Prostate cancer (PCa) affects approximately 12.5% of men in the US during their lifetime,¹ and radiotherapy (RT) remains a mainstay of PCa management in clinical practice. RT is used alone or in combination with hormone therapy and/or surgery in more than 50% of PCa cases.² Despite its clinical benefits, RT causes persistent radiation cystitis (RC) and related forms of late bladder toxicity in roughly 25% of treated patients.^3,4 There is no Food and Drug Administration–approved preventative therapy for this debilitating form of radiotoxicity, which is characterized by hematuria, leading to a pronounced decline in quality of life and decisional regret in many cases. The discovery of biomarkers capable of predicting RC in PCa patients before it develops has the potential to inform treatment planning and elucidate the molecular basis for this condition, enabling the design of appropriate pharmacological interventions that can mitigate the risk of this devastating bladder radiotoxicity.

Extracellular vesicles (EVs) are small, membrane-enclosed vesicles released from all nucleated cells that contain a variety of biologically active macromolecular cargoes specific to their cells of origin. Our group recently reported that urinary EV (uEV) particle counts increased significantly at the end of RT in PCa patients who developed subsequent hematuria 6 months to 5 years post RT, whereas such induction by RT was not found in patients who did not develop hematuria over the course of similar follow-up.⁵ This suggests that uEVs may serve as a valuable biomarker for predicting future RC risk, potentially enabling timely and earlier intervention for at-risk patients to mitigate the development of late bladder toxicities. Given the functional role of EVs in many biological systems, we performed a differential proteomics analysis of paired uEV samples collected before RT and at the end of RT from 6 PCa patients, 3 of whom developed post-RT hematuria and 3 who did not. This approach revealed the enrichment of neutrophil-related proteins in the uEVs from the donors who developed hematuria, with some of these proteins having been enriched even before RT (Figure 1), raising the tantalizing possibility of predicting RC risk even before RT. Given these findings and a growing body of evidence suggesting a link between neutrophil functions and adverse post-RT outcomes in cancers and other preclinical settings,^6-8 we conducted a series of in vitro analyses aimed at clarifying the effects of irradiation on neutrophils.

We generated neutrophil-like cells suitable for experimental use by using either dimethyl sulfoxide or all-trans retinoic acid to differentiate human promyelocyte-like HL60 cells for 5 days, confirming their successful differentiation based on flow cytometry and quantitative reverse transcription polymerase chain reaction analyses of neutrophil markers. We then evaluated the longitudinal responses of these cells to irradiation in an effort to determine whether irradiation can directly activate neutrophils or induce their release of EVs. Following irradiation (2 Gy or 10 Gy), differentiated HL60 (dHL60) cells exhibited time-dependent changes in neutrophil effector gene expression that coincided with increased oxidative burst activity and the release of significantly higher numbers of EVs. Strikingly, the co-culture of these irradiated dHL60s with SV-HUC bladder urothelial cells led to a significant decline in SV-HUC cell viability and higher rates of apoptotic urothelial cell death, while the knockout of the RAB27A gene required for EV biogenesis or the use of the Rab27a inhibitor Nexinhib20 reversed this irradiated neutrophil-mediated cytotoxicity, supporting a role for neutrophil-derived EVs and their cargoes as mediators of urothelial damage (Figure 2).

Our in vitro findings support a functional link between RT-associated neutrophil EV release and late bladder toxicity in PCa patients, suggesting that neutrophil-derived EVs may offer value as predictive biomarkers for RC and that neutrophils or their EV release pathways may be viable targets for efforts to prevent this form of radiotoxicity. Given our enticing data and the growing interest in neutrophils as RT-related biomarkers in various cancers,⁹ we are in the process of conducting further detailed bioinformatics analyses of our uEV and matched serum EV proteomic datasets from PCa patients with and without late hematuria and corresponding functional analyses that we hope will inform efforts to mitigate the risk of late bladder radiotoxicity, ultimately leading to better patient outcomes.