PROJECT 3 SUMMARY Bladder cancer is the second most common urologic malignancy affecting 573,278 people worldwide in 2020. Pathologically, bladder cancer (BC) can be classified into non-muscle-invasive (NMI) and muscle-invasive (MI) disease. Here we define early bladder lesions as non-muscle-invasive bladder cancer (NMIBC). Major clinical gaps in NMIBC include i) lack of mechanistic insights defining NMIBC progression, and ii) lack of platform for risk stratification of NMIBC that recur but never progress (i.e., “non-progressors”), from those that progresses into MIBC (i.e., “progressors”) and consequently demonstrate poor prognosis. The goal of our Center is to tackle this clinical issue by deciphering the underlying mechanisms restraining or promoting the progression of early lesions (Project 1 & 2), and to leverage this novel biology as candidate biomarkers to risk stratify aggressive NMIBC (Project 3). Project 3 is unique to test the hypothesis that a bladder cancer-associated “fingerprint” exists, which can be leveraged to risk stratify aggressive bladder cancer in a non-invasive assay as urine biomarkers prior to its current clinical presentation. Specific Aims: 1) To prospectively evaluate a bladder cancer-associated fingerprint” for its ability to risk stratify aggressive bladder cancer, with the propensity to progress, before their clinical presentation; and 2) To evaluate the relationship of our bladder cancer-associated “fingerprint” to early microenvironment changes within the stromal compartment, using a relevant and established carcinogen-induced bladder cancer mouse model. Significance: We believe the BC “fingerprint” can be exploited to identify aggressive bladder cancers that are more likely to be progressors from non- progressors. Project Integration & Benchmark of Success: Project 3 will leverage a clinical grade, BC “fingerprint” as a non-invasive assay. This will serve as an initial biomarker panel, while biology-backed insights from Project 1 & Project 2 will reveal novel biomarkers associated with corresponding tumor-restraining and tumor-promoting mechanisms, respectively. Such project integration will open the door for optimizing the non- invasive method for risk stratification of aggressive NMIBC, and as such, it will have a marked impact on patient care as the long-term goal.