned interdigital transducers fingers on the surface Methods: Antibodies against tetraspanins and IP con- of piezoelectric materials. Due to such a field, biopar- dition were established and used to isolate exosomes ticles are deflected (and hence sorted) at different from serum, plasma and urine. Obtained exosomes points along the microchannel depending on their were subjected to immunoblotting, nanoparticle track- volumes. Soft lithography and etching processes are ing analysis (NTA), proteomic analysis, internalization used for fabrication of microchannel and transducers assay and 3D-Gene miRNA microarray. of the platform. Results: Immunoblotting and NTA revealed the recov- Results: To optimize the geometry and operational ery of highly pure exosomes from serum and plasma parameters of the platform, polystyrene (PS) particles with increased efficiency by our IP method. Our are first used as they have similar size, density and method was successful in recovering exosomes from compressibility of the components in the body fluid urine specimens, whereas commercialized antibodies samples. The results showed that 90% of PS particles failed to do so. Internalization assay showed that are deflected at a frequency of 26.5 MHz and the input uptake rate of exosomes isolated from conditioned voltage of 10 Vpp. Using these parameters, we are then medium using our method were similar to that of able to sort EVs from cell culture media into size exosomes isolated using conventional method. ranges between 500–1000 nm. The size of each sorted Number of identified proteins has increased, whereas vial is characterized by nanoparticle tracking analysis the detection of nonspecific proteins decreased by our and shown a size separation resolution of 500 nm and a method. Expression profiles of miRNAs from our throughput of 4 uL/min. obtained exosomes differed from that obtained by con- Summary/Conclusion: Acoustofluidics-based separa- ventional isolation method. tion results show the size separation resolution of Summary/Conclusion: Our established exosome pur- 500 nm and a throughput of 4 uL/min, indicating the ification methods are capable of efficiently recovering protentional of such a technique as a non-invasive, exosomes from serum and plasma in addition to urine label-free and effective EV purification method. specimens. Our approach can be readily automated to Funding: This work was supported by the University isolate exosomes from specimens, which could contri- of British Columbia Eminence fund. bute to therapeutic application of exosomes and biomarker detection. PS04.11 PS04.12 Proteomic and miRNA analysis of highly purified extracellular vesicles recovery using immunoaffinity purification and ultracentrifugation from serum, plasma and urine Capture and release of extracellular vesicles in tens of μL samples for Ayako Kurimoto, Yuki Kawasaki and Tatsutoshi Inuzuka ocular neuroprotection studies a b a Miraca Research Institute G.K., Hachioji-shi, Japan Yi-Hsun Chen , Rong-Kung Tsai and Chihchen Chen aInstitution of NanoEngineering and MicroSystems, National Tsing Hua Introduction: Exosomes, one of extracellular vesicles, University, Hsinchu, Taiwan (Republic of China); bInstitute of Eye Research, Buddhist Tzu Chi General Hospital, Hualien, Taiwan (Republic are secreted into extracellular fluids from all types of of China) cells via endosomal pathway and found in most body fluids including blood and urine. Exosomes are report- Introduction: The incidence of eye diseases is on the edly associated with various disease conditions includ- rise with increasing longevity and use of 3C products. ing cancer metastasis and vascularization. Although However, treatments for several eye diseases, such as exosomes seem to be promising biomarkers, methods vision-threatening glaucoma and age-related macular to isolate and quantify exosomes still remain contro- lesions, offer only symptomatic control with no cura- versial. Conventionally used methods include ultracen- tive options. Extracellular vesicles (EVs) are cell- trifugation (UC), polymer precipitation and derived vesicles that have been shown to play a role immunoaffinity purification (IP) using surface marker in intercellular communication, immune regulation, antibodies. In addition, obtained exosomes from cer- extracellular matrix turnover, stem cell division/differ- tain types of specimens, urine in particular, is extre- entiation, neovascularization and cellular waste mely difficult. removal. At present, ocular EV studies remain rare 312 ISEV2019 ABSTRACT BOOK mainly due to the challenges associated with accessing intercellular communication. Exosomes are 30–150 nm and processing minute ocular samples. membrane vesicles and are also shed by both normal Methods: In this work, we collected EVs from Sprague and cancer cells. Cancer cells are known as very het- Dawley rat intraocular samples after non-arteritic ante- erogeneous, so exosomes are also heterogeneous and rior ischaemic optic neuropathy (NAION) induction. have different surface expression markers. Cancer- 30 μL ocular fluid collected at day 0, 0.25, 1, 3 and 7 derived exosomes contain unique cargo determined after NAION induction was applied to each paper- by the molecular characteristics of cancer cells. based device. Long-wavelength UV light (360 nm) Therefore, it is very important to selectively separate was utilized to break the photolabile crosslinker and exosomes depending on surface expression for down- release captured EVs for subsequent analyses. stream analysis. We designed an integrated microflui- Results:RNAmoleculescontainedincapturedCD63 dic chip for selective exosome isolation. The +EVswereextracted,andthenextgeneration microfluidic chip consists of Hoof Structure (HS) for sequencing (NGS) results showed that more anti- mixing exosomes and two different sized aptamer- inflammatory M2 miRNAs were present in NAION coated particles and Multi-Orifice Flow Fractionation samples than in sham controls. In addition, we have (MOFF) for separating each particle. identified 53 miRNAs that showed more than two- Methods: Biotinylated EpCAM aptamer was immobi- fold changes in expression during the natural course lized on the surface of 7 μm streptavidin-coated poly- of recovery after NAION. These miRNAs included styrene particle and HER2 on 15 μm. The HS has the pro-inflammatory M1-related miRNAs (miR-184, circular expansion channel on the 1st layer to generate miR-3473, let-7c-5p, miR-124, miR-125a-5p, miR- expansion vortices and the two curvature channels on 210-3p) and anti-inflammatory M2-related miRNAs the 2nd layer to make chaotic advection. It makes (miR-31a-5p, miR-99a-5p, let-7i-5p, miR-204-5p, transverse flow and mixes two particles without parti- miR-16-5p). Interestingly, M1-related miRNAs exhib- cle focusing phenomenon. The 100-nm (exosome), 7- ited a biphasic expression that peaked at day 1 and μm and 15-μm fluorescence particles were used to test then elevated again at day 7, whereas M2-related mixing performance between exosomes and particles in miRNAs were upregulated at day 7 from NAION to the HS. The MOFF was designed by a series of con- achieve putative neuroprotection effects. traction/expansion microchannels for continuous size- Summary/Conclusion: We have developed an easy and based separation. Separation performance was tested by fast method capable of collecting and releasing EVs using the 7-μm and 15-μm fluorescence microparticles from low-volume samples. The quantity and quality in the MOFF. of miRNA extracted is enough for NGS analysis. Results: The mixing efficiency was the highest at the Funding: Taiwan Ministry of Science & Technology flow rate 150 μl/min. Each exosome was continuously (MOST 106–2628-E-007–010-MY3) and the Taiwan captured by aptamer-conjugated particle in the HS Ministry of Education (Higher Education Sprout channel. The capture efficiency of EpCAM positive Project: Grant No. 107Q2713E1). exosome was 96.9% and HER 2 was 68.09%. Two particles were separated in the integrated microfluidic PS04.13=OWP3.04 device at the same flow rate. 96.26% of 15 μm microparticles were positioned into the centre of the channel, and 89.48% of 7 μm microparticles were separated on An integrated microfluidic device for selective exosome isolation from human plasma both sides of the channel. Hogyeong Gwaka, Junmoo Kimb, Leila Kashefi-Kheyrabadib, Seung-Il Kimb, Summary/conclusion: Each exosome was continuously b b Kyung-A Hyun and Hyo-Il Jung captured by mixing aptamer-conjugated particle in the a School of Mechanical Engineering, Yonsei University, Seoul, Republic of HS. Exosome-conjugated microparticles were success- Korea; bYonsei University, Seoul, Republic of Korea fully separated by inertial force in MOFF. This analysis Introduction: Extracellular vesicles released by many of each exosome will shed light on diagnosis and ther- cell types circulate in blood vessel and play a key role in apy of cancers. JOURNAL OF EXTRACELLULAR VESICLES 313

PS05: EV Protein Biomarkers Chairs: Seiko Ikezu; Yusuke Yoshioka Location: Level 3, Hall A 15:00–16:00

When cancer cells were co-cultured with EVs, the PS05.03 movement of cancer cells stimulated by antibody- blocked EVs was increased. Summary/Conclusion: Our study indicated Caveolin-1 Caveolin-1 reduces in extracellular vesicles derived from lung cancer tissue and plasma and associates with cancer cell migration on EVs can inhibit cancer cell movement. Cancer cells Taixue Ana, Lei Zhengb, Han Zhangc and Yiyao Huangc promote the movement of other cells by reducing aNan Fang Hospital, Southern Medical University, Guangzhou, China Caveolin-1 on EVs. Caveolin-1 significantly decreased (People’s Republic); bClinical Laboratory Department, Nanfang Hospital, c ’ Southern Medical University, Guangzhou, China (People’s Republic); Nan in cancer tissues and patients plasma. Caveolin-1 may Fang Hospital, Southern Medical University, Guangzhou, China (People’s serve as a potential marker for the diagnosis in lung Republic) cancer diagnosis. Funding: This study was supported by grants from the Introduction: Early diagnosis is of significance mean- National Natural Science Foundation of China ing for lung cancer. Extracellular vesicles (EVs) are a (81371901). new kind of diagnostic biomarkers with great potential. However, the discovery of biomarkers based on EVs remains disturbed by EVs from cells disassociated with PS05.04 lung cancer. If biomarkers, we suggest, can be screened based on EVs from cancer tissue and validated in DNA assembly assisting magnetic fluorescence nanosensor based on plasma, discovered biomarkers may combine good spe- aggregation-induced emission probe/graphene oxide for cancerous exosome analysis cificity and practicability in clinical practice. Bo Li, Chunchen Liu, Weilun Pan and Lei Zheng Methods: Thirteen Lung cancer tissues and 71 plasma Clinical Laboratory Department, Nanfang Hospital, Southern Medical samples (47 early stage lung cancer patients, 9 University, Guangzhou, China (People’s Republic) advanced stage lung cancer patients and 15 healthy controls) were collected from Nang Fang Hospital. Introduction: Exosomes are emerging as non-invasive Our research was approved and supervised by the diagnostic biomarkers of cancer because they carry Medical Ethics Committee of Nan Fang Hospital. EVs biomolecules that include proteins and nucleic acids were purified from lung cancer tissues and paracancer- for intercellular communication. Assessing special exo- ous tissues and characterized by LC MS/MS; protein some surface proteins provides a powerful means of profiles of two groups were compared and Caveolin-1 identifying the origins of their cancerous parental cells. was picked out in differentially expressed proteins. Glypican-1 (GPC-1), an exosomal membrane protein, With high-sensitivity flow cytometry, the diagnostic was discovered to have much higher expression on the performance of Caveolin-1 was validated in 79 plasma cancerous exosomes than the noncancerous for the samples. In cell line experiments, Caveolin-1 on EVs early diagnosis of pancreatic, breast and colorectal was blocked by antibody, and the migration of EVs cancer. However, quantification of low concentrations stimulating cancer cells was evaluated by transwell. of specific exosomes present in very small volumes of Results: We determined profiles of EVs in lung cancer clinical samples remains a challenge. tissue and paracancerous tissue separately. Combined Methods: Herein, we proposed a magnetic fluorescence bioinformatics analysis and western blotting verifica- nanosensor based on GPC-1 antibody-functionalized tion, Caveolin-1 was chosen as candidate biomarker magnetic microcarriers for GPC-1(+) exosome subpo- and verified by western blotting in six plasma samples. pulation isolation and detection. And the recognition Subsequently, Caveolin-1 was evaluated in 79 plasma signal was transformed into the formation of free-state samples. Caveolin-1 was significantly decreased in lung DNA nanostructure by the trigger containing CD63 cancer patients and the area under curve of ROC aptamer. To increase the sensitivity of this method, a reached 0.958 in diagnosis of cancer patients and “Y” shape DNA self-assembly nanostructure amplifica- healthy controls. Furthermore, we observed the biolo- tion strategy was adapted to assist aggregation-induced gical function of Caveolin-1 on EVs with cell line. emission probe/graphene oxide (AIE/GO) “turn-on” 314 ISEV2019 ABSTRACT BOOK fluorescence reporting system to realize the label-free nanoparticles of known particle concentration as the and ultracentrifugation-free quantitative analysis of internal standard, particle concentration of uEVs was GPC-1(+) exosome subpopulation of breast cancer in measured via single particle enumeration. The purity of a homogeneous. uEVs in the isolates was examined by measuring the Results: We optimized the reaction conditions and particle concentration before and after Triton X-100 evaluated the detection performance of the method, treatment. Lipid-membrane was labelled with PKH26 such as specificity, sensitivity and linear range. Under and PKH67. Subpopulation of uEVs expressing specific optimal conditions, the results show that this magnetic surface proteins were analysed via immunofluorescent fluorescence nanosensor could distinguish breast can- staining. SYTO 16, a cell-permeant stain, was used to cer exosomes from five other types of cancerous exo- stain the nucleic acids of uEVs before and after DNase somes, and the linear range of detection for breast I treatment. cancer exosomes is estimated to be 7.8 ×104– Results: The concentration of uEVs was determined 3.9 ×109 exosomes/µL with a detection of limit around 10^9 particles/mL in urine, and the purity of (LOD) of 6.56 ×104 exosomes/µL. We demonstrated isolated uEVs via UC was above 90%. Comparing with the application of the magnetic fluorescence nanosen- the light scattering signal of single EVs, the lipid dye sor in quantitative detection of exosomes in plasma labelling efficiency was found to be around 90%. samples directly from breast cancer patients. Considering the purity of EVs, we conclude that almost Summary/Conclusion: This magnetic fluorescence all the uEVs can be stained by lipid dyes. We also nanosensor is expected to become a powerful tool for found that ~30% of uEVs expressing CD9, CD63, rapid and simple cancer liquid biopsy. CD81 or TSG101 on their surface. The ratio of uEVs Funding: This study was financed by grants from the lightened up by SYTO 16 decreased from 16% to 10% National Natural Science Foundation of China after DNase I treatment, which indicates that part of (81371901, 81702100) and the Science and the DNA resides on the outer membrane surface of Technology Planning Project of Guangdong Province uEVs. (2017A020215123) Summary/Conclusion: The laboratory-built nFCM is applicable to the multiparameter biochemical analysis PS05.05 of individual uEVs via protein, lipid and nucleic acid staining. We expect nFCM will facilitate more in-depth studies of uEVs and aid the development of clinical Quantitative multiparameter analysis of individual urinary diagnosis with uEVs. extracellular vesicles via a laboratory-built nano-flow cytometer Haisheng Liua, Ye Tianb, Shaobin Zhuc and Xiaomei Yana aDepartment of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China (People’s Republic); PS05.06 bDepartment of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China (People’s Republic); cNanoFCM Inc., Xiamen, China (People’s Republic) Proteomic profiling of urinary exosomes for potential predictors of albuminuria in subjects with diabetes Introduction: Urinary extracellular vesicles (uEVs) Rajni Sharma, Manju Kumari, Bhuvnesh Rai, Aradhana Mohan and Swasti have attracted much attention as a source of non-inva- Tiwari sive biomarkers. To exploit their prominent potential Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India in the diagnosis of urinary tract diseases including Introduction: Albuminuria is considered to be an urinary cancers, an in-depth study of uEVs at the important clinical hallmark for renal diseases. single-particle level is important. Employing a labora- However, it has limited ability to predict the earliest tory-built nano-flow cytometer (nFCM) that facilitates stages of diabetic nephropathy. Early biomarker poten- multiparameter analysis of single EVs as small as tial of urinary exosomes (UE) for renal disease has 40 nm, here we report quantitative measurement of been highlighted by us and others. We carried out size distribution, particle concentration, purity, lipid proteomic profiling of UE followed by a longitudinal membrane, nucleic acids and surface proteins of uEVs. follow-up study to determine potential predictors of Methods: uEVs were isolated from mid-stream urine albuminuria in subjects with type-1 diabetes (T1D). samples collected from healthy donors via differential Methods: In study-1, proteomic profiling of UE from ultracentrifugation (UC). Monodisperse silica nanopar- T1D with or without albuminuria (urine albumin to ticles were used as the size reference standards for the creatinine ratio between 30–300 mg/g, n = 3/group) size distribution measurement of uEVs via light scat- was performed using two-dimensional differential gel tering detection. By using fluorescent silica electrophoresis (2D-DIGE). Diagnostic potential of one JOURNAL OF EXTRACELLULAR VESICLES 315 of the identified UE protein to predict incidence of candidates VCAM1, IL18BP and S100A6 were selected microalbuminuria was determined. For this, 29 T1D for further validation in urine exosome samples from a subjects without albuminuria were followed for separate cohort of CaP patients and CaP cell lines ~4.5 years (study-2). Urine microalbumin, serum crea- by WB. tinine and HbA1C were analysed. Results: We successfully isolated exosomes from Results: 2D-DIGE revealed a total number of 592 dif- human urine, which were further validated by TEM, ferential protein spots between T1D subjects with or NTA, WB and FC. In total, 1330 proteins were identi- without albuminuria. The MASCOT search for 26 fied through LC-MS/MS. Among them, 596 proteins selected spots revealed 14 proteins associated with were differentially expressed between CaP and normal nephropathy, including Wilms’ tumour 1 (WT1) pro- controls. According to statistical analysis, a focus list of tein. At the end of 4.5 years of follow-up, 9 subjects 37 proteins, including 17 upregulated and 20 down- (out of total 19) progressed to albuminuria in WT1- regulated proteins was revealed as dysregulated candi- positive group (presence of WT-1 in UE at the time of dates in urinary exosomes for CaP. The validation of recruitment) as opposed to 1 in WT1 negative group. potential biomarkers including VCAM1, IL18BP and Both groups had statistically similar diabetes duration, S100A6 showed that the levels of these proteins were age, % HbA1c and estimated GFR at the baseline. higher in CaP cell lines including PC-3, PC-3M, Summary/Conclusion: Urinary exosomal protein DU145 and LNCaP compared to the normal prostate could help in categorizing diabetic subjects that may cell line RWPE-1. In addition, the expression level of go on to develop nephropathy. IL18BP was higher in urinary exosomes from CaP Funding: Funded by intramural, DST, Govt. of India. patients compared to healthy controls. Summary/Conclusion: Urinary exosomes harbour PS05.07 informative proteins that might be used for the early detection of CaP or monitoring its progression through a non-invasive way. Identification of exosomal biomarkers in urine for human prostate Funding: ARC-Linkage Grant cancer Duojia Wu, Ying Zhu, Jie Ni, Julia Beretov, Paul Cozzi, Mark Willcox, Valerie Wasinger, Bairen Pang, Xupeng Bai, Peter Graham and Yong Li UNSW (The University of New South Wales), Sydney, Australia PS05.09

Introduction: Prostate cancer (CaP) is the second lead- Optimization of exosome isolation and ELISA method for ing cause of cancer-related death in males. identification of novel cancer biomarkers Identification of novel biomarkers is important for Ju-Hyun Baea, Hee-Kyung Jangb, Eun-Ju Imc, Chan-Hyeong Leec and Moon- c the early detection of CaP. Exosomes are small mem- Chang Baek brane-bound vesicles released from most cell types aSchool of Medicine, Kyungpook National University, Daegu, Republic of Korea; bSchool of medicine, KyungPook National University, Daegu, including cancer cells. Exosomes play a key role in Republic of Korea; cSchool of medicine, Kyungpook National University, intercellular signalling and potentially play a role in Daegu, Republic of Korea tumorigenesis and cancer progression. In this study, Introduction: Exosomes are a type of extracellular we investigated differential urinary exosomal proteins vesicles with diameter of 30–150 nm secreted by cell in CaP patients compared to healthy controls by mass and circulate in blood abundantly. Especially, cancer- spectrometry. cell-derived exosomes contain oncogenic molecules Methods: Midstream spot urine samples from CaP that can be novel biomarker for cancer diagnosis. (n = 20) and benign prostatic hyperplasia (BPH; Recent compelling issue of cancer patients is the n = 10) patients were obtained, as well as urine from immune system that is negatively regulated by cancer- age- and sex-matched controls (n = 10). Urinary exo- cell-derived exosomes. Therefore, first we have to opti- somes were isolated using the Total Exosome Isolation mize exosome isolation methods and ELISA methods Reagent (invitrogen). The presence of exosomes was to analyse exosome’s constituents precisely. Through evaluated by transmission electron microscopy (TEM) this method, we can screen several candidates which and nanoparticle tracking analysis (NTA). Exosomal contain in cancer-cell-derived exosomes to identify markers including TSG101, CD9, CD63 and CD81 novel biomarkers for cancer prediction. were validated by western blotting (WB) and flow Methods: Exosomes were isolated from cancer patients’ cytometry (FC). High-throughput LC-MS/MS-based plasma using serial centrifugation method. For western label-free quantification was performed on Q Exactive blot analysis, we loaded exosomes to observe existence to identify proteins in the exosomes. Three biomarker and difference in the expression of protein between 316 ISEV2019 ABSTRACT BOOK cancer patients’ and healthy controls’. And using exo- analysis was performed to detect TSHR in cell lysates somes each well in 96-well plate, sandwich ELISA was and exosomes. Human embryonic kidney HEK293 performed to measure protein level of exosomes from cells (HEK) overexpressing TSHR (HEK/TSHR) were cancer patients’ and healthy controls’. We also made established for the functional analysis of TSHR exo- mouse xenograft models to find the correlation between somes. Using exosomes isolated from HEK and HEK/ exosomal protein level and tumour burden. TSHR cells, in vitro binding capacity of a human Results: We optimized isolation method to purify exo- monoclonal autoantibody (M22) to TSHR exosomes somes and to minimize sample variation, and we opti- and their effect on M22-mediated stimulation of intra- mized ELISA method using well-known exosomal cellular cAMP production in HEK/TSHR cells were surface biomarkers and confirmed assay stability. By studied. Human recombinant TSHR chimera capable optimization of exosome isolation and ELISA method, of binding to M22 was used as a positive control. we built finding system for novel cancer biomarker Results: TSHR was detected in exosomes from cancer which is expected significantly overexpressed in exo- cells as well as normal epithelial cells. The binding somes from cancer patients‘ plasma compared to assay demonstrated that M22 dose-dependently healthy controls’. In addition, we checked the level of bound to TSHR exosomes. M22 stimulated intracellu- exosomal surface protein’s correlation with tumour lar cAMP production in HEK/TSHR cells in a dose- burden, therefore prove possibility as novel cancer dependent manner. Exosomes from HEK/TSHR cells biomarkers. but not those from HEK cells significantly reduced Summary/Conclusion: Based on our results, we opti- cAMP production activated by M22 in HEK/TSHR mized our own finding system and identified novel cells. A similar inhibitory effect was observed for cancer biomarkers. human recombinant TSHR chimera. Funding:ThisresearchwassupportedbytheBio& Summary/Conclusion: Our results suggest that TSHR Medical Technology Development Program of the exosomes may be secreted from normal and cancerous National Research Foundation (NRF) funded by the thyroid epithelial cells. In the thyroid gland of patients Ministry of Science & ICT (2017M3A9G8083382) with GD, TSHR exosomes may exert a decoy effect by and by the National Research Foundation of Korea sequestering M22, alleviating autoantibody-stimulated (NRF) grant funded by the Korea government cAMP production. (2014R1A5A2009242). Funding: There is nothing to disclose.

PS05.10 PS05.11=OWP3.06

Thyrotropin receptor-positive exosomes alleviate autoantibody- mediated stimulation of cAMP production Naoki Edoa, Kyojiro Kawakamib, Yasunori Fujitab, Koji Moritaa, Kenji Unoa, In vitro and in vivo investigation of extracellular vesicles (EVs) as Kazuhisa Tsukamotoa, Hiroyuki Onosec, Toshio Ishikawaa, Masafumi Itob biomarker carriers in the diagnosis of early Alzheimer’s disease a b b b a Soraya Moradi-Bachiller , Miriam Ciani , Roberta Zanardini , Luisa Benussi , Department of Internal Medicine, Teikyo University School of Medicine, Roberta Ghidonib, J. Mark Cooperc, Gianluigi Forlonia and Diego Albania Tokyo, Japan; bResearch Team for Mechanism of Aging, Tokyo Metropolitan Institute of Gerontology, Itabashi-ku, Japan; cDepartment of Internal aDepartment of Neurosciences, Mario Negri Institute for Pharmacological Medicine, Kanaji Hospital, Tokyo, Japan Research IRCCS, Milan, Italy; bMolecular Markers Laboratory, IRCCS Istituto Centro San Giovanni di Dio Fatebenefratelli, Brescia, Italy; cDepartment of Clinical Neurosciences, Faculty of Brain Sciences, Introduction: Exosomes or extracellular vesicles University College London – Institute of Neurology, London, United secreted from cells play a variety of roles in both Kingdom physiological and pathological processes. In Graves’ disease (GD), autoantibodies bind to thyrotropin Introduction: Extracellular vesicles (EVs) represent an receptor (TSHR) on thyroid follicular epithelial cells, ideal source of biomarkers due to their role in cellular stimulating thyroid growth and thyroid hormone communication and their ability to carry protein aggre- synthesis and secretion through cAMP production. In gates. The most investigated EVs are exosomes, active this study, we examined if exosomes expressing TSHR entities secreted from cells and able to cross the blood– are secreted from thyroid cells and defined their roles brain barrier. Several neurodegeneration-involved in GD. molecules may undergo intercellular spreading through Methods: Exosomes by differential centrifugation from exosome release. In Alzheimer’s disease (AD), before the culture medium of NTHY-ori 3-1 human thyroid clinical signs appear, several proteins implicated in follicular epithelial cell line and 8305C, 8505C and exo- and endocytic pathways are altered. In this sce- FTC133 thyroid carcinoma cell lines. Western blot nario, the identification of a correlation between JOURNAL OF EXTRACELLULAR VESICLES 317 variations in proteins carried by EVs and the progres- All the samples were collected after ethical committee sion of AD is the main aim of our project. approval respecting Helsinki’s declaration. Informed Methods: We performed exosome isolation and char- consents were provided by all the subjects. acterization from H4-SW glioma cells (a cell model Results: Our preliminary results show that APP, featuring mutated β-amyloid overexpression), as well PGRN, sTREM2 are carried by H4- and human as in mouse- (triple-transgenic mouse model for famil- plasma-derived EVs. H4-SW cell-culture medium and ial AD) and human-plasma samples (Mild Cognitive 3Tg mouse plasma had a decrease in the EVs number Impairment (MCI) and AD subjects). In every case a release (≈1*10e8 EVs/ml) in comparison to control differential centrifugation protocol was applied and (≈7*10e8 EVs/ml). This decrease was not found in exosomes were then characterized using Nanoparticle human plasma samples. Tracking Analysis with the NanoSight. We then Summary/conclusion: EVs purified from H4-glioma explored exosome content, specifically Amyloid cellular AD model, 3xTg mouse-, MCI- and AD- Precursor Protein (APP) and its proteolytic fragments, plasma samples carry proteins relevant for neurodegen- Microtubule Associated Protein Tau (tau), Progranulin erative diseases (NDs). EVs release is reduced in cellu- (PGRN protein), Soluble Triggering Receptor lar and animal AD-models. Expressed on Myeloid Cells 2 (sTREM2) and α-synu- Funding: Horizon 2020 Marie Sklodowska-Curie clein (α-syn), using Western blot and ELISA. L1CAM Innovative Training Networks – Blood Biomarker- and CD63 were evaluated to define the neural-derived based Diagnostic Tools for Early Stage Alzheimer’s exosomes amount in human samples. Disease. 318 ISEV2019 ABSTRACT BOOK

PS06: Advancing EV Studies in Biological Samples Chairs: Peter Kurre; J. Bryan Byrd Location: Level 3, Hall A 15:00–16:00

PS06.01 9053.6 copies/mL, respectively). Also, we found that AR-V7 transcript levels and the AR-V7/AR-FL ratio in urinary EVs were higher in patients with advanced AR-V7 in urinary EVs of patients with prostate cancer prostate cancer. Hyun-Kyung Wooa, Juhee Parkb, Ja Yoon Kuc, Chan Ho Leed, Vijaya Sunkaraa, Hong Koo Hac and Yoon-Kyoung Choa Summary/Conclusion: This study demonstrates that aUlsan national institute of science and technology (UNIST), South Korea, mRNA of urine-derived EVs is a reliable source for Ulsan, Republic of Korea; bCenter for soft and living matter, institute for AR-V7 expression analysis, suggesting a simple and c basic science (IBS), South Korea, Ulsan, Republic of Korea; Pusan National promising approach to liquid biopsy with a great University Hospital (PNUH), South Korea, Busan, Republic of Korea; dDepartment of Urology, Inje University Busan Paik Hospital, South Korea, potential for therapeutic impact on prostate cancer. Busan, Republic of Korea Funding: This study is funded by Ministry of Health and Welfare (HI12C1845) and Institute for Basic Introduction: Prostate cancer is the most common Science (IBS-R020-D1), Republic of Korea. cancer affecting men and a leading cause of cancer deaths. Almost all patients initially respond to androgen deprivation therapy but inevitably progress to a PS06.02 lethal stage of disease, termed castration-resistant prostate cancer (CRPC). Androgen-receptor splice variant (AR-V7) is associated with CRPC and resistance to Microvesicles are absorbed on the surface of extracorporeal membrane oxygenation circuit tubing anti-androgen therapy. Despite its clinical importance, Yoichi Ikia, Shotaro Matsumotob, Michiko Abea, Kieran O’Deac, Hidenobu the lack of efficient methods for AR-V7 analysis Shigemitsub, Masao Takatac and Kenji Wakabayashia remains a challenge for broader use of this marker in aDepartment of Intensive Care Medicine, Tokyo Medical and Dental routine clinical practice. Here we suggest a practical University, Tokyo, Japan; bDepartment of Intensive Care Medicine, Tokyo Medical and Dental University, Tokyo, Japan; cSection of Anaesthetics, Pain and non-invasive liquid biopsy method for analysis of Medicine, and Intensive Care, Imperial College London, London, UK AR-V7 in the RNA of urine-derived extracellular vesicles (EVs) without the need for blood withdrawal. Introduction: There is an increasing interest in poten- Methods: Urine samples were collected from patients tial role of microvesicles (MVs) as non-invasive bio- at Pusan National University Hospital (PNUH). The markers for acute critical care diseases. Clinically, study protocol was reviewed and approved by the extracorporeal membrane oxygenation (ECMO) draws Institutional Review Board of PNUH and UNIST, and attention in critical care, and MVs are considered to be written informed consent was obtained from all sub- involved in the development of adverse events in criti- jects. All patients that progressed to CRPC underwent cally ill patients. However, the dynamics of MVs within docetaxel-based chemotherapy. Using a newly ECMO circuit, in particular MV absorption on the upgraded centrifugal microfluidic device for size- biomaterial, has not been systematically studied. The based EV isolation, rapid enrichment of EVs (< purpose of this study was to explore whether MVs are 30 min) from each 4 mL of urine was accomplished. absorbed by ECMO circuit tubing. Followed by mRNA extraction, and AR-V7 and andro- Methods: Granulocytes were isolated from healthy gen-receptor full-length (AR-FL) mRNA levels were volunteer blood using density-gradient centrifugation. quantified by droplet digital polymerase chain reaction MV production was induced by treatment with cal- (ddPCR). Furthermore, protein and mRNA expression cium ionophore for 20 min, and MVs were quantified of EVs isolated from blood plasma are compared on flow cytometry (FACS) based on their size and together. surface expression of CD11b and CD66b. MVs were Results: Higher AR-V7 and lower AR-FL expression then purified by differential centrifugation and dis- were detected in urine-derived EVs from 14 patients solved with DMEM. Thereafter, 0.5 to 1.0 ×107 MVs with CRPC (0–217.0 and 12.9–562.5 copies/mL, were administered to either ECMO circuit tubing respectively) than in those from 22 patients with hor- (material group) or Eppendorf tubes (control group) mone-sensitive prostate cancer (HSPC, 0–24.1 and 6.2– with their lid removed and re-sealed with paraffin film. JOURNAL OF EXTRACELLULAR VESICLES 319

MV solution was rotated for 6 h, and small volume of Results: Physical properties of exosome were measured sample was regularly taken in 1–2 h interval for MV using AFM observation and force curve measurement. quantification by FACS. Exosomes derived from low metastatic strains derived Results: The number of MVs in the material group was from human breast cancer cell lines showed a relatively significantly decreased over time, compared to the con- smooth surface. Exosomes derived from highly meta- trol group (64% reduction vs. 22% reduction at 6 h. static strains have layers or chain-like substances hav- Significant interaction (n =3,p < 0.05) between time ing a thickness of 10–20 nm. In addition, its physical and treatment by 2-way repeated measures ANOVA). property was soft. Furthermore, it was found that exo- Summary/Conclusion: ECMO circuit is known to somes derived from nSMase2 knock-down 4T1 cells absorb bioactive drugs and mediators, and our results are harder than exosome from wild type or nSMase2 showed that ECMO circuit also impacts on the over-expressing 4T1 cells. dynamics of circulating MVs. Further investigation Summary/Conclusion: Now, we are investigating how should explore the effects and mechanisms of MV these properties are related to exosome characteristics absorption/production induced by the biomaterial-cell in cancer malignancy. interaction. PS06.04 PS06.03 Stability of human salivary extracellular vesicles under gastrointestinal tract conditions Yuko Ogawaa, Mamoru Ikemotoa, Yoshihiro Akimotob, Hayato Kawakamic Measurement of physical properties of exosome by atomic force and Ryohei Yanoshitaa microscope a b Akinori Kogurea, Noriyuki Motohashib, Satoshi Otsukab, Yurika Sawac, Teikyo Heisei University, Tokyo, Japan; Kyorin University, Tokyo, Japan; Nobuyoshi Kosakad and Takahiro Ochiyad cKyorin University, Tokyo, Japan aShimadzu Techno-Research, INC, Hadano, Japan; bShimadzu Corporation, Chiyodaku, Japan; cDepartment of Molecular and Cellular Medicine, Institute Introduction: Human saliva plays an important role as of Medical Science, Tokyo Medical University, Tokyo, Japan; dDepartment of front-line of body defence. Extracellular vesicles (EVs) Molecular and Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Shinjyuku-ku, Japan are secreted from various types of cells and it is recognized that they are involved in intercellular commu- Introduction: It has been shown that exoxome can be nication via delivering their contents. We have isolated used as a drug delivery system (DDS), and exosome in EVs with dipeptidyl peptidase IV (DPP IV) from blood / body fluid has been studied as a new diagnosis human whole saliva, whereas little is currently known method for various diseases. The existence of various about the fate of secreted salivary EVs. In the present proteins and sugar chains such as CD antigen, integrin study, we investigated morphological stability of sali- and ligand has been confirmed in the membrane of vary EVs and chemical stability of proteins associated exosome. However, physical properties of exosome and with the EVs in simulated gastrointestinal (GI) fluids. their contribution for exosome-mediated phenomenon Methods: Human whole saliva was collected from have not been clarified yet. There are many reports healthy volunteers. Salivary EVs were separated by showing the morphology of exosomes by electron size-exclusion chromatography. For simulated gastric microscopy; however, it is necessary for chemical fixa- fluids or intestinal fluids treatment, indicated concen- tion of sample and vacuum environment. Therefore, tration of pepsin (pH3.0) or pancreatin (pH7.4) was the form of exosome by electron microscopic observa- added to EV samples, respectively. For bile acid treat- tion and the form of exosome in liquid may be differ- ment, sodium cholate (pH7.4) was added. After the ent. The AFM is an equipment that can observe incubation, the treated samples were then subjected to exosome in liquid (physiological saline, culture liquid). SDS-PAGE, western blot analysis, DPP IV activity In recent years, biomolecular property measurement by measurement, dynamic light scattering study or obser- AFM has been increasing (Kogure et al., BPS, 2018). vation by a transmission electron microscope. Methods: We used SPM-9700HT (Shimadzu Results: Salivary EVs were morphologically stable Corporation) for AFM measurement. Measurement under simulated gastric fluids with pepsin and simu- mode is dynamic mode and force curve mapping mea- lated intestinal environment using pancreatin. While surement. Exosomes were isolated from human breast some proteins associated with surface of the EVs, cancer cell line (MDA-MB-231) and mouse breast can- such as mucin 5B and CD9, were digested with these cer cell line (4T1) (Kosaka et al., JBC, 2013) by treatments, inside components such as Alix and ultracentrifugation. TSG101 were resistant. Even though DPP IV is 320 ISEV2019 ABSTRACT BOOK oriented outside, it was not digested and retained its Results: We found that most of the extracellular small enzymatic activity. Thus, membrane integrity was ncRNAs in serum consisted of miRNA, isomiRs and intact and internal components were retained in diges- tRFs. Especially, most of ncRNAs in EVs were tRFs. tive enzymes. Morphological changes and solubiliza- Several isomiRs and tRFs were expressed specifically in tion of proteins in the EVs scarcely occurred after serum from cancer patients. Some of them were also treatment with physiological concentration of sodium observed in EVs from cultured cancer cell lines. EV- cholate. Membrane integrity was destroyed with free ncRNAs were decreased, and ncRNAs with EVs increasing concentration of sodium cholate. However, were increased in blood during long 4°C storage after components of the vesicles were not completely solu- blood sampling. bilized at higher concentration of sodium cholate. Summary/Conclusion: The expression profile of the Summary/Conclusion: These results suggest that sali- extracellular small ncRNAs is changed during storage vary EVs are stable and functional in GI tract. This at 4°C after blood sampling. It may affect the accuracy study would help to elucidate their potential pathophy- of extracellular small non-coding RNA biomarkers. siological roles in GI tract. Funding: This research is partially supported by the Funding: This work was supported by Japan Society “Development program of microRNA measurement for the Promotion of Science (JSPS) KAKENHI Grant technology foundation in body fluid” from Japan Number 16K08348. Agency for Medical Research and development, AMED.

