ABSTRACT/SUMMARY Radiotherapy (RT) is an effective treatment modality for pelvic malignancies. However, radiation cystitis (RC) is a widely recognized irreversible and chronic condition reported in 8-11% of cancer patients treated with pelvic RT. The symptoms of RC can include hematuria, increased urinary frequency and urgency, incontinence, and dysuria. Few effective treatments exist to alleviate these adverse symptoms, and there are no FDA approved preventative agents. Hyperbaric oxygen therapy does alleviate some symptoms, but its use is restricted by inaccessibility, cost, and contraindications, leading to poor patient compliance. The ill-defined pathophysiology and mechanisms of RC thwart the development of new therapies. Our GWAS in six large prostate cancer (PCa) RT cohorts identified SNPs, tagging AGT, correlated with patient-reported hematuria, a defining symptom of RC. AGT encodes angiotensinogen, part of the renin-angiotensin system (RAS). Our subsequent multi-site clinical study supports the hypothesis that angiotensin-converting enzyme inhibitors (ACEi) are radioprotective in the bladder; RC was seen in 16.5% of patients not taking an ACEi vs only 4.8% of those taking an ACEi during RT (p=0.01). In the same clinical cohort, release of extracellular vesicles into the urine (uEVs) and increased circulating levels of the pro-inflammatory chemokine CCL2 were significantly associated with symptoms of RC. In our murine model, ACEi protected against micturition changes, immune cell recruitment, and urothelial injury after RT. Based on these data, we hypothesize that RAS modulation and preventing CCL2-dependent immune cell recruitment will prevent bladder injury after RT. Our objectives are to characterize and investigate the mechanistic role of RAS and its pharmacologic modulation in tissue inflammation and injury in the bladder after RT and develop a urine-based biomarker of clinical RC. Our four specific aims use a combined preclinical and translational approach: Aim 1: To characterize mechanisms of bladder inflammation and immune cell recruitment in the development of bladder injury after single dose and fractionated RT, as a function of dose and irradiated volume; Aim 2: To determine optimal duration of ACEi or angiotensin receptor blockers (ARBs) to elicit maximal radioprotection, and the mechanism of prevention of progressive bladder injury, and determine the therapeutic ratio using an orthotopic PCa model; Aim 3: To assess bladder uEV release kinetics by nanoparticle tracking analyses as a predictive biomarker of RT injury, and characterize EV cargo proteins and functional ability to induce cellular stress/damage response in our mouse models; and Aim 4: To validate uEV kinetics as a biomarker of late RC, and predictor of RAS modulator response, using biosamples in two clinical studies of men receiving RT for PCa. An EV biomarker could be used to identify patients needing early mitigating interventions, such as an ACEi, to avoid progr...