The Pennsylvania State University
The Graduate School
Department of Neurosurgery
EXOSOMES AND THEIR IMPLICATIONS IN GLIOBLASTOMA
A Dissertation in
Biomedical Sciences
by
Oliver D. Mrowczynski
2018 Oliver D. Mrowczynski
Submitted in Partial Fulfillment of the Requirements for the Degree of
Doctor of Philosophy
May 2018
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The dissertation of Oliver D. Mrowczynski was reviewed and approved* by the following:
James R. Connor University Distinguished Professor Vice-Chair of the Department of Neurosurgery Dissertation Advisor Chair of Committee
Jennifer W. Baccon Professor and Chair Department of Pathology, Northeast Ohio Medical University Chair, Department of Pathology and Laboratory Medicine, Akron Children’s Hospital
Barbara A. Miller Professor of the Department of Pediatrics
Achuthamangalam B. Madhankumar Assistant Professor of the Department of Neurosurgery
Brad E. Zacharia Assistant Professor of the Department of Neurosurgery Special Member
Jong K. Yun Associate Professor of the Department of Pharmacology
Ralph L. Keil Chair, Biomedical Sciences Graduate Program Associate Professor of Biochemistry and Molecular Biology
*Signatures are on file in the Graduate School
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ABSTRACT
Exosomes are 20-100 nm cellular derived vesicles that when discovered, were initially thought to be a form of cellular recycling of intracellular contents. More recently, these vesicles are under study for their purported significant roles in intercellular communication in both healthy and diseased states. Exosomes are secreted by all cancer types and have a major impact on the tumor microenvironment. Exosomes contain protected intracellular content, including
DNA, RNA, and proteins that can be transferred to recipient cells. This transfer subsequently leads to enhanced tumorigenic properties including angiogenesis, cancer progression, and therapeutic resistance. Genetic components of the cell of origin can be included in the secreted exosomes. The presence of genetic material could serve as a biomarker for the presence of mutations in tumors leading to modification of treatment strategies. In the last decade, exosomes have been identified as having major implications in many aspects of medicine and tumor biology and appear to be primed to take a critical position in cancer diagnosis, prognosis, and treatment.
The first aspect of my thesis studies the effects of changes in genotype on exosome phenotype by studying neuroblastoma cancer and hemochromatosis mutations. Neuroblastoma is the third most common childhood cancer, and timely diagnosis and sensitive therapeutic monitoring remain major challenges. Tumor progression and recurrence is common in advanced stages with little understanding of mechanisms. Although exosomes have been demonstrated to contribute to the oncogenic effect on the surrounding tumor environment and also mediate resistance to therapy, the effect of genotype on exosomal phenotype had not been explored. I interrogated exosomes from human neuroblastoma cells that express wild-type or mutant forms of the HFE gene. HFE, one of the most common autosomal recessive polymorphism in the
Caucasian population, originally associated with hemochromatosis, has also been associated with increased tumor burden, therapeutic resistance boost, and negative impact on patient survival.
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Herein, I demonstrated that changes in genotype cause major differences in the molecular and functional properties of exosomes; specifically, HFE mutant derived exosomes have increased expression of proteins relating to invasion, angiogenesis, and cancer therapeutic resistance. HFE mutant derived exosomes were also shown to transfer this cargo to recipient cells and cause an increased oncogenic functionality in those recipient cells. This has major implications because it allows clinicians to profile and stratify cancer patients with HFE mutations and adapt therapeutic strategies to provide patients with the optimal survival outcome.
The second aspect of my thesis assessed the effects of radiation on exosome function and profile. Radiation therapy is essential in the arsenal of cancer treatment and is utilized in the therapeutic regimen of more than 50% of all cancer patients. Unfortunately, many aggressive malignancies may become resistant to radiation over time, rendering treatment futile. This mechanism of acquired radiation resistance is not understood. I investigated the hypothesis that acquired radiation resistance may occur through cellular communication via exosomes. Three aspects were analyzed: 1) exosome function, 2) exosome profile, and 3) exosome uptake/blockade. Radiation-derived exosomes increased cellular proliferation and radiation resistance in recipient tumor cells in vitro in cell culture. Furthermore, radiation-derived exosomes increased tumor burden and decreased survival in vivo in a murine model of glioblastoma. The mechanism underlying this phenomenon is that radiation-derived exosomes exhibit specific miRNA, mRNA, and protein expression changes favoring a resistant/proliferative profile. Radiation-derived exosomes upregulate oncogenic miR-889, CCND1, ANXA2, DERL1,
WWC1, NPM1, SCD, ACTG1, FUT11, VAMP8, ZFR, DNM2, CISD1, RPL15, PPIC, and proteins involved in the proteasome, Notch, Jak-STAT, and cell cycle signaling pathways.
Radiation-derived exosomes also downregulate tumor-suppressive miR-516, miR-365, TPM1,
STAT4, LRRFIP1, TSPAN5, and CGGBP1. Ingenuity pathway analysis revealed the top upregulated networks included cell growth, cell cycle, and cell survival. The top increased
v upstream regulator was the MYC oncogene. Upregulation of Dynamin 2 correlated with increased uptake of radiation-derived exosomes. In addition to inducing changes in recipient cells that promote a cancerous phenotype, I evaluated exosome blockade as a potential therapeutic.
Heparin and simvastatin blocked uptake of radiation-derived exosomes in recipient cells and inhibited induction of cellular proliferation and radiation resistance, both in vitro and in vivo. In conclusion, I provide a novel exosome-based mechanism that may underlie acquired radiation resistance in patients harboring malignant cancers. Furthermore, I elucidate key factors carried by exosomes that may lead to tumor recurrence and subsequent therapeutic resistance. I also show the potential for advancement of cancer treatment, or understanding existing treatments, whose mechanism may be through exosome inhibition. By interrogating this mechanism, I ultimately aim to pave the way for the development and implementation of targeted exosome blocking agents to inhibit the acquired radiation resistance that inevitably leads to tumor recurrence and the devastating prognosis that follows.
The last aspect of my thesis assessed the effects of glioma cell stemness on exosome phenotype. One reason proposed for the ineffectiveness of the current therapeutic regimen of glioblastoma is glioma stem cells (GSCs). We investigated the hypothesis that the communication of GSCs to their microenvironment through exosomes is a key factor to the enhanced tumor burden and the development of resistance to therapeutics in glioblastoma. Two properties of exosomes were analyzed: 1) exosome function and 2) exosome profile. Exosomes secreted by patient derived-glioma stem cells (GSC-exosomes) increased cellular proliferation, radiation resistance, temozolomide resistance, and doxorubicin resistance. We further profiled the GSC- exosomes to elucidate the underlying mechanism of this phenomenon. Profiling showed specific changes to RNA and protein favoring therapeutic resistance and cellular proliferation. GSC exosomes have increased expression of proteins involved in radiation and chemotherapeutic resistance (E.g. CDK4 and Notch), cellular proliferation (E.g. Cyclin B1 and Cyclin D2),
vi angiogenesis (E.g. VEGF-A and EGFR), glioma cell stemness and de-differentiation (E.g.
EPHA2, Cathepsin B), and cell invasion and metastasis (E.g. ITGA3, COL4A2). The results of our study provide a novel exosome-based mechanism that may underlie the aggressiveness of glioma cancer stem cells. Furthermore, we elucidate key factors carried by glioma stem cell derived exosomes that may lead to enhanced therapeutic resistance and increase in tumor burden.
Together, these data demonstrate the impact that exosomes have on multiple aspects of tumor biology. Exosomes are affected due to genotype, and thus genotype must be taken into consideration for cancer patient stratification. Exosomes are also are critical for radiation resistance and are reprogrammed due to radiation. Glioma stem cells secrete exosomes which have increased oncogenic contents and have an enhanced functional impact on recipient cancer cells. My studies have shown that treatment with heparin and simvastatin inhibits uptake of these cancer-derived exosomes which may lead to enhanced therapeutic efficacy in the cancer patient population. My thesis also suggests that our current therapies to treat glioblastoma may be ineffective due to exosomes secreted by glioma stem cells in conjunction with exosomes secreted by glioma cells stressed with radiation. These secreted exosomes confer therapeutic resistance and may lead to subsequent tumor recurrence in the glioblastoma patient population. The results of my thesis offer direct recommendation for clinical studies that suggests utilizing our standard therapies in combination with exosome uptake inhibitors may lead to optimal patient outcomes.
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TABLE OF CONTENTS
List of Figures ...... x
List of Tables ...... xii
List of Abbreviations ...... xiii
Acknowledgements ...... xvi
Chapter 1. Exosomes and their implications in central nervous system tumor biology ...... 1
1.1. Abstract ...... 2 1.2. Introduction ...... 2 1.3. Extracellular Vesicle Formation, Secretion, and Uptake ...... 6 1.3.1. Exosome Biogenesis ...... 7 1.3.2. Exosome Secretion ...... 11 1.3.3. Exosome Uptake ...... 12 1.4. Exosome Purification and Identification ...... 17 1.5. Exosome Function in Oncogenesis ...... 20 1.5.1. Exosome Effect on Treatment Resistance ...... 21 1.6. Current Role of Exosomes as Cancer Biomarkers ...... 22 1.6.1. Use of Exosomes for Molecular Profiling ...... 23 1.6.2. Use of Exosomes as Diagnostics ...... 24 1.6.3. Use of Exosomes as Markers for Treatment Effectiveness ...... 25 1.7. Therapeutic Potential of Exosomes ...... 26 1.8. Future Implications ...... 28 1.9. References ...... 28
Chapter 2 HFE Genotype Affects Exosome Phenotype in Cancer ...... 54
2.1. Abstract ...... 54 2.2. Introduction ...... 55 2.3. Materials and Methods ...... 56 2.3.1. Cell Culture and Exosome Isolation ...... 56 2.3.2. Exosome Confirmation and Size Analysis ...... 56 2.3.3. Immunoblot Analysis ...... 57 2.3.4. Invastion/Migration Assay ...... 57 2.3.5. Tubulogenesis Assay ...... 58 2.3.6. Cellular Proliferation Assay ...... 58 2.3.7. Apoptosis Assay ...... 59 2.3.8. Statistical Analysis ...... 59 2.4. Results ...... 59 2.5. Discussion ...... 73 2.6. References ...... 75
Chapter 3 Exosomes and Acquired Radiation in Cancer ...... 82
3.1. Abstract ...... 82
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3.2. Introduction ...... 83 3.3. Materials and Methods ...... 84 3.3.1. Cell Culture, Materials, and Exosome Isolation ...... 84 3.3.2. Exosome Confirmation ...... 85 3.3.3. Immunoblot Analysis ...... 86 3.3.4. Cellular Proliferation Analysis ...... 86 3.3.5. Apoptosis Assay ...... 86 3.3.6. Reactive Oxygen Species Assay ...... 87 3.3.7. Exosome Blockade Analysis ...... 87 3.3.8. Exosome Fluorescent Tagging and Blockade Analysis ...... 88 3.3.9. In Vivo Studies ...... 89 3.3.10. Immunohistochemistry of Tumor Tissue Sections...... 90 3.3.11. RNA Analysis ...... 90 3.3.12. Proteomic Analysis ...... 92 3.3.13. Statistical Analysis ...... 93 3.4. Results ...... 93 3.5. Discussion ...... 112 3.6. References ...... 117
Chapter 4. The Impact of Glioma Cancer Cell Stemness on Exosome Phenotype ...... 129
4.1. Abstract ...... 129 4.2. Introduction ...... 130 4.3. Materials and Methods ...... 131 4.3.1. Cell Culture and Exosome Isolation ...... 131 4.3.2. Exosome Size Analysis and Quantification ...... 132 4.3.3. Immunoblot Analysis ...... 132 4.3.4. Cellular Proliferation Analysis ...... 132 4.3.5. Apoptosis Assay ...... 132 4.3.6. Proteomic Analysis ...... 133 4.3.7. Statistical Analysis ...... 134 4.4. Results ...... 134 4.5. Discussion ...... 141 4.6. References ...... 144
Chapter 5. Overarching Themes of Exosomes in Cancer Biology ...... 151
5.1. Introduction: Summary of Main Findings of Dissertation ...... 151 5.2. Theme One ...... 152 5.2.1. Exosome Use for Molecular Profiling ...... 152 5.2.2. Exosome Monitoring of Treatment Response ...... 153 5.2.3. Exosomes to Diagnose and Predict Brain Metastases ...... 155 5.3. Theme Two ...... 157 5.3.1. Effects of Chemotherapy on Exosome Composition and Function ...... 157 5.3.2. Stress-Induced Exosomes and Their Effects on Immune Function ...... 158 5.3.3. Stress-Induced Exosomes and Their Effects on Invasion and Migration ...... 158 5.3.4. Radiation Treatment Margins and Leading Edge Radiotherapy ...... 159 5.4. Theme Three ...... 162 5.4.1. Exosome Blockade as a Novel Therapeutic Strategy ...... 162
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5.4.2. Exosome Structure to Inform Optimal Liposome Formulation ...... 163 5.5. Limitations and Challenges ...... 164 5.6. Conclusion ...... 165 5.7. References ...... 166
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LIST OF FIGURES
Figure 1-1. Exosome biogenesis...... 10
Figure 1-2.Visualization of Glioblastoma exosomes...... 19
Figure 2-1. Transmission electron microscopy visualizing exosome morphology from SH-SY5Y neuroblastoma variants...... 60
Figure 2-2. Exosome size distribution analysis with dynamic light scattering ...... 62
Figure 2-3. Immunoblot analysis of the ferrome contained within exosomes ...... 64
Figure 2-4. Invasion and migration analysis ...... 66
Figure 2-5. Angiogenesis functionality determined with HUVEC incubation with exosomes from all genotypes...... 68
Figure 2-6. Analysis of cellular proliferation and therapeutic resistance ...... 71
Figure 3-1. Exosome confirmation analysis...... 95
Figure 3-2. Cellular Proliferation and Radiation Resistance Effects of Exosomes ...... 97
Figure 3-3. Exosome blockade analysis...... 100
Figure 3-4. In Vivo analysis of radiation derived exosome effect and therapeutic blockade...... 103
Figure 3-5. Immunohistochemistry of Glioblastoma tumor samples from each group...... 105
Figure 3-6. Analysis and comparison of miRNA contents within the non-radiation and radiation derived glioma exosomes...... 107
Figure 3-7. Analysis and comparison of mRNA contents within the non-radiation and radiation derived glioma exosomes...... 109
Figure 3-8. Analysis and comparison of protein contents within the non-radiation and radiation derived glioma exosomes...... 111
Figure 3-9. Proposed mechanism of exosomes in acquired radiation resistance ...... 116
Figure 4-1. Exosome confirmation analysis in stem and non-stem glioma cells ...... 135
Figure 4-2. Cellular proliferation and therapeutic resistance effects of glioma stem cell derived exosomes ...... 137
Figure 4-3. Analysis and comparison of protein contents within the stem and non-stem cell derived glioma exosomes ...... 139
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Figure 4-4. Schematic representation of the proposed model for the mechanism of glioma stem cell derived exosome induced acquired tumorigenicity on recipient cells...... 143
Figure 5-1. Schematic representation of the mechanism of acquired therapeutic resistance in cancer...... 160
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LIST OF TABLES
Table 1-1. Types of Extracellular Vesicles ...... 4
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LIST OF ABBREVIATIONS
ACTG1; Actin Gamma 1
ANXA2; Annexin A2
APNG; alkylpurine-DNA-N-glycosylase
CCND1; Cyclin D1
CISD1; mitoNEET
CD81; cluster of differentiation 81
CD63; cluster of differentiation 63
CD9; cluster of differentiation 9
CD19; Cluster of differentiation 19
CD20; Cluster of differentiation 20
CD151; cluster of differentiation 151
CDK4; Cyclin dependent kinase 4 c-Myc; V-Myc Avian Myelocytomatosis Viral Oncogene Homolog
CGGBP1; CGG Triplet Repeat Binding Protein 1
CNS; central nervous system
COL4A2; Collagen Type IV Alpha 2 Chain
COTL1; Coactosin Like F-Actin Binding Protein 1
CREBBP; cAMP-response element binding protein
CSF; cerebrospinal fluid
DERL1; Derlin 1
DNM2; Dynamin 2
EFEMP2; EGF Containing Fibulin Like Extracellular Matrix Protein 2
EGFR; endothelial growth factor receptor
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EGFRvIII; endothelial growth factor receptor variant three
EPHA2; Ephrin A2
FUT11; Fucosyltransferase 11
Gy; Gray
H & E; Hematoxylin and Eosin
HFE; Human hemochromatosis protein
IDH1; Isocitrate dehydrogenase one
IL-8; interleukin 8
IL-10; interleukin 10
IL-13; interleukin 13
IL13Rα2; interleukin 13 receptor alpha 2 iRANO; Immunotherapy Response Assessment in Neuro-Oncology
ITGA3; Integrin alpha 3
ITGB1; Integrin Beta 1
LRRFIP1; LRR Binding FLII Interacting Protein 1
MDR-1; Multi-drug resistance 1
MGMT; O6-methylguanine DNA methyltransferase
MMP2; Matrix metalloproteinase 2
MVBs; multi-vesicular bodies
NG2; Neuronal-glial antigen 2
NPM1; Nucleophosmin 1
PDGFR; Platelet derived growth factor receptor
PKN2; Serine-threonine protein kinase 2
PPIC; Peptidylprolyl Isomerase C
PTEN; Phosphatase and tensin homolog
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RL15; Ribosomal Protein L15
ROS; Reactive oxygen species
SCD; Stearoyl-CoA desaturase
STAT4; Signal Transducer And Activator Of Transcription 4
TfR1; Transferrin receptor 1
TfR2; Transferrin receptor 2
TGF beta; Transforming growth factor beta
TGF-B2; Transforming Growth Factor-Beta 2
TMZ; temozolomide
TPM1; Tropomyosin 1
TSG101; Tumor susceptibility 101
TSPAN5; Tetraspanin
VAMP8; Vesicle Associated Membrane Protein 8
VCAN; Versican core protein
VEGF-A; Vascular endothelial growth factor receptor-A
WWC1; Kibra
ZFR; Zinc Finger RNA Binding Protein
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ACKNOWLEDGEMENTS
First and foremost, I would like to sincerely thank Dr. James Connor for the opportunity and invaluable experience I have attained by working in his laboratory. Dr. Connor’s mentorship is truly unparalleled, and this has allowed me to grow exponentially as a scientist and person over the past years. Dr. Connor has treated me not only as a student, but as a colleague, and provided a laboratory environment that really has no limitations other than the amount of work one is willing to do. I cannot thank Dr. Connor enough for all that he has done to make this PhD work a success.
I would also like to thank the members of the Connor Laboratory for all of their endless support throughout my PhD. Becky Webb for being a true champion and spending numerous hours helping me with my work and making sure the work was a success. Madhan for always having an open door and spending many hours discussing experiments and results with me in gritty detail. Wint Nandar for taking her time to teach me how to do much of the basic culture work. Beth Neely for always keeping the work in proper protocol. Mandy Snyder for her support for all things important outside of work. Dr. Russell Payne for his comradery and being a sounding board for many of the ideas. Dr. Elias Rizk for being a mentor to me and not only always having ideas of future projects but also inspiring me to work hard, achieve my goals, and teach me that you truly can do anything you put your mind to. Lisa Harman for always being willing to help with any logistics. Vagisha, Anne, Kari, Brian, Insung, Doug, Dasha, Christine,
Sang, Mark, Stephanie, Andrew, and all the other members of the Connor laboratory for their support and for being great friends during this time.
I would also like to thank my committee members for their effort and time in being part of my PhD committee. Dr. Barbara Miller for asking clinically relevant questions and keeping my thesis focused on helping patients. Dr. Jong Yun for always questioning me to think outside of the
xvii box. Dr. Jennifer Baccon for being a guiding light, not only during the PhD but during the whole
MD/PhD program. Dr. AB Madhankumar as a committee and lab member for always providing novel insight into the questions of my PhD. Dr. Brad Zacharia for always keeping the research in the translational and clinical realm, showing me what it means to be a great physician-scientist, and most importantly, being a mentor and role model to me for my life and career.
Lastly, I would like to thank my friends and family for their continuous love, encouragement, and support during my years as a graduate student and throughout this thesis. My mother for her truly endless positivity and optimism. My father for teaching me the most important thing is and always will be an eternal work ethic. Dee and John Berry for always taking me in and providing me with a home away from home. And last but not least, Katherine Berry for her laughs, love, and ceaseless support of all of my ambitions.
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Chapter 1
Exosomes and Their Implications in Central Nervous System Tumor Biology
1.1. Abstract
Exosomes are 20-100nm cellular derived vesicles that when discovered, were initially thought to be a form of cellular recycling of intracellular contents. More recently, these vesicles are under study for their purported significant roles in intercellular communication in both healthy and diseased states. In this review we focus primarily on the secretion of exosomes associated with all forms of brain tumors; although most studies come from glioblastoma.
Exosomes contain protected intracellular content that can be transferred to recipient cells and subsequently lead to enhanced tumorigenic properties including angiogenesis, cancer progression, and therapeutic resistance. Genetic components of the cell of origin can be included in the secreted exosomes. The presence of genetic material could serve as a biomarker for the presence of mutations in tumors leading to modification of treatment strategies. In the last decade, exosomes have been identified as having major implications in many aspects of medicine and tumor biology and appear to be primed to take a critical position in cancer diagnosis, prognosis, and treatment.
2 1.2. Introduction
Tumors that reside in either the brain or spinal cord are termed central nervous system
(CNS) tumors and are further categorized as primary if they originated within the CNS, or secondary if they arose due to metastasis from cancer in a different location of the body. Primary and metastatic CNS tumors make up between two and four percent of all adult cancer diagnoses, respectively 1. The numerous CNS tumor types including glioblastoma, medulloblastoma, and oligodendroglioma secrete extracellular vesicles, called exosomes, which contain protected, intracellular content that has functional implications including angiogenesis, cancer progression, and therapeutic resistance through vesicle-mediated cell-to-cell communication. Given the recent increase in interest regarding exosomes, they appear to be primed to take a critical role in cancer diagnosis, prognosis, and treatment. As such, we provide a targeted review of the key aspects of exosome biogenesis, secretion, and uptake, exosome purification and identification, exosome functions in oncogenesis, current roles of exosomes as CNS cancer biomarkers, and their therapeutic potential.
Extracellular vesicles are formed as lipid bilayers and are 20-1000nm in size. They play important roles in both healthy and diseased states 2–8. These vesicles were first discovered 30 years ago and initially thought to be a form of cellular recycling of intracellular contents during reticulocyte maturation 9. Upon further research, extracellular vesicles were found to originate in many different forms, and have thus been subcategorized based upon size and mechanism of release from the cell 10, shown in table 1. Microvesicles range in size from 100nm to 1um and are released through budding from the plasma membrane 11. The largest vesicles are apoptotic bodies released by dying cells and range in size from 400nm to 1um 12,13. Exosomes, the smallest of the extracellular vesicles, range in diameter from 20-100nm and are formed through inward budding of the endosomal membrane. These endosomes with exosome vesicles inside are termed multi-
3 vesicular bodies, or MVBs, which then fuse with the plasma membrane to release exosomes into the extracellular space 14.