PS06.05 PS06.06

The factor affecting to the accuracy of extracellular small non-coding RNA biomarkers Generation of reference material for flow cytometric detection of Yukie Nishiyama, Yumiko Koi, Genki Nishimura, Eri Kojima, Morihito extracellular vesicles Okada and Hidetoshi Tahara Anna Nowocin

Hiroshima University, Hiroshima, Japan NIBSC, London, UK

Introduction: Extracellular small non-coding RNAs Introduction: Extracellular vesicle (EV)-related tech- (ncRNAs), such as microRNAs (miRNAs), isoforms nologies have been developing rapidly over the past of microRNAs (isomiRs), tRNA-derived fragments few years and substantial growth is expected for the (tRFs) and others, are known as regulator of gene market as they get integrated into the fields of liquid expression for cell metabolism. They are released into biopsy, precision and regenerative medicine. NIBSC as body fluid from various cells with extracellular vesicles a designated WHO standardization laboratory is (EVs) including exosomes. In recent studies, some actively developing methods that in the future may extracellular miRNAs and tRFs in blood were reported allow the production of diagnostic and therapeutic as novel biomarkers for diseases. In this study, we EV reference material for clinical and pre-clinical use. investigated the factor affecting to the accuracy of As flow cytometry enables characterization of EV extracellular small ncRNA biomarkers such as populations down to single-event level, it has been miRNA and tRFs for next generation sequencing adapted as a meaningful tool in characterizing EV (NGS)-based detection. isolates. High-throughput and multiparameter analysis Methods: Blood was collected from the patients who of EV are crucial to further advance the ability to provided written informed consent to participate in the characterize these particles. study (approved by IRB of Hiroshima University). Methods: EVs from plasma samples were isolated Serum were isolated and stored at –80°C. EVs in the using several methods and their morphology and mole- cell culture supernatant were collected after culture in cular content was assessed. The effects of freeze-drying DMEM with FBS followed by one-day additional cul- were investigated to explore a possibility of long-term ture without FBS. Total small RNAs were purified by storage of EV-reference material that has been labelled using miRNeasy Mini Kit (Qiagen). EVs, including in that way for flow cytometric analysis. exosomes, were isolated by using Total Exosome Results: The populations of submicron EVs could be Isolation Kit (Thermo Fisher Scientific). NGS was per- detected using commercially available flow cytometers formed by using Ion S5 (Thermo Fisher Scientific). We only when fluorescence and not light scatter triggered analysed the sequence data of small ncRNAs (15-55 nt) detection was used. The labelling with 5-(and-6)-car- with software, CLC Genomics and JMP. boxyfluorescein diacetate succinimidyl ester followed JOURNAL OF EXTRACELLULAR VESICLES 321 by removal of unbound dye was efficient enough to platelet-associated proteins were specifically detected robustly label single EVs without generating label-asso- in serum-derived EVs. ciated artefacts. Freeze-drying process had some effects Summary/Conclusion: We found that serum has the on morphology but not molecular content of EV larger number of EVs than plasma, despite of the same preperations. volume of blood. The existence of the platelet-specific Summary/Conclusion: Efficient labelling and preser- proteins detected in serum-derived EVs implies that vation of pure populations of EVs present a viable serum may be contaminated with platelet-derived option for the development of a stable monodispersed nanoparticles, which are reported to be produced dur- reference material that can be used as positive control ing coagulation. or calibrant of flow cytometers used for analysing submicron populations. PS06.08 PS06.07 Evaluation of stability maintenance of extracellular vesicles upon storage temperature and period Comparison of serum and plasma as a source of blood extracellular Eun Kyoung Shina, Jae Min Chab, Mi Jeong Oha, Eun Hee Kima and Oh vesicles reveals possible contamination of serum with platelet- Young Bangc derived particles produced during coagulation a b Xiaoman Zhanga, Toshihide Takeuchib and Yoshitaka Nagaib Samsung medical center, Seoul, Republic of Korea; Department of Mechatronics, College of Engineering, Incheon National University, aDepartment of Neurotherapeutics, Osaka University Rraduate School of Incheon, Republic of Korea; cSamsung medical center, Seoul, Republic of Medicine, Osaka, Japan; bOsaka University, Suita, Japan Korea

Introduction: Extracellular vesicles (EVs), including Introduction: Extracellular vesicles (EVs) secreted exosomes and microvesicles, are released from cells to from stem cells are bilipid-layered and nano-sized, extracellular environment, and can be found in several retaining medicinal potency equivalent to that of stem biological fluids, such as blood, cerebrospinal fluid and cells. As much interest in clinical use of therapeutic urine. Among them, blood-derived EVs are expected to EVs has been increasingly gained in the fields, how- offer a more efficient and faster diagnostic method for ever, few studies have been conducted regarding opti- neurologic diseases than conventional diagnosis. mal storage and shipping conditions for EVs, which are Although serum and plasma are utilized as a source critical to commercialize EVs as a medicinal product. of blood EVs, it still remains unknown whether there In this study, we tested the maintenance efficiency of are differences in EVs derived from serum and plasma. EVs in terms of physical stability and proteomic/geno- In this study, we performed a series of experiments to mic contents of EVs in the following storage condi- see the differences between serum and plasma EVs. tions: (1) 4°C during the 28-day of short-term period Methods: Whole blood was obtained from 9-week-old and (2) −80°C during the one-year of long-term period. mice. Serum was collected from the supernatant of the Methods: Comprehensively characterized stem cell- clotted blood. Plasma was collected from the blood derived EVs were stored at 4°C for 28 days and −80° treated with EDTA. EVs were isolated from serum C for one year. During given periods, preserved density and plasma using ultracentrifugation method. The and differing sizes of EVs were evaluated by nanopar- morphology of EVs was analysed by electron micro- ticle tracking analysis (NTA) along with quantitative scopy, and the particle numbers and the diameter were measurement of variations in total protein and RNA measured by nanoparticle tracking analysis (NTA). The concentrations. protein contents of EVs were analysed by LC-MS/MS Results: At the 4°C storage condition, concentration and western blotting. and size of EVs were relatively unvarying for 28 days. Results: NTA measurement revealed that the particle In terms of total protein and RNA concentrations, numbers in the EV fraction isolated from serum are about 3–40% of decreasing rates were shown during ~2-fold larger than those derived from plasma the first week of period, but rest of the amounts were (p < 0.01, Student’s t-test), while the particle diameter stably preserved until day 28. At −80°C, EV concentra- showed no difference between serum and plasma EVs. tion decreased about 10% from the initial level during LC-MS/MS analysis of EVs identified total 520 pro- the first two weeks, but rest of the amounts were stably teins, of which 317 proteins were detected in both preserved for one year. Size of EVs was not changing serum- and plasma-derived EVs, while 189 proteins during the long-term period. In terms of total protein and 14 proteins were detected only in serum- and and RNA concentrations, about 50% of decreasing plasma-derived EVs, respectively. Interestingly, rates were shown during the first two weeks, but rest 322 ISEV2019 ABSTRACT BOOK of the amounts were stably preserved during the one methods to deplete culture media of EVs are lacking as year of period. they do not guarantee an RNA-free preparation. Summary/Conclusion: While a variety of studies are Methods: In this study we have addressed the RNA actively ongoing to reach effective cell-free treatments contamination issue of EVs in FBS, ultracentrifugation using EVs, the information of EV storage provided by EV-depleted FBS, commercially available EV-depleted our study would support safe and reliable use of EVs in FBS, and in our recently developed filtration-based EV- clinic depleted FBS. Commercially available serum-free, Funding: This study was supported by a grant from the xeno-free defined media were also screened for RNA Korean Healthcare Technology R&D Project, Ministry contamination. of Health & Welfare (HI17C1256) and Basic Science Results: Our small non-coding (nc) RNA sequencing Research Program, the Ministry of Science, ICT and data emphasized that all EV-depleted media contained Future Planning (2018M3A9H1023675). RNA contaminants. Additionally, defined media contained miRNAs and other small RNAs, albeit at a much lower level than in serum preparations. Out of the PS06.09 different FBS preparations studied, our ultrafiltration EV-depleted FBS performed the best in depleting miRNAs. Certain miRNAs, such as miR-122 and Questioning the purity of the media – extracellular small non-coding RNA contaminants in foetal bovine serum and serum-free media miR-203a, proved difficult to remove and were present Bettina I. Mannerströma, Riku Paananenb, Ahmed Abu-Shahbac, Riitta in all media. As compared to miRNAs, other small a a Seppänen-Kaijansinkko and Sippy Kaur RNAs (snRNA, Y RNA, snoRNA and piRNA) were a Department of Oral and Maxillofacial Diseases, University of Helsinki and difficult to eliminate from the media. Helsinki University Hospital, Helsinki, Finland; bHelsinki Eye Lab, Ophthalmology, University of Helsinki and Helsinki University Hospital, Summary/Conclusion:Ourstudyshowedthateven Helsinki, Finland; cDepartment of Oral and Maxillofacial Diseases, defined media contained trace amounts of small ncRNA. University of Helsinki and Helsinki University Hospital, Helsinki, Finland Therefore, in order to screen for baseline RNA contamina- Introduction: Extracellular vesicles (EVs) behave as tion in culturing media, RNA sequencing data should be paracrine effectors as they are released from cells to carefully controlled by adding a media sample as a control. deliver signals to other cells. They control a diverse This should be a mandatory step before performing cell range of biological processes by transferring proteins, culture experiments in order to eliminate the confounding lipids and nucleic acids between cells and are secreted effects of media. by a wide spectrum of cell types and are found in Funding:ThisresearchwassupportedbyUniversityof various biological fluids. In the research field of EV Helsinki project funding, Helsinki University Hospital research, the use of EV-depleted foetal bovine serum State funding for university-level health research, the (FBS) for in vitro studies is crucial to eliminate the Finnish Dental Society Apollonia, Business Finland grant. confounding effects of media-derived EVs. The current JOURNAL OF EXTRACELLULAR VESICLES 323

PS07: Cellular Uptake of EVs and Membrane Function Chairs: Quan Lu; Nobuyoshi Kosaka Location: Level 3, Hall A 15:00–16:00

PS07.01 vesicles with a fluorescent dye allows for quantification of GFP signal decay specifically from those compartments. A tunable system to visualize retrofusion, a major pathway for Results: Using this chemically tuneable system, we exosome uptake Priscillia C. Perrina, Lennert Janssena, Daphne van Elslandb and Jacques found that knocking out the lysosomal integral mem- Neefjesc brane protein Limp2 partially hampers retrofusion, aLeiden University Medical Center, Leiden, Netherlands; bLeiden University suggesting that Limp2 may be a major player in this Medical Center, Leiden, Netherlands; cLeiden University Medical Center, process. Leiden, Netherlands Summary/Conclusion: We further aim to identify Introduction: Exosomes constitute a crucial mode of other proteins implicated in retrofusion in order to intercellular communication, as they can travel through propose a suitable mechanistic model. extracellular space to transfer various cellular components from one cell to another. While we understand, to some extent, how exosomal contents can affect reci- PS07.02 pient cells, the molecular mechanisms governing exosome uptake are yet to be unravelled. Upon encounter with a target cell, exosomes may be internalized and Uptake of EVs derived from cervical cancer patients with transported to late multivesicular compartments. To precancerous induces HeLa cell proliferation Piyatida Molikaa and Raphatphorn Navakanitworakulb avoid imminent degradation in lysosomes, exosomes aFaculty of Medicine. Department of Biomedical Science. Prince of Songkhla must escape the endocytic pathway and fuse back to University, Maung, Thailand; bFaculty of Medicine. Department of the limiting membrane of multivesicular bodies (MVB) Biomedical Science. Prince of Songkhla University, Hat Yai, Thailand through a process referred to as “back-fusion” or “retrofusion”. Within MVBs, retrofusion of intraluminal Introduction: Precancerous lesion is defined as early vesicles (ILV) can notably allow recycling of membrane biological effects of cells which occur prior to invasive proteins and also lead to cytoplasmic release of endo- carcinomas. The lesion is not cancerous and exhibits cytosed viruses. As retrofusion is poorly understood, variations at the cellular and molecular levels in the deciphering its workings would help unfold a major pathway leading to cancer. Current evidence indicates pathway for exosome uptake. that extracellular vesicles (EVs) can release from most Methods: To enable exploration of this process and of the cell types and affect adjacent or distant cells by ultimately reveal the molecules responsible, we created circulating in all bodily fluids. an inducible system allowing quantification of retro- Methods: We collected serum of healthy persons and fusion in real time. CD63, a tetraspanin protein loca- cervical cancer patients with precancerous lesions, lized on both the limiting (LM) and intraluminal stage I, stage II and stage III and then counted con- membranes (ILM) of late endosomes, was fused to centration and size distribution of the EVs using nano- GFP and stably expressed in MelJuso cells, along with particle tracking analysis (NTA). Differential two inactive fragments of the tobacco etch virus (TEV) ultracentrifugation incorporated with size exclusion protease. Upon addition of “dimerizer” to the cells, the chromatography was used to isolate and purify EVs TEV protease regains activity and cleaves the GFP off from pooled serum of each sample groups. Moreover, of CD63 exposed on the cytosolic side of the LM. A isolated EVs were investigated their characteristic nuclear localization signal then directs this newly lib- based on morphology using transmission electron erated GFP to the nucleus. When retrofusion occurs, microscope (TEM) and the expression of CD63, intraluminal GFP-CD63 repopulates the LM from ILV CD81, CD9, and Alix protein markers using western stores and becomes accessible for TEV protease clea- blot analysis. Live cell imaging machine was used to vage, resulting in the increase of nuclear GFP fluores- monitor uptake of EVs derived from pooled serum of cence over time. Concomitant labelling of acidic healthy persons or precancerous lesion on HeLa cells. 324 ISEV2019 ABSTRACT BOOK

Results: NTA shows that the concentration of EVs is of intracellular AA concentrations were reflected in increased in patients with precancerous lesion and exosomes. stage I, and declined in the later stages. We also Summary/Conclusion: We developed the optimized found that EVs isolated from serum of healthy and pre-analytical method for AA quantification in exo- precancerous group are capable of uptake into the somes. This method would be applicable to metabolo- cells within 4 h. Nonetheless, only EVs isolated from mics approaches to identify disease biomarkers or precancerous can stimulate HeLa cell proliferation surrogate biomarkers for the metabolic status of cells compared to those isolated from healthy and no EVs of origin. treatment group. Summary/Conclusion: This induction would associate with the biomolecules inside of EVs. Our further study PS07.04 is addressing to find out both proteins and regulatory molecules which contribute to cancer progression. Funding: This work was financially supported by Metabolome analysis of pancreatic cancer-derived extracellular vesicles Faculty of Medicine, Prince of Songkhla University Ryosuke Hayasaka, Akiyoshi Hirayama, Sho Tabata, Tomoyoshi Soga and and TRF research grant for new scholar. Masaru Tomita Keio university, Tsuruoka, Japan

PS07.03 Introduction: Extracellular vesicles (EVs) are facilitators of cell-to-cell communication. Cancer-derived EVs contribute to cancer progressions such as distant metastasis,

Optimized protocol for the quantification of amino acid angiogenesis and immunosuppression. EVs contain concentrations in exosomes functional cellular components including DNA, Hidehiro Nakamura, Satoko Ueno and Asami Hagiwara mRNA, microRNA and protein. However, metabolome Ajinomoto Co., Inc., Kawasaki-shi, Japan profiling in cancer-derived EVs remains largely unexplored. The purpose of this study is to explain compre- Introduction: Exosomes contain parent cell-derived hensive metabolite profiling of pancreatic cancer- molecules including nucleic acids and metabolites, derived EVs. As a model for studying cancer metabo- which are useful as potential biomarkers serving as lism, we evaluate the difference between metabolomic surrogates of their cells of origin. Accurate quantifica- profiles in EVs obtained from cancer cells cultured in tion of these molecules in exosomes requires to mini- normoxic or hypoxic conditions. mize the carryover contamination of residual condition Methods: Pancreatic cancer cell line Panc-1 was culti- medium (CM) or biological fluids, as they also contain vated under normoxic (20% O2) and hypoxic (1% O2) these molecules in high amount. Here, we developed a conditions. Cells were sampled using methanol, and method for accurate quantification of amino acids EVs were isolated from conditioned medium using (AAs) in exosomes by optimizing pre-analytical sample ultracentrifugation. The amount of EVs was deter- preparation and applying highly sensitive analytical mined by nanoparticle tracking analysis, and the pro- system. The method enabled us to evaluate the AA tein level of the CD9 exosomal marker was measured profiles of exosomes in comparison with those of CM using enzyme-linked immunosorbent assay (ELISA). and cell extracts or biological fluids. Metabolomic analysis was performed by using capillary Methods: Exosomes were isolated from CM of human ion chromatography-mass spectrometry and liquid pancreatic cancer cell line, PANC-1, or rat serum by chromatography-mass spectrometry. combination of ultrafiltration and ultracentrifugation. Results: We identified more than 180 kinds of meta- AAs were extracted by methanol and analysed by LC- bolites in pancreatic cancer-derived EVs. Principal MSMS after pre-column derivatization. AAs concentra- component analysis (PCA) of metabolites in EVs tion and profile were compared among exosomes, CM showed somewhat differentiated results between nor- and parental cells or serum. moxia and hypoxia. Further, the metabolite profiles Results: Ultrafiltration was introduced to minimize the contained in the cells and EVs may be different. effect of carryover contamination of residual AAs from Summary/Conclusion: In conclusion, we optimized CM or serum. A minimal amount of exosomes the collection protocol of EVs from cultured cell sam- required for AAs quantification was determined. AA ples for metabolomic analysis. Our results suggested profiles of exosome were different from those of CM that the metabolic character in EVs may vary that in and parental cells or serum. In contrast, some changes cells. JOURNAL OF EXTRACELLULAR VESICLES 325

Funding: This study was supported by the Japan PS07.06 Society for the Promotion of Science KAKENHI Grants and research funds from the Yamagata Prefecture Government and Tsuruoka City. Unrevealed mystery of cell dust: extracellular vesicles and tumour derived exosomes a b c PS07.05 Deanna Ayupova , Thomas Nann and Renee Goreham aThe MacDiarmid Institute for Advanced Materials and Nanotechnology, Victoria University of Wellington, Wellington, New Zealand; bThe c Exosomal miR-141-3p regulates osteoblast activity to promote the Univeristy of Newcastle, Callaghan, Australia; Victoria University of osteoblastic metastasis of prostate cancer Wellington, Wellington, New Zealand Yun Ye The First Affiliated Hospital of Xi’an Medical University, Xi’an, China Introduction:Bindingexosomestotheirtargetcellsis (People’s Republic) more likely to be determined by specific interaction(s) of protein(s) enriched in membrane of extracellular vesicles Introduction: Exosomes from cancer cells, which con- (EVs) including tumour-derived exosomes (TEX) and cel- tain microRNA and reach metastasis loci prior to can- lular proteins. Although the exact mechanism of EVs cer cells, stimulate the formation of a metastatic action is not fully understood, several studies reported microenvironment. Previous studies have shown that that TEX can modify tumour microenvironment by trans- exosomal miR-141-3p is associated with metastatic ferring non-coding RNAs and miRNA (particularly prostate cancer (PCa). However, the role and regula- miRNA-21 and −29) to target cells. Several studies have tory mechanism of miR-141-3p in the microenviron- showed comparative proteomic analyses of exosomes and ment of bone metastases require further study. circulating EVs from different biological fluids validating Methods: In this study, we performed a series of their respective role as novel biomarkers offering an early, experiments in vivo and in vitro to determine whether non-invasive method for cancer diagnosis. Protein profil- exosomal miR-141-3p from MDA PCa 2b cells regu- ing and detection conducted by label-free quantification lates osteoblast activity to promote osteoblastic will contribute to understanding the contents of healthy metastasis. and tumour-derived exosomes by identifying their con- Results: We demonstrate that extracts obtained from centration, size distribution, population and composition cell culture supernatants contained exosomes and that enabling characterization of peptide peak intensity. miR-141-3p levels were significantly higher in MDA Collected data combined into a protein library for early PCa 2b cell exosomes. Via confocal imaging, numerous diagnosis will result in the improvement of cancer immu- MDA PCa 2 bexosomes were observed to enter osteo- notherapy and new therapeutic targets. blasts, and miR-141-3p was transferred to osteoblasts Methods: AB; SEC; IF; NTA;LC-MS; TEM; Flow through MDA PCa 2b exosomes in vitro. Exosomal Results: According to cell viability assay, exosomes miR-141-3p from MDA PCa 2b promoted osteoblast increase cell proliferation after 24 h of treatment for activity and increased osteoprotegerin OPG expression. both cell lines: MCF 10 A and A549 for all concentra- miR-141-3p suppressed the protein levels of the target tions of exosomes (0.125, 0.25, 05, 1 mg/mL). Annexin gene DLC1, indicating its functional significance in V/ PI assay was used to define the percentage of necro- activating the p38MAPK pathway. In animal experi- tic and apoptotic cell death after treating cancerous and ments, exosomal miR-141-3p had bone-target specifi- non-tumorigenic cell lines. Interestingly, the higher city and promoted osteoblast activity. Mice injected percentage of necrotic death (6.45%) was recorded for with miR-141-3p-mimics exosomes developed appar- THP-1 exosomes conditioned with MCF 10 A, while ent osteoblastic bone metastasis. the smallest number of necrotic death was noticed for Summary/Conclusion: Exosomal miR-141-3p from A549 exosomes condition with the same cell line A549 MDA PCa 2b cells promoted osteoblast activity and (1.99%). The TEM analysis showed roughly same size regulated the microenvironment of bone metastases, (40–50 nm) of exosomes for all various types of exo- which plays an important role in the formation of somes used in this study (THP-1, MCF 10 A and A549 bone metastases and osteogenesis damage in PCa. exosomes). Clarifying the specific mechanism of bone metastasis Summary/Conclusion: In summary, we can conclude will help generate new possibilities for the treatment that extracellular vesicles (including exosomes) derived of PCa. from mammalian cell including cancerous and non- 326 ISEV2019 ABSTRACT BOOK tumorigenic cell line increase cell proliferation. That Summary/Conclusion: In conclusion, a lectin array being said, cancerous cell line secrets quiet a larger method is a powerful tool for comprehensively glycan amount of extracellular vesicles compared to non- analysis of EVs towards biomarker discovery. tumorigenic cell line. PS07.08 Funding: No external fundings.

Tomato fruit-derived vesicles: isolation, biocargo characterization and the dissection of different vesicle types PS07.07 Ramesh Bokkaa;, Gabriella Pocsfalvia, Teresa Silvestrea, Immacolata Fiumea, Lilla Turiakb and Tamás Csizmadiac aExtracellular Vesicles and Mass Spetrometry Group, Institute of Bioscience and BioResosrces (IBBR) – CNR, Naples, Italy; bHungarian Academy of Surface glycan profiling of extracellular vesicles by lectin array Sciences, Research Centre for Natural Sciences, Budapest, Hungary; cEötvös system for biomarker discovery Loránd University, Budapest, Hungary Asako Shimodaa, Shin-ichi Sawadab, Yoshihiro Sasakib and Kazunari Akiyoshib Introduction: Plant-derived vesicles are receiving con- aKyoto Univerisity, Kyoto, Japan; bKyoto University, Kyoto, Japan siderable attention due to their potential applications as Introduction:Extracellularvesicles(EVs)areknown vectors for the delivery of biologically active substances as cellular communicators that carry their contents in the nutraceutical, cosmetic and pharmaceutical including proteins, lipids and nucleic acids. Since fields. Here, in the first time, we report the in depth cells handover their biological information to EVs, characterization of micro (MVs) and nanovesicles they can be applicable to cell biomarkers. We showed (NVs) enriched fractions isolated from the pericarp that glycans on mesenchymal stem cells (MSCs)- tissue of Solarium lycopersicum with the aim to develop derived EVs play important roles in cellular recogni- a new generation, natural vesicles-based delivery vec- tion using an evanescent-field fluorescence-assisted tors. This includes the setup of a novel GC-MS/MS ’ lectin array system [1]. Most remarkable feature of platform suitable for the characterization of vesicles this method is that simple, sensitive and real-time metabolites. detection of surface glycan patterns on intact EVs. Methods: MV and NV fractions were isolated by dif- In this study, surface glycan profiling on EVs from ferential centrifugation. NVs were further purified by many types of cells was analysed using the lectin array sucrose gradient ultracentrifugation method. Isolation system. of NVs resulted to be troublesome due to the co-pur- Methods: EVs were isolated from various kinds of ifying pectin substances. Physiochemical properties of mouse and human cells including cancer cells, undif- the vesicles were analysed by TEM and DLS, while ferentiated and differentiated MSCs, and immune cells biocargo composition was studied by mass spectrome- by differential ultracentrifugation. Cy3-labelled EVs try-based proteomic and metabolomics workflows. and their originating cell membranes (CMs) were Functional annotation and data mining were per- applied to a glass slide with 45 lectins, and fluorescence formed using Blast2Go software package including intensities were detected using an evanescent-field InterPro, enzyme codes, KEGG pathways and GOSlim fluorescence scanner. functions. Results: Most types of EVs showed higher binding to Results: The isolation method was improved by differ- sialic acids-recognizing lectins and weaker binding to ential solubilization using 0.1M phosphate 10 mM mannose-binding lectin as compared with their origi- EDTA buffer pH 8, to keep pectin substances in solu- nating CMs. Hierarchical clustering analysis and prin- tion allowing by the efficient purification of NVs. In – cipal component analysis were performed to evaluate each sample, approximately 600 800 proteins and whether surface glycans on EVs have their cell specific approximately 50 metabolites could be identified. A patterns. The results indicated that glycan profiling of novel method based on GC-MS/MS metabolomic pro- EVs can be used to classify cell types (normal or can- filing of plant-derived vesicles has been developed. cer) and they can be further divided into each type of Summary/Conclusion: Protein biocargo of tomato cancer, MSC sources and cell lineages, indicating that pericarp tissue-derived vesicles reveals heterogeneous surface glycans on EVs may act as potential biomarkers transport and extracellular vesicle subpopulations. of cell state. More than 340 enzymes comprising 43 antioxidants identified in tomato nanovesicles may count for its JOURNAL OF EXTRACELLULAR VESICLES 327 health-promoting effects in the diet, i.e. protection innovation programme under the Marie Skłodowska- against cancer, maintenance of healthy blood pressure Curie grant agreement number 798576 and FET Open and reduction of blood glucose in diabetic patients. grant agreement number 801338. Funding: This project has received funding from the European Union’s Horizon 2020 research and 328 ISEV2019 ABSTRACT BOOK

PS08: Advances in EV Quantification and Characterization II Chairs: Cecilia Lässer; Li Min Location: Level 3, Hall A 15:00–16:00

PS08.01 PS08.02

Electrical characterization of individual exosomes secreted from Taxonomy of individual EVs by nanomechanics amyloid beta-treated neuroblastoma cells via electrostatic force Andrea Ridolfia, Marco Brucaleb, Lucia Paolinic, Costanza Montisd, Debora microscopy Bertid, Francesco Valleb and Paolo Bergesee Yeseong Choia, Sumi Kima, Dae Sung Yoonb and Ji Yoon Kanga a ISMN-CNR and CSGI; Department of Chemistry, University of Florence, a b b c Korea Institute of Science and Technology, Seoul, Republic of Korea; School Firenze, Italy; ISMN-CNR and CSGI, Bologna, Italy; Department of of Biomedical Engineering, Korea University, Seoul, Republic of Korea Molecular and Translational Medicine and CSGI, Università degli Studi di Brescia, ITALY, Firenze, Italy; dDepartment of Chemistry and CSGI, University of Florence, Firenze, Italy; eDepartment of Molecular and Introduction: Exosomes are cell-derived nanovesicles Translational Medicine and CSGI, Università degli Studi di Brescia, ITALY, Brescia, Italy known to provide information about the state of parent cells. Recently, it has been found that the pathogenic Introduction: Probing and understanding the physical amyloid beta oligomers (oAβs), known as a biomarker properties of individual EVs – as a whole and of their of Alzheimer’s disease (AD), can be propagated separate components – are fundamental aspects in EV between neighbouring neurons through exosomes. At research that still need to be addressed. We will present the same time, there is an increasing need to introduce our latest results regarding the nanomechanical char- a new technique for the characterization of individual acterization of individual EVs via Atomic Force exosomes because of their diversity. In this paper, we Microscopy-based Force Spectroscopy (AFM-FS) and used electrostatic force microscopy (EFM) to demon- discuss their significance and perspectives. strate the effect of oAβ on electrical properties of Methods: Our experimental approach entails adsorp- individual exosomes. tion of EVs (separated from cell culture media) on Methods: Different concentrations (30, 150, 750 nM) inorganic substrates with controlled surface properties. of oAβs were treated to mouse neuroblastoma (N2a) The response of each individual EV to an applied cells, and exosomes were harvested from cell culture mechanical deformation in physiological buffer is media through ultracentrifugation. The electrical prop- then sampled via liquid AFM-FS. The obtained force erties of exosomes were investigated by using EFM. For curves are finally quantitatively analysed by dedicated EFM experiment, the 10 μL of each exosome solution models to obtain the EV “nanomechanical fingerprint”. was deposited on a fresh mica substrate for 15 min, Results: The reversible elastic deformation behaviour washed in PBS and DW order and dried under pure of an EV in response to the AFM tip indentation nitrogen gas. resulted to be the convolution of several characteristics Results: EFM can visualize the electrostatic force gra- of the EV. We found that the overall apparent stiffness dient corresponding to the surface potential of single of an intact EV effectively recapitulates its mechanical exosomes. The scatter plot resulted from EFM data behaviour. We also found first evidences that this analysis showed a correlation between the size and property can be exploited to sort single EVs and that the charge of exosomes. Moreover, charge density it relates them to other organic envelopes of similar values, which excludes the influence of size by dividing size and composition, such as viruses and synthetic the charge value by height, decreased by up to four liposomes. times depending on the concentration when compared Summary/Conclusion: These results proof that a with the control (−5.95 μV/nm at control, −9.17, −11.1, nanomechanics-based taxonomy might be an impor- −23.85 μV/nm at 30, 150, 750 nM, respectively). It tant tool for advancing characterization and under- implies that exosomes from oAβ-treated N2a cells standing of EVs at the single vesicle level. have significantly higher negative surface potential Funding: This research has received funding from the than those from untreated N2a cells. Horizon 2020 Framework Programme under the grant Summary/Conclusion: This paper proposes a new FETOPEN-801,367 evFOUNDRY. nano-electrical characterization to differentiate neuronal exosomes treated by oAβs from untreated ones. It is JOURNAL OF EXTRACELLULAR VESICLES 329 possible to use EFM as imaging and analysis tool for label-free sensing of EVs. High chemical specificity single exosome characterization. Furthermore, it is afforded by Raman spectroscopy rapidly identified expected that exosomes associated with AD are isolated tumour EVs from healthy controls in clinical samples. from plasma in the diagnosis of AD according to a Our nanocomposites are inexpensive, reusable, stable surface potential of exosome. and suitable for low resource environments, with high potential for translational application of clinical diagnostics using EVs. PS08.03 Funding: The authors acknowledge funding from the Ovarian Cancer Education and Research Network (OCERN). Hybrid plasmonic biomaterial nanofilter scaffold for cancer EV diagnostics based on surface-enhanced Raman scattering (SERS) a a b Randy Carney , Tatu Rojalin and Sebastian Wachsmann Hogiu PS08.04 aUC Davis, Davis, USA; bMcGill University, Montreal, Canada

Introduction:Newanalyticalapproachesareneeded Electrochemical quantification of EVs at physiological concentrations that account for the vast molecular heterogeneity of Pepijn Beekmana, Dilu Mathewb and Séverine Le Gacc nanoscale extracellular vesicles (EVs). Raman spec- aWageningen University, Wageningen, Netherlands; bNanoElectronics, troscopy is an attractive technology capable of sensi- University of Twente, Enschede, The Netherlands, Enschede, Netherlands; cApplied Microfluidics for BioEngineering Research, University of Twente, tive molecular fingerprinting of chemical changes The Netherlands, Enschede, Netherlands associated with disease. Surface-enhanced Raman Spectroscopy (SERS) overcomes the inherent weak Introduction: Tumour-derived extracellular vesicles nature of spontaneous Raman scattering and is prov- (tdEVs) are promising markers for cancer patient man- ing to be a promising tool for next-generation clin- agement. An advantage of tdEVs over circulating ical diagnostics. The principle of SERS is based on tumour cells is their higher concentration in patient amplification of Raman scattering using metal sur- blood by 3–4 orders of magnitude (∽103–105 tdEVs faces that have a nanoscale roughness with features /ml), giving more robust information while requiring of ~20–200 nm. We introduce an inexpensive and smaller sample sizes. However, their small size and flexible SERS substrate based on a novel biosilica complex composition of blood samples require sensi- plasmonic nanocomposite that acts as a simulta- tive and selective detection methods. Here, we report neous nanofilter and detection platform for sensitive electrochemical detection of tdEVs using a nano-inter- characterization of tumour-associated EVs. digitated electrode array (nIDE) functionalized with Methods: A porous biosilica scaffold doped with plas- cancer-specific antibodies and an antifouling coating. monic silver nanoparticles can be simply and easily The detection mechanism is based on enzymatic con- prepared on office-grade adhesive tape. This nanocom- version of aminophenyl phosphate (APP) by alkaline posite deposition requires no chemical modification of phosphatase (ALP) followed by redox cycling of the the raw materials. Particles larger than ~100 nm con- cleaved substrate, yielding a double signal amplifica- centrate on the top surface in close proximity to clus- tion. The proposed sensing scheme is 10 times more ters of plasmonic nanoparticles, affording usability as a sensitive than state-of-the-art detection approaches, SERS-based sensing platform. giving a physiologically relevant limit of detection Results: We tested our platform with dozens of sam- (LOD) of 10 EVs/μl. ples of tumour-associated EVs enriched from ovarian Methods: nIDEs (120 nm width, 80 nm spacing, 75 nm cancer patients and healthy controls to demonstrate height) were functionalized with an amino-undeca- that SERS imaging can sensitively detect and identify nethiol monolayer, and reacted with poly(ethylene gly- disease profiles. We found enhancement factors of col) diglycidyl ether. Anti-EpCAM antibodies were more than 10^8-fold compared to spontaneous next immobilized to subsequently capture tdEVs. Raman signatures. Sensitivity and specificity exceeding Anti-EpCAM-alkaline phosphatase conjugates were 90% was found for human clinical samples using less then introduced to yield ALP-tagged tdEVs. The non- than 1 μL of minimally processed plasma, all in just a electroactive pAPP was finally used to quantify the few seconds using a commercial Raman imaging ALP concentration. system. Results: With increasing tdEV concentration, an Summary/Conclusion: We introduce a simple plasmo- increase in redox current was measured, from 0.35 nic composite using readily available biomaterials and nA for 10 tdEV/μl to 12.5 nA for 10^5 tdEV/μl (avg., metallic nanoparticles, and demonstrate its efficacy for n = 3). Current is produced by the electroactive 330 ISEV2019 ABSTRACT BOOK cleavage product of APP, which redox cycles between particles in the mixture and showed quantification electrodes. The short migration distance in our nanoe- errors at 150 nm diameter. lectrode array yielded a factor ∽8 improvement com- Experiment 2: MRPS showed the particle size dis- pared to micro-electrodes (3 μm width, spacing). As a tribution expected: Concentration increased with negative control, the experiment was performed with decreasing particle size with an approximate power- incubation of platelet derived EVs, whereby the signal law dependence on diameter reported elsewhere in did not significantly increase (background current the literature. MRPS was in excellent agreement with ∽0.15 nA). TEM. NTA reported misleading results: A loss of Summary/Conclusion: A sensitive sensor was devel- counting efficiency was apparent as high as 200 nm oped for the detection of EVs at unprecedented low diameter, and led to a 10,000-fold discrepancy by concentrations. With an LOD of 10 tdEVs/μl and high 65 nm. Critically, NTA reported a prominent peak selectivity towards tdEVs, our platform opens new that does not in fact exist. avenues for screening patient blood samples. Summary/Conclusion: These experiments expose a Funding: Funded by NWO Perspectief critical failure mode of NTA: Its LOD depends strongly on the composition of the sample, with enormous impact for EV measurements. Critically, a researcher PS08.05 could be severely led astray by the NTA results in isolation, without an orthogonal technique for reference.

The Importance of Orthogonal Techniques in EV Quantification Jean-Luc Fraikina, Franklin Monzonb, Lew Brownb, Mac Baileyb and Ngoc Dob PS08.06 aSpectradyne LLC, Torrance, USA; bSpectradyne, Torrance, USA

Introduction: As EV research matures, so must mea- Fourier-transform Infrared Spectroscopy (FT-IR) to fingerprint EV subpopulations as a whole surement technologies. Two simple experiments are Lucia Paolinia, Stefania Federicib, Giovanni Consolic, Diletta Arceric, reported that expose a critical failure mode of Annalisa Radeghierid, Ivano Alessandrie and Paolo Bergesef Nanoparticle Tracking Analysis (NTA) for quantifying aDepartment of Molecular and Translational Medicine and CSGI, Università b ’ degli Studi di Brescia, ITALY, Firenze, Italy; Department Mechanical and EVs: NTA s small size limit of detection (LOD) Industrial Engeneering, University of Brescia, Italy, Brescia, Italy; depends strongly on the composition of the sample, cDepartment Molecular and Translational Medicine, University of Brescia, d causing 10,000-fold errors within the EV size range Italy, Brescia, Italy; Department of Molecular and Translational Medicine and CSGI, Università degli Studi di Brescia, ITALY, Brescia, Italy; relative to Microfluidic Resistive Pulse Sensing eDepartment of Information Engineering, University of Brescia, Italy, (MRPS) and Tunnelling Electron Microscopy (TEM). Brescia, Italy; fDepartment of Molecular and Translational Medicine and CSGI, Università degli Studi di Brescia, ITALY, Brescia, Italy Results show orthogonal methods for EV quantification are critical. Introduction: Characterizing EV subpopulations Methods: Experiment 1: Three sizes of polystyrene remains a challenge, which up-to-date has been tackled particles – 94, 150 and 208 nm diameters – were through analysis that assess for a single biochemical or measured by NTA and MRPS separately and after biophysical component of the target subpopulation. mixing in equal parts. The relative concentration accu- However, these approaches may be unsuitable to racy of NTA and MRPS was assessed as a function of describe EV subpopulations defined by higher level of size, and the LOD evaluated for each sample. heterogeneity. In our contribution, we will discuss how Experiment 2: The striking implications of Fourier-transform Infrared Spectroscopy (FT-IR) Experiment 1 were demonstrated in a real-world sam- allows to fingerprint EV subpopulations as a whole, ple. Urinary exosomes were measured by NTA, MRPS presenting itself as a promising complement/alternative and the gold standard, Tunnelling Electron Microscopy to describe EV subpopulations (TEM). The accuracy of relative concentration mea- Methods: Medium from murine prostate cancer surements was assessed for each method. (TRAMP-C2) and skin melanoma (B16) cell lines Results: Experiment 1: Polystyrene standards were were processed with serial centrifugation: 800g 30’ to accurately quantified by MRPS: Each component was enrich large EVs (LEVs), 16,000g 45’ to enrich medium clearly detected, and the relative concentrations of all EVs (MEVs) and 100,000g for 4 h to enrich small EVs were measured to be approximately equal as intended. (SEVs). LEVs, MEVs and SEVs were characterized for NTA showed similar results for the separate compo- size, purity and EV markers with Atomic Force nents. However, NTA was unable to detect the 94 nm Microscopy, colloidal nanoplasmonic assay and JOURNAL OF EXTRACELLULAR VESICLES 331

Western Blot, respectively. FT-IR measurements were purified OMVs were incubated with either cholesteryl performed on LEVs, MEVs and SEVs re-suspended in PEG 2000 FITC or sulpho cyanine7 NHS ester. For milliQ water and deposited onto a diamond cell. diazo transfer the pellet after UC was incubated with a Spectral regions between 3100–2800 cm-1 and 1880– diazo transfer agent and the OMVs subsequently con- 900 cm-1, corresponding to lipids and proteins, respec- jugated with DBCO-AF594. Unincorporated dye was tively, were considered, and processed by Principal removed by SEC. Liposomes were composed of DMPC Component Analysis (PCA) and DPPC in 2:3 molar ratio. Results represent corre- Results: PCA was applied to data set of FT-IR spectra lated fluorescence intensity and particle number. (five replicates for each EV subpopulations) collected Results: Treatment with sulpho cyanine7 NHS ester led for TRAMP and B16 cell line and visualized with scores to the modification with 547 ± 163 molecules per plots. LEVs, MEVs and SEVs resulted grouped sepa- OMVs, compared to 18 ± 1 for the control using rately for both considered cell lines. Moreover, spectra sulpho cyanine7 acid. Cholesterol insertion introduced from the same subpopulation, but from different cells 4 ± 1 molecules per OMV, compared to 101 ± 23 for are reported in two distinct groups liposomes. First results for the diazo-transfer showed Summary/Conclusion:EVsubpopulationsofdiffer- 71 dye-molecules per OMV, with 32 for the control. ent sizes and cellular origin are characterized by spe- Summary/conclusion: Of the three methods, NHS cific FT-IR fingerprint. This offers a proof of concept ester-modification displayed the highest efficiency, that FT-IR could be effectively translated in real sce- similar to published results for mammalian EVs. In narios to characterize EVs with different content and comparison, diazo transfer only yielded ~13% of the origin dye-molecules per particle. However, there are still Funding: LP acknowledges the BIOMANE grant many parameters to be optimized for this method, (University of Brescia) and evFOUNDRY grant including OMV concentration and incubation period. (H2020-FETOPEN-2016–2017 – Project ID: 801367) Cholesterol insertion was unsuccessful for OMVs, for the financial support probably owing to their membrane structure. In this study, we aim to get important insights into the mod- PS08.07=OWP1.07 ification of OMVs for bacterial targeting and EV-surface engineering in general. Funding: This project was funded by Studienstiftung Exploration of the surface modification of outer membrane vesicles des Deutschen Volkes and Bundesministerium fuer Maximilian Richtera, Eleonora Diamantib, Anna Hirschb and Gregor Fuhrmannc Bildung und Forschung. aHelmholtz-Institute for Pharmaceutical Research Saarland, Biogenic Nanotherapeutics, Saarbruecken, Germany; bHelmholtz-Institute for Pharmaceutical Research Saarland, Drug Design and Optimization, Saarbruecken, Germany; cHelmholtz-Institut for Pharmaceutical Research PS08.08=OWP2.01 Saarland (HIPS), Saarbrücken, Germany

Introduction: Introducing bacteria-binding small Identification of common EV markers in plasma using high-resolution molecules to the surface of outer membrane vesicles flow cytometry Anders Askelanda, Jaco Bothab, Rikke Wehner Rasmussenb and Aase (OMVs) could greatly improve their potential for anti- b Handberg microbial drug delivery to difficult to treat bacteria. aAalborg University Hospital, Aalborg, Denmark; bDepartment of Clinical Among the small number of studies on surface mod- Biochemistry, Aalborg University Hospital, Aalborg, Denmark, Aalborg, ification of OMVs, very few deal with small molecules. Denmark The aim of the present study is to evaluate different methods of introducing bacteria-specific targeting moi- Introduction: Recent advancements in flow cytometry eties to OMVs. We assessed the modification of surface (FCM) have led to the development of high-resolution proteins using N-hydroxysuccinimide (NHS) esters, FCMs dedicated to the analysis of small particles well established for mammalian extracellular vesicles (hFCM). hFCM studies have predominantly focused (EVs), cholesterol insertion, mainly applied for lipo- on the analysis of EVs expressing phosphatidylserine somes and the novel application of diazo-transfer fol- (PS). PS is enriched in microvesicles (MVs), wherein it lowed by click-chemistry. is involved in lipid rearrangements responsible for MV Methods: OMVs were obtained from model budding. While PS also is expressed on exosomes, it is Myxobacteria by differential ultracentrifugation (UC) unknown whether it can be used as a universal marker followed by size exclusion chromatography (SEC). For for smaller EVs. In this study, we attempted to char- cholesterol insertion and NHS ester-modification, acterize proteins enriched in smaller EVs (CD9, CD63, 332 ISEV2019 ABSTRACT BOOK

CD81 and ADAM 10) and the relative co-expression of Biomedical Engineering and Physics, Amsterdam, Netherlands, Amsterdam, Netherlands PS with each of these markers. Methods: Flow cytometry analysis was performed on Introduction: In search of new biomarkers, flow cyt- an Apogee A60 Micro-PLUS. In brief, platelet-poor ometers are used in clinical studies to measure the plasma (PPP) from healthy individuals was stained concentration of specific extracellular vesicles (EVs). with lactadherin-FITC (PS+) and one of several EV Flow cytometers measure light scattering and fluores- surface markers enriched in smaller EVs. To evaluate cence of single EVs in a fluid stream. However, to the precise differences in PS and specific EV marker realize data interpretation and comparison, light scat- expression, the analysis was performed twice, (1) trig- tering and fluorescence signals and the flow rate gering on lactadherin and (2) each EV marker (CD9- require calibration. Moreover, flow cytometers gener- PE, CD81-PE, CD63-PE, ADAM10-PE), separately. All ate large datasets. For example, a clinical study invol- antibodies were matched with appropriate isotope con- ving 60 patients, 30 controls and 8 antibody labels trols and centrifuged at 17,000g for 10 min. prior to covers 1224 data files, >33 gigabytes of data and >0.3 antibody labelling. EVs were defined as lactadherin or billion events. To manually calibrate and analyse such a ≤ EV surface marker positive events 1000 nm. dataset would take days if not weeks and is prone to Results: Initial results indicate that CD9 is highly human mistakes. Therefore, an urgent need exists for expressed on EVs and is not universally associated to software to automate calibration and processing of flow PS. Triggering on PS revealed that 34.7% of all events cytometry data. were CD9 positive (CD9+|PS+). Conversely, triggering Methods: We have developed software (MATLAB on CD9 resulted in a 2.1-fold increase in total events, R2018a) to automatically process multiple .fcs files where 17.0% of events were PS+ (CD9+|PS+). Inferring and (1) relate two scatter signals to the diameter in size from silica nanospheres, it appeared that popula- nm and refractive index (RI) of EVs, (2) express fluor- tions containing CD9 (CD9+|PS+ and CD9+|PS-) were escence signals in terms of molecules of equivalent – smaller (94.4 99.7% <180 nm) compared to popula- soluble fluorochrome (MESF), (3) export calibrated tions that did not (PS+|CD9-; 85.6% <180 nm & 95.2% channels to new .fcs files, (4) recognize unstable flow <300 nm). Interestingly, we did not detect CD81, CD63 rates, (5) determine fluorescence thresholds, (6) apply or ADAM10 on EVs. We hypothesize that this is due to gates, (7) create PDFs with scatter plots and (8) report a low abundance of these markers in PPP from healthy statistics. We are using clinical studies to validate and individuals. apply the software. Summary/conclusion: Our findings demonstrate that Results: Compared to manual thresholding, automatic hFCM can be used for the characterization of smaller thresholding results in a systematic decrease in counts EVs in PPP. Furthermore, we find that CD9+ EVs does of 10% and a maximum difference of 14% (n = 5). not universally express PS. From this point on, we plan Using a high-end laptop, data processing takes typically to study enrichment of these EV phenotypes following a minute or several seconds per .fcs file with or without a number of EV purification protocols and determine PDF reporting, respectively. Flow rate monitoring is whether EV isolation enable a more extensive charac- useful for 61% of the data. The platelet marker CD61 terization of smaller EVs. stains 7% of the events with an RI>1.42, which are lipoproteins, and the concentration of these lipoproteins differed 4000-fold between individuals. PS08.09=OWP2.02 Summary/conclusion: We have developed software to automate calibration and processing of flow cytometry data in clinical studies, thereby reducing analyses time, Software to automate calibration and processing of flow cytometry data in clinical studies preventing human mistakes and providing new Edwin van der Pola, Frank Coumansb, Leonie de Rondc, Aleksandra insights. For example, non-specific labelling of antibo- d e b f Gasecka , Najat Hajji , Rienk Nieuwland and Ton van Leeuwen dies to lipoproteins together with variations in lipopro- aAmsterdam UMC, University of Amsterdam, Department of Biomedical tein concentrations emphasize the relevance of fasting Engineering and Physics, Amsterdam, Netherlands, Amsterdam, Netherlands; bAmsterdam UMC, University of Amsterdam, Laboratory of before venipuncture. Our next step is to extend the Experimental Clinical Chemistry, Amsterdam, Netherlands, Amsterdam, software with machine learning. Netherlands; cAmsterdam University Medical Centers, Amsterdam, USA; d1st Chair and Department of Cardiology, Medical University of Warsaw, Funding: NWO-TTW VENI 15924 Warsaw, Poland; eAmsterdam University Medical Centers, Amsterdam, Netherlands; fdAmsterdam UMC, University of Amsterdam, Department of JOURNAL OF EXTRACELLULAR VESICLES 333

PS08.10=OWP2.03 presentation provide useful strategies for circumventing these.

