Abstract Recent immunotherapy failures in clinical trials have elucidated the affects of the tumor stroma on attenuating therapeutic response. In some cancers, the stroma consists of a collagen-rich fibrotic zone on the tumor periphery that traps infiltrating CD8+ T cells, allowing the tumor to evade immune cell-mediated death. Typically, animal models are employed to study complex physiology. However, known challenges to these methods include cost, duration and throughput. Thus, we sought to develop a 3D in vitro model that recapitulates the immune exclusion phenotype in a high throughput manner. We utilized a novel 3D cell culture platform, VersaGel and Symphony, to pattern tumor-stroma-immune compartments in a layer-by-layer fashion in 96-well plates. VersaGel is a chemically modified extracellular matrix that is amenable to light-induced crosslinking using the Symphony apparatus. Moreover, Symphony allows for discrete layers to be formed with any VersaGel/cell mixture, with defined 3D gel thicknesses (eg. 100µm, 250µm, 500µm) and areas in the plate (eg. 2mm, 4mm, or 8mm diameter discs). GFP-labeled MC38-ova (ovalbumin antigen) colon carcinoma cells were grown in 100µm thick 3D VersaGel with or without CFP-labeled mouse embryonic fibroblasts (MEF) layered on top for 4 days. After, OT-1-RFP CD8+ cells were added to the culture medium to assess their penetration depth and tumor cell death over 3 days. 3D cell analysis through the optically clear VersaGel was performed in situ using high content confocal microscopy, effectively quantifying RFP/CFP/GFP-labeled cells and DRAQ7-labeled dead cells using Z-stack analysis. In the control (tumor-only condition), OT-1 cells fully penetrated the tumor layer and induced cell death. In the experimental condition (with fibroblasts), it was found that the fibrotic stromal layer limited OT-1 penetration to the periphery of the 3D tumor, and, moreover, the total OT-1 cells increased relative to the control. Future analysis may include the utility of wild-type T cells and novel therapeutics targeting the stroma to activate the immune response. The results suggest a novel 3D immune exclusion model that integrates with high content confocal analysis and is amenable to immunotherapeutic screening and translational assessment. Citation Format: Kolin C. Hribar, Tracy Kleinheinz, Joanna Klementowicz, Susan Kaufman, Robert A. Blake, Celine Eidenschenk. An in vitro 3D immune exclusion tumor model engineered in a layer-by-layer fashion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4522.