The Hypoxic Tumor Microenvironment Regulates Fibroblast-Mediated Epithelial-Mesenchymal Transition in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy characterized by a complex tumor microenvironment (TME) containing phenotypically diverse cancer-associated fibroblasts (CAFs) and regions of hypoxia. This thesis investigates how hypoxia and chemotherapy influence CAF-malignant cell interactions to drive epithelial-mesenchymal transition (EMT), a process linked to chemoresistance. We first aimed to characterize hypoxia's ability to modulate CAF phenotype and alter CAF-malignant cell communication. By analyzing publicly available PDAC patient data, we revealed that the inflammatory CAF subtype was enriched in hypoxia-regulated transcripts and displayed distinct cytokine and growth factor expression patterns. Cell culture experiments further confirmed that hypoxia independently induces a cell-autonomous inflammatory CAF-like state, enhancing the ability of CAFs to induce EMT in PDAC cells through secretion of IGF-2 and HGF. We characterized the underlying cell-autonomous mechanism in hypoxic CAFs, demonstrating that hypoxia-induced reactive oxygen species accumulation activates the NF-κB signaling pathway and cooperates with a histone methylation mark to promote IGF-2 secretion. Furthermore, in an autochthonous PDAC mouse model, we observed mesenchymal cancer cells colocalizing with CAFs in hypoxic regions, within the characteristic length scale for paracrine signaling. Importantly, in tumors engineered with hypoxia fate-mapped CAFs, once-hypoxic re-oxygenated CAFs did not colocalize with mesenchymal cancer cells, which indicates that the hypoxia-induced phenotype is reversible. Next, we investigated the combined effects of hypoxia and gemcitabine on CAF-malignant cell crosstalk within a PDAC mouse model by using spatial transcriptomics. We found that gemcitabine induced significant tumor microenvironment alterations, evidenced by a decrease in the overall number of predicted ligand-receptor interactions but a notable increase in CAF-enriched and T cell-enriched niches. We identified that ephrin signaling between CAFs and malignant cells was increased by gemcitabine treatment and further bolstered by hypoxia. Inhibition of ephrin signaling antagonized EMT in gemcitabine-treated co-cultures of PDAC cells and CAFs. Collectively, this thesis emphasizes the intricate and dynamic nature of cell-cell signaling within the PDAC microenvironment in response to hypoxia and chemotherapy treatment, thereby highlighting specific, targetable pathways to antagonize CAF-regulated EMT in pancreatic cancer.