Conventional, high-resolution and imaging flow cytometry: potentials, pitfalls and solutions for EV characterization PS08.11=OWP2.04 Jaco Botha, Rikke Wehner Rasmussen, Mathilde Sanden and Aase Handberg Department of Clinical Biochemistry, Aalborg University Hospital, Aalborg, Denmark, Aalborg, Denmark Convolutional neural networks for classification of tumour derived extracellular vesicles Wooje Leea, Aufried Lenferinka, Cees Ottob and Herman Offerhausa Introduction: Flow cytometry (FCM) has long been a aUniversity of Twente, Enschede, Netherlands; bMedical Cell Biophysics, preferred method for characterizing EVs, however their University of Twente, Enschede, Netherlands small size have limited the applicability of conventional FCM to some extent. Thus, high-resolution and ima- Introduction: Raman spectroscopy probes molecular ging FCMs have been developed but not yet system- vibration and thus reveals chemical information of a atically evaluated. The aim of this presentation is to sample without labelling. This optical technique can be describe the applicability of high-resolution and ima- used to study the chemical composition of diverse EVs ging FCM in the context of EV characterization and subtypes. EVs have a complex chemical structure and the most significant pitfalls potentially influencing data heterogeneous nature so that we need a smart way to interpretation. analyse/classify the obtained Raman spectra. Machine Methods: First, we present a side-by-side comparison learning (ML) can be a solution for this problem. ML is of three different cytometry platforms on characteriz- a widely used strategy in the field of computer vision. It ing EVs from blood plasma regarding sensitivity, reso- is used for recognizing patterns and images as well as lution and reproducibility: a conventional FCM, a classifying data. In this research, we applied ML to high-resolution FCM and an imaging FCM. Next, we classify the EVs’ Raman spectra. demonstrate how different pitfalls can influence the Methods: With Raman optical tweezers, we obtained interpretation of results on the different cytometry Raman spectra from four EV subtypes – red blood cell, platforms. Finally, we propose controls, solutions or platelet, PC3 and LNCaP – derived EVs. To classify workarounds for understanding and limiting the influ- them by their origin, we used a convolutional neural ence of each of these pitfalls. network (CNN). We adapted the CNN to one dimen- Results: (1) High-resolution FCM and imaging FCM sional spectral data for this application. displayed greater sensitivity and resolution compared The ML algorithm is a data hungry model. The model to conventional FCM when measuring a mixture of requires a lot of training data for accurate prediction. nanospheres. Equally, both methods could detect larger To further increase our substantial dataset, we per- concentrations of specific EV phenotypes than conven- formed data augmentation by adding randomly gener- tional FCM, where imaging FCM outperformed high- ated Gaussian white noise. resolution FCM. Within day variability (n = 20 ali- The model has three convolutional layers and fully quots) was similar for conventional and high-resolu- connected layers with five hidden layers. The Leaky tion FCM, while imaging FCM had a markedly larger rectified linear unit and the hyperbolic tangent are variability. Between day variability (n =5×5 aliquots) used as activation functions for the convolutional was similar for all three platforms. (2) The three most layer and fully connected layer, respectively. substantial pitfalls variably influencing interpretation Results: In previous research, we classified EV Raman of results on the three platforms are non-specific bind- spectra using principal component analysis (PCA). ing of labels, antibody aggregates and entities in the PCA was not able to classify raw Raman data, but it sample (i.e. lipoproteins) binding EV-defining dyes. (3) can classify preprocessed data. CNN can classify both The most important strategies for circumventing these raw and preprocessed data with an accuracy of 93% or pitfalls are stringent matching, gating and comparison higher. It allows to skip the data preprocessing and of antibodies and isotype controls, high-speed centri- avoids artefacts and (unintentional) data biasing by fugation of antibodies and labels prior to staining, and data processing. the use and interpretation of stained buffer controls Summary/conclusion: We performed Raman experi- and detergent-treated samples. ments on four different EV subtypes. Because of its Summary/conclusion: High-resolution and imaging complexity, we applied a machine learning technique FCM hold great potential for EV characterization. to classify EV spectra by their cellular origin. As a However, increased sensitivity also leads to new arte- result of this approach, we were able to classify EVs facts and pitfalls. The solutions proposed in this by cellular origin with a classification accuracy of 93%. 334 ISEV2019 ABSTRACT BOOK

Funding: This work is part of the research program This tool holds great potential for early cancer diagno- [Cancer-ID] with project number [14197] which is sis in clinical applications. financed by the Netherlands Organization for Scientific Research (NWO). PS08.13=OWP2.06

PS08.12=OWP2.05 A software suite allowing standardized analysis and reporting of fluorescent and scatter measurements from flow cytometers Joshua Welsh and Jennifer C. Jones

Translational Nanobiology Section, Laboratory of Pathology, National Cancer Microfluidic electrochemical aptasensor for detection of breast Institute, National Institutes of Health, Bethesda, USA cancer-derived exosomes in biofluids Leila Kashefi-Kheyrabadi, Sudesna Chakravarty, Junmoo Kim, Kyung-A Hyun, Seung-Il Kim and Hyo-Il Jung Introduction: Single vesicle analysis using flow cyto- Yonsei University, Seoul, Republic of Korea metry is an extremely powerful technique to allow identification of unique proteins in biological samples, Introduction: Exosomes are nanosized extracellular as well as enumerating the changes in concentrations. vesicles, which are emerging as potential non-invasive While small particle analysis (for viruses and large biomarkers for early diagnosis of cancer. However, the microparticles) using flow cytometry has been con- small size and heterogeneity of the exosomes remain ducted for several decades, there is no comprehensive significant challenges to their quantification in the bio- method for standardization of such studies. Therefore, fluids. In the present research, a microfluidic electro- we developed a suite of flow cytometry post-acquisition chemical biosensing system (MEBS) is introduced to analysis software (FCMPASS) tools that enable the detect ultra-low levels of breast cancer cell-derived conversion of scatter and fluorescent axes to standar- exosomes (BCE). dized units using appropriate controls, writing standar- Methods: Fabrication procedure of MEBS comprises dized units to .fcs files for sharing upon publication three main steps: first, biosensing surface was prepared with open repositories, and exporting templates of by immobilizing EPCAM binding aptamer (EBA) on a obtained data. nanostructured carbon electrode. The nanostructured Methods: Standalone software packages for scatter and surface (NS) consists of 2D nanomaterials including fluorescent standardization were built using MATLAB. MoS2 nano-sheets, graphene nano-platelets and a The scatter software is based upon Mie modelling and well-ordered layer of electrodeposited gold nanoparti- is capable of predicting the optical collection angle of cles. The NS was well characterized with FESEM and the instrumentation and reporting the Mie modelling EDX. FESEM analysis showed a well-ordered gold criteria in a standardized way, making it possible to nano-structuring for 50 nM of gold solution. reproduce the models and flow cytometry settings. Furthermore, EDAX analysis confirmed >60% coverage Fluorescent standardization data uses least-squares lin- of gold nanoparticles on nano-structured surface com- ear regression to enable conversions of arbitrary unit pared to bare carbon electrode. At the second step, a scales to molecules of equivalent soluble fluorophore herringbone structured microfluidic channel, which is (MESF) using MESF calibration beads. able to enrich BCE was designed and fabricated. Results: The FCMPASS software converts arbitrary Finally, microfluidic channel was integrated to biosen- fluorescence units to MESF units and writes them to sing surface. Different concentrations of exosome solu- data files for clearer reporting and sharing of data. tions was introduced and enriched to biosensing FCMPASS also converts arbitrary scatter units to a surface (SPCE/NS/GNP/EBA) using microchannel. measurement of scattering cross-section using model- After capturing BCEs on the sensing surface a second- ling software that predicts the collection angle of the ary aptamer labelled with silver nanoparticles (SNPs) as instruments and normalizes the data automatically. redox reporter was introduced to the sensing surface. Summary/conclusion: Utilization of our FCMPASS Results: Direct electro-oxidation of SNPs was moni- software can help the EV flow cytometry more easily tored as analytical signal. The unique design of micro- implement standardization into their experimental channel in combining with high-specific interaction analysis and the use of the output templates can make between BCE and EBA provided a high-sensitive detec- reporting more consistent. While currently available tion of BCE as low as ~100 exosomes/μl. MESF controls can be further optimized for small Summary/conclusion: The unique design of MEBS particles, we believe their utilization along with the provides a highly sensitive accurate platform for detec- other controls and can bring a new era to the reporting tion of ultra-low levels of cancer-derived exosomes. of EV research using flow cytometry. This will be JOURNAL OF EXTRACELLULAR VESICLES 335 particularly useful for future comparison and valida- Methods: ZP measurements were performed on EVs tion of translational studies and will enable better collected from the conditioned medium of human understanding and utilization of EVs across a broad choriocarcinoma (JAr) cells grown in EV-depleted range of disciplines. media. EVs were purified using size exclusion chromatography. EV populations were incubated with EV surface membrane-specific antibodies and the change in PS08.14 the electrokinetic mobility upon the binding of surface EV proteome with an antibody was measured using nanoparticle tracking analysis (Zetaview;

The effect of antibody binding on the zeta potential of extracellular Particlemetrix, Inning, Germany). vesicles secreted by cultured human choriocarcinoma cells Results: The mean+SEM ZP was −22.1 ± 0.8 mV and a b a a Getnet B. Midekessa , Kasun Godakumara , Ene Reimann , Janeli Viil , −20.5 ± 0.8 mV for non-treated JAr EVs and immu- Freddy Lättekivia, Keerthie Dissanayakea, Sergei Kopanchukc, Lisa Thurstond, Stephen Ebbense, Ago Rinkenc and Toonika Rinkenc noglobulin G isotype antibody (control)-treated EVs, aDepartment of Pathophysiology, Institute of Biomedicine and Translational respectively, indicating the absence of influence of Medicine, University of Tartu, Estonia, Tartu, Estonia; bDepartment of nonspecific binding. Whereas the ZP distribution of Pathophysiology, Institute of Biomedicine and Translational Medicine, University of Tartu, Tartu, Estonia, Tartu, Estonia; cInstitute of Chemistry, EVs incubated with surface exosomal marker antibo- University of Tartu, Estonia, Tartu, Estonia; dAcademic Unit of Reproductive dies showed a significant positive shift in the measured and Developmental Medicine, Department of Oncology and Metabolism, values compared to EVs incubated with control anti- Medical School, University of Sheffield, UK, Sheffield, United Kingdom; eDepartment of Chemical and Biological Engineering, University of body. The mean+SEM ZP values of EVs bound with Sheffield, UK, Sheffield, United Kingdom CD63 and CD81 were 17.2 ± 1.1 mV and −17.8 ± 0.9 mV respectively (N = 3 biological replicates of mini- Introduction: Research on extracellular vesicles (EVs), mum 1000 particles measured in each replicate). which include exosomes and microvesicles, has wit- Western blot analysis showed particles carrying EV- nessed an exponential increase in the past decade. specific surface markers. Furthermore, we investigated EVs are membrane-derived vesicles, which play vital the other factors that may have a potential effect on the role in transporting functional molecules to nearby or changes in EV’s electrokinetic mobility such as the distant cells, thus being involved in the intercellular concentration of particles and concentration of the communications. Developing a reliable and quantita- antibody. tive method for confirming a nanoparticle as an EV is Summary/conclusion: The measured antibody-specific still challenging. Nanoparticles carry a net surface changes in ZP values provide an insight into the nature charge due to the nature of their surface molecules. of the nanoparticle surface antigens in a biological We have hypothesized that EVs, which normally carry sample. ZP measurement is a simple, cost-effective a negative zeta potential (ZP), can be identified by the and reliable method for profiling EV surface change of net surface charge when bound to EV-spe- composition. cific antibodies. 336 ISEV2019 ABSTRACT BOOK

PS09: EV Cancer Pathogenesis Chairs: Marta Prieto Vila; Judy Yam Location: Level 3, Hall A 15:00–16:00

cyclodextrin resulted in marked reduction of exosomes PS09.01 and TSG101 in them. Summary/Conclusion: GD3 expression in cancer cells resulted in increased levels of integrins in ECVs, sug- Extracellular vesicles secreted from ganglioside GD3-expressing gesting that GD3 and integrins play roles in the malig- cancer cell lines contain high levels of integrins: Roles of lipid rafts nant properties of cancers by forming molecular Koichi Furukawaa, Iori Kobayashi, Yoshiki Kodamab, Yuhsuke Ohmic, Satoko Yamamotod, Yuki Ohkawa, Mariko Kambe, Keiko Furukawaa complexes on ECVs. Lipid rafts may play roles as aDepartment of Biomedical Sciences, College of Life and Health Sciences, sites for the complex formation. Chubu University, Nagoya, Japan; bDepartment of Biomedical Sciences, Funding: Grants-in-aid from the Ministry of College of Life and Health Sciences, Chubu University, Kasugai, Japan; cDepartment of Biochemical Sciences, College of Life and Health Sciences, Education, Culture, Sports, Science and Technology Chubu University, Kasugai, Japan; dKanazawa Medical University, Uchinada, of Japan Japan

Introduction: Cancer-associated glycosphingolipids PS09.02 have been utilized as tumour markers and targets of cancer therapy. We have analysed functions of gang- Amniotic Epithelial Exosomes Result In Reversal of Epithelial to liosides in cancers, and reported that cancer-associated Mesenchymal Transition in Hepatocellular Carcinoma Cell Lines Daniel Huanga, Fiona Leeb, Lei Zhouc, Nur Halisah Jumatc, Wan Xin Tand, gangliosides enhance malignant properties of cells by d e f forming complexes with various membrane molecules. Madelaine Theardy , Ramanuj Dasgupta , Yock Young Dan a b In this study, we have examined contents of extracel- National University Health System, Singapore, Singapore; Genome Institute of Singapore, SIngapore, Singapore; cDepartment of Medicine, National lular vesicles (ECVs) secreted from ganglioside GD3- University of Singapore, SIngapore, Singapore; dDepartment of Medicine, expressing cancer cells to investigate roles of ganglio- National University of Singapore, singapore, Singapore; dGenome Institute of Singapore, singapore, Singapore; fDepartment of Medicine, National sides in the regulation of ECVs, leading to the induc- University Hospital, singapore, Singapore tion of cancer microenvironments and metastasis. Methods: GD3-positive cells as well as GD3-negative Introduction: Mesenchymal type hepatocellular carci- control cells were established by transfection of GD3 noma (HCC) with epithelial to mesenchymal transition synthase (ST8SIA1) cDNA into melanoma, glioma and (EMT) constitutes the most aggressive HCC. Our work small cell lung cancer (SCLC) cell lines. ECVs were has shown that exosomes from amniotic epithelial cells collected from culture supernatants by repeated ultra- (AECs), an intriguing cell from the epiblast which can centrifugation. Contents in ECVs were analysed by switch between epithelial and mesenchymal phenotype, Western blotting. Roles of lipid rafts were analysed by contain a myriad of growth and signalling factors that treating cells with 1 mM methyl β-cyclodextrin. regulate cell differentiation and has immunomodula- Results: In ECVs from GD3-positive melanoma cells, tory and antiproliferative properties. We hypothesize GD3 and ST8SIA1 mRNA were detected in TLC and that modulation of HCC differentiation into more dif- by RT-qPCR, respectively. In Western blotting, ferentiated epithelial phenotype via amniotic epithelial increased levels of integrin families were detected in cell exosomes will abrogate aggressive biology. ECVs from GD3-positive melanoma cells compared Methods: Size exclusion chromatography via the use of with those from GD3-negative cells. Similar increase qEV columns was used to separate AEC media into of integrins was also found in glioma and SCLC cells. exosome (less than 100 nm) and non-exosome frac- This was contrastive with integrin levels in cell lysates tions (more than 100 nm). Using the MACSPlex exo- from GD3-positive and – negative cells, showing some kit, we showed the abundant expression of CD63, almost equivalent levels of integrins regardless of CD9 and CD81 in these AEC exosomes. HUH-7, SK GD3 expression. Particularly in melanoma cells, levels Hep-1 and HLF cell lines were seeded into plates trea- of integrin α2, β1 and β2 showed marked increase in ted with exosomes, non-exosome fractions and control GD3-positive cell-derived ECVs. Treatment of GD3- daily. Proliferation and migration were assessed over positive melanoma cells by 1 mM methyl β- 72 h by Alamar blue, Glo and wound healing assays. JOURNAL OF EXTRACELLULAR VESICLES 337

Immunofluorescence for vimentin, E cadherin, KDR tested the significance of EV on EGFRTKI sensitivity of and EPCAM were performed to assess for epithelial CL1-5 (EGFR-wild) in co-culture system with PC9 to mesenchymal transition (EMT). (EGFR-mutant) pretreatment with or without Results: The proliferation of all three cell lines were GW4869. To further evaluate the role of EV in gefitinib significantly reduced in the exosome and non-exosome resistance, we harvested EV from PC9 cells and eval- arms compared with control, on both Alamar Blue uated their effect on gefitinib sensitivity of CL1-5 in stain and Glo assay (all p < 0.05). Wound healing was orthopedic animal model. We further compared the reduced significantly in the exosome arm vs. control in EV miRNAs from PC9 to those from CL1-5 and iden- Sk-Hep1 and HLF (p = 0.016 and 0.004, respectively), tified a panel of discriminative miRNAs. but not in HUH-7 (p = 0.156). Results: The CL1-5 uptake of PKH26 labelled exo- On immunofluorescence, there was upregulation of somes derived from PC9 cell can be recorded by the epithelial marker E cadherin in the exosome and time-lapse microscope. And the EGFRDel19 DNA non-exosome arms in SK-Hep1 and HUH7, but it was and specific protein can be detected in recipient wild- not expressed in the control arm. E cadherin was type EGFR cells by digital PCR and Western blotting upregulated in the cells treated with exosomes com- respectively. We demonstrated that wild-type EGFR pared to non-exosomes in SK-Hep1 and HUH7. There lung cancer cell became sensitive to EGFR-TKI after was downregulation of the mesenchymal marker co-culture with PC9 cell for 48 h and then subjected to vimentin in the HLF cells treated with exosomes and gefitinib for 72 h. However, the pretreatment with non-exosomes as compared to control. GW4869 for 48 h reversed the sensitivity to EGFR- Summary/Conclusion: Exosomes have the ability to TKI in co-culture system with PC9. In CL1-5 animal modulate HCC tumour biology, possibly by pushing model, neither gefitinib nor exosome treatment alone HCC cell lines into mesenchymal epithelial transition inhibited tumour growth compared to control group. to become less proliferative and motile. Only combination treatment with exosome and gefitinib delayed tumour growth. Some miRNA among the panel such as miR-200 family have been identified PS09.04 associated with resistance to EGFR-TKI Summary/Conclusion: Our study proposed that in Extracellular vesicles miRNA in mediating EGFR-TKI sensitivity in heterogeneous EGFR-mutant NSCLC, tumour cells heterogeneous EGFR-mutant NSCLC share biomolecules such as through local and systemic Chien-Chung Lina, Chin-You Wub, Wei-Yuan Changb, Yu-Ting Huangc, Mei-Ling Tsai and Wu-Chou Sud transfer of EVs, which may affect cell sensitivity. a Funding − Department of Internal Medicine, National Cheng Kung University : MOST-107-2314-B-006 069 - Hospital, Tainan,Taiwan, Tainan, Taiwan (Republic of China); bInstitute of Clinical Medicine, National Cheng Kung University College of Medicine and Hospital, Tainan, Taiwan; cDepartment of Seafood Science, National Kaohsiung University of Science and Technology, Kaohsiung Taiwan; PS09.05 d1Center of Applied Nanomedicine, 2Department of Internal Medicine, College of Medicine and Hospital, National Cheng Kung University, Tainan, Taiwan, Tainan, Taiwan (Republic of China) Senescent cells-derived extracellular vesicles repress tumour growth Introduction: Tumour heterogeneity has impacts on by transferring miR-127-3p and miR-134-5p. Megumi Okadaa, Kimiyoshi Yanoa, Shigeyuki Teranishib, Mariko Ikuoc and targeted drug resistance. At lung cancer, the discor- Hidetoshi Taharac dance rates of EGFR mutation implying tumour het- aHiroshima university, Hiroshima, Japan; bHiroshima university, Yokohama, erogeneity in metachronous and synchronous settings Japan; cHiroshima University, Hiroshima, Japan were 14.3% and 9.1%, respectively. Extracellular vesicles (EVs) serve as the transporter of bioactive mole- Introduction: The mechanism called cellular senescence cules between cells and become one of the major avoids tumourigenesis by arresting DNA-damaged cells mechanisms contributing intratumoural heterogeneity growth. The microRNAs are about 20-nt non-coding via transferring genetic information. Since most RNAs. MiRNAs complementary bind to target mRNA patients harbouring EGFR mutation showed excellent and suppress their translations and/or stabilities. Cellular response, we hypothesized that EVs mediate the cross- miRNAs play important roles in cellular senescence talk between EGFR mutant cell and EGFR wild type induction, and termed as senescence associated cell contributing the change of sensitivity of EGFR wild miRNAs. MicroRNAs are transferred by extracellular type cell to EGFR-TKI in heterogeneous NSCLC vesicles (EVs), and regulate phenotypes of recipient Methods: We used ultrafiltration (UF) method to iso- cells. However, the roles of EV-miRNAs secreted from late the EV. To mimic tumour heterogeneity, we next senescent cells are still unclear. In this study, we examined 338 ISEV2019 ABSTRACT BOOK whether EVs and EV-miRNAs secreted from senescent associated cells, and clinical biofluids using the classical cells regulate cancer cell’s activities. ultra-centrifugation (UC) method and alternative ultra- Methods: The normal fibroblast TIG-3 was continu- filtration (UF) method. The EVs could be uptake by ously cultured to establish replicative senescent cells. lung cancer cells and trigger oncogenic signals such as EVs were collected by ultracentrifugation. Particle Stat3 and Akt. Previously, we have shown that IL-6/ numbers and their size distributions were analysed by Stat3/tissue factor (TF)/VEGF pathway plays an impor- a tunable resistive pulse sensing instrument (qNano; tant role in lung cancer angiogenesis and metastasis. IZON Science). The expressions of exosomal marker Here, we showed that EVs from lung cancer samples proteins were analysed by western blot. MicroRNA carried high level of VEGF and TF and triggered vas- expression profiles were analysed by next-generation cular permeability changes in both in vitro and in vivo sequencing. MicroRNA and mRNA expressions were models. quantified by quantitative reverse transcription poly- Summary/Conclusion: Using the UC as well as the UF merase chain reaction. methods, we isolated EVs not only from culture super- Results: EV secretion was elaborated in replicative natants but also lung cancer associated clinical samples senescent TIG-3 cells. Senescent cell-derived EVs (S- and showed that the EVs triggered oncogenic signals in EVs) treatment repressed growth of breast cancer cell an autocrine/paracrine fashion and increased vascular line MDA-MB-231. The expression of miR-127-3p and permeability. These results may help the understanding miR-134-5p were enriched in S-EVs. Mir-127-3p and of potential roles of cancer derived extracellular vesicles miR-134-5p expressions were increased in S-EVs trea- in lung cancer metastasis and progression. ted cancer cells. Growth arrest activity of S-EVs was Funding: This work was financially supported by the inhibited by pretreatment of LNA-miRNA inhibitor for Centre of Applied Nanomedicine from The Featured miR-127-3p and miR-134-5p in MDA-MB-231. Areas Research Centre Program within the framework Summary/Conclusion: Senescence cell-derived extra- of the Higher Education Sprout Project by the Ministry cellular vesicles inhibited tumour growth by transfer- of Education in Taiwan, MOHW 106-TDU-B-211– ring miR-127-3p and miR-134-5p. 144004 and MOHW 105-TDU-B-211–133016 from the Ministry of Health and Welfare in Taiwan, MOST PS09.06 106–2314-B-006–040-MY2, and MOST 104-2314-B- 006-046-MY3 from the Ministry of Science and Technology in Taiwan. Potential roles of cancer derived extracellular vesicles in lung cancer metastasis and progression Wei-Lun Huanga and Wu-Chou Sub PS09.07 aCenter of Applied Nanomedicine, National Cheng Kung University, Tainan, Taiwan, Tainan, Taiwan (Republic of China); b1Center of Applied Nanomedicine, 2Department of Internal Medicine, College of Medicine and Whole transcriptome and miRNome profiling of plasma-derived Hospital, National Cheng Kung University, Tainan, Taiwan, Tainan, Taiwan extracellular vesicles cargo in haematological malignancies. (Republic of China) Maddalena Arigonia, Federica Riccardoa, Antonella Padellab, Luca Alessadric, Neha Kulkarnic, Martina Oliveroa, Ana Rodriguez-Vicented, Jesus d b a Introduction: Cells release different types of nano- Hernandez-Rivas , Giovanni Martinelli and Raffaele A. Calogero metre sized extracellular vesicles (EVs) of endosomal aUniversity of Torino, Torino, Italy; bUniversity of Bologna, Bologna, Italy; cUniversity of Torino, Torino, USA; dUniversity of Salamanca, Salamanca, and plasma membrane origin consisting into the extra- Spain cellular environment to mediate intercellular communication. EVs have been shown to play important roles Introduction: Extracellular vesicles (EVs) role in in many diseases including tumour. However, the role patients with haematological malignancies has not of EVs in lung cancer is still not fully understood. In been investigate as extensively as in solid cancers. In this study, we tried to find out the biological functions this study, the overall composition of RNA species of EVs in lung cancer. content of plasma derived EVs isolated from lymphoid Methods: EVs were isolated from culture supernatants, and myeloid malignancies (B-cell chronic lymphocytic serum, and malignant pleural effusion (MPE) using – CLL, acute myeloid – AML, acute lymphoid – ALL ultra-centrifugation (UC) and ultra-filtration (UF) leukemia, monoclonal B-cell lymphocytosis – MBL, and then evaluated by TEM, cryo-EM, and Nanosight. myelodisplastic syndrome – MDS, myeloproliferative The biological functions of EVs were analysed in both neoplasms – MPN) was investigated. in vitro cell line model and in vivo animal model. Methods:Participantsgavewritteninformedconsentin Results: EVs were isolated from culture supernatants accordance with the Declaration of Helsinki. EVs were from both cell lines and ex vivo cultured cancer isolated with ExoquickTM (System Biosciences) from JOURNAL OF EXTRACELLULAR VESICLES 339 plasma collected from patients and then analysed with an unknown mechanism of cancer spread. Tumour Nanosight. Whole transcriptome (WTS) and small RNA inflammation is the most critical processes of cancer sequencing were performed respectively on 123 and 256 onset, growth and metastasis. By inflammation, tumour samples. TruSeq stranded mRNA library preparation kit cells can establish an immunosuppressive microenviron- (Illumina) was used to detect coding and long non-coding ment to induce cancer progression. RNAs. Small RNA libraries were prepared using the Hypothesis: We hypothesize that the release of extracel- NebNext kit (NEB). Differential expression (DE) analysis lular vesicles (EVs) by tumour endothelial cells (TEC) of RNA species was done with EdgeR Bioconductor pack- induce reprogramming of immune cells as well as stromal age (ANOVA-like) and DESeq2 implemented in docker4- cells to create an immunosuppressive microenvironment seq package using as reference the expression values that favour tumour spread. We call this mechanism as detected in HD. non-metastatic contagious carcinogenesis. Results:TheanalysedEVshavesizerangingbetween80 Methods: EVs were collected from primary HNSCC- and 250 nm. WTS generated, on average, more than 10 derived endothelial cells (TEC-EVs) and were used for million mapped reads/samples. The RNA cargo was mainly stimulation of peripheral blood mononuclear cells composed of protein coding genes (95%), and the remain- (PBMC) and primary adipose mesenchymal stem cells ing fraction by lincRNAs and processed pseudogenes. 48 (ASCs). Regulation of ASC gene expression was inves- RNAs were detected as DE comparing diseases to HD. tigated by RNA sequencing and protein array. PBMC Among them 14 were mitochondrial pseudogenes over- stimulated with TEC-EVs were analysed by ELISA and expressed in all diseases with respect to HD and their FACS. The effect of ASCs or PBMC, treated with TEC- expression is higher in chronic versus acute diseases. EVs, we demonstrated on tumour cells using various in Small-RNA seq generated at least 100,000 mapped reads/ vitro assays, such as invasion, adhesion or proliferation. samples. Sets of miRNAs able discriminate each disease Results: We found and confirmed that TEC-EVs were from HD were also detected. Further, analysis to detect able to change ASC inflammatory gene expression disease-specific and disease-predictive signatures are in within 24–48 h. TEC-EVs were also able to enhance progress. the secretion of TGFb1 and IL-10 by PBMC and to Summary/Conclusion: This study gives an overview of increase T regulatory cell (Treg) expansion. TEC-EV plasma derived EVs RNAs cargo in haematological carries specific proteins and RNAs relevant for Treg diseases. The analysis of the common/unique RNA differentiation and immune suppression. ASCs and biotypes and the evaluation of their expression levels PBMC, treated with TEC-EVs, enhanced proliferation among samples, can guide the identification of patients’ of tumour cells, their adhesion, and invasion, therefore stratification markers. Moreover, this study provides a driving non-metastatic cancer spread. collection of EVs-associated RNAs/miRNAs to be used Summary/Conclusion: Conclusions. These data indi- as reference in different applications in liquid biopsy cate that TEC-EVs are a mechanism of non-metastatic research. contagious carcinogenesis that regulates tumour micro- Funding: FP7 NGS-PTL European grant environment and reprogrammes immune cells to sustain tumour growth and progression. Funding: NIH fund R21DE025398, Grants from the PS09.08 Associazione Italiana per la Ricerca sul Cancro (AIRC) projects IG 2015.16973 and IG 2015.17630

The mechanism of non-metastatic contagious carcinogenesis Tatiana Lopatinaa, Enrica Favarob, Benedetta Bussolatic, Ludmila Danilovad, Tiziana Martonee, Elana J Fertigd, Renato Romagnolif, Alexander V Favorovd, PS09.09 Maria Felice Brizzig, Giovanni Camussig and Daria A Gaykalovad aPostdoc, Turin, Italy; bDepartment of Medical Sciences, University of Turin, Turin, Italy, Torino, Italy; cDepartment of Molecular Biotechnology and Exosomes from mitotic slippage-induced senescent cells stimulate Health Sciences, University of Turin, Turin, Italy, Turin, Italy; dDepartment inflammatory response of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Johns Rekha Jakhar, Joycelyn Teo and Karen Crasta Hopkins University School of Medicine, Baltimore, MD, USA; eUniversity of Turin, Turin, Italy, Torino, Italy; fGeneral Surgery 2U, Liver Nanyang Technological University Singapore, Singapore, Singapore Transplantation Center, AOU Città della Salute e della Scienza di Torino, University of Turin, Turin, Italy; gDepartment of Medical Sciences, University of Turin, Turin, Italy Introduction: Microtubule-targeting drugs are the most-commonly used first-line chemotherapy. We pre- Introduction: viously showed nocadazole treatment can lead to para- Background: Head and neck squamous cell carcinoma crine pro-tumorigenic effects via mitotic slippage- (HNSCC) has a high recurrence and metastatic rate with induced senescence. Senescent cells exosomes, which 340 ISEV2019 ABSTRACT BOOK role in non-cell autonomous cell-cell communication. Methods: To analyse the cell types taking up EVs from The aim of this study was to decripher effect of exo- tumour cells, we created breast cancer cell lines secreting somes released from senescent-inflammatory breast can- fluorescent EVs, with CD63-GFP fusion protein or with cer cells post-slippage on recipient normal breast cells. surface mCherry. The cells were implanted in the mouse Methods: MDA-MB-231 and MCF-10A breast cancer mammary fat pad or tail vein and the uptake of EVs were cell lines treated with Noc (100 ng/µl) for 72 h. analysed in different cell populations of the tumours and Conditioned media (CM) was prepared after Noc and the lungs using FACS. We then purified EVs from breast DMSO treatment by incubating cells in growth media cancer cells using ultracentrifugation and profiled containing exosome-depleted FBS for 72 h. CM was miRNAs using sequencing. The abundance of miR-125b then collected and centrifuged at 500 ×g 10 min, was validated in size exclusion chromatography -purified 2000 ×g 30 min and 15,000 ×g 30 min at 4°C to remove EVs. The function of miR-125b was analysed by knock- cells and large debris. Supernatant was filtered, exo- down or overexpression experiments. somes pelleted at 120,000 ×g, 2 h, 4°C, washed with Results: We found that fluorescent EVs from tumour PBS, centrifugation at 100,000 ×g,1 h, 4°C. Exosomes cells are taken up most robustly by fibroblasts in the were dissolved in PBS for whole exosome experiments tumours or the metastatic lungs. Our RNA sequencing or processed for total RNA, miRNA and protein isola- data revealed that miR-125b is one of the most abun- tion for microRNA profiling, RNA-seq and mass spec. dant microRNAs in the EVs from mouse 4T1 and Results:Mitotic-slippage-inducedsenescent(MIS)cells 4TO7 cells. Treatment with 4T1 EVs promotes fibro- activate NFκBpathwayandincreaseexosomeproduction, blast activation in isogenic 4TO7 tumours. This is assessed via immunoblots of cytoplasmic and nuclear pro- rescued by knocking down miR-125b in 4T1 EVs; tein fraction, and IF for p65 localization. We characterized therefore, miR-125b transfer by EVs is responsible for exosomal proteins using TMT labelling and detected sig- the fibroblast activation. Similarly, we found that miR- nificant upregulation of caveolin-1 in Noc treated exo- 125b is abundant in EVs from human breast cancer somes. Exosomal microRNA also showed significant cells. The uptake of EVs from human breast cancer upregulation of inflammatory pathway-related genes cells increases cellular levels of miR-125b in the resi- upon Noc-treatment. Exosomes were transferred from dent fibroblasts hence upregulates several markers of MDA-MB-231 cells after Noc treatment to the recipient cancer-associated fibroblasts in vivo. miR-125b over- MCF-10A cells. Uptake of MIS-derived exosomes resulted expression also upregulates alpha-SMA and promotes in transfer of NFκBresponseinrecipientcells. invasion of isolated fibroblasts in vitro. We further Summary/Conclusion: Noc treatment leads to MIS identified Tp53 and Tp53inp1 as the targets of miR- and inflammation in MDA-MB-231 cells. Exosomes 125b that are responsible for the phenotype. released from senescent-inflammatory breast cancer Summary/Conclusion: In summary, our study shows cells contribute to transfer of soluble factors which that the delivery of miR-125b in EVs from breast activate inflammatory pathway in recipient cells. cancer cells to resident fibroblasts promotes the devel- Hence, senescence-induced exosomes can transfer opment of cancer-associated fibroblasts in the tumour therapy-induced immune signalling via non-cell auton- microenvironment. omous mechanisms. Funding: This study is supported by City University of Funding: National Research Foundation Fellowship Hong Kong (grant 9610343, 9667133 and 7200475), the Singapore MOE AcRF Tier 2015-T1-002-046-01. Hong Kong Health and Medical Research Fund (03141186), the Hong Kong Research Grants Council PS09.10 (21106616) and the National Natural Science Foundation of China (81602514 and 81773246).

