Immuno-Reactive Cancer Organoid Models to Examine Microbiome Metabolite Effects on Immune Checkpoint Blockade Efficacy Ethan Shelkey1, David Soto-Pantoja2, Yong Lu3, Shay Soker1,4 1Wake Forest Institute for Regenerative Medicine, Wake Forest School of Medicine, Winston-Salem, NC; 2Hypertension and Vascular Research Center, Wake Forest School of Medicine, Winston-Salem, NC; 3Department of Microbiology and Immunology, Wake Forest School of Medicine, Winston-Salem, NC; 4Comprehensive Cancer Center at Wake Forest Baptist Medical, Winston-Salem, NC Introduction: As the number of available immunotherapies for solid tumors increase, their prevalence in the clinic continues to rise as well. While the results are promising, a sizable percentage of patients are non-responders to all types of immunotherapy. Yet, there has been limited 3D in-vitro models to assess tumor immune-reactivity. These systems are ideal for isolating mechanisms that dictate cell behavior and interactions. Our goal was to create an organoid model containing cancer cells paired with T-cells to model immune checkpoint blockade (ICB) efficacy. This model could then be used to examine novel microbiome-ICB interactions shown by recent research to alter therapeutic responses in patients. Methods: We created organoids consisting of tumor and immune cells, embedded in extracellular matrix-like hydrogels. Organoids were treated with therapeutic equivalent doses of αPD-1 and αCTLA-4 or single dose of αCD-47 and viability assays, flow cytometry, RT-qPCR, and IHC staining were performed to determine the effects of ICB. Physiologic concentrations of metabolites derived from likely effector bacterial species were then evaluated for their effects on genetic expression and immunotherapy efficacy. Results and Discussion: We showed that ICB therapy stimulated internally localizing T-cells, inducing T-cell-mediated tumor cell killing. ICB treated samples resulted significant loss of viability with corroborating readings from the other methods of characterization. RT-qPCR resulted in some elucidation of potential cellular changes due to bacterial metabolites with further quantification ongoing. Conclusion: We have created an ex-vivo tumor immune-reactive organoid model for studying immunotherapy. We can now continue to change individual microenvironment factors, such as microbiome metabolites, and observe their impacts on immunotherapy efficacy to better understand what conditions are conducive to ICB treatment.