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Tumor-Stromal Interactions in a Co-Culture Model of Human Pancreatic Adenocarcinoma Cells and Fibroblasts and Their Connection with Tumor Spread.

ABSTRACT: One key feature of pancreatic ductal adenocarcinoma (PDAC) is a dense desmoplastic reaction that has been recognized as playing important roles in metastasis and therapeutic resistance. We aim to study tumor-stromal interactions in an in vitro coculture model between human PDAC cells (Capan-1 or PL-45) and fibroblasts (LC5). Confocal immunofluorescence, Enzyme-Linked Immunosorbent Assay (ELISA), and Western blotting were used to evaluate the expressions of activation markers; cytokines arrays were performed to identify secretome profiles associated with migratory and invasive properties of tumor cells; extracellular vesicle production was examined by ELISA and transmission electron microscopy. Coculture conditions increased FGF-7 secretion and α-SMA expression, characterized by fibroblast activation and decreased epithelial marker E-cadherin in tumor cells. Interestingly, tumor cells and fibroblasts migrate together, with tumor cells in forming a center surrounded by fibroblasts, maximizing the contact between cells. We show a different mechanism for tumor spread through a cooperative migration between tumor cells and activated fibroblasts. Furthermore, IL-6 levels change significantly in coculture conditions, and this could affect the invasive and migratory capacities of cells. Targeting the interaction between tumor cells and the tumor microenvironment might represent a novel therapeutic approach to advanced PDAC.

SUBMITTER: Prieto-Garcia E

PROVIDER: S-EPMC8065458 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:IntroductionPancreatic ductal adenocarcinoma (PDAC) is projected to rise to the second leading cause of U.S. cancer-related deaths by 2020. Novel therapeutic targets are desperately needed. MicroRNAs (miRs) are small noncoding RNAs that function by suppressing gene expression and are dysregulated in cancer. miR-21 is overexpressed in PDAC tumor cells (TC) and is associated with decreased survival, chemoresistance and invasion. Dysregulation of miR regulatory networks in PDAC tumor-associated fibroblasts (TAFs) have not been previously described. In this study, we show that miR-21 expression in TAFs promotes TC invasion.MethodsIn-situ hybridization for miR-21 was performed on the 153 PDAC patient UCLA tissue microarray and 23 patient-matched lymph node metastases. Stromal and TC histoscores were correlated with clinicopathologic parameters by univariate and multivariate Cox regression. miR-21 positive cells were further characterized by immunofluorescence for mesenchymal/epithelial markers. For in vitro studies, TAFs were isolated from freshly resected human PDAC tumors by the outgrowth method. miR-21 was overexpressed/inhibited in fibroblasts and then co-cultured with GFP-MiaPaCa TCs to assess TC invasion in modified Boyden chambers.ResultsmiR-21 was upregulated in TAFs of 78% of tumors, and high miR-21 significantly correlated with decreased overall survival (P = 0.04). Stromal miR-21 expression was also significantly associated with lymph node invasion (P = 0.004), suggesting that it is driving TC spread. Co-immunofluorescence revealed that miR-21 colocalized with peritumoral fibroblasts expressing ?-smooth muscle actin. Moreover, expression of miR-21 in primary TAFs correlated with miR-21 in TAFs from patient-matched LN metastases; evidence that PDAC tumor cells induce TAFs to express miR-21. miR-21 expression in TAFs and TCs promotes invasion of TCs and is inhibited with anti-miR-21.ConclusionsmiR-21 expression in PDAC TAFs is associated with decreased overall survival and promotes TC invasion. Anti-miR-21 may represent a novel therapeutic strategy for dual targeting of both tumor and stroma in PDAC.

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Tumor-Stromal Interactions in a Co-Culture Model of Human Pancreatic Adenocarcinoma Cells and Fibroblasts and Their Connection with Tumor Spread.

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