Abstract 1449: A live-cell imaging approach for assessing efficacy of immune-targeting therapies using high content imaging and analysis of 3Din vitrotumor models

Background: Immunotherapy has revolutionized cancer research, however limited clinical success to date underscores the need for novel therapeutic targets. Assessment of new targets has been challenging due to limitations of ex vivo animal platforms that only partially recapitulate tumor-intrinsic features and cell interactions. Here, we present a live-cell imaging approach for assessing efficacy of immune-targeting therapies using a 3D in vitro oncology platform in combination with high-content imaging and analysis (HCA) techniques. This study shows how effects of immune-targeting agents on tumor infiltration and killing by peripheral blood mononuclear cells (PBMCs) can be visualized and quantified with high-content endpoints. Aim: To develop and test a robust live-cell-imaging methodology for assessing efficacy of immuno-modulatory cancer therapies. Material & Methods: Lung carcinoma cell-line models were generated using GFP-A549 cells in coculture with dermal fibroblasts and PBMCs. PDX cell suspensions of lung, breast and melanoma origin were self-aggregated with cancer-associated fibroblasts (CAFs) and labeled with live-cell dye for specific monitoring of tumor cell viability. Tumor killing activity and T-cell effector function were evaluated by measuring tumor model size using automatic stage fluorescence microscopy or confocal HCA instrument. Release of inflammatory cytokines IL-6, TNF, IFNγ and GM-CSF was measured with a multiplex cytokine panel. T cell infiltration into tumor models was quantified using fluorescent staining of CD3+ cells. To generate a pro-inflammatory tumor microenvironment, PBMCs were stimulated prior to coculture either with cytokines or anti-CD3/CD28. We monitored immune cell attack on tumors in realtime (56 timepoints) and in 3 dimensions without any discernable phototoxicity. Using cell centroid tools, we mapped physical locations of individual cells belonging to each population. Finally, we calculated tumor and immune cell volumes from individual image stacks to evaluate tumor killing and proliferation/infiltration of immune cells. Results: Our realtime confocal analysis of tumor-immune cell cocultures showed activation of PBMCs with either cytokines or CD3/CD28 increased proliferation/infiltration of immune cells and enhanced tumor killing. Conclusion: Proinflammatory tumor models combined with our novel, high-content imaging analysis endpoints offer a versatile solution for preclinical translational research and a promising approach for high-content screening of immune-targeting therapeutic agents and I-O studies involving engineered T cells (e.g., CAR-T) as well as for testing immunomodulatory agents, such as bispecific antibodies and immune checkpoint inhibitors. Citation Format: Irina Agarkova, Mahomi Suzuki, Silvan Strebel, Armin Wolf, Judith Wardwell-Swanson, Francesca Chiovaro. A live-cell imaging approach for assessing efficacy of immune-targeting therapies using high content imaging and analysis of 3D in vitro tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1449.