Extracellular vesicles from breast cancer cells deliver microRNA-125b to activate cancer-associated fibroblasts PS09.11 Minh T. Lea, Luyen Vua, Boya Penga and Judy Liebermanb aCity University of Hong Kong, Kowloon, Hong Kong; bBoston Children’s Hospital, Boston, USA Carnitine palmitoyltransferase 1 regulates proliferation of prostate cancer cells under hypoxia via extracellular vesicles-mediated Introduction: Extracellular vesicles (EVs) are often removal of oxidized proteins Gagan Deep, Leslimar Rios-Colon, Gati Panigrahi, Yixin Su, Kiran Kumar released by tumour cells for intercellular communica- Solingapuram Sai, Isabel Schlaepfer, Jingyun Lee, Cristina Furdui and Deepak tion with other cell types in the tumour niches. Kumar However, it is unclear which cell type is the most Wake Forest Baptist Medical Center, Winston Salem, USA frequent recipient of tumour EVs in vivo. JOURNAL OF EXTRACELLULAR VESICLES 341

Introduction: Prostate cancer (PCa) is the most com- cells showed increased lipid uptake and β-oxidation mon cancer in men with 164,690 new cases and 29,430 under hypoxia. CPT1 is the main regulator of β-oxida- deaths estimated in the USA in 2018. Studies have tion, and CPT1 knock-down in PCa cells significantly shown that hypoxia determines PCa aggressiveness, reduced the viability, clonogenicity and stemness under and poor outcome. We have recently reported that hypoxia; while CPT1 overexpression increased the PCa increased extracellular vesicle (EV) secretion under cells proliferation, clonogenicity and stemness. Both hypoxia promotes survival of aggressive PCa cells; hypoxia and β-oxidation are known to promote oxida- here, we focused on underlying molecular mechan- tive stress, and we also observed high ROS levels in ism/s. PCa cells under hypoxia. We also observed higher Methods: PCa cells were cultured under normoxia amount of oxidized proteins in hypoxic PCa cells, (~21% O2) or hypoxia (1% O2), and EVs (exosomes) measured by BP1 labelling and immunoblotting. MS isolated from the conditioned media by ultracentrifu- analyses identified the signature of oxidized proteins gation. β-oxidation was measured using a novel PET that were altered in PCa cells under hypoxia. tracer (18[F]Fluoro-4-thia-oleate), reactive oxygen spe- Interestingly, PCa cells proliferating under hypoxia cies (ROS) levels measured by DCF-DA staining, and secreted increased concentration of EVs, loaded with oxidized proteins in PCa cells and EVs were measured high amount of oxidized proteins; while treatment with using an oxidative stress sensor (BP1:biotin-1,3-cyclo- either exosomes biogenesis inhibitors (GW4869 and pentanedione) followed by immunoblotting and mass DMA) or antioxidants (N-acetylcysteine and ascorbic spectrometry (MS). CPT1 knock-down and overex- acid) strongly reduced the growth of PCa cells under pressing PCa cells were generated using lentiviral hypoxia. particles. Summary/Conclusion:PCacellscouldproliferateunder Results: We identified PCa cell lines (E006AA-hT, hypoxia through CPT1-mediated increased β-oxidation MDA PCa 2b, 22Rv1 and WFCB17) with higher pro- and via managing high intracellular oxidative stress liferation under hypoxia compared to normoxia. These through EV-mediated removal of oxidized proteins. 342 ISEV2019 ABSTRACT BOOK

PS10: EV Cancer Pathogenesis II Chairs: Hiroshi Ageta; Ming Jer Tang Location: Level 3, Hall A 15:00–16:00 bone metastasis compared to non-metastatic PCa patients. PS10.01 Summary/Conclusion: PCa-EVs synergistically activate osteoclastogenesis with RANKL. PCa-EVs will be the novel diagnostic and therapeutic target for BM in Prostate cancer cells promote bone metastasis through extracellular vesicles PCa, leading the great improvement of quality of life in Fumihiko Urabea, Nobuyoshi Kosakab, Yusuke Yamamotoc, Takahiro PCa patients. Kimurad, Shin Egawad and Takahiro Ochiyab aDivision of Molecular and Cellular Medicine, National Cancer Center Research Institute, Tokyo, Japan; bDepartment of Molecular and Cellular PS10.02 Medicine, Institute of Medical Science, Tokyo Medical University, Shinjyuku-ku, Japan; cDivision of Molecular and Cellular Medicine, d National Cancer Center Research Institute, Tokyo, USA; Department of Novel Exosomal miRNAs-891-5p as an Indicator of Chemoresistance in Urology, The Jikei University School of Medicine, Tokyo, Japan Ovarian Cancer Mona G. Alharbia, Carlos Salomona, Dominic Guanzona, Andrew Laib, Alexis c b b d e Introduction: Bone metastasis (BM) is one of the Salas , Carlos Palma , Katherin Scholz-Romero , Yaowu He , Felipe Zuniga , Lewis Perrinf and John Hooperf major concerns that causes skeletal-related events and aExosome Biology Laboratory, Centre for Clinical Diagnostics, University of increases mortality in prostate cancer (PCa) patients. Queensland Centre for Clinical Research, Royal Brisbane and Women’s Vicious cycle paradigm has been proposed to describe Hospital, The University of Queensland, Brisbane, Australia; bExosome Biology Laboratory, Centre for Clinical Diagnostics, University of how PCa cells educate osteoblasts and osteoclasts Queensland Centre for Clinical Research, Royal Brisbane and Women’s (OCs) to benefit the survival and growth of the PCa Hospital, The University of Queensland, Brisbane, Australia; cFaculty of cells in the metastatic site. However, the underlying Biological Science, Department of Pharmacology, Universidad de Concepción, Concepción, Chile; dMater Research Institute-University of mechanisms of BM in PCa remain obscure. Here, we Queensland, Translational Research Institute, Woolloongabba, Australia; show that extracellular vesicles (EVs) from PCa cells eDepartment of Clinical Biochemistry and Immunology, Faculty of Pharmacy, University of Concepción, Concepción, Chile; fMater Health (PCa-EVs) are involved in the vicious cycle, and con- Services, South Brisbane, Australia tribute to the progression of BM. Methods: PCa-EVs and normal prostatic epithelial cell Introduction: Ovarian cancer patients usually have a (NPE)-derived EVs (NPE-EVs) were isolated by ultra- poor prognosis and low five year’s survival rate because centrifugation and evaluated their effect on OC differ- it predominantly presents at late stages of the disease. entiation by Tartrate-resistant acid phosphatase New approaches are required to develop more effective (TRAP) stain. PCa-EVs and NPE-EVs were analyzed early detection strategies and real-time response mon- using LC-MS/MS to identify candidate proteins which itoring to the available treatments. Thus, this study promote OC differentiation. Then, a small-scale aimed to identify an exosomal signature which can be screening was conducted using siRNA in PCa cells to used to determine a patient’s response to the determine proteins essential for osteoclastogenesis. The chemotherapy. expression level of the specific molecule on EVs was Methods: A panel of ovarian cancer cell lines were evaluated in clinical samples. used in this study. Cell migration, proliferation and Results: We found that PCa-EVs promoted OC differ- apoptosis in response to different concentrations of entiation in the presence of RANKL. In addition, RNA carboplatin (0-100 µM) were evaluated using a real- sequence analyses confirmed the drastic change of gene time monitoring system (Incucyte). The miRNA profile expression essential for osteoclastogenesis in OC pre- was determined using TruSeq® SmallRNA Library cursors. Moreover, we found a specific molecule on (Illumina). Hierarchical clustering and principal com- EVs which promote OC differentiation. Elimination ponent analysis (PCA) were used for multi-omics ana- of the molecule on PCa-EVs led to the attenuation of lyses. Subsequently, candidate miRNAs inducing OC differentiation. In addition, overexpression of this chemoresistance was confirmed in cells and their exo- molecule promoted OC differentiation. Finally, we somes. Candidate miRNAs (mimic) were incubated on found the molecule on EVs was specifically detected sensitive ovarian cancer cells (CAOV-3) and cells in plasma-derived exosomes from PCa patients with response to carboplatin was determined. Finally, a set JOURNAL OF EXTRACELLULAR VESICLES 343 of miRNAs were validated in circulating exosomes expression by MTT assay, trans-well assay and flow- obtained from a small cohort of patients who experi- cytometry. Cells were inoculated into the mice subcu- ence cancer relapse. taneously or via tail vein, then tumour and metastatic Results: The migration capacity of these cells were tissues were observed by H&E stain. Cells from tumour associated with cell apoptosis in response to carbopla- sites were cultured then examined about proliferation tin with EC50 (concentration of a drug that gives half- and invasion ability. Exosomes were isolated from cell maximal response) of 12.1 ± 2.6, 9.4 ± 2.2, 4.4 ± 1.5, culture medium by differential centrifugation, and used 4.1 ± 1.6, 4.0 ± 1.9, 2.8 ± 0.9, 1.5 ± 0.6, 0.9 ± 0.2 and for Western blotting. Cells treated by exosomes were 0.7 ± 0.1 for HEY, SKOV-3, OVACR-429, OV90, analysed for malignant properties as described above. OVTOKO, OVCAR-420, OVCAR-3, CAOV-3 and Results: In proliferation, migration, and invasion assay, TOVII-2D, respectively. In contrast, the proliferation low metastatic subline showed lower proliferation, of these cells was inversely correlated (p < 0.005) with migration, invasion activity than high metastatic sub- their migration and EC50. Based on migration, prolif- lines. In flow-cytometry, high metastatic sublines eration and response to carboplatin PCA separated into showed decreased GM1 and GD1a expression levels four distinct groups. Using miRNA approach, we suc- compared with low metastatic subline. To examine cessfully identified miR-21-5p, 3p and miR-891-5p that metastatic ability, the cells were inoculated into mice. were enriched in resistant cells and their exosomes. After 2–4 weeks, invasive- and metastatic- foci to dis- Transfected CAOV-3 cells (sensitive cells) with tant tissues such as thigh muscle and lung were miRNAs showed a reduction in cells sensitivity to observed. To examine effects of exosomes on culture carboplatin. Finally, we were able to confirm the cells, cells were treated with isolated exosomes. As a expression of these miRNAs in plasma from ovarian result, low metastatic subline treated with high meta- cancer patients. static cell-derived exosomes showed increased prolif- Summary/Conclusion: We suggest that exosomal eration, migration, and invasion activity, and increased cargo may be used as prognostic biomarkers to moni- phosphorylation of intracellular signalling molecules tor the response to treatments in patients with ovarian such as paxillin and Erk1/2. In turn, high metastatic cancer. subline treated with low metastatic cell-derived exosomes showed reduced proliferation, migration, and invasion activity, and phosphorylation of intracellular PS10.03 signalling molecules. Summary/Conclusion: High metastatic subline- derived exosomes enhanced malignant properties in Functional analysis of exosomes in cancer metastasis Yoshiki Kodamaa, Yuhsuke Ohmib, Zhang Qingc, Satoko Yamamotod, Keiko low metastatic sublines. Furukawad and Koichi Furukawad aDepartment of Biomedical Sciences, College of Life and Health Sciences, Chubu University, Kasugai, Japan; bDepartment of Biochemical Sciences, PS10.04 College of Life and Health Sciences, Chubu University, Kasugai, Japan; cDepartment of Biochemistry II, Nagoya University Graduate School of Medicine, Tokyo, Japan; dKanazawa Medical University, Uchinada, Japan; eDepartment of Biomedical Sciences, College of Life and Health Sciences, Profiling of circulating exosomal content across epithelial ovarian Chubu University, Nagoya, Japan cancer and the role of exosomes in tumour progression Shayna Sharmaa, Andrew Laia, Dominic Guanzonb, Terry Morganc, Lewis Introduction: The development of metastasis is a cause Perrind, John Hooperd and Carlos Salomonb of death in many human cancers. Mechanisms for the aExosome Biology Laboratory, Centre for Clinical Diagnostics, University of acquisition of metastatic potential remain unknown. Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane, Australia; bExosome Recently, it has been reported that exosomes are a Biology Laboratory, Centre for Clinical Diagnostics, University of trigger of cancer metastasis. Exosomes are small vesi- Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane, Australia; cDepartment cles that are secreted from cells and have been found to of Pathology and Obstetrics, Oregon Health and Science University, Portland, mediate signal transduction between neighbouring or OR, USA; dMater Health Services, Brisbane, Australia distant cells. They have the tendency to specifically Introduction: A significant proportion of patients with interact with target cells. In the future, it may be epithelial ovarian cancer (EOC) often present with possible that exosomes can be used as biomarkers to advanced stage disease, when treatment options are predict the metastatic destination. limited. Therefore, it is essential to gain a better under- Methods: Established mouse Lewis lung cancer cells standing of the tumour microenvironment to identify (low or high metastatic sublines) were examined potential therapeutic targets. We profiled the exosomal about proliferation, migration, invasion and gaglioside content (miRNAs and proteins) of patients with EOC 344 ISEV2019 ABSTRACT BOOK and examined the effect of these exosomes on cells Introduction: Acute Myelogenous Leukaemia (AML) is within the tumour microenvironment. an aggressive cancer originating from abnormal white Methods: A cohort of 127 patients were included in blood cells of the bone marrow (BM). AML modifies this study. Exosomes were isolated and characterized the BM into a pro-leukaemic niche in part through the from plasma obtained at different stages of EOC. A release of extracellular vesicles (EVs). We previously small RNA library was prepared, and the expression of demonstrated that AML EVs decrease mature blood cell specific miRNAs was validated using RT-qPCR. The production, and traffic to stromal cells to induce osteo- protein profile was determined using Mass genesis. We hypothesized that AML cells utilize EVs to Spectrometry (MS/MS) and SWATH Analysis. transmit regulatory factors to recipient BM cells to Exosomal proteins and miRNAs were subjected to lin- change the cellular composition of the BM and support ear mixed modelling analysis using the lme4 package in cancer progression. Our studies confirmed that AML EVs “R”. Fibroblast cells were incubated with patient- contain bone morphogenic protein (BMPs) – historically derived exosomes and monitored using the IncuCyte though to be secreted growth factors – involved in for- (TM), a live-cell imaging system. Cell proliferation and mation of bone and maintenance of stem cells. migration was determined over the course of the Methods: To identify the association of BMPs with experiment and RNA and proteins were extracted AML EVs, we used both in vitro and in vivo xenograft after 48 hours. The expression of nine specific models, and a combination of ELISA, flow cytometry, miRNAs was confirmed using RT-qPCR and the pro- and super resolution microscopy. tein profile determined using MS/MS. Results: AML cells explanted from the BM display Results: Exosomal miRNAs and proteins demonstrated marked ER-stress in comparison to in vitro cultured cell differential expression with advancing cancer progres- types as an adaptive response to the tumour microenvir- sion, following at least three distinct patterns of change: onment. In AML blasts, the expression of BMP-2,4,6,7 increasing, constant or decreasing rate. Ingenuity mRNA strongly correlated with the activation of the Pathway Analysis analysis revealed that the exosomal unfolded protein response pathway (which acts to miti- content was associated mainly with cell–cell communica- gate ER-stress). Inducing ER-stress in AML cells in vitro tion and cell migration. Functional analysis showed that resulted in both an increase in BMP protein as well as exosomes increase fibroblast migration and proliferation total EVs produced. EVs released from these cells con- in association with EOC progression (i.e. Stages I to IV). tained ~3-fold more BMP-2,6 over non-stressed cells, MS/MS identified 115 proteins differentially expressed whereas the level of free-BMP-2,6 in supernatant between early stage and advanced stage-exosome treated remained unchanged. Exposing these purified EVs to cells. A comparison between control cells (no treatment) BM stromal cells induced osteogenic differentiation and and treated cells showed a difference in the expression of apoptosis. Additionally, in ER-stressed AML cells, BMP-2 126 proteins, with tumour suppressor, Paired Box 1 and localizes into CD63+ intracytoplasmic vesicles – indica- lysosomal trafficking protein, VPS41 expression, signifi- tive of pre-exosomal multivesicular bodies – further con- cantly lower in the treated cells (p <0.05). firming the EV-BMP association. Thus far, AML cells Summary/Conclusion: We propose that exosomes have been found to release EVs that contain BMP-2 and present in the circulation of EOC patients transfer −6, while additional BMP types remain to be tested. oncogenic cargo to cells present in the tumour micro- Summary/Conclusion: Since we have shown that AML environment to promote cancer progression. EVs rapidly accumulate within stromal cells in vitro and in vivo, altering cellular phenotypes, we propose that EVs concentrate and target functional BMPs directly to these PS10.05 cells to create a pro-leukaemic environment. EV trafficking of BMPs is a novel signalling mechanism that may explain phenotypic changes seen in leukaemic BM, and

Extracellular vesicle-mediated transmission of bone morphogenic their role in leukaemic progression merits further study. proteins in Acute Myelogenous Leukaemia John Butlera, Ben Doronb, Sherif Abdelhamedc, Peter Kurred and Daniel Markse PS10.06 aMedical Scientist Training Program, Oregon Health & Science University, Portland, USA; bHuman Biology Division, Fred Hutch Center for Cancer Research, Seattle, USA; cKnight Cancer Institute, Oregon Health & Science University, Portland, USA; dChildren’s Hospital of Philadelphia, Philadelphia, Exosomes derived from differentially invasive ovarian cancer cells USA; eDepartment of Pediatrics, Oregon Health & Science University, modulate tumour growth and metastasis in vivo Portland, USA Mona G. Alharbia, Carlos Salomona, Andrew Laib, Yaowu Hec, Felipe Zunigad and John Hoopere JOURNAL OF EXTRACELLULAR VESICLES 345 aExosome Biology Laboratory, Centre for Clinical Diagnostics, University of PS10.07 Queensland Centre for Clinical Research, Royal Brisbane and Women’s Hospital, The University of Queensland, Brisbane, Australia; bExosome Biology Laboratory, Centre for Clinical Diagnostics, University of Cancer-associated fibroblast accelerate cancer metastasis through Queensland Centre for Clinical Research, Royal Brisbane and Women’s exosomes Hospital, The University of Queensland, Australia; 3Mater Research Kim Kimina, Yeon Ju Hunb, Sohn Yeh Jooa, Ruri Leea, Yoo Hye Jua

Institute-University of Queensland, Translational Research Institute, a b Brisbane, Australia; dDepartment of Clinical Biochemistry and University of brain education, Cheon-an, Republic of Korea; University of Immunology, Faculty of Pharmacy, University of Concepción, Concepción, British Columbia, Cheonan, Republic of Korea Chile, Concepción, Chile; eMater Health Services, South Brisbane, Brisbane, Australia Introduction: Exosomes are known to be important mediators between the primary and secondary sites for Introduction:Exosomeshavebeenlikenedtoa tumour progression and metastasis with their micro- “fingerprint” of their originating cells, as they con- environment. Exosomes released by cancer cells induce tain several bioactive molecules which can be deliv- the cancer-associated fibroblasts, which create a niche ered to the target cells at either local or distant to development cancer progression, making it more locations. The delivery of these biomolecules can permissive cancer metastasis. cause changes in gene expression and signalling in Methods: We have developed 3D tumour microenvir- the target cells. Hence, in this study, we aimed to onment model mimicking the interactions between explore the impact of exosomes on tumour growth/ cells and ECM by injecting of collagen gel for ECM metastasis in a xenograft model and assess the to, and then, the formation of monolayer of cells for changes in the proteomic content of tumour cells blood vessel. The exosomes were isolated from three and exosomes from mice. different malignant cancer cells (i.e. from A431, Methods: Exosomes were isolated from highly invasive B16BL6 and MDAMB231), and delivered into the SKOV-3 (exo-SKOV-3) and less invasive OVCAR-3 channel in microfluidic device, then created a unidir- (exo-OVCAR-3) ovarian cancer cell lines. Exosomes ectional flow by the difference in pressure gradient. We (10 ug/mL) were injected into a xenograft model profile mRNAs of normal cell, CAFs with and without (n = 8/ group), twice a week for 6 weeks and the cancer cells in genetic analysis. tumour growth was monitored using In Vivo Results: We confirmed that various cancer-derived exo- Bioluminescence Imaging (IVIS). Tissue and circulat- somes differentiated CAFs, facilitating metastasis in reca- ing exosomes obtained from the mice were subjected to pitulating the 3D tumour microenvironment in real time. a quantitative mass spectrometry approach SWATH The three difference CAFs have commonly enriched MS/MS, followed by ingenuity pathway analysis genes related to extracellular region for cellular response, (IPA). Finally, we compared between the protein and fibrinolysis to degrade ECM for biological process in expressions profiles from the circulating exosomes genetic analysis. The migrated cancer cells followed by and metastatic nodes or tumour growth. CAFs showed different specific molecular mechanisms, Results: IVIS imaging indicated that the tumour bur- suggesting that the melanoma cells had MAPK related den in mice injected with exo-OVCAR-3 was higher signalling, the squamous cancer cells had cell adhesion than in mice injected with exo-SKOV-3 (p = 0.004). related signalling, and the breast cancer cells had inflam- However, mice injected with exo-SKOV-3 had more mation, cytokine related signalling, which may contribute tumour nodules throughout the peritoneal cavity. to the invasive progression of cancer. Proteomic analysis of the cancer tissue obtained from Summary/Conclusion: The cancer-derived exosomes mice injected with exo-SKOV-3 compared to exo- play an important role in modulating the tumour OVCAR-3 identified the differential expression microenvironment, and induce CAFs to promote (p < 0.05) of 105 proteins. Interestingly, the protein metastasis. The 3D microfluidic model showed the profile in tumour tissue obtained from mice injected relationship between the CAFs and cancer cells inva- with exo-SKOV-3 was associated with the Wnt cano- sion in real time in physiological manner and specific nical pathway (β-catenin). Moreover, we found 36 pro- mechanism in a genetic manner. teins with differential expression in exosomes from Funding: This work was supported by the Basic mice treated with exo-SKOV-3, (p < 0.04). Finally, we Science Research Program through the National identified 29 exosomal proteins that are highly asso- Research Foundation of Korea (NRF) funded by the ciated with cancer metastasis and 21 proteins are asso- ministry of Education, Science and Technology (NRF- ciated with tumour growth. 2016R1C1B2013345) and Samsung Research Funding Summary/Conclusion: These observations suggest that Center of Samsung Electronics under Project Number exosomal signalling plays an important role in ovarian SRFC-IT1701-00 cancer metastasis. 346 ISEV2019 ABSTRACT BOOK

PS10.09 Introduction:Extracellularvesicles(EV)playakey role in cancer development and metastasis by influencing the behaviour of the primary tumour and by The miR-27b in breast cancer exosomes aiding the establishment of a pre-metastatic niche in Wen-Hung Kuo distant organs. This process is due to the EV- National Taiwan University Hospital, Taipei, Taiwan (Republic of China) mediated functional transfer of biologically active molecules including microRNA (miRNA). miR-1227 Introduction: miR-27b has been shown to possess is a poorly characterized miRNA that is enriched in anti-tumour growth and anti-drug resistance activities EV secreted by prostate cancer (PC) cells in compar- in associated with breast cancer progression. Loss of ison to non-tumourigenic prostate epithelial cells. miR-27b existed in the cancer cells can lead to the However, the role of miR-1227 in cancer is poorly promotion of cancer cells. However, the precise understood. Our objective is to determine the role of mechanism of miR-27b loss is unclear, in particular, miR-1227 in PC. involving in tumour microenvironments and Methods:RNAsequencingfrommiR-1227stably metastasis. expressing PC cells, RISCTRAP Methods: Here, we attempted to elucidate tumour- Immunoprecipitation of miR-1227 bound mRNA, derived exosomes bearing miR-27b in regulating and five different in silico miRNA target prediction tumour microenvironments via modulation of cancer methods were used to identify putative miR-1227 stem cell growth and migration. targets. Exosomes and large oncosomes (LO) were Results: The expression level of miR-27b was decreased isolated by differential ultracentrifugation followed in tumour-derived exosomes in coincidence with pro- by density gradient purification. Atomic force micro- gression of breast cancer, suggesting its negative role in scopy and TRPS were used to quantify exosomes and tumour progression via modulating tumour microen- LO secreted by PC cells stably expressing miR-1227 vironments. Consistently, miR-27b showed a dimin- or vector control. ished trend in malignant breast cancer cell lines Results: A comparative analysis between different EV compared with the control cell line. To further examine subtypes indicates that miR-1227 is enriched in LO, a the impact of miR27b in tumour microenviroments, we class of EV that are secreted by highly invasive and found that the formation of tumour associated fibro- metastatic amoeboid-migrating cells. LO carry more blasts (TAFs) and tumour associated macrophages RNA than the more widely studied exosomes indicat- (TAMs) were impacted by miR-27+ exosomes. ing that LO may be a more robust source of EV- Moreover, the increases in tumour migration and inva- encapsulated miRNA. Gene ontology analysis from sion were observed by miR-27b+ exosomes treated miR-1227 targets identified by RNA sequencing from fibroblasts. miR-1227 stably expressing PC cells, RISCTRAP Summary/Conclusion: Therefore, we illustrated a sim- Immunoprecipitation of miR-1227 bound mRNA, and ple mechanism of miR-27b attending in the progres- in silico miRNA target prediction highlighted several sion of breast cancer. In the future, the manipulating genes related to EV secretion. miR-1227 alters the the existence of miR-27b may be a novel strategy for localization of exosome and LO markers in multiple breast cancer therapeutic. cancer cell lines, and induces the shedding of LO while inhibiting the shedding of exosomes. Furthermore, miR-1227 induces the migration of poorly migratory PS10.10=OWP1.01 cancer cells and increases the expression of tumour supportive cytokines. Summary/conclusion: Together these data hint that Mir-1227 alters extracellular vesicle shedding Andrew R. China, Minyung Kimb, Valentina R. Minciacchic, Tatyana miR-1227 may promote prostate cancer progression Vagnerb, Javier Mariscalb, Cristiana Spinellia, Mandana Zandianb, Paolo through several mechanisms including alteration of Gandellinid, Nadia Zaffaronid, Shivani Sharmae, Sungyong Youb and Dolores Di Vizioa EV shedding. Funding: 2017–2022 R01CA218526 Chesapeake urol- aCedars Sinai Medical Center, West Hollywood, USA; bCedars Sinai Medical Center, Los Angeles, USA; cCedars Sinai Medical Center, Frankfurt, ogy associates Sanford J. Siegel, MD prostate cancer Germany; dFondazione IRCCS Istituto Nazionale Tumori, Milan, USA; research scholarship Luke wu-jei chang discovery fund e University of California, Los Angeles, Los Angeles, USA PI dod PCRP award PC150836 JOURNAL OF EXTRACELLULAR VESICLES 347

PS11: Stem Cells Chairs: Kyoko Hida; Noriko Watanbe Location: Level 3, Hall A 15:00–16:00 PDLSCs might be a potential therapeutic target for PS11.02 the inflammation in the periodontium. Funding:ThisworkwassupportedbytheBasicScience Research Program through the National Research Bacterial endotoxin-preconditioned periodontal ligament stem cells Foundation of Korea (NRF-2017R1A2B4002405). induce M1 polarization of macrophage through extracellular vesicles Hyejong Kanga, Myung-Ju Leeb, Sang Jung Parkb and Myung-Shin Leeb aDankook University Sejong Dental Hospital, Sejong, Republic of Korea; PS11.03 bEulji University School of Medicine, Daejeon, Republic of Korea

Hypoxia enhances the angiogenic properties of adipose stem cell- Introduction: Periodontitis is a common disease that derived extracellular vesicles in culture characterized by chronic inflammation and tissue Jolene Phelps, David Hart, Alim Mitha, Neil Duncan and Arindom Sen destruction of gums. To resist pathogenic microbes, University of Calgary, Calgary, Canada gingival epithelial cells and inflammatory cells produce various pro-inflammatory cytokines, chemokines and Introduction: The widely recognized benefits of adi- enzymes. Human periodontal ligament stem cells pose stem cells (ASCs) in regenerative medicine have at (PDLSCs) derived from mature periodontal ligaments least in part been attributed to the extracellular vesicles have stem cell properties similar to mesenchymal stem (EVs) that they secrete, which are known to deliver cells. PDLSCs possess not only differentiation potential bioactive cargo to target cells. EVs can be isolated from to other tissues but also immunomodulatory abilities. spent medium following ASC population expansion in Therefore, PDLSCs might be a vital role in the mod- culture. It has been shown that manipulating the cul- ulation of immune response. In this study, we investi- ture environment may impact the biological character- gated the effect of PDLSCs on the polarization of istics of EVs. Here we examined if the angiogenic macrophages. properties of ASC-derived EVs are impacted by culture Methods: The polarization of macrophage cell line, oxygen level, and tested their effect on cerebral micro- THP-1 cells, was investigated on the conditioned vascular endothelial cells (CMECs). media or extracellular vesicles (EVs) from PDLSCs Methods: Ethically obtained human ASCs were cul- that were pretreated with or without lipopolysacchar- tured for 3 days in PPRF-msc6 serum-free medium ide. EVs were isolated from the conditioned media of under 3% (hypoxic) or 21% (normoxic) headspace O2 PDLSCs by differential centrifugation and character- conditions. EVs were isolated from media via ultracen- ized. The functions of EVs on macrophage polarization trifugation and evaluated for concentration (nanopar- and underlying mechanisms were analysed by RT- ticle tracking analysis), and angiogenic factor content qPCR and ELISA, (Luminex technology). Functional assays (proliferation, Results: While the conditioned media from PDLSCs in tube formation) were carried out by culturing human normal culture condition did not affect the polarization CMECs in endothelial basal medium (EBM-2) supple- of macrophage, lipopolysaccharide (LPS)-precondi- mented with 2 different concentrations of ASC derived tioned PDLSCs induce significant changes in M1 polar- EVs. CMEC proliferation in tissue culture flasks was ization of macrophages. Extracellular vesicles (EVs) quantified using a Cyquant Proliferation Kit. Tube isolated from the conditioned media of LPS- precondi- formation on Matrigel coated plates was quantified tioned PDLSCs by centrifugal filter device (MWCO using ImageJ software. RT-qPCR was used to measure 100 kD) or differential centrifugation methods showed angiogenic gene expression levels in ASCs and CMECs strong M1 polarization effect of macrophages. for each test condition. All studies and analyses were Additionally, M1 polarization was abolished by carried out in at least triplicate. DNase I treatment on EVs. Results: Hypoxia upregulated VEGF expression in Summary/Conclusion: Our results demonstrated that ASCs 4.47 ± 0.24 fold (p < 0.0015) compared to nor- LPS-stimulated PDLSCs induce M1 polarization of moxia and induced higher EV secretion. EVs obtained macrophage through EVs, suggesting EVs from from hypoxic ASC cultures contained higher 348 ISEV2019 ABSTRACT BOOK concentrations of angiogenic proteins VEGF, HGF, Methods: First, a Human Plasma Lysate (HPL) is pro- PLGF and follistatin; and reduced concentrations of duced from which the EV are removed by tangential- bFGF, endoglin, IL-6 and IL-8. The presence of ASC- flow-filtration resulting in an EV-FREE HPL (EV derived EVs enhanced angiogenesis of CMEC cultures depletion > 99%). Second, cells (grown in HPL-supple- in a dose dependent manner as measured via enhanced mented medium) are rinsed and placed in medium proliferation, tube formation and upregulation of added with EV-FREE HPL. After 72 h, the medium is ANG-1, ET-1, TGF-β and VEGF expression. collected for EV quantification and replaced by fresh Summary/Conclusion: The angiogenic properties of EV-FREE HPL supplemented media for a new produc- ASC-derived EVs may be enhanced through hypoxic tion cycle. culture. These EVs are able to promote angiogenesis of Results: This method allows multiple production cycles CMECs in vitro and may have utility in the treatment and improved cell survival, cellular morphology and of ischemic injury. EV production. Following 3 ×72 h consecutive pro- Funding: Natural Sciences and Engineering Research duction phase, MSCs amplification would produce 2.4 Council of Canada and 2.7 more EV when incubated in the presence of, respectively, 5% and 8% EV-free HPL compared to HPL-free medium. PS11.06 Summary/Conclusion: This process, compatible with the production of large volumes of conditioned media including in bioreactors, will allow large-scale produc-

Production and use of extracellular vesicles-depleted human platelet tion of therapeutic EV. lysate to improve large, clinical grade-compatible production of therapeutic human cell-derived extracellular vesicles Philippe Mauduita, Sylvie Goulinetb, Juliette Peltzerc, Bastien Rivalc, Sebastien Banzetc, Jean-jacques Latailladec and Georges Uzanb PS11.07 aInserm, Villejuif, France; bINSERM, villejuif, France; cCTSA, CLAMART, France; dINSERM, Villejuif, France Synchronized cell differentiation via exosomes Introduction: Human cells use multiple and sophisti- Tomohiro Minakawa; Kae Nakamura and Jun K. Yamashita a cated modes of communication. These include direct Laboratory of Stem Cell Differentiation, Center for iPS Cell Research and Application (CiRA), Kyoto University, Kyoto, Japan cellular communication, secretion of cytokines, chemokines or growth factors and also production of extra- Introduction: Embryonic development proceeds in a cellular vesicles (EV) containing proteins, DNA, highly orchestrated manner. It is assumed that syn- mRNA, miRNA. On the other hand, cell therapy chronization of a timing of differentiation and cell using Mesenchymal Stromal Cells (MSCs) is getting a fate among neighbouring cells is necessary for proper growing interest in a wide range of indications in tissue development. However, the mechanism of syn- human. In many cases, a substantial part of the ther- chronization is still largely unknown. apeutic effects relies on cell-secreted factors and the Methods: A mouse embryonic stem cell (ESC) line extracellular vesicles (EV) are proposed as a cell-free PKA-ESC, which can inducibly express constitutively surrogate for MSCs therapy. However, culture media active protein kinase A (CA-PKA), rapidly differenti- commonly used for culturing cells requires serum or ates into mesoderm with PKA activation (depletion of platelet lysate that contains large amounts of EV that doxycycline (Dox-)). We established a cell-chimeric cannot be distinguished and separated from the cell- culture system using two mouse ESC lines, PKA-ESC secreted EV. Purification and characterization of EV and Control-ESC to artificially generate a gap of timing therefore needs the prior elimination of contaminant in differentiation. We cocultured Control ESCs with EV contained in serum or Human Platelet Lysate PKA-ESCs to observe how they synchronously differ- (HPL). Serum-free media to produce EV may not be entiate by overcoming the gap of timing in differentia- fully satisfactory since they often limit cell survival. tion. Exosomes were collected from PKA-ESCs and Since regulatory authorities recommend avoiding ani- added to Control-ESCs or mouse embryos. miRNA mal components and xenobiotic-free culture condi- sequencing was performed comparing contents in exo- tions have to be considered for EV production. HPL somes from PKA-ESCs under Dox+ condition: control offers such a possibility as it is useful substitute to FBS or Dox- condition: PKA activation, accelerated differ- to isolate, amplify and maintain human cells. entiation. We also established several ESC lines that Therefore, we describe a new procedure for GMP- encode miRNAs and performed coculture experiments compatible production of human cells-derived EV. with control-ESCs. JOURNAL OF EXTRACELLULAR VESICLES 349

Results: After Dox-inducible activation of PKA, PKA- and electron microscopy. MSC and EV surface markers ESCs differentiate faster than Control-ESCs. In the were identified by bead-based flow cytometry. To study coculture system, the timing of mesoderm differentia- the EV contend, the presence of a panel of regulatory tion of Control-ESCs were synchronized with faster molecules was verified by qPCR and Western blot. differentiating PKA-ESCs (synchronized cell differen- Results: We found that both MSC treatment generate tiation). Furthermore, addition of exosomes purified population of EV heterogeneous in size, with main from PKA-ESCs promoted the differentiation of range between 100 and 200 nm and bigger vesicles Control-ESCs. The exosomes also promoted mesoderm (>500 nm) present in apoptotic MSC-EV samples. differentiation in postimplantation-stage mouse Apoptosis induction significantly increased the particle embryos. We found several miRNAs as the functional release. MSC-derived EV share mRNA and protein molecules in exosomes, and confirmed that miRNAs with their parental cells, and the different environment overexpressing cells can promote the differentiation of where the MSC is cultivated interfere in the EV con- Control-ESCs in the coculture system. tent. Moreover, our preliminary data shown that Summary/Conclusion: We unveiled a novel cellular GvHD patients receiving MSC have increased EV con- synchrony phenomenon and its mechanisms regulated taining MSC-related suppressive molecules straight by exosome-mediated cell communication, which after cell infusion. would be broadly involved in tissue development. Summary/conclusion: In summary, our results show Funding: This work was supported by JST CREST that the different environment where MSC is cultivated Grant Number [JPMJCR17H5 Japan]. interfere on their EV content, and can provide a signature of the “licensed” MSC. This was further tested PS11.08 in patients undergoing MSC treatment with a view of identifying biomarkers for pharmacokinetics studies. Funding: This work was supported by the Bloodwise Effects of mesenchymal stromal cells licensing on profile of Specialist Programme and by CAPES – Brazil. extracellular vesicles Giuliana Minani Bertolinoa, Tik Shing Cheungb, Chiara Giacominic, Martin Bornhauserd and Francesco Dazzie PS11.09 aKing’s College London, London, United Kingdom; bKing’s College London, London, United Kingdom; cKing’s College London, London, United Kingdom; dKing’s College London; Technische Universität Dresden, Dresden, Germany; eKing’s College London, London, United Kingdom Effects of mesenchymal stromal cells licensing on profile of extracellular vesicles Giuliana Minani Bertolinoa, Tik Shing Cheunga, Chiara Giacominia, Martin Abstract: The roles of mesenchymal stromal cells Bornhauserb and Francesco Dazzia (MSC) in the immune system are subject of increasing aKing's College London, London, United Kingdom; bKing's College London; interest and of widening clinical applications. Recent Technische Universität Dresden, Dresden, Germany evidences has shown that extracellular vesicles (EV) secreted by MSC can share some of the functional Abstract: The roles of mesenchymal stromal cells roles of their parental cells, among them the immuno- (MSC) in the immune system are subject of increasing suppression ability. Prior to exert immunomodulation, interest and of widening clinical applications. Recent MSC effects rely on the presence of inflammatory evidences has shown that extracellular vesicles (EV) mediators in the microenvironment: (1) proinflamma- secreted by MSC can share some of the functional tory cytokines such as IFN-γ and TNF-α, and (2) by roles of their parental cells, among them the immuno- the action of inflammatory effector cells which culmi- suppression ability. Prior to exert immunomodulation, nates on MSC apoptosis without the loss of immuno- MSC effects rely on the presence of inflammatory modulatory property. Therefore, we propose that mediators in the microenvironment: (i) proinflamma- different licensing of MSC can generate EV with dis- tory cytokines such as IFN-γ and TNF-α, and (ii) by tinct profiles and aspects on the immunomodulation. the action of inflammatory effector cells which culmi- Methods: To test this hypothesis, we characterized EV nates on MSC apoptosis without the loss of immuno- population derived from untreated MSC, MSC licensed modulatory property. Therefore we propose that by pro-inflammatory cytokines (IFNγ and TNFα) and different licensing of MSC can generate EV with dis- from MSC undergoing apoptosis (anti-Fas antibody). tinct profiles and aspects on the immunomodulation. We also isolated and characterized EV from plasma of Methods: To test this hypothesis, we characterized EV Graft-versus-Host Disease (GvHD) patients receiving population derived from untreated MSC, MSC licensed MSC as therapy (0, 4, 24, 48 h after MSC injection). EV by pro-inflammatory cytokines (IFNγ and TNFα) and size, shape and concentration were accessed by NTA from MSC undergoing apoptosis (anti-Fas antibody). 350 ISEV2019 ABSTRACT BOOK

We also isolated and characterized EV from plasma of with their parental cells, and the different environment Graft-versus-Host Disease (GvHD) patients receiving where the MSC is cultivated interfere in the EV con- MSC as therapy (0h, 4h, 24h, 48h after MSC injection). tent. Moreover, our preliminary data shown that EV size, shape and concentration was accessed by NTA GvHD patients receiving MSC have increased EV con- and electron microscopy. MSC and EV surface markers taining MSC-related suppressive molecules straight were identified by bead-based flow cytometry. To study after cell infusion. the EV contend, the presence of a panel of regulatory Summary/Conclusion: In summary, our results show molecules was verified by qPCR and western blot. that the different environment where MSC is cultivated Results: We found that both MSC treatment generate interfere on their EV content, and can provide a sig- population of EV heterogeneous in size, with main nature of the ‘licensed’ MSC. This was further tested in range between 100 and 200 nm and bigger vesicles patients undergoing MSC treatment with a view of (>500 nm) present in apoptotic MSC-EV samples. identifying biomarkers for pharmacokinetics studies. Apoptosis induction significantly increased the particle Funding: This work was supported by the Bloodwise release. MSC-derived EV share mRNA and protein Specialist Programme and by CAPES – Brazil. JOURNAL OF EXTRACELLULAR VESICLES 351

LBS01: Late Breaking- EV Therapeutics Chairs: Xabier Osteikoetxea; Akiko Takahashi Location: Level 3, Hall A 15:00–16:00 kinase (p38) molecules given in response to LPS stimulation. LBS01.01 Summary/conclusion: MSC-EVs are strong modulators of microglia activation. The modulatory activity of MSC-EVs can be of major impact in the treatment Mesenchymal stromal cells derived-extracellular vesicles effect on microglia cells of neuroinflammatory diseases. Dorota Kaniowskaa, Kerstin Wenkb, Franziska Langea, Sebastian Greisera, Funding: This project is co-financed with tax money Ulf-Dietrich Braumanna and Yarua Jaimesc from the state of Saxony, Germany. High Performance aFraunhofer IZI, Leipzig, Germany; bInstitute for Clinical Immunology, University of Leipzig, Leipzig, Germany; cISEV, Leipzig, Germany Center of Chemical and Biosystem Technology: Grant 100312141, Grant 100321061. YJ is financed by a Introduction: Mesenchymal stromal cells (MSCs) are a TALENTA Financing award from the Fraunhofer heterogeneous population of cells with very high self- Society. renewal properties and the capacity to induce tissue regeneration and reduce inflammation. Extracellular vesicles (EVs) from MSCs have shown to have immune LBS01.02 modulatory properties and given their small size, are good candidates as therapeutic agents for tissues of Porcine milk exosomes protect intestine against deoxynivalenol difficult access, such as the central nervous system damage a a b (CNS). Microglia cells are the CNS immune cells and Mei-Ying Xie , Ting Chen and Yong-Liang Zhang are involved in the progression of the degeneration in aSouth China Agricultural University, Guangzhou, USA; bcollege of animal science, south china agricultural university, Guangzhou, China (People’s many neuroinflammatory diseases. We evaluated the Republic) interaction of MSC-EVs with microglia cells and their effect as regulators of activation. Introduction: Deoxynivalenol (DON) serious damage Methods: We have used an in vitro model for stimula- intestinal vulnerable structures and intestinal integrity. tion of the BV-2 microglia cell line and primary cells Our previous study showed that exosomes could facil- with lipopolysaccharides (LPS) and amyloid β aggre- itate intestinal cell proliferation and neonate intestinal gates. Real time PCR methods were used to assessed tract development, but the protection of milk exosomes the transcripts upregulation of tumour necrosis factor of damage caused by DON is unclear. (TNF)-α, Interleukin (IL)-1β, IL-6, nitric oxide Methods: Neonatal Kunming mice were given 0.4 ml synthases (iNOS), Prostaglandinendoperoxide synthase porcine milk exosomes or saline for 3 weeks and then 2 (PTGS2) and chemokine ligand (CCL)-22. Protein given 2.5 mg/kg bw/day DON for 7 days. Intestinal levels of TNF-α, IL-1β and IL-6 were evaluated by morphology was assessed using H&E. Cells viability ELISA and cytometric bead arrays. Live cell imaging are tested by MTT, Edu and cell counting assay. WB, approaches were used to evaluate the interaction of qRT-PCR and immunofluorescence were used to show MSC-EVs with microglia cells in vitro. the effects of porcine milk exosomes on the damages of Results: We demonstrated that MSC-EVs are actively intestine and IPEC-J2 cells caused by DON. At last, internalized by microglia cells. Moreover, that presence bioinformatics Analysis, luciferase reporter assay was of MSC-EVs prevents transcription and protein expres- to verify the potential targeting relationship between sion of pro-inflammatory cytokines tumour necrosis miRNAs and mRNAs. factor (TNF)-α, interleukin (IL)-1β, IL-6, inducible iso- Results: Porcine milk exosomes significantly alleviated form of nitric oxide synthases (iNOS) and prostaglan- the negative effects of DON on body weight and the dinendo peroxide synthase 2 (PTGS2) upregulation by damage degree of intestinal epithelial. In addition, microglia cells towards LPS and amyloid β. these exosomes significantly reversed the inhibition of Furthermore, MSC-EVs suppressed the phosphoryla- DON on cell proliferation and intercellular tight junction of the extracellular signal kinases 1/2 (ERK1/2), tion-associated proteins, such as levels of β-catenin, p- c-Jun N-terminal kinases (JNK) and the p38 MAP Akt, cyclinD1 and claudin1, and decreased the 352 ISEV2019 ABSTRACT BOOK apoptosis-related protein p53 and p21. In vitro, porcine preserve exosomal PD-L1 throughout the wound. Flow milk exosomes significantly attenuated the damage of cytometry analysis, qPCR, HE staining and immuno- DON on cell viability, proliferation and tight junctions, histochemical analysis were performed to further consistent with the results in vivo. Our results also explore the therapeutic effect of exosomal PD-L1 at indicated that porcine milk exosomes up-regulate the the tissue levels. expression of miR-181a, miR-30c, miR-365-5p and Results: Exosomal PD-L1 in thermoresponsive gel led miR-769-3p in cells and downregulated their targeting to a decreased T cell activation, indicated by CD4, genes in p53 pathway, such as FAS, TP53, SERPINE1. CD8, and IL-2 markers. In the presence of exosomal Summary/conclusion: Porcine milk exosomes pro- PD-L1, there was also an increased expression of tected intestine and IPEC-J2 cells against DON growth factors, which significantly promoted wound damage, and encapsulated miRNAs play a role in reg- contraction and wound re-epithelialization. ulating p53 pathway. Our study opened a new sight in Summary/conclusion: Collectively, our current find- breast milk exosomes, which may contribute to intest- ings suggest that exosomal PD-L1 speeds up wound inal health during the neonatal period healing when applying into a novel thermoresponsive Funding: This work was supported by grants from the gel on top of the injured skin, which provides a new National Natural Science Foundation of China [grant perspective for using immunotherapy to promote tis- numbers 31472163], and The Chinese National Key sue repair and regeneration. Scientific Project (2016YFD0500503). Funding: F. Cheng would like to thank Sigrid Jusélius foundation, the National Natural Science Foundation of China (Grant no. 81702750) and the Basic Research LBS01.03 Project of Shenzhen (Grant no. JCY20170818164756460) for funding.

