Abstract Introduction: Integrin signaling plays an important role in cellular proliferation and migration via interactions with extracellular matrix proteins. Prior studies indicate that integrin signaling facilitates tumor invasion and metastasis, and there are several ongoing clinical trials using agents that modulate this pathway. We recently identified clonal enrichment in missense mutations in the integrin cell surface interactions pathways in advanced chemotherapy-resistant urothelial carcinoma. An ideal strategy for investigating integrin signaling is via 3D organoid culture, maintaining intercellular interactions that replicate the epithelial microenvironment. We hypothesize that pharmacologic integrin signaling modulation will impair organoid growth in human bladder cancer cells and demonstrate a potential therapeutic utility for this approach. Methods: RT4 human bladder cancer cell line was used as well as a second cell line established from a patient-derived bladder cancer sample (PM748). Cells were grown in 3D organoid culture as previously described. For in vitro integrin modulation, defactinib (VS-6063), an orally-bioavailable selective inhibitor of focal adhesion kinase (FAK, a convergent and conserved enzyme activated by integrin ligand binding), was used. SDS-PAGE and immunoblotting were performed to show in vitro FAK inhibition. Single-cell suspensions and formed organoids were plated in the presence of various concentrations of defactinib to determine the impact on organoid formation and regression. Results: Both RT4 and PM748 bladder cancer cell lines demonstrated consistent organoid growth in three-dimensional culturing conditions. Addition of defactinib to cultured cells showed a dose-dependent decrease in autophosphorylation of FAK for both cell lines, demonstrating effective FAK inhibition. 3D culture of single cells in the presence of defactinib produced a dose-dependent decrease in organoid size after 96 hours (mean size for DMSO only, 100nM, 1uM, and 10uM were 128um, 75um, 48um, and 26um, respectively; p<0.0001 versus DMSO for all dilutions). Established bladder cancer organoids showed a dose-dependent regression in size after 72 hours of defactinib exposure (mean size for DMSO, 100nM, 1uM, and 10uM were 225um, 96um, 70um, and 34um, respectively; p<0.0001 versus DMSO). Experiments utilizing Crispr-Cas9-mediated FAK knock-out as well as in vivo studies with FAK inhibitors in mouse xenograft models are currently underway. Conclusions: Integrin modulation via FAK inhibition with defactinib causes both inhibition of organoid formation as well as regression of formed organoids, and the effects are seen at concentrations well below the cytotoxic range for the drug. This study suggests a utility for these agents in bladder cancer treatment. Citation Format: LaMont Barlow, Rebecca Meyer, Ethan Shelkey, Bishoy Faltas, Mark Rubin. Integrin signaling modulation demonstrates potential therapeutic strategy in bladder cancer using three-dimensional organoid culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5941. doi:10.1158/1538-7445.AM2017-5941