Pancreatic ductal adenocarcinoma (PDAC) is characterized by an extensive fibroinflammatory stroma and often experiences conditions of insufficient oxygen availability or hypoxia. Cancer-associated fibroblasts (CAF) are a predominant and heterogeneous population of stromal cells within the pancreatic tumor microenvironment. Here, we uncover a previously unrecognized role for hypoxia in driving an inflammatory phenotype in PDAC CAFs. We identify hypoxia as a strong inducer of tumor IL1ɑ expression, which is required for inflammatory CAF (iCAF) formation. Notably, iCAFs preferentially reside in hypoxic regions of PDAC. Our data implicate hypoxia as a critical regulator of CAF heterogeneity in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with a dense desmoplastic stroma and an immunosuppressive tumor microenvironment that contribute to therapeutic resistance. Here, we identify plasminogen activator inhibitor 1 (PAI1) as a stroma-derived mediator of immune evasion and tumor progression in PDAC. PAI1 is predominantly produced and secreted by cancer-associated fibroblasts, and its genetic ablation in the stromal compartment impairs tumor growth. Mechanistically, hypoxia induces PAI1 expression in fibroblasts, which in turn shifts macrophages toward immunosuppressive phenotypes and suppresses CD8 ⁺ T cell infiltration and function. We further show that tissue plasminogen activator (tPA), a direct PAI1 target, is also secreted by fibroblasts and supports antitumor CD8 ⁺ T cell responses. Notably, elimination of stromal tPA promotes immunosuppressive macrophage phenotypes, reduces CD8 ⁺ T cell infiltration, and accelerates PDAC progression. These findings define a previously unrecognized PAI1-tPA regulatory axis within the tumor stroma that modulates antitumor immunity. Targeting this pathway may provide a therapeutic opportunity to overcome stroma-driven immune suppression in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy characterized by a dense fibroinflammatory stroma and profound hypoxia. Using pancreatic stellate cell-tumor organoid coculture models and single-cell RNA sequencing analyses, we uncover that hypoxia-driven fibroblast reprogramming promotes immunosuppressive macrophage phenotypes in PDAC. Mechanistically, hypoxia acts through tumor-fibroblast crosstalk to increase IL-6 expression in fibroblasts; in turn, fibroblast-derived IL-6 induces expression of arginase 1 (ARG1), a key mediator of immunosuppression, in macrophages via activation of the JAK/STAT signaling pathway. Consistent with these findings, macrophages enriched for hypoxia signatures are strongly associated with elevated immunosuppression programs and IL6/JAK/STAT3 signaling signatures in PDAC. Our study reveals a paracrine mechanism by which hypoxia coordinates tumor cell, fibroblast, and macrophage interactions to promote immune suppression in PDAC.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense stromal microenvironment and profound hypoxia, which contribute to therapeutic resistance. Using an in vitro system incorporating pancreatic cancer cells and cancer-associated fibroblasts (CAFs), we show that hypoxia suppresses CD8⁺ T cell accumulation and, in combination with cancer cell- and CAF-derived factors, further impairs T cell fitness by increasing cell death and reducing proliferation. Although the combination of hypoxia and cancer cell/CAF-derived factors enhances IFNγ and granzyme B expression in CD8⁺ T cells on a per-cell basis, the overall number of functional effector T cells is markedly reduced. Analysis of human PDAC single-cell RNA sequencing data corroborates these findings, revealing that CD8⁺ T cells enriched for hypoxia signatures exhibit elevated apoptosis and stress-response pathways. Furthermore, hypoxia is associated with downregulation of stemness-related genes and upregulation of terminal differentiation markers. Together, these data suggest that the integration of intrinsic hypoxic responses and extrinsic cues from tumor cells and CAFs impairs CD8⁺ T cell fitness and correlates with a terminally differentiated, dysfunctional T cell state.