Hypoxia and Chemotherapy Promote Secretion of EMT-Driving Growth Factors By Fibroblasts in Pancreas Cancer

Pancreatic ductal adenocarcinoma (PDAC) has a five-year survival rate of just 12%. PDAC is characterized by a dense fibrotic stroma laden with cancer-associated fibroblasts (CAFs) and numerous hypoxic tissue subdomains due to hypovascularity. Interactions between CAFs and malignant PDAC cells remain poorly understood, but their elucidation will undoubtedly nominate new targets for precision medicine approaches to curb chemoresistance. We hypothesized that hypoxia and chemotherapy enhance CAF secretion of growth factors that promote malignant cell epithelial-mesenchymal transition (EMT), which leads to tumor chemoresistance and invasiveness. We first explored how chemotherapy alters paracrine signaling between CAFs and malignant cells by using publicly available single-cell RNA sequencing data from human PDAC tumors. CellChat analysis of the annotated data predicted that ligand-receptor interactions were generally increased between CAFs and malignant cells in response to chemotherapy. In particular, AREG-EGFR and HGF-MET signaling between CAFs and malignant cells were elevated. Importantly, EGFR and MET signaling cooperate to promote EMT. To investigate the possibility that malignant cells regulate growth factor secretion by CAFs, we next explored how juxtacrine interactions between CAFs and malignant cells impact CAF phenotypes. We focused on CAF phenotype regulation via NF-κB and histone 3 lysine 36 dimethylation (H3K36me2) because we recently showed that these pathways cooperate to promote CAF secretion of EMT-promoting growth factors. PDAC CAFs were cultured with paraformaldehyde-fixed cancer cells (to prevent paracrine interactions), and immunofluorescence imaging and quantitative image analysis were performed. CAFs in contact with malignant cells exhibited increased NF-κB nuclear localization and elevated H3K36me2. CAFs in contact with cancer cells while cultured in hypoxia displayed even greater NF-κB nuclear translocation, suggesting hypoxia and cancer cell-CAF interactions had cooperative effects that may be crucial for regulation of CAF subtype. Our findings highlight the ability of chemotherapy and tumor hypoxia to enhance CAF secretion of EMT-promoting growth factors and identify drug targets whose antagonism is predicted to sensitize PDAC to chemotherapy by impeding CAF-cancer cell crosstalk.