Abstract 315: Multifaceted functional assessment of checkpoint inhibitor efficacy using 3D tumor spheroids

Three-dimensional (3D) culture of immortalized cell lines and patient-derived primary cells has been shown to be a more representative in vitro model of tumor biology compared to standard 2D techniques. For immune-oncology, animal models continue to lack the ability to fully recapitulate the human immune system. The use of 3D models to study immunotherapies provides the opportunity to mimic the complex interactions between immune cells and the tumor microenvironment in a fully human system. However, standard well-based assays that measure cell viability prevent the obtainment of useful knowledge on tumor-immune cell interactions and immunotherapy effect on immune cell numbers and viability. To address these limitations, we have developed an in vitro based assay for the visualization and quantitation of T-cell-mediated cell death using fluorescently labeled live tumor cells in a 3D spheroid platform. Enhanced T-cell mediated tumor cell killing using immune checkpoint inhibitors can be detected allowing for real-time evaluation of tumor-specific apoptosis in the presence of cytotoxic T-cells. Moreover, the cytotoxic effects can be inhibited by co-treatment with a blocking anti-CD8 antibody, demonstrating a specific requirement for CD8+ T-cells. In order to detect changes to tumor biology induced by T-cells as well as immune-oncology drug treatments, we monitored dynamic fluctuation of PD-L1 and MHC-I expression using immunofluorescence in the spheroids. Finally, we detected changes in T-cell populations, such as T-cell activation, and changes in CD8+:Treg ratios, induced by tumor spheroids with or without drug treatment using flow cytometry. In summary, we can observe T-cell mediated tumor cell killing and relate drug efficacy back to identified changes in tumor cell and T-cell phenotypes. This multifaceted approach is ideal for the functional evaluation of preclinical I/O agents and for identifying drug combinations as it represents a holistic perspective of drug response. Citation Format: Katy A. Lassahn, Kathryn M. Appleton, Ashley Elrod, Aaron L. Carlson, Teresa M. DesRochers. Multifaceted functional assessment of checkpoint inhibitor efficacy using 3D tumor spheroids [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 315.