PROJECT SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States despite representing only 2% of all cancer cases. PDAC is characterized by an extensive fibroinflammatory stroma and a hypoxic microenvironment, which contributes to disease progression and therapeutic resistance. Macrophages and cancer-associated fibroblasts (CAFs) are the predominant cell types within the PDAC stroma. Macrophages are a major immunosuppressive population in PDAC. These cells are highly plastic and, as a result, their environment plays an important role in regulating their function. Macrophages adapt to hypoxia, a condition of low oxygen availability, primarily through the stabilization of oxygen-liable transcription factors called hypoxia-inducible factors (HIFs). Although PDAC is extremely hypoxic and both the tumor cells and stromal cells experience hypoxia, the effects of hypoxia and HIFs on macrophages and their communication with other stromal cells in PDAC remain largely unknown. Our group has recently found that hypoxia promotes the acquisition of inflammatory cancer-associated fibroblasts (inflammatory CAFs), a recently defined PDAC fibroblast subtype that produces high levels of inflammatory cytokines and chemokines. By injecting a hypoxia probe into mice bearing PDAC, we have observed that inflammatory CAFs and macrophages predominantly reside in hypoxic tumor regions compared with normoxic regions whereas T cells are largely excluded from hypoxic tumor areas. Based on these preliminary data, I hypothesize that inflammatory fibroblasts induced by hypoxia promote macrophage infiltration into hypoxic tumor regions and facilitate an immunosuppressive macrophage phenotype. This hypothesis will be investigated with the following two Aims: (1) to determine the role of hypoxia-induced fibroblast-secreted factors in regulating macrophage migration and function in PDAC and (2) to define the role of macrophage HIFs in regulating macrophage migration and function in PDAC. To complete Aim 1, I will culture macrophages with conditioned media derived from mono- and co- cultures of pancreatic tumor cells and fibroblasts under either hypoxia or normoxia, and then assess the expression of immunosuppressive macrophage markers as well as their migration. To complete Aim 2, I will culture HIF1α-deficient and HIF2α-deficient macrophages under hypoxia with fresh media or conditioned media from co-cultures of pancreatic tumor cells and fibroblasts under hypoxia. Then I will assess the expression of immunosuppressive markers in these macrophages and evaluate their migration. Finally, I will define the role of macrophage HIFs in mediating pancreatic tumorigenesis using orthotopic tumors from mice lacking myeloid expression of HIF1ɑ or HIF2ɑ. My proposal will provide new insights into how hypoxia promotes an immunosuppressive PDAC microenvironment by modulating the macrophage-fibroblast crosstalk, and ultimately infor...