Abstract Background: Better models of the tumor microenvironment (TME) are needed in order to reduce the number of anticancer drug failures in clinical trials and to reduce animal use. Current models fail because of 1) reliance on single cell systems; 2) reliance on artificial 2 dimensional (2D) monolayers; and 3) a focus on high throughput assays that pre-select drugs affecting growth and apoptosis. To more accurately model the TME, we developed a 3D perfusion co-culture system for purposes of anti-cancer testing. Materials & Methods: The system consisted of 3DKUBE™ 3D Cell Culture Plasticware, membranes, closed flow circuits, connectors, syringes and gas exchange reservoir bags. 3D static culture experiments combining a variety of cell types, scaffolds, media and reagent conditions, and analytical techniques were performed to define the co-culture conditions. Flow rates and cycling times are used to demonstrate: 1) fibroblast priming, 2) drug effect, 3) intra-spheroid cell viability and 4) long term culture conditions. Multiple methods of in situ and endpoint analytical techniques are shown to be compatible with the 3DKUBE including resazurin reduction, Hoechst staining, immunohistochemistry and 3D confocal imaging. Enhanced kinase signaling is demonstrated by immunoblotting and phosphor-protein signaling. 6-21 day experiments are performed in the 3DKUBE Tumor-Fibroblast model with cytotoxic agents (cisplatin[CP], 5-flourouricil) and small molecule inhibitors (PI3K inhibitors) to demonstrate utility. A polyHEMA-based technique was used to preform standardized spheroids of consistent size among many tumor cell lines (MCF-7, DLD-1, HepG2, A2780). Fibra-Cel® disc scaffolds permit stable stromal cell (MRC-5, human foreskin fibroblasts) adhesion and growth, and accommodate varying analytical methods. 3D perfusion co-cultures demonstrate larger spheroids (confocal) with more viable cells (confocal, hoechst p<0.001) and more sensitivity to CP as compared with static cultures. Perfusion permits “priming” of fibroblasts prior to establishing co-cultures, which further promotes spheroid maintenance and growth. Perfusion demonstrated enhanced maintenance of cell proliferation in primary mouse mammary tumor slices cultured in the 3KDUBE. LY294002 demonstrated enhanced activity in 3D co-culture as compared to 2D monoculture, which correlates with increased p-Akt signaling in 3D. A panel of PI3K and c-Met pathway inhibitors is being tested to discriminate activity in the 3DKUBE co-culture system. Conclusions: The 3DKUBE Segregated Co-culture system is a universally applicable method across multiple different tumor types for modeling the 3D tumor microenvironment for evaluation of anti-cancer drug efficacy. Citation Format: Hal Crosswell, Rebecca D. Widener, Anindya Desgupta, Stephen Shuford, Matthew R. Gevaert, David E. Orr, John Langenheim. The 3DKUBE™ segregated co-culture system: optimal tumor-stromal interactions for modeling the tumor microenvironment in anticancer therapeutic development. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1102. doi:10.1158/1538-7445.AM2013-1102