Genetically encoded biosensor of apoptosis and necrosis reveals the dynamics and biphasic cytotoxicity by Anti-EGFR CAR-T cells

This study presents the development and validation of a dual genetically encoded fluorescence biosensor system capable of distinguishing apoptosis and necrosis in live cells with high spatial and temporal resolution. The system integrates a nucleus-localized FRET-based caspase-3/7 reporter (RealCas3) with a mitochondria-targeted DsRed marker, enabling simultaneous monitoring of caspase activation and membrane integrity during cell death. Using EGFR-positive tumor cell lines and anti-EGFR CAR T cells, we demonstrate the utility of this platform in quantifying and phenotyping immune-mediated cell death dynamics at single-cell resolution.