Abstract 1844: Development of a high content screening (HCS) platform for novel cancer immunotherapeutics

Abstract

Abstract There has been dramatic progress in the understanding of fundamental causes of cancer, giving rise to novel treatments which harness the power of the immune system to combat cancer progression. Despite this, no two cancers have the same pathogenesis and there is significant variation in patient response rates. Understanding mechanisms of carcinogenesis in model cell culture systems, such as aberrant signaling, as well as how tumor immunity can be initiated, retargeted or reinvigorated upon incorporation of the immune system will fuel drug discovery. High-Content Screening (HCS) platforms allow single-cell imaging analysis and are used to interrogate these models to enable drug development and understand mechanisms of action. They provide an opportunity to test novel cancer therapeutics in a translationally relevant setting that integrates primary immune effector function. Here we outline several macrophage-based HCS assays which allow assessment of therapeutics targeting;1) early aspects of immune cell activation 2) macrophage mitochondrial health 3) macrophage activation and re-polarization 4) immune cell infiltration and tumor killing. To test efficacy of drugs targeting immune signaling or activation, macrophages were treated with a canonical NF-ƙB activator +/- TCPA-1, a known inhibitor for benchmarking as a model therapeutic. Robust NF-ƙB translocation was observed and reduced in the presence of TCPA-1 rendering this an appropriate assay to screen therapeutic cytokines or TLR agonists activating NF-ƙB signaling. A macrophage inflammasome activation assay was developed to analyze levels of ASC and speck formation. CRID3i, an established inflammasome inhibitor was validated as a robust control therapeutic that prevented NLRP3 activation suggesting this is a relevant platform to assess cancer drug efficacy. Interrogating mitochondrial health and dynamics in primary macrophages is vital to understanding the impact of accumulating ROS species or metabolites in the tumor microenvironment. Assays modelling the effects of these molecules on macrophage polarization and activation as well as mitochondrial integrity were used to test macrophage repolarizing therapeutics, a key approach in immuno-oncology. Finally, a complex multi-cellular 3D spheroid tumor killing assay was optimized to model immune cell infiltration following exposure to IO-targeted therapeutics and robust enhancement of tumor killing was directly observed. Overall, these assays demonstrate the power of imaging in performing HCS to assess the efficacy and mechanisms of action of a range of IO-targeted therapeutics. This suite of assays is applicable to IO drug screens targeting macrophage function, immune cell activation and immune cell infiltration. Citation Format: Rebecca Roberts, Henry Leonard, Ilona Aylott, Douglas Best, Dan Rocca, Ben Thompson, Nunan Robert, Louise Brackenbury. Development of a high content screening (HCS) platform for novel cancer immunotherapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1844.