Project Summary The presence of tumor invasion into the submucosal muscle of the bladder defines the progression from nonlethal to lethal bladder cancer; however, this process is not well understood at the molecular level. Genomic and transcriptomic profiling of muscle invasive bladder cancer (MIBC) has recently identified mutations and gene expression profiles associated with established MIBC. Although a major advancement in the bladder cancer field, these analyses were unable to determine the unique transcriptional programs activated in individual tumor, stromal and immune populations at the invasive interface of MIBC. This was primarily due to the limitations of accurate spatial collection of infiltrating tumor and adjacent/intermingled stroma and infiltrating immune cells. As a result, earlier transcriptomic studies and even recent studies using single cell RNAseq relied on heterogeneous bulk tumor, which includes both noninvasive as well as normal stroma and immune components spatially distant from the invasive border. This heterogeneity makes it difficult to accurately de-convolute gene expression signatures unique to tumor cells, stroma and infiltrating immune cells in the immediate invasive micro- environment. We hypothesize that distinct gene networks are activated in individual tumor, stromal and immune populations at the invasive interface of MIBC. RNAseq profiling and bioinformatic analysis should reveal enriched and distinct transcriptomic signatures from the individual populations. This research would provide positive impact by identifying cellular and molecular signatures of distinct cellular populations driving invasive progression. These findings could then lead to examination of candidate gene networks that will serve as the basis for biological function, potential biomarker identification and new therapeutic targets that are needed for bladder cancer patients who have been diagnosed with or have relapsed with muscle invasive disease.