4 Vesicle Type Diameter Formation
Exosomes 20nm – 100nm Formed through inward budding of the endosomal
membrane. These endosomes with exosomes inside are
termed Multi-vesicular bodies, or MVBs, which then fuse
with the plasma membrane and release exosomes into the
extracellular space
Microvesicles 100nm – 1um Formed and released through budding from the plasma
membrane
Apoptotic bodies 400nm - 1um Released by dying cells
Table 1.1. Types of Extracellular Vesicles
5 Exosomes are released by all cells 15 and their protective lipid bilayer, permits protected travel throughout the blood stream in the body 16. Exosomes are taken up by endocytosis or membrane fusion17. They have been shown to have critical functions in healthy states including communication between placental and maternal tissues for proper biological growth 7 and embryonic development 18 as well as in pathological states ranging from cardiac disease 19 to cancer 20,21 and neurodegenerative disorders including Alzheimer’s 22 and Parkinson’s disease 23.
For example, tau has been identified in the CSF exosomes of patients with Alzheimer’s disease and is suggested to play a role in Alzheimer’s disease progression 24,25. Similarly, α-synuclein has been found increased in plasma exosomes of Parkinson’s patients 26 and levels of CSF exosomal
α-synuclein was able to differentiate Parkinson’s disease from a similar disease, dementia with
Lewy bodies 27. A full description of exosomes in neurodegenerative diseases is beyond the scope of this chapter that is focused on exosomes in neuro-oncology. A number of excellent reviews on this topic exist 28–30.
Exosomes have been purified from many different cancers including breast 6,31, brain32,33,34, and lung 35,36. Exosome release is reportedly increased in cancer patients 37,38 and some studies estimate that in a single day one glioma cell can release five thousand exosomes
39,40. Numerous studies have reported on the process of tumor metastasis being mediated in part by the critical interaction that a cancer cell has on regions both throughout the body and its microenvironment through the secretion of exosomes 41,42. Proteins, RNA, and DNA, have all been detected within exosomes and have the ability to be transferred to recipient cells through direct uptake of the exosomes. These transferred contents then may become functional in the recipient cell, contributing to the tumorigenic properties in the microenvironment surrounding the tumor 43,44,45. Exosomal contents have also been shown to mediate resistance to a multitude of cancer therapies 46,47,48.
6 The genetic components in the cell of origin are also found within the secreted exosomes, which provides a unique opportunity to develop exosomes as optimal biomarkers 39,49,50.
Exosomes have been shown to contain mutations of critical targets such as EGFRvIII 51 and IDH1
52 that importantly reflect the mutation status of the parent tumor. Interestingly, there have also been cases where actionable mutations such as EGFRvIII were found in the exosomes from CSF and serum and not in a biopsy of the parent tumor itself 51, suggesting the potential for exosomes to recapitulate the entire biology of the parent tumor in contrast to an anatomically restricted surgical biopsy of only one portion of a heterogeneous tumor. Balaj et al. have reported single- stranded DNA of MYC oncogene was found in exosomes derived from cultured medulloblastoma cells52. The effect of germline genotype mutations on exosome profile has not been fully explored. Studies I perform in my second chapter demonstrate the effect of germline changes in the HFE gene, and show subsequent enhanced tumorigenicity in exosome phenotype 53. HFE mutations in cancer cells lead to the release of exosomes which contain increased levels of proteins related to invasion, angiogenesis, and therapeutic resistance, and these exosomes transfer enhanced tumorigenicity to recipient cells 53.
1.3. Extracellular vesicle formation, secretion and uptake
As shown in table 1, extracellular vesicles are differentiated based upon size and mechanism of release. The largest form of vesicles are apoptotic bodies, which are vesicles released by dying cells that range in size from 400nm to 1um54,55. The process of apoptosis occurs in multiple stages including chromatin condensation, blebbing of the plasma membrane, and apoptotic body formation by intracellular content dispersion into enclosed vesicles 54. These apoptotic vesicles are distinctly differentiated from the other types of vesicles based on the inclusion of include cellular organelles 55. These apoptotic bodies are then cleared by macrophages through phagocytosis and do not appear to be available, in general, for uptake by normal or other cancer cells because macrophages recognize specific markers on the apoptotic
7 bodies. These apoptotic body markers include complement protein C3b 56, annexin V 57, and thrombospondin 58.
Microvesicles are in the middle range of extracellular vesicles with sizes from 100nm to
1um. Microvesicles are generally released through budding from the plasma membrane (Kahn et al., 2017). The mechanism behind microvesicle release combines the contraction of the actin cytoskeleton with the changing of distribution of plasma membrane phospholipids 59. This redistribution is catalyzed by aminophospholipid translocases, including floppases and flippases.
Floppases redistribute phospholipids from the inner to outer leaflet of the plasma membrane, while flippases redistribute phospholipids from the outer to inner leaflet 60. A critical point in microvesicle formation and budding is the translocation of phophatidylserine to the outer leaflet of the plasma membrane. This process is underlined by the initial activation of phospholipase D by ADP-ribosylation factor 6 (ARF6). Myosin light chain kinase (MLCK) is then activated by extracellular regulated kinase (ERK), which then leads to the subsequent secretion of microvesicles. Other factors have also been linked to microvesicle release, including tumor susceptibility gene 101 (TSG101) 61, Arrestin 1 domain containing protein 1 (ARRDC1) 62, and calcium induced release 63. The proteomes of glioblastoma derived microvesicles may be clinically useful as they are reported to contain tumor specific mutations including EGFR, EGFR variant III, and IDH1 64.
1.3.1. Exosome Biogenesis
Exosomes, the smallest of the extracellular vesicles, range in diameter from 20-100nm and are formed through inward budding of the endosomal membrane into the endosomes, thus forming intraluminal vesicles (Figure 1). These endosomes with exosome vesicles inside are termed multi-vesicular bodies, or MVBs. MVBs are then sorted into three different categories: early endosomes, late endosomes, and recycling endosomes 65. Early endosomes are termed as such because at this stage of the internalization process they do not have a final destination. Once
8 they fuse with endocytic vesicles, the early endosomes are then sent for recycling, degradation by the lysosome, or secretion into the extracellular space. Subsequently, late endosomes are formed and intraluminal vesicles can be created within the late endosome through inward budding of the late-endosomal membrane. This inward budding is catalyzed by multiple factors, including the tetraspanin proteins, such as CD63, as well as the Endosomal Sorting Complexes Required for
Transport (ESCRTs). The tetraspanin proteins, thus named because of their four transmembrane domains, become highly expressed on the late endosomal membrane and aid to bring together necessary factors for exosome formation 66. This high expression on exosomal membranes has also led to utilization of the tetraspanins as exosome markers.
There are four types of Endosomal Sorting Complexes Required for Transport (ESCRTs), including ESCRT 0, ESCRT I, ESCRT II, and ESCRT III 67. First, ubiquitinated proteins are found by ESCRT 0 outside of the membrane of the endosome 68 and delivered to ESCRT I and
ESCRT II. The ESCRT I-ESCRT II complex then interacts with phosphatidylinositol 3 phosphate
(PIP3) 69, hepatocyte growth factor regulated tyrosine kinase substrate (HRS) 70, and the curved endosomal membrane 71 which initiates intraluminal vesicle formation. ESCRT III becomes activated by the ESCRT I- ESCRT II complex and the protein Alix. ESCRT III subsequently binds to TSG101 and MVB protein 4A (CHMP4A), completing exosome formation 72. TSG101 and Alix are thus highly expressed on the exosomal membrane, making both TSG101 and Alix exosome biomarkers.
Although this endosomal sorting complex required for transport (ESCRT) pathway has been most avidly studied, other pathways for exosome formation have also been found. These studies began after Stuffers et al. determined that multivesicular endosomes are still formed even when the ESCRT pathways is inactivated 73. One such alternative pathway is the syndecan- syntenin-Alix pathway, as described by Baietti et al. 74. In this pathway, Alix and syntenin interact with syndecan heparin sulfate proteoglycans, ADP ribosylation factor 6 (ARF6), and
9 phospholipase D2 (PLD2) which initiate the inward budding of the endosomal membrane to form intraluminal vesicles, or exosomes 74. PLD2 and ARF6 then mediate the process of heparan sulfate side chains on syndecan to be enzymatically degraded by heparanase, which further activates and completes the process of exosome formation 75,76. Other methods of exosome formation include lipid mediated exosome formation by the sphingolipid ceramide 77. Ceramide is produced by neutral sphingomyelinases and it was further demonstrated that exosome release was diminished when neutral sphingomyelinases were inhibited 77 The ESCRT pathway has been the most well characterized with regards to exosome formation in glioblastoma, but a combination of these previous pathways for exosome formation cannot be ruled out, as they are most likely utilized in combination. Interestingly, heparin and ceramide have been suggested to have anti- tumor effects7879, and the heparin/ceramide lipid aspects of these pathways also provide opportunities for potential therapeutic manipulation, as discussed later.
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Figure 1-1. Exosome biogenesis. The early endosome is formed through the inward budding of the plasma membrane. These early endosomes have proteins in their membranes that were originally bound to the plasma membrane of the cell. Subsequently, intraluminal vesicles, or exosomes, are formed through inward budding of the endosomal membrane which occurs by the
Endosomal Sorting Complexes Required for Transport family of proteins. ESCRT proteins are shown in route to the endosomal membrane. Through this process, exosomes contain membrane bound proteins. Exosomes also contain multiple components including DNA, RNA, and intracellular proteins. These endosomes with exosome vesicles inside are termed multi-vesicular bodies, or MVBs. Exosomes range in diameter from 20-100nm. With the help of the RAB
GTPase family of proteins on the way to the plasma membrane, these MVBs subsequently fuse with the plasma membrane to release exosomes into the extracellular space.
11 1.3.2. Exosome secretion
Multiple mechanisms have been proposed for facilitation of exosome secretion, most of which include the RAB GTPases protein family (Figure 1). RAB7 was shown to be facilitate exosome release in breast cancer cells 74. RAB11 was found to be utilized in exosome secretion by leukemia cells in vitro and was dependent on calcium 80. RAB27A and RAB27B have both been shown to be critical for the secretion of exosomes with upregulated major histocompatibility complex class II (MHCII) from HeLa cells 81. Furthermore, when synaptotagmin-like 4 (SYTL4) and exophillin 5 (EXPH5), known mediators of RAB27 activation, were inhibited, exosome secretion was also diminished 81. RAB35 was demonstrated to be critical for the secretion of exosomes from oligodendrocytes, and when RAB35 was inhibited, exosome secretion decreased
82. Other mechanisms of exosome secretion involve the Soluble NSF (N-ethylmaleimide soluble factor) Attachment Protein Receptor (SNARE) proteins, vehicle associated membrane protein 3
(VAMP3) and vehicle associated membrane protein 7 (VAMP7), which increase the release of exosomes containing acetylcholine esterase 83. Nakano et al. interestingly reported that the neural, proneural, classical, and mesenchymal subtypes of glioblastoma have different expression levels of these secretory proteins84. The neural and proneural subtypes both had downregulated
RAB27A and unchanged levels of RAB27B, whereas the classical subtype has downregulation of both RAB27A and RAB27B84. The mesenchymal subtype in contrast had upregulated RAB27A and unchanged levels of RAB27B84. These findings suggest that the secretory mechanism of exosomes is quite complex, even within one tumor type. Thus it may be possible that different subtypes of glioblastoma can be profiled based upon their exosome protein contents, providing diagnostic and prognostic value.
As mentioned earlier, much work has been performed to determine the content of the secreted exosomes. Commonly found content within exosomes include RNA, lipids, and proteins, most of which are used for exosome biogenesis. Tetraspanin proteins and proteins involved in the
12 ESCRT process have all been found to be highly expressed in exosomes, for example, CD63,
CD81, CD9, Alix, and TSG101. Proteins important for exosome secretion are also highly expressed within exosomes and include the RAB family of proteins, for example RAB11,
RAB27A/B, and RAB35. The lipids that make up the membranes of exosomes have a similar make up to the cells of origins of the exosome. Although, glycosphingolipids, sphingomyelin, cholesterol, and phosphatidylserine are all increased in the exosome membrane whereas diacylglycerol and phosphatidylcholine seem to be decreased85,86, the functional implications of these lipid alterations are not well understood. DNA and RNA have both been found within exosomes 40,87. Interestingly, 3’ untranslated region fragments were highly associated with exosomal mRNA compared to cellular mRNA 88. This suggests that the three prime untranslated region fragment may act as delivery address for RNA into the exosome. RNAs within the exosomes are enriched in a GGAG nucleotide sequence 89. This four nucleotide motif seems to bind to heterogeneous nuclear ribonucleoprotein A2B1 (hnRNPA2B1) which aids in miRNA loading into exosomes 89. Although this GGAG repeat is enriched in exosomal mRNA, the mechanism behind this enrichment and the reason these specific mRNAs become enriched is not understood. A potential area for study is the possibility that exosomes play a role in transfer of polynucleotide repeats between cells90. Although membrane bound proteins such as the tetraspanins are known to be enriched in exosomes through the exosome formation process, the inclusion of specific intracellular proteins has not been well described. It is clear that an important research direction is to elucidate the mechanism behind why certain exosomes are packaged by the cell with certain proteins, lipids, and nucleic acids.
1.3.3. Exosome Uptake
Numerous studies have demonstrated direct evidence of exosome uptake into recipient cells and, furthermore, the transfer of the exosomal contents into those cells 91. Fluorescent dyes that stain the lipid membrane have also been used to visualize exosome uptake into recipient
13 cells, and include DiI 92, rhodamine b 93, DiD 94, and most commonly, PKH67 95, and PKH26 96.
GFP-tagging of the tetraspanin proteins CD63 and CD9 has also been used to prove that cells internalize exosomes 97,98.
Various mechanisms of exosome uptake have been studied and include membrane fusion, phagocytosis, clathrin mediated endocytosis, lipid raft mediated endocytosis, caveolin mediated endocytosis, and macropinocytosis 99. Other factors that have been linked to exosome uptake include heparin sulfate proteoglycans 100, protein interactions 101,102, and lipid 103. Multiple studies have suggested that energy is required for exosome uptake and when incubating exosomes and cells at four degrees Celsius, exosome uptake is decreased significantly 93,96. Fixing cells with paraformaldehyde and subsequently incubating these cells with exosome also inhibits uptake of exosomes, demonstrating exosome uptake is not occurring through a process of diffusion 104.
A variety of proteins have been implicated in membrane fusion including the RAB proteins and SNARE proteins 105. The pH of the environment also seems to be important for this process of membrane fusion between the cell and exosome. Parolini et al. show that an acidic pH increases exosome secretion and exosome uptake in a fusion dependent manner 106. This may be critical in the microenvironment and within a tumor, where the pH is increasingly acidic 106.
Phagocytosis is a process whereby macrophages engulf matter, including apoptotic debris and bacteria. Thus, it has also been implicated in exosome uptake. Feng et al. show that macrophages engulf exosomes from leukemia cells which is mediated through
Phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K) 107. Furthermore, exosome internalization was dose dependently decreased by blocking PI3K with PI3K inhibitors 107. Phosphatidylserine is important in the process of phagocytosis, and utilizing phosphatidylserine inhibitors decreases exosome internalization 108. Outcompeting phosphatidylserine with a mimic molecule also reduced exosome uptake 96, implying that phosphatidylserine on the plasma membrane of exosomes is used for internalization by recipient cells.
14 Endocytosis is a term that encompasses multiple pathways of particle uptake and includes clathrin mediated endocytosis, caveolin mediated endocytosis, and micropinocytosis. Clathrin mediated endocytosis is mediated through clathrin creating a coated pit which then vesiculates, pinches off, and subsequently diffuses intracellularly away from the plasma membrane 109.
Inhibition of the clathrin mediated uptake pathway with multiple agents has suggested that this process is important for exosome uptake 110,111. The initiation stage of clathrin mediated endocytosis begins with the formation of clathrin coated pits. This process can be inhibited with chlorpromazine 110. Dynamin 2 is a protein that participates in clathrin mediated endocytosis by aiding in the vesiculation and pinching of vesicles off of the plasma membrane 111. Using chlorpromazine or inhibiting dynamin 2, both causing decreased clathrin mediated endocytosis, also reduces exosome uptake 110,111.
Caveolin mediated endocytosis has also been shown to mediate particle uptake 112.
Caveolin 1 is a critical protein that is sufficient for caveolin dependent endocytosis and causes the formation of a hairpin structure on the plasma membrane which creates the caveolae, or plasma membrane invaginations of this endocytosis method 112. Caveolin 1 inhibition was demonstrated to decrease exosome uptake in cells infected with the Epstein barr virus 113. As the caveolae pits have high levels of cholesterol, agents that inhibit cholesterol synthesis have also been used to demonstrate exosome uptake can be mediated through caveolin dependent endocytosis. The use of filipin, methyl beta cyclodextrin, and simvastatin were all able to reduce the uptake of exosomes in recipient cells 103. Dynamin 2 has also been implicated in caveolin mediated endocytosis and the blockade of dynamin 2 decreases exosome uptake 113.
Macropinocytosis is the process where cup-shaped invaginations are created on the plasma membrane of cells with the actin cytoskeleton 114. These cups form ruffles, or cell membrane sheet extensions, which then pinch at their distal margins and internalize particles within their milieu 114. Although macropinocytosis is similar to phagocytosis, the sodium-
15 hydrogen exchanger, actin, and cholesterol, are all necessary parts of the macropinocytosis process 115. Also, particles must come into contact with the membrane during the process of phagocytosis, which is not necessary during macropinocytosis 114. Reduced exosome uptake by microglia was seen when blocking the sodium-hydrogen transporter 104.
Numerous proteins and families of proteins have been implicated in the process of exosome uptake. Tetraspanin proteins, including CD81 and CD9 are highly expressed on the surface of exosomes and because they have been shown to be involved in the fusion of the phagocytes 116 and sperm-egg fusion 117, it is also believed they play a role in exosome-membrane fusion 118. Furthermore, tetraspanin antibodies were able to partially inhibit exosome uptake in recipient cells 96. Proteoglycans like heparin sulfate have also been implicated in exosome uptake, as this process is also utilized for virus internalization into recipient cells 119. Using heparin as a proteoglycan analogue, exosome uptake was able to be outcompeted and internalization into recipient cells was reduced 100. Immune related proteins including integrins and immunoglobulins have also been studied as a mechanism for exosome uptake. Intercellular Adhesion Molecule 1
(ICAM-1) has been shown to mediate exosome uptake, and ICAM-1 blockade was able to reduce exosome internalization 96. Lymphocyte function associated antigen 1 (LFA-1) and the T-cell receptor (TCR) are also mediators of exosome uptake in T-cells 120, while dendritic cells utilize
LFA-1-ICAM-1 and TCR-MHC binding for exosome internalization 121. Blockade of the integrins CD51 and CD61 on the surface of dendritic cells has also been shown to decrease exosome internalization by dendritic cells 96. Lectins are a class of proteins important for the engulfment of particles by macrophages. Galectin-5 is a lectin shown to be upregulated in exosomes and incubating exosomes and cells with excess external galectin-5 was able to decrease cellular internalization of exosomes 122. The use of antibodies to block the lectins DC-SIGN and
DEC-205, both led to a subsequent decrease in exosome uptake 123,124.
16 Lipids and lipid rafts also seem important for exosome internalization. Lipid rafts are areas of densely packed lipids such as sphingomyelin and cholesterol which can be found in areas with expression of flotillin and caveolin-1 125. These areas have been shown to mediate viral endocytosis 126. Svensson et al. show that when using simvastatin, filipin, and methyl beta cyclodextrin, all agents that lower cholesterol, exosome internalization was reduced 103. The annexin proteins have also been implicated in exosome uptake through their interaction with lipid rafts by anchoring exosomes 98. Plebanek et al. demonstrate that the internalization of exosomes by recipient cells can be reduced by binding scavenger receptor type 1 (SR-B1) with a synthetic nanoparticle mimic of HDL, which normally functions to remove cholesterol through SR-B1 127.
Exosomes impart their functions on recipient cells either by being internalized, as stated previously, or by the interaction of proteins on the exosome surface with proteins on the surface of cells. Multiple studies have demonstrated that a receptor or ligand on the exosome surface can bind with its ligand or receptor counterpart on the recipient cell, triggering downstream effects in that cell 128,129. For example, Cossetti et al. showed that interferon gamma on the surface of neural stem cell derived exosomes was able to activate Stat 1 in recipient cells 128. Exosomes were also able to activate the Notch and Rheb signaling in recipient cells mediated through the activation of
Notch1 and Rheb from the exosomes themselves 129.
The method utilized for exosome internalization by recipient cells, whether membrane fusion, phagocytosis, or a type of endocytosis, is an underserved area of exosome research.
Further work needs to be performed to determine the predominant internalization process used for a specific cell type and exosome type. For example, exosomes derived from cancer cells may be taken up through a different mechanism than exosomes derived from immune cells due to the lipid and protein composition of the exosomal membrane. Furthermore, the lipid/protein composition of the recipient cell may also influence the predominant method of exosome internalization. Regardless of the predominant pathway a specific cell utilizes for exosome
17 uptake, multiple pathways are most likely used as inhibiting one type of internalization pathway does not completely inhibit exosome uptake.
1.4. Exosome purification and identification
Exosomes are shed into bodily fluids, such as blood, urine, and CSF, paving the way for a non-invasive “liquid biopsy”, allowing frequent molecular profiling and real-time monitoring of treatment response 49,130,131. With the vast array of functions critically affected by exosomes, they can be considered major players in the realm of cancer diagnosis, prognosis, and therapy.
Exosomes can be purified using a myriad of techniques including differential ultracentrifugation, sucrose or iodixanol gradients, immunoaffinity capture, lipid nanoprobes, and commercial kit based isolation 132–135. Differential ultracentrifugation protocols consist of a series of centrifugations starting from low speeds to pellet out cells and cellular debris and culminating with high speed spins of ~100,000 X G to isolate exosomes 136,137. Density gradients with sucrose or iodixanol utilize the buoyancy of exosomes in combination with ultracentrifugation for isolation 138. The immunoaffinity capture method utilizes antibodies of proteins highly enriched on exosomes attached to magnetized microbeads for exosome isolation through a column 139.
Commercial kit based isolation including Exo-Quick (System Biosciences), Total Exosome
Isolation Kit (Invitrogen), and others utilize polyethylene glycol to create a polymer lattice and gently precipitate out exosomes from solution 140. Ultracentrifugation and density gradient purification have shown the purest exosome sample isolation, but the compromise is a decreased yield. A recently developed method of exosome isolation by Wan et al. demonstrated similar efficacy to ultracentrifugation through utilizing lipid based nanoprobes to isolate and purify exosomes 141. With these methods, exosomes have been purified from blood 142, saliva 143, urine
144, CSF 145, semen 146, and breast milk 147.
Once isolated, multiple methods for exosome confirmation have been used including dynamic light scattering, nanoparticle tracking analysis, immunoblots of exosome markers, flow
18 cytometry, and electron microscopy. Dynamic light scattering provides information on exosome diameter while nanoparticle tracking analysis provides vesicle diameter, concentration, and visualization (Figure 2A) 148. Immunoblots and flow cytometric analysis of exosome markers including CD81, CD9, and CD63, and HSP70 have also been used to confirm exosome isolation
149,150. Electron microscopy also allows for direct visualization of exosomes (Figure 2B) 151.