Exosomal PD-L1 embedded with thermoresponsive gel promotes wound healing Dandan Sua, Zhanxue Xub, Hongbo Chenb, Fang Chengb and Xiangyi Caic LBS01.05 aSchool of pharmaceutical sciences(Shenzhen), Sun Yat-sen University, Guanghzou, China (People‘s Republic); bSchool of pharmaceutical sciences (Shenzhen), Sun Yat-sen University, Guangzhou, China (People‘s Republic); Intranasal delivery of mesenchymal stem cell derived exosomes c School of pharmaceutical sciences(Shenzhen), Sun Yat-sen University loaded with PTEN siRNA repairs complete spinal cord injury Shawei Goua, Nisim Peretsb, Oshra Betzerc, Shahar Ben-Shauld, Anton e f c Introduction: Wound healing is a complex process Sheinin , Izhak MichaelevskiMichaelevski , Rachela Popovtzer , Daniel Offeng and Shulamit Levenbergh involving multiple cell types with different roles, aDepartment of Biomedical Engineering, Technion-Israel Institute of which is divided into stages of haemostasis, inflamma- Technology, Israel; bSagol School of neuroscience, Tel Aviv University, tion, proliferation and remodelling. As many chronic Israel, Tel aviv, Israel; cFaculty of Engineering and the Institute of Nanotechnology & Advanced Materials, Bar-Ilan University, Israel, Ramat wounds are the result of excessive and chronic inflam- Gan, USA; dDepartment of Biomedical Engineering, Technion-Israel Institute mation, we hypothesized that effective wound repair of Technology, Haifa, 3200002, Israel, Haifa, Israel; eSagol School of f could be achieved by inhibiting overactive immune Neuroscience, Tel Aviv University, Israel., Haifa, Israel; Department of Molecular Biology, Ariel University, Israel.; gSagol School of neuroscience, cells to the injured skin. The PD-1/PD-L1 immune Tel Aviv University, Israel, Sacklar school of medicine, department of human checkpoint pathway prevents excessive tissue destruc- genetics and biochemistry Tel Aviv University, Israel., Tel Aviv, USA; hDepartment of Biomedical Engineering, Technion-Israel Institute of tion during inflammatory states, and PD-L1 expression Technology, Israel, Haifa, Israel is induced by pro-inflammatory factors in multiple cell types throughout the body. Interestingly, recently PD- Introduction: Complete spinal cord injury (SCI) is a L1 is found to exists in extracellular vesicles (EVs) as a debilitating disease which usually leads to permanent transmembrane protein. Thus we would like to test if functional impairments, with various complications exosomal PD-L1 would regulate the immunity and and limited spontaneous recovery or efficient treat- inflammatory response to promote proper wound ment. Here, we report that in rats with complete SCI, healing. intranasal administrations of mesenchymal stem cells- Methods: Exosomal PD-L1 were isolated from mela- derived exosomes (MSC-Exo) could penetrate the noma cells stimulated with IFN-γ by differential cen- blood brain barrier, home selectively to the spinal trifugation and were characterized by flow cytometry, cord lesion, and show affinity to neurons within the TEM, DLS, zeta potential, Western blot and confocal lesion. When these exosomes were loaded with phos- microscopy. Exosomal PD-L1 were administered in a phatase and tensin homolog small interfering RNA, mouse skin injury model through a thermoresponsive termed ExoPTEN, they migrated from the nose and hydrogel, which was gelatinized at body temperature to silenced PTEN expression in the lesion. Furthermore, JOURNAL OF EXTRACELLULAR VESICLES 353 the loaded exosomes promoted robust axonal regen- overcome the limitations of MSC easily and become eration and angiogenesis, accompanied with decreased powerful alternative therapeutics. Here, we investigated astrogliosis and microgliosis. Moreover, the intranasal the therapeutic effects of exosome from adipose tissue- ExoPTEN treatment partially restored electrophysiolo- derived MSC (ASC-EXOSOME) on atopic dermatitis gical and structural integrity, and most importantly, in two in vivo models. enabled remarkable functional recovery. This rapid, Methods: ASC originated from adipose tissue of a non-invasive approach, using cell-free nano-swimmers healthy donor. ASC-EXOSOME was isolated from carrying molecules to target pathophysiological ASC conditioned media through a sequential filtration mechanisms, suggests novel strategy for clinical trans- method. AD-like skin lesions were induced in mice by lation to SCI and beyond. applying house dust mite antigen or a chemical irritant. Methods: MSC-exo were extracted from Human bone After administration of ASC-EXOSOME either subcu- marrow mesenchymal stem cells. All rats had complete taneously or intravenously the anti-inflammatory transection of the spinal cord. MSC-exo were loaded effects were demonstrated by measuring serum IgE with co-incubation together with siRNA for PTEN level, immunostaining of immune cells, real-time conjugated to cholesterol. The MSC-exo were given PCR, etc. by intranasal administration 1–4 h post SCI. Results: Systemic administration of ASC-EXOSOME Results: Here we show that SCI rats that were intrana- dose-dependently lowered serum IgE level and the sally treated with MSC-exo present functional number of eosinophils in AD mice blood, and reduced improvement in motor and sensory output. The mast cell infiltration and up-regulated mRNA levels of MSC-exo were homed in the SCI area and led to IL-4, IL-31, IL-23 and TNF-α in the skin lesions com- reduction in inflammatory markers, increased angio- pared to AD control. Skin barrier function was also genesis and regrowth of transected axons. MRI and improved by ASC-EXOSOME. electrophysiological measurements were done to show Summary/conclusion: Systemic administration of the axonal recovery and signal transduction ASC-EXOSOME dose-dependently lowered serum IgE Summary/conclusion: Exosomes derived from Human level and the number of eosinophils in AD mice blood, bone marrow mesenchymal stem cells and loaded with and reduced mast cell infiltration and up-regulated inhibitor molecule for PTEN pathway were found effi- mRNA levels of IL-4, IL-31, IL-23, and TNF-α in the cient in ameliorating complete transection of the spinal skin lesions compared to AD control. Skin barrier cord via intranasal administration, including remark- function was also improved by ASC-EXOSOME. able functional improvement. LBS01.07 LBS01.06

Porcine milk exosome miRNAs attenuate lipopolysaccharide-induced apoptosis by inhibiting TLR4/NF-κB and P53 pathways ASC-EXOSOME as a potential therapeutic for atopic dermatitis Yong-Liang Zhanga, Mei-Ying Xieb and Ting Chenb Byong Seung Choa, Jin Ock Kimb, Dae Hyun Haa and Yong Weon Yia aCollege of Animal Science, South China Agricultural University, Guangzhou, aExocobio Inc., Seoul, Republic of Korea; bExocobio Inc, Seoul, Republic of China (People‘s Republic); bSouth China agricultural university, Korea Guangzhou, USA

Introduction: Atopic dermatitis (AD) is an inflamma- Introduction: Intestinal epithelial cells are important tory disease that has rapidly increased in the prevalence for pathogen infection. LPS is an endotoxin and in recent decades. Despite the high demand for AD induces intestine inflammation. Milk exosomes therapy, current treatment options are limited and have improve the intestine development and immune sys- potentially harmful side effects. Recently, several clin- tem of newborn. The objective of this study is to ical studies highlighted human mesenchymal stem cells investigate the protective mechanisms of porcine milk (MSCs) as novel potential therapeutics for suppressing exosomes in rescuing LPS-induced intestinal epithe- allergic progress in the AD, and the majority of their lium injuries. therapeutic effects is mediated their secretome which Methods: Both in vivo and in vitro tests were carried contains exosomes. There are, however, several draw- out to confirm protection of porcine milk exosome on backs for the therapeutic use of MSCs, such as poor LPS induced injury to intestine. engraftment efficiency, non-specific differentiation, Results: In vivo, exosomes protected the jejunum integ- and short half-life, etc. Otherwise, exosomes can be rity and health from LPS damage through H&E results off-the-shelf since they are not live, expecting to and attenuated LPS-induced pro-inflammatory factors 354 ISEV2019 ABSTRACT BOOK secretion through ELISA results. In vitro, we got simi- by size exclusion chromatography and characterized by lar results in the intestinal epithelial cell line IPEC-J2. Nanoparticle tracking analysis. MSC-EVs were evalu- Bioinformatics analyses and cell experiments results ated in vitro in an inflammatory assay using human shown exosome miR-4334, miR-219 reduced pro- monocytic THP-1 cells treated with lipopolysaccharide, inflammatory responses and miR-338 inhibited LPS- with or without co-culture with MSCs or EVs. The induced apoptosis of intestinal epithelial cells via level of pro-inflammatory TNFα in the culture super- TLR4/MyD88/NF-κB and P53 pathway, respectively. natant was measured by ELISA assay. EVs were also Co-transfection of those three miRNAs had the best evaluated in vivo using a mouse model of acute hind effect on resisting LPS-induced IPEC-J2 apoptosis than limb radiation injury. Cell therapy products (1x106 any one of these three miRNAs. MSCs or a range of 2.45E+10, 4.90E+10 or 9.80E+10 Summary/conclusion: In conclusion, porcine milk MSC-EVs/animal) were intramuscularly injected exosomes protected the intestine against LPS-induced 14 days post-irradiation. Macroscopic analysis of injury injury through decreasing cell inflammatory and resist- was performed at regular intervals. ing cell apoptosis by exosome miRNAs. This study Results: Preliminary results showed an immunomodu- expands our understanding of bioactive molecules in latory effect of MSCs-EVs, as shown by their ability to milk and provides new strategies for developing func- reduce TNFα secretion by THP-1 cells in response to tional foods in the future. LPS. Moreover, in vivo results showed a decrease of Funding: This work was supported by grants from the injury score in animals injected with the highest EV National Natural Science Foundation of China [grant concentration at day 10 and 14 post-injection. numbers 31472163], and The Chinese National Key Summary/conclusion: These preliminary results sug- Scientific Project (2016YFD0500503). gest a beneficial effect of MSC-EVs on the healing process of cutaneous radiation syndrome and could LBS01.08 represent a valuable therapeutic alternative in the context of radiological emergency. Further exploration of the molecular mechanisms is now necessary. Extracellular vesicles from mesenchymal stromal cells for the Funding: French Direction Générale de l‘Armement, treatment of radiological burns Juliette Peltzera, Stephane Flamantb, Philippe Mauduitc, Sylvie Goulinetc, under contract ANR-16-ASTR-0024 Bastien Rivala, Jean-jacques Latailladed, Georges Uzanc, Sebastien Banzete and Radia Tamaratb aInstitut de Recherche Biomédicale des Armées, INSERM UMR-MD-1197, LBS01.09 Clamart, USA; bInstitut de Radioprotection et Sûreté Nucléaire (IRSN), Fontenay aux Roses, France; cINSERM UMR-MD-1197, Villejuif, France; dInstitut de Recherche Biomédicale des Armées, INSERM UMR-MD-1197, Clamart, France; eInstitut de Recherche Biomédicale des Armées, INSERM Adipose-derived stem cells enhance chondrogenesis and UMR-MD-1197, CLAMART, France cartilaginous matrix synthesis of articular chondrocytes is mediated by extracellular vesicles Shun-Cheng Wu, Jhen-Wei Chen, Che-Wei Wu, Chung-Hwan Chen, Je-Ken Introduction: High-dose acute radiation accidents of Chang and Mei-Ling Ho industrial and medical origin and the risk of a terrorist Orthopaedic Research Center, College of Medicine, Kaohsiung Medical act (NRBC) have been taken into consideration for University, Kaohsiung, Taiwan (Republic of China) some years. The work carried out by our teams led to a new therapeutic approach for the management of Introduction: To date, mesenchymal stem cells includ- victims of accidental irradiation, consisting of autolo- ing adipose-derived stem cells (ADSCs) have been gous Mesenchymal Stromal Cells (MSCs) injection intensively investigated as a cell-based therapy to treat associated with reparative surgery. Preclinical studies articular cartilage damages in both animal and human showed that MSCs, mainly by their secretory activity, studies. However, the detailed mechanism of how contribute to control inflammation, promote angiogen- ADSCs regenerate the damaged articular cartilage esis and tissue regeneration. MSC-derived extracellular remains unclear. Increasingly, studies present evidence vesicles (MSC-EVs) might be key mediators of MSC that ADSCs mediate tissue repair via secretion of function. This project aims to propose an innovative trophic factors on damaged tissue. In this study, we therapy product based on the use of Extracellular test the hypothesis that ADSCs-derived extracellular Vesicles (EVs) for the treatment of radiological burns vesicles (EVs) enhances chondrogenesis and matrix following accidental irradiation. synthesis of human articular chondrocytes. Methods: MSCs were grown until reaching 80% con- Methods: Human ADSCs were labelled with CM-DiI fluence, then moved to EV collection medium for 72 h. and then pre-cultured in DMEM supplemented with EVs were purified by tangential flow filtration followed 2% FBS for 48 h to induce EVs release. After induce JOURNAL OF EXTRACELLULAR VESICLES 355

EVs release, the conditioned medium derived from antimicrobial activity test, Staphylococcus aureus (S. pre-cultured with ADSCs were isolated, and then was aureus) was cultured in LB broth medium at 37℃,O/ used to treat articular chondrocytes. There were three N. The seed culture ratio (1/100, 1/1000) and different groups in the study: (1) Control: articular chondrocytes exosome concentration were inoculated and growth treated with DMEM supplemented with 2% FBS with- was confirmed by time. out pre-cultured with ADSCs, (2) Conditioned med- Results: The average size of the MiExo obtained was ium: articular chondrocytes treated with DMEM 120 ~ 140 nm. Both TEM and cryo-EM image showed supplemented with 2% FBS, which is pre-cultured a typical exosome shape morphology. The Western with ADSCs, (3) Conditioned medium remove EVs: blotting confirmed the detection of TSG101 marker, articular chondrocytes treated with conditioned med- which is a representative marker of MiExo. The anti- ium, which the EVs were removed by ultracentrifuga- microbial activity of S. aureus was determined at dif- tion. At the indicated time point, the chondrocytes ferent conditions. It exhibited 2.5 times antimicrobial were harvested for further analysis including cell pro- effect when the MiExo and the bacteria were inoculated liferation, chondrogenic gene expressions (Collagen together at an early stage in log phage (10^8 CFU/mL). type II), and cartilaginous matrix synthesis Based on the inoculation dilution factor(DF), very high (Glycosaminoglycan synthesis). antimicrobial effect of approximately 19 times was Results: Intercellular communication occurs through observed for 1/1000 DF as compared to the 1/100 DF. EVs. EVs transferred into chondrocytes can be found S. aureus hardly grew in the experiment group with 1/ in the conditioned medium group. However, there is 1000 DF. The antimicrobial efficacy based on the no EVs transfer in the conditioned medium removed amount of exosome was 13 times higher for 10^11 EVs. There is no significant difference in cell prolifera- particles as compared to 10^6 particles. tion of chondrocytes among three groups. The chon- Summary/conclusion: The extraction of MiExo and its drogenic gene expression and cartilaginous matrix antimicrobial effect was determined. The antimicrobial synthesis of chondrocyte is significantly enhanced in effect of MiExo performed in this study is considered conditioned medium group when compared with con- to be stable with low side effects and has great potential trol group. Moreover, there is no significant difference as a superior natural material in the future cosmeceu- between control and conditioned medium removed EV tical market. groups. Funding: This work was carried out with the support Summary/conclusion: ADSCs enhances chondrogen- of “Cooperative Research Program for Agriculture esis and matrix synthesis of human articular chondro- Science & Technology Development (Project No. cytes is mediated by EVs PJ012653)” Rural Development Administration Republic of Korea. LBS01.10 LBS01.11 Application of milk exosome for leaping cosmeceutical materials. Gna Ahna, Yang-Hoon Kimb and Ji-Young Ahnb aChungbuk National University, Cheong-ju, Republic of Korea; bSchool of Control of neural stem cell differentiation to generate defined Biological Sciences, Chungbuk National University, Cheongju, Republic of exosome populations Korea Nicola Goddarda, Daniel Bracewellb, Randolph Cortelingc, Simon Youltonc and Ivan Walld Introduction: Milk is one of the best exosome materi- aUniversity College London, Brentwood, United Kingdom; bUniversity als widely used as an ingredient in various foods. College London, London, United Kingdom; cReNeuron Limited, Pencoed Although the antibacterial effect present in milk has Business Park, Pencoed, Bridgend, Wales, CF35 5HY, United Kingdom, Bridgend, United Kingdom; dUniversity College London, Birmingham, been long known, however studies related to the anti- United Kingdom bacterial activity associated with milk exosomes are fairly limited. The purpose of this study is to suggest Introduction: Exosomes derived from the clinical the possibility of using the antimicrobial effect of milk grade neural stem cell line CTX (ReNeuron) are the exosomes in cosmeceutical field. basis of a new class of therapy for the treatment of Methods: Commercially available non-fat milk-based degenerative disorders. Since exosomes contain a sub- on Pasteur treatment was used. Milk was centrifuged at set of molecules derived from their parent cell, pro- 210,000 g for 70 min at 4℃. TEM and cryo-EM was genitor and differentiated CTX may generate exosomes used to determine the shape of milk exosomes and its with diverse phenotypes. It is vital that these are well size was measured using qNano (iZon, Australia). For characterized to allow robust manufacture and 356 ISEV2019 ABSTRACT BOOK isolation of particular exosome populations and to Introduction:Facedwithvariouslimitationsinhow understand their implications in therapeutic functionalizationofnanoparticles‘ surfaces with syn- applications thetic organic materials, researchers have begun to Methods: Screening of support matrices (microcar- envision strategies for using bioinspired materials as riers) and substrates for growing CTX was performed an alternative. In particular, nanoparticles require in a bespoke microfluidic device for 7 days. Cells were many improvements in vivo to increase stability, then fixed and stained before applying automated ima- enhance circulation timeanddistributionand ging and analysis to determine the differentiated state improve dissolution. For this reason, directly trans- of the cells. The process was repeated with a reduced ferring the cell membrane to the surface of the panel of matrix/substrate combinations to study differ- nanoparticles, the complexity of the lipid, protein entiation and exosome agonists for a period of 6 weeks and carbohydrate-containing cell membranes can be as a means to accelerate CTX differentiation and faithfully maintained, allowing the coated nanoparti- increase exosome production. The conditions selected cles to have characteristics that appear to the source for each cell type were validated in a model bioreactor cell. The behaviour of cells in vivo is usually deter- system at the 0.1L scale and the resultant exosomes mined by cell membrane proteins. This method of characterized in terms of particle number, size distri- directly coating the cell membrane also has the same bution, miRNA content and CD markers characteristics of nanoparticles depending on the Results:Themicrofluidicscreeningapproachallows characteristics of the source cells. the study of a panel of 336 matrix, substrate, differ- Methods:First,inordertocoatthecellmembrane, entiation agonist and exosome agonist/antagonist the capped CTAB for the surface stabilization of the combinations enabling the experimental space to be gold nanorod was replaced with a negatively charged reduced by >98% prior to any scale-up activities, citrate. And, extracted stem cell membranes are engi- thereby minimising experimental time, cost and risk neered using lipid-tethered peptide for enhancing of failure. Our validation successfully achieved our targeting specificity to cancer cell. Engineered cell target cell population of 60,000 cells/cm2 in 4 days membrane was cloaked onto the surface of gold and found that the resultant exosomes had miRNA nanorods using simple bath sonication. It can be and CD marker profiles dependent on stage of dif- confirmed that the stem cell membrane is well- ferentiation of the culture coated on the surface of gold nanorod through var- Summary/conclusion: CTX were successfully adapted ious analysis including, zeta potential, TEM, PAGE for growth on microcarriers in a suspension bioreactor gel and Western blotting, etc. system to provide a scalable platform for progenitor Results: Citrate-capped gold nanorods are character- and differentiated CTX-derived exosome production. ized using zeta potential and TEM. And, zeta potential The exosome characteristics change in terms of both of well-established cell membrane-cloaked gold nanor- CD markers and miRNA profile according to the dif- ods is similar to that of cell membrane. Cell membrane ferentiated state of their parent cell. This has implica- coating onto gold nanorods is also visualized using tions on not only their therapeutic function and TEM with negative staining. After cell membrane coat- potency but also the design of processes for their man- ing, the protein marker in the cell membrane is ufacture and purification in order to deliver consistent detected identically to the cell membrane, but the product profile nuclear marker is not detected in Western blotting Funding: Innovate UK analysis. Summary/conclusion: Cell membrane coating is a platform technology that provides an easy top-down LBS01.12 method for designing nanocarriers with surfaces that replicate very complex and diverse functions needed for effective bio-interfacing and give nanoparticles Engineered stem cell membrane-cloaked gold nanorods for efficient more functional. cancer therapy Won-Kyu Rhima, Kyungwon Koa, Soo-Hong Leeb, Wooram Parka and Dong Funding: This study was supported by the National Keun Hanc Research Foundation of Korea (NRF) Grants funded aBiomedical science, CHA university, seongnam, Republic of Korea; by MEST (NRF-2017R1A6A3A040123620) bDongguk University, goyang, Republic of Korea; cBiomedical science, seongnam, Republic of Korea JOURNAL OF EXTRACELLULAR VESICLES 357

LBS02: Late Breaking- EVs in Intercellular and Interorganism Communication Chairs: Hanne Winther-Larsen; Kallen Sullivan Location: Level 3, Hall A 15:00–16:00

Summary/conclusion: In conclusion, both MSC-EVs and An5-MSC-EVs shift the macrophage phenotype LBS02.01=OWP1.14 from M1 to M2. The combined induction of TGFbeta1 and IL-10, observed only in An5-MSC-EV- stimulated macrophages, might be related to the Annexin V binding modulates the response of macrophages to mesenchymal stromal cell-derived extracellular vesicles immune-modulating characteristics of these modified Michele Grassia, Michela Pozzobonb, Melania Scarpac, Damiana Incendid, EVs that contribute to the therapeutic effects observed Alessia Giarraputoe, Anna Maria Tolomeoa, Marcin Jurgaf, Andrea Porzionatog and Maurizio Muracah in vivo. aDepartment of Women’s and Children’s Health – University of Padova, Funding: The BROAD MEDICAL RESEARCH Padova, Italy; bUniversity of Padova, Padova, Italy; cIstituto Oncologico PROGRAM AT CCFA supported this work Veneto IOV-IRCCS, Padova, Italy; dUniversity di Padova Department DNS, Padova, Italy; eUniversity of Padova, Padova, Italy; fThe Cell Factory, Niel, Belgium; gDepartment of Neuroscience, University of Padua, Padova, Italy, Padova, Italy; hUniversity of Padova, Italy, Padova, Italy LBS02.02 Introduction: We have previously shown that Annexin a5 (An5) binding to mesenchymal stromal cell-derived PD-L1/CTLA-4 nanovesicles have an immunosuppressive effect on a extracellular vesicles (MSC-EVs) enhances the anti- mouse skin graft model a b a a inflammatory properties of these nanoparticles in an Zhanxue Xu , Xiangyi Cai , Fang Cheng and Hongbo Chen animal model of colitis. However, the mechanisms aSchool of pharmaceutical sciences(Shenzhen), Sun Yat-sen University, Guangzhou, China (People‘s Republic); bSchool of pharmaceutical sciences underlying these effects are unknown. Here, we inves- (Shenzhen), Sun Yat-sen University tigated the immunoregulatory effect of MSC-EVs with and without An5 binding on activated macrophages in Introduction: Skin transplantation has been employed vitro. to serious injuries, but a potent inflammatory immune Methods: Macrophages were isolated from mouse bone response often leads to rejection of allogeneic skin marrow and activated by INFgamma and LPS. Clinical grafts. T-cell activation by immune allorecognition is grade Wharton Jelly-derived MSC-EVs were obtained a major cause to trigger acute rejection. Immune from The Cell Factory (Esperite NV, Niel, Belgium) checkpoint pathways such as the programmed death- and quantified by Resistive Pulse Sensing analysis. 5,0E 1 (PD-1)/programmed death-ligand 1 (PD-L1) and +05 macrophages were incubated with PBS (vehicle cytotoxic T-lymphocyte-associated protein 4 (CTLA- only, control, group 1) 5,0E+08 MSC-EVs (group 2), 4)/Cluster of differentiation 80 (CD80) provide an 5,0E+08 MSC-EVs added with 2 ug An5 (group 3) or immunosuppressive environment, preventing excessive with 2 ug free An5 (group 4). After 24 h, the cells were tissue destruction due to inflammatory immune analysed by flow cytometry and RNA was extracted for response. Thus we would like to see if bioengineering RT-PCR analysis. cell membrane derived nanovesicles (NVs) to display Results: Incubation with MSC-EVs significantly PD-L1 and CTLA-4 would reduce immunological increased only the expression of IL-10 in IFNgamma/ rejection through enhancing PD-1/PD-L1 and CTLA- LPS-activated macrophages. Incubation with An5- 4/CD80 immune inhibitory axis. MSC-EVs resulted in a significant induction in the Methods: We established HEK 293T cells that stably expression of both pro- and anti-inflammatory cyto- express PD-L1/CTLA-4 on the cell membranes and kines, including TNFalfa, IL-1Beta, IL-6, IL-10 and prepared cell membrane nanovesicles. Confocal micro- TGFbeta1. Incubation with free An5 induced only scopy and immunoprecipitation analysis were used to pro-inflammatory cytokines without affecting IL-10 determine the interaction of PD-1/PD-L1 and CTLA-4/ and TGFbeta1 expression. The iNOS2/Arg1 ratio was CD80 on the cell membrane. After that, T-cell activa- reduced in both EV-treated groups, indicating a shift tion and proliferation were examined by flow cytome- from M1 to M2 polarization. try analysis of a panel of markers. Finally, we tested the 358 ISEV2019 ABSTRACT BOOK effect of PD-L1/CTLA-4 NVs on relieving skin grafting Results: To determine the origin of bovine DNA frag- in vivo. ments, we used goat serum, rabbit serum, and exo- Results: We successfully engineered cell membrane some-free FBS instead of FBS in the cell culture derived nanovesicles to display PD-L1/CTLA-4, which medium. Goat BovB and rabbit LINE1 sequences were characterized by transmission electron micro- were horizontally transferred to DSB sites, however, scopy and Western blotting In addition, confocal almost no bovine DNA sequences were captured, sug- microscopy showed that PD-L1/CTLA-4 NVs can gesting that these horizontal gene transfers were interact with PD-1 of and CD80 of target cells. mediated by exosomes. Furthermore, PD-L1/CTLA-4 NVs led to reduction of Summary/conclusion: We demonstrated that horizon- T cells activation and proliferation. Finally, compared tal gene transfer assisted by CRISPR-Cas9 occurs in to control mice, the skin-grafting mice had a high NIH-3T3 cells and mouse embryos. This phenomenon response rate to relieve immunologic rejection when might be the driving force behind mammalian genome treated with PD-L1/CTLA-4 NVs. evolution. In fact, mice with fusions between the mur- Summary/conclusion: As a summary, NVs containing ine Peg10 gene and a bovine SINE were obtained. A dual molecular targets PD-L1/CTLA-4 exhibit strong number of possible trans-species horizontal gene trans- immune inhibitory effect, promoting the healing of fer events have been reported in mammals. Exosomes grafting skin. Thus, PD-L1/CTLA-4 dual immune are present in all fluids from living animals, including blockade by nanovesicles provides a promising strategy seawater and breathing mammals, suggesting that exo- to inhibit skin graft rejection. some-mediated horizontal gene transfer is the driving force behind mammalian genome evolution. The findings of this study also highlight an emerging new risk LBS02.03 for this leading-edge technology. Funding: AMED (18mk0104073j0103 &18ak0101093j001), Health Sciences Research Grants Exosome-mediated horizontal gene transfer: a possible driving force behind mammalian genome evolution & a new risk for genome from the Ministry of Health, Labor, and Welfare, Japan editing (H30-KAGAKU-IPPAN-002 & H30-KAGAKU- Ryuichi Onoa, Yukuto Yasuhikob, Ken-ichi Aisakib, Satoshi Kitajimac, Jun Kannod and Yoko Hirabayashib SHITEI-001), and JSPS (26430183 and 18K19315) aDivision of Cellular & Molecular Toxicology, Biological Safety Research Center, National Institute of Health Sciences (NIHS), Kawasaki, Japan; bNational Institute of Health Sciences (NIHS), Kawasaki, Japan; cNational LBS02.04 Institute of Health Sciences (NIHS), Kawasaki, USA; dJapan Bioassay Research Center, Kawasaki, USA Comparative studies on in vitro and in vivo inflammatory activities of extracellular vesicles and soluble factors derived from bacteria Introduction: The CRISPR-Cas9 system has been suc- Kim Sang sooa, Jaewook Leeb, Park Kyong-Suc and Yong Song Ghoa cessfully applied in many organisms as a powerful aDepartment of Life Sciences, Pohang University of Science and Technology, genome editing tool. We have previously reported Pohang, Republic of Korea; bDepartment of Life Sciences, Pohang University that unintentional DNA sequences derived from retro- of Science and Technology (POSTECH), Pohang, Republic of Korea; cDepartment of Life Sciences, Pohang University of Science and Technology transposons, genomic DNA, mRNA and vectors are (POSTECH) (graduate), Pohang, Republic of Korea captured at double-strand breaks (DSBs) sites when DSBs are introduced by the CRISPR-Cas9 system. Introduction: Soluble factors released by cells play Therefore, it is possible that unintentional insertions important roles in intercellular communication. associated with DSB repair represent a potential risk However, extracellular vesicles (EVs) have recently for human genome editing gene therapies. To address attracted much attention as intercellular communica- this possibility, comprehensive sequencing of DSB sites somes, complex extracellular organelles that mediate was performed, and we found that bovine DNA frag- intercellular communication. While it has been ments were captured at DSB sites in fertilized mouse reported that EV-associated molecules elicit greater eggs and cell lines. activities than soluble forms, no studies have compared Methods: We determined the lengths of the indels the activities of EVs as a whole with soluble factors. In introduced by the CRISPR-Cas9 system in vivo and in this study, EVs and soluble factors derived from bac- vitro by deep sequencing of PCR products amplified teria were compared with regard to local and systemic with two primers across the target DSB site. inflammatory activities. All animal studies were conducted in accordance with Methods: Escherichia coli was cultured in a chemically the guidelines approved by the animal care committee defined medium, and conditioned medium (CM) was of the National Institute of Health Sciences. harvested from the culture. EVs and soluble factors JOURNAL OF EXTRACELLULAR VESICLES 359

(CM-EVs) were isolated from 3 kDa cut-off concen- However, no studies have assessed the effects of trated CM by ultracentrifugation. RAW264.7 cells were Gram-negative bacterial EVs on angiogenesis. treated with EVs and CM-EVs, then the release of Methods: Escherichia coli EVs were subcutaneously TNF-α and IL-6 were measured with ELISA. In addi- administered to wild-type mice, along with Matrigels. tion, wild-type mice were intraperitoneally adminis- The Matrigels were subjected to whole mount immu- tered with EVs and CM-EVs, and septic signs were nostaining, and vascular area was measured. As macro- observed. Inflammatory indices including the concen- phages are involved in angiogenesis, macrophage trations of TNF-α and IL-6 as well as the numbers of infiltration was also assessed in the Matrigels. infiltrated immune cells were also assessed from the Peritoneal macrophages from wild-type mice were peritoneal lavage fluid, serum and bronchoalveolar treated with E. coli EVs, and the conditioned media lavage fluid. were treated to endothelial cells to measure cell migra- Results: EVs mediated the release of IL-6 from tion. Furthermore, to show the role of interleukin-6 RAW264.7 cells in vitro, with greater extent than (IL-6) on angiogenesis, E. coli EVs were subcutaneously CM-EVs. Unlike CM-EVs, EVs mediated systemic sep- administered to wild-type and IL-6 knock-out mice, tic symptoms including hypothermia, eye exudate for- along with Matrigels. Then, the Matrigels were sub- mation and leukopenia. While both EVs and CM-EVs jected to whole mount immunostaining, and vascular mediated immune cell infiltration into the peritoneum, area was measured. In addition, peritoneal macro- EVs mediated the elevation of the concentrations of phages from wild-type and IL-6 knock-out mice were TNF-α and IL-6 in the peritoneal lavage fluid, more treated with E. coli EVs, and the conditioned media efficiently than CM-EVs. In addition, EVs mediated from the macrophages were treated to endothelial cells the elevation of the concentrations of TNF-α and IL-6 to measure cell migration. in the serum, whereas, CM-EVs did not. More impor- Results: E. coli EVs promoted in vivo angiogenesis and tantly, EVs mediated immune cell infiltration as well as macrophage infiltration in wild-type mice. Peritoneal the elevation of the concentrations of TNF-α and IL-6 macrophages from wild-type mice, treated with E. coli in the bronchoalveolar lavage fluid, whereas CM-EVs EVs, mediated endothelial cell migration in vitro. did not. However, E. coli EVs did not promote angiogenesis Summary/conclusion: Although EVs and soluble fac- and macrophage infiltration in IL-6 knock-out mice. tors mediated local inflammatory responses, only EVs Furthermore, peritoneal macrophages from IL-6 can act as long-range effectors in systemic inflamma- knock-out mice, treated with E. coli EVs, did not med- tory responses, suggesting EVs as new therapeutic tar- iate endothelial cell migration. gets for infectious diseases. Summary/conclusion: Gram-negative bacterial EVs have potent angiogenic activities by promoting macrophage infiltration and inducing IL-6. These findings LBS02.05 provide insights into the effects of Gram-negative bacterial EVs on bacterial infection-related pathological diseases including bacterial infection, inflammatory Gram-negative bacterial extracellular vesicles promote angiogenesis diseases, and bacterial sepsis. by inducing interleukin-6 Jaemin Leea, Jaewook Leeb, Tae-Young Rohc and Yong Song Ghod aPohang University of Science and Technology, Pohang, Republic of Korea; bDepartment of Life Sciences, Pohang University of Science and Technology LBS02.06 (POSTECH), Pohang, Republic of Korea; cDiv. of IBB, Dept of Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, Republic of Korea; dDepartment of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea Dendritic cell derived-exosomes activate immune systems by transferring exosome involved factors to T cell Masakatsu Takanashia, Shinobu Uedaa, Katsuko Sudob and Masahiko Introduction: Angiogenesis, the formation of blood Kurodaa vessels from pre-existing vasculature, is an essential aDepartment of Molecular Pathology, Tokyo Medical University, Tokyo, complex process for multiple pathophysiological con- Japan; bAnimal Research Center, Tokyo Medical University, Tokyo, Japan ditions including bacterial infection, inflammatory diseases and bacterial sepsis. Several pathological Introduction: Exosomes released from dendritic cells functions of Gram-negative bacterial extracellular vesi- (DCs) are responsible for the persistence of antigen cles (EVs), also known as outer membrane vesicles presentation. So, we considered that whether DCs- have been shown to induce local inflammation, sys- derived exosomes could induce suppress cancer cells temic inflammation, and septic shock, and so on. and more effective response of an immune system and 360 ISEV2019 ABSTRACT BOOK what factors in exosomes-involved DCs can activate T or TG induces autophagy using immunofluorescence cells. microscopy, transmission electron microscopy and Methods: Luciferase gene transferred-3LL cells (mur- Western blot analysis. Also, to investigate TM or TG ine lung cancer cell line derived C57BL/6) were inhibits oxidative stress through the induction of injected into C57BL/6J mice by intraperitoneal admin- autophagy. In addition, we established an adenine- istration. And then, DCs, DCs-exosomes or 3LL-exo- induced chronic kidney disease (CKD) mice model. somes were weekly administrated to lung cancer- The effectiveness of TM or TG was investigated in bearing mice. The exosomes derived from DCs CKD mice model. decreased lung cancer cell growth compared with Results: Low concentrations of TM and TG did not DCs, DCs-exosomes and non-treated. We evaluated affect cell viability in HK-2 cells. TM and TG induced mRNA expressions differences in between LPS-treat- UPR pathway and autophagy. Furthermore, TM and ment DCs and none treatment DCs with DNA micro- TG inhibited oxidative stress-induced cell death. The array analysis. inhibition of autophagy can increase cytotoxicity in Results: DNA microarray analysis data showed that 44 HK-2 cells. Therefore, TM- and TG-induced autophagy genes increased as ratio LPS-treated DC mRNA vs palys a protective role. The inflammasome and cyto- non-treatment DC one. Western blot analysis showed kine synthesis were suppressed after treatment with one of the genes contained higher in exosomes derived TM and TG. In addition, HK-2 stimulated with TM from LPS-treatment DCs than that derived from non- or TG had increased production of exosomes. In the treatments. model of adenine diet-induced CKD, TM and TG Summary/conclusion: This gene induces T cell prolif- ameliorated renal dysfunction and injury through the eration and signals for T cell maturation. We con- induction of ER stress and autophagy. cluded that DCs derived-exosomes activate anticancer Summary/conclusion: These results suggest that TM immune systems by transferring exosome-involved the and TG protected kidney cells against oxidative stress- factor to T cells. induced cell death and inhibited inflammatory effect. ER stress-induced autophagy may be a pro-survival role. However, the complete mechanism of how TM LBS02.07 and TG regulate exosomes is yet unknown and need further investigation.

