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

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Evaluating CAR-T cell-induced cytotoxicity requires precise discrimination of cell death phenotypes.
• Current live cell assays lack the sensitivity to differentiate apoptosis from necrosis in real-time.
• Understanding cell death mechanisms is vital for predicting immunotherapy outcomes.

Purpose of the Study:

• To develop a genetically encoded dual-biosensor system for real-time, single-cell resolution of apoptosis and necrosis.
• To enable precise quantification of cell death phenotypes induced by CAR-T cell therapy.
• To establish a mechanistic framework for evaluating and designing next-generation immunotherapies.

Main Methods:

• Development of a dual-biosensor system combining a FRET-based caspase-3/7 sensor (RealCas3) and a mitochondria-targeted DsRed reporter (MitoDsRed).
• Application of the biosensor platform for live cell imaging and flow cytometry analysis.
• Functional profiling of anti-EGFR CAR-T cells to assess cytotoxicity patterns.

Main Results:

• The dual-biosensor system successfully distinguished between apoptosis and necrosis in real-time.
• A biphasic cytotoxicity pattern was observed in CAR-T cell therapy, with early apoptosis followed by delayed necrosis.
• The system enabled time-resolved detection of secondary necrosis, a phenotype often misclassified.
• Demonstrated a robust and scalable approach for functional phenotyping of immune effector responses.

Conclusions:

• The developed dual-biosensor platform addresses a critical gap in evaluating cell death mechanisms in immunotherapy.
• Provides a tool for time-resolved quantification of tumor cell demise and mode of death.
• Facilitates the mechanistic evaluation and rational design of safer and more effective immunotherapies.