Abstract Introduction Broad application of immunotherapy to treat cancer has been hampered by low response rates in the clinics (~20%) and high failure rates of clinical trials. The lack of translational preclinical models that accurately replicate human immunological complexity is one of the reasons leading to ineffective candidates in clinical trials. There is an urgent need of advanced preclinical models that can enhance clinical trial success rate and improve clinical impact of immunotherapies. We present a novel 3D Ex vivo Patient Tissue platform combining short-term 3D ex vivo tumor culture system with high content image (HCI)-based analysis. The platform preserves tumor heterogeneity and tumor immune context, including (exhausted) tumor infiltrating lymphocytes. We report a quantification of ex vivo tumor responses to a panel of immunotherapies, including checkpoint inhibitors, tested on patient tissues from various cancers. Methods Patient tumor tissues were obtained from hospitals and tissue providers and processed within 24 hours to preserve the tumor microenvironment (TME). Tumor clusters from ovarian, cervical, breast, and non-small cell lung cancer patients were embedded in a protein-rich hydrogel and exposed to panels of immunomodulatory drugs in a 384-well format for 5-7 days. Phenotypic effects of the drugs on physiologically relevant morphological changes, such as tumor cell killing and immune cell proliferation, were measured using our proprietary automated HCI analysis platform. In addition, IHC and FACS analysis of primary samples as well as cytokine measurements were performed. Results Responses of ex vivo tissues to immunotherapies targeting various pathways (e.g., ipilimumab, pembrolizumab and STING agonists), combination treatments and selected controls were evaluated by automated phenotypic analysis platform. Based on their sensitivity, patient tissues were classified as immunotherapy responders or non-responders. A deeper investigation of the responder tissues was performed with IHC and FACS to pinpoint the critical TME components necessary for immunotherapy sensitivity. In addition, cytokine profiling was done in supernatants from treated ex vivo cultures to confirm the functional HCI readouts. Accurate and reproducible response evaluation demonstrated the feasibility of preclinical immunotherapy drug testing on primary patient material using this platform. Conclusion The 3D ex vivo patient tissue platform successfully combined drug testing protocols using fresh patient tumor tissue with preserved TME and advanced 3D HCI analysis. The platform offers a rapid, reliable and patient-relevant approach to test (clinical) immunotherapies for different solid tumours. It has the potential to significantly improve the preclinical evaluation of immunotherapies and support the decision-making process during progression of drug candidates to the clinic. Citation Format: Nataliia Beztsinna, Niels Meesters, Lidia Daszkiewicz, Fanny Grillet, Donny van der Meer, Kuan Yan, Emma Spanjaard, Willemijn Vader, Leo Price. Immunotherapy testing in 3D Ex vivo Patient Tissue Platform with preserved tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2059.