19
Figure 1-2. Visualization of glioblastoma exosomes. (A) Nanoparticle tracking analysis
(NTA) of glioma exosomes providing visualization. NTA uses a laser beam to analyze the
Brownian motion and light scattering of exosomes in a PBS suspension. The subsequent video capture by the mounted camera shows the exosomes moving in Brownian motion (B) Electron
Microscopy of exosomes. The glioma exosome resuspension is plated on Formvar coated copper grids, fixed with paraformaldehyde, and contrasted with uranyl acetate before visualization on a
JEOL-1400. Exosomes can be seen with an average diameter of 30 nanometers.
20 1.5. Exosome function in oncogenesis
Exosomes appear to play a role in many facets of tumorigenesis. Exosomes have been implicated in metastasis through the creation of a pre-metastatic niche 16; preparing an area of the body for cancer cell seeding and subsequent progression. Tominaga et al. demonstrated that exosomes cause destruction of the blood brain barrier through miR181c, which enhances CNS metastasis 152. Fong et al. demonstrated that breast cancer cell derived exosomes use miR122 to prepare a metastatic niche in the brain and facilitate disease progression by suppressing glucose uptake in non-tumor cells, and thus increasing available nutrients for cancer cells themselves 153.
Interestingly, the pro-metastatic effect of exosomes has been shown to not only occur by cancer cell derived exosomes, but also of non-tumor cell secreted exosomes. For example, Zhang et al. demonstrated that exosomes secreted by astrocytes target and inhibit the PTEN tumor suppressor gene in tumor cells once disseminated into the brain, leading to enhanced oncogenesis 154.
Moreover, when these cancer cells were then subsequently taken out of the brain, their PTEN suppression was reversed 154. This initially seems counterintuitive, as astrocytes would be thought to attempt to decrease tumorigenesis, but what may be occurring is initial cross-talk from the tumor cells to the astrocytes with the goal of the tumor changing the astrocyte phenotype into a malignant one. These now tumorigenic astrocytes then subsequently send reciprocal feedback responses back to the tumor cells to suppress PTEN expression, allowing the tumor cells to increase their malignancy.
Exosomes may also exert an effect immune function and lead to an immunosuppressed phenotype 155. De Vrij et al. demonstrated that glioblastoma derived exosomes induced macrophages into a M2 phenotype 156, while van der Vos et al. reported that microglia/macrophages take up glioblastoma derived exosomes readily and lead to an immunosuppressive phenotype mediated by delivery of miR21 and miR451 157. Van der Vos et al. subsequently show that this transfer decreases the c-Myc mRNA in the recipient microglia and
21 macrophages which may be the causative factor of the immunosuppressed phenotype seen 157.
Harshyne et al. analyzed the serum of glioblastoma patients and found that the serum exosomes promote a T-helper cell type two environment, which may be promoting an oncogenic phenotype beyond the confines of the central nervous system 158. These studies suggest that cancer cell derived exosomes may aid in facilitating tumor progression through immune suppression of the immunomodulatory cells in the CNS.
Skog et al. demonstrated that glioblastoma derived exosomes increase angiogenesis by increasing human umbilical vein endothelial cell tubule length through transfer of angiogenic proteins 51. In fact, the exosomes derived from EGFRvIII glioblastoma cells can transfer this
EGFR variant to non-EGFR mutant cells, which then enhance their tumorigenicity by subsequently expressing EGFRvIII themselves 159.
1.5.1. Exosome effect on treatment resistance
Exosomes and treatment resistance have been a topic of research and exosomes have been implicated in promoting resistance in multiple cancer types including glioblastoma 160, gastric 161, breast 162,47,163, prostate 164, and others 165. Micro RNA play a significant role in this therapeutic resistance effect. For example, exosomes from glioblastoma cells can transfer miR221 which subsequently targets DNM3 and leads to increased resistance to TMZ 160. Transfer of miR222 via exosomes from Adriamycin resistant cells has also been demonstrated to confer treatment resistance 163. Chemotherapeutic-related protein transfer by exosomes has also been linked to increased therapeutic resistance. Exosomal transfer of multi-drug resistance (MDR-1) plays a role in increasing chemoresistance in recipient cells. Chemoresistant breast cancer and prostate cancer cell lines can both release exosomes with increased levels of MDR-1 that are then taken up by recipient, chemo sensitive cells, and subsequently promote a chemoresistant phenotype 47,164.
22 Stressors such as radiation exposure and hypoxia affect the composition and functionality of exosomes. As a tumor becomes larger, areas of the tumor become hypoxic, beginning the process of angiogenesis. Kucharzewska et al. demonstrated that exposing glioblastoma cells to a hypoxic environment caused the release of exosomes that have an altered composition with increased expression of proteins integral for angiogenesis, such as IL-8 and IGFBP 4. It was subsequently demonstrated that these hypoxia-derived exosomes enhance the functional aspect of angiogenesis as well, and lead to the recruitment of increased vasculature 4. The stress of radiation has also been shown to cause cancer cells to release exosomes with altered composition
166. Arscott et al. demonstrated that radiation leads to increased exosome release and that exosomes derived from radiated glioblastoma cells caused a subsequent increase in recipient cancer cell migration 167. Chemotherapeutic stress reportedly will cause changes in exosomal contents. For example, treatment with Paclitaxel caused increased exosome expression of the protein Survivin, which was transferred to recipient cancer cells and subsequently enhanced survival of those recipient cells162.
1.6. Current role of exosomes as cancer biomarkers
Exosomes are under evaluation as biomarkers for numerous cancer types. Silva et al. demonstrated a unique miRNA signature of plasma derived exosomes in patients with lung cancer in comparison to controls, which also correlated to survival 168. Melo et al. were able to differentiate and diagnose patients with pancreatic cancer from control with exosomal levels of glypican-1 169. Hannafon et al. show that patients with breast cancer have plasma exosomes with increased levels of miR21 and miR1246 when compared to controls 170. Bryzgunova et al. detected levels of miR19b in urinary exosomes of patients with prostate cancer and were able to differentiate cancer patients from healthy controls 171. The use of exosomes as biomarkers in CNS tumors is extremely attractive because biopsy of tumors within the CNS can be complex and carry the potential for morbidity and mortality. This has prompted an active area of exosome
23 research assessing their utility as a liquid biopsy for not only tumor detection, but also molecular profiling of the tumor itself. CNS tumor exosomes have been detected in the CSF 172,173 and plasma 174, and although the functional biological implications of these exosomes has not yet been fully elucidated, isolating these cancer derived exosomes have demonstrated biomarker potential with regards to assessing tumor prognosis and determining the molecular profile of the parent tumor.
1.6.1. Use of exosomes for molecular profiling
Isocitrate dehydrogenase 1 (IDH1) mutations are found in 80% of secondary glioblastomas that arise from a glioma of a lower grade and 10% of all primary glioblastomas that arise spontaneously 175. IDH1 is an enzyme that normally functions in the citric acid cycle, but when mutated, produces 2-hydroxyl-glutarate, which is involved in DNA methylation and the pathogenesis of glioblastoma 176. Chen et al. were able to isolate exosomes from the CSF of glioma patients and determine whether these patients had a IDH1 mutated tumor through an ultra- sensitive PCR technique 52, while Garcia-Romero et al. isolated exosomes from the plasma of patients with IDH1 mutated gliomas and were able to detect this IDH1 mutation with the plasma exosomes alone 174.
Endothelial growth factor receptor (EGFR) is a protein integral in angiogenesis, and the mutant form, EGFRvIII has enhanced oncogenic functionality 177. Skog et al. analyzed exosomal contents from the exosomes isolated from the serum of glioblastoma patients with EGFRvIII and were able to detect this variant in these patients 51. The authors also detected the EGFRvIII variant in patients who did not have a mutation diagnosed through surgical biopsy, prompting the question whether exosomes have the potential to capture the entire picture of tumor biology instead of the surgical snapshot from biopsy of only one portion of a heterogeneous tumor.
MicroRNAs, specifically miR21, have also been interrogated for biomarker potential in glioblastoma. MiR21 is overexpressed in glioblastoma and reportedly enhances glioblastoma
24 tumorigenicity by inhibiting insulin like growth factor binding protein 3, a tumor suppressor gene
178. Multiple studies have analyzed miR21 contents within CSF exosomes and have correlated levels of exosomal miR21 with tumor grade and decreased survival in glioblastoma patients
172,173.
1.6.2. Use of exosomes as diagnostics
Brain metastases are more common than primary CNS tumors and the ability to differentiate between these types of tumors is essential for optimal patient care. Differentiating primary from secondary tumors as well as differentiating from non-neoplastic processes such as intraparenchymal hemorrhage and inflammatory conditions adds further complexity to patient diagnosis. The diagnostic differentiation of primary glioblastoma from CNS lymphoma or secondary metastasis due to lung, breast or melanoma, all of which radiographically may present similarly, is currently accomplished via a surgical biopsy. Exosomes may provide a non-invasive methodology to differentiate these CNS tumor types. Manterola et al. demonstrate that glioblastoma can be diagnosed from exosomes isolated from the serum of glioblastoma patients by using PCR and finding levels of a small non-coding RNA, RNU6-1 and 2 microRNAs, miR-
320 and miR-574-3p in 75 total patients 179. Furthermore, RNU6-1 was seen as an independent predictor of a glioblastoma diagnosis 179. B-cell lymphoma derived exosomes express B-cell specific antigens, such as CD19 and CD20 180. Metastatic breast cancer cell lines have been shown to secrete exosomes that contain upregulated miR210, downregulated miR19a and 29c 181, upregulated annexin II 182, and upregulated integrin beta 3 3. Exosomes derived from metastatic lung cancer cell lines expressed increased levels of TGF beta and IL-10 183. As previously mentioned, Tominaga et al. show that CNS metastasis can be enhanced by miR181c which causes blood brain barrier disruption 152, suggesting that miR181c may be a potential metastatic biomarker. One major problem with utilizing exosomes as biomarkers is that all cells secrete exosomes, not only cancer cells, which leads to significant noise in exosome profiles. The
25 specific profiling of tumor cell derived exosomes may come in the form of utilizing cancer- specific markers, for example IL13Rα2, to purify the tumor cell derived exosomes from the exosomes secreted from all other cell types. IL13Rα2 is a decoy receptor for IL-13 that inhibits the downstream functionality of IL-13 and is selectively expressed on gliomas 184,185. Utilizing
IL13Rα2 targeted nanovesicles encapsulated with doxorubicin has demonstrated to be effective for the treatment of glioblastoma 186,187. IL-13 conjugated quantum dots have also been utilized to analyze exosomes from glioblastoma patients’ CSF and were found to have a unique pattern in comparison to non-targeted quantum dots 188. These findings prompt the further development of tumor-derived exosome purification methods and analysis of CSF or serum isolated exosomes to non-invasively differentiate primary glioblastoma from other CNS tumors.
1.6.3. Use of exosomes as markers of treatment effectiveness
Being able to utilize exosomes from the CSF or plasma as a marker of treatment effectiveness also has the potential to provide critical patient data for optimal therapeutic regimens. Isolating exosomes and profiling them before and after treatment with chemotherapeutics as well as radiation could assess treatment effect on tumor progression.
Longitudinal exosome profiling would be especially important during times when imaging findings may not accurately reflect present tumor biology. This would be critical in the scenarios of radiation necrosis, where it is difficult to differentiate pseudoprogression from tumor progression based upon imaging alone, as well as with current immunotherapy regimens 189.
Numerous obstacles have been encountered in immunotherapy clinical studies when assessing the benefit of these therapies. The radiological changes may be delayed due to inflammation associated with therapy 190. The assessment of tumor progression and long-term survival is also complicated because even with development of new lesions, there may be clinical benefit 190.
Exosomes may prove to be critical in the assessment of immunotherapy effectiveness and the evolution of the immunotherapy Response Assessment for Neuro-Oncology (iRANO) criteria.
26 Temozolomide (TMZ) therapy has increased survival significantly in glioblastoma, but not all patients have a good response, and thus a predictive marker for therapeutic efficacy is needed. TMZ causes DNA strand breakage and MGMT (O6-methylguanine DNA methyltransferase) and APNG (alkylpurine-DNA-N-glycosylase) are both enzymes that are necessary for DNA repair 191 and levels of these enzymes are used as treatment response predictors. Shao et al. demonstrate that MGMT levels can be found in exosomes and correlate with levels in the original tumor cells themselves. Furthermore, the levels of MGMT and APNG change throughout the course of patient treatment 191. In another study, glioblastoma patients were enrolled in an anti-tumor vaccination trial and serum exosomes were isolated before and after vaccination treatment 192. Exosomal levels of IL-8 were shown to correlate to overall survival in patients receiving the vaccine 192. These initial studies suggest that exosomes may be utilized as predictive markers on initial response to therapy as well markers for therapeutic response throughout the course of treatment.
1.7. Therapeutic potential of exosomes
Exosomes have a lipid bilayer and mimic other content carriers, such as liposomes, allowing them to travel throughout the body without degradation. This characteristic has rendered exosomes as novel drug delivery vehicles. Zhang et al. demonstrated in a zebrafish model that injecting brain endothelial cell derived exosomes previously loaded with doxorubicin were able to pass through the blood brain barrier and reach brain tissue 193. Exosomes have also provided a novel method of anti-tumor vaccination through their stimulation of enhanced cytotoxic T cell activity as well as enhancing dendritic cell antitumor activity 194,195,196,197. Exosomes may also be used as an adjuvant therapy to work synergistically with current treatments. Munoz et al. demonstrated the mesenchymal stem cell derived exosomes transfer anti-miR9 to glioblastoma cells and confer TMZ sensitivity to these cancer cells 198. Drugs that effect nucleic acids, such as siRNA and miRNA, currently have limited in vivo translatability due to the non-targeted side
27 effects and the instability of the RNA therapeutics in vivo due to the presence of endogenous
RNAses. Exosomes are emerging as therapeutic vehicles for these types of treatments. Ohno et al. were able to produce exosomes that not only had EGFR ligands on their surface, but were also loaded with the tumor suppressor miR-let-7 199. These EGFR-targeted exosomes were able to be injected in vivo and targeted to cancer tissue over-expressing EGFR, subsequently delivering miR-let7 to that tissue and decreasing tumor growth 199. Targeted nanoplatforms for drug delivery, especially to brain tumors, is an area of intense investigation. Although beyond the scope of this review to compare, for example, liposome versus exosome delivery of therapeutic agents, liposomes are artificially created and thus allow for large yield of treatment, while exosomes are purified from biological fluids or cell media, thus producing quite a low yield, creating an obstacle for therapeutic exosomes to be used in the human setting. The study of exosome targeted delivery of therapeutic agents will likely inform our knowledge on the optimal type of treatment vehicle, providing us with a basis with which to create the ideal liposome formulation.
Exosomes are taken up by recipient cells through a multitude of mechanisms 99 and due to the variety of oncogenic effects that exosomes have on recipient cancer cells, the blockade of exosome uptake is being investigated as a novel therapeutic avenue. CD81 and CD63 are tetraspanin proteins and common exosome markers that are highly expressed on the surface of exosomes and thought to aid with exosome targeting to recipient cells 96. Antibodies to these proteins may thus block tetraspanin mediated uptake into recipient cells 96. Another potential exosome uptake mechanism is through proteoglycans; a family of proteins used by virus particles for internalization into recipient cells 200. Multiple studies have demonstrated that the use of
Heparin, a proteoglycan substituted with glycosaminoglycans, may inhibit this recipient cell- exosome interaction and exosome internalization 200,201,202. Atai et al. demonstrate that heparin diminishes the exosomal transfer of EGFRvIII to recipient glioma cells 201, while Christianson et
28 al. demonstrate that using heparin inhibits the increased invasiveness that exosomes provide to recipient glioblastoma cells in vitro 200. Exosomes may also be internalized through a lipid raft mediated mechanism, and the use of cholesterol depletion agents has been shown to decrease exosome uptake 103. For example, Simvastatin may be of particular interest for CNS malignancies due to its hydrophobicity and thus increased uptake into the brain through the blood brain barrier
203. Heparin and statins have been suggested to have anti-tumor effects 78,204,205,206, and although the mechanism is unclear, the data suggest they could be blocking exosome uptake.
1.8. Future implications
In the last decade, exosomes have been identified as having major implications in many aspects of medicine and tumor biology. Exosomes have an impact on a tumor locally and can have an influence at distant sites in the body, and their potential use as biomarkers and therapeutics is beginning to be established. As technology improves and more advanced and rapid methods of exosome isolation and profiling become available, so the use of exosomes as non- invasive liquid biopsies will become increasingly sensitive and specific for actionable tumor targets. The use of exosomes as therapeutics in the form of exosome blockade or as therapeutic nano-carriers is in its infancy, but proving to be an exciting avenue of research. Exosomes and extracellular vesicles may prove to play an integral role in this time of personalized medicine.
Further research is warranted to uncover their full potential for clinical applications.
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54 Chapter 2
HFE Genotype Affects Exosome Phenotype in Cancer
This works has been published previously: Oliver D. Mrowczynski, A.B. Madhankumar, Becky Slagle-Webb, Sang Y. Lee, Brad E. Zacharia, James R. Connor. HFE Genotype Affects Exosome Phenotype. Biochim. Biophys. Acta - Gen. Subj. (2017).
2.1. Abstract
Neuroblastoma is the third most common childhood cancer, and timely diagnosis and sensitive therapeutic monitoring remain major challenges. Tumor progression and recurrence is common in advanced stages with little understanding of mechanisms. A major recent focus in cancer biology is the impact of exosomes on metastatic behavior and the tumor microenvironment. Exosomes have been demonstrated to contribute to the oncogenic effect on the surrounding tumor environment and also mediate resistance to therapy. The effect of genotype on exosomal phenotype has not yet been explored. We interrogated exosomes from human neuroblastoma cells that express wild-type or mutant forms of the HFE gene. HFE, one of the most common autosomal recessive polymorphisms in the Caucasian population, originally associated with hemochromatosis, has also been associated with increased tumor burden, therapeutic resistance boost, and negative impact on patient survival. Herein, we demonstrate that changes in genotype cause major differences in the molecular and functional properties of exosomes; specifically, HFE mutant derived exosomes have increased expression of proteins relating to invasion, angiogenesis, and cancer therapeutic resistance. HFE mutant derived exosomes were also shown to transfer this cargo to recipient cells and cause an increased oncogenic functionality in those recipient cells.
55
2.2. Introduction
Neuroblastoma is a tumor derived from neural crest cells and is the most common extra- cranial malignant neoplasm during infancy, making it the third most common childhood cancer 1,
2. Ninety percent of children with neuroblastoma are diagnosed before the age of 4 3. A subset of neuroblastoma tumors are deemed high risk, and timely diagnosis and sensitive therapeutic monitoring remain major challenges 3-9. Adjuvant therapy is driven by histologic and molecular analysis of tissue obtained via surgical biopsy. High risk cases have the unfortunate but common event of tumor progression and recurrence. A major recent focus in cancer biology is the impact of exosomes on metastatic behavior and the tumor microenvironment.
Multiple exosome properties have been elucidated, including functional aspects they provide to recipient cells like contributing to the oncogenic effect on the surrounding tumor environment 14-16, as well as mediating resistance to therapy 17-19. The genetic components contained within exosomes are representative of the cell of origin, rendering exosomes an ideal target for biomarker development20-23. However, the effect of genotype on exosomal phenotype has not yet been explored. My work elucidates the molecular and functional changes in exosomes due to changes in genotype by using stable human neuroblastoma cell lines that express wild type or mutant forms of the HFE gene. HFE stands for “high iron”, originally found associated with hemochromatosis, but considerable research in our laboratory and others has demonstrated that
HFE mutations are also associated with increased tumor burden, therapeutic resistance boost, and negative impact on patient survival in many cancer types, including neuroblastoma 28-29, 35. HFE variants are one of the most common autosomal recessive polymorphisms in the Caucasian population, with a prevalence of 1:200 to 1:500 25-27. With such an immense prevalence and major impact on oncogenesis, studying the genotype specific changes in exosome phenotype has major implications on how cancer of patients with an HFE mutation progress, as well as
56 impacting therapeutic strategies for these patients’ optimal treatment and positive outcome. We demonstrate that changes in HFE genotype are associated with major differences in the molecular and functional properties of exosomes, which elucidates key driving factors that underlie the aggressiveness of cancer associated with the HFE gene variants, and may pave the way to new and promising forms of cancer diagnosis and personalized treatment.
2.3. Materials and Methods
2.3.1. Cell Culture and Exosome Isolation
Human neuroblastoma SH-SY5Y cells that were stably transfected to express wild type
(WT), H63D, C282Y, as well as vector control forms of HFE as described previously (28, 30) were used. Cells were cultured in DMEM/F12 (Life Technologies by Gibco) supplemented with
10% FBS, 1% non-essential amino acids (Gibco), 1% Penicillin-Streptomycin (Gibco), and 200
μg/ml Geneticin (Gibco). Before exosome isolation experiments, cell culture media was switched to media supplemented with 10% Exosome-free FBS (System Biosciences). Cells were then cultured for 48 hours until 80-90% confluency. Media was then aspirated and centrifuged at
3000xG for 15 minutes to purify out cellular debris. The supernatant was then incubated with
Exo-Quick-TC exosome isolation polymer (System Biosciences) for a minimum of 12 hours at 4 degrees Celsius. The media-ExoQuick combination was then centrifuged at 1500xG for 30 minutes. The supernatant was then aspirated and the purified exosome pellet was resuspended in
150-300μl dPBS (Gibco).
2.3.2. Exosome Confirmation and Size Analysis
Size analysis was performed using the ZetaSizer particle size analyzer (Malvern
Instruments) by taking 10 μl of each exosome solution at a concentration in the range 0.5 mg/mL
-2 mg/mL, and resuspending in 1mL of dPBS in a cuvette, which was subsequently placed into the instrument and read. Transmission electron microscopy was performed by taking 10 μl of exosome solution and placing them on parafilm. Formvar coated copper grids were then placed
57 on top of the drops and incubated for 20 minutes. The copper grids were then incubated with a
4% solution of paraformaldehyde in 0.1M PBS for 20 minutes, washed thrice with PBS for 1 minute each, incubated with 1% glutaraldehyde in .1M PBS for 5 minutes, washed with distilled water for 2 minutes, washed thrice with PBS for 2 minutes each, negatively stained with 1%
Uranyl acetate for 20 seconds, and then observed by transmission electron microscopy (JEOL-
1400). Acetyl choline esterase assay was performed by taking 25 μl of each exosome solution and adding 100μl of 12.5mM acetyl choline(M.W. 181.66) and 100μl of 1mM 5,5-dithiobis(2- nitrobenzoic acid)(M.W. 396.35). The solution was then placed in 200 μl quadruplicates in a 96 black walled plate and incubated for 10, 20, 30, and 60 minutes and absorbance measured at
412nm.