Crosstalk between endoplasmic reticulum stress and autophagy in kidney diseases Yuh-Feng Lina and Hui-Wen Chiub LBS02.08 aTaipei Medical University, Taipei, Taiwan (Republic of China); bGraduate Institute of Clinical Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (Republic of China) Extracellular Vesicle-induced protein phosphorylation: Rapid activation of epithelial-mesenchymal transition pathways in lung Introduction: The endoplasmic reticulum (ER) regu- epithelial cell a b c d lates several cellular functions, including the protein Ganesh Shelke , Yin Yanan , Cecilia Lasser , Hjalmar Brismar and Jan Lötvalle biosynthesis, folding, trafficking and modification. aSahlgrenska Cancer Center/Department of Surgery, Institute of Clinical The accumulation of unfolded or misfolded proteins Sciences, University of Gothenburg, Gothenburg, Sweden., Gothenburg, causes a form of cellular stress that has been termed ER Sweden; bDepartment of Biochemistry and Molecular Cell Biology, Shanghai Jiao Tong University, School of Medicine 80 South Chongqing stress. ER stress activates the unfolded protein response Road, Shanghai 200025 China, Shenghai, China (People‘s Republic); (UPR) signalling network which serves as an adaptive cKrefting Research Centre/University of Gothenburg1 Krefting Research response. The potential benefit of maintaining ER Centre, Dept of Internal medicine and clinical nutrition, Institute of Medicine, University of Gothenburg, Sweden, Gothenburg, Sweden; homeostasis modulates ER stress status to protect the dScience for Life Laboratory, Dept. of Applied Physics, Royal Institute of kidney against various pathogenic environments. Technology, PO Box 1031, 17121, Solna, Sweden, Solna, Stockholm, Sweden; eKrefting Research Centre, Institute of Medicine at the Sahlgrenska Academy, Furthermore, ER stress induces autophagy in mamma- University of Gothenburg, Göteborg, Sweden, Gothenburg, Sweden lian cells. The ER stress-induced autophagy offers protection from oxidative-induced cytotoxicity and Introduction: Extracellular vesicles are important ameliorated kidney injury. In this study, we understand mediators of cell-to-cell communication. With their the mechanism modulated the regulation of UPR and bioactive cargos including proteins, lipids and nucleic autophagy in kidney cells. acids, they can alter the fate of a recipient cell. Mast- Methods: We examined cytotoxicity of ER stress indu- cells and lung epithelium exists in close physical proxi- cers (tunicamycin (TM) or thapsigargin (TG)) in mity and activity in mast cells is reflected in epithelial human kidney cells HK-2. To analyse low doses TM cells. In this study, we hypothesized that mast cell- JOURNAL OF EXTRACELLULAR VESICLES 361 derived EVs alter recipient epithelial cells by inducing Introduction:Idiopathicpulmonaryfibrosis(IPF)is phosphorylation of multiple proteins. achronicandprogressivelungdiseaseforwhichno Methods: Mast cells derived-EVs (HMC1.1) were treatment is capable of providing a complete cure. obtained by differential ultracentrifugation. We deter- The median survival for IPF patients from the time mined the early protein phosphorylation induced by of diagnosis is approximately 3 years. IPF patients EVs, in recipient cell A549 cells using phospho-protein differ in terms of the disease progression rate and microarray (Sciomics), and determined the longer- prognosis, complicating the prediction of survival. term effects on RNA transcripts and protein changes The identification of prognostic predictors for IPF in epithelial cells. is important for determining who requires the most Results: Prolonged exposure of EVs altered cellular intensive therapies. In this study, we explored the morphology of recipient epithelial A549 cells. This possibility that microRNAs of serum EVs changed was in line with changes in the transcript that are during lung fibrosis and could serve as prognostic known to activate epithelial-mesenchymal transition biomarkers of IPF. (EMT), including increased levels of TWIST1, MMP9, Methods: To determine target microRNAs in IPF, we TGFB1, and BMP-7. This was also reflected at the measured serum EV microRNA expression profiles protein levels in recipient cells; e.g downregulation of using microRNA PCR arrays in a bleomycin mouse CDH1 and upregulation of MMP. By contrast, EMT lung fibrosis model. Secondly, we enrolled 41 IPF inducing transcription factor Slug-Snail was upregu- patients and conducted a 60-month prospective cohort lated. To determine any rapid responses 30 minutes study. Expression of serum EV miR-21-5p was normal- after EV treatment we performed phospho-protein ized by dividing by the EV amount. The relative microarray of recipient cells. In-silico analysis of phos- amount of EVs was measured using the ExoScreen pho-proteome revealed proteins in signalling networks method. We calculated the correlations between base- that are part of the PI3K-Akt pathway or cytokine line serum EV miR-21-5p expression and other clinical receptor interactions. Interestingly, a protein involved variables. Furthermore, we determined if serum EV in regulating focal adhesion and tight junctions was miR-21-5p can predict mortality during 60 months phosphorylated in these experiments; e.g. CLDN1, using the Cox hazard model. According to the median OCLN, and ACTN1. Finally, we validated one of the level, we divided the IPF patients into two groups. well-studied EMT-regulating pathway (TGFβ signal- Then we compared the survival rate during 60 months ling) in both A549 and BEAS-2B cell lines. between the two groups using the Kaplan-Meier Summary/conclusion: Mast cell-derived EV facilitates method. activation of EMT in lung epithelial cells, which is Results: Serum EV miR-21-5p was elevated in both the closely associated to EMT-associated protein phos- acute inflammatory phase (day 7) and the chronic phorylation. This study highlights the component of fibrotic phase (day 28) in the mouse model. In the signalling pathways that are rapidly phosphorylated in clinical setting, serum EV miR-21-5p was significantly recipient cells with the contact of EVs. higher in IPF patients than in control subjects. The Funding: VBG group Herman Krefting Foundation, baseline serum EV miR-21-5p was correlated with the Swedish Cancer Foundation, Swedish Research rate of decline in vital capacity over 6 months. Council, and Heart and Lung Foundation, EAACI, Furthermore, serum EV miR-21-5p was independently AG Foundation, Lundgren Foundation, Sahlgrenska associated with mortality during the following University Hospital, and Sahlgrenska Academy. 60 months, even after adjustment for other variables. In the survival analysis, IPF patients whose baseline LBS02.09 serum EV miR-21-5p was high had a significantly poorer prognosis over 60 months. Summary/conclusion: Our results may suggest that Serum extracellular vesicular miR-21-5p is a predictor of the prognosis in idiopathic pulmonary fibrosis serum EV miR-21-5p has potential as a prognostic Mitsuhiro Yamadaa, Tomonori Makiguchia, Yusuke Yoshiokab, Takahiro biomarker for IPF. c a Ochiya and Masakazu Ichinose Funding: This work was supported by Grants-in-Aid aDepartment of Respiratory Medicine, Tohoku University Graduate School of for Scientific Research (24591148 and 15K09206 to M Medicine, Sendai, Japan; bTokyo Medical University, Tokyo, Japan; cDepartment of Molecular and Cellular Medicine, Institute of Medical Yamada) from the Japan Society for the Promotion of Science, Tokyo Medical University, Tokyo, Japan Science (JSPS). 362 ISEV2019 ABSTRACT BOOK

LBS02.10 markers (CDs and ICAM-1). Protein arrays were used to discover the immunomodulatory content of subsets. In addition, functional integrity of the EV subpopula- Extracellular vesicles subpopulations: who are the key players in tions was assessed using migration cell based assays. vascular inflammation? Baharak Hosseinkhania, Sören Kuypersa, Nynke Van den Akkerb, Daniel Results: We demonstrated that HUVEC upon inflam- Molinb and Luc Michielsa mation release two distinct populations of heteroge- aHasselt University, Martelarenlaan 42, B-3500 Hasselt, Belgium, Diepenbeek, neous EV, differing in size and quantity. The Belgium; bMaastricht University, dept. Of Physiology, Cardiovascular Research Institute Maastricht (CARIM), Universiteitssingel 50, 6200 MD immunoaffinity of these two populations towards EV Maastricht, The Netherlands, Maastricht, Netherlands classical markers (a cocktail of CD9, CD63 and CD81) and an inflammatory-associated marker revealed that Introduction: Current conceptual insights on the the circulating form of ICAM-1 is abundantly docked mechanistic aspect of cell signalling by discovering on the membrane of large EV, thus offering a poten- extracellular vesicles (EV) have shed light on the patho- tially promising biomarker for immunocapturing of EV logical understanding of chronic inflammatory dis- subsets. Moreover, protein profiling of EV size-based eases. The biological function of EV bulk in the populations and their inflammation-associated EV sub- progression of inflammation-associated disorders has sets showed that the patterns of cytokines and adhesion become clear, the protein composition and function of markers were significantly different. In cell-based the disease-specific subsets are often hampered by assays, EV of different sizes work synergistically in undesirable EV co-isolates. Thus, there is an urgent accelerating the vascular inflammation. need to isolate certain subpopulations to generate a Summary/conclusion: A procedure of two purification disease-relevant signature while retaining their func- steps resulted in purer inflammation-associated EV tional integrity. Therefore, we aimed to fractionate isolates, allowing a better understanding of their biol- the inflammation associated-EV subsets based on two ogy and functions at the onset of vascular important characteristics (sedimentation and surface inflammation. markers) and subsequently profiling the immunomo- Funding: This work was co-financed by the EU dulatory protein content. through the Interreg IV Flanders-the Netherlands pro- Methods: TEM, NTA and Western blot were used to ject Interreg V Flanders-the Netherlands project Trans characterize the purified inflammation-associated EV Tech Diagnostics (TTD). subsets from TNF-α treated HUVEC based on their sedimentation speeds (10K and 110K) and surface JOURNAL OF EXTRACELLULAR VESICLES 363

LBS03: Late Breaking- EV Biogenesis, Loading, and Uptake Chairs: Samarjit Das; Wang Jiang Location: Level 3, Hall A 15:00–16:00

labelling approach rendered its result more reliable and LBS03.01=OWP1.15 was used to compare ExoB16 biodistribution in melanoma-bearing immunocompromized (NSG) mice. Similar biodistribution profile was observed in both Membrane-radiolabelled exosomes for comparative biodistribution C57BL/6 and NSG mice, where prominent accumula- analysis in immunocompetent and immunodeficient mice – A novel and universal approach tion was seen in liver and spleen, apart from the lower Farid N. Faruqua, Julie Wanga, Lizhou Xub, Luke McNicklea, Ming-Yiu tumour accumulation observed in the NSG mice. a c c d a Chong , Mark Gurney , Aled Clayton , Lesley A. Smyth , Robert Hider , Summary/conclusion: Membrane radiolabelling of Jane Sosabowskie and Khuloud Al-Jamala exosomes is a reliable approach that allows for both aKing‘s College London, London, United Kingdom; bSchool of Cancer and Pharmaceutical Sciences, King‘s College London, London, United Kingdom; live imaging and quantitative biodistribution studies to cCardiff University, Cardiff, United Kingdom; dUniversity of East London, be performed on potentially all exosome types without London, United Kingdom; eQueen Mary University of London, London, United Kingdom engineering parent cells.

Introduction: Exosomes have gained interest as novel LBS03.02 drug nanocarriers due to their biological origin and role in intercellular biomolecule delivery. In-depth Rala and ralb finely tune EVs biogenesis and promote metastasis knowledge of their in vivo biodistribution is therefore Vincent Hyennea, Shima Ghoroghib, Olivier Lefebvreb and Jacky G. Goetzb essential. This work aimed to develop a reliable and aINSERM U1109/CNRS, Strasbourg, France; bINSERM U1109, Strasbourg, universal method to radiolabel exosomes to study in France vivo biodistribution in mice. Methods: Melanoma (B16F10 cells)-derived exosomes Introduction: Tumour extracellular vesicles (EVs) pro- (ExoB16) were isolated and characterized for size, mote tumour progression. However, their behaviour in yield, purity, exosomal markers and morphology body fluids remains mysterious. In addition, further using Nanoparticle Tracking Analysis (NTA), protein understanding of molecular mechanisms driving their measurements, flow cytometry and electron micro- biogenesis is needed to develop strategies aiming to scopy. Two radiolabelling approaches were explored – impair their tumorigenic potential. We recently intraluminal labelling (111Indium entrapment via tro- showed that the zebrafish embryo can be used to polone shuttling); and membrane labelling (111Indium track and assess the function of circulating tumour chelation by covalently attached bifunctional chelator). EVs in vivo and provide a high-resolution description Labelling efficiency and stability was assessed by gel of their dissemination and uptake (Hyenne et al., Dev filtration and thin layer chromatography. Melanoma- Cell, 19). We provided a first description of tumour bearing immunocompetent (C57BL/6) and immunode- EVs’ hemodynamic behaviour and showed that they ficient (NSG) mice were injected intravenously with are rapidly taken up by endothelial cells and blood radiolabelled ExoB16 (1x1011 particles) followed by patrolling macrophages and subsequently stored in metabolic cages study, whole body SPECT-CT imaging degradative compartments. and ex vivo gamma counting at 1, 4 and 24 h post- Methods: In addition, we recently investigated the injection. molecular mechanisms of EV release in a tumorigenic Results: Membrane-labelled ExoB16 (ML-ExoB16) context, using a mouse model of breast cancer showed superior radiolabelling efficiency and radioche- carcinoma. mical stability compared to intraluminal-labelled Results: We observed that depletion of either RalA or ExoB16 (IL-ExoB16). Both IL- and ML-ExoB16 RalB GTPases decreases levels of EVs’ secretion showed prominent accumulation in liver and spleen. (Hyenne et al. JCB 15) and modifies their protein and IL-ExoB16 showed higher tumour accumulation than RNA content. We further showed that RalA and B are ML- ExoB16 (6.7% and 0,6% ID/g tissue, respectively), required to properly localize PLD1 on MVBs thereby with the former showing similar value as its free tracer inducing EVs biogenesis. Interestingly, EVs secreted ([111]Trop). The superior stability of the membrane- from RalA and RalB depleted cells are less prone to 364 ISEV2019 ABSTRACT BOOK endothelial permeabilization in vitro. Finally, RalA and LBS03.04 RalB depletion significantly impairs lung metastasis in a syngeneic model of breast carcinoma suggesting that RalA/B controls lung metastatis by tuning the levels New products from donated blood – unravelling the potential of and contents of tEVs. blood cell derived EVs a b b Summary/conclusion: Overall, our recent works Ulla Impola , Sami Valkonen and Saara Laitinen a b proves the usefulness and prospects of zebrafish Blood Service, Finnish Red Cross, Helsinki, Finland; Blood Service Finnish Red Cross, Helsinki, Finland embryo to track tumour EVs and dissect their role in metastatic niches formation in vivo. It further provides Introduction: Extracellular vesicles (EV) originated new mechanistic information as to how RalA and RalB from different cell types have recently been under control the biogenesis of potent tumour-promot- intense investigation. Platelet EVs constitute the ing EVs. major fraction of EVs in the circulating plasma, however, there are only a few studies characterizing the LBS03.03 populations of platelet concentrate derived EVs in more detail. Few recent publications show that plasma EVs can target specifically into certain mononuclear In vivo visualization of extracellular vesicles released from mature cell populations but little is known about their biologi- osteoblasts by intravital multiphoton microscopy Hiroki Mizuno, Maki Uenaka and Masaru Ishii cal function, signalling and communication. As just

Department of Immunology and Cell Biology, Graduate School of Medicine recently addressed by Onodi et al., EV purification and Frontier Biosciences, Osaka University, Osaka, Japan, Suita, Japan has major challenges as majority of EVs from plasma has lipoprotein particles and the abundant plasma pro- Introduction: Bone remodelling is essential for main- teins as impurities complicating the study of the role of taining bone architecture and systemic mineral home- pure EVs. We have previously shown that platelet ostasis throughout life. In the process, the formation of concentrates used for transfusions contain increasing bone matrix by osteoblasts follows the removal of amount of EVs after longer storage period. It is impor- mineralized bone by osteoclasts. Despite intensive tant to study these platelet-EVs in more detail in order investigations on understanding their functions, the to understand their role in product functionality. detailed mechanisms on their dynamic nature in vivo Methods: Excess platelet concentrates not needed for remain unknown. In this study we especially focus on the clinical use were obtained from the Finnish Red the dynamics of mature osteoblasts which replenish Cross Blood Service. All donated blood products used bone matrices during homeostasis. for research were obtained from healthy volunteers Methods: To understand the cellular dynamics of who had given their informed consent. In our ongoing mature osteoblasts in vivo, here we established a repor- work we compared ultracentrifugation based isolation ter system where mature osteoblasts express enhanced methods and size exclusion chromatography in order cyan fluorescent protein (ECFP). We could visualize to collect differing populations of platelet concentrate their dynamic nature in vivo by using intravital multi- derived EVs. EVs are further labelled with fluorescent photon microscopy for live bone tissues which we have surface protein, lipid and RNA markers and studied originally developed so far. using Amnis ImageStream®X Mark II Imaging Flow Results: We detected that mature osteoblasts sponta- Cytometer. Purity and characteristics of these isolated neously release large extracellular vesicles (EVs), whose EVs are compared and their targeting into different sizes are from 0.2 to 1 µm, and those are also taken up mononuclear cells as well as their immunological rele- by mature osteoblasts. Such phenomenon could also be vance are investigated. reconstituted in mature osteoblasts cultured ex vivo. Results: Based on our results we are able to say that we Further analyses are currently ongoing in order to get a pure population of EVs with low contamination analyse the physiological and pathophysiological func- of lipid or plasma protein impurities. The main popu- tions of these vesicles. lation of the platelet concentrate derived EVs are plate- Summary/conclusion: This is the first study detecting let derived and thus CD41 positive, however, the origin the real dynamic nature of microvesicles in vivo, which of EVs differ as well as their cargo indicating differ- are actively released from mature osteoblasts in the ences in their immunological functions. bone cavity. We think these microvesicles are impor- Summary/conclusion: Our aim is to find previously tant regulators for normal bone homeostasis as well as ignored, new applications for donated blood compo- pathological remodelling. nents and to identify the potential EV population to be JOURNAL OF EXTRACELLULAR VESICLES 365 utilized either as therapeutic components in tissue 3CD/3C protease activities during HRV16 infection. repair or as drug delivery vehicles. Therefore, these proteases may be hypothesized to be embedded in HRV16MV suggesting that they could potentially be hijacked by the virus to spread infection. LBS03.05

The effect of rhinovirus type 16 derived microvesicles on the growth LBS03.06 of hela cells Roberta Cordeiro Freezora, Gary McLeanb and Sheelagh Heughc aLondon Metropolitan University, London, United Kingdom; bSupporting A highly efficient cell-free protein synthesis system from PhD supervisor, London, United Kingdom; cPhD supervisor, London, plasmid DNA. United Kingdom Hyangsu Nam University of Seoul, Seoul, Republic of Korea Introduction: Belonging to group A, Rhinovirus Type 16 (HRV16) uses the receptor Intercellular Adhesion Introduction: Protein is essential molecules that play Molecule (ICAM) 1 to enter cells. Studies demon- many critical roles in the body. Activity and interaction strated Extracellular Vesicles (EV) discharge from of gene encoding proteins are recognized as a key viral infected cells harbour and distribute regulatory element of pharmaceutical and biomaterials. However, factors to recipient cells. These include viral RNA and since the general protein production method is per- proteins, viral and cellular miRNA, as well as host formed through microorganisms and cell culture pro- functional genetic elements to nearby cells, leading to cesses that require a lot of time and labour, it is not the production of infections particles and modulating only decomposed and deformed by enzymes produced cellular responses including the spread or limitation of by cell growth but also has a limit to producing a large infection conditional on the type of pathogen and amount of protein. This study reports a DNA hydrogel target cells. Here, we demostrate that HRV16 derived that can induce excessive protein without lives cells microvesicles (HRV16MV) infect HeLa cells at a higher using denatured DNA plasmid. This novel synthesis rate than HRV16 particles. method improved the efficiency of protein expression Methods: HRV16MV were extracted from HeLa cells through Rolling circle amplification that repetitive after 24 h of HRV16 infection (MOI 0.2) via ultracen- sequencing of nucleotides for protein expression in trifugation. Quantified by Flow cytometry, HeLaMV plasmid DNA that codes a specific gene. Notably, our control (HelaMVc) and HRV16MV were added to synthesized DNA plasmid gel expressed large scale each well containing the HeLa cells treated with CGM green fluorescence protein (GFP) than wild type. In (exosome free). The plates were incubated at 37°C in a addition, DNA gel has the strength of having a physical 5% CO2 and left untreated for 24 h. HeLa cells control characteristic and being able to express the specific samples were observed against MV treated cells and the protein in a specific area. Novel large-scale protein concentration/mL and % viability were determined expression method using cell-free DNA gel has great every 4 h after 12 h incubation period to determine potential as a biomaterial for therapeutic protein deliv- the effect of MV on the growth of the cell line. ery. In addition, this method can be applied mRNA Results: HRV16MV treated cells showed a growth manipulation, which plays a major role in transmitting decline after 16 h into the experiment, which suggests proteins, can be used to transfer particles using RCA a faster infection rate (P**) when compared to HRV16 method and to produce vesicles containing proteins by infection. Both, HRV16 and HRV16MV treated cells putting them in vesicles. demonstrated a decline in % viability (P>**) after 16 h Methods: The plasmid pT7CEF1-Chis was prepared of infection in comparison to HeLaMVc. However, Thermofisher. Linear single stranded circular DNA despite a slight decrease in in the growth rate of oligonucleotides from denatured DNA plasmid were HRV16MV treated cells no statistical significance was incubated with T4 polymerase at 30℃ 72 h with observed in % viability between samples. dNTPs. Summary/conclusion: HRV16MV treated cells showed Results: We developed the periodically repeated an advanced infection rate of treated HeLa cells. nucleotides through RCA process band its cell-free HRV16 genome encodes two proteases specifically, largescale protein expression system. The synthesized 2A and 3C as well as a precursor protease, 3CD. plasmid DNA hydrogel has stable formation and hav- These proteases are necessary for adequate virus repli- ing a high efficiency to express specific protein. cation, for the precise localisation of proteins during Summary/conclusion: In this study, we developed cell- infection and for the temporal regulation of 2A and free protein expression method, which were 366 ISEV2019 ABSTRACT BOOK synthesized by a uniquely designed RCA method, as a Summary/conclusion: Our results describe for the first protein therapeutics platform technology. The cell-free time a novel anticancer potential of VPA, being able to protein producing DNA plasmid gel can be considered impact cancer cell to cell communication by reducing as a potential candidate for protein delivery. the shedding of large EVs. Funding: AIRC FIRC LBS03.07 LBS03.08

Targeting mevalonate pathway with low doses of valproic acid The role of glycogen synthase kinase 3 beta in the biogenesis of reduces large EVs shedding in different solid tumours extracellular vesicles by modulating microtubule dynamics Chiara Ciardielloa, Rossella Migliorinoa, Federica Iannellia, Maria S. Rocaa, Seoyoon Baea, Eun-Jeong Choia, Kwan Soo Hongb and Yong Song Ghoa Francesca Bruzzesea, Biagio Puccia, Susan Costantinia, Elena Di Gennaroa, a b Alessandra Leone and Alfredo Budillon aDepartment of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea; bBioimaging Research Team, Korea Basic Science aExperimental Pharmacology Unit, Istituto Nazionale Tumori – IRCCS- Institute, Cheongju, Republic of Korea Fondazione G. Pascale, Napoli, Italy; bIstituto Nazionale Tumori – IRCCS- Fondazione G. Pascale, Napoli, Italy Introduction: Extracellular vesicles (EVs) are spherical, Introduction: Epithelial to mesenchymal transition bilayered membranous vesicles secreted by all living (EMT) as well as mesenchymal to amoeboid transition cells. EVs harbour various bioactive materials, and (MAT) are linked with increased cancer cell motility play diverse roles in biological processes such as and stemness, MAT being also described to favour tumour progression. There are several reports studied large extracellular vesicles (EVs) shedding. Recently, on the proteins involved in EV biogenesis mainly both these phenotypic changes were associated to focused on the proteins involved in vesicle trafficking. metabolic control involving the mevalonate pathway However, proteins regulating EV biogenesis are still (MVP), a key controller of lipid metabolism but also unclear. As most cellular processes are regulated by a regulator of cell structure and signalling. valproic acid protein phosphorylation, which is regulated by kinases (VPA), an antiepileptic and a well-known histone dea- and phosphatases, identifying kinases and phospha- cetylase inhibitor, showed antitumor activity and cap- tases involved in EV biogenesis helps to understand ability to augment anticancer efficacies of other EV-mediated pathophysiological functions. therapeutic approaches (i.e. ionizing radiation, che- Methods: To identify kinases and phosphatases motherapy, immunotherapy). involved in EV biogenesis, a total of 76 kinase inhibi- Methods: Two different isogenic models developed by tors and 33 phosphatase inhibitors were treated to our group were used: prostate cancer DU145 cells and A549 cells. The amounts of CD81, an EV-enriched their derived more aggressive subline DU145R80 protein, were quantified from the conditioned media selected as resistant to MVP-pathway inhibitors and to show alterations in EV biogenesis. To further verify enriched in stem markers; the colorectal cancer the role of glycogen synthase kinase 3 beta (GSK3β)in CO147 primary cell line, cultured either as differen- EV biogenesis, stable cell lines expressing wild-type, tiated cells or as cancer stem cells enriched spheres. constitutively active mutant, and dominant-negative Western blotting and metabolomics were performed to mutant GSK3β were established, and alterations in monitor MVP modulation upon VPA treatment (0.5- EV biogenesis were measured in these cell lines. As 1 mM). Large EVs were isolated from cell media by microtubule dynamics affects EV biogenesis, changes discontinuous density gradient ultra-centrifugations in microtubule dynamics were also assessed in these and measured by Tunable resistive pulse sensing or cell lines. flow cytometry VPA-treated or untreated cells. Results: Among the kinase and phosphatase inhibitors, Results: Both DU145R80 cells and CO147 cultured as an inhibitor of GSK3β and calcineurin decreased and spheres showed enriched stem like features and higher increased EV biogenesis, respectively. EV biogenesis large EVs shedding, compared to parental DU145 and was increased in the conditioned media from cells differentiated CO147 cells, respectively. At very low expressing constitutively active mutant GSK3β, and doses, VPA reduced large EVs shedding in both decreased in the conditioned media from cells expres- DU145R80 and CO147 sphere cultures, compared to sing dominant-negative mutant GSK3β, when com- the untreated cells, without affecting cells viability. pared with cells expressing wild-type GSK3β. Mechanistically, preliminary data suggest that VPA- Microtubules were more disorganized in cells expres- induced effect is mediated by MVP pathway sing constitutively active mutant GSK3β, and more modulation. aligned in cells expressing dominant-negative mutant JOURNAL OF EXTRACELLULAR VESICLES 367

GSK3β, when compared with cells expressing wild-type inhibitor 4-MU) blocked the shedding of all transfected GSK3β. HAS2 and its mutants. Summary/conclusion: By high-throughput screening Summary/conclusion: Our data show that an enzyma- of kinase/phosphatase inhibitors, we identified GSK3β tically inactive HAS2 residing in PM (K190R) as a positive regulator of EV biogenesis by modulating enhanced EV secretion to the same extent as microtubule dynamics. These observations suggest that HAS2 wt, while it did not induce the PM protrusions. GSK3β as a novel therapeutic target against several Just the insertion of HAS2 in PM must, therefore, diseases by modulating EV biogenesis. trigger a signal or structural alteration in the membrane that facilitates its inclusion in, and shedding of the EVs. LBS03.09 Another interesting finding was that while HA was not necessary for EV formation, the HA synthesis inhibitor 4-MU blocked HAS2 insertion in the EVs. This may Post-translational modifications affects trafficking of hyaluronan represent yet another mechanism of HA synthesis inhi- synthase 2 and the release of extracellular vesicles Raquel Maria. Meleroa, Uma Thanigai Arasub, Riikka Kärnäb, Sanna Oikarib, bition by 4-MU. Exploring the mechanism of the block Kirsi Rillab, Davide Vigettic, Alberto G. Passic, Heldin Paraskevid, Tammi and its importance in HA synthesis and EV shedding e e Markku and Ashik Jawahar Deen will be interesting targets of future studies, especially in a b CEU-San Pablo, Boadilla, Spain; Institute of Biomedicine, University of cancer epidemiology. Eastern Finland, Kuopio, Finland., Kuopio, Finland; cDepartment of Medicine and Surgery, University of Insubria, Varese, Italy., Varese, Italy; dDepartment of Medical Biochemistry and Microbiology, Uppsala University, Uppsala, Sweden, Uppsala, Sweden; eInstitute of Biomedicine, University of LBS03.10 Eastern Finland, Kuopio, Finland, Kuopio, Finland

Introduction: Hyaluronan synthase 2 (HAS2) is the Improving the stability of the large extracellular loop of human tetraspanin CD81 major producer of Hyaluronan (HA) in adult verte- Stefan Vogta, Gordana Wozniak-Knoppb, Gerhard Stadlmayrb, Katharina brates. Its enhanced expression has been lately related Stadlbauerb, Florian Rükerc and Johannes Grillarid within the apical filopodia growth and the budding of aDepartment of Biotechnology, University of Natural Resources and Life Sciences (BOKU), Vienna, Muthgasse 18, 1190 Vienna, Austria, Vienna, extracellular vesicles (EVs). Moreover, a fraction of Austria; bChristian Doppler Laboratory for Innovative Immunotherapeutics, HAS enzymes are secreted from PM into extracellular Department of Biotechnology, University of Natural Resources and Life vesicles (EVs), often covered by HA. We studied Sciences, Vienna (BOKU),Muthgasse 18, A-1190 Vienna, Austria, Wien, Austria; cChristian Doppler Laboratory for Innovative Immunotherapeutics, whether the mutations blocking post-translational Department of Biotechnology, University of Natural Resources and Life modifications on HAS2 also affected the EVs released. Sciences, Vienna (BOKU), Muthgasse 18, A-1190 Vienna, Austria, Wien, Austria; dEvercyte GmbH, Muthgasse 18, A-1190 Vienna, Austria, Wien, Methods: Site-directed mutagenesis was used to block Austria ubiquitination (K190R), and phosphorylation (T110A) HA was measured using ELSA Introduction: Members of tetraspanin protein family Isolation of EV secreted by HAS2-transfected cells was are abundant on the surface of nearly every type of performed using ultracentrifugation extracellular vesicles (EVs) and are therefore attractive Analysis of extracellular vesicles (EV) was performed targets for modification, leading to transformation of with a Nanoparticle Tracking Analyzer and 3D culture the EVs into a targeted drug delivery system. The Results: Cell cultures transfected with HAS2 wt engineering of tetraspanin extracellular domains as secreted ~50% more EVs as compared to mock con- independent folding units towards specific antigen trols. Similar stimulation of EV secretion was found recognition is therefore of particular interest. with K190R, while non-increase of EVs occurred with Methods: We have applied rigid body protein model- T110A. These results lead us to two conclusions. First, ling approach to design more stable mutants of large PM residence of HAS2 is likely required for the stimu- extracellular loop (LEL) of human tetraspanin protein lation of EV secretion. And second, HA synthesis is not CD81. Proteins were expressed in ExpiCHO expression strictly necessary for EV secretion, since K190R is system and IMAC-purified. Their stability was exam- enzymatically inactive. Cells were grown in a 3D ined using DSC and the protein fold integrity assessed matrix to check if K190R was entering itself in the with HPLC-SEC in native conditions and reactivity vesicles. The data show that HAS2 wt and K190R, but with structurally dependent binding anti-CD81 anti- not T110A were present in the EVs. This indicates that body. Mutants based on such stabilized scaffolds were the mechanism of HAS2 stimulation of EVs involves engrafted with human transferrin receptor (hTfr) spe- HAS2 incorporation in them, and without the involve- cific peptide at different positions, tested for their bio- ment of HA. Unexpectedly, 4-MU (HA synthesis physical properties and internalization in vitro. 368 ISEV2019 ABSTRACT BOOK

Results: In order to enhance the tolerance for modifi- supernatants of transfected HEK293T cells were har- cation we successfully identified positions that could vested and subjected to a serial centrifugation protocol accommodate pairs of point mutations to cysteine resi- (300 ×g for 10 min, 2000 ×g for 10 min and 10,000 × g dues, leading to de novo disulphide bridges in the for 30 min) to remove debris. Then, exosomes were human CD81 LEL. We achieved an increased thermal isolated from the cell culture medium by ultracentrifu- stability with a shift in melting temperature (Tm) of up gation (150,000×g for 2 h). Ferritin-SIRPα and mono- to 25°C in mutants with one additional disulphide mer SIRPα proteins were purified through an Ni-NTA bridge. Mutants harbouring a combination of 2 engi- chromatography step. For the impartial comparison, neered disulphide bonds showed an increased Tm of we adjusted the same amount of SIRPα proteins of 2 up to 43°C. nanocages in all experiments. The graft of a hTFR-binding peptide into the D-Helix Results: Exo-SIRPα exceeds Ferritin-SIRPα in all of the wild-type LEL resulted in a protein that still experiments, cell binding ability, enhancing phagocytic exhibited a compact fold. When the same peptide function of bone marrow derived macrophage, in vivo sequence was inserted between the helices A and B, anti-tumour effect and tumour specific immune the mutant showed an aberrant profile in SEC, which response. Exosome-SIRPα shows better feasibility com- could be cured by using a scaffold variant with a pared to ferritin-SIRPα; five-folds higher in the aspect stabilized LEL backbone. Additionally, both peptide- of cell binding ability, three folds higher of phagocyto- grafted proteins revealed increased internalization sic activity and 4 folds higher in the case of tumour into hTFR-overexpressing SK-BR-3 breast cancer cells growth inhibition. compared to the respective wild-type proteins. Summary/conclusion: We compared the efficacy of Summary/conclusion: These results define important two nanoparticles and concluded that exosome has requirements for improving the amenability of tetra- more advantages in delivering membrane proteins for spanins, in particular CD81 LEL, for their engineering therapeutic purpose. Our findings highlight the ability into a more versatile protein scaffold, which should of exosomes to display native membrane proteins on empower the design of antigen-binding tetraspanins their surface – a significant advantage of this delivery as targeting moieties of EVs and functionalize them system – and suggest that CD47 blockade by exosome- as a drug delivery vehicle. mediated SIRPα delivery is superior to that mediated by a protein scaffold. LBS03.11 LBS03.12

Comparison of exosomes and ferritin protein nanocages for the delivery of membrane protein theraqeutics Cell-specific growth surface topography optimization for extracellular Eunji Cho, Gi-Hoon Nam, Jiyoung Goo, Cherlhyun Jeong, Yoosoo Yang and vesicle studies In-San Kim Colin L. Hiseya, Cherie Blenkironb and Larry Chamleyc

Center for Theragnosis, Korea Institute of Science and Technology, Seoul, aUniversity of Auckland, Grafton, New Zealand; bThe University of Republic of Korea Auckland, Auckland, New Zealand; cThe University of Auckland

Introduction: Exosomes are small membrane vesicles Introduction: While patient fluid samples provide secreted by most cell types that plays an important role valuable insight into the role of EVs in human health, in intercellular communication. Due to the character- their limited supply and heterogeneous nature make istic of transferring their biomacromolecules, exosomes them impractical for basic studies. Conditioned media have potential as a new alternative for delivering pro- provides a consistent and limitless supply of EVs from tein therapeutics. Here, we investigate whether exo- a known cell type, but large volumes are required to somes provide crucial advantages over other generate adequate numbers of EVs. Also, little is nanoparticles, in particular protein nanocage formula- known about how factors in the cellular microenviron- tions, as a delivery system for membrane protein ther- ment, like surface topography, affect the EVs due to a apeutics. We characterized membrane-scaffold–based lack of accessible biomimetic cell culture systems. We exosomes and protein-scaffold–based ferritin nano- present a unique cell culture dish covered in micro- cages, both harbouring SIRPα (signal regulatory pro- track patterns and demonstrate that this biomimicry tein α), an antagonist of CD47 on tumour cells. affects the EVs produced by cancer cells. Methods: For preparing exo-SIRPα, HEK293T cells Methods: Microtrack patterns were fabricated using were transiently transfected with desirable plasmid photolithography. Soft lithography was used to create DNA. Following a further incubation for 48 h, the microtrack moulds, which were spincoated with JOURNAL OF EXTRACELLULAR VESICLES 369 polystyrene and stamped onto 150 mm petri dishes. new hybrid EVs expressing immune checkpoint pro- Oxygen plasma and UV sterilisation were used to pre- tein PD-1 by this method and evaluation of the func- pare the surfaces for cell growth. MCF7 breast cancer tions such as the specific interaction with cancer cells cells were seeded and cell viability and morphology Methods: The cDNA of PD-1 on a baculovirus vector were quantified. Live cells stained with Calcein-AM was transfected into Sf9 insect cells, and EVs that were were imaged and their morphology was quantified expressed PD-1 on the surface were collected by ultra- using FIJI. Cytoskeletal structure was imaged using centrifugation. The hybrid EVs were prepared by DAPI, TRITC-phalloidin and anti-vinculin/FITC-IgG. membrane fusion between PD-1 EVs and FITC- Cells were cultured in EV-depleted media for the last Dextran loaded-liposomes at the acidic condition. 48h and EVs from smooth (control) and patterned PD-1 and gp64 expression on PD-1 EVs and PD-1 dishes were isolated using Vivaspin ultrafiltration and hybrid EVs were detected by Western blotting. PD-1 sequential ultracentrifugation. Finally, EV structural hybrid EVs were incubated with Hela cells, and cellular integrity, concentration and size distribution were uptake of PD-1 hybrid EVs was observed by confocal characterized using TEM and nanoparticle tracking laser scanning microscopy (CLSM). analysis. Results: As results of Western blotting, PD-1 and gp64 Results: MCF7 cells cultured on microtrack dishes were detected on EVs and also hybrid EVs prepared at demonstrated similar viability to smooth surfaces. acidic pH. Membrane fusion between EVs containing Cell morphologies on microtracks had higher average gp64 and liposomes proceeded only under the acidic aspect ratios and less circularity (p < .05), as well as pH. Interaction between PD-1 hybrid EVs and PD-L1- greater actin cytoskeletal alignment. Early nanoparticle expressing cancer cells was investigated by CLSM. The tracking analysis results indicate that cells cultured on PD-1 hybrid EVs effectively internalized into the cells fibrous surfaces release more EVs than EVs from via interaction with PD-L1, and FITC-dextran (as a smooth surfaces and these results are currently being model of drug) loaded into PD-1 hybrid EVs was further corroborated. efficiently delivered into the cells. Summary/conclusion: This type of patterned growth Summary/conclusion: In summary, we prepared PD-1 surface could have implications in both EV biomimicry hybrid EVs by using baculovirus-expression system and biomanufacturing. While it appears that simple and membrane fusion with functional liposomes. This surface patterning with microtracks could simply and method provides a new strategy for engineering EVs. inexpensively improve EV-yield from cell cultures, we are now exploring whether it also affects their LBS03.14 biomimicry.

