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

Biosensors & Bioelectronics

Volume 301, 1 June 2026, 118471 doi.org/10.1016/j.bios.2026.118471

Aijaz Ahmad Rather, Aman Munirpasha Halikar, Ritika Sachdeva, Shivanshu Kumar Tiwari, T R Santhoshkumar

Abstract

Precise and quantitative evaluation of CAR-T cell-induced cytotoxicity by resolving distinct cell death phenotypes in live cells remains a critical unmet need in the development of immunotherapies. Discriminating between apoptosis and necrosis induced by CAR-T therapy at single-cell resolution is essential for elucidating cytotoxic mechanisms and predicting therapeutic outcomes. Yet, most live cell assays lack the spatiotemporal sensitivity to do so. Here, we present a genetically encoded dual-biosensor system that concurrently monitors caspase-3/7 activation and mitochondrial integrity, enabling the real-time distinction between apoptosis and necrosis. The platform combines a FRET-based caspase sensor (RealCas3) and a mitochondria-targeted DsRed reporter (MitoDsRed) that exhibits compatibility with both microscopy imaging and flow cytometry. We applied this platform to functionally profile anti-EGFR CAR-T cells and uncover a biphasic cytotoxicity pattern, marked by early caspase-3/7 induced apoptosis, followed by a delayed necrosis. Notably, the system enables the time-resolved detection of secondary necrosis, a delayed immunogenic death phenotype that is often misclassified in conventional assays. The developed platform offered a robust and scalable approach for real-time functional phenotyping of immune effector responses, addressing a key gap in evaluating cell death mechanisms during immunotherapy development. By enabling the time-resolved quantification of tumor cell demise along with the mode of death, this dual-sensor system established a mechanistic framework for evaluation and rational design of safer and more effective next-generation immunotherapies.