2.3.3. Immunoblot Analysis
Protein expression was determined with immuno slot blot analysis. Exosome concentration was determined using BCA assay (Thermo-Scientific). 5 μg of exosomes was solubilized on nitrocellulose membrane. The membrane was then blocked with 5% milk in TBS-T for 1 hour. Membranes were then incubated overnight at 4 degrees Celsius with primary antibodies for CD81 (1:200 sc-166029, Santa Cruz), CD63 (1:200 Ab134045, Abcam), tsg101
(1:200 sc-7964, Santa Cruz), Transferrin Receptor 1 (1:500 136800, Invitrogen), Transferrin
Receptor 2 (1:500 sc-32271, Santa Cruz), H-Ferritin (1:500 D1D4, Cell Signaling), MMP-9
(1:500 sc-21733, Santa Cruz), EGFR (1:500 sc-03, Santa Cruz), VEGF (1:500 sc-7269, Santa
Cruz), MDR-1 (1:200 sc-55510, Santa Cruz), Beta-Actin (1:3000 AC-15, Sigma). Secondary antibodies were then incubated for 1 hour and imaged with GE Amersham Imager 600. Optical density was measured using Image J software. Data are expressed as a ratio of WT exosome concentration, normalized to actin.
2.3.4. Invasion/Migration Assay
58 Invasion/Migration was determined by using a kit from Trevigen and followed according to manufacturer’s protocol (Trevigen). Briefly, 2.5 X 10^5 Wild type SH-SY5Y cells were plated on 96 trans-well plate inserts that were previously coated with matrigel. For the invasion assay, the apical chamber of the trans-well plate was supplemented with 10ug/mL of exosomes in serum-free media from all genotypes separately done in quadruplicate. Thus the WT cells were exposed to WT, vector, H63D, and C282Y derived exosomes separately, and incubated for 48 hours to allow for transfer of exosomal proteins as well as invasion of the plated WT cells into the basal chamber. For the migration assay, WT cells were plated in the apical chamber and the bottom chamber of the trans-well plate was supplemented with 10ug/mL of exosomes in serum- free media from all genotypes separately done in quadruplicate. The WT cells were allowed to migrate for 48 hours to allow for the chemo attractant properties of the exosomes to take effect.
The cells were then incubated with Calcein AM (Trevigen) for 1 hour and read at 485/520 nm.
Data are expressed as a ratio of WT cells exposed to WT exosomes.
2.3.5. Tubulogenesis Assay
A tubulogenesis assay was performed as reported in Skog et al 20. Briefly, four sets of
30,000 human umbilical vein endothelial cells (HUVECs) were incubated for 16 hours with
30ug/mL of exosomes derived from WT, Vector, H63D, and C282Y cells separately, and then tubule formation was analyzed from microscope images of the tubules using Image J software
Angiogenesis Analyzer. Data are expressed as a ratio of HUVECs exposed to WT exosomes.
2.3.6. Cellular Proliferation Assay
Four sets of 4 X 10^5 wild type SH-SY5Y cells were plated on 96 well plates and allowed to adhere overnight. The following day, these cells were incubated with WT, vector,
H63D, and C282Y cell derived exosomes, individually in quadruplicate. These cells were then allowed to proliferate for 48 hours and analysis of cell proliferation was performed using an
MTS-PMS Assay (Promega) according to manufacturer’s protocol. WT cells are incubated with
59 exosomes at a concentration of 30μg/mL. Data are expressed as a ratio of WT cells exposed to control.
2.3.7. Apoptosis Assay
Four sets of 4 X 10^5 wild type SH-SY5Y cells were plated on 96 well plates and allowed to adhere overnight. The following day, these cells were incubated with WT, vector,
H63D, and C282Y cell derived exosomes, individually in quadruplicate. The exosomes were allows to incubate with the cells for 24 hours. After the 24 hour incubation, 96 well plates were radiated. 24 hours after radiation, analysis of cell death was performed using an MTS-PMS Assay
(Promega) according to manufacturer’s protocol. WT cells were incubated with exosomes at a concentration of 30μg/mL. Radiation dosages of 3 Gy and 12 Gy were used. Data are expressed as ratio of WT cells exposed to control.
2.3.8. Statistical Analysis
All of the data generated in the proposed experiments were subjected to statistical analysis. GraphPad Prism 4.03 (GraphPad Software, San Diego, CA) was used for statistical analysis. At least 3 replicates were done and groups were analyzed using one-way ANOVA with
Tukey-Kramer post-test for all analyses. A p value < .05 was deemed significant.
2.4. Results
Characterization of exosomes from HFE mutant and WT neuroblastoma cells
First, we show in figure 2-1 that vesicles which appear to be exosomes can be detected in media from all genotypes of the SH-SY5Y neuroblastoma cells as visualized using electron microscopy (Fig 2-1A-D). Common surface protein markers that are highly expressed on exosomes are the tetraspanin family of proteins, which include CD81, CD63, and TSG101.
Immunoblot assays were performed on the exosomes from all genotypes of SH-SY5Y neuroblastoma cells and all genotypes demonstrated exosomes that expressed these three confirmatory protein markers (Fig 2-1E-G).
60
Figure 2-1. Transmission electron microscopy visualizing exosome morphology from
SH-SY5Y neuroblastoma variants. (Exosomes highlighted with arrows) (A) WT (B) VEC (C)
H63D (D) C282Y; all demonstrating sizes averaging 30nm. The 27nm exosome in the bottom left corner is an example of the classic lipid bi-layer morphology. Common surface protein markers that are highly enriched on exosome membrane surface are the tetraspanin family of proteins, which include CD81, CD63, and TSG 101. All four genotypes demonstrated substantial
61 expression of these three exosomal markers, confirming exosome isolation and purification (E)
CD81 (F) CD63 (G) TSG101. Representative images shown (n=4)
After visualization with transmission electron microscopy, the isolated exosomes were evaluated with dynamic light scattering on the Zetasizer particle size analyzer (Malvern Nano
ZS). The average sizes of exosomes purified from all SH-SY5Y cell lines were between 29 and
32 nanometers (Fig 2-2A-D). The data indicate there are no apparent changes in size distribution with regards to HFE genotype.
To confirm that the vesicles we isolated are exosomes, we performed an Acetylcholine esterase assay. Acetylcholine esterase is expressed on the surface of exosomes (31), thus this assay is commonly used as an exosome confirmatory test. Exosomes from all genotypes of SH-
SY5Y cells demonstrated Acetylcholine esterase activity when compared to dPBS control (Fig 2-
2E-I).
62
Figure 2-2. Exosome size distribution analysis with dynamic light scattering. There were no apparent changes in exosome size distribution as a function of HFE genotype (A) WT
(B) VEC (C) H63D (D) C282Y; average size of 30nm. Acetylcholine esterase assay demonstrating exosome activity after 10 minutes, 20 minutes, 30 minutes, and 60 minutes of incubation. Exosomes from all genotypes of SH-SY5Y cells demonstrated activity (measured in
AchE enzymatic activity units) when compared to PBS control (E) dPBS control (F) WT (G)
VEC (H) H63D (I) C282Y
63
Changes in HFE genotype affects ferrome related proteins within the exosomes secreted by SH-SY5Y neuroblastoma cells.
Elevated levels of iron have been shown to increase cancer aggressiveness and cancer mortality 32, 33. Increased expression levels of H-Ferritin has been shown to increase oncogenic properties of cancer cells, including increased therapeutic resistance, increased angiogenesis, and increased tumor cell growth 35, 36. These factors demonstrate the critical importance of the ferrome with regards to tumorigenesis. Because HFE mutations cause changes in iron content and ferrome proteins in cells, we thus analyzed the protein expression of iron related proteins in the exosomes released by all the genotypes of the SH-SY5Y neuroblastoma cells.
Exosomes secreted from C282Y cells expressed 124% higher levels of transferrin receptor 1, while H63D cell derived exosomes had an 11% increase in transferrin receptor 1 when compared to WT derived exosomes (Fig 2-3A), although these values were not statistically significant. C282Y exosomes had a significant 39% increase (p<.001) in transferrin receptor 2 when compared to wild type and H63D exosomes (p<.001) (Fig 2-3B). Exosomes secreted from
H63D cells expressed a 37% increase (although not statistically significant) and C282Y exosomes had a significant 83% increase (p<.05) in H-ferritin when compared to wild type cell derived exosomes (Fig 2-3C).
64
Figure 2-3. Immunoblot analysis of the ferrome (proteins involved in iron homeostasis) contained within exosomes, shown as percentage protein expression relative to
WT exosomes. All genotypes of the exosomes demonstrated substantial expression of ferrome proteins. (A) Transferrin Receptor-1 (TfR1) expression (n=3). Exosomes secreted from H63D cells had an 11% increase, while C282Y exosomes expressed 124% increased levels of TfR1, when compared to WT exosomes (B) Transferrin Receptor-2 (TfR2) expression (n=4). C282Y exosomes had a 39% increase in TfR2 when compared to WT and H63D exosomes (C) H-ferritin expression (n=6). Exosomes secreted from H63D cells expressed a 37% increase and C282Y derived exosomes had an 83% increase in H-ferritin when compared to WT exosomes. Data are shown as mean +/- SEM. (*p<.05), (***p<.001), (###p<.001 compared to H63D). Representative images shown. Statistical analysis performed with ANOVA and Tukey-Kramer post-test.
65
Exosomes demonstrate an HFE genotype dependent effect on Invasion and Migration
Exosomes increase the epithelial to mesenchymal transition (EMT) process, thus enhancing invasion and migration of recipient cells 39. MMP-9 is a specific proteinase involved in
EMT that is contained within exosomes 40, thus it may be transferred to recipient tumor cells and increase their invasive and migratory properties. MMP-9 is secreted from all genotypes of SH-
SY5Y cells and increased by 38% in H63D (although not statistically significant) and significantly increased (p<.05) by 97% in C282Y cell derived exosomes (Fig 2-4A). We then determined the functional impact of these exosomes with an invasion/migration assay, described previously. The WT cells incubated with H63D exosomes increased invasiveness by 35% (p<.05) when compared to WT cells incubated with WT exosomes (Fig 2-4B). In the migration assay, the
WT cells exposed to H63D exosomes had a 60% increase, while WT cells exposed to C282Y exosomes had a 107% increase in migration when compared to WT cells exposed to WT exosomes (Fig 2-4C), although these values were not statistically significant.
66
Figure 2-4. Invasion and migration analysis (A) Immunoblot analysis of exosome
MMP-9 shown as percentage protein expression relative to WT exosomes (n=5). MMP-9 is contained within exosomes of all genotypes of SH-SY5Y cells and increased by 38% in H63D and increased by 97% in C282Y exosomes. (B) To assess the functionality of this increase in protein expression, an invasion/migration study was performed. WT cells that were incubated with H63D exosomes Increased invasiveness by 35% when compared to WT cells that were incubated with WT exosomes (C) Migration analysis with WT cells incubated with exosomes of all genotypes (n=4). WT cells that were exposed to the H63D exosomes had 60% increase and to
C282Y exosomes had 107% increased migration when compared to WT cells exposed to WT exosomes. Data are shown as mean +/- SEM. Representative image shown. (*p<.05) Statistical analysis performed with ANOVA and Tukey-Kramer post-test.
67
Exosomes demonstrate an HFE genotype dependent effect on Angiogenesis
Tumors require a blood supply to survive and grow, and we have thus analyzed protein expression of EGFR and VEGF in exosomes secreted by different genotypes of SH-SY5Y cells.
Immunoblot analysis demonstrated exosomes from H63D had a 16% increase (although not statistically significant) while C282Y exosomes had a significant 115% increase (p<0.001) in levels of EGFR expression when compared to WT (Fig 2-5F). C282Y exosomes also had a significant increase (p<.001) in EGFR level when compared to H63D. Exosomes from C282Y cells had a significant 51% increase (p<0.001) in levels of VEGF expression when compared to exosomes secreted from WT cells (Fig 2-5G). To determine if the HFE genotype dependent changes in exosome angiogenic proteins are functionally significant, a tubulogenesis assay was performed. Four sets of human umbilical vein endothelial cells (HUVECS) were incubated with exosomes derived from WT, Vector, H63D, and C282Y cells separately (Fig 2-5A-D). Tubule formation was then analyzed. Tubule analysis showed an increase of 14% when incubated with
H63D exosomes (although not statistically significant), and a significant 41% (p<.05) increase when incubated with C282Y exosomes (Fig 2-5E). The degree of tubule formation demonstrates that the exosomes derived from HFE mutant cells that have increased expression levels of angiogenic proteins can transfer these proteins to endothelial cells and increase their angiogenic properties.
68
Figure 2-5. Angiogenesis functionality determined with HUVEC incubation with exosomes from all genotypes (A) WT (B) VEC (C) H63D (D) C282Y (E) Quantification of angiogenesis, shown as percentage angiogenesis relative to WT exosomes. HUVECs incubated with H63D exosomes had a 14% increase while incubation with C282Y exosomes caused a 41% increase. Immunoblot analysis of exosome angiogenic proteins shown as percentage protein expression relative to WT (F) EGFR expression (n=6). Exosomes from H63D cells had a 16% increase while C282Y cell derived exosomes had a 115% increase in EGFR levels when compared to WT cell derived exosomes as well as H63D cell derived exosomes (G) VEGF expression (n=6). Exosomes from C282Y cells also had a 51% increase in levels of VEGF expression when compared to exosomes secreted from WT and H63D cells. Data are shown as mean +/- SEM. N is greater than or equal to 4. (*p<.05) (***p<.001) (###p<.001 compared to
H63D) Representative images shown. Statistical analysis performed with ANOVA and Tukey-
Kramer post-test.
69 Exosomes have a HFE genotype dependent effect on cellular proliferation and therapeutic resistance
Another functional aspect of the exosomes is the possibility of transfer of proteins related to cellular proliferation and therapeutic resistance. The question interrogated here is whether therapeutic resistance may be in part due to exosome transfer of proteins such as H-ferritin and
MDR-1 37, 38.
First, we determined the expression levels of MDR-1 from the exosomes of the neuroblastoma cells. Immunoblot analysis demonstrated that exosomes secreted from H63D mutant cells had a 47% increase in MDR-1 expression (although not statistically significant) while C282Y exosomes had a significant 293% increase (p<.01) when compared to WT and
H63D (p<.05) (Fig 2-6A).
We then addressed the functional significance of exosome exposure by performing cell proliferation and apoptosis assays (described previously) to interrogate the possible transfer of proliferative and therapeutic resistance functionality from the exosomes to the neuroblastoma cells. The cellular proliferation assay demonstrated that WT cells incubated with H63D exosomes had a 33% increase (p<.01) and the WT cells incubated with C282Y exosomes had a 40% increase (p<.001) in cellular proliferation when compared to control (Fig 2-6B). This demonstrates that the HFE mutant derived exosomes increase cancer cell proliferation and growth. For the apoptosis assay, WT cells were incubated with exosomes derived from all HFE genotype cells, individually, and then radiated at 3Gy or 12Gy. The apoptosis assay demonstrated that WT cells radiated at 3Gy after being incubated with H63D exosomes had a
33% increase (p<.01) and the WT cells incubated with C282Y exosomes had a 39% increase
(p<.01) in cell survival when compared to control (Fig 2-6C). The apoptosis assay also demonstrated that WT cells radiated at 12Gy after being incubated with H63D exosomes had a
40% increase (although not statistically significant) and the WT cells incubated with C282Y
70 exosomes had a significant 53% increase (p<.05) in cell survival when compared to control (Fig
2-6D).
71
Figure 2-6. Analysis of cellular proliferation and therapeutic resistance. Immunoblot analysis of MDR-1 shown as percentage protein expression relative to WT exosomes (n=5). (A)
Exosomes secreted from H63D mutant cells had a 47% increase while C282Y exosomes had a
293% increase expression of MDR-1 when compared to WT and H63D exosomes. (B) The cellular proliferation assay demonstrated that WT cells incubated with H63D exosomes had a
33% increase while C282Y exosomes had a 40% increase in cellular proliferation when compared to control (n=5). (C-D) For the apoptosis assay, WT cells were incubated with exosomes derived from all HFE genotype cells, individually, and then radiated at 3Gy or 12Gy.
(C) The apoptosis assay demonstrated that WT cells radiated with 3Gy after being incubated with
H63D exosomes had a 33% increase while C282Y exosomes had a 39% increase in cell survival when compared to control (n=4). (D) The apoptosis assay demonstrated that WT cells radiated
72 with 12Gy after being incubated with H63D exosomes had a 40% increase while C282Y exosomes had a 53% increase in cell survival when compared to control (n=3). (*p<.05)
(**p<.01) (**p<.01) (#p<.05 compared to H63D) Data are shown as mean +/- SEM. Statistical analysis performed with ANOVA and Tukey-Kramer post-test.
73 2.5. Discussion
Our study focuses on the effects of genotype on exosome phenotype, which have not been adequately explored. In this study, we demonstrate HFE genotype specific changes in exosome contents also have a functional impact on recipient cells. We analyzed the exosome size, invasive/migratory properties, angiogenic potential, cellular proliferation, and therapeutic resistance aspects of exosomes in relation to HFE genotype. The model for this study was SH-
SY5Y neuroblastoma cells that were stably transfected with WT and mutant forms of the HFE gene. Using this cell line enabled us to interrogate the specific impact of the HFE gene variant, thus providing us with a model to analyze how germline mutations can affect the contents and functionality of exosomes. As mentioned, HFE is one of the most common autosomal recessive polymorphism in the Caucasian population 18-20, and well-known to impact iron regulation, cancer outcomes, and neurodegenerative diseases, making these results relevant to a wide range of cancers.
Elevated levels of iron, which can occur through the HFE mutation, have been shown to increase cancer aggressiveness and cancer mortality 32, 33. We have previously shown that the presence of an HFE mutant protein conveys therapeutic resistance to neuroblastoma cells 34.
Increased expression levels of the protein H-Ferritin, intimately involved in iron regulation, have been shown to increase oncogenic properties of cancer cells, including increased angiogenesis, increased tumor cell growth, and increased therapeutic resistance 35, 36. Chemotherapeutic or radiation resistance properties are innate in many cancer cells, including neuroblastoma, but resistance can also develop over the course of treatment 37, 38. Our laboratory has demonstrated increased levels of H-ferritin in glioma cells increase resistance to radiation, and decreased expression of H-ferritin is associated with an increase in radiation sensitivity 35. Another protein involved in therapeutic resistance is Multi-Drug Resistance 1 (MDR-1) 37. Increased MDR-1 expression has been shown to negatively impacts neuroblastoma patient prognosis 38. Our study
74 demonstrates elevated levels of both resistance proteins H-Ferritin and MDR-1 in the HFE mutant derived exosomes (Fig 2-3C, Fig 6A). Furthermore, when WT cells were incubated with mutant derived exosomes, an increase in radiation resistance is seen (Fig 2-6C and D). The increase in resistance in WT cells may be due to HFE mutant genotype specific exosomal transfer of these proteins which are involved in therapeutic resistance.
Elevated levels of H-Ferritin have also been implicated in increased cancer angiogenesis
35, 36. The increased expression of H-Ferritin in the HFE mutant derived exosomes further prompted an angiogenic analysis. VEGF and EGFR are proteins critical to angiogenesis, which we also show to be increased in the mutant exosomes (Fig 2-5F and G). The elevated levels of H-
Ferritin, VEGF, and EGFR may be transferred to recipient cells to enhance angiogenesis, which we tested through a tubulogenesis assay. The degree of increased tubule formation suggests that the exosomes derived from HFE mutant cells that have increased expression levels of angiogenic proteins can transfer these proteins to endothelial cells and enhance their angiogenic properties
(Fig 2-5A-E).
A key component to an aggressive cancer is the process of cancer cell invasion. Matrix metalloproteinases (MMPs) are a family of proteins that are integrally involved in tumor cell migration and invasion. MMP-9 is a specific proteinase in this family that is contained within exosomes 40, which we show to be elevated in the HFE mutant cell derived exosomes (Fig 2-4A).
The functional changes in invasion and migration we saw are consistent with the levels of increase in expression of MMP-9 in the HFE mutant derived exosomes (Fig 2-4). These data provide evidence supporting our hypothesis that these exosomes contain genotype specific components that have effects on invasion and migration, which can also be transferred to recipient cells and become functional.
In conclusion, our study has demonstrated that exosomes derived from HFE mutant cancer cells have genotype specific increased expression of proteins related to integral aspects of
75 oncogenesis, which has major implications on not only HFE variant patients with neuroblastoma, but all types of cancer. This further suggests that the aggressive phenotype of cancer patients with
HFE mutations 34 may be due to HFE mutant cancer cell derived exosome uptake in recipient cells. This study implicates the importance of considering the HFE genotype for evaluating patient prognosis, planning treatment strategies, and having successful outcomes in cancer patients with the HFE variant.
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82 Chapter 3
Exosomes and Acquired Radiation Resistance in Cancer
3.1. Abstract
Radiation therapy is essential in the arsenal of cancer treatment and is utilized in the therapeutic regimen of more than 50% of all cancer patients. Unfortunately, many aggressive malignancies may become resistant to radiation over time, rendering treatment futile. The mechanism of acquired radiation resistance is not fully understood. We investigated the hypothesis that acquired radiation resistance may occur through cellular communication via exosomes. Exosomes are cell-derived vesicles containing DNA, RNA, and protein instrumental to a cell’s interaction with its microenvironment. Three properties were analyzed: 1) exosome function, 2) exosome profile, and 3) exosome uptake/blockade. Radiation-derived exosomes increased cellular proliferation and radiation resistance in recipient tumor cells in cell culture.
Furthermore, radiation-derived exosomes increased tumor burden and decreased survival in an in vivo murine model of glioblastoma. To address the mechanism underlying this phenomenon we obtained a profile of radiation-derived exosomes and found they exhibit specific miRNA, mRNA, and protein expression changes favoring a resistant/proliferative profile. For example, radiation- derived exosomes contain elevated levels of oncogenic miR-889, multiple oncogenic mRNAs, and proteins involved in the proteasome pathway, Notch, Jak-STAT, and cell cycle signaling pathways. Radiation-derived exosomes also contain decreased levels of tumor-suppressive miR-
516, miR-365, and multiple tumor-suppressive mRNAs. Ingenuity Pathway analysis revealed the most represented networks included cell growth, cell cycle, and cell survival. The most represented upstream regulator was the MYC oncogene. Upregulation of DNM2 correlated with increased uptake of radiation-derived exosomes. Heparin and simvastatin blocked uptake of radiation-derived exosomes in recipient cells and inhibited induction of cellular proliferation and
83 radiation resistance, both in vitro and in vivo. Because these latter two agents have shown some success as a cancer therapy, our data suggest that their mechanism of action could be to limit exosome communication between cells. The results of our study identify a novel exosome-based mechanism that may underlie acquired radiation resistance in cancer.
3.2. Introduction
More than fifty percent of cancer patients, including patients with the most devastating central nervous system malignancy, glioblastoma1,2,3, receive radiation as a critical component of their standard treatment regimen4. One reason for the dire prognosis of cancer is its ability to elude standard radiotherapy5. Glioblastoma is molecularly heterogeneous and this intratumoral heterogeneity and environmental modification traits of cancer are accentuated during treatment.
