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Broadening Adenoviral Oncolysis in PDAC

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Broadening Adenoviral Oncolysis in PDAC Broadening Adenoviral Oncolysis in PDAC: Interrogation of Patient-Derived Organoids for personalized virotherapy and modulation of miRNA content to boost adenoviral potency Giulia Raimondi Aquesta tesi doctoral està subjecta a la llicència Reconeixement 4.0. Espanya de Creative Commons. Esta tesis doctoral está sujeta a la licencia Reconocimiento 4.0. España de Creative Commons. This doctoral thesis is licensed under the Creative Commons Attribution 4.0. Spain License. UNIVERSITAT DE BARCELONA FACULTAT DE FARMÀCIA I CIÈNCIES DE L’ALIMENTACIÓ Tesi dirigida per la Dra Cristina Fillat Fonts Grup de Teràpia Gènica i Càncer - IDIBAPS Broadening Adenoviral Oncolysis in PDAC: Interrogation of Patient-derived Organoids for personalized virotherapy and modulation of miRNA content to boost adenoviral potency Giulia Raimondi 2020 Abbreviations 5-FU 5-FluoroUracil Adwt Adenovirus Wild Type Specie C Serotype 5 ATCC American Type Culture Collection BAC Bacterial Artificial Chromosome BiTE Bi-specific T cell Engager bp Base Pair CAF Cancer Associated Fibroblasts CAR Coxsackie and Adenovirus Receptor cDNA copy DNA CK19 CytoKeratin 19 CMV CytoMegaloVirus CPE Cytoplasmatic Polyadenylation Element CRC Colorectal Cancer CSC Cancer Stem Cell DAPI 4,6-DiAmidino-2-PenilIndole DMEM Dulbecco’s Modified Eagle Medium DMSO DiMethyl SulfOxide DNA DeoxyriboNucleic Acid dNTPs deoxyNucleotides DTT DiThioThreitol E. Coli Escherichia Coli EBV Epstein-Barr Virus ECM Extra Cellular Matrix eGFP enhanced Green Fluorescence Protein EMT Epithelial Mesenchimal Transition FC Fold Change FNA Fine Needle Aspiration Fw Forward G Gauge g Gravity Gem Gemcitabine GM-CSF Granulocyte Macrophage Colony Stimulating Factor h Hours H&E Hematoxilyin and Eosin HBS HEPES Buffer Saline HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HIV Human Immunodeficiency Viruse HSV Herpex Simplex Virus ID50 Infectious Dose 50 iFBS inactivated Fetal Bovine Serum IFU Infectious Forming Unit IHC ImmunoHistoChemistry IPMN Intraductal Papillary Mucinous Neoplasm ITR Inverted Terminal Repeats Kb Kilobase KO Knock Out KRAS Kirsten RAt Sarcoma viral oncogene homolog LB Luria Broth M Molar MCN Mucinous Cystic Neoplasm miRISC miRNA-Induced Silencing Complex miRNA/miR microRNA MLP Major Late Promoter MMP Matrix MetalloProteinase mRNA messenger RNA MTT 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide nab-paclitaxel nanoparticle albumin-bound paclitaxel nm nanometer ºC Celsius Grade OD Optical Density ORF Open Reading Frame OV Oncolytic adenoVirus PanIN Pancreatic Intraepithelial Neoplasia PBS Phosphate Buffer Saline PBS++ Phosphate Buffer Saline supplemented withCaCl2 and MgCl2 PCR Polymerase Chain reaction PDAC Pancreatic Ductal AdenoCarcinoma PDO Patient-Derived organoid PI Post-Infection Rb Retinoblastoma RE Restriction Enzyme RGD Arginine - Glycine - Aspartic Acid RLU Relative Light Units RNA RiboNucleic Acid rpm revolutions per minute Rspo R-spondin RT-qPCR Real Time quantitative PCR Rv Reverse SDS Sodium Dodecyl Sulfate SEM Standard Error of the Mean SPF Specific Pathogen Free SPS Sequence Paired binding Sites Taq Thermophilus aquaticus TCF TransCription Factor TIC Tumor Initiating Cells UTR Un-Translated Region VA-RNA Viral Associate RNA vp viral particle VSV Vescicular Stomatitis Virus Wnt Wingless-related integration site Presentation Pancreatic ductal adenocarcinoma is a malignancy with a very dismal prognosis, predicted to become the second leading cancer- related death in 2030. Available treatments do not contribute to improve patients’ life expectancy, with a 5 years survival lower than 10%. The enormous genetic variability, the dense desmoplastic stroma, and the immunosuppressed microenvironment make PDAC an unsolved clinical challenge. Oncolytic virotherapy has been envisioned as a therapeutic alternative for clinical management of PDAC. Attenuated viruses, orientated to specifically eliminate cancer cells, offer multiple advantages over current treatments. The therapeutic viruses self- amplify upon tumor lysis, propagating to adjacent cells with the release of tumor associated antigens that can awake the immune system against the tumor. Nowadays, many oncolytic adenoviruses are undergoing clinical trials for the treatment of solid tumors, including PDAC. However, patients’ response to oncolytic viruses is lower than expected, and their administration as monotherapy is not sufficient to abrogate tumor mass. This can be partially attributed to the lack of relevant models of pre-clinical screening, performed in in vitro systems or in xenografts derived from cell lines that do not recapitulate the genetic heterogeneity of PDAC and its complex microenvironment. PDAC organoids are 3D structures established