Spatial and mechanistic assessment of the role of stromal fibroblasts in driving emergence of aggressive prostate and bladder cancer

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ABSTRACT This application is being submitted in response to the Notice of Special Interest (NOSI) identified as NOT-CA- 23-045. Prostate and bladder cancers are the two most frequent genitourinary cancers, and their progression from low to high aggressiveness remains poorly understood. Stromal-epithelial-immune interactions, particularly involving fibroblast activation protein (FAP)-expressing fibroblasts, may contribute to tumor aggressiveness. However, the precise role of these cells in the tumor microenvironment (TME) and the molecular mechanisms driving cancer progression remain unclear. We hypothesize that FAP+ fibroblasts engage in cell-cell interactions and paracrine signaling, supporting cancer cell growth and invasion, leading to aggressive cancer formation. To test this hypothesis, we propose two specific aims: (1) Integrate spatial transcriptomics and multiplex immunohistochemistry/in situ hybridization (IHC/ISH) analyses of human prostate and bladder cancer tissues to define the molecular phenotype and spatial relationships between FAP+ fibroblasts, cancer cells, and other stromal cell compartments in the TME; and (2) Investigate the functional role of FAP+ fibroblasts in aggressive prostate and bladder cancer using genetically engineered mouse models (GEMMs) and spatial transcriptomic alterations with and without FAP genetic disruption. We will perform cutting-edge spatially resolved transcriptomic and multiplex IHC/ISH analysis of human prostate and bladder cancer tissues using advanced platforms. These data will be integrated using the AstroPath platform, which will be extended to handle the spatial transcriptomics data alongside the multiplex in situ methods. To investigate the functional role of FAP+ fibroblasts in aggressive cancer development, we will employ GEMMs and evaluate spatial transcriptomic alterations with and without FAP genetic disruption. These data will augment phenotypic studies and allow investigation of FAP's role in cell-cell spatial relationships and paracrine signaling mechanisms. Findings will be validated using multiplex IHC/ISH panels. Expected Results and Impact: Our study will provide insights into FAP's role in prostate and bladder cancer and its potential as a therapeutic and theranostic target. The use of spatial transcriptomics and multiplex in situ immunohistochemistry will enable identification of FAP-expressing cells and their spatial relationship with other TME components. The use of GEMMs will facilitate investigating FAP's functional role in tumor growth, angiogenesis, and metastasis. Validating findings in human tissues will provide clinical relevance. Overall, this study will contribute to understanding the molecular mechanisms underlying prostate and bladder cancer, potentially leading to novel therapeutic strategies targeting FAP. This pilot study will establish the feasibility of these methods and models in our groups, allowing comparison of FAP+ fibroblasts' role in both cancer types and laying the foundation for longitudinal collaboration beyond the Supplement award to facilitate inter-institutional collaboration through our U54 TBEL consortium.