Even in the face of surgical resection and adjuvant chemoradiation, recurrence and progression are nearly universal. While the inherent heterogeneity of aggressive cancers likely mediates part of therapeutic resistance, the mechanisms are largely unknown. We propose that cellular communication via exosomes is critical to the development of radiation resistance. Exosomes are nanometer-sized vesicles6–9 released by cells that contain genetic components of the parent cancer cell from which they were derived10–15, and have broad ranging effects on the tumor microenvironment 16,17,18. They have a protective lipid bilayer and are small enough to permit travel throughout the body without being degraded19,20,21,22. It has been demonstrated that stressors such as hypoxia can change exosomal content and functionally impact the local cell population23,24. Recent studies have also demonstrated that ionizing radiation increases the release of exosomes from glioblastoma cells and alters their contents rendering the exosomes more oncogenic25. Although changes in exosome content due to radiation have been identified25,26,27,28, the mechanisms underlying resistance and proliferation in recipient cancer cells induced by these radiation-derived exosomes have not been explored. Moreover, the functional impact of these radiation-derived exosomes in vitro and in vivo have not been investigated.
84 To that end, we undertook studying the impact of radiation on exosome profile and the effects of radiated exosomes on recipient cells in the surrounding tumor environment. We explore whether exosomes secreted by radiated cancer that are subsequently taken up by recipient cells render those recipient cells resistant to radiation therapy. This concept may be especially critical for inherently radiation resistant cancer cells, as well as cancer cells at the border of the radiation treatment field where instead of causing cell death, sub-lethal doses of radiation may act as a stressor causing the release of oncogenic exosomes and the malignant functional effects that follows. Although highly controversial, a recent study by Duma et al. 29 demonstrated that utilizing a technique coined “Leading edge radiation” and expanding the radiation treatment margins led to significantly better outcomes in glioblastoma patients when compared to standard radiation protocols.
In addition to inducing changes in recipient cells that may promote a cancerous phenotype, exosomes are also being evaluated as potential therapeutic targets. Exosomes are taken up by a multitude of mechanisms, mediated by tetraspanin (CD81), proteoglycans, and/or lipid rafts. The use of antibodies to the CD81 protein on the cell surface of exosomes as well as using heparin, and simvastatin to block exosome uptake are just beginning to be investigated30,31,32,33,34,35. Heparin and statins have also been suggested to have anti-tumor effects36–40,41,42, but the underlying mechanism is unclear. Simvastatin is of particular interest for
CNS malignancies due to its hydrophobicity and thus increased uptake into the brain through and intact blood brain barrier41. By interrogating this mechanism, we ultimately aim to advance cancer treatment and help understand existing treatments whose mechanism may be through exosome inhibition.
3.3. Methods
3.3.1. Cell Culture, Materials, and Exosome Isolation
85 SH-SY5Y human neuroblastoma cells, U87 glioma cells from ATCC, and STS26T human malignant peripheral nerve sheath tumor cells generously gifted by Dr. Daniel Scoles,
University of Utah, were used. All cell lines were maintained at 37˚C in a humidified incubator with 5% CO2. The SH-SY5Y cell line was cultured in Dulbecco’s Modified Eagle Medium
(DMEM, Life Technologies by Gibco) supplemented with 10% FBS, 1% non-essential amino acids (Gibco), 1% Penicillin-Streptomycin (Gibco), and 200 μg/ml Geneticin (Gibco). The U87 and STS26T cell lines were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin (Gibco). Heparin was purchased from Sigma and simvastatin was purchased from Med Chem Express. Before exosome isolation experiments, cell culture media was switched to media supplemented with
10% Exosome-free FBS (System Biosciences). Cells were then immediately either not radiated
(control), or radiated at a dosage of 3Gy or 12Gy. The cells were subsequently cultured for 48 hours until 80-90% confluency. Media was aspirated and centrifuged at 3000xG for 15 minutes to purify out cells and cellular debris. The resulting supernatant was incubated with Exo-Quick-TC exosome isolation polymer (System Biosciences) for a minimum of 12 hours at 4°C. The media-
ExoQuick combination was centrifuged at 1500xG for 30 minutes. The supernatant was aspirated and the purified exosome pellet was resuspended in 150-300μl dPBS (Gibco).
3.3.2. Exosome Confirmation
Size analysis was performed using the ZetaSizer particle size analyzer (Malvern
Instruments) by taking 10 μl of each exosome solution and resuspending in 1mL of dPBS in a cuvette, which was subsequently placed into the instrument and read. Exosomes were quantified using a BCA Assay (Thermo-Scientific) or with nanoparticle tracking analysis (NanoSight
NS300). Transmission electron microscopy was performed by taking 10 μl of exosome solution and placing them on parafilm. Formvar coated copper grids were then placed on top of the drops and incubated for 20 minutes. The copper grids were incubated with a 4% solution of
86 paraformaldehyde in 0.1M PBS for 20 minutes, washed thrice with PBS for 1 minute each, incubated with 1% glutaraldehyde in 0.1M PBS for 5 minutes, washed with distilled water for 2 minutes, washed thrice with PBS for 2 minutes each, negatively stained with 1% Uranyl acetate for 20 seconds, and observed by transmission electron microscopy (JEOL-1400).
3.3.3. Immunoblot Analysis
Protein expression was determined with immunoblot analysis. Exosome concentration was determined using BCA assay (Thermo-Scientific). 5μg of exosomes was solubilized on nitrocellulose membrane. The membrane was then blocked with 5% milk in TBS-T for 1 hour.
Membranes were incubated overnight at 4°C with primary antibodies for CD81 (1:200 sc-
166029), CD63 (1:200 Ab134045), tsg101 (1:200 sc-7964. Secondary antibodies were incubated for 1 hour and imaged with GE Amersham Imager 600.
3.3.4. Cellular Proliferation Analysis
4 X 10^5 SH-SY5Y cells, 4 X 10^3 U87 cells, and 4 X 10^3 STS26T cells were plated on 96 well plates and allowed to adhere overnight. The following day, these cells were incubated with PBS control, non-radiation derived exosomes, or radiation derived exosomes individually, in quadruplicate. These cells were allowed to proliferate for 48 hours and analysis of cell proliferation was performed using an MTS-PMS Assay (Promega) according to manufacturer’s protocol. Naïve cells were incubated with exosomes at a concentration of 30μg/mL. Data is expressed as a ratio of naïve cells exposed to control.
3.3.5. Apoptosis Assay
4 X 10^5 wild type SH-SY5Y cells, 4 X 10^3 U87 cells, and 4 X 10^3 STS26T cells were plated on 96 well plates and allowed to adhere overnight. The following day, these cells were incubated with PBS control, non-radiation derived exosomes, or radiation derived exosomes individually, in quadruplicate. After a 24 hour incubation, 96 well plates were radiated. 24 hours after radiation, analysis of cell death was performed using an MTS-PMS Assay (Promega)
87 according to manufacturer’s protocol. Naïve cells were incubated with exosomes at a concentration of 30μg/mL. Radiation dosages of 3Gy and 12Gy were used. Data is expressed as a ratio of naïve cells exposed to control.
3.3.6. Reactive Oxygen Species Assay
25 X 10^3 U87 cells and 25 X 10^3 STS26T cells were plated on 96 well plates and allowed to adhere overnight. The following day, these cells were incubated with PBS control, non-radiation derived exosomes, or radiation derived exosomes individually, in quadruplicate.
Exosomes were treated at a concentration of 30ug/mL. After a 24 hour incubation, media was removed and two 5 minute PBS washes were performed. PBS was removed and analysis of reactive oxygen species was performed using a H2DCFDA (2′,7′-Dichlorodihydrofluorescein diacetate) assay (Promega) according to manufacturer’s protocol, where the H2DCFDA will be oxidized by the ROS to highly fluorescent DCFDA. Briefly, 3mg of H2DCFDA was solubilized in 300uL DMSO, which was subsequently mixed with 20mL of PBS. 100uL of the H2DCFDA solution was added to wells. 96 well plates were radiated. Plates were incubated for 30 minutes to
3 hours and read on a plate reader (Gemini EM) at 492-495nm excitation and 517-527nm emission. Data is expressed as a ratio of naïve cells exposed to control.
3.3.7. Exosome Blockade Analysis
2 X 10^3 U87 cells and 2 X 10^3 STS26T cells were plated on 96 well plates and allowed to adhere overnight. The following day, one set of each type of cells was incubated with simvastatin (2uM)35. Simvastatin was chosen for its blood brain barrier permeability making it relevant for gliomas43. 24 hours after incubation, another set of cells was incubated with heparin
(20ug/mL) for 30 minutes32,34,44.The radiation derived exosomes were aliquoted and one aliquot was incubated with heparin (20ug/mL) for 30 minutes at room temperature and another exosome aliquot was incubated with anti-CD81 antibodies (20ug/mL) (sc-166029) for 30 minutes at room temperature30. The cells were incubated with PBS control, non-radiation derived exosomes,
88 radiation derived exosomes, radiation derived exosomes plus heparin, radiation derived exosomes plus simvastatin, radiation-derived exosomes plus anti-CD81 antibodies, anti-CD81 antibody alone, simvastatin alone, or heparin alone, individually, in quadruplicate. Exosomes were treated at a concentration of 30ug/mL. Cellular proliferation analysis and cell survival analysis was performed 24 hours later as previously described in 2.4 and 2.5, respectively.
3.3.8. Exosome Fluorescent Tagging and Blockade Analysis
Exosomes were isolated as described previously in 2.1. The exosomes were fluorescently labeled with PKH67 following manufacture’s protocol. Briefly, exosome pellets were resuspended in 1 mL Diluent C. Separately, 1 mL Diluent C was mixed with 4 μL PKH67. The
Diluent C-PKH67 solution was incubated with the exosomes for four minutes. The fluorescent labeling reaction was stopped by adding an equal volume of 1% BSA. Labeled exosomes were ultracentrifuged at 110,000 X G for 60 minutes, washed with PBS, and ultracentrifuged again at
110,000 X G for 60 minutes.
5 X 10^3 U87 cells and 5 X 10^3 STS26T cells were plated in 8 well chamber slides and allowed to adhere overnight. The following day, simvastatin (2uM) was added as described in 2.7 and allowed to incubate for 24 hours. After 24 hours, heparin (20ug/mL) or anti-CD81 antibodies
(20ug/mL) were added to cells and exosome aliquots and incubated for 30 minutes, as described in 2.7. The cells were incubated with PBS control, non-radiation derived exosomes, radiation derived exosomes, radiation derived exosomes plus heparin, radiation derived exosomes plus simvastatin, or radiation derived exosomes plus anti-CD81 antibodies. Exosomes were treated at a concentration of 10ug/mL. 24 hours after incubation, media was removed and wells were washed twice with PBS for 5 minutes each. The wells were incubated with DAPI (1:1000) for 10 minutes. The wells were washed twice with PBS for 5 minutes each wash. Subsequently, the wells were fixed with 10% formaldehyde for 20 minutes and again washed twice with PBS for 5 minutes each wash. A drop of gel mount media was placed on the slides and they were mounted
89 with coverslips and allowed to dry in the dark overnight. Slides were viewed under a fluorescence microscope (Nikon Eclipse 80i).
3.3.9. In Vivo Studies
Five groups of nude mice were injected with 1 X 10^6 U87-Luciferase cells subcutaneously in the mouse flank. This well-established glioma cell line transfected with luciferase allows for accurate in vivo tumor quantification. One week after injection of tumor cells, the mice were imaged using Intravital Imaging Spectroscopy (IVIS). A linear relationship between the bioluminescent intensity and the tumor weight is evident by earlier reports 45 and is also able to accurately monitor tumor progression over time46,47.The mice were first anesthetized within an induction chamber using a concentration of 5% isoflurane. Next, a subcutaneous injection of 100μL of luciferin-D Substrate (purchased from Caliper LS and diluted in 35 mL of dH2O with a final concentration of 28.57 mg/mL) was administered. These mice were weighed and then transferred into the imaging chamber where anesthesia was maintained with a concentration of 1-2% isoflurane emitted through nose cones. Five minutes post-injection of luciferin-D, imaging utilizing the IVIS 50 (Perkin-Elmer) was performed according to manufacturer’s protocol (Perkin Elmer). The IVIS was run for 0.5 seconds and bioluminescence was recorded. Once it was confirmed that there was a measurable signal from the tumor cells, the intensity of bioluminescence was measured utilizing LivingImage software. All values were ranked in order or bioluminescent signal intensity and then normalized and evenly distributed into five homogeneous groups. Groups consisted of mice treated with weekly intra-tumoral injections of either saline control (n=6), non-radiation derived exosomes (n=7), radiation derived exosomes
(n=6), radiation derived exosomes plus subcutaneous heparin (n=6), radiation derived exosomes plus oral simvastatin (n=6), heparin alone (n=6), or simvastatin alone (n=6). 50ug of exosomes in
PBS were given48. Heparin was solubilized in PBS and given daily subcutaneously at a concentration consistent with the current prophylactic dose in humans, 100IU/kg49. Simvastatin
90 was solubilized in Ora-Plus (Perrigo) and given daily as an oral gavage at a concentration of
10mg/kg. This dosage was shown previously to not affect tumor progression in mice50 and was chosen to isolate the exosome blockade effect. Mice were treated with 3Gy radiation weekly by covering their bodies except for the site of the tumor with lead. Tumor growth was analyzed through IVIS imaging and quantification weekly. Mouse survival was assessed through end point analysis which was defined as mouse death from tumor by cachexia or the veterinarians deeming the tumor to be large enough that necessitated mouse sacrifice.
3.3.10. Immunohistochemistry of Tumor Tissue Sections
In order analyze expression of tumor proliferation and apoptosis proteins after tumor treatment, tumor tissues were obtained from mice in each group and H&E and immunohistochemistry was performed. The tumor tissues were frozen and sectioned to 10μm using a cryostat and subjected to immunohistochemistry to identify protein expression. The sections were fixed with 4% paraformaldehyde for 20 minutes and washed with PBS for 5 minutes. The non-specific binding sites on the tissues were blocked with 10% normal goat serum for 1 hour at room temperature. Subsequently, the tumor tissues were incubated with Ki-67 antibody (1:500 ab15580, Abcam) or Cleaved Caspase 3 antibody (1:500 9661S, Cell Signaling) overnight at 4°C and washed with phosphate buffered saline (PBS) three times for 5 minutes each wash. The sections were treated with DAPI (1:1000) and secondary Alexa Fluor 488 conjugated anti-rabbit IgG (1:200) for 60 minutes and washed with PBS 3 times for 5 minutes before gel mounting, drying overnight, and viewing under a fluorescence microscope (Nikon Eclipse 80i).
3.3.11. RNA Analysis
Exosomes were isolated as previously described in 4.1. Briefly, cells flasks were either not radiated, radiated at 3Gy, or radiated at 12Gy. Exosomes from flasks were isolated and run for RNA exploration analysis. Total exosomal RNA was extracted using mirVana kit (Ambion, cat#:AM1560). The extracted RNAs were quantified and quality checked using a BioAnalyzer
91 RNA 6000 Pico Kit (Agilent Technologies). QuantSeq 3' mRNA-Seq Library Prep Kit FWD for
Illumina (Lexogen) was used to generate mRNA-seq libraries as per manufacturer’s recommendation, followed by deep sequencing on an Illumina HiSeq 2500 as per the manufacturer’s instructions. Briefly, 0.5-1 ng of total RNA was subjected to the first cDNA strand which is initiated by oligodT priming. The synthesis of the second cDNA strand is performed by random priming, in a manner that DNA polymerase is efficiently stopped when reaching the next hybridized random primer, so only the fragment closest to the 3′ end gets captured for later indexed adapter ligation and PCR amplification. The processed libraries were assessed for fragment size distribution and quantity using a BioAnalyzer High Sensitivity DNA kit (Agilent Technologies). Pooled libraries were denatured and loaded onto a TruSeq Rapid flow cell on an Illumina HiSeq 2500 and run for 50 cycles using a single-read recipe according to the manufacturer's instructions. De-multiplexed sequencing reads passed the default purify filtering of Illumina CASAVA pipeline (released version 1.8) were subjected to QuantSeq data analysis pipeline on a Bluebee genomics analysis platform (Bluebee). Small RNA-seq libraries was generated by NEXTflex Small RNA Library Prep Kit v3 for Illumina (BioO Scientific), followed by deep sequencing on an Illumina HiSeq 2500 as per the manufacturer’s instructions. Briefly, 1-
2 ng of total RNA was ligated with chemically modified 3’- and 5’- adapters that can specifically bind to mature micro RNAs, followed by reverse transcription and PCR amplification. Unique index sequence tags were introduced during PCR to enable multiplexed sequencing. Each library was assessed for the presence of desired micro RNA population and approximate library quantity by Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies). Pooled libraries were denatured and loaded onto a TruSeq Rapid flow cell on an Illumina HiSeq 2500 and run for 50 cycles using a single-read recipe according to the manufacturer's instructions. De-multiplexed sequencing reads passed the default purify filtering of Illumina CASAVA pipeline (released version 1.8) were quality trimmed/filtered using The FASTX-Toolkit
92 (http://hannonlab.cshl.edu/fastx_toolkit). The filtered reads were further trimmed with both 5’ and 3’ adapter sequences and subjected to Chimira suite to align and count miRNA expression92.
For both mRNA and small RNA-seq datasets, TCC v1.14.0 R package93 was used to identify differentially expressed genes (DEG) between the non-radiated (0Gy) and radiated (3Gy and
12Gy) exosomal RNA read counts. We used edgeR as a test method94. Significantly DEX between control and radiated samples (n=2) were defined to be those with q-value < 0.1. We used
Ingenuity Pathway Analysis (IPA) to identify overrepresented mRNA in the pathways and their effects in various functional contexts, such as subcellular location, functional gene family, association with drugs, pathways, and disease relevance.
3.3.12. Proteomic Analysis
Exosome protein analysis was performed utilizing the Tandem Mass Tag (TMT)-10plex kit according to manufacturer’s protocol (Thermofischer Scientific). This kit provides multiplexed protein identification and quantitative analysis by tandem mass spectrometry.
Briefly, exosomal proteins were normalized to 25ug of exosome proteins in 40uL of PBS for each of the groups (non-radiation derived exosomes, 3Gy radiation derived exosomes, and 12Gy radiation derived exosomes) (n=3). The exosomal proteins were incubated with lysis buffer and then centrifuged at 16,000 X G for 10 minutes. The supernatant was then mixed with 100mM
TEAB, 200mM TCEP, for one hour, and subsequently 5µL of the 375mM iodoacetamide for 30 minutes. Acetone was then added and allowed to precipitate overnight. The samples were centrifuged at 8000 X G for 10 minutes and the pellets were then resuspended in 100mM TEAB.
Trypsin storage solution and trypsin were added to the samples and digested overnight at 37˚C.
These samples were then labeled with the TMT label reagents with 1 hour incubation at which point 5% hydroxylamine was then added to quench the reaction. The samples were then run on the Orbitrap Velos Mass Spectrometer (Thermofischer Scientific) and the data was subsequently processed using Proteome Discoverer (version 2.1). The search engine is SequestHT. PSMs are
93 filtered using Percolator. Proteome Discoverer uses its own algorithm for protein grouping.
Samples were searched against human database and filtered to retain proteins/peptides with <1%
FDR. Adavita iPathwayGuide (Advaita Bioinformatics) Next-gen pathway analysis software with the Impact Analysis method was then used to identify dysregulated protein relationships and their effects in various functional contexts, such as subcellular location, gene ontology, association with drugs, pathways, and disease relevance.
3.3.13. Statistical Analysis
All of the quantified data generated in the proposed experiments were subjected to statistical analysis. GraphPad Prism 4.03 (GraphPad Software, San Diego, CA) was used for statistical analysis and groups were analyzed using one way ANOVA with Tukey-Kramer posttest unless otherwise noted in previous methods. Survival analysis was performed using
Kaplan-Meier curves and curves were compared by means of a log rank test. At least three replicates were performed unless otherwise noted in previous methods. A p value <0.05 was deemed significant.
3.4. Results
Characterization of exosome size and quantity released from radiated and non-radiated glioblastoma cells
Figure 3-1 shows the purification of exosomes between 20-200 nanometers with dynamic light scattering on the Zetasizer particle size analyzer (Malvern Nano ZS). Two populations of exosomes were released from the U87 glioma cells, with average sizes of 24 nm and 93 nm respectively (Fig 3-1. A-C). The data indicate there are no apparent changes in size distribution with regards to radiation treatment. However, we did note an increase in exosomal release following radiation treatment in a dose-dependent manner, as shown by the number of particles, or exosome release intensity (Figure 3-1A-C). This increase in radiation-induced exosome release was also confirmed with BCA protein analysis as well as nanoparticle tracking analysis
94 (Fig 3-1 D,E). Both analyses showed a 2-fold increase in exosome secretion after exposure to
3Gy radiation and a 3-fold increase in exosome secretion after exposure to 12Gy compared to control. Common surface protein markers that are highly expressed on exosomes include the tetraspanin family of proteins, which include CD81 and TSG101. These markers are expressed by exosomes from glioma cell lines with and without radiation treatment (Fig. 3-1F,G). Exosomes from glioma cells were visualized and confirmed with electron microscopy (Fig.3-1H).
95
Fig. 3-1. Exosome confirmation analysis. Zetasizer analysis in Panels A-C demonstrate that size of the exosomes was not affected by radiation exposure but there is a dose dependent increase in release intensity (A) at 0Gy radiation, exosomes had a release intensity of 8.5 and 7.1 respectively. (B) At 3Gy radiation the release intensity was 7.98 and 9.1. (C) At 12Gy radiation the release intensity was 11.3 and 12.2. Exosomes were then quantified with (D) BCA assay and
(E) nanoparticle tracking analysis. Demonstration of exosome release from glioblastoma cells before and after exposure of radiation at 3Gy and 12Gy with immunoblots of exosome confirmation markers (F) CD81 and (G) TSG101. (H) Electron microscopy visualization of exosomes from glioblastoma cells. (*p<.05, **p<.01, ***p<.001)
96 Functional impact of exosomes derived from radiated and non-radiated cancer cell lines in vitro
Naïve cancer cells incubated with exosomes purified from each of the cancer cell types radiated with 3Gy and 12Gy had a significant increase in cellular proliferation when compared to control (Fig. 3-2A-F). Furthermore, naïve cancer cells incubated with exosomes derived from radiated cancer cells had a significant increase in radiation resistance and survival when compared to control in all cell types (Fig. 3-2 G-L). We then determined whether a decrease in production of reactive oxygen species (ROS) by these cell lines incubated with exosomes was a potential mechanism underlying the increase in cellular resistance after radiotherapy. No changes in ROS production were found after radiation exposure by the cells incubated with and without exosomes (Fig.3-2 M, N).
97
Fig. 3-2. Cellular Proliferation and Radiation Resistance Effects of Exosomes. In the graphs, U87 glioma cells are represented with a “U”, STS26T MPNST cells with an “S”, and SH-
SY5Y neuroblastoma cells with a “B”. The number following the cell line letter is the dosage of radiation used; either 3R or 12R. The cells were either exposed to exosomes from not irradiated cells “NR” or to exosomes from cells that received one of the two doses of radiation (3R or 12R).