from patient tumors that are representative of various features of PDAC, displaying inter-patient variability and characterized by an easy handling manipulation. In the current thesis we have explored the use of organoids in virotherapy for the screening of personalized adenoviral treatments, either as monotherapy or in combination with actual gold standard chemotherapy. We identified patient- specific sensitivities to adenoviruses and showed that oncolytic virus response in patient-derived organoids (PDOs) from primary tumors might predict metastases sensitivity. The results from this part of the thesis contributed to the characterization of the organoids as a feasible and personalized model for the pre-clinical study of oncolytic adenoviruses. With the aim to generate oncolytic adenoviruses with improved tumor potency and enhanced therapeutic index, we studied the influence of the deregulated miRNome in PDAC on adenoviral replication. We identified several miRNAs that strongly impacted adenoviral propagation. Furthermore, we approached a miR-222 sponge design to inhibit miR-222 and the restoration of miR-29c as strategies to promote adenoviral replication, exploring some mechanistic insights. Studies with the novel oncolytic viruses AdNuPAR-E-miR-222-S and ICOVIR15-E-miR29c revealed their increased potency, positioning these viruses as candidates for future development. Index 1. Introduction .................................................................................. 1 1.1 The Pancreas ........................................................................... 2 1.2 Pancreatic cancers and Pancreatic Ductal Adenocarcinoma .. 3 1.2.1 Histopathological alteration in PDAC ............................. 4 1.2.2 Molecular characterization of PDAC tumors .................. 6 1.2.3 PDAC microenvironment .............................................. 12 1.2.4 PDAC treatment ............................................................ 16 1.3 microRNAs ........................................................................... 17 1.3.1 microRNAs biogenesis and regulation .......................... 18 1.3.2 Target recognition.......................................................... 22 1.3.3 Regulation of mRNA translation ................................... 23 1.3.4 miRNAs and cancer ....................................................... 24 1.4 Patient-derived organoids (PDOs) ........................................ 27 1.4.1 Organoids and cancer .................................................... 28 1.5 Oncolytic Virotherapy .......................................................... 35 1.6 Oncolytic adenoviruses......................................................... 37 1.6.1 Adenoviral Biology ....................................................... 38 1.6.2 Design of oncolytic adenoviruses .................................. 43 2. Objectives ................................................................................... 48 3. Materials and Methods ............................................................... 51 3.1 DNA Manipulation ............................................................... 52 3.1.1 Plasmid DNA isolation from prokaryotic cells ............. 52 3.1.2 Isolation of genomic and viral DNA from cells ............ 53 3.1.3 DNA quantification ....................................................... 53 3.1.4 DNA digestion ............................................................... 54 3.1.5 DNA amplification via Polymerase Chain reaction (PCR) ................................................................................................ 55 3.1.6 DNA sequencing ........................................................... 56 3.2 DNA constructs generation .................................................. 56 3.2.1 Plasmids expressing miRNA sponges ........................... 56 3.2.2 Generation of plasmid vectors with sgRNAs for miRNA KO .......................................................................................... 59 3.3 RNA manipulation .......................................................... 64 3.3.1 RNA extraction and retro-transcription to cDNA ......... 64 3.3.2 RNA Expression ............................................................ 65 3.4 Eukaryotic cell lines manipulation ....................................... 67 3.4.1 Cell lines. Maintenance and culture conditions. ............ 67 3.4.2. Transient Transfection .................................................. 69 3.4.3 Lentiviral transduction ................................................... 69 3.5. Patient-derived pancreatic organoids (PDOs) ..................... 72 3.5.1 Organoids generation from human pancreatic tissues ... 72 3.5.2. Organoids follow up after seeding ............................... 75 3.5.3. Organoids culture routine ............................................
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