Abstract Introduction: Tumor growth is dramatically affected by the microenvironment, including supporting cells such as the stroma and vasculature, mechanical factors, such as interstitial flow and extracellular matrix and aspects of the tumor mass itself, including shape. However, current tumor growth models, including xenograft models, 2D and/or simplistic 3D cultures, are unable to address these interactions in a high throughput human-derived system. We have developed a novel microfluidic platform that combines human derived perfused microvessels, stroma, and interstitial flow with 3D culture. This platform was used to quantitatively compare the role of these microenvironmental factors on tumor growth. Methods: A microfluidic device was fabricated consisting of two supply channels on either side of a central tissue compartment. The inner stromal compartment consists of normal human fibroblasts (NHLFs) and GFP-labeled human colorectal adenocarcinoma tumor cells, SW620, seeded in a fibrin matrix. To simulate a vasculogenic-like process, human cord blood endothelial colony forming cells endothelial cells (ECFC-ECs) were distributed throughout the stromal channel with the fibroblasts and tumor cells. Tumor growth rate and area was compared across day, interstitial flow rates and tumor shape (fractal dimension, perimeter to area) with ANOVA. Results: Cell viability within the device was maintained under interstitial flow conditions for a period of 21 days. Within one week of culture, microvessel formation and significant tumor growth into spheroids (n=636) were observed. On average, tumor growth rate was 26% ±62% per day with the highest growth rates observed on the first days. By day 7, many tumor masses had died off, with 2-3 large, fast growing tumors remaining per chamber. Highest tumor growth rates and areas were observed in tumor masses with a characteristic morphology of high perimeter to area and lower cohesion. Interstitial flow rates ranging from essentially static to supraphysiologic were generated. Differences in tumor growth rates were not statistically significant across chambers with different mean flow rates. To demonstrate intraluminal flow within the vascular network, fluorescently labeled dextran (40, 70, and 150 kDa) was introduced into the microfluidic lines. Dextran was retained in the vessel network, and showed tumor cells residing in the intraluminal space of the formed vasculature. By day 14, the network eroded, as the tumor masses overgrew and encompassed more than 60% of the chamber volume. Discussion: We have developed a novel 3D microfluidic system of the tumor microenvironment that features perfused capillaries and controlled interstitial flow. Tumor growth was affected by tumor characteristic shape in this model though interstitial flow appeared to play a lesser role. Vascular development was observed and its interaction with tumor growth will be analyzed in future work. Citation Format: Luis F. Alonzo, Claire J. Robertson, Monica L. Moya, Marian L. Waterman, Christopher C. Hughes, Steven C. George. Recapitulating the microenvironment in vitro for comparative study of factors affecting tumor growth and vascularization. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3930. doi:10.1158/1538-7445.AM2014-3930