Extracellular vesicle proteomics identify neutrophils as potential mediators of bladder radiotoxicity in prostate cancer.

Radiation cystitis (RC) is a chronic debilitating complication that impacts 8-11% of patients with prostate cancer (PCa) after radiotherapy (RT), reducing quality of life and contributing to decisional regret. Reliable treatments to alleviate the symptoms of RC, which include gross hematuria, are lacking, emphasizing the need for predictive biomarkers of RC to support therapeutic decision-making. We performed paired liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses of the protein cargos in extracellular vesicles (EVs) from the urine of PCa patients who did or did not develop late hematuria following standard-of-care RT. We identified a 60-protein RT-toxicity-associated signature enriched for neutrophil-related proteins, several of which, including myeloperoxidase (MPO), were elevated even before RT in urinary EVs from patients who later developed hematuria. In healthy mice, focal bladder irradiation was sufficient to elicit a localized neutrophil response in the bladder, while in vitro co-culture assays confirmed the ability of neutrophils to induce urothelial cell killing following irradiation, a process that was abrogated by the CRISPR/Cas9-mediated deletion of MPO or the EV release regulator Rab27α. Serum EV proteomics from these same patients further validated the neutrophil-related RC risk signature, and enzyme-linked immunosorbent assays (ELISAs) gave additional support to MPO and other neutrophil-related proteins, including defensin α3 (DEFA3) and neutrophil elastase (ELANE), as possible predictors of late hematuria development following RT in a larger cohort of PCa patients. Neutrophils may thus at least partially mediate bladder radiotoxicity in PCa patients. The multi-protein risk signatures and specific biomarkers identified herein may provide an opportunity for risk stratification in PCa.