Best practices of 2D and 3D CAR T cytotoxicity assay using high throughput plate based image cytometry

Best practices of 2D and 3D CAR T cytotoxicity assay using high throughput plate based image cytometry

Scott Cribbes3, Yu-Jun Sun1, Yi-Chun Chen1, Wei-Kai Hua1, Sareina Chiung-Yuan W1u, David Zurowski2, Daniel Hui2, Mignane Ka2, Andrea Moore2, Charles Hernandez3, Andrea Love3,
Bo Lin3, Samir Patel3, and Leo Li-Ying Chan3
1Department of Research and Development, Genome Frontier Therapeutics, Taipei City, Taiwan
2Resilience, Inc. East Norriton, PA 19403, USA
3Department of Advanced Technology R&D, Revvity, Lawrence, MA 01843, USA

In the recent decade, chimeric antigen receptor (CAR)-T cell therapy has revolutionized strategies for cancer treatments due to its highly effective clinical efficacy and response for B cell malignancies. Currently, there are six CAR-T cell products available in the market including Kymriah, Yescarta, Tecartus, Breyanzi, Abecma, and Carvykt. The success of CAR-T cell therapy has stimulated the increase in the research and development of various CAR constructs to target different tumor types. Therefore, a robust and efficient in vitro potency assay is needed to quickly identify potential CAR gene design from a library of construct candidates. In the first part of this work we will expose the drawbacks of the the numerous traditional in vitro CAR-T cell-mediated cytotoxicity assays and demonstrate why image cytometry methodologies have been utilized for various CAR-T cell-mediated cytotoxicity assay using different fluorescent labeling methods, mainly due to their ease-of-use, ability to capture cell images for verification, and higher throughput performance.
The success of CART has been limited to hematological blood born cancers and the transition into solid tumors has not been as effective as hoped. Immune cell trafficking and immunosuppressive factors within the tumor microenvironment are but two methods that increase the relative difficulty in developing a robust CAR-T cell therapy against solid tumors. In the second part of this work we will highlight why , with the development of 3D spheroid models, image cytometry may provide the necessary tools and applications for CAR T cell therapy discovery geared towards solid tumors.