Imaging the interactions of chimeric antigen receptor-expressing T-cells with colorectal cancer cells

ABSTRACT Chimeric antigen receptor (CAR) T-cell therapy has shown unprecedented success in haematological cancers but faces challenges in solid tumours. Although carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5) is differentially expressed in many solid tumours, CEACAM5 CAR T-cells are ineffective. Here, we have studied the interaction of CEACAM5 targeting CAR primary T-cells with colorectal cancer (CRC) cells using fluorescence microscopy. We find that CRC cells’ glycocalyx is much thicker than the CAR T-cell and likely contributes to immune-escape. Oscillating calcium flux, a signature of non-sustained triggering and decreased killing, was observed when CAR T-cells interacted with CRC, which increased with increasing cell-seeding time. This was because CEACAM5 became increasingly unavailable on the CRC cell monolayer, as revealed by fluorescence imaging. Local proteolytic treatment with trypsin to disrupt the CRC cell monolayer, using a micropipette, increased CEACAM5 availability, decreased glycocalyx thickness, and restored sustained CAR T-cell calcium fluxes, increasing the killing of CRC cells. Our results reveal why CAR T-cells targeting CEACAM5 are ineffective and suggest possible routes for improved therapy. One Sentence Summary CEACAM5 unavailability and the thick CRC cell glycocalyx are major barriers to CAR T-cells, which local proteolysis helps to overcome.