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Abstract A hallmark of effective immunotherapy is the reliance on the cytotoxic effects of immune cells, such as T-cells and natural killer (NK) cells, on tumor lesions. Cell cluster formation is an important sign of activation, and proliferation, as well as an indication of the killing capability of immune cells. Understanding and modulating the mechanisms underlying the activation, proliferation, and clustering of immune cells is essential for the development of innovative approaches to promote immune cell-induced cytotoxicity and address tumor progression. Here, we report a novel kinetic immuno-oncology assay using the Agilent xCELLigence RTCA eSight, which can simultaneously and quantitatively assess effector/immune cell clustering and proliferation through brightfield live cell time-lapse imaging and immune cell-mediated cytotoxicity of target/cancer cells through real-time monitoring cancer cell viability over a chronic treatment. In this study, we validated the assay using 2 cell models, including CD19 CAR T-cells against Raji cells and NK-92 cells against A549 cells, in a 96-well plate format. The activation, clustering, and proliferation of CD19 CAR T-cells and NK-92 at different Effector: Target (E: T) ratios, as well as the sequential killing of the target cancer cells, were plotted using imaging and cellular-impedance-based readouts throughout the entire assay, respectively. Our results showed 1) the clustering and proliferation of the effector cells could be accurately assessed by brightfield without the facilitation of fluorescent label using the image analysis module of the system; 2) the killing efficacy of the effector cells quantified by the impedance readout highly correlated to their clustering and proliferation assessed by the live-cell imaging; 3) cellular impedance provided a sensitive readout CAR-T killing of immobilized Raji cell count, independent of fluorescent labeling; 4) the kinetics of cluster formation/proliferation and killing efficacy of CD19 CAR T-cells were different from NK cells. The capability of multiparametric assessment of immune cell-medicated cytotoxicity from both effector and target cell perspectives of the assay provides a simple and real-time workflow to understand the correlation and interaction between effector and target cells, which could be an addition of immense value to the development of innovative immunotherapies. Citation Format: Grace Yang, Yan Lu, Tian Wang, Peifang Ye, Xiaoyu Zhang. Real-time multiparametric assessment of immune cell-mediated cytotoxicity abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 77.
Yang et al. (Fri,) studied this question.