Abstract 3739: An in vitro fluorescence-based co-culture model identifies tumour microenvironment-mediated chemoresistance in pancreatic cancer
Authors: Xu Ruiling , Richards Frances M. , Lin Yao , Jodrell Duncan I. 
Published: May 2015

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis, with poor responses to cytotoxic or targeted agents. PDAC is characterised by abundant desmoplasia and this tumor microenvironment is a major determinant of intrinsic resistance to anticancer therapies. In particular, pancreatic stellate cells/cancer-associated fibroblasts (PSCs/CAFs) are periacinar stromal cells which promote epithelial-mesenchymal transition (EMT) and radioresistance of PDAC. However, this unique tumour microenvironment is not recapitulated in conventional cell monoculture or xenografts, which might attribute to the poor predictivity of these preclinical drug screening models. To address this, we have developed a PDA tumor cell-specific in vitro Fluorescence-Based Co-culture (PDA-FBC) platform to investigate potential mechanisms of stroma-mediated chemoresistance and to evaluate novel agents and combinations. Using K8484 (PDA cell line derived from the LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-Cre (KPC) mouse) stably expressing GFP, we selectively measured tumor cell viability in response to gemcitabine, with and without the presence of stromal cells. Monolayers of CAFs (primary fibroblast-like cells isolated from PDA tumour in KPC mice) or NFs (primary fibroblast-like cells from the normal pancreas of paired PC mice) were grown in 96 well plates and K8484 cells were seeded on top, prior to 72 hour drug exposure. When co-cultured with CAFs, K8484-GFP cells were significantly less sensitive to gemcitabine than cells grown in absence of CAFs: gemcitabine GI50 (50% growth inhibition concentration) is 19+/-3.2nM (n=4) in K8484 cell monolayer, 690+/-250nM (n=4) in the presence of CAFs (p = 0.03). Transwell assays revealed that this CAF-mediated chemoresistance was only induced in a cell-cell direct contact-dependent manner. Moreover, similar stroma-mediated gemcitabine resistance was not detected in co-culture of K8484 with NFs from normal pancreas (GI50=21+/-3.3nM). We are now investigating whether the chemoresistance phenotype extends to other cytotoxic agents. To evaluate potential mechanisms of resistance, RNA sequencing is being conducted to compare molecular profiles of K8484-GFP cells co-cultured with CAFs or NFs (with/without gemcitabine), to identify the genes modulated by the tumor-stroma interaction and potentially responsible for the decreased gemcitabine sensitivity in co-culture. In conclusion, we have identified that cancer-associated fibroblasts induce resistance to gemcitabine in KPC PDAC tumour cells, in contrast to fibroblasts from normal pancreas. This platform will have utility in the assessment of novel drug combinations and a mechanistic understanding of the tumor-stroma interactions may identify new candidate drug combinations.

Citation Format: Ruiling Xu, Frances M. Richards, Yao Lin, Duncan I. Jodrell. An in vitro fluorescence-based co-culture model identifies tumour microenvironment-mediated chemoresistance in pancreatic cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3739. doi:10.1158/1538-7445.AM2014-3739