Bladder cancer, the fifth most common malignancy in the U.S. with approximately 70,000 new cases diagnosed each year, is essentially incurable after it has begun to progress. Much of the difficulty lies in the scarcity of mechanistic insightsin the functional involvement of the fibroblastic microenvironment, and its interactions with the epithelial cancer compartment in modulating early bladder cancer invasion. The applicant and his collaborators have made considerable strides in closing this gap. They were among the first to isolate and characterize human bladder cancer stem cells. Recently, they showed that an expansion of phenotypic cancer stem cells correlates with a poorer clinical outcome and bladder cancer invasion. Their proposal aims to extend these findings by elucidating the functional contribution of cancer-associated fibroblasts (CAFs) in the modulation of bladder cancer stem cells and tumor progression. This application has its foundation via demonstrating that bladder cancer patients with an elevated expression of CAF genes also have muscle-invasive cancer and a poorer survival. Moreover, activated CAFs localize immediately adjacent to phenotypic cancer stem cells, suggesting a possible functional interaction between these two cell types. Additional findings indicate an important role of collagen I (COL1) excreted by CAFs, which can bind to discoidin domain receptor tyrosine kinase 1 (DDR1) on bladder cancer cells and modulate their tumorigenic properties. This progress has led to a novel working hypothesis - that COL1-DDR1 signaling between activated CAFs and bladder cancer cells is one of the mechanisms exerted by CAFs to regulate cancer stem cells and tumor progression. Three specific research aims will be pursued. Aim 1 seeks to establish the functional involvement of CAFs in the regulation of cancer stem cells and tumor progression, with an emphasis on using primary patient specimens. To further define the clinical significance of CAFs, we will utilize tw independent patient cohorts to assess the expression of CAFs/COL1 in relation to bladder cancer differentiation and selected clinical prognostic information. Aim 2 will employ both gain-of-function and loss-of-function strategies to define the functional contributions of DDR1 on bladder cancer cells to mediate the biological properties promoted by CAFs. These studies will be aided by the availability of newly isolated bladder CAFs and the applicant's expertise in isolating cancer stem cells for subsequent analysis. Finally, Aim 3 will access Stat3 and additional downstream mechanisms related to DDR1, using established proteomics profiling approach. The applicant intends to uncover druggable targets within this pathway for early bladder cancer intervention, as the long-term goal of this proposal.