PROJECT SUMMARY Carcinomas are the most common cancers. In tumors arising in hollow organs, two major events during tumor progression are disruption of the basement membrane and invasion of the smooth muscle layer. In bladder cancer, muscle-invasion is associated with lymph node and distant metastases and poor prognosis. Despite extensive interest in the tumor microenvironment, virtually nothing is known about how tumor cells and smooth muscle cells (SMC) interact. Preliminary observations support our central hypothesis that the smooth muscle layer may promote, rather than restrain, tumor progression and that it can impact on the tumor cell phenotype in a context- dependent manner. We aim to address this significant knowledge gap through a top-down systems and bottom-up experimental approach. In Aim 1, we propose combining high-throughput and spatially-resolved single cell expression profiling with innovative computational analyses of cell-cell interactions in samples from patients with muscle-invasive bladder cancer to identify cellular interactions that modulate tumor cell phenotype at the muscle-invasive front. Special emphasis will be placed on interrogating tumor cell-SMC crosstalk at this interface, although all autocrine and paracrine interactions will be analyzed at a systems level in an unbiased fashion. In Aim 2, we propose using co-culture systems to mechanistically dissect tumor cell-SMC crosstalk, including identifying transcriptomic changes induced by SMC on tumor cells, determining whether this cross- talk is mediated by contact or soluble factors, and identifying candidate pathways that underlie this effect for downstream validation. The amalgamation of these approaches will reveal markers of SMC activation with prognostic and/or predictive value and may lead to the discovery of new therapeutic targets for muscle-invasive bladder cancer. We expect to uncover a yet unexplored role of the tumor microenvironment in bladder cancer phenotypes and progression.