The molecular mechanisms that regulate progression of bladder cancer to invasion and metastasis are poorly understood, in part due to the lack of suitable model systems that reflect the biology of the human disease. In recent work, we have used three-dimensional culture approaches to establish a novel biobank of human patient-derived organoids from primary bladder tumors. These organoid lines recapitulate the histopathological and molecular diversity of non-muscle invasive bladder cancer as well as muscle invasive bladder cancer, and can be readily converted into xenografts by orthotopic transplantation. We hypothesize that these patient-derived organoid lines represent a suitable model for functional analyses of bladder cancer metastasis, and will therefore pursue three specific aims: 1) Investigation of tumor invasiveness in organoid culture to examine the roles of extracellular matrix and tumor-specific mutations in conferring invasive properties to bladder tumor cells; 2) Molecular analysis of metastasis by orthotopic transplantation of organoids to analyze the metastatic ability of organoid-derived xenografts in vivo; and 3) Investigation of collective invasion and dissemination using multi-color organoids to determine whether metastasis is mediated by single cells or multicellular (collective) clusters. Overall, this proposal represents a highly innovative project that uses human patient-derived tumor organoids to identify tumor cell autonomous and microenvironmental factors that regulate progression to metastatic bladder cancer.