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Background Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with a survival rate under 13%. Treatments like immunotherapy and chemotherapy are ineffective in PDAC due to the immunosuppressive tumor microenvironment, in which tumor-associated neutrophils (TANs) are a major component. The immunosuppressive function of TANs has been shown to depend upon increased production of reactive oxygen species (ROS), with myeloperoxidase (MPO) being a major source of it. Our study investigated the contribution of MPO in mediating TANs immunosuppression and chemotherapy response in PDAC.Methods An orthotopic PDAC mouse model was established using KPCY6419 CBG-GFP cells injected into the pancreas of wild-type and MPO-deficient mice. Wild-type mice were also treated with a pharmacological MPO inhibitor starting on day 5 post-injection. Tumor burden was quantified on day 24 followed by flow cytometry and trichrome staining analysis. Neutrophils isolated from spleen of tumor-bearing mice were used for CD8 + T and NK cell suppression assays, neutrophil extracellular traps (NETs) formation, and ROS production assays. MPO activity in neutrophils from PDAC patient blood and tissue was assessed using a luminol-based reporter assay.Results MPO deficiency or inhibition significantly delayed PDAC tumor growth and improved mouse survival. Trichrome staining showed reduced collagen level in MPO-deficient or inhibitor-treated tumors, indicating a role for MPO in modulating the tumor stroma. Flow cytometry analysis revealed a significant increase in CD8 + T and NK cells in MPO-deficient tumors, accompanied by higher granzyme B expression and elevated activation/memory markers in CD8+ and CD4+ T cells. Ex vivo studies using isolated TANs from tumor-bearing mice showed reduced CD8+ T cell and NK cell suppression, NETs production and ROS level in the absence of MPO, indicating that MPO is essential for TAN immunosuppressive activity. Further, depletion of NK and CD8+ T cells in MPO-deficient mice abolished the anti-tumor effects observed with MPO loss, underscoring the critical role of these cells in host protection. Moreover, MPO inhibition or deficiency sensitized tumors to gemcitabine, highlighting a synergistic effect with chemotherapy. Complementing these murine studies, elevated MPO activity was detected in PDAC patient tumor tissue and circulating neutrophils compared to adjacent normal pancreas and non-PDAC blood-isolated neutrophil controls. Interestingly, higher MPO activity in circulating neutrophils correlated with chemotherapy resistance.Conclusions Overall, our study demonstrates the critical function of MPO in regulating PDAC tumor progression, TAN immunosuppression, immune cell infiltration, and treatment response in an orthotopic PDAC model, as well as the correlation of MPO with poor patient outcomes and treatment resistance.