# Project 2 > **NIH NIH U54** · CEDARS-SINAI MEDICAL CENTER · 2022 · $446,235 ## Abstract PROJECT SUMMARY Bladder cancer (BC) patients present with non-muscle (NMI) or muscle invasive (MI) 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 mechanisms of NIMBC progression; 2) risk stratification of NMIBC to patients likely to progress. Project Integration: Project 1 & 2 address the first gap and provide new markers for Project 3 to address the third. The premise of Project 2, based on prior literature, is that progressing NMIBC harbor similar cancer cell populations as MIBC. To test this hypothesis, we carried out single nuclei RNA sequencing of MIBC and identified 18 distinct epithelial and stromal populations based on their unique gene expression profiles. When gene signature scores of each population were calculated for NMI (N=327) and MI (TCGA) BC based on their bulk RNA data, high scores of only one malignant epithelial and 2 cancer-associated fibroblast (CAF) populations associated with progression and survival, respectively. Only prevalence of CAFs expressing fibroblast activation protein (FAP) stratified outcomes and progression of NMI to MI BC, suggesting FAP+ CAFs cooperate with this epithelial population to drive progression. Spatial profiling found this epithelial population proximal to CAFs, and ligand-receptor analysis suggested communication with CAFs, leading us to name these cells “C3”, for “Cell-Cell Communication”. Interestingly, RNA velocity analysis to computationally decern lineage relationships in the normal urothelium found cells with C3 gene signatures in a trajectory following basal cells. Thus, C3 tumor cells may arise from transformation of normal C3 cells during differentiation. Our Specific Aims test the Hypothesis that NMIBC progression is driven by C3 tumor cell intrinsic properties and C3 interactions with FAP+ CAFs. In Aim 1 we use lineage tracing of our novel iPSC-based model of normal bladder development which finds C3 cells and FAP+ fibroblasts emerge during bladder differentiation and spatial proteomic data from normal bladder to test the hypothesis that C3 cells emerge from basal cells and promote the appearance of FAP+ fibroblasts. In Aim 2 we examine the intrinsic determinants of transformation/progression of C3 cells using functional genomics in an autochthonous murine model and human xenograft models of BC to test the hypothesis that expression of some of 14 candidate C3 driver genes is necessary for carcinogenesis, tumor growth and progression. In Aim 3 we co-culture human tumor cells with known levels of C3 with normal bladder fibroblasts and BC CAFs and use murine BC cells implanted intravesically in mice with fibroblast defects in FAP to test the hypothesis that C3 cells promote FAP+ CAFs, which reciprocally drive C3 expansion in a vicious cycle to drive tumor growth a... ## Key facts - **NIH application ID:** 10519083 - **Project number:** 1U54CA274375-01 - **Recipient organization:** CEDARS-SINAI MEDICAL CENTER - **Principal Investigator:** Dan Theodorescu - **Activity code:** U54 (R01, R21, SBIR, etc.) - **Funding institute:** NIH - **Fiscal year:** 2022 - **Award amount:** $446,235 - **Award type:** 1 - **Project period:** 2022-09-22 → 2022-12-20 ## Primary source NIH RePORTER: https://reporter.nih.gov/project-details/10519083 ## Citation > US National Institutes of Health, RePORTER application 10519083, Project 2 (1U54CA274375-01). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10519083. Licensed CC0. --- *[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*