Repetitive Tumor Challenge Assay: An In Vitro Culture Assay to Evaluate Chimeric Antigen Receptor (CAR) T Cells for Repetitive Tumor Killing Potential

Cancer immunotherapy employs chimeric antigen receptor T cells or CAR T cells, genetically engineered T cells expressing specific CAR, which attach to tumor cells causing cell death.  To evaluate CAR T cells' tumor-killing potential in vitro, begin with a brain tumor spheroid culture. Treat the culture with a suitable enzyme solution to obtain a single-cell suspension. Centrifuge and resuspend the cell pellet in a suitable media.

Pipette the desired volume of tumor cells followed by CAR T cells in a multi-well plate. In culture, specific receptors on CAR T cells recognize and bind to specific antigens on tumor cells. This binding activates CAR T cells and releases cytokines, resulting in tumor cell death. The activation also leads to increased CAR T cell proliferation.

Replace the media with an equal volume of tumor cells. Repeat cell addition on alternate days. Following repeated antigen exposure, few CAR T cells may undergo cell exhaustion. These cells display limited cytokine production and proliferation capabilities with increased inhibitory receptor expression. Harvest the cells from the plate and centrifuge.

Resuspend the cell pellet in FACS buffer containing an antibody cocktail. The antibodies bind to their target receptors on these cells. Add a fluorescent DNA-binding dye to stain the nuclei of permeabilized cells. Using flow cytometry, determine the different populations of cells via analysis of fluorescent cell surface markers.