Abstract A037: Linking reactive oxygen species and neutrophil extracellular traps formation to pancreatic ductal adenocarcinoma progression

Abstract Neutrophils, key effector cells of the innate immune system, combat pathogens through mechanisms including the production of reactive oxygen species (ROS) and the release of neutrophil extracellular traps (NETs). While these responses are critical for host defense, prolonged elevation of ROS and dysregulated NETosis mediated by neutrophils have been implicated in autoimmune diseases, chronic inflammation, and cancer. In pancreatic ductal adenocarcinoma (PDAC), a highly aggressive and inflammatory malignancy, higher level of tumor-associated neutrophils (TANs) infiltrates the tumor microenvironment and promote cancer progression, invasion and metastasis through increased ROS production and NET release. However, the interplay between ROS and NETosis remains poorly understood. This study investigates the correlation between ROS levels and NET formation in pancreatic cancer, exploring whether ROS could serve as a surrogate marker for NETosis. Using the L-012 bioluminescence reporter and NET assays, we assessed ROS and NET release induced by phorbol myristate acetate and platelet-activating factor in bone-marrow isolated neutrophils from wild-type (WT) and myeloperoxidase-deficient (MPO -/- ) mice. MPO has been shown to be a major drive of ROS and NETs. We observed a positive correlation between ROS and NET formation. In vivo analyses using subcutaneous, orthotopic, and spontaneous PDAC tumor murine models revealed elevated ROS levels in tumors, which were significantly reduced upon genetic deletion of MPO, Peptidyl arginine deiminase 4 (PAD4), an essential enzyme for NET formation, and after treatment with hydroxychloroquine, a NET inhibitor, in a subcutaneous PDAC model. TANs isolated from WT orthotopic tumor-bearing mice showed significantly higher ROS and NET release compared to MPO-deficient mice. To test further whether MPO in TANs supports PDAC growth, we co-injected PDAC tumor cells with either WT or MPO -/- TANs (1:1 ratio) into the pancreas of WT or MPO -/- recipient mice. The addition of WT TANs restored tumor growth that was otherwise inhibited by MPO deficiency, indicating that TAN-derived ROS and NETs promote tumor progression. Furthermore, loss of MPO-derived ROS and NETs in TANs influenced collagen remodeling within orthotopic tumors, potentially enhancing drug delivery to the tumor microenvironment. Lastly, PDAC tissues and patient blood-isolated neutrophils exhibited elevated ROS compared to controls ex vivo. Importantly, ROS levels correlated strongly with NET formation in these patient samples. These findings reveal a bidirectional relationship between ROS and NETs and highlight the potential utility of L-012 for monitoring ROS and NET-associated inflammation and tumor progression in pancreatic cancer. Citation Format: Angisha Basnet, Kaitlyn M. Landreth, Brian A. Boone, Tracy W. Liu. Linking reactive oxygen species and neutrophil extracellular traps formation to pancreatic ductal adenocarcinoma progression [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr A037.