Clinical and pre-clinical investigation of extracellular vesicles as a mechanism of toxicity in the bladder of prostate cancer patients treated with radiotherapy - We propose to study extracellular vesicles (EVs) as novel mediators of the pathogenesis of radiation cystitis (RC), which affects approximately 15% of prostate cancer (PCa) patients treated with radiotherapy (RT). Bladder radiotoxicity impacts the lives of PCa survivors, most notably older patients, and there are no predictive markers associated with its incidence, nor are there durable molecular therapies capable of preventing RC. There is an urgent need for the development of effective medical countermeasures against RC and the identification of molecular diagnostics to aid in mitigating this debilitating treatment-related complication. EVs are lipid-bound nanoparticles that mediate intercellular communication by delivering cargo molecules to neighboring or distant cells. Studies have shown that RT induces EV release and alters the composition of these EVs. However, most research to date has been conducted in cell lines, with limited preclinical studies and no human research. Using preserved samples collected from a genome-wide association study (GWAS), we reported for the first time a link between urinary EV (uEV) levels and the future onset of hematuria, providing a promising biomarker that may enable more timely treatment of at-risk patients to mitigate the development of late bladder toxicities. The prognostic utility of EVs in serum was less robust. This is further supported by an animal study showing a correlation of uEV particle counts with RT-induced bladder toxicity. Critically, post-RT uEVs derived from PCa patients who developed late hematuria induced substantial oxidative stress in normal bladder recipient cells, further supporting a functional link between EVs and radiotoxicity. Proteomic analyses of 12 uEV samples revealed that those EV cargo proteins were profoundly altered by RT. Notably, we identified 60 RT-toxicity signature uEV proteins including many with functions associated with innate immunity and neutrophil activity, highlighting potential mechanism(s) of action. Based on these studies, we hypothesize that RT induces the release of EVs and alters their composition, and the resultant RT-EVs carry immunologically active biomolecules, thereby inducing additional cellular damage. The kinetics of RT-EV release and RT-EV cargo molecules can serve as predictive biomarkers for RT-induced toxicity that will allow for early intervention to mitigate RT toxicity. We propose three Specific Aims. Aim 1: To define the roles of radiation-induced extracellular vesicles in mediating RT-induced bladder damage in vitro and in vivo. Aim 2: Characterization of RT-EV cargo molecules and their roles in mediating radiotoxicity. Aim 3: To establish body fluid-derived EV-based predictive biomarkers for RT-induced toxicity. Accomplishing the proposed studies will define the functional roles of RT-EVs as mediators of RT toxicity. RT-induced EV release kinetics and alterations in EV cargos will represent sensitive, noninvasive urine/blood-based EV biomarkers that can predict clinically relevant RT outcomes before the onset of late toxicities.
