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.

本研究报道了一种双组分基因编码荧光生物传感器系统的开发与验证，该系统可在活细胞中以高时空分辨率精准区分细胞凋亡与坏死。该系统将核定位的基于荧光共振能量转移（Fluorescence Resonance Energy Transfer, FRET）的半胱天冬酶-3/7报告探针（RealCas3）与线粒体靶向DsRed标记物相结合，可实现细胞死亡过程中半胱天冬酶激活与细胞膜完整性的同步监测。本研究采用表皮生长因子受体（Epidermal Growth Factor Receptor, EGFR）阳性肿瘤细胞系与抗EGFR嵌合抗原受体T（CAR-T）细胞，验证了该平台在单细胞分辨率下对免疫介导的细胞死亡动力学进行定量分析与表型分型的应用价值。