Cellular proliferation effect of exosomes is shown in Panels A-F: (A,B) U87 glioma cells, (C,D)
STS26T MPNST cells, (E,F) SH-SY5Y neuroblastoma cells. Radiation resistance effect of exosomes in Panels G-L: (G,H) U87 glioma cells, (I,J) STS26T MPNST cells, (K,L) SH-SY5Y neuroblastoma cells) (the numbers on the y axis are too crunched I redid the graphs to change neuroblastoma to “B” and made the y axis less crunched. (*p<.05, **p<.01). Reactive oxygen
98 species of cells incubated with exosomes compared to control (M) 3Gy (N) 12Gy. The results shown increased proliferation and increased resistance to radiation when cells are exposed to exosomes from irradiated cells. There was no effect of production of ROS.
99 Inhibition of enhanced cellular proliferation and radiation resistance in vitro
The addition of heparin or simvastatin blocked the oncogenic effects of the radiation- derived exosomes. Recipient cellular proliferation (Fig. 3-3A-D) and radiation resistance (Fig. 3-
3E-H) were both inhibited. The attempt to block exosome uptake with an antibody to the tetraspanin protein CD81 was unsuccessful, corroborating previous studies30. To determine if these functional effects were due to decreased uptake of exosomes we performed microscopic analysis of exosome uptake. The radiation-derived exosomes (Fig. 3-3J) are internalized more readily by recipient cells than their non-radiation derived counter parts (Fig. 3-3I). Simvastatin and Heparin decreased the uptake of exosomes consistent with the effect of these compounds on the functional measures (Fig. 3-3L, M).
100
Fig. 3-3. Exosome blockade analysis. We demonstrate that the previous increases in cellular proliferation (Fig.3-2 A-F) and radiation resistance (Fig.3-2 G-L) following exposure to radiation derived exosomes can be inhibited through the use of heparin (Hep) and simvastatin
(SMV) but not to CD81 antibody to (Ab) in U87 and STS26T cell lines. Panels A-D show Hep and SMV were able to decrease the proliferation caused by the radiation derived exosomes in
(A,B) U87 and (C,D) STS26T cells Panels E-H show Hep and SMV were able to decrease radiation resistance in (E,F) U87 and (G,H) STS26T cells. We microscopically examined uptake of exosomes labeled with green PKH67 fluorescence under the various conditions in U87 glioma cells. (I) exosomes from non-radiation cells show minimal uptake whereas (J) exosomes derived
101 from irradiated cells are taken up robustly (arrows on positive cells). (K) radiation derived exosomes plus anti-CD81 antibodies (L) radiation derived exosomes plus heparin (M) radiation derived exosomes plus simvastatin. Hep (Fig. 3-3L) and SMV (Fig. 3-3M) both decreased uptake of fluorescently labeled radiation derived exosomes in recipient naïve cancer cells when compared to fluorescently labeled radiation derived exosomes without treatment (Fig. 3J).
(*p<.05, **p<.01, ***p<.001 significant decrease compared to radiation derived exosomes alone without treatment)(# denotes that the significant increase caused by radiation derived exosomes seen previously in Fig. 2 is inhibited and not significant) (*** alone denotes significant decrease p<.001 in comparison to both radiation derived exosomes alone and control)
102 In Vivo Studies
Representative images of the mice and their tumors are shown with IVIS (Fig. 3-4A-E).
Though all seven groups started with similar average bioluminescent signals, there was increased radiation resistance and enhanced tumor burden in the mice treated with radiation-derived exosomes (Fig. 3-4F). This effect was abrogated with daily treatment of heparin or simvastatin
(*p<0.05). Survival was consistent with the in vivo imaging results. Mice treated with radiation- derived exosomes showed a decrease in survival and co-treatment with heparin or simvastatin conferred a survival advantage (Fig. 3-4G).
103
Figure 3-4. In Vivo analysis of radiation derived exosome effect and therapeutic blockade. Representative IVIS images of (A) Control (B) Non-radiation exosomes (C) Radiation derived exosomes, (D) Radiation derived exosomes plus daily heparin, (E) Radiation derived exosomes plus daily simvastatin. Mice treated with radiation derived exosomes had visually larger tumors when compared to control. When treating mice with radiation derived exosomes plus heparin or simvastatin, the tumor size was similar to control levels. (F) Tumor progression over time was quantified with IVIS counts. Mice treated with radiation derived exosomes had an increase in tumor progression, and when treated with heparin or simvastatin tumor progression was similar to baseline (*p<0.05) (L) Mice treated with radiation derived exosomes had a decrease in survival time but when co-treating with heparin (Hep) or simvastatin (SMV) the mouse survival increased. (n=6-7 mice per group)
104 Immunohistochemistry of Tumor Samples
Immunohistochemical analysis of tumor tissue for markers of tumor growth, proliferation, and apoptosis was performed (Figure 3-5A-C). H&E staining of tumor tissues showed increased amount of necrosis in the control saline treated tumors, when compared to tumors treated with radiation-derived exosomes. This phenotype reverted back to control with co- treatment of heparin or simvastatin (Fig. 3-5A). Ki-67 cellular proliferation marker analysis showed less proliferation in the control tumors compared to tumors treated with non-radiation and radiation-derived exosomes. The amount of Ki67 staining was similar to control in the tumors co- treated with radiation-derived exosomes and heparin or simvastatin (Fig. 3-5B). Cleaved caspase
3 marker for cell death increased in control tumors, to a lesser extent in the tumors treated with non-radiation derived exosomes, and even less in the tumors treated with radiation-derived exosomes. (Fig. 3-5C). Adding heparin and statin therapy to the tumors treated with the radiation- derived exosomes caused those tumors to have increased cell death (Fig. 3-5C).
105
Figure 3-5. Immunohistochemistry of glioblastoma tumor samples from each group.
(A) H & E staining revealed increased necrotic tissue in the control saline treated tumors when compared to the radiation-derived exosome treated tumors. (B) Ki-67 cellular proliferation marker analysis showed decreased proliferation in the control tumors when compared to the radiation-derived exosome treated tumors. (C) Cleaved caspase 3 marker for cell death increased in control tumors when compared to tumors treated with radiation derived exosomes. All of the effects associated with radiation-derived exosomes seen by immunohistochemical analysis were not present in tissue from tumors co-treated with heparin or simvastatin. The tumors from the heparin and simvastatin treated animals appeared similar to controls.
106 Analysis of RNA and proteomic contents within exosomes
A total of 516 miRNAs were found within the exosomes. Heat maps generated show differential miRNA profiles based upon the dose of radiation (Fig. 3-6A). Fig. 3-6B shows the 4 miRNAs that were identified as statistically significantly changed (p<0.05) and includes miR-
516, miR-365, miR-889, and miR-5588. Moreover, it is noteworthy that the tumor suppressive miRNAs (miR-516 and miR-365) decrease when exposed to increasing radiation stress, while the oncogenic miR-889 increases when exposed to increasing radiation stress (Fig. 3-6B).
107
Figure 3-6. Analysis and comparison of miRNA contents within the non-radiation and radiation derived glioma exosomes. (A) Distinct heat map profiles were generated for exosomes derived from cells exposed to 0Gy (control glioma exosomes), 3Gy (low radiation), and 12Gy (high radiation). A total of 516 miRNA were dysregulated following irradiation (B)
Table showing the 4 statistically significantly dysregulated exosomal miRNAs following irradiation. The oncogenic miRNAs and tumor suppressive miRNAs were up and down regulated, respectively.
108 A total of 59 mRNAs were statistically significantly dysregulated in response to increasing radiation. Heat map profiles show differential expression based upon radiation dosage
(Fig. 3-7A). Oncogenic mRNAs significantly (p<0.05) upregulated following irradiation included
Nucleophosmin 1 (NPM1), Actin Gamma 1 (ACTG1), Vesicle Associated Membrane Protein 8
(VAMP8), Ribosomal Protein L15 (RPL15), fucosyltransferase 11 (FUT11), Zinc Finger RNA
Binding Protein (ZFR), Cyclin D1 (CCND1), Annexin A2 (ANXA2), Stearoyl-CoA desaturase
(SCD), Dynamin 2 (DNM2), Derlin 1 (DERL1), mitoNEET (CISD1), Kibra (WWC1), and
Peptidylprolyl Isomerase C (PPIC). Tumor-suppressive mRNAs found to be significantly downregulated following irradiation include Tropomyosin 1 (TPM1), LRR Binding FLII
Interacting Protein 1 (LRRFIP1), Tetraspanin 5 (TSPAN5), Signal Transducer And Activator Of
Transcription 4 (STAT4), CGG Triplet Repeat Binding Protein 1 (CGGBP1)54–58. The most highly represented molecular and cellular function pathways in the radiation-derived exosomes include cellular assembly and organization, cell morphology, cellular development, cellular growth and proliferation, and cell cycle (Fig. 3-7B). The most represented networks in the radiation-derived exosomes include cell cycle, cancer, cell death and survival, and organismal injury (Fig. 3-7C). The most represented upstream regulator was the MYCN oncogene (Fig. 3-
7D).
109
Figure 3-7. Ingenuity Pathway Analysis and comparison of mRNA contents within the non-radiation and radiation derived glioma exosomes. (A) The change in expression levels of 59 mRNA were identified following irradiation. Distinct heat map profiles were generated for exosomes derived from cells exposed to 0Gy (control glioma exosomes), 3Gy (low radiation), and 12Gy (high radiation). mRNA that have been demonstrated to have oncogenic or tumor suppressive functionality are highlighted with a red box. There is clearly a dose response to the patterns of expression. (B) Molecular and cellular function pathways most highly represented in the radiation derived exosomes (C) The mRNA networks most represented in the radiation derived exosomes
110 Over 1000 proteins were found within the glioma exosomes, 50 of which were unique to the 3Gy-derived exosomes, 92 which were unique to the 12Gy-derived exosomes, and 195 that were in both radiation dose-derived exosomes but not found in to non-radiation derived exosomes (Fig. 3-8A). iPathwayGuide analysis revealed 4 significantly (p<0.05) dysregulated biological pathways (Fig. 3-8B) included the Proteasome pathway, the Notch signaling pathway, the Jak-STAT signaling pathway, and the cell cycle pathway (Fig. 3-8C-F). The proteasome alpha and beta subunits were both upregulated within the radiation-derived exosomes in comparison to their non-radiation derived counterparts (Fig. 3-8C). iPathwayGuide analysis also revealed an upregulation of the oncoproteins STAT3, Notch1/2, Cullin1, Transforming Growth Factor-Beta 2
(TGF-B2), and cAMP-response element binding protein (CREBBP) (Fig. 3-8D-F).
111
Figure 3-8. Analysis and comparison of protein contents within the non-radiation and radiation derived glioma exosomes. (A) 50 proteins were unique to the 3Gy derived exosomes, 92 were unique to the 12Gy derived exosomes, and 195 were overlapping in both radiation dose derived exosomes in comparison to non-radiation derived exosomes (B) Table showing the 4 most statistically significantly represented protein profiles in the exosomes following radiation were (C) Proteasome pathway proteins (D) Notch signaling pathway proteins
(E) Jak-STAT pathways proteins (F) Cell cycle pathway proteins. All of the proteins upregulated in the exosomes are known to be associated with increased resistance to radiation and increased cellular proliferation.
112 3.5. Discussion
Exosomes are instrumental in a cancer cell’s interaction with its microenvironment. In the present study, we explored whether the stress of radiation alters the dynamics of exosomes released from multiple cancer cell types. We provide evidence that exposure to radiation treatment results in dose dependent increased secretion of exosomes and that these radiation derived exosomes have upregulated oncogenic and downregulated tumor-suppressive contents.
We further show that these radiation-derived exosomes alter recipient cancer cells in vitro and in vivo by increasing cellular proliferation, radiation resistance, and tumor burden, and that these effects can be abrogated, in part, via blockade of exosome uptake with heparin and simvastatin.
Three properties are analyzed: 1) exosome function, 2) exosome profile, and 3) exosome uptake/blockade.
In the first aspect of this study, we demonstrate that radiation-derived exosomes have an oncogenic impact on recipient cancer cells. Exosomes derived from cancer cells stressed with radiation cause a subsequent increase in cellular proliferation and radiation resistance in naïve recipient cells.
The functional impact of the exosomes on naïve recipient cells suggests there are alterations in exosome composition due to radiation. Thus, in the second aspect of this study, we assessed the changes in exosomal contents due to radiation. We show that the radiation-derived exosomes have upregulation of oncogenic and downregulation of tumor suppressive miRNA, mRNA, and protein. Multiple RNA species changed as a result of radiation. Downregulation of miR-516, a tumor suppressive miRNA, decreases metastasis by decreasing sulfatase-1 expression leading to a decrease in the Wnt-Beta catenin pathway59. Downregulation of miR-365, a tumor suppressive miRNA, increases cancer cell proliferation, therapeutic resistance, and decreases apoptosis by disinhibiting expression of Cyclin-D1, BCL-2, and PI3K, while decreasing expression of PTEN60–63. Upregulation of miR-889, an oncogenic miRNA, increases cancer cell
113 proliferation, radiation resistance, metastasis, and decreases apoptosis by inhibiting DAB2IP expression64–66. The upregulated oncogenic mRNA found have a functional influence on cancer cells by increasing cellular proliferation (e.g. CCND1), radiation resistance (e.g. WWC1), and exosome uptake (e.g. DNM2)67–80. The downregulated tumor-suppressive mRNA found have a functional influence on cancer cells by decreasing cellular proliferation (e.g. STAT4) and decreasing radiation resistance (e.g. TPM1). Upregulated oncogenic contents predominated over the downregulasted tumor suppressive contents. IPA analysis demonstrated the most highly represented upstream regulator was the MYCN onogene. MYCN has numerous effects in cancer including enhancing tumor progression81 and radiation resistance82, and MYCN inhibition leads to decreased tumor burden and increased survival81.
Based on our findings we propose that one factor underlying the mechanism of acquired radiation resistance may thus be as follows: radiation exposure causes the release of exosomes that have an increase in oncogenic and decrease in tumor-suppressive cargo (Fig 3-9A).
Subsequently, neighboring cancer cells internalizing these re-programmed radiation-derived exosomes are activated/disinhibited. The recipient cell receives more oncogenic RNA (e.g. miR-
889, Cyclin D1, Annexin A2) and less tumor-suppressive RNA (e.g. miR-516, miR-365, TPM1), which then act in that recipient cell to increase its proliferation and resistance to radiation. The recipient cell also receives more oncogenic proteins involved in the proteasome pathway, Notch pathway, Jak-STAT signaling pathway, and cell cycle pathway. Upregulation of the proteasome pathway has been implicated in glioma aggressiveness and radiation resistance, and proteasome inhibitors are being developed for cancer treatment83,84. STAT3, Notch1/2, Cullin1, TGF-B2, and
CREBBP mediate tumor cell proliferation and therapeutic resistance83–90, and inhibition of the
Notch and TGF families sensitizes cancer cells to radiation therapy88,91. The RNA and protein data show that when a cancer cell is stressed with radiation it secretes exosomes with upregulated oncogenic and downregulated tumor-suppressive cargo. This re-programmed cargo is
114 communicated via exosomes and internalized by recipient cells, which subsequently positions the cells to be more resistant to radiation treatment.
In the third aspect of this study, we assess exosome uptake with regards to radiation.
Radiation increased DNM2 in the secreted radiation-derived exosomes. DNM2 is critical for exosome uptake, and knockdown of DNM2 decreases exosome internalization76. Upregulation of
DNM2 is consistent with the increased uptake of radiation-derived exosomes seen when compared to control. The combination of our uptake data and the composition data indicate that a compounding effect occurs that results in the recipient cells not only internalizing increased amounts of radiation-derived exosomes, but these radiation-derived exosomes are also re- programmed to contain and transfer increased oncogenic and decreased tumor-suppressive cargo.
Moreover, radiation increased exosome release in a dose-dependent manner. This increase was quantified based upon protein through BCA analysis, as well as with vesicle number based upon nanoparticle tracking analysis. The fold increases in exosome secretion due to radiation were similar in both assays: 3Gy (two-fold increase) and 12Gy (three-fold increase), suggesting that quantification of exosomes through BCA and NTA may be similar.
In a further effort to understand exosome uptake mechanisms we hypothesized that treatment with heparin or simvastatin would block uptake of exosomes. Exosomes are taken up by multitude of mechanisms. The first being through proteoglycans; a family of proteins used by virus particles for internalization into recipient cells. This proteoglycan mediated mechanism is thought to be critical for exosome uptake as well, and the use of Heparin, a proteoglycan substituted with glycosaminoglycans, may inhibit this recipient cell-exosome interaction and exosome internalization. Lipid raft mediated internalization is also believed to be an important method of exosome uptake, and the use of statins to decrease cellular production of cholesterol and lipids to decrease exosome uptake is being investigated. Simvastatin is of particular interest for CNS malignancies due to its hydrophobicity and thus increased uptake into the brain through
115 the blood brain barrier. Heparin and simvastatin were effective in inhibiting the effects of radiation-derived exosomes in recipient cells in vitro in cell culture and in vivo in a murine model of glioblastoma (Fig. 3-9B). Although off-target effects of these medications are possible, the fluorescent uptake data suggest that these treatments are can directly decrease exosome uptake.
Heparin and simvastatin have been shown to decrease cancer cell metastasis and tumor cell proliferation, respectively37,39,50,92. The mechanism behind these effects may be mediated through exosome inhibition. Both therapeutics have minimal side effect profiles and further studies must be performed with these agents to elucidate their full potential in this context. Attempted blockade of exosome uptake with an antibody to the tetraspanin protein CD81 was unsuccessful, corroborating previous studies30. The blockage of uptake data suggests exosome uptake, which is proteoglycan-mediated, and lipid raft-mediated may be more critical than tetraspanin-mediated uptake.
116
Figure 3-9. (A) Proposed model for the mechanism of exosome induced acquired resistance in cancer (B) Proposed model for the therapeutic blockade of exosome uptake
117 In the present study we utilized the U87 glioblastoma, STS26T malignant peripheral nerve sheath tumor, and SH-SY5Y neuroblastoma cell lines. However, in vivo, the tumor is highly heterogeneous and includes cancer stem cells (CSCs). When a tumor is treated with radiation, the non-stem cancer cells in the periphery may be stressed with sublethal doses of radiation causing them to secrete increasingly oncogenic exosomes. Furthermore, the cancer stem cells (CSCs) within the tumor are also exposed to radiation therapy. CSCs are inherently more radioresistant than normal cancer cells, and thus these CSCs may release exosomes that are able to transfer therapeutic resistance functionality to recipient cells. The combination of exosomes secreted by both cancer cells that survive radiation exposure in the margins of the tumor and
CSCs may provide recipient tumor cells the enhanced cellular proliferation and acquired radiation resistance, leading to increased tumor burden.
In conclusion, we interrogated three aspects of radiation effects on exosomes including exosome function, exosome profile, and exosome uptake/blockade. We provide a novel exosome- based mechanism that may underlie acquired radiation resistance in patients harboring malignant cancers. Furthermore, we elucidate key factors carried by exosomes that may lead to tumor recurrence and subsequent therapeutic resistance. We also show the potential for advancement of cancer treatment, or understanding existing treatments, whose mechanism may be through exosome inhibition.
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129 Chapter 4
The Impact of Glioma Cancer Cell Stemness on Exosome Phenotype
4.1. Abstract
Glioblastoma is the most common central nervous system malignancy in adults with a devastating median survival of only 14 months. The current arsenal of treatment modalities including surgical resection and combination chemoradiation have been largely ineffective. One reason proposed for the ineffectiveness of our current therapeutic regimen of glioblastoma is glioma stem cells (GSCs). We investigated the hypothesis that the communication of GSCs to their microenvironment through exosomes is a key factor to the enhanced cellular proliferation and the development of resistance to therapeutics. Exosomes are nanometer sized vesicles released by cancer cells that contain DNA, RNA, and protein critical to the interaction of a cell with its microenvironment. Two properties of exosomes were analyzed: 1) exosome function and
2) exosome profile. Exosomes secreted by patient derived-glioma stem cells (GSC-exosomes) increased cellular proliferation, radiation resistance, temozolomide resistance, and doxorubicin resistance. We further profiled the GSC-exosomes to begin to probe the underlying mechanism of this phenomenon. Profiling showed specific changes to protein favoring therapeutic resistance and cellular proliferation. GSC exosomes have increased expression of proteins involved in radiation and chemotherapeutic resistance (E.g. CDK4 and Notch), cellular proliferation (E.g.
Cyclin B1 and Cyclin D2), angiogenesis (E.g. VEGF-A and EGFR), glioma cell stemness and de- differentiation (E.g. EPHA2, Cathepsin B), and cell invasion and metastasis (E.g. ITGA3,
COL4A2) compared to non-stem cell derived exosomes. The results of our study provide a novel exosome-based mechanism that may underlie the aggressiveness of glioma cancer stem cells.
130 Furthermore, we elucidate key factors carried by glioma stem cell derived exosomes that may lead to enhanced therapeutic resistance and increase in tumor burden.
4.2. Introduction
Glioblastoma is the most common central nervous system malignancy in adults with a devastating median survival of only 14 months1. The current arsenal of treatment modalities including surgical resection and a combination of chemotherapy and radiation, both of which have been largely unsuccessful. One reason proposed for the ineffectiveness of our current therapeutic regimen of glioblastoma is glioma stem cells (GSCs)2,3. Glioma stem cells, also known as glioma-initiating cells, display CD133 positivity4 and are thought to propagate tumor growth through tumor regeneration and self-renewal 5. GSCs also have inherent resistance to chemotherapy6 and radiation7, both hallmarks of glioblastoma. Due to the inherent therapeutic resistance of GSCs and their ability to repopulate the cells of the tumor, it has been suggested that targeting the glioma stem cells would lead to enhanced treatment efficacy. Thus, a critical component necessary to develop novel and effective therapeutic strategies, is understanding the mechanism underlying how GSCs promote cancer cell proliferation and therapeutic resistance.
We propose that the communication of GSCs to their microenvironment through exosomes is a key factor to the enhanced cellular proliferation and the development of resistance to therapeutics.
Exosomes are 20-200 nm cell derived vesicles which contain DNA, RNA, and protein, and increasing evidence demonstrates the importance of exosome communication in cancer8.
Exosomes transfer their contents to recipient cells in the tumor microenvironment enhancing numerous aspects critical to tumor progression9. Exosome have been demonstrated to enhance angiogenesis, metastasis, therapeutic resistance, and cancer cell proliferation10,11,12,13. However, the biology and dynamics of glioma stem cell-derived exosomes is not well understood. Glioma stem cell-derived exosomes have been shown to enhance angiogenesis14,15 and modulate a tumor suppressive phenotypes in T cells16, but their effects on cellular proliferation and therapeutic
131 resistance have not been explored. Due to the inherent therapeutic resistance and aggressive nature of GSCs, we hypothesize that communication of GSCs to recipient tumor cells in the local tumor milieu via exosomes to be integral in enhancing therapeutic resistance and increasing tumor burden in the tumor as a whole. We interrogate the effects of GSC exosomes on proliferation and therapeutic resistance. Furthermore, we perform RNA and protein profiling to highlight factors within the GSC exosomes that may be the cause of their demonstrated oncogenic effects.
