Imaging Reactive Oxygen Species with L-012 Reveals Neutrophil Extracellular Trap Formation in Pancreatic Ductal Adenocarcinoma.

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, an increase in neutrophils infiltrating the tumor microenvironment promotes cancer progression and metastasis through increased ROS production and NET release. Using bioluminescence imaging with the reporter L-012 and NET assays, we assessed ROS and NET release, respectively, induced by phorbol myristate acetate and platelet-activating factor in bone-marrow-isolated neutrophils from wild-type and syngeneic myeloperoxidase (MPO)-deficient mice ex vivo. MPO deficiency impaired both ROS generation and NET release, establishing a positive correlation between these processes. In vivo analyses using subcutaneous and spontaneous murine PDAC models revealed elevated ROS in tumors, which were significantly reduced upon genetic deletion of host MPO or peptidyl arginine deiminase 4, an essential enzyme for NET formation, or after treatment with hydroxychloroquine, a NET inhibitor. Furthermore, luminol and 4-[