Project 1

PROJECT 1 SUMMARY Bladder cancer (BC) patients present with non-muscle (NMI) or muscle (MI) invasive disease, and 20% of NMI progress to MI during follow up with a major reduction in survival. Hence, for this proposal we define early bladder lesions as non-muscle-invasive bladder cancer (NMIBC) and seek to address the two major clinical gaps in NMIBC: 1) defining actionable mechanism of NIMBC progression; 2) risk stratification of NMIBC to patients likely to progress. Project 1 is unique to investigate a novel and understudied tumor restraining mechanism, i.e., how “immunofibroblasts” instruct the formation of ectopic immune cell microarchitectures known as “tertiary lymphoid structures” to mount a local anti-tumoral response. Progress in studying immunofibroblasts and their regulation of tertiary lymphoid structure (TLS) formation is hampered by the lack of preclinical study models recapitulating the TLS phenotype. Project 1 is significant to i) reveal TLS phenotype in a published cohort of human NMIBCs, ii) overcome a technical hurdle in the field by describing a consistent TLS phenotype within early bladder lesions from a carcinogen-induced mouse model, and iii) discover a candidate immunofibroblast population that co- organize TLSs with other lymphoid tissue inducer cells by single-cell RNA sequencing. Project 1 is innovative to dissect the understudied roles of these immunofibroblasts and their interactions with lymphoid tissue inducers and B cells to instruct TLS formation. Project Integration & Benchmark of Success: Project 1 will reveal biological insights into the tumor restraining role of immunofibroblasts/TLSs and how they oppose the tumor- promoting mechanisms proposed in Project 2. Furthermore, secreted proteins and factors from these immunofibroblasts/TLSs will provide new candidate biomarkers for evaluation in Project 3. Success of Project 1 will validate immunofibroblast-mediated TLS formation as a tumor-restraining mechanism in early bladder lesions and provide relevant targets for future precision intervention.