An In Vitro Assay to Assess CAR T Cell Cytotoxicity against Tumor Spheroids

Take a multi-well round-bottom plate containing tumor spheroids, three-dimensional clusters of transduced cancer cells expressing a specific surface antigen, and a green fluorescent protein.

Carefully replace the media with Annexin-V red, a fluorescent apoptosis probe.

Now, introduce transduced chimeric antigen receptor T cells (CAR T cells) expressing chimeric antigen receptors to the wells.

These engineered receptors on CAR T cells recognize and bind to antigens on cancer cells within the spheroids, activating the CAR T cells.

This binding triggers the release of granules containing cytotoxic molecules, inducing cancer cell apoptosis.

In apoptotic cells, phosphatidylserine residues translocate to the cell membrane's outer leaflet.

Further, Annexin-V red probes with high affinity for phosphatidylserine bind to exposed residues, facilitating apoptotic cell detection.

Acquire time-lapse fluorescent images of the tumor spheroids following CAR T cell addition.

A decrease in green fluorescence from cancer cells in spheroids, coupled with an increase in red fluorescence indicative of apoptosis, indicates CAR T cell cytotoxicity against tumor spheroids.