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

Authors

Angisha Basnet
Kaitlyn M Landreth
Michael Sestito
Kristen Ranson
Seth T Gammon
David Piwnica-Worms
Brian A Boone
Tracy W Liu

Language

English

Publication Date

12-8-2025

Journal

Antioxidants

DOI

10.3390/antiox14121473

PMID

41462673

PMCID

PMC12729264

PubMedCentral® Posted Date

12-8-2025

PubMedCentral® Full Text Version

Post-print

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, 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-[18F]fluoro-1-naphthol ([18F]4FN), functional L-012 analogs, also enabled non-invasive detection of intratumoral ROS by bioluminescence and PET imaging in vivo, respectively; [18F]4FN PET showed a three-fold increased uptake in PDAC tumors versus muscle. PDAC tissues and blood-isolated neutrophils obtained from PDAC patients exhibited elevated ROS compared to controls ex vivo. Importantly, ROS levels correlated strongly with NET formation in patient samples. These findings reveal a bidirectional relationship between ROS and NETs and highlight the potential utility of L-012- and [18F]4FN-based PET imaging for monitoring NET-associated inflammation in PDAC in vivo.

Keywords

reactive oxygen species, neutrophil, neutrophil extracellular traps, pancreatic ductal adenocarcinoma, bioluminescence imaging, L-012, 4-[18F]fluoro-1-naphthol ([18F]4FN), PET imaging

Published Open-Access

yes

Recommended Citation

Basnet, Angisha; Landreth, Kaitlyn M; Sestito, Michael; et al., "Imaging Reactive Oxygen Species with L-012 Reveals Neutrophil Extracellular Trap Formation in Pancreatic Ductal Adenocarcinoma" (2025). Faculty, Staff and Student Publications. 6426.
https://digitalcommons.library.tmc.edu/uthgsbs_docs/6426