Carcinogenesis and exosome packaging LBS03.13 Parul Katocha, Jessica Rodrigueza, Mark Floryb, Randall Armstrongb and Thuy Ngob

aOregon Health and Science University, Portland, USA; bOHSU, port- Development of engineered extracellular vesicles expressing immune land, USA checkpoint protein PD-1 by fusion with liposomes Raga Ishikawaa, Shosuke Yoshidab, Shin-ichi Sawadaa, Sada-atsu Mukaia, Yoshihiro Sasakia and Kazunari Akiyoshia Introduction: Identification of cancer-specific biomarkers on exosomes has evaded researchers for years and aKyoto University, Kyoto, Japan; bNara Institute of Science and Technology, Ikoma, Japan poses an enormous roadblock towards developing effective diagnostic tools for early cancer detection. Introduction: Extracellular vesicles (EVs) can poten- This is mainly due to the lack of a unique exosome tially be used in biomedical applications in drug deliv- marker. Therefore, if we intend to use exosomes as ery system. However, many problems still remain, such cancer biomarkers in liquid biopy we need to identify as equipping EVs with active targeting ability and con- unique markers present on the exosomes or associated trolling the encapsulation of therapeutic drugs in EVs. with them in cancer. Thus, we aim to delineate mole- We have developed a new method for the preparation cular mechanisms employed during exosome packa- of the functional EVs that display exogeneous mem- ging and cargo-loading. We hypothesize that cancer brane proteins using the baculovirus-expression sys- cell employ distinct molecular machineries to package tem. In addition, we have proposed that EV – protein-cargo vs RNA-cargo into exosomes. We further liposome hybrids were prepared by membrane fusion postulate that understanding the packaging proteins between liposomes and EVs containing fusogenic viral involved in this cargo-specific packaging will reveal glycoprotein 64 (gp64). Here, we report preparation of key stages of biogenesis and will aid in identifying 370 ISEV2019 ABSTRACT BOOK unique markers associated with exosomes during myristoylation sequence), transmembrane cargo pro- carcinogenesis. tein (EGFP-tagged α5β6 integrin) and an RNA mole- Methods: Recombinant DNA technology, confocal, cule (metabolically labelled). Exosomes generated by super-resolution imaging, ultracentrifugation, ultrafil- the system are isolated and characterized. tration and size exclusion columns, nanoparticle-track- Summary/conclusion: Thus, we have generated a ing analysis electron microscopy, high-resolution flow unique tool, with which we aim to identify – a) unique cytometry and Western blotting patterns of cargo packaging within these exosomes and Results: We have generated an in-vitro cell culture b) to elucidate the proteins involved in this directed system comprised of lung-cancer cell lines BEAS2-B packaging. Research undertaken in this project will and A549. The former represents early stage indolent provide new and exciting avenues to detect signs of disease while the latter represents late stage aggressive early cancer progression holds immense potential for disease. The culture system is genetically engineered to identifying cancer biomarkers. stably express tagged components of an exosome, Funding: CEDAR (Cancer Early Detection Advanced namely- lipid bilayer (Td-Tomato-tagged Research Center), Knight Cancer Institute) JOURNAL OF EXTRACELLULAR VESICLES 371

Symposium Session 26: Flow Cytometric Analysis of EVs Chairs: Xiaomei Yan; Joshua Welsh Location: Level 3, Hall B 16:00–17:00

when human plasma is used in generic fluorescent- OS26.01 based FC-detection of EV. Summary/Conclusion: In order to perform reliable

Influence of lipoprotein particles in extracellular vesicle analysis by and reproducible fluorescent-based FC-analysis of sin- single particle flow cytometry gle EV from human plasma, either EV-specific fluor- a b b Estefanía Lozano-Andrés , Sten F. Libregts , Ger Arkesteijn and Marca H. escent dyes or labels should be used or plasma samples M. Waubenc should be carefully cleared from particles prone to aDeaprtment of Biochemistry & Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands; bFaculty of Veterinary Medicine, incorporate the generic dye. Utrecht University, Utrecht, Netherlands; cDepartment of Biochemistry & Funding: European Union’s Horizon 2020 research Cell Biology, Faculty of Veterinary Medicine, Utrecht University, Utrecht, Netherlands and innovation programme under the Marie Skłodowska-Curie grant agreement No [722148] and Introduction: High-resolution flow cytometry (FC) STW-Perspectief Cancer-ID grant [14,191]. allows for the detection of single extracellular vesicles (EV) and enables quantitative and qualitative charac- OS26.02 terization. EV in plasma has been associated with diseases, making them attractive for diagnosis and prognosis of patients. However, the presence of lipo- Single-particle analysis of exosome DNA/RNA abundance, identity and location via a laboratory-built nano-flow cytometer protein particles (LPP) in plasma may hamper robust Xiaomei Yana, Haisheng Liua, Ye Tianb and Shaobin Zhuc flow cytometric analysis of EV. We here investigated aDepartment of Chemical Biology, College of Chemistry and Chemical the interference of these particles when generic fluor- Engineering, Xiamen University, Xiamen, China (People’s Republic); escent dyes are used for labelling and detection of EV bDepartment of Chemical Biology, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen, China; cNanoFCM Inc., Xiamen, by FC. China (People’s Republic) Methods: To define the impact of LPP on fluorescence- based FC–detection of EV, commercially available LPP Introduction:Throughpackingandtransferringnucleic preparations, EV isolated from conditioned media of acids including genomic DNA, mitochondrial DNA, the mouse 4T1 mammary carcinoma cell line, and microRNA, mRNA and long noncoding RNA, exosomes platelet-poor plasma samples from healthy fastened play important roles in maintaining cellular homeostasis, human donors were stained with PKH67 and CFSE. priming immune system and regulating tumour progres- EV was isolated from samples by differential ultracen- sion. However, the abundance, identity (single stranded or trifugation or size-exclusion chromatography (SEC). double stranded) and location (surface-bound or inside) of Stained LPP, plasma EV and 4T1 EV were succumbed nucleic acids in single exosomes is still a conundrum. to density gradient floatation, after which FC-analysis Herein, a laboratory-built nano-flow cytometer (nFCM) was performed using a BD Influx that was optimized that enables multiparameter analysis of single exosomes as for detection of submicron-sized particles. small as 40 nm is used to investigate the features of exoso- Results: We found that both PKH67 and CFSE have mal nucleic acids. the capacity to label various types of LPP. When ana- Methods: Exosomes derived from a colorectal cancer lysed by FC, fluorescently labelled LPP and EV are cell line (HCT15) and a normal colon fibroblast cell hard to discriminate based on fluorescent and light line (CCD-18Co) were isolated by differential ultracen- scatter signals. Interestingly however, both dyes show trifugation. The location and identity of DNA/RNA a different staining pattern for LPP and are indicative was examined by measuring the fluorescence signals for the type of LPP analysed. In addition, we demon- of single exosomes upon nucleic acid labelling with strated that LPP show different sensitivity to detergent SYTO 16 or SYTO RNASelect dye before and after lysis when compared to EV. Finally, using spike-in enzymatic digestion with DNase I, dsDNase, S1 nucle- experiments we found that the presence of LPP can ase and RNase A, respectively. To achieve a selective obscure generic fluorescent labelling of EV, highlight- labelling for DNA/RNA, ethynyl-modified dUTP ing the need for proper EV isolation and purification (EdU)/ethynyl-modified UTP (EU) were incorporated 372 ISEV2019 ABSTRACT BOOK into the newly synthesized DNA/RNA by metabolic cell types or malignant cells also will display distinct biosynthetic pathway followed by chemoselective cou- surface profiles. Until today, only few EV surface mar- pling with azide-AF488 via click chemistry. kers have been related to specific cell sources. Results: We found that the majority of exosomal DNA Methods: We have recently optimized two flow cyto- reside on the outer surface of exosomes via bound with metry based methods for EV surface marker analysis, a membrane proteins and most of DNA are dsDNA. multiplex bead-based approach which allows robust Meanwhile, almost all the RNA are encapsulated inside identification of co-expressed surface marker combina- exosomes. Through correlation analysis with side scat- tions (Wiklander et al, 2018) and a method using tering signals of single exosomes, it was identified that imaging flow cytometry to quantify EV subsets at the most of the luminal DNA are associated with large size single vesicle level (Görgens et al, in revision). exosomes and surface-adhering DNA mainly exist on Results: Here, we combined both flow cytometric small size exosomes; yet the particle size of RNA con- approaches aiming to identify EV surface marker com- taining exosomes ranges from small to large sizes. binations being specific for EVs from specific cell types Because exosomes maintain cellular homeostasis by and/or disease-related cells such as cancer cells. We excreting harmful cytoplasmic DNA from cells, the first used the multiplex bead-based assay to screen effect of anti-tumour agents such as etoposide, topote- EVs isolated from 40+ different immortalized human can, SN-38 and cisplatin on the abundance and loca- cell lines from different tissues. Next, we have applied tion change of exosomal DNA will be reported. the same screening technology to assess surface signa- Summary/Conclusion: nFCM provides a straightfor- tures of EVs derived from diverse biological fluids of ward and practical approach for the location and iden- human healthy donors in order to identify differential tity studies of nucleic acids in individual exosomes, surface marker combinations between different body which will be very helpful in the illustration of exo- fluids and estimate general donor-to-donor variation some-mediated, nucleic acids-based intercellular within respective sample groups. Validation of identi- communication. fied EV surface signatures by high resolution single vesicle imaging flow cytometry and other methods is OS26.03 currently ongoing. Summary/Conclusion: We will show preliminary data resulting from this approach and propose that the Using a combination of bead-based flow cytometry and imaging flow identification of specific EV surface marker combina- cytometry to understand Extracellular Vesicle heterogeneity tions will be highly relevant to further understand the André Görgensa, Beklem Bostancioglub, Daniel Hageyb, Joshua Welshc, Maximilian Kordesd, Björn Evertssone, Manuela Gustafssonb, Jennifer C. molecular content and related functions of subsets of Jonesc, Oscar Wiklanderb and Samir El Andaloussif EVs in health and disease. aKarolinska Institutet, Department of Laboratory Medicine, Stockholm, Sweden; bClinical Research Center, Department for Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden; cTranslational Nanobiology Section, Laboratory of Pathology, National Cancer Institute, National OS26.04 Institutes of Health, Bethesda, MD, USA; dDepartment of Clinical Sciences, Intervention and Technology (CLINTEC), Karolinska Institutet, Stockholm, Sweden; eDepartment of Clinical Neuroscience, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden; fDepartment of A single extracellular vesicle (EV) flow cytometry approach to reveal Laboratory Medicine, Clinical Research Center, Karolinska Institutet, EV heterogeneity Sweden, Stockholm Wenwan Zhong and Kaizhu Guo University of California, Riverside, CA, USA Introduction: Extracellular vesicles (EVs) are secreted by all cell types and can be found in all body fluids. Introduction: To reveal the clear correlation between They can be roughly classified based on their size and extracellular vesicle (EV) functions and molecular sig- origin as exosomes (70–150 nm) and microvesicles natures, the only effective approach is to analyse the (100 nm to 1 µm). However, it is nowadays commonly molecular profile of individual EVs. Flow cytometry accepted in the field that there is a much higher degree (FC) has been widely employed to distinguish different of EV heterogeneity within these two subgroups. Also, cell types in mixed populations, but the sizes of EVs fall their content, protein composition and surface signa- well below the detection limit of conventional flow ture likely is dependent on multiple parameters like the cytometers, making it impossible to do single-EV ana- cell’s metabolic or immunological status. Moreover, the lysis without significant instrumentation development. protein composition and surface marker signature of Methods: We innovatively solve this difficulty by EVs is further dependent on the cell type releasing amplifying the size of each EV by DNA nanostructures them. Accordingly, EVs secreted by different normal so that they can be analysed in conventional flow JOURNAL OF EXTRACELLULAR VESICLES 373 cytometers. In this approach, either an aptamer or an applying this technique to analyse EVs produced from antibody is employed to recognize the specific surface different breast cancer cell lines, as well as the EVs in marker on each EV, and initiate construction of a large patients’ sera. DNA nanostructure by hybridization chain reaction. Summary/Conclusion:Insummary,wehavedevel- The resultant structure not only enlarges the overall oped a single-EV FC analysis technique to visualize size of the single EV, but also can bind to multiple single EV in a conventional flow cytometer. Our fluorophores to amplify the signal from the few num- technique enables study of single EVs using this ber of molecules on the EV surface, enabling visualiza- widely available instrument to gain in-depth insights tion of single EVs in a conventional flow cytometer. into the molecular signatures of EV sub-populations Results: We have successfully demonstrated counting at the single EV level. Targeting multiple markers single EVs in the FACSCanto after a one-pot reaction, greatly improves differentiation of EV sub-popula- and multiple surface markers can be simultaneously tions. The high simplicity of our method and its targeted to differentiate EV sub-groups based on their good adaptivity to clinical labs will be highly bene- surface protein signature. While aptamers provide a ficial for screening for effective EV markers for cleaner background for detection, the large selection liquid biopsy applications. of antibodies makes it applicable for diverse surface Funding: NIH-NCI markers on the EVs for sub-grouping. We have been 374 ISEV2019 ABSTRACT BOOK

Symposium Session 27: Non-mammalian EVs Chairs: Richard Ferrero; J. Max Silverman Location: Level B1, Hall B 16:00–17:00

amino acids, alcohols, ketones and monosaccharides. OS27.01 Proteomic analysis showed the presence of several bacterial proteins in EVs that may be associated with the

Extracellular vesicles released by commensal Lactobacillus suppress anti-HIV effect. HIV-1 infection Summary/Conclusion: Our findings demonstrate that Rogers A. Nahui Palominoa, Christophe Vanpouillea, Peter Backlundb, Carola Parolinc, Luca Laghid, Beatrice Vitalic and Leonid Margolisa the protective effect of Lactobacillus against HIV transmission is, in part, mediated by EVs released by these aSection of Intercellular Interaction, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of commensal bacteria. This finding may lead to new Health, Bethesda, MD, USA; bBiomedical Mass Spectrometry Facility, Eunice strategies to prevent male-to-female sexual HIV Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA; transmission. cDepartment of Pharmacy and Biotechnology, University of Bologna, Bologna, Italy; dCentre of Foodomics, Department of Agro-Food Science and Technology, University of Bologna, Bologna, Italy OS27.02 Introduction: The vaginal microbiota, mostly domi- nated by Lactobacillus spp. plays a key role in prevent- Extracellular vesicles of the human gut microbiota: do you hear me ing from numerous uro-pathogens’ infections, in host? particular from HIV-1. Recently, we demonstrated Anna Kaisanlahtia, Anatoliy Samoylenkob, Genevieve Bartb, Johanna Korvalaa, Annastiina Rytkönenc, Artem Zhyvolozhnyic, Ilkka Miinalainenc, that Lactobacillus of various strains inhibit HIV-1 Leo Lahtid, Seppo Vainioe and Justus Reunanenf replication in human cervico-vaginal and tonsillar tis- aBiocenter Oulu/Cancer and Translational Medicine Research Unit, sues ex vivo providing an experimental system to study University of Oulu, Oulu, Finland; bUniversity of Oulu, Biocenter Oulu, c mechanisms of this phenomenon. A growing body of Laboratory of developmental Biology, Oulu, Finland; Biocenter Oulu, University of Oulu, Oulu, Finland; dDepartment of Mathematics and evidences suggest that any kind of cells, including Statistics, University of Turku, Turku, Finland; eUniversity of Oulu, bacteria communicate to each other through extracel- Biocenter Oulu, Laboratory of Developmental Biology, Oulu, Finland; fUniversity of Oulu, Biocenter Oulu, Cancer and Translational Medicine lular vesicles (EVs). Here, we investigated whether the Research Unit, Oulu, Finland protective anti-HIV effect of lactobacilli is mediated by EVs released by these bacteria. Introduction: Microbial populations colonize the Methods: EVs were isolated from four strains of whole length of the human gastrointestinal track. Lactobacillus cultures, previously isolated from vaginas Changes in composition and function of the gut micro- of healthy women, by ultracentrifugation. Vesicles’ biota have been linked with numerous pathologies, sizes and concentrations were evaluated using underlining the importance of the host-microbiota NanoSight. Human cervico-vaginal and tonsillar tissues co-operation, although quite little is known of the ex vivo, as well as cell lines were treated with mechanism of communication between microbiota Lactobacillus-derived EVs, infected with HIV-1 and and distal organs. Our aim was to describe EV secre- virus replication was assessed by measuring the tion in healthy human gut, explore the contribution of released capsidic protein p24 using Luminex. Protein different bacteria to EV secretion and characterize the and metabolite cargo of bacterial EVs were detected by cargo of gut microbiota EVs, our hypothesis being that LC/MS/MS and 1H-NMR analysis, respectively. EVs are one of the major communication systems Results: EVs released by L. crispatus BC3 and L. gasseri between human gut microbiota and the host. BC12 protected human cervico-vaginal and tonsillar Methods: Gut microbiota EVs were isolated with a tissues ex vivo as well as isolated mammalian cells combination of commercial kits and centrifugation from HIV-1 infection by at least 50%. This protection methods from 20 faecal samples from healthy donors. was not due to cytostatic or cytotoxic EV-effects but Presence of EVs was assessed with transmission elec- rather was associated with the decrease of viral attach- tron microscopy (TEM). Proteins and RNA were iso- ment to the target cell and viral entry as demonstrated lated from the obtained vesicles and analysed with LC- in TZM-bl and MT-4 cell assays. Metabolomic analysis ESI-MS/MS (Turku Proteomics Facility) and showed 42 molecules associated with EVs including Illumina550 sequencing (Biocenter Oulu Sequencing JOURNAL OF EXTRACELLULAR VESICLES 375

Centre). DNA was isolated from the faecal samples and yield of the cNPs was evaluated by the protein amount analysed with 16S rRNA sequencing (Institute of measured using Bradford assay. The size and zeta Biotechnology, University of Helsinki) along with potential of the cNPs were measured by a zeta sizer. intact faeces-derived vesicles to allow comparison of To evaluate the effect of the cNPs on cells, three types taxonomic profiles. of cell lines, i.e. murine fibroblast NIH3T3 cells, mur- Results: Populations of faecal EVs were detected with ine macrophage-like RAW264.7 cells, and murine TEM, with a size ranging from 50 to 200 nm. On colon adenocarcinoma colon26 cells, were selected. average, 184 bacterial proteins and 56 human proteins Cells were added with cNPs and incubated at 37°C were identified per sample. Taken together, the data for 24 h. The cell viability was evaluated by using describes presence of 1194 distinct bacterial proteins CCK8 assay. Separately, the cNPs were labelled with and 264 human proteins in faecal EVs. On functional DiI and labelled cNPs were added to cells. After incu- level, the majority of bacterial EV proteins of the gut bation, we observed the cells by confocal microscopy. seem to consist of outer membrane proteins relating to Results: About 10 mg cNPs were obtained from 100 g metabolism, bacterial invasion and transport. Data for plants, indicating that cNPs can be obtained with high RNA cargo analysis is pending. In terms of bacterial yield compared with EVs. The size of the cNPs was EV proteins, the data suggests the most diverse secre- about 200 nm. In addition, the zeta potential was a tion from phyla bacteroidetes and firmicutes. negative charge (about −15 mV), which is comparable Taxonomic profiles analysed by 16S rRNA sequencing to that of EVs. Low concentrations of cNPs hardly demonstrated differences in the bacterial composition affected the viability of the cells. Confocal microscopy of the faecal samples and faeces-derived EVs: proteo- showed that DiI-labelled cNPs were taken up by bacteria, while present in small abundancies in faeces, RAW264.7 cells. The results of onion- or orange- was one of the most predominant phyla found in derived NPs will also be presented. faeces-derived EVs. Summary/Conclusion: We succeeded in preparing Summary/Conclusion: Human gut microbiota actively cNPs in large scale and revealed that the particulate secretes EVs with range of protein and RNA cargo properties of the cNPs are comparable to those of EVs. which biological significance in human health and dis- We also demonstrated that cNPs can be efficiently ease needs to be studied further. taken up by RAW264.7 cells. These results raise a Funding: Academy of Finland possibility that cNPs can be used as carriers for bioactive molecules to such cells. OS27.03 OS27.04 Preparation, characterization and cellular interaction of edible plant- derived nanoparticles Daisuke Sasakia, Kosuke Kusamorib and Makiya Nishikawab aFaculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Biophysical and electrochemical characterization of redox-active Japan; bTokyo University of Science, Noda, Japan extracellular vesicles from Shewanella oneidensis Lori Zacharoffa,Shuai Xua, Grace Chonga, Lauren Ann Metskasb, Poorna Subramanianb, Grant Jensenb and Moh El-Naggara Introduction: Nanoparticles, including liposomes, aUniversity of Southern California, Los Angeles, CA, USA; 2California polymeric micelles and animal cell-derived extracellu- Institute of Technology, Pasadena, CA, USA lar vesicles (EVs), are promising carriers for bioactive molecules. Recently, edible plant-derived nanoparticles Introduction: Production of bacterial extracellular are expected to be a novel class of nanoparticles, vesicles has been observed in marine and freshwater because they have advantages in terms of mass produc- systems and in laboratory cultures. However, little is tion and cost-effectiveness. However, their pharmaceu- known about the function and mechanism of vesicula- tical and biological characteristics need to be evaluated tion in these nonpathogenic contexts. In addition to prior to their application and use in clinical practice. In vesicles, the Gram-negative bacterium, Shewanella this study, we selected corn as an edible plant, and oneidensis also produces chains of outer-membrane prepared corn-derived nanoparticles (cNPs). Then, we vesicles that are proposed to function as bacterial nano- evaluated their property and interaction with cells. wires for electron transport to solid-phase electron Methods: Corn was put in a blender with distilled acceptors ranging from minerals to electrodes. A pre- water to obtain juice. The juice was separated by cen- vious report demonstrated mineral reduction by iso- trifugation and ultra-centrifugation (UC), and the pel- lated S. oneidensis vesicles. Many basic questions let after UC at 100,000 g was collected as cNPs. The remain about the function and biogenesis of these 376 ISEV2019 ABSTRACT BOOK structures, particularly during metal and electrode vesicles reveals the signatures of known outer-mem- respiration. brane multiheme cytochromes. Methods: Here we report the purification and charac- Summary/Conclusion:Theseresultshaveimplica- terization of outer membrane vesicles from S. oneiden- tions for the role of vesicles and vesicle chains sis. Preliminary analyses using dynamic light scattering, during respiration of iron oxides and anodes. fluorescence microscopy, cryoelectron microscopy, and Excitingly, this research suggest that a BAR domain proteomics, confirm the size, content and reproduci- protein provides the mechanistic relationship bility of purified vesicles. between vesicles and the outer membrane exten- Results:Proteomicdatasuggestthatsomeproteins sions known as nanowires are selectively loaded into vesicles with the assis- Funding: US DOE Division of Chemical Sciences, tance of a novel BAR domain protein. Geociences and Biosciences, Office of Basic Energy Electrochemical analysis of surface deposited Science DE-FG02-13ER16415 National Science Foundation grant DEB-1542527 JOURNAL OF EXTRACELLULAR VESICLES 377

Symposium Session 28: EVs in Kidney and Urological Diseases Chairs: Uta Erdbrügger; Juan Falcon-Perez Location: Level B1, Hall A 16:00–17:00

positive for multiple markers varied depending on the OS28.01 isolation methods. The relationship between therapeutic effectiveness and EV subpopulation marker expression were tested using an AKI model. EV Single MSC EV analysis for characterizing a subpopulation having subpopulation using four different EV-specific markers therapeutic effects in AKI model Hyejin Kanga, Chungmin Hanb, Jongok Pyoc and Jaesung Parkd might be a useful tool for assessing the quality of aPohang University of Science and Technology, Pohang, Republic of Korea; isolated EVs in terms of their therapeutic effectiveness. bPohang University of Science and Technology, Pohang, Republic of Korea; Funding:ThisworkwassupportedbytheKHIDIgrant cEXOSOMEplus, Seoul, Republic of Korea; dDepartment of Mechanical Engineering, POSTECH, Pohang, Republic of Korea [HI16C2221] and supported by NRF grant [NRF- 2018R1A2B3006280] funded by the Korean government. Introduction: Therapeutic applications of MSCEVs have been extensively studied. Previous MSCEV studies demonstrated that MSCEVs showed various effects OS28.02 depending on how they were prepared. Recent studies suggested that this diversity might result from the Urinary microvesicular biomarkers for delayed graft function and heterogeneity of isolated EV populations. However, overall outcome after living donor kidney transplantation because of the absent of a proper EV subpopulation Fabian Brauna, Markus Rinschenb, Ingo Plagmannb, Corinna Kleinc, Denise Buchnerd, Roger Wahbad, Dirk Stippeld, Christine Kurschatb, Bernhard analysis method, no studies have succeeded to charac- Schermerb, Andreas Beyerc, Thomas Benzingb and Roman-Ulrich Müllerb terize an effective subpopulation from whole EV popu- aIII. Department of Medicine, University Medical Center Hamburg- lations. We analysed the subpopulations of MSCEVs Eppendorf, Hamburg, Germany; bDepartment II of Internal Medicine and prepared by different isolation methods using a single Center for Molecular Medicine Cologne, University of Cologne, Germany, Cologne, Germany; cCologne Excellence Cluster on Cellular Stress Responses EV analysis method. We assessed the correlation in Aging-Associated Diseases, University of Cologne, Germany, Cologne, between the therapeutic effectiveness and MSC EV Germany; dDepartment of General, Visceral and Cancer Surgery, Division of Transplantation Surgery, Transplant Center Cologne, University of subpopulations using mouse acute kidney injury Cologne, Cologne, Germany (AKI) model Methods: EVs were prepared from hMSC conditioned Introduction: With a cargo of specific proteins and media using different isolation methods: differential nucleic acids, urinary microvesicles represent a poten- centrifugation, density gradient centrifugation and tial source for cellular material, that can be isolated polymeric methods. A part of EVs were analysed easily and non-invasively. Yet, their clinical implemen- using a TIRF microscopy based single EV analysis tation in nephrology remains scarce with kidney biop- method, which can provide quantitative subpopulation sies still being the gold standard procedure in most information characterized by up to four different mar- diagnoses. We hypothesize that the addition of non- ker expressions. EVs were applied to an AKI model to invasive biomarkers could benefit this invasive method assess their therapeutic effectiveness. with the potential risk of a sampling error. Results: EVs prepared by different isolation methods Methods: With differential (ultra-)centrifugation, we showed different subpopulation characteristics. The isolated urinary microvesicles from living kidney trans- numbers of lipid marker positive EVs were different plant recipients and their donors over the course of 40 depending on their isolation method. Overall expres- kidney transplantations. Whole urine samples were sion profile of three representative EV specific marker collected on day −1 (donor sample), 0, 1 and 3 months (CD9, 63 and 81) were also different depending on after transplantation (recipient sample). Microvesicular their isolation methods. EVs expressing more EV-spe- protein content was measured using quantitative mass cific markers were found to be more effective in mouse spectrometry. We detected proteins, which linearly AKI models. change their abundance in correspondence to clinical Summary/Conclusion: We demonstrated that the sub- parameters, e.g. glomerular filtration rate (GFR) at 6 population composition of EVs prepared by different and 12 Months after transplantation in a set of 20 isolation methods were different. The numbers of EVs transplantations, by linear regression models. These 378 ISEV2019 ABSTRACT BOOK results were validated in a targeted proteomic screen in were added to macrophages or intrarenal injected to a cohort of 20 additional transplantations. mice to determine its effects both in vitro and in vivo. Results: We identified >1500 proteins present in at Results: Global miRNA expression profiling on renal least 50% of the first sample set. Hierarchical clustering exosomes was examined in LPS-induced AKI model analysis depicted a clear clustering by time point of and miR-19b-3p was identified as the most remarkable urine collection. Microvesicular proteins of glomerular miRNA increased in TEC-derived exosomes compared (e.g. nephrin, podocin) or tubular origin (e.g. V- with controls. Similar results were found in ADR- ATPase and Slc transporters) were regulated distinctly induced chronic proteinuric kidney disease model in over the course of transplantation. Overall, specific which exosomal miR-19b-3p was markedly released. proteomic time course patterns were apparent over Interestingly, once released, TEC-derived exosomal the course of transplantation. Depending on low sta- miR-19b-3p was internalized by macrophages, leading tistical error and high stability in a leave-one-out cross- to M1 phenotype polarization through targeting NF- validation of the linear models correlating to GFR κB/SOCS-1. Importantly, the pathogenic role of exoso- values after transplantation, we created a list of 64 mal miR-19b-3p in initiating renal inflammation was candidate proteins. Validation of these revealed revealed by the ability of adoptive transfer of purified PEPCK as a urinary microvesicular protein associated TEC-derived exosomes to cause tubulointerstitial with GFR 12 months after transplantation. inflammation in mice, which was reversed by inhibi- Summary/Conclusion: With this study, we present the tion of miR-19b-3p. Clinically, high levels of miR-19b- first analysis of the changes in the human urinary 3p were found in urinary exosomes and correlated with microvesicular proteome over the course of kidney the severity of tubulointerstitial inflammation in transplantation. We believe, the validated biomarkers patients with diabetic nephropathy. Thus, our studies of all 40 Transplantations to hold the potential to demonstrated exosomal miR-19b-3p mediated the further aid the diagnosis of graft survival. communication between injured TECs and macro- Funding: MIWF Nachwuchsgruppen.NRW phages, leading to M1 macrophage activation. Summary/Conclusion: Exosome/miR-19b-3p/SOCS1 axis played a critical pathologic role in tubulointersti- OS28.03 tial inflammation that might represent a new therapeutic target for kidney disease.

Exosomal miRNA-19b-3p of tubular epithelial cell promotes M1 macrophage activation in kidney injury Ye Fenga, Linli Lvb, Taotao Tangc and Bi-Cheng Liua OS28.04 aInstitute of Nephrology, Southeast University, Nanjing, China (People’s Republic); bInstitute of Nephrology, Zhongda Hospitial, Southeast University, Nanjing, China (People’s Republic); cInstitute of Nephrology, A urine exosome RNA signature for prediction of high-grade prostate Zhongda Hospital, Southeast University School of Medicine, Nanjing, cancer: clinical validation in over 1,000 biopsy naïve patients China (People’s Republic) Robert Kitchena, Phillipp Torklerb, James McKiernanc, Michael Donovand, Mikkel Noerholmb, Peter Carrolle and Johan Skogf Introduction: Tubulointerstitial inflammation is a aExosome Diagnostics, Inc, Waltham, MA, USA; bExosome Diagnostics, common characteristic for acute and chronic kidney GmbH, Martinsried, Germany; cColumbia University, Department of injury. However, the mechanism by which the initial Urology, New York, NY, USA; dDepartment of Pathology, Mount Sinai, New York, NY, USA; eUniversity of California San Francisco, San injury on tubular epithelial cells (TECs) drives inter- Francisco, CA, USA; fExosome Diagnostics, Inc., Waltham, MA, USA stitial inflammation remains unclear. Here we set out to characterize the miRNA profile of kidney exosomes Introduction: Discriminating indolent from clinically and aim to explore the role of exosomal miRNAs significant prostate cancer (PCa) prior to initial biopsy derived from TECs in the development of tubulointer- remains an important clinical and health economic stitial inflammation. issue. We have previously described the ExoDx Methods: Exosomes were isolated from kidney and Prostate(IntelliScore) (EPI) assay for discriminating characterized via electron microscopy and nanoparticle high- vs low-grade prostate cancers using RNA analysis. We examined expression profiles of miRNAs extracted from urine exosomes. However proving effi- in kidney exosomes from LPS-induced kidney injury cacy and facilitating clinical adoption of a diagnostic model by Exiqon microarray. Putative targets of assay requires extensive validation in prospectively col- miRNA were predicted by TargetScan. Chronic protei- lected patient cohorts. Here we compare performance nuric kidney disease model was induced by adriamycin of the EPI urine exosome assay vs. the Prostate Cancer (ADR) administration. Exosomes purified from TECs Prevention Trial-Risk Calculator 2.0 (PCPT-RC) for JOURNAL OF EXTRACELLULAR VESICLES 379 discriminating high-grade from low-grade PCa and 51% positive biopsy rate (30% GS≥7, 13% GS≥4 + 3). benign disease on initial biopsy. Performance of the EPI test (AUC = 0.70) was superior Methods:Wecollecteddatafromtwodistinctvalida- to PSA (AUC = 0.56), and PCPT-RC (AUC = 0.6; all p- tion cohorts (N =519and503,respectively)repre- values<0.001) for discriminating high- from low-grade senting 1022 subjects and compared EPI test results PCa and benign disease. Using the previously validated with biopsy outcomes. Eligible subjects were selected cut-point of 15.6 (or alternative 20) would avoid 30% by age (>50-years) and PSA concentration (2–10 ng/ (or 43%) of unnecessary biopsies, with an NPV of 90% mL), and were scheduled for initial prostate needle for both cut-points and miss only 7.5% (or 12%) of biopsy. Test performance was reported using the area high-grade PCa patients. under the receiver operating characteristic curve Summary/Conclusion: EPI is a non-invasive 3-gene (AUC), negative and positive predictive value (NPV; urine exosome RNA expression assay that we have PPV), sensitivity, and specificity. Outcome was based now successfully validated in over 1000 patients to on Gleason Score (GS) for discriminating high- discriminate high- from low-grade PCa and benign (GS≥7) from low-grade (GS = 6) and benign disease disease. EPI identifies high-risk patients better than on initial biopsy. any current standard of care and provides a valuable Results: In this diverse cohort of 1022 biopsy naïve tool for shared decision making so the right patients patients (mean age: 64 years, mean PSA: 5.6 ng/mL, are sent for biopsy. ethnicity: 16% African, 71% Caucasian) we observed a 380 ISEV2019 ABSTRACT BOOK

Symposium Session 29: Late Breaking- EV Therapeutics Chairs: Masahiko Kuroda; Carolina Soekmadji Location: Level B1, Lecture Room 08:30–09:30 Methods: We have manufactured GMP-compliant LB01.01 umbilical cord (UC)-MSC- derived extracellular vesicles/exosomes (EVs) and subjected such preparations to a series of in vivo and in vitro assays. Animal studies First-in-human application of umbilical cord mesenchymal stromal cell-derived exosomes for the prevention of fibrosis following included both systemic and local injection for the cochlear implant surgery improvement of seemingly unrelated indications such a a b Athanasia Warnecke , Jennifer Schulze , Julia Hollerweger , Teresa as critical size bone defects, partial tendon rupture and Lassacherb, Karin Pachlerb, Heide-Marie Binderb, Alexandre Desgeorgesb, Gerhard Weidlerb, Magdalena Mayrb, Pasquale Romanellic, Sebastien spinal cord injury in a rat contusion-model. Couillard-despresc, Hinrich Staeckerd, Jennifer Nelson-Brantleyd, Andreas Results: In all cases EV application resulted in a sig- Trawegere, Eva Rohdeb and Mario Gimonaf nificant modulation of immune reaction, overall aKlinik für Hals-, Nasen-, Ohrenheilkunde, Hannover Medical School, Hannover, GERMANY, Hannover, Germany; bSCI-TReCS GMP Unit at reduced inflammation and scar reduction as evidenced Paracelsus Medical University, Salzburg, AUSTRIA, Salzburg, Austria; by a reduction in ECM deposition. In an in vitro spiral c Institute of Experimental Neuroregeneration, Paracelsus Medical ganglion neuron protection assay UC-MSC-EVs out- University, Salzburg,, Salzburg, Austria; dAuditory & Vestibular Neuroscience Laboratory, University of Kansas Medical Center, Kansas performed the current best-in-class soluble neuropro- City,, Kansas City, USA; eInstitute of Tendon and Bone Regeneration, tective factor, BDNF. In vivo application of EVs in mice Paracelsus Medical University, Salzburg, AUSTRIA, Salzburg, Austria; fGMP Unit at Paracelsus Medical University, Salzburg, AUSTRIA, Salzburg, challenged by noise trauma resulted in significant pro- Austria tection of hearing when compared to untreated controls. Reduction of impedance as a measure for current Introduction: Cochlear implantation (CI) can restore resistance and fibrotic tissue formation around the hearing perception by bypassing the auditory hair cells electrode array were observed in a model of implanta- (HC) and directly stimulating the spiral ganglion neu- tion trauma in guinea pigs implanted with an electrode rons (SGN). Insertion of an electrode array into the array. After careful consideration of the medical history cochlea is associated with robust early and chronic of a patient requiring CI surgery an experimental heal- inflammatory responses that promote intra-cochlear ing attempt was performed and the patient received a fibrosis and loss of HC and SGN. Conservation of single intra-cochlear injection of 5 ×109 EVs (total) residual hearing and prevention of fibrous tissue prior to electrode insertion. The application was toler- deposition around the electrode are thus major chal- ated well, no adverse reactions were recorded and lenges in CI surgery. JOURNAL OF EXTRACELLULAR VESICLES 381 robust auditory sensation was detected 6 weeks post ARMMs through direction fusion whereas the full surgery. length ARRDC1 failed to. Summary/conclusion: Based on these initial safety Summary/conclusion: These results indicate that data on the local application of EVs in the inner ear, ARMMs formed by the minimal ARRDC1 may be a phase 1/2a clinical trial is currently in preparation to used as a novel efficient therapeutic delivery platform further evaluate the neuroprotective, immunomodula- for the Cas9-based gene editors. tory and anti-fibrotic potential of UC-MSC-EVs. Funding: “Exothera” IT-AT 1036 (EU), Project “ExtraNeu” (Land Salzburg) LB01.04

Microvesicle-mediated delivery of minicircle DNA results in effective gene-directed enzyme prodrug cancer therapy LB01.03 a b c d Masamitsu Kanada , Bryan Kim , Jonathan Hardy , John Ronald , Michael Bachmanne, Matthew Bernardf, Gloria Perezg, Ahmed Zareag, Jessie Geh, Alicia Withrowi, Sherif Ibrahimg, Victoria Toomajianj, Sanjiv Gambhirk, Ramasamy Paulmuruganl and Christopher Contagm Engineering of ARMMs for efficient delivery of Cas9 genome editors Qiyu Wanga and Quan Lub aInstitute for Quantitative Health Science and Engineering (IQ), Dept. of Pharmacology & Toxicology, Michigan State University, East Lansing, USA; a b Qilu Pharma, Boston, USA; Harvard University, Boston, USA bDept. of Chemistry, UC Santa Cruz, Santa Cruz, USA; cInstitute for Quantitative Health Science and Engineering (IQ), Dept. of Microbiology & d Introduction: Our previous studies have shown that Molecular Genetics, Michigan State University, East Lansing, USA; Robarts Research Institute, Western University, Lawson Health Research Institute, the arrestin domain containing protein 1 (ARRDC1) London, Canada; eAssociate Professor, Institute for Quantitative Health drives the formation of extracellular vesicles known as Science and Engineering (IQ), Dept. of Microbiology & Molecular Genetics, Michigan State University, East Lansing, USA; fAssistant Professor, Institute ARMMs (ARRDC1-mediated microvesicles) (Nabhan J for Quantitative Health Science and Engineering (IQ), Dept. of Pharmacology et al., PNAS 2012) and that these vesicles can be har- & Toxicology, Michigan State University, East Lansing, USA; gInstitute for Quantitative Health Science and Engineering (IQ), Michigan State University, nessed to package and deliver a variety of molecular East Lansing, USA; hDept. of Radiology, Stanford University, Palo Alto, USA; cargos such as protein, RNA and the genome editor iCenter for Advanced Microscopy, Michigan State University, East Lansing, USA; jInstitute for Quantitative Health Science and Engineering (IQ), Dept of Cas9 (Wang Q and Lu Q, Nat Commun 2018). In the Biomedical Engineering, Michigan State University, East Lansing, USA; published packaging and delivery study, we used the kDepts. of Radiology, Bioengineering, and Materials Science, and Molecular full-length ARRDC1 protein (433 amino acids at ~46 Imaging Program at Stanford (MIPS), Stanford University, East Lansing, USA; lDept. of Radiology, Molecular Imaging Program at Stanford (MIPS), kD) to recruit the molecular cargos into the vesicles, Stanford University, Palo Alto, USA; mInstitute for Quantitative Health either through a direct fusion or via a protein-protein Science and Engineering (IQ), Depts of Microbiology & Molecular Genetics, Biomedical Engineering, Michigan State UniversityMichigan State interaction module. Because ARRDC1 protein itself is University, East Lansing, USA packaged into ARMMs and because the size of the vesicles is limited (~80–100 nm), a smaller ARRDC1 Introduction: An emerging approach for cancer treat- protein that can still function in driving budding would ment employs the use of extracellular vesicles (EVs), potentially increase the number of cargos that can be specifically exosomes and microvesicles, as delivery packaged into the vesicles. Moreover, a smaller vehicles. ARRDC1 may allow the recruitment of a relatively Methods: We previously demonstrated that microvesi- large cargo molecule. cles can functionally deliver plasmid DNA to cells and Methods: We used protein engineering to identify a showed that plasmid size and sequence determine, in minimal ARRDC1 protein that can drive the formation part, the efficiency of delivery. Delivery vehicles com- of ARMMs. We then fused the minimal ARRDC1 to prised of microvesicles loaded with engineered mini- multiple proteins including the genome-editor Cas9 circle DNA (MC) encoding prodrug converting and tested the packaging and delivery efficiency of enzymes were developed here as a cancer therapy in the fusion protein. mammary carcinoma models. Results: Here we will present new data that identified a Results: We demonstrated that MCs were loaded into minimal ARRDC1 protein that contains an arrestin shed microvesicles with greater efficiency than their domain, PSAP and PPXY motifs. The minimal parental plasmid counterparts and that microvesicle- ARRDC1 is able to drive ARMM budding as efficiently mediated MC delivery led to significantly higher and as the full-length ARRDC1. We further present evi- more prolonged transgene expression in recipient cells dence that the minimal ARRDC1 protein can effi- than did microvesicles loaded with the parental plas- ciently package cargos such as the relatively large mid. Microvesicles loaded with MCs encoding a thy- Cas9/gRNA complex. In particular, we showed that midine kinase (TK)/nitroreductase (NTR) fusion the minimal ARRDC1 can package Cas9/gRNA into protein produced TK-NTR expression in mammary 382 ISEV2019 ABSTRACT BOOK carcinoma cells. In vivo delivery of TK-NTR and approach to cancer therapy. To understand the administration of prodrugs led to the effective killing mechanism of this microvesicle-mediated enzyme proof both targeted cells and surrounding tumour cells via drug therapy, we are currently assessing recipient cells TK-NTR-mediated conversion of prodrugs to active in the tumour microenvironment. cytotoxic agents. The efficiency of killing non-trans- Funding: This work was funded in part through a fected bystander/neighbouring cells was assessed in generous gift from the Chambers Family Foundation mouse models and determined to require one in 100 for Excellence in Pediatrics Research (to C.H.C.), Grant cancer cells to be targeted. 1UH2TR000902-01 from the National Institutes of Summary/conclusion: These results suggest that MC Health (to C.H.C.), and the Child Health Research delivery via microvesicles can mediate gene transfer to Institute at Stanford University (to C.H.C.). Start-up an extent that enables effective prodrug conversion and fund from Michigan State University (to M.K.) tumour cell death such that it comprises a promising JOURNAL OF EXTRACELLULAR VESICLES 383

Symposium Session 30: Late Breaking- EVs and Cancer Chairs: Suvendra Bhattacharyya; Vincent Hyenne Location: Level B1, Hall B 08:30–09:30

for cell dissemination and ELEVs production using LB02.01 vast genetic tools available in Drosophila.

