PROJECT SUMMARY Bladder cancer is one of the top 10 most frequent cancers, yet the most underfunded cancer by NCI based on funding dollars and ratio of funding/mortality. There has been no significant improvement in overall survival and prognosis over the last thirty years except for the recent development of immunotherapy. After initial diagnosis and treatment, over 60% of the BC patients will relapse within two years and progression into advanced stages in up to 25% of patients. Therefore, almost all patients will need long-term expensive cystoscopy which makes bladder cancer the costliest cancer (per case) of all cancer types. If a sensitive but less expensive method to detect cancer were available, it would improve the treatment outcomes and decrease the cost. Dr. Xuefeng Liu (PI) and his team recently invented a robust method of conditional reprogramming (CR) technology to establish patient-derived cell models from cystoscopy or needle biopsies and urine samples of BC patients. The overall success rate was near 100%. These urine cancer cell cultures (UCCC) provide a simple, non-invasive, comfortable, reliable method to detect BC recurrence, and represent a living biomarker to predict patients’ response to chemotherapy and targeted therapy. Several cancer centers and laboratories including PI’s lab are actively involved in clinical trials to validate clinical utility of the UCCC in BC clinics. Since a variety of patient- context and collection, storage and transportation of urine samples to laboratories for cell cultures affect enrichment and viability of cancer cells in urine samples, it is urgent needed to evaluate pre-analytical factors of urine samples for UCCC and optimize these non-Invasive and living liquid biopsies as a routine approach in monitoring recurrence and prediction of response of BC patients. In this application, we propose to address the gaps by extending the work done by our team members on evaluating effects of sample processing protocols on UCCC analysis, to further investigate effect of patient-specific context. In this application, we will first further optimization of UCCC system to establish a culture kit for clinical applications, specifically we will optimize and simplify conditions with co-culture kits, conditioned medium (CM), hypoxic condition for maximal efficiency and robustness. Second, we will establish a Standard of Procedure - SOP- for urine collection and transportation in clinics: we will test pH, temperatures, minimal cell number, a cocktail with anti-biotics and Y-27632 affect UCCC cultures and success rate. Last, we will determine patient-context factors that affect UCCC cultures: we will evaluate how urine timing (days before surgery, and after surgery, early morning, noon, night, drinking water before urine collection, etc), urine volume affect cell viability and UCCC. Last, we will optimize a self- sampling procedures for BC patients. The overall goal is to expedite UCCC clinical assay development...