4.3. Materials and Methods
4.3.1. Cell Culture and Exosome Isolation
Patient derived CD133 positive 3691 glioma stem cells (GSCs) and CD133 negative
3691 non-stem glioma cells (NSCs) were generously donated from Dr. Jeremy Rich, UC San
Diego. U87 glioma cells from ATCC were also used for functional analyses. All cell lines were maintained at 37˚C in a humidified incubator with 5% CO2. The U87 cell line was cultured in
Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum and 1% Penicillin-Streptomycin (Gibco). GSCs and NSCs were cultured in neurobasal medium supplemented with 2% B-27 (Gibco), 1% Pen strep (Gibco), 1% sodium pyruvate (Gibco), 1%
GlutaMax (Gibco), 0.004% fibroblast growth factor, and 0.004% endothelial growth factor.
Exosomes were isolated from both the GSCs and NSCs. The cells and media were centrifuged at
3000 X G for 3 minutes to pellet the cells. The media was then aspirated, transferred to a new tube, and centrifuged at 3000xG for 15 minutes to purify out cells and cellular debris. The resulting supernatant was incubated with Exo-Quick-TC exosome isolation polymer (System
Biosciences) for a minimum of 12 hours at 4°C. The media-ExoQuick combination was centrifuged at 1500xG for 30 minutes. The supernatant was aspirated and the purified exosome pellet was resuspended in 150-300μl dPBS (Gibco). Doxorubicin and Temozolomide were both purchased from Cayman Chemicals.
132 4.3.2. Exosome Size Analysis and Quantification
Size analysis and exosome distribution was performed with nanoparticle tracking analysis
(NTA)(NanoSight NS300). 10 μl of each exosome solution was resuspended in 1mL of dPBS and subsequently placed into the NTA and read. Exosomes were quantified using a BCA Assay
(Thermo-Scientific) or with nanoparticle tracking analysis. Exosomes were visualized by NTA.
4.3.3. Immunoblot Analysis
Protein expression was determined with immunoblot analysis. Exosome concentration was determined using BCA assay (Thermo-Scientific). 5μg of exosomes was transferred to a nitrocellulose membrane. The membrane was then blocked with 5% milk in TBS-T for 1 hour.
Membranes were incubated overnight at 4°C with primary antibodies for CD81 (1:200 sc-
166029), CD63 (1:200 Ab134045), tsg101 (1:200 sc-7964). The membranes were then washed thrice for 5 minutes each with 1X TBST. Secondary antibodies (1:5,000) were incubated for 1 hour and the blots were again washed thrice for 3 minutes with 1X TBST. The blots were then imaged with GE Amersham Imager 600.
4.3.4. Cellular Proliferation Analysis
5 X 10^3 CD133 negative non-stem glioma cells (NSCs) or 4 X 10^3 U87 cells were plated on 96 well plates and allowed to adhere overnight. The following day, these cells were incubated with PBS control, CD133 positive glioma stem cell (GSC) derived exosomes, or
CD133 negative non-stem glioma cell (NSC) derived exosomes individually, in triplicate. These cells were allowed to proliferate for 48 hours and analysis of cell proliferation was performed using an MTS-PMS Assay (Promega) according to manufacturer’s protocol. Naïve cells were incubated with exosomes at a concentration of 30μg/mL. Data is expressed as a ratio of naïve cells exposed to control.
4.3.5. Apoptosis Assay
133 5 X 10^3 CD133 negative non-stem glioma cell (NSC) or 4 X 10^3 U87 cells were plated on 96 well plates and allowed to adhere overnight. The following day, these cells were incubated with PBS control vehicle, CD133 positive glioma stem cell (GSC) derived exosomes, or CD133 negative non-stem glioma cell (NSC) derived exosomes individually, in triplicate. After a 24 hour incubation, 96 well plates were radiated with 12Gy, or incubated with 2uM doxorubicin, or incubated with 500uM temozolomide. 24 hours after radiation/doxorubicin/temozolomide exposure, analysis of cell death was performed using an MTS-PMS Assay (Promega) according to manufacturer’s protocol. Naïve cells were incubated with exosomes at a concentration of
30μg/mL. Data is expressed as a ratio of naïve cells exposed to control.
4.3.6. Proteomic Analysis
Exosome protein analysis was performed utilizing the Tandem Mass Tag (TMT)-10plex kit according to manufacturer’s protocol (Thermofischer Scientific). This kit provides multiplexed protein identification and quantitative analysis by tandem mass spectrometry.
Briefly, exosomal proteins were normalized to 25ug of exosome proteins in 40uL of PBS for each of the groups (GSC-exosomes and NSC-exosomes). The exosomal proteins were incubated with lysis buffer and then centrifuged at 16,000 X G for 10 minutes. The supernatant was then mixed with 100mM TEAB, 200mM TCEP, for one hour, and subsequently 5µL of the 375mM iodoacetamide for 30 minutes. Acetone was then added and allowed to precipitate overnight. The samples were centrifuged at 8000 X G for 10 minutes and the pellets were then resuspended in
100mM TEAB. Trypsin storage solution and trypsin were added to the samples and digested overnight at 37˚C. These samples were then labeled with the TMT label reagents with 1 hour incubation at which point 5% hydroxylamine was then added to quench the reaction. The samples were then run on the Orbitrap Velos Mass Spectrometer (Thermofischer Scientific) and the data was subsequently processed using Proteome Discoverer (version 2.1). The search engine is SequestHT. PSMs are filtered using Percolator. Proteome Discoverer uses its own algorithm
134 for protein grouping. Samples were searched against human database and filtered to retain proteins/peptides with <1% FDR. Adavita iPathwayGuide (Advaita Bioinformatics) Next-gen pathway analysis software with the Impact Analysis method was then used to identify dysregulated protein relationships and their effects in various functional contexts, such as subcellular location, gene ontology, association with drugs, pathways, and disease relevance.
4.3.7. Statistical Analysis
All of the data generated in the proposed experiments were subjected to statistical analysis. GraphPad Prism 4.03 (GraphPad Software, San Diego, CA) was used for statistical analysis unless otherwise noted in previous methods. At least 3 replicates were performed unless otherwise noted and groups were analyzed using one way ANOVA with Tukey-Kramer posttest.
A p value <0.05 was deemed significant.
4.4. Results
4.4.1 Characterization of exosomes
Figure 1 shows that exosomes could be purified from both the GSCs and NSCs. No significant differences in exosome diameter between the two groups were noted (Fig.1A,B). GSC exosomes had a diameter of 142nm while the NSC exosomes had a diameter of 130nm
(Fig.1A,B). Common exosome markers include the tetraspanin family of proteins, which include
CD81 and CD63, as well as TSG101. These exosome proteins are expressed by both the GSC- exosomes and NSC-exosomes (Fig.1C). Although no changes in exosome diameter was found, we did note a trending increase in exosomal release per cell from the stem cells when compared to non-stem cells. This data is shown by the ug of exosomal protein released and nanoparticle tracking analysis with the number of exosomes released (Fig.1D,E). Exosomes released from glioma cells were also confirmed with NTA visualization (Fig.1F).
135
Figure 4-1. Exosome confirmation analysis in stem and non-stem glioma cells.
Nanoparticle tracking analysis in Panels A and B demonstrate that size of the exosomes was not affected by stemness. (A) GSC exosomes had a diameter of 142nm while the (B) NSC exosomes had a diameter of 130nm. Panel C demonstrates exosome release from stem and non-stem glioma cells with immunoblots of exosome confirmation markers CD81, CD63, and TSG101. Exosomes were then quantified with (D) BCA assay and (E) nanoparticle tracking analysis and there was a trend of increased release of the GSC-exosomes (F) Visualization of glioma stem cell derived exosomes by Nanoparticle tracking analysis
136
4.4.2. Impact of cancer cell stemness on exosome functionality in vitro
Incubation of naïve non-stem 3691 cancer cells with exosomes purified from GSCs resulted in a significant increase in cellular proliferation, cell survival after radiation exposure, and temozolomide resistance in the NSCs compared to NSCs incubated with non-stem glioma exosomes (Fig.2A-C). Naïve NSCs incubated with exosomes from GSCs had a significant increase in resistance to doxorubicin when compared to control vehicle (Fig.2D). Naïve non-stem
U87 cancer cells incubated with exosomes purified from GSCs had a significant increase in cellular proliferation and cell survival after radiation exposure when compared to U87 cells incubated with non-stem glioma exosomes (Fig.2E,F). Naïve U87 cells incubated with GSCs had a significant increase in resistance to temozolomide and doxorubicin when compared to control vehicle (Fig.2G,H).
137
Figure 4-2. Cellular proliferation and therapeutic resistance effects of glioma stem cell derived exosomes. The effect of the glioma stem cell derived exosomes (GSCs) and glioma non-stem cell derived exosomes (NSCs) on naïve non-stem PDx glioma cells is shown in Panels
A-D. GSC-exosome effects on (A) cellular proliferation, cellular survival after exposure to (B) radiation, (C) temozolomide, and (D) doxorubicin. The effect of the glioma stem cell derived exosomes (GSCs) and glioma non-stem cell derived exosomes (NSCs) on naïve non-stem U87 glioma cells is shown in Panels E-H. GSC-exosome effects on (E) cellular proliferation, cellular survival after exposure to (F) radiation, (G) temozolomide, and (H) doxorubicin. The results shown increased proliferation and increased resistance to therapeutics when cells are exposed to exosomes from glioma stem cells. (*p<0.05, **p<0.01, ***p<0.001, in comparison to non-stem cell derived exosomes)(#p<0.05, ##p<0.01, in comparison to control vehicle)
138
4.4.3. Analysis of protein contents within exosomes
4048 proteins were found within the glioma stem and non-stem cell derived exosomes;
616 of which were differentially dysregulated based on stemness (Fig 3A). iPathwayGuide analysis revealed 4 significantly (p<0.05) represented biological pathways included the extracellular matrix-signaling pathway, cell adhesion molecule pathway, pathways in cancer,
PI3K-Akt pathway, and the p53 signaling pathway(Fig.3B). Panels C-G show the levels of exosomal proteins categorized based on tumorigenic function that were differentially expressed significantly. Panel C shows proteins which have been implicated in therapeutic resistance. Panel
D shows proteins which have been implicated in angiogenesis. Panel E shows proteins which have been implicated in cellular proliferation. Panel F shows proteins which have been implicated in glioma cell stemness and de-differentiation. Panel G shows proteins which have been implicated in invasion and metastasis.
139
Figure 4-3. Analysis and comparison of protein contents within the stem and non- stem cell derived glioma exosomes. (A) Out of 4048 proteins found with the stem and non-stem glioma cell derived exosomes, 616 were differentially expressed. (B) Table showing significantly represented pathways. Panels C-G show the levels of exosomal proteins categorized based on tumorigenic function that were differentially expressed significantly. Panel C shows proteins in purple which have been implicated in therapeutic resistance including cyclin dependent kinase
4(CDK4), Notch 2, Notch 3, Integrin Beta 1(ITGB1), Neuronal-glial antigen 2(NG2), and Serine- threonine protein kinase 2(PKN2). Panel D shows proteins in red which have been implicated in angiogenesis including Vascular endothelial growth factor receptor-A (VEGF-A), matrix metalloproteinase 2(MMP2), and endothelial growth factor receptor (EGFR). Panel E shows proteins in blue which have been implicated in glioma cellular proliferation including Cyclin B1,
Cyclin D2, platelet derived growth factor receptor (PDGFR), EGF Containing Fibulin Like
140 Extracellular Matrix Protein 2 (EFEMP2), Coactosin Like F-Actin Binding Protein 1 (COTL1),
Frizzled 1, and Transferrin receptor 1(TfR1). Panel F shows proteins in grey which have been implicated in glioma cell stemness and dedifferentiation including Ephrin A2 (EphA2), Cathepsin
B, Thymosin Beta 4, and Versican core protein (VCAN). Panel G shows proteins in green which have been implicated in glioma invasion and metastasis including Integrin alpha 3 (ITGA3),
Collagen Type IV Alpha 2 Chain (COL4A2), CD151, and Tenascin-C.
141 4.5. Discussion
Exosomes have a critical impact on tumor progression in the tumor microenvironment as well as distant sites in the body. In the present study, we explored whether glioma cell stemness alters the dynamics of exosomes secreted by glioma cells, including function and profile. Two properties of the exosomes were analyzed: 1) exosome function, 2) protein profile. We provide evidence that glioma stem cell derived exosomes have upregulated oncogenic contents. We further show that these glioma stem cell derived exosomes alter recipient non-stem glioma cells in vitro by increasing cellular proliferation, therapeutic resistance to radiation, temozolomide, and doxorubicin to a greater extent than their non-stem cell derived exosome counterparts. The functional impact of the exosomes on naïve recipient cells suggests there are alterations in exosome composition due to parent cell stemness. Thus, we assessed the changes in exosomal contents due to stemness.
We show that the glioma stem cell-derived exosomes have upregulation of oncogenic and downregulation of tumor suppressive miRNA, mRNA, and protein. GSC exosomes have higher levels of proteins involved in radiation and chemotherapeutic resistance (E.g. CDK4 and
Notch)20–26, cellular proliferation (E.g. Cyclin B1 and Cyclin D2)27–34, angiogenesis (E.g. VEGF-
A and EGFR)35–37, cell stemness and de-differentiation (E.g. EPHA2, Cathepsin B)38–41, and cell invasion and metastasis (E.g. ITGA3, COL4A2)42–45. CDK4, Notch2/3, and other proteins in purple have been demonstrated to cause enhanced therapeutic resistance and knockdown of these proteins leads to therapeutic sensitivity. The increase in therapeutic resistance in our data may be due to the transfer of these proteins from the GSC exosomes to recipient cancer cells. The increase in EGFR and VEGF in the GSC-exosomes in comparison to the NSC-exosomes support previous studies by other groups suggesting that these angiogenic factors are critical components that exosomes transfer to recipient cells14,15. In addition to the previous work of other groups, our data shows MMP-2 to be another factor involved in the increased angiogenesis of GSC
142 exosomes. Furthermore, we show that not only are these angiogenic factors found with the GSC exosomes, but they are increased due to stemness of the parent glioma cell. Cyclin B1/D2,
PDGFR, and other proteins in blue have been demonstrated to be critical for cell cycle progression and cellular proliferation. These proteins are significantly upregulated in the GSC exosomes in comparison to the NSC exosomes and thus the increase in proliferation we have shown may be due to transfer of these factors from the GSC exosomes to the recipient cancer cells. Furthermore, the transfer of EPHA2, Cathepsin B, and other proteins in grey involved in glioma cell stemness and dedifferentiation from the glioma stem cell derived exosomes to the non-stem glioma cells may cause the non-stem cells to become more “stem-like” by becoming more aggressive and more resistant to therapies. Although we did not test invasion and migration, our data suggest that the GSC exosomes would confer enhanced invasion to recipient cells through the transfer of ITGA3, COL4A2, and other proteins in green.
Based on our findings we propose that one factor underlying glioma stem cell aggressiveness and a mechanism of enhanced tumorigenicity in glioma may thus be as follows: glioma stem cells release exosomes that have an increase in oncogenic cargo. Subsequently, cancer cells in the tumor microenvironment take up these glioma stem cell-derived exosomes and are activated (Fig. 4). These recipient tumor cells receive more oncogenic contents (e.g. CDK4,
Notch, Cyclin B1, VEGF-A, EPHA2, etc) which then act in those recipient cells to increase their proliferation, resistance to therapies, and make the cells more stem-like. In effect, this leads to tumor propagation and growth through tumor cell regeneration and self-renewal.
143
Figure. 4. Schematic representation of the proposed model for the mechanism of glioma stem cell derived exosome induced acquired tumorigenicity on recipient cells.
144 The data in figure 2 suggests that cells may have a tropic paracrine effect in the uptake of their own released exosomes. We show that the U87 cells seem to have less of a response to the
GSC and NSC exosomes in comparison to the NSCs taking up the GSC and NSC exosomes. The
U87 cell proliferation, resistance to radiation, doxorubicin, and temozolomide after incubation with GSC/NSC-derived exosomes is less than that seen when NSCs are incubated with
GSC/NSC-derived exosomes. This may be due to factors on or within the exosome that cause enhanced targeting and uptake into the cell of which it is derived. The U87 cell itself must also be factored into this as the cell may be less adept at internalizing exosomes. It also cannot be ruled out that the U87 cell is inherently less resistant to radiation and chemotherapy than the PDx cells.
Further studies are warranted to elucidate the mechanism that causes this enhanced tropism of the exosome for its parent cell. Our findings of enhanced proliferation and therapeutic resistance in not only the PDx glioma cells but even in the less than optimal U87 cells suggest that we are isolating the exosome effect.
In conclusion, we interrogated two properties of glioma stem cell derived exosomes including exosome function and exosome profile. We provide a novel exosome-based mechanism that may underlie the aggressiveness of glioma cancer stem cells. Furthermore, we elucidate key factors carried by glioma stem cell derived exosomes that may lead to enhanced therapeutic resistance and increase in tumor burden.
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151 Chapter 5
Overarching Themes of Exosomes in Cancer Biology
5.1. Introduction: Summary of Main Findings of Dissertation
My work has led to three overarching themes including exosome use for tumor profiling, the effects of stressors on exosome profile and function, and the potential for exosome blockade.
In my first chapter I review the significant potential of exosomes to influence tumor progression, biomarker development, and novel therapeutic avenues. In my second chapter I have found that germline changes in genotype can influence exosome impact on recipient cells1. Exosomes from
HFE mutant cells transfer tumorigenic properties to recipient cells and thus HFE genotype can significantly impact cancer outcomes, specifically with regards to exosomes1. In my third chapter
I have shown that exosomes provide a RNA and protein signature that can underlie radiation resistance. In my fourth chapter I demonstrate that glioma stem cells have a specific exosome profile and that these stem cell derived exosomes provide increased oncogenic functionality to recipient non-stem cancer cells. These points have led to my first theme: exosomes provide molecular information allowing us to profile the tumor and its characteristics, both at initial diagnosis and longitudinally over time. In my third chapter, I also demonstrate that exosomes respond to radiation stress by upregulating oncogenic contents that provide enhanced ability to combat radiation and survive its damaging effects. This has led to my second theme: a tumor responds to any stress by secreting exosomes that have upregulated contents useful to combat that specific stress and giving the tumor the ability to survive that stress. In my third chapter, I also demonstrate that the use of simvastatin and heparin to block exosome uptake may mitigate the radiation-induced oncogenic effects that exosomes have on recipient cancer cells. This finding has led to my third theme: there is a potential to use exosome blockade to inhibit the oncogenic
152 effects that exosomes provide to recipient tumor cells. By blocking this exosome effect, there may be lasting positive effects for cancer patients.
5.2. Theme One
5.2.1. Exosome Use for Molecular Profiling
Exosomes have the immense potential to be used as a biomarker for tumor molecular profiling2–6. In chapter two, I describe that germline changes in HFE genotype alter the exosome contents and subsequent exosome effects on recipient cells. I demonstrate that HFE mutations caused the release of exosomes that have increased ferrome proteins, increased VEGF, EGFR,
MMP9, and MDR1. All of these proteins have tumorigenic functions and demonstrate a malignant exosome profile. In chapter three, I interrogated whether exposing cancer cells to radiation treatment stress alters the exosome profile. I demonstrate that the radiation derived exosomes have dysregulated levels of miRNAs including miR-516, miR-365, miR-889, and miR-
5588. They also have dysregulated levels of mRNAs including NPM1, ACTG1, VAMP8, RPL15,
FUT11, ZFR, CCND1, ANXA2, SCD, DNM2, DERL1, CISD1, WWC1, and PPIC. This underlies the notion that exosomes can be utilized for profiling. The heat maps created from this data provides a visualization of the molecular tumor landscape at that specific point in time. This profiling is may not be limited to cases of radiation resistance, but also chemotherapeutic resistance as well7–10.
A major obstacle physicians face in cancer treatment is whether a patient’s specific tumor will respond to a specific therapy. To address this, physicians use a tumor biopsy and subsequently molecularly analyze and profile that piece of tumor tissue11–13. Adjuvant therapy of glioblastoma is currently being driven by histologic and molecular analysis of tissue obtained via surgical biopsy14,15. There are problems with a tumor biopsy, however, in that a biopsy only obtains one portion of tissue from one small area of a large and significantly heterogeneous
153 tumor. A biopsy thus only allows a physician to observe a miniscule surgical snapshot of the current molecular tumor dynamics. Due to the fact exosomes contain genetic factors from the parent tumor cell, and are secreted by all cells that make up the tumor, there is a potential to obtain the complete picture of the current tumor dynamics, and do so non-invasively through serum or CSF analysis16–18. Interestingly, multiple cases have demonstrated that actionable mutations such as EGFRvIII were found in the exosomes and not in a biopsy of the parent tumor themselves16. This finding suggests that exosomes have the potential to recapitulate the entire biology of the parent tumor in contrast to a surgical biopsy snapshot of only one portion of a heterogeneous tumor. This is critical to optimal patient care. For example, patients with glioblastoma that are found to harbor EGFR variants and utilize these variants as their driver mutation are more responsive anti-angiogenic treatments13,19,20. If a biopsy is not sensitive enough to pick up an EGFR variant and the patient is thus not started on anti-angiogenic therapy, they will not survive as long as if this mutation was found initially. The opposite is also true. It may not be the most effective to start a patient on a specific targeted therapy if the patient does not harbor that specific mutation because this would put the patient through arduous chemotherapy with multiple side effects without an effective tumor response.
5.2.2. Exosome Monitoring of Treatment Response
Another obstacle during patient care is determining whether a patient is still responding to their initial therapy weeks after being started on that treatment. Cancer is known to change and evolve over time, especially in response to a chemotherapy21–24. The tumor cells will actually evolve to become resistant to a therapy that at the start of the patient treatment, worked effectively. In the previous example, the tumor of a patient harboring an EGFR variant treated with anti-angiogenic therapy may evolve and rely on a new oncogenic driver. Subsequently, this patient begins to not respond to anti-angiogenic therapy, and imaging is not adequately sensitive to determine when a treatment has stopped working. Thus the patient stays on an ineffective
154 therapy for months until their tumor progresses to such a point that imaging is finally able to diagnose progression, at which time changing the patient’s therapy is not as effective. Taking repeat biopsies of a tumor is not possible due to morbidity, and even if it were possible, there is still the problem of tumor heterogeneity. Exosome profiles would provide the clinician with a less invasive and more global means of garnering the genetic and molecular profile the evolved tumor and monitor the evolving mutational tumor landscape25. This would provide the physician with real-time knowledge of the new driver mutation, allowing the physician to adapt the patient’s therapeutic regimen to target the new driver. This is especially critical in glioblastoma, when the subsequent weeks to months after treatment the differentiation of progression from pseudoprogression is unclear based solely on imaging. Exosome profiles may be able to accurately and non-invasively determine whether a patient is actually progressing or whether the imaging is showing pseudoprogression, inevitably leading to better outcomes.
The exosome heat map profiles that I have found in chapter three provide an initial backbone of what RNAs and proteins to analyze in patient samples with regards to initial treatment effectiveness and to monitor treatment response over time. For example, a patient with an exosome heat map profile that looks similar to the heat map of 0Gy exosomes would respond effectively to radiation therapy. In contrast, patients with exosome heat map profiles resembling
3Gy or 12Gy may not respond as well to radiation therapy. This may also be true for chemotherapy as different exosome profiles may correlate to chemotherapeutic sensitivities. To monitor treatment response over time, a patient would have an initial heat map profile generated from their exosomes before treatment, which would represent the exosome profile of the greatest tumor burden. Exosome samples will then be obtained serially after treatment with surgical resection, radiation, and chemotherapy. These longitudinal exosome profiles would be less malignant than the initial profile obtained, suggesting effective tumor response. If the longitudinal exosome heat map profile begins to resemble the initial malignant heat map of greatest tumor
155 burden, the clinician would be hinted at the fact that the current therapeutic regimen is failing and the patient’s tumor is progressing. The clinician should then start the patient on a new treatment strategy for optimal care.