Extremely-large extracellular vesicles (elevs) aid invasiveness of LB02.02 rasv12 tumour cell dissemination Jiae Lee and Young Kwon

University of Washington, Seattle, USA House dust extracellular vesicles promote tumour metastasis to the lungs by inducing tumour necrosis factor-α Introduction: Cancer cell dissemination has been Nhung Thi Hong. Dinha, Jaewook Leeb, Jaemin Leec, Gyeongyun God, Kim Sang sood, Seoyoon Baed, Yein June, Tae Young Rohf and Yong Song Ghod recognized for the association with cancer recurrence, aPostech, Pohang, Republic of Korea; bDepartment of Life Sciences, Pohang invasion and metastasis, however, the exact molecular University of Science and Technology (POSTECH), Pohang, Republic of mechanism is not fully understood. Most of the pre- Korea; cPohang University of Science and Technology, Pohang, Republic of d vious studies were conducted in cell culture, which is Korea; Department of Life Sciences, Pohang University of Science and Technology, Pohang, Republic of Korea; ePohang University of Science and difficult to track the consequence of disseminated cells. Technology (POSTECH), Pohang, Republic of Korea; fDepartment of Life Moreover, the lack of a simple yet conserved model Sciences, Pohang University of Science and Technology (POSTECH), 77 Cheongam-ro, Nam-gu, Pohang 37673, Republic of Korea, Pohang, system deferred genome-wide screening. Therefore, we Republic of Korea established an in vivo cell dissemination model in Drosophila. Introduction: Air pollution is associated with multiple Methods: We express mutant Ras (RasV12) in adult pulmonary disorders. As a part of pollutant air, house Drosophila midgut intestinal stem cells (ISCs) and dust harbours several biological contaminants includ- enteroblasts (EBs) using the conditional GAL4 driver ing extracellular vesicles (EVs). House dust EVs have esgts (esg-GAL4, tub-GAL80ts, UAS-GFP). been shown to induce pulmonary inflammation, but no Results: When RasV12 is expressed in ISCs and EBs, studies have assessed the effect of dust EVs on tumour tumour rapidly proliferates, then become eliminated. metastasis to the lungs. Cellular processes protrude while damaging and invad- Methods: EVs were isolated from house dust using ing the surrounding visceral muscle fibres, and intact buoyant density gradient ultracentrifugation. Isolated cells can completely disseminate. Interestingly, we dust EVs were characterized with transmission electron observed with ex vivo live imaging that RasV12 cells microscopy and dynamic light scattering. To assess the produce large blebs and release extracellular vesicles. role of dust EVs in tumour metastasis, dust EVs were The average size of these vesicles was bigger than exo- intranasally administered to mice, followed by intrave- somes (<100 nm) and microvesicles (100–1000 nm), so nous injection of tumour cells after 1 day. At 2 weeks we refer them as extremely-large extracellular vesicles after tumour introduction, lungs were harvested from (ELEVs). Additionally, GFP-positive particles were mice to measure metastasis by counting metastatic detected in haemolymph prepared from RasV12 flies colonies. To investigate the mechanism, the lungs but not from controls assuring that ELEVs were also were collected at 12 h or 24 h after tumour cell intro- produced in vivo. Of note, we found that metastatic duction to access tumour cell infiltration into the lungs RasV12, scrib-/- disc tumours also produced ELEVs. by immunohistochemistry. Furthermore, lung lysates Thus, we propose that the generation of ELEVs is a were prepared from mice intranasally administered characteristic of invasive tumours in Drosophila. with dust EVs to examine tumour necrosis factor-α Interestingly, these ELEVs are reminiscent of large (TNF-α) production and their effect on tumour cell oncosomes or cytoplasts, which have been implicated migration. Finally, TNF-α knock-out mice were used in the invasive behaviour of cancer cells. to show the importance of TNF-α in dust EV-induced Summary/conclusion: This model shares many known tumour metastasis. aspects of tumour cell dissemination implied from the Results: House dust EVs had membrane-enclosed studies in mammalian systems. We plan to utilize this structures with an average diameter of unique system to elucidate the molecular mechanism 129.6 ± 4.5 nm, as observed by transmission electron 384 ISEV2019 ABSTRACT BOOK microscopy and dynamic light scattering. Dust EVs Results: In all models, including prostate, breast, color- significantly promoted tumour metastasis to the ectal and gastric cancer, 3D environment caused a lungs. The mechanism study showed that these EVs considerable shift in EV size distribution. Specific enhanced tumour cell infiltration into the lungs. changes in EV surface protein profiles and distribution Although dust EVs did not directly mediate tumour of oncogenic DNA and RNA among EV populations cell migration, lung lysates from dust EV-treated mice was observed. These changes varied between different could promote this migratory effect. In addition, the tumour types. Comparison with the delivery of concentration of TNF-α was increased in lung lysates mutated oncogenes among EV populations in the by treating dust EVs. Finally, TNF-α knock-out mice blood of patients revealed that EVs released from in treated with dust EVs could not promote tumour 3D resemble closer the content of EVs isolated from metastasis to the lungs. the blood of tumour patients. Summary/conclusion: House dust harboured signifi- Summary/conclusion: Here, we present a unique cant amounts of EVs which could promote tumour model applicable to study the EV heterogeneity under metastasis by inducing TNF-α. These findings provide conditions mimicking physiological tumour microen- mechanistic insights into the effect of house dust on vironment. It can serve as a new tool for drug screen- tumour metastasis to the lungs. ing and biomarker search, representing a new reliable model for EV-based liquid biopsy. Funding: H2020 MSCA-ITN „Train-EV“, Project LB02.03 722148; BMBF “EXONANSENS”, Project 01DJ51206.

Modeling tumour: key issues of cell communication by mean of EVs in LB02.04 a three-dimensional environment and the impact on biomarker discovery Richa Khandurya, Liliia Paniushkinaa, Andreas Kellerb, Tanja Gajney- Schleichera and Irina Nazarenkoc Secretion mechanisms of wnt proteins Alena Ivanovaa, Jan Wintera, Oksana Voloshanenkoa, Kathrin Glaesera, aMedical Center Univeristy of Freiburg, Freiburg, Germany; bSaarland Steffen Scholppb, Ulrike Engelc, Clotilde Theryd and Michael Boutrose c University, Saabruecken, Germany; Medical Center University of Freiburg, a Freiburg, Germany German Cancer Research Center, Division of Signaling and Functional Genomics, Heidelberg, Germany, Heidelberg, Germany; bLiving Systems Institute, School of Biosciences, College of Life and Environmental Sciences, Introduction: The impact of growth architecture on University of Exeter, Exeter, United Kingdom, Exeter, United Kingdom; c intercellular communication, including EV release, Nikon Imaging Center at Heidelberg University and Centre for Organismal Studies (COS), Heidelberg University, Heidelberg, Germany, cargo, and function was not examined in many details. Heideberg, Germany; dInstitut Curie, PSL Research University, INSERM In our recent work, we described a new model for U932, Immunity and Cancer, Paris, France, Paris, France; eDepartment of Cell and Molecular Biology, Medical Faculty Mannheim, Heidelberg efficient EV production in a three-dimensional envir- University, Heidelberg Germany, Heidelberg, Germany onment. Cells growing in 3D produced an increased number of small EVs, which differed in their miRNA Introduction: Wnt signalling pathways play important and protein profiles from the EVs, released by the cells roles in development and diseases of multicellular grown under conventional conditions. In the current organisms. Key players in intercellular signalling – work, we describe the impact of growth architecture on Wnt proteins – travel through extracellular space, but EV heterogeneity and their content respecting recruit- since lipid modifications renders them insoluble, they ment of activated oncogenes and mutated products use special carriers. According to the current under- on EVs. standing of Wnt secretion, to contact neighbouring Methods: EVs of different size were isolated by subse- cells Wnt proteins can diffuse using heparan sulphate quent sedimentation by 5000 ×g (EV5), 12000 ×g proteoglycan chains on the cell surface or they can be (EV12) and small EVs, purified by size exclusion chro- transported on filopodia. Additionally, Wnt ligands can matography (SEC). Subsequently, iodixanol gradient be solubilized and travel in the intracellular space by centrifugation was performed. These EV populations creating lipoprotein particles. Wnts can be recycled were separated from 2D and 3D cell cultures and through the endosomal compartment and secreted on characterized respecting their size using NTA, DLS, exosomes. It is been a long debate which secretory and TRPS. Surface proteins were examined using mechanism is preferred by Wnt ligands and which beads-assisted flow cytometry. Subsequently, DNA forms of secreted Wnt proteins maintain signalling and RNA were isolated, and the number of mutated activity. We aimed to dissect the very complex Wnt oncogenes to different EV populations was assessed secretion network to identify new regulators specific using ddPCR. for the certain secretion roots. JOURNAL OF EXTRACELLULAR VESICLES 385

Methods: We first, performed focused RNAi screen to and length of cell filopodia. Beyond this, only a few identify new components of Wnt secretory pathway. By other proteins have been described so far to regulate activating Wnt pathway either in the secreting cell or at specialized filopodia like cytonemes. Additionally, we the receptor level we manage to distinguish between observed that Wnt proteins travel across filopodia proteins that involved only in the secretory part of the being packed on vesicle-like structures. pathway or play role in the receiving cells. At the next Summary/conclusion: For the first time a forward step, we used array CRISPR/Cas9 screening approach genetic screen allowed to identify new components for targeted disruption of genes together with canonical that are important for filopodia associated Wnt signal- Wnt activity assay to confirm that identified genes are ling. Surprisingly, Wnt ligands use vesicles as carries required for the secretion of functional Wnt proteins. for transport across cell protrusions. These findings Results: With the described approach, a panel of 83 add another piece of evidence that microvesicles and possible secretory factors have been tested. Among the filopodia plays a significant role in the distribution of others we found a protein that involved in the filopodia Wnt ligands. formation process. Identified protein regulates number 386 ISEV2019 ABSTRACT BOOK

Symposium Session 31: Late Breaking- EV Biomarkers Chairs: Johannes Grillari; Mariko Ikuo Location: Level B1, Hall A 08:30–09:30 whereas in total plasma, gDNA was increased in cancer LB03.02 patients. In addition, several membrane proteins were significantly enriched or exclusively present in cancer patients EVs compared to healthy donors. Assessing the value of extracellular vesicles‘ DNA and proteins as Summary/conclusion: Our findings provide evidence biomarkers in metastatic breast cancer that the detection of DNA within total circulating EV Mercedes Tkacha, Caroline Hegob, Clotilde Therya and Charlotte Proudhonb does not provide added value as compared to the whole aInstitut Curie, PSL Research University, INSERM U932, Immunity and b plasma. However, analysis of a subtype of EV-asso- Cancer, Paris, France, Paris, France; Institut Curie, PSL Research University, SiRIC, Laboratory of circulating tumor biomarkers, Paris, ciated proteins may reliably identify cancer patients. France, Paris, France Funding: INCa-11548, ARC PGA1 RF20180206962 Introduction: Analysis of cell-free circulating tumour DNA (ctDNA) and cancer-specific extracellular vesicles LB03.03 (EVs) in patients’ blood have been widely explored as biomarkers for cancer detection and disease follow up. These non-invasive biomarkers represent a promising A novel strategy for early detection of clinically significant prostate cancer by high-throughput palmitoyl-proteomics of extracellular tool for real-time monitoring of treatment efficacy. vesicles Particularly, tumour-derived EVs contain specific pro- Dolores Di Vizioa, Javier Mariscalb, Tatyana Vagnerb, Minyung Kimb,Bo Zhouc, Desmond PINKd, Andrew Chinb, Mandana Zandianb, John Lewise, tein cargo and nucleic acids, which are protected from Michael Freemanb, Stephen Freedlandb, Sungyong Youb, Wei Yangb and degradation. However, most of the protocols used to Andries Zijlstraf isolate EVs co-isolate other nucleic acids carriers and aCedars Sinai Medical Center, West Hollywood, USA; bCedars Sinai Medical the actual value of EV-associated nucleic acids as Center, Los Angeles, USA; c1Cedars Sinai Medical Center, Los Angeles, USA; dNanostics and University of Alberta, Nashville, USA; eNanostics, Nashville, robust biomarkers remain unclear. Here, we assessed USA; fVanderbilt University Medical Center, Nashville, USA the clinical validity of nucleic acids specifically derived from EV-enriched fractions in comparison to non-EV Introduction: Early diagnosis of lethal prostate cancer fractions and total plasma as a source of specific and (PC) is critical for treatment stratification. Extracellular sensitive biomarkers in breast cancer. Vesicles (EVs) are an appealing source of circulating Methods: Healthy donors or metastatic breast cancer biomarkers. We sought to perform a state-of-the-art patient’s plasma (collected under patient written con- palmitoyl proteome to identify markers of aggressive sent) was subjected to size exclusion chromatography PC because we noticed an enrichment for putative to separate EVs (EV fraction) from other circulating palmitoylated proteins in EVs in comparison with components (soluble fraction). We quantified different cells, and because most of the plasma proteins that DNA species present in these fractions as compared to contaminate the EV preps are not palmitoylated. total plasma. Nuclear and mitochondrial DNA (gDNA Palmitoylation is a post-translational modification and mtDNA) were quantified by qPCR. Tumour spe- that anchors proteins transiently to the membrane. cific nuclear alleles were detected by droplet digital We reasoned that this could be a mechanism to anchor PCR targeting known point mutations (previously proteins temporary to the membrane and shed them identified from the tumour of each patient). Finally, in EVs. 37 EV proteins were analysed using the MACSPlex Methods: Discontinuous centrifugation gradient, tun- Exosome Kit (Miltenyi). able resistive pulse sensing (QNano), next-generation Results: gDNA and mtDNA were both detected in EV PalmPISC for highly selective enrichment of palmitoyl- fractions. However, gDNA content (total or mutant proteins, 2D LC-MS/MS for deep proteomics profiling, alleles) detected in the EV fractions was lower than in Nano-Flow Cytometry (Apogee), Western blotting. the soluble fractions and total plasma. In contrast, Results: We isolated large and small EVs from PC3 mtDNA was preferentially enriched in EV fractions. cells and confirmed their biochemical and biophysical We observed similar levels of mtDNA or gDNA in identity. We observed enrichment of distinct palmi- cancer patients and healthy donors in the EV fractions, toyl-proteins in both populations of EVs versus the JOURNAL OF EXTRACELLULAR VESICLES 387 cells of origin. Pathway analysis demonstrated a strong need to implement the use of circulating biomarkers association between large EV cargo and protein locali- in the clinic to facilitate personalized therapy and pre- zation and small EV cargo and metabolic activity. dict treatment response. We conducted a prospective Interestingly, palmitoyl-CD63 was enriched in large study to demonstrate the involvement of circulating EVs while the total protein is enriched in small EVs. PD-L1 exosomes in melanoma patients. Similarly, palmitoyl-HSPA5 was enriched in small EVs, Methods: One hundred melanoma patients were while the total protein is enriched in large EVs. This included. Exosomes were isolated by ultracentrifuga- result suggests that the palmitoyl proteome might tion and evaluated by nanoparticle tracking analysis reveal a pool of markers that would not be identified using a NTA technology. Isolated exosomes were tested otherwise. The Six Transmembrane Epithelial Antigen for the expression of exosomal markers such as Prostate 1 (STEAP1) was enriched in EVs from aggres- TSG101. PD-L1 expression in plasma and in melanoma sive cancer cells but not in the cell themselves, suggest- plasma-derived exosomes (ExoPD-L1) was measured ing that it might be shed and thus identified in plasma using an enzyme-linked immunosorbent assay. of patients with aggressive disease even if it is not Results: First, ExoPD-L1 was assessed in melanoma enriched in the tumour tissue. We interrogated a cell lines. ExoPD-L1 have a role in cancer immunosup- cohort of benign (n = 30), low Gleason Score (GS) pression mediated by T-cells since they were as effi- (n = 30) and high GS (n = 30) patients. The number cient as cancer cells to inhibit T-cells activation. In of samples with detectable STEAP1 expression was melanoma patients, ExoPD-L1 (median 64,26 pg/mL) negligible in men with benign disease, and a signifi- was significantly higher than free PD-L1 in the plasma cantly more frequent event in patients with high vs which was barely detectable (0,1 pg/mL). Furthermore, low GS. ExoPD-L1 was detected in all patients whereas only Summary/conclusion: This study suggests that identi- 67% of the tumours were positive for PD-L1. fication of bonafide palmitoylated proteins in EVs Although baseline ExoPD-L1 levels were not associated represents a viable liquid biopsy to identify lethal pros- with clinicopathologic characteristics and tumour bur- tate cancer. den, ExoPD-L1 variations (ΔExoPD-L1) after treatment correlated with tumour response and survival. A ΔExoPD-L1 cut-off of > 100 was defined, yielding a LB03.04 83% sensitivity, a 70% specificity, a 91% positive predictive value and a 54% negative predictive values for disease progression. The use of this cut-off allowed Circulating exosomal PD-L1 as a marker for the follow up of melanoma patients stratification in two groups of patients statistically dif- Jessica Gobboa, Marine Cordonnierb, Charlée Nardinc, Gaetan Chanteloupb, ferent in terms of overall survival and progression free Valentin Derangéred, Marie-Paule Algrose, Aurelie Bertautd, Laurent d b c survival. Arnould , Carmen Garrido and François Aubin Summary/conclusion: PD-L1 level in circulating exo- aCentre Georges-Francois Leclerc, dijon, France; binserm1231, dijon, France; cCHU Besançon, besançon, France; dCGFL, dijon, France; eCHU Besançon, somes may be a more reliable marker than PD-L1 besancon, France expression in tumour tissue. Circulating exosomal PD-L1 monitoring may be a promising biomarker to Introduction: In the era of effective molecular targeted predict tumour response and the clinical outcome. treatments and immunotherapies, there is an urgent 388 ISEV2019 ABSTRACT BOOK

Symposium Session 32: Late Breaking- EV Labeling, Separation, and Detection Chairs: Elisa Lazaro-Ibanez; Ryou-u Takahashi Location: Level B1, Lecture Room 09:30–10:15

Summary/conclusion: This new platform suggests that LB04.01 MAF of EV-derived DNA can have large patient variability that may depend on cancer type, stage, progression, or other pathophysiological factors. These results A microfluidic device with nanoscale surface topology and functionalized with lipid nanoprobes for extracellular vesicle isolation support the need for a rapid and reliable EV isolation and clinical cancer diagnosis technique, such as this reported device. Yuan Wana, Mackenzie Maurerb, Hong-Zhang Heb, Yi-Qiu Xiab, Wen-Long Zhangb, Si-Jie Haob, Nelson Yeec and Siyang Zhengb Funding: This work was supported by the National Cancer Institute of the US National Institutes of aBinghamton University, State University of New York, Binghamton, USA; bThe Pennsylvania State University, University Park, USA; cPenn State Health under grant number 1R01CA230339 to S. Y. College of Medicine, Hershey, USA Zheng.

Introduction: Extracellular vesicles (EVs) are cell- derived, lipid membrane enclosed particles. Tumour LB04.02 cell-derived are increasingly recognized for their pathophysiological contributions and potential towards can- Asparagine-linked glycosylation amplifies the heterogeneity of cer diagnosis and treatment monitoring. However, tumour extracellular vesicles clinical translation of EVs has been limited by techno- Yoichiro Haradaa, Kazuki Nakajimab, Nobuyoshi Kosakac, Tomoko Fukushiged, Kiyotaka Kondoa, Junichi Seinoe, Tadashi Suzukie, Hiromasa logical challenges for EV isolation. A rapid, high- Inouea, Takuro Kanekuraf, Takahiro Ochiyac and Ikuro Maruyamaa throughput, and on-chip EV isolation technology is aKagoshima University Medical and Dental Sciences, Kagoshima, Japan; critical for EV-based cancer diagnosis. bFujita Health University, Aichi, Japan; cDepartment of Molecular and Methods: We report a lipid nanoprobe-functionalized Cellular Medicine, Institute of Medical Science, Tokyo Medical University, Tokyo, Japan; dKagoshima Univeristy Medical and Dental Sciences, nanostructured silica microfluidic device that can be Kagoshima, Japan; eRIKEN, Saitama, Japan used in combination with nucleic acid extraction, and digital droplet polymerase chain reaction (ddPCR) for Introduction: Tumour cells secrete heterogeneous EV isolation, enrichment, and DNA mutation detec- populations of extracellular vesicles (EVs) carrying dis- tion from clinical plasma samples for cancer diagnosis. tinct proteins. However, the molecular underpinnings The device consists of EV-size-matched silica nanos- that regulate such EV heterogeneity remain largely tructures, surface-grafted lipid nanoprobes and a poly- elusive. Tumours consume a large quantity of glucose dimethylsiloxane (PDMS) herringbone micromixer through glycolysis for the synthesis of various bioactive chamber. Plasma samples are collected from either metabolites. cell lines or clinical samples (IRB approved and Methods: EVs were prepared from conditioned med- patients consented). As plasma flows through the ium of mouse B16-F10 melanoma cells by differential microfluidic device, the EVs are isolated. EV DNA is centrifugation. The number of EVs secreted, their then extracted and pathological mutations are detected cargo proteins and intracellular carbohydrate metabo- with ddPCR. lism were analysed. Results: The microfluidic device removes 96.5% Results: Here, we show that 2-DG, a glycolysis inhibi- plasma proteins. The limit of detection of a KRAS tor, suppressed secretion of melanoma EVs indepen- mutation from plasma EV by ddPCR is 0.01% mutant dently of its glycolysis blockade action. 2-DG-sensitive allele fraction (MAF). The device is validated in a pilot EVs were enriched with asparagine (N)-linked glycosy- clinical study for pancreatic cancer diagnosis. Clinical lated proteins, while 2-DG-resistant EVs contained samples with known KRAS mutations in the tissue intrinsically non-glycosylated proteins. Metabolic con- were validated with the device. ddPCR indicated MAF version of 2-DG to artificial nucleotide sugars via gly- of 1.8%, 10.1%, and 22.3%, respectively, from DNA colysis branches induced degradation of N-linked extracted from plasma EV, while none were detected glycan precursors and hypoglycosylation of multiple in healthy controls. glycoproteins. Mutagenesis at N-linked glycosylation JOURNAL OF EXTRACELLULAR VESICLES 389 sites of an EV cargo protein or pharmacological inhibi- analysis known as Amnis ISX mkII imaging flow cyt- tion of N-glycosylation reaction by oligosaccharyltrans- ometer to measure fluorescent signals from individual ferase was sufficient to suppress secretion of N-linked nanoparticles with the added value of being able to glycosylated proteins by EVs. individually visualize particles being measured. Summary/conclusion: This study establishes N-linked Results: We first compared the rate of EVs released glycosylation as a key posttranslational modification from glioma cells treated with 5-ALA and determined a that influences the heterogeneity of tumour- significant number of fluorescent EVs released within derived EVs. hours of exposure to 5-ALA, while the healthy human brain microvascular endothelial cells (HBMVEC) did LB04.03 not release any fluorescent EVs. We also compared the direct analysis of conditioned media to that of EVs purified by a commercial kit and determined that the Characterization of fluorescent plasma evs following 5-ALA use in extra exposure to light of EVs with the commercial kit malignant gliomas. leads to a significant loss of fluorescent EVs. To con- Leonora Balaja, Pamela Jonesb, Anudeep Yekulab and Bob Carterb firm our findings we exposed 5-ALA EVs to white light aMassachusetts General Hospital, Boston, USA; bMGH, Boston, USA for 20 min and compared the number of fluorescent Introduction: Malignant gliomas are rapidly progres- events before and after exposure to light, and deter- sive brain tumours with very high morbidity and mor- mined a > 98% loss of fluorescent EVs. Finally, a tality. The recent FDA approval of 5-aminolevulinic comparison of the plasma samples from glioma acid (5-ALA, Gliolan) provides the neurosurgeon with patients collected upon administration of 5-ALA real-time fluorescent delineation of malignant tissue revealed that we can reliably detect fluorescent EVs in which allows a significantly higher rate of complete the plasma of these patients when the primary tumour resections of malignant gliomas and longer progres- fluoresces, while these events were undetectable in the sion-free survival compared to conventional white- cases where the primary tumour did not fluoresce. light resections. We sought to determine whether fluor- Furthermore, these events were undetectable upon escent EVs would be released in the plasma of these tumour resection. patients. Summary/conclusion: This study is as a proof of con- Methods: Here, we characterize EVs isolated from cept to determine our ability to utilize fluorescent glioma cell lines treated with 5-ALA for 24 h. We based tumour-specific EV characterization to aid in also evaluated plasma-derived EVs from glioma the diagnostics and prognostics of gliomas. patients following preoperative oral administration of Funding: CA069246 CA230697 TR000931 5-ALA. We used a highly sensitive fluorescence-based 390 ISEV2019 ABSTRACT BOOK

Symposium Session 33: Late Breaking- From Biogenesis to Uptake Chairs: Yutaka Naito; Ganesh Shelke Location: Level B1, Hall B 09:30–10:15

Funding: This study was part of the NIH Extracellular RNA Communication Consortium paper package and LB05.01 was supported by the NIH Common Fund’s exRNA Communication Program. The work was funded by NIH grants The work was funded by NIH grants F31 Reassessment of exosome composition Dennis Jeppesena, Aidan Fenixb, Jeffrey Franklina, James Higginbothama, Qin HL136081 to Aidan M. Fenix, R35 GM125028 to Dylan Zhanga, Leonard Romec, Dylan Burnetteb and Robert Coffeya T. Burnette, and R35 CA197570 and U19 CA179514 to aVanderbilt University Medical Center, Nashville, USA; bVanderbilt Robert J. Coffey University School of Medicine, Nashville, USA; cDavid Geffen School of Medicine, University of California, Los Angeles, USA LB05.02 Introduction: The heterogeneity of extracellular vesicles (EVs) and presence of non-vesicular extracellular Biofunctional peptide-modified extracellular vesicles for targeted nanoparticles pose major obstacles to our understand- intracellular delivery ing of the composition and functional properties of Ikuhiko Nakase distinct secreted components. Greater precision in Graduate School of Science, Osaka Prefecture University, Sakai-Shi, Japan assigning RNA, DNA and protein to their correct extracellular compartments and identifying their Introduction: Our research group is developing ther- mechanisms of secretion is crucial for identification apeutic techniques based on extracellular vesicles (exo- of biomarkers and design of future drug interventions. somes, EVs) and peptide chemistry to deliver Methods: We have employed high-resolution density therapeutic/diagnostic molecules into targeted cells. gradient fractionation and direct immunoaffinity cap- Because of pharmaceutical advantages of the EVs as ture (DIC) to precisely characterize the RNA, DNA, carriers for intracellular delivery of therapeutic mole- and protein constituents of exosomes and other non- cules, we are trying to develop methodology to easily vesicle material. Proteomics and RNA-Seq analyses modify biofunctional peptides on exosomal mem- were performed on purified small EVs and extracellular branes for receptor target and enhanced cellular uptake non-vesicular material. DIC was used to specifically of the EVs. In this presentation, modification techni- isolate exosomes from other types of small EVs and ques using biofunctional peptides such as arginine-rich was performed without ultracentrifugation and with cell-penetrating peptides (CPPs, macropinocytosis capture beads targeting the classical exosomal tetraspa- induction) [1], artificial coiled-coil peptides (receptor nins CD63, CD81 and CD9. Biochemical analysis and target) [2], membrane fusion peptides (cytosolic structured illumination microscopy were used to exam- release) will be introduced [3, 4]. And newly developed ine secretion and presence of extracellular DNA. exosomes decorated with cell-penetrating sC18 pep- Results: Extracellular RNA, RNA-binding proteins and tides [5], which are derived from cationic antimicrobial other cellular proteins are differentially expressed in protein, CAP18, will be also presented and discussed exosomes and non-vesicle compartments. Argonaute for cancer targeting. 1–4, glycolytic enzymes and cytoskeletal proteins were Methods: For cellular uptake assessments of EVs, we not detected in exosomes. We further demonstrate that used CD63 (EV marker protein)-GFP-fusion protein small EVs are not vehicles of active DNA release. expressed EVs. All biofunctional peptides were synthe- Instead, we propose a new model for active secretion sized by Fmoc solid-phase methods. of extracellular DNA through an autophagy- and mul- Results: Macropinocytosis has been shown to be very tivesicular endosome-dependent but exosome-indepen- important for cellular EV uptake [1]. Therefore, our dent mechanism. research group developed the methods for modification Summary/conclusion: This study demonstrates the of arginine-rich CPPs on EV membranes using chemi- need for a reassessment of exosome composition and cal linkers or acylation technique, which can induce offers a framework for a clearer understanding of EV clustering of proteoglycans (e.g. syndecan-4) and and extracellular nanoparticle heterogeneity. macropinocytosis signal transduction [1]. In the JOURNAL OF EXTRACELLULAR VESICLES 391 research of artificial coiled-coil peptides, the artificial ing analysis, and immune-electron microscopy were leucine zipper peptide-modified EVs recognize the used to analyse EVs and HAV virus particles. peptide-tagged receptor expression on targeted cells Fluorescence microscopy in live-cell and immune-elec- [2]. Stearylation of branched sC18 peptides were easily tron microscopy was used to identify the exosome-like modified on the EVs by their insertion of hydrophobic biogenesis of eGFP-pX. Co-IP was performed in 293T moiety in EV membranes, resulted in effective induc- cells. Amino-acids truncation and mutation in pX were tion of macropinocytosis and cancer cellular uptake. performed in order to find the novel functional domain Summary/conclusion: These experimental techniques of pX. will contribute to development for the EV-based tar- Results: Fusing pX to eGFP could guide eGFP into geted intracellular delivery systems. exosomes through directing eGFP into multivesicular Reference: [1] I. Nakase, et al. Sci. Rep. 6, 34937 (2016), bodies (MVBs). Simultaneously, the release of ALIX, an [2] I. Nakase, et al. Chem. Commun. 53, 317 (2017), [3] MVBs maker protein, is enhanced and co-IP assay I. Nakase, et al. Sci. Rep. 5, 10112 (2015), [4] M. confirms the direct interaction between pX and V Akishiba, et al. Nat. Chem. 9, 751 (2017), [5] A. domain of ALIX. Furthermore, deleting C-terminal Gronewold, et al. ChrmMedChem. 12, 42 (2017) half of pX abolishes eHAV release and the interaction between HAV virion and ALIX. Consistently, the C- terminal half of pX alone is sufficient to load eGFP into LB05.03 exosomes by interacting with ALIX. On the other side, we find there is a novel nucleus export signal (NES)

Virus protein pX facilitates naked particles of hepatitis A virus to motif MMSRIAAGD in its N-terminal 12 amino-acids, acquire an exosome-derived membrane by interacting with ESCRT- which was previously reported essential for virion associated protein ALIX assembly Wang Jianga, Pengjuan Mab, Libin Dengb and Gang Longb Summary/conclusion: Here, we find that pX is aInstititut Pasteur of Shanghai, Shanghai, USA; bInstitut Pasteur of Shanghai, Shanghai, China (People‘s Republic) required for eHAV release and contains a novel nucleus export signal (NES) in its N-terminal. To con- Introduction: Hepatitis A virus (HAV), a classically- clude, pX plays important roles in HAV assembly and thought non-enveloped virus, has recently been found release through two independent domains: the N-term- to release majorly in the form of quasi-enveloped HAV inal domain containing a novel NES, and the C-term- (eHAV) by hijacking the host’s endosomal sorting inal half domain associated with the membrane complexes required for transport (ESCRT) complexes. envelopment via the interaction with ALIX. Thus, pX Compared to the non-enveloped virion, eHAV exclu- may have the potential for protein loading into exo- sively contains a viral protein pX. somes in the future. Methods: Differential centrifugation and iodixanol- Funding: This work was supported by the national based gradient centrifugation were used to isolate dif- natural Science Foundation of China (81772200). ferent types of EVs. Western-blot, Nanoparticle track- 392 ISEV2019 ABSTRACT BOOK

Symposium Session 34: Late Breaking- EV Signatures and Function Chairs: Ter-Ovanesyan; Yusuke Yoshioka Location: Level B1, Hall A 09:30–10:15

muscle protein expression profiles to identify miRNA- targeted networks. LB06.01 Results: A total of 279 (NGT), 308 (GDM) and 175 (NGT vs GDM) miRNAs were significantly changing in expression across gestation. 6 miRNAs (hsa-miR- Proteomic and miRNA transcriptome analysis revealed an association 92a-3p, hsa-miR-10a-5p, hsa-miR-151b, hsa-miR-16– between circulating exosomal miRNAs and insulin sensitivity in gestational diabetes mellitus during gestation 2-3p, hsa-miR-1910-5p and hsa-miR-423-5p) were Soumyalekshmi Naira, Dominic Guanzonb, Andrew Laic, Katherin Scholz- confirmed to be differentially expressed in GDM. Romeroc, Carlos Palmac, Jezid Mirandad, Fatima Crispie, David McIntyref, Martha Lappasg and Carlos Salomonb Proteomic characterization revealed 55 proteins to be differentially expressed in GDM skeletal muscles com- aExosome Biology Laboratory, Centre for Clinical Diagnostics, UQ centre for Clinical Research, Royal Brisbane and Women‘s Hospital, Faculty of pared to NGT. The exosomal miRNAs upregulated in Medicine + Biomedical Sciences, The University of Queensland, Brisbane, GDM target some of these differentially expressed pro- Australia; bExosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and teins (Serine/Threonine Protein Phosphatase 6 (PPP6), Women’s Hospital, The University of Queensland, Brisbane QLD 4029, Chloride Intracellular Channel Protein 4 (CLIC4) and Australia., Brisbane, Australia; cExosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Actin Related Protein Complex 2 (ARPC2)) in skeletal Royal Brisbane and Women’s Hospital, The University of Queensland, muscles in GDM and associated with pathways regu- d Brisbane QLD 4029, Australia, Brisbane, Australia; Fetal i + D Fetal lating glucose metabolism and insulin signalling (such Medicine Research Center, BCNatal – Barcelona Center for Maternal-Fetal and Neonatal Medicine (Hospital Clínic and Hospital Sant Joan de Deu), as STAT 3 pathway). ICGON, IDIBAPS, Universitat de Barcelona, Spain, Barcelona, Spain; eFetal Summary/conclusion: The miRNA content in mater- i + D Fetal Medicine Research Center, BCNatal – Barcelona Center for Maternal-Fetal and Neonatal Medicine (Hospital Clínic and Hospital Sant nal circulating exosomes differs across gestation in Joan de Deu), ICGON, IDIBAPS, Universitat de Barcelona, Spain., Barcelona, GDM patients compared to NGT and target specific Spain; fMater Research, Faculty of Medicine, University of Queensland, Mater Health, South Brisbane, Australia., Brisbane, Australia; gUniversity of proteins and pathways in skeletal muscle. This suggests Melbourne, Department of Obstetrics and Gynaecology, Australia. Mercy that exosomes may be involved in maternal metabolic Hospital for Women, 163 Studley Road, Heidelberg, Victoria 3084, Australia., Brisbane, Australia adaptation to pregnancy through the delivery of bioactive miRNAs. Introduction: Gestational Diabetes Mellitus (GDM) is Funding: Diabetes Australia, Lions Medical Research the most common medical complication in pregnancy, Foundation, NHMRC; 1114013, and FONDECYT with short and long term metabolic effects in mothers 1170809. and offsprings. We comprehensively analysed the exosomal miRNA profile across gestation in normal glucose tolerant (NGT) and women with GDM and LB06.02 determined the signalling pathways associated with changes in miRNA profile. Extracellular vesicles from induced neurons trigger epigenetic Methods: Exosomes were isolated from plasma sam- silencing of a brain neurotransmitter a b c d ples collected at three time points during pregnancy Glenn McConkey , Isra Alsaady , Ellie Tedford and Norhidayah Badya from NGT and GDM women. Using a small RNA aUniversity of Leeds, Leeds, United Kingdom; bUniversity of King Abdulaziz, Leeds, United Kingdom; cUniversity of Cambridge, Cambridge, United library and linear mixed modelling analysis, the Kingdom; dUniversity of Leeds, Leeds, United Kingdom miRNA profiles across gestation in NGT, GDM and NGT vs GDM were identified in a discovery cohort Introduction: Our new breakthrough finding is that and the expression of candidate miRNAs were mea- extracellular vesicles (EVs) injected into the brain spe- sured using qRT-PCR in a validation cohort. Further, cifically down-regulated production of the neurotrans- we characterized the changes in the proteomic profile mitter norepinephrine suppressing transcription of the in skeletal muscles obtained from GDM patients com- DBH gene and hypermethylation of the gene’s promo- pared to NGT controls, using a quantitative, data-inde- ter. DBH produces norepinephrine from dopamine in pendent acquisition mass spectrometric approach and neurons. Previous studies found EVs regulate immune finally integrated the exosomal miRNA and skeletal responses via PTGS but regulating neurons and JOURNAL OF EXTRACELLULAR VESICLES 393 epigenetic changes have not been described. DNA Paris, France; iAssistance Publique – Hôpital Européen Georges Pompidou- Cardiology and INSERM U970 – PARCC, Paris, France methylation in neurons is involved in memory and neurological disorders (Science 2018 361 (6409)). Introduction: Extracellular vesicles (EV) recapitulate These observations concur with our recent study that most of the cardioprotective effects of stem cells but found central noradrenergic signalling is suppressed in their immunological impact remains poorly the brains of infected rodents and in neurons (Infect understood. Immun 2019 87(2)) for this parasite that causes move- Hypothesis: Immune response to EV may be beneficial ment disorders and is associated with neurological rather than deleterious for the infarcted heart. disorders. Methods: EV secreted from human-induced pluripo- Methods: Neuronal cells were induced by infection with tent stem cells [EV-hPg-iPS] were first assessed in vitro the neurotropic protozoan Toxoplasma gondii and EVs for the expression of immune and stem cell markers by purified on sucrose gradients. EVs, characterized by flow cytometry and their cross-talk with allogeneic T TEM, were used to treat rat and human neuronal cells and NK cells, was determined by mixed lymphocyte and DBH mRNA and nascent DBH gene transcription reactions (MLR). Then, 70 immunocompetent mice were measured. DNA methylation was measured by underwent a myocardial infarction and surviving MSRE-qPCR. Induced EVs were injected into the locus mice were injected intramyocardially (under echo gui- co-eruleus of rats and DBH gene expression was dance) with EV-hPg-iPS, hPg-iPS or PBS either acutely monitored. (n = 6) or chronically (n = 6), i.e., 3 days and 3 weeks Results: We found that EVs purified from infected neu- after infarction, respectively. Immune responses were ronal cultures (43 ± 5 nm) specifically caused transcrip- monitored 3 days after treatment in all mice. Eighteen tional gene silencing (TGS) and DNA methylation in additional animals were sham-operated and also noradrenergic neurons. The induced EVs down-regu- injected after 3 weeks with EV-hPg-iPS, hPg-iPS or lated DBH gene expression >200-fold and, surprisingly, PBS. Pro- and anti-inflammatory cytokines were mea- the down-regulation was at transcriptional level. The EVs sured in heart tissue and plasma by a bead-based multi- also caused an epigenetic change; specifically inducing plex immunoassay (n = 6/group). DNA hypermethylation of the DBH gene. Intracerebral Results: EV-hPg-iPS expressed stem cell markers injection of induced EVs into rats down-regulated DBH (SSEA-1, CD15, CD133) and low levels of HLA class expression. We are currently identifying the RNA I and PD-L1. MLR and in vivo studies demonstrated responsible as the down-regulation was disabled by that EV do not activate an adaptive allogeneic immune degradation of the small RNAs in the EVs. response since they failed to induce proliferation of Summary/conclusion: This is the first study to find allogeneic CD8+ or CD4 + T cells. In contrast to transcriptional gene silencing of a neurotransmitter in their parental cells, EV did not induce NK cell degra- the brain by EVs and DNA hypermethylation in the nulation either. While injection of hPg-iPS or their EV neurons. This research will enhance our understanding at the chronic post infarction stage did not affect the of neurological disorders (ie. schizophrenia, epilepsy, number of T cells, B cells, and macrophages in cardiac drug addiction) and how memory works. The role of tissue, spleen, bone marrow and blood, they signifi- EVs in regulating neurotransmission in the brain will cantly decreased the circulating levels of the probe presented. inflammatory cytokines IL-1α and IFN-γ compared to the control group (PBS). However, at the acute stage, and in contrast to PBS, EV significantly reduced the LB06.03 number of monocytes High (M1 macrophage precursor), M1 macrophages, and neutrophils as well as the circulating levels of the pro-inflammatory cytokines IL- Extracellular vesicles from human iPS-derived cardiovascular progenitors do not trigger an immune response in the infarcted heart 1α, IL-2 and IL-8 while it significantly increased those Bruna Lima Correaa, Nadia EL-Haraneb, Ingrid Gomezb, Hocine Rachidc, of IL-10. José Vilard, Manon Desgrese, Valérie Bellamye, Laetitia Pidiale, Paul Alayrace, Dominique Charronf, Nisa Renaultg, Reem Al-Daccakh, Jean-Sebastien Summary/conclusion: EV-hPg-iPS look immunologi- Silvestree and Philippe Menaschéi cally neutral in vitro and in vivo and seem even able to aINSERM U970 – PARCC, Paris, France; bINSERM U970 – PARCC, PARIS, mitigate the infarct-related inflammatory response. c d France; INSTITUT CURIE, Paris, France; INSERM U970 – PARCC, Paris, Funding: INSERM, LabexRevive, APHP, University French Guiana; eINSERM U970 – PARCC, paris, France; fAssistance Publique – HÔPITAL SAINT-LOUIS -Immunology, Paris, France; Paris Descartes, Fondation de France, FRM gFUJIFILM Cellular Dynamics, Inc., Madison, USA; hINSERM· UMRS 976,