Project Summary: Most men diagnosed with prostate cancer (PCa) are 65 years of age or older. Radiotherapy (RT) is an effective and curative treatment for early-stage PCa. However, 20% of PCa patients can develop late, often irreversible, chronic adverse side-effects after their RT. Radiation cystitis (RC) is a clinically significant chronic toxicity. Symptoms of RC include hematuria and urinary dysfunction, including increased urinary frequency and urgency, incontinence, and dysuria, and these can be significant clinical concerns for the ~3.1 million PCa survivors in the US. These symptoms can be more pronounced in older PCa patients because of preexisting urinary symptoms and/or reduced functional reserve, putting them at increased risk of dysfunction after RT induced injury. RT dose-volume constraints are used to lower the risk of developing a treatment-related toxicity, but the bladder cannot be avoided completely in the PCa RT treatment plans, and significant variation in the incidence and severity of RC exists amongst PCa patients which suggests an important role for host factors personal to each PCa patient. The urinary microbiome is altered in urologic diseases common in older adults, and dysbiosis has been linked with urinary dysfunction unrelated to infection. But, no studies have investigated the effect of RT on the urinary microbiome, or the effect of dysbiosis on RC, among PCa patients. Therefore, we hypothesize that RT-mediated urothelial damage in the bladder that leads to disruption of routine urinary function, and increases urinary frequency, will produce urinary microbiome dysbiosis, and therefore microbiome dysbiosis could be a non-invasive urine-based early biomarker of RC. Our clinical genetic studies identified signaling pathways linked to the microbiome and immune cell recruitment that support this hypothesis. Using banked urine from our prospective clinical study of RC with up to 5 years of follow-up, as well as our preclinical mouse model of RC, we have demonstrated we can detect a urinary microbiome despite the low abundance, and we show evidence of dysbiosis after RT. This project will use banked urine from our clinical study to test the hypothesis that (i) the microbial composition and/or metabolic potential of the urinary tract is changed by RT, and (ii) dysbiosis and/or metabolic dysregulation will correlate with patient reported outcomes (PROs) capturing bladder dysfunction following RT. Then, using our mouse model of RT-induced bladder injury, we will test the hypothesis that (iii) changes in the microbiome may serve as a potential biomarker of RT injury to the urothelium, and if changes in microbiome are a response to tissue injury and recruited immune cells. This will be achieved by comparing dysbiosis with functional micturition, and local and systemic biomarkers of bladder injury, after focal RT to the entire bladder volume or just bladder trigone. Finally, using banked urine from both our human and mouse studie...