Immunotherapy has been one of the fastest growing areas of cancer therapeutics but numerous obstacles have been encountered in immunotherapy clinical studies when assessing the benefit of these therapies11. When treating a patient with immunotherapy, the radiological changes may be delayed due to inflammation associated with therapy. The assessment of tumor progression and long-term survival is also complicated because even with development of new lesions, there may be clinical benefit. Exosomes may prove to be critical in the assessment of immunotherapy effectiveness and the evolution of the immunotherapy Response Assessment for
Neuro-Oncology (iRANO) criteria11. The changing of exosome burden as well as exosome heat map profile is primed to take a prominent role in determining whether immunotherapy is effective or not, in real time.
This ability to identify emerging therapeutic resistance in real time would provide the clinician with the knowledge of which second line therapy would be best for their patient. Such a methodology, which would allow clinicians to track treatment efficacy, differentiate tumor progression from treatment effect, and determine the patient’s overall tumor status, is highly desired. Exosomes may take a pivotal role in this effort and may offer tangible benefits in the management of these devastating diseases.
5.2.3. Exosomes to Diagnose and Predict Brain Metastases
Brain metastases are more common than primary CNS tumors and the ability to differentiate between these types of tumors is essential for optimal patient care. Differentiating primary from secondary tumors as well as differentiating from non-neoplastic processes such as intraparenchymal hemorrhage and inflammatory conditions adds further complexity to patient diagnosis. The diagnostic differentiation of primary glioblastoma from CNS lymphoma or
156 secondary metastasis due to lung, breast or melanoma, all of which radiographically may present similarly, is currently accomplished through biopsy. Exosomes may provide a non-invasive methodology to differentiate these CNS tumor types. B-cell lymphoma derived exosomes express
B-cell specific antigens, such as CD19 and CD2026,27. Metastatic breast cancer cell lines have been shown to secrete exosomes that contain upregulated miR210, downregulated miR19a and
29c, upregulated annexin II, and upregulated integrin beta 328–31. Exosomes derived from metastatic lung cancer cell lines expressed increased levels of TGF beta and IL-10. One may envision a patient being diagnosed with a brain tumor by imaging and subsequently, instead of biopsy, take an exosome profile. If the patient’s exosome profile has specific factors for breast or lung cancer, etc, then the tumor would not only be treated differently, but the clinicians may also be able to identify a primary tumor when they would not have previously due to small primary tumor size and insufficient resolution on imaging.
One major problem with utilizing exosomes as biomarkers is that all cells secrete exosomes, not only cancer cells, which thus leads to a significant number of noise in exosome profiles. The specific profiling of tumor cell derived exosomes may come in the form of utilizing cancer-specific markers, for example IL13Rα2, to purify the tumor cell derived exosomes from the exosomes secreted from all other cell types. IL13Rα2 is a decoy receptor for IL-13 that inhibits the downstream functionality of IL-13 and is selectively expressed on gliomas32–35. IL-13 conjugated quantum dots have been utilized to analyze exosomes from glioblastoma patients’
CSF and were found to have a unique pattern in comparison to non-targeted quantum dots36.
These findings prompt the further development of tumor-derived exosome purification methods and analysis of CSF or serum isolated exosomes to non-invasively differentiate primary glioblastoma from other CNS tumors.
Exosomes secreted from primary cancer cells have been shown to travel to distant sites in the body without degradation. These traveling exosomes may prepare a distant site for metastasis
157 and create a “pre-metastatic niche” by inhibiting immune function and increasing angiogenesis at that site37–40. This primes the area for circulating cancer cells to seed, subsequently proliferate, and create a metastatic tumor. The mechanism of exosome-induced metastasis may be able to be utilized to predict which patients develop secondary tumors in another location of their body. For example, a patient with breast cancer would have breast cancer derived exosomes in the CSF much in advance to an actual secondary metastatic tumor in the CNS. This provides an opportunity to predict tumor metastasis. If a breast cancer patient is found to have a CSF exosome profile that resembles breast cancer and resembles a metastatic profile, this would suggest that this patient would develop eventual brain metastasis. Some of the RNA and proteins that I have found in the radiation derived exosomes in chapter 3 are critical for invasion and migration.
These genetic factors may be used as a starting point for what exosome components to analyze in the metastatic exosome profile. This is powerful information that may lead to more frequent CNS imaging and thus earlier CNS metastatic tumor diagnosis before the patient develops any symptoms. The patient would then be treated much earlier than he/she otherwise would have, leading to significantly increased survival.
5.3. Theme Two
5.3.1. Effects of Chemotherapy on Exosome Composition and Function
In chapter three I demonstrated that radiation derived exosomes contain upregulated contents that act to aid in cancer cell survival through radiation resistance and cellular proliferation. Exosomes released by cancer cells after chemotherapy may also have similar downstream effects. This may be due to the cancer cells being exposed to chemotherapeutic stress, similar to the stress radiation exposure. The findings I have seen in chapter three suggest exosomes secreted from cancer cells stressed with chemotherapy would have upregulated contents that aid in chemotherapeutic resistance and increase cellular proliferation. Multi-drug
158 resistance 1 (MDR-1) is a possible candidate component within exosomes that may be upregulated in response to chemotherapeutic stress which would be transferred to recipient cancer cells41–43. Once transferred, MDR-1 would become functional in those recipient cancer cells and decrease their sensitivity to the chemotherapy. Future studies would interrogate this hypothesis by determining whether chemotherapy-induced exosomes released by cancer cells have upregulated oncogenic contents that are transferred to recipient cancer cells in the local environment, increasing therapeutic resistance and cancer malignancy.
5.3.2. Stress-Induced Exosomes and Their Effects on Immune Function
Exosomes derived from cancer cells have also been shown to affect immune function and lead to an immunosuppressed phenotype. Multiple studies have suggested that cancer cell derived exosomes may aid in facilitating tumor progression through immune suppression of the immunomodulatory cells in the CNS38–40,44. They may induce the M2 macrophage phenotype as well as diminished immune function over all in immunomodulatory cells. The radiation derived exosomes in chapter three had many oncogenic functions, and may also effect immune cells. An important study to perform would be to assess how the radiation derived exosomes effect immune cells to determine whether the stress of radiation leads to exosomes that aid in overall tumor progression by suppressing the immunomodulatory cells of the CNS. This same study can also be performed with exosomes derived from cancer cells after exposed to the stress of chemotherapeutics. We predict that the cancer cell derived exosomes, especially when derived from stressed cells, would lead to a significantly immunosuppressed phenotype. Macrophages would be in their M2 phenotypes and T cells may also become less active.
5.3.3. Stress-Induced Exosomes and Their Effects on Invasion and Migration
Exosomes have also been shown to enhance the invasion and migration of recipient cancer cells. In chapter three I found the radiation derived exosomes to have upregulated contents important for invasion and migration, including annexin A2, cyclin D1, and others. Further
159 studies could be performed with the radiation derived exosomes to determine whether the stress of radiation produces exosomes that transfer enhanced invasive functionality to recipient cancer cells. To test this, in vitro invasion analysis with transwell plates could be used. The transwells could be plated with cancer cells and incubated with radiation or non-radiation derived exosomes, subsequent invasion could be analyzed. This could be also done in vivo by injecting metastatic cancer cells into the tail vein of mice along with non-radiation or radiation derived exosomes. The mice treated with radiation derived exosomes and cancer cells may have an increased tumor burden when compared to control.
5.3.4. Radiation Treatment Margins and Leading Edge Radiotherapy
The overall concept of chapter three was that when cancer cells are exposed to radiation they release exosomes that subsequently provide acquired resistance to recipient cancer cells in the environment. This mechanism may be integral for inherently radiation resistant cancer cells, as well as cancer cells at the border of the radiation treatment field. The cells at the border of the treatment field may be exposed to sub-lethal dosages of radiation that instead of causing cell death, may act as a stressor causing the release of oncogenic exosomes and the malignant functional effect that follows (Figure 1). Indeed, a recent study by Duma et al. demonstrated that utilizing a technique coined “Leading edge radiation” and expanding the radiation treatment margins led to significantly better outcomes in glioblastoma patients when compared to standard radiation protocols45. This suggests that by increasing the dosage of radiation the cancer cells at the border of the radiation field are exposed to through the increase in treatment margins may limit the secretion of these oncogenic-resistance enhancing exosomes. Further clinical trials utilizing “leading edge radiation” are necessary to accurately determine its clinical benefit.
160
Figure 5-1. Schematic representation of the mechanism of acquired therapeutic resistance in cancer. Cancer stem cells may be stressed with radiation or chemotherapy and subsequently release increased amounts of highly oncogenic exosomes that then cause enhanced oncogenicity in recipient cancer cells. Non-stem cancer cells on the periphery of the radiation treatment field may also be stressed with sub-lethal doses of radiation treatment which subsequently causes those cells to release increased oncogenic exosomes that are taken up by recipient cells leading to a similar effect as described by the cancer stem cells.
161 This is compounded with the effect cancer stem cells may have on this tumor microenvironment. Cancer stem cells are known to be increasingly resistant to radiation and chemotherapeutic treatment46,47. This suggests the cancer stem cells may also be able to release exosomes that cause increased therapeutic resistance and cellular proliferation to recipient cancer cells in the tumor microenvironment (Figure 1).
This data is shown in chapter 4 of my thesis which demonstrates that glioma stem cell derived exosomes do in fact have a unique signature in comparison to non-stem glioma cell derived exosomes. This data suggests that the renewal and replenishment properties of cancer stem cells as well as their increased tumorigenicity may be in part due to the exosomes the stem cells secrete. These effects may be further compounded when a cancer stem cell is stressed with radiation or chemotherapy. It may respond to an even greater extent than what I have shown in chapter 3 with the non-stem glioma cells.
My work in chapter 4 demonstrates glioma stem cell derived exosomes provide increased cellular proliferation, radiation, and chemotherapeutic resistance. The protein profiling data show that in addition to having increased levels of proteins involved in proliferation and therapeutic resistance, there are increased levels of proteins important for angiogenesis, invasion, and cancer cell stemness. Studying the effects of the cancer stem cells on human umbilical vein endothelial cells as I have performed in chapter 2 of my thesis, would be critical to determine whether the increased levels of angiogenic proteins in the glioma stem cell exosomes can be transferred and become functional. Also, it would be important to determine whether the glioma stem cell derived exosomes can transfer the invasion proteins leading to increased invasion in recipient glioma cells.
Further interesting work to explore whether the glioma stem cell derived exosomes can cause non-stem cells to de-differentiate and become more “stem-like” would be critical to interrogate the full extent of the stem cell derived exosomes. An experiment to determine whether
162 CD133 negative non-stem glioma cells which are exposed to glioma stem cell derived exosomes begin to display increased CD133 positivity, thus becoming more “stem-like”, would begin to answer this question.
5.4. Theme Three
5.4.1. Exosomes Blockade as a Novel Therapeutic Strategy
Exosomes are taken up by a multitude of mechanisms. My work in chapter three elucidated how the use of simvastatin and heparin may be used to block the proteoglycan mediated and lipid raft mediated methods of exosome uptake. Furthermore, this blockade was able to significantly decrease the enhanced radiation resistance and cellular proliferation that was caused by the radiation derived exosomes both in vitro and in vivo. This work is a critical stepping-stone for the identification of exosome blocking agents to inhibit the acquisition of radiation resistance and enhance therapeutic efficacy in patients harboring malignant cancers. The in vivo data in chapter three suggests that heparin and simvastatin may work to decrease tumor burden in cancer patients being treated with radiation therapy. Thus, translating this data from bench to bedside would also be an important step to helping cancer patients who are not responding well to radiation therapy. As heparin and simvastatin are both fairly innocuous drugs already used in many patients, performing a clinical trial on glioblastoma patients undergoing radiation therapy with these therapies may be of great clinical benefit. Glioblastoma patients would take heparin and simvastatin one week before radiation therapy, and then continue the heparin and simvastatin up until one week after their last day of radiation. By doing this, the exosome blocking agents may aid in decreasing the acquired radiation resistance that inevitably develops in cancer patients, thus making radiation therapy more effective.
In theme two I describe interrogating the effects of radiation and chemotherapeutic stress on invasion and immune function. As stated previously, exosomes can travel to distant sites in the
163 body and create a metastatic niche as well as decrease functions of the immunomodulatory cells of the human body. It would thus be logical to determine whether exosome uptake blockade with heparin or simvastatin would be able to inhibit these oncogenic sequelae of stress-induced exosomes on recipient cancer cells, both locally on immunosuppression, and at distant sites by decreasing metastasis. Similar studies as outlined in theme 2 could be performed with the added exosome blockade groups to determine whether exosome blockade could inhibit the potential immunosuppression and enhanced invasion caused by stress-induced exosomes.
The Rab GTPase family of proteins has been shown to be critical for exosome creation and exosome release48–50. Without the Rab GTPases, especially Rab 27, exosomes would be shuttled to the lysosome for degradation rather than out of the cell for export. Some studies have begun to analyze the efficacy of utilizing siRNAs for the Rab GTPase family to decrease exosome secretion. This is an interesting idea and it may be of benefit to follow up on this initial idea with our studies. We could utilize siRNAs for the Rab GTPases and determine, first, if the side effect profile in mice with tumors treated with RAB GTPases is minimal. Secondly, we could assess the potential for siRNAs for the Rab GTPase family to be used in the treatment of acquired radiation resistance as shown in chapter three. This could be an additional therapeutic modality to be used in conjunction with heparins and statins to thus block exosome secretion as well as exosome uptake, and ultimately block all exosome effects on enhancing tumorigenesis.
5.4.2. Exosome Structure to Inform Optimal Liposome Formulation
Liposomes are double lipid bilayer vessels utilized in cancer therapy to encapsulate and target chemotherapy to tumor cells. Although more targeted than chemotherapy alone, liposomes still have inefficiencies in uptake. Exosome mediated delivery of therapeutics is an area of research in its infancy. Comparing the delivery of chemotherapeutics by liposomes to the delivery by exosomes, liposomes are artificially created and thus allow for a large yield of treatment, while exosomes are purified from biological fluids or cell media, thus producing quite a low
164 yield, creating an obstacle for therapeutic exosomes to be used in the human setting. The study of exosome targeted delivery of therapeutic agents will likely inform our knowledge on the optimal type of treatment vehicle, providing us with a basis with which to create the ideal liposome formulation. My finding in chapter three that the radiation derived exosomes have upregulation of
Dynamin 2, a factor critical in exosome uptake, suggests that utilizing dynamin 2 in a liposome formulation would yield liposomes that have enhanced uptake. This ideal formulation would be more readily taken up than traditional liposomes and thus would be more effective in therapeutic delivery of their chemotherapeutic payload. Inevitably, patients would have increased survival and a decreased side effect profile because the enhanced targeted liposomes would go specifically to the tumor and deliver increased chemotherapy to the cancer cells rather than normal cells in the patient. Further studies on exosome uptake must be performed to determine other factors, such as dynamin 2, that are critical for exosome uptake, which can then be transferred into the enhanced liposome formulations.
5.5. Limitations and Challenges
The data in previous chapters is showing a proof on concept demonstrating the potential for exosomes to be used in the clinical realm. Although exciting, there are some limitations and challenges for the translation of this data into the clinic. One limitation is that these data were all performed in cell lines. Although we performed our studies in multiple lines, including patient derived cell lines, the use of cell lines creates a view of a homogeneous tumor, when in fact we know that tumors are incredibly heterogeneous. This is especially true in the human condition of glioblastoma, where not only is there intratumoral heterogeneity, but also intertumoral heterogeneity between different patients and their different glioblastomas. Many different types of tumor cells are contained within one tumor, thus a significant challenge in translating this data to the clinic will be purifying all of the profiling data to the core of factors which are up and down regulated within the exosomes that provide an accurate view of patient status. Overcoming this
165 obstacle is critical in a day where personalized medicine has repeatedly shown to be crucial to optimal patient care and patient outcome.
Furthermore, another challenge arises when choosing which specimens to use, serum or
CSF. Serum is optimal because it is minimally invasive and easily garnered, but serum has limitations. The largest of these limitations being that serum contains exosomes from a vast array of cell types, making the exosome sample impure. Differentiating the “normal” exosomes from the cancer-derived exosomes is a technical obstacle which will be able to be addressed in the future when more effective and specific exosome isolation techniques are discovered. CSF is currently a better medium with which to isolate glioblastoma-derived exosomes for multiple reasons. The first reason is CSF is in close proximity to the tumor itself, so the medium will be enriched with tumor-derived exosomes. Secondly, CSF is a much more pure fluid than serum, making the exosome profiling significantly more specific for tumor-derived exosomes. An obstacle with CSF is the route to obtain this fluid is a lumbar puncture, which is more invasive.
To address this for initial studies, CSF will be taken from patients who have recurrent glioblastoma. The reason for this is because patients with recurrent glioblastomas have ommaya reservoirs in place allowing simple access to CSF fluid. The initial studies on human CSF will thus be performed on these recurrent glioblastoma patients until significant exosome profiles are found. Only after this step can the data be taken further to primary glioblastoma patients who do not have an ommaya reservoir in place.
Although there are limitations and challenges to the work presented in this thesis, we provide exciting proof of concept data that suggests exosome profiling can be translated to the clinic. Further exploratory clinical trials with patient CSF profiling must be performed to adequately determine their full potential for translation to patient medicine.
5.6. Conclusion
166 The long-term goal of my work is to exploit exosomes for both diagnostic and therapeutic purposes. Cancer derived exosomes may provide an accurate picture of the native tumor biology where a simple biopsy snapshot cannot. Exosomes may also be used to longitudinally monitor and identify impending treatment resistance and subsequent tumor recurrence in real time.
Exosomes also seem to be central to tumorigenesis. Determining the effects of chemotherapy induced exosome release on recipient cancer cells on oncogenic functions including invasion, immunosuppression, and therapeutic resistance would continue to shed light on how cancer cells respond to stressors by releasing exosomes to combat that specific stress type. Further evaluation of exosomes as therapeutic agents is paramount. Blockade of exosomes may lead to enhanced cancer patient survival. The analysis of exosome uptake and the factors not only contained within the exosomes, but also expressed on the exosome surface is critical to inform the optimal liposome formulation for cancer chemotherapy. These studies build the stepping-stones for understanding the clinical interplay and therapeutic potential of exosomes in cancer biology.
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173 VITA Oliver D. Mrowczynski
Education 2013 – Present Penn State University, MD/PhD Candidate, Biomedical Sciences Program 2012 University of California, San Diego. M.S. Neuroscience 2011 University of California, San Diego. B.S. Neuroscience Awards 2017 International BioIron Society Conference Travel Grant 2017 Award for Excellence and Innovation, Penn State College of Medicine 2016 Best Presentation Award, PA Neurosurgical Society Conference 2016 Blue Ribbon Best Presentation Award, Society for NeuroOncology 2016 Award for Excellence and Innovation, Penn State College of Medicine 2015 Best Presentation Award, PA Neurosurgical Society Conference Selected Publications Howell KK, Monk BR, Carmack SA, Mrowczynski OD, Clark RE, Anagnostaras SG. Inhibition of PKC disrupts addiction-related memory. Frontiers in Behavioral Neuroscience. (2014) Oliver D. Mrowczynski, Joshua Chern, Dan Barrow, Elias Rizk. Pediatric Neurosurgery: Encephalocele. Journal of Neurosurgery. (2017). The Society of Neurological Surgeons Handbook 1st Edition. 7:7 Oliver D. Mrowczynski, A.B. Madhankumar, Becky Slagle-Webb, Sang Y. Lee, Brad E. Zacharia, James R. Connor. HFE Genotype Affects Exosome Phenotype. Biochim. Biophys. Acta . (2017) A.B. Madhankumar, Oliver D. Mrowczynski, Suhag R. Patel, Cody L. Weston, Brad E. Zacharia, Michael J. Glantz, Christopher A. Siedlecki, Li-Chong Xu, James R. Connor., Interleukin-13 conjugated quantum dots for identification of glioma initiating cells and their extracellular vesicles, Acta Biomaterialia (2017) A.B. Madhankumar, Oliver D. Mrowczynski, Becky Slagle-Webb, Vagisha Ravi, Alexandre Bourcier, Russell A. Payne, Kimberly Harbaugh, Elias B. Rizk, James R. Connor. Tumor targeted delivery of doxorubicin in Malignant Peripheral Nerve Sheath Tumors. Plos One. (2017) Russell A. Payne* and Oliver D. Mrowczynski*, Becky Slagle-Webb, A.B. Madhankumar, Christine Mau, Dawit Aregawi, Alexandre Bourcier, Kimberly Harbaugh, James R. Connor, Elias Rizk. MLN8237 Treatment in an Orthoxenograft Murine Model of MPNSTs. Journal of Neurosurgery. (2017) *Both authors contributed equally and are co-first authors* Oliver D. Mrowczynski, Sara T. Langan, Elias B. Rizk. Infant brachial neuritis following a viral prodrome: A case in a 6 month old child and review of the literature. Child Nerv. System. (2017) Oliver D. Mrowczynski, Achuthamangalam B. Madhankumar, Becky Slagle-Webb, Elias Rizk, Brad Zacharia, James Connor. Exosomes and Acquired Therapeutic Resistance in Cancer. Oncotarget. (2017). In Revisions. Oliver D. Mrowczynski, Brad Zacharia, James Connor. Exosomes and their Implications on Central Nervous System Tumor Biology. Progress in Neurobiology. (2017). Submitted. Oliver D. Mrowczynski, Sara T. Langan, Elias B. Rizk. Craniopharyngiomas: Evaluation of the Current Evolving Intratumoral Treatment Landscape. Neurosurgery. (2017). Submitted. Oliver D. Mrowczynski, Sara T. Langan, Elias B. Rizk. Intrathecal antibiotic agents: A Review of the current status. Neurosurgery. (2017). Submitted. Oliver D. Mrowczynski, Christine Mau, Sara T. Langan, Dan T.D. Nguyen, Kimberly S. Harbaugh. Thermal ablation of paraspinal neurofibromas: A Case Report. JNS: Spine. 2017. Submitted. Oliver D. Mrowczynski, Russell A. Payne, Alexandre Bourcier, Christine Mau, Becky Slagle-Webb, Ganesh Shenoy, A.B. Madhankumar, Kimberly Harbaugh, Elias Rizk, James R. Connor. Intratumoral Mutant IL-13 Conjugated Pseudomonas Toxin Treatment of Malignant Peripheral Nerve Sheath Tumors. Cancer Letters. (2017) Submitted. Oliver D. Mrowczynski, Alexandre J. Bourcier, Jason Liao, Charles Specht, Elias Rizk. Hyponatremia as a Predictive and Prognostic Factor in Glioblastoma Multiforme. Journal of Clinical Oncology. (2017). Submitted