Abstract Background: Drug screening is a key component for drug development and optimizing anti-tumor therapies. Traditionally, in vitro drug testing has been conducted in monolayer systems that are not capable of recapitulating the tumor complexity. Recently, the field has witnessed the rise of interest in 3D culture systems which are capable of reproducing tumor complexity while circumventing the cost associated with in vivo drug testing. Our access to fresh patient samples has enabled us to establish a novel 3D culture system consisting of bladder cancer patient derived cell lines. Using a wide range of matrices and co-culture conditions with tumor associated stromal cells we were able to establish a unique high throughput drug testing tool. Methods: Matrigel and collagen based matrices were used to establish 3D culture systems of bladder cancer patient derived cells. Tumor cells were cultured in 3D conditions either alone or in coculture with tumor associated stromal cells. Response to Cisplatin and PI3K pathway targeted agents (i.e. LY LY3023414) was tested in both conditions. High throughput imaging via Thermo ArrayScan XTI was used to assess the biological behavior of spheroids as well as their response to therapies overtime. Confocal microscopy was used to validate the biological mimicry of tumor derived spheroids to the original patient tumors. Integration of RNA-seq data from the patient-derived tumor cells with the biological behavior and therapeutic response in 3D culture is ongoing for the purpose of characterizing the 3D model Results: In 3D culture conditions; bladder cancer derived cells were able to re-express E-cadherin that was suppressed upon propagation in monolayer. The re-expression of the epithelial marker (E-cadherin) observed in 3D accurately mirrors the original tumors; which are of epithelial origin. Phenotypic differences were observed across different matrix conditions and also among different tumor derived cells. Bladder 3D organoids of luminal origin were more sensitive to both cisplatin and PI3K pathway inhibitors as compared to those of basal origin. This drug response profile was reminiscent of what we observed in vivo using patient derived xenograft (PDX) models derived from the same tumors. The phenotypic as well as the drug response variations observed in our 3D culture correlated with variable gene expression profiles (luminal vs basal) that were detected in our RNA-seq data. Conclusion: As compared to monolayer, 3D culture is more capable of recapitulating tumor complexity and accurately reflects the drug resistance / sensitivity profiles that are observed in PDX models in vivo. Therefore, a 3D culture system provides an invaluable tool for high throughput screening of drugs in bladder cancer and providing a better understanding of tumor biology in the search of more effective treatments for bladder cancer patients. Citation Format: May Elbanna, Sreenivasulu Chintala, Eric Ciamporcero, Remi Adelayie, Ashley Orillion, Sreevani Arisa, Nur Damayanti, Michelle Grimard, TJ Puls, Sherry Harbin, Melissa Fishel, Roberto Pili. In vitro modeling of patient derived bladder cancer cell lines in 3D culture systems [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 5783. doi:10.1158/1538-7445.AM2017-5783