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Updated: Aug 11, 2025
A Spheroid Killing Assay by CAR T Cells
Published on: December 12, 2018
High-throughput method to analyze the cytotoxicity of CAR-T Cells in a 3D tumor spheroid model using image cytometry
David Zurowski1, Samir Patel2, Daniel Hui1
1Department of Analytical Development, Cell Therapy, Resilience, Inc. East Norriton, PA 19403, USA.
Abstract:
Solid tumors account for approximately 90% of all adult human cancers. As such, the development of novel cellular therapies has become of increasing importance to target solid tumor malignancies, such as prostate, lung, breast, bladder, colon, and liver cancers. One such cellular therapy relies on the use of chimeric antigen receptor T cells (CAR-T cells). CAR-T cells are engineered to target specific antigens on tumor cells. To date, there are six FDA-approved CAR-T cell therapies that have been utilized for hematologic B cell malignancies. Immune cell trafficking and immunosuppressive factors within the tumor microenvironment increase the relative difficulty in developing a robust CAR-T cell therapy against solid tumors. Therefore, it is critical to develop novel methodologies for high-throughput phenotypic and functional assays using 3D tumor spheroid models to assess CAR-T cell products against solid tumors. In this manuscript, we discuss the use of CAR-T cells targeted towards PSMA, an antigen that is found on prostate cancer tumor cells, the second most common cause of cancer deaths among men worldwide. We demonstrate the use of high-throughput, plate-based image cytometry to characterize CAR-T cell-mediated cytotoxic potency against 3D prostate tumor spheroids. We were able to kinetically evaluate the efficacy and therapeutic value of PSMA CAR-T cells by analyzing the cytotoxicity against prostate tumor spheroids. In addition, the CAR-T cells were fluorescently labeled to visually identify the location of the T cells as cytotoxicity occurs, which may provide more meaningful information for assessing the functionality of the CAR-T cells. The proposed image cytometry method can overcome limitations placed on traditional methodologies to effectively assess cell-mediated 3D tumor spheroid cytotoxicity and efficiently generate time- and dose-dependent results.
Insights
Researchers developed a new high-throughput image cytometry method to assess the effectiveness of chimeric antigen receptor T cells (CAR-T cells) against solid tumors, specifically prostate cancer. This method uses 3D tumor spheroids to evaluate CAR-T cell potency and function.
Area of Science:
- Oncology
- Immunotherapy
- Cellular Therapy
Background:
- Solid tumors represent approximately 90% of adult human cancers, necessitating advanced therapeutic strategies.
- Chimeric antigen receptor T cell (CAR-T cell) therapies are effective against hematologic malignancies but face challenges in solid tumors due to the tumor microenvironment.
- Developing robust CAR-T cell therapies for solid tumors requires novel high-throughput assays using 3D models.
Purpose of the Study:
- To introduce and validate a novel high-throughput image cytometry method for assessing CAR-T cell efficacy against solid tumors.
- To evaluate the cytotoxic potency of prostate-specific membrane antigen (PSMA)-targeted CAR-T cells against 3D prostate cancer spheroids.
- To demonstrate the utility of fluorescently labeled CAR-T cells for visualizing T cell localization and function during cytotoxicity assays.
Main Methods:
- Utilized 3D prostate tumor spheroids as a model system for solid tumors.
- Employed high-throughput, plate-based image cytometry to quantify CAR-T cell-mediated cytotoxicity.
- Incorporated fluorescent labeling of CAR-T cells to enable visual tracking of their location and interaction with tumor spheroids.
Main Results:
- Successfully demonstrated the ability to kinetically evaluate the efficacy and therapeutic value of PSMA CAR-T cells against 3D prostate tumor spheroids.
- Characterized CAR-T cell-mediated cytotoxic potency using the developed image cytometry method.
- Visual tracking of fluorescently labeled CAR-T cells provided insights into their functional behavior during the killing of tumor spheroids.
Conclusions:
- The proposed image cytometry method effectively overcomes limitations of traditional assays for evaluating 3D tumor spheroid cytotoxicity.
- This approach enables efficient generation of time- and dose-dependent results for assessing CAR-T cell functionality against solid tumors.
- The methodology holds promise for advancing the development of CAR-T cell therapies for solid tumor malignancies, including prostate cancer.
