Tumor-Stroma Co-Cultures Modify the Invasive Properties of Human Pancreatic Adenocarcinoma Cells Through Different Patterns of Cytokine Secretion and Depending on the Type of Cell Lines.

Abstract

\n Background: Pancreatic ductal adenocarcinoma (PDAC) is among the most lethal cancer worldwide. One key feature of PDAC is a dense desmoplastic reaction that has become recognized to play important roles in tumor growth, metastasis, and therapeutic resistance. We aim to study tumor-stromal interactions in an in vitro co-culture model between human PDAC cells and fibroblasts.Methods: Capan-1 or PL-45 human pancreatic tumor cell lines and LC5 fibroblasts were grown in direct or indirect co-cultures, and their relationship regarding migratory and invasive properties were studied by wound-healing and transwell migration assays. Confocal immunofluorescence, ELISA, and western blotting were used to evaluate the expression of activation markers. Cytokines arrays were performed to identify secretome profiles associated to migratory and invasive properties of tumor cells. Extracellular vesicles production was examined by ELISA and transmission electron microscopy. Results: Co-cultures conditions increased FGF-7 secretion and α-SMA expression, characterizing fibroblasts activation; and decreased epithelial marker E-cadherin in tumor cells, suggesting that they suffer epithelial-to-mesenchymal transition. The expression pattern of cytokine secretion differed under co-culture conditions versus monocultures, with GM-CSF, GRO a/b/g, GRO-α, IL-6, IL-8, MCP-1, and RANTES being the most affected. A greater number of exosomes with higher amount of proteins was quantified in co-cultures assays. Interestingly, tumor cells and fibroblasts migrate as a package, with tumor cells in the middle surrounded by fibroblasts, and maximizing the contact between cell to cell. Conclusions: We show different migratory properties in tumor and stromal cells, and we draw a different mechanism for tumor spread through a cooperative migration between tumor cells and activated fibroblasts. Furthermore, we found that the level of IL-6 mainly, in addition to other cytokines, change significantly in co-cultures conditions with respect to monocultures, and this could affect the invasive and migratory capacities of cells. Targeting the interaction between tumor cells and tumor microenvironment might represent a novel therapeutic approach to advance pancreatic carcinoma.