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Cancer cell invasion is a highly orchestrated and complex biological process, which is influenced by a plethora of biophysical and biochemical signals. Despite its significance, the mechanistic understanding of cellular invasion within the tumor microenvironment has been largely limited due to the lack of physiologically relevant 3D models. Microfabrication of organized cellular constructs offers numerous applications in tracking cell-cell and cell-matrix interactions as well as anti-cancer drug screening. In this regard, the majority of the previously reported 3D tumor models lack specific organization and therefore cannot effectively isolate individual effects of microenvironmental cues on cellular invasion. In this project, by building upon our expertise in microscale tissue engineering and cancer biology, we aim to develop highly organized 3D tumor model, using gelatin methacrylate (GelMA) hydrogel, and to study breast cancer cell invasion in response to various microenviromental cues (e.g. cancer associated fibroblasts, CAFs, and matrix stiffness). Breast cancer MDA-MB-231 cells were encapsulated in GelMA hydrogel and micropattered in the form of organized circular constructs (500μm diameter, 100 μm height) using bench-top photolithography techniques. Subsequently, pure GelMA was patterned in between the constructs and crosslinked for a significantly lower time compared to the circular tumor region (4s vs. 18 s). This process created a 3D microenvironment with high-stiffness circular constructs (representative of tumors) surrounded by a lower-stiffness stroma. After days 1, 3, and 5 of culture, cancer cell invasion was characterized through quantifying the number of cells that disseminated from the tumor region and migrated towards the surrounding stroma. Atomic Force Microscope (AFM) based nanoindentation measurements confirmed significantly higher stiffness within the micropatterned circular constructs (∼1.2 kPa) compared to the surrounding stroma (∼ 0.3 kPa) resembling a distinct physiological condition. Preliminary studies demonstrated high cellular viability (84.36±5.96%) at day 5 of culture. Furthermore, MDA-MB-231 cells proliferated within the model at an average rate of 32.19% per day and displayed invasive behavior from day 1. Particularly, by day 5, 13.89 ± 0.94% of total cells invaded the surrounding stroma. Our findings demonstrate a promising approach to create highly organized tumor models to mechanistically study cancer cell invasion within a 3D microenvironment. We are currently using a similar method to create tumor models which track various cell-cell and cell-matrix interactions_one of which incorporates stromal cells to particularly study the effects of CAFs signaling on cellular invasion. Citation Format: Nitish Peela, Feba S. Sam, Wayne Christenson, Adam W. Watson, Robert Ros, Ghassan Mouneimne, Mehdi Nikkhah. Breast cancer cell invasion in a highly organized three dimensional (3D) microengineered tumor model. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 310. doi:10.1158/1538-7445.AM2015-310