ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest and most aggressive cancer types, and thus new treatment strategies are urgently needed. Pancreatic cancer cells are surrounded by a dense stroma consisting of extracellular matrix and noncancerous cells, including fibroblasts and immune cells. Interactions between tumor cells and the associated stroma are critical for tumor progression and resistance to therapy. Due to the unusually low vascular density of PDAC, both the tumor and stromal cells experience hypoxia, a condition of insufficient oxygen availability. To cope with hypoxic stress, both the tumor and stromal compartments undergo adaptive changes primarily mediated by hypoxia-inducible factor 1ɑ and 2ɑ (HIF1ɑ and HIF2ɑ). Although PDAC is known to be severely hypoxic and hypoxia predicts poor clinical outcome, the effects of hypoxia and stromal HIF activation on tumor-stroma interactions in PDAC are not fully understood. Based on our preliminary observation that inflammatory fibroblasts and macrophages are preferentially located in hypoxic tumor regions whereas CD8+ T cells are largely excluded from hypoxic regions in PDAC, we hypothesize that hypoxia drives an immunosuppressive microenvironment by promoting an inflammatory fibroblast phenotype and in turn modulating infiltration and function of macrophages and CD8+ T cells. In Specific Aim 1, we will determine how hypoxia and HIFs regulate an inflammatory fibroblast phenotype in PDAC using a three- dimensional (3D) coculture system of pancreatic tumor cells and fibroblasts and exposing the coculture to either hypoxia or normoxia, as well as using mouse models lacking fibroblast expression of HIF1ɑ or HIF2ɑ. In Specific Aim 2, we will define how hypoxia-induced fibroblast-secreted factors and macrophage-intrinsic hypoxic responses regulate macrophage recruitment and function in PDAC by treating macrophages with conditioned media derived from 3D tumor cell-fibroblast cocultures under hypoxia, and using mouse models lacking myeloid expression of HIF1ɑ or HIF2ɑ. In Specific Aim 3, we will determine how macrophages and fibroblasts contribute to CD8+ T cell exclusion in hypoxic tumor niches by depleting macrophages or blocking a factor secreted from fibroblasts in syngeneic orthotopic transplanted tumors, injecting a hypoxia probe into mice, and assessing CD8+ T cell infiltration and activation in hypoxic and normoxic tumor regions. Together, the proposed studies will provide new knowledge on hypoxic regulation of the tumor-stroma crosstalk and inform the optimal design of stroma-targeting therapeutic strategies for PDAC.