Establishing three-dimensional ex-vivo culture platforms of human pancreatic ductal adenocarcinoma as pre-clinical models for the development of oncolytic virotherapy

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest human neoplasms which remains poorly understood. There is a pressing need to develop a research platform that better recapitulates the in-vivo tumour microenvironment in order to develop better therapeutics and understand the biologics of PDAC. In this study we evaluate two such models; i) organotypic tissue slices and ii) multicellular tumour spheroids (MCTS). Our aims were to i) optimize the culture conditions for the tissue explants and devise a comprehensive method for their analysis and ii) generate MCTS using three PDAC cell lines, Panc-1, Capan-1 and MiaPaCa-2, both as homotypic MCTS or heterotypic MCTS, the latter upon co-culture of the PDAC cell lines with either fibroblasts (MRC5) or epithelial cells (DEChTERT). We also aim to infect both the tissue slices and spheroids with Ad5-based oncolytic viruses and evaluate their response. Ultra-thin tissue slices were cut from freshly retrieved Whipple’s resections. Several variables were modified to establish their ideal culture conditions. We assessed tissue i) integrity using immunohistochemical analysis ii) viability using a TUNEL assay and iii) functionality using an amylase assay. We found that tissue explants can be maintained in culture for ≥ 3 days, longer for tumour tissue, whilst retaining excellent morphology and viability. We co-cultured PDAC cell lines with fibroblasts or epithelial cells, to form heterotypic MCTS and observed them over a 7-days using immunofluorescence microscopy. These were infected with a wild type and recombinant Adenovirus 5 (Ad5)-based viruses in order to evaluate the i) oncolytic efficacy of Ad5, ii) selectivity for cancer cells, and iii) effect of fibroblasts and epithelial cells on the response of the heterotypic MCTS to viral oncolysis. Homotypic MCTS were blown apart by both viruses with minimal-to-no residual spheroid, with the exception of MiPaCa-2. Heterotypic MCTS showed a subverted response to viral oncolysis highlighting the importance of the tumour microenvironment