A Dual-Locked β-Galactosidase/pH Near-Infrared Sensor Enables High-Fidelity Monitoring of Therapy-Induced Senescence

Abstract

Therapy-induced senescence is increasingly recognized as a critical determinant of chemotherapy efficacy, tissue toxicity, and senolytic intervention. However, conventional senescence-associated β-galactosidase (SA-β-Gal) assays and single β-Gal-responsive probes often suffer from limited specificity because elevated β-Gal activity is not exclusive to senescent cells. Here, we report ECD-Gal, a dual-locked near-infrared fluorescent sensor that integrates β-Gal enzymatic activity with lysosomal pH variation for high-fidelity sensing therapy-induced senescence. ECD-Gal shows weak emission in β-Gal positive cells with an acidic lysosomal microenvironment but is strongly activated in senescent cells where β-Gal accumulation coincides with lysosomal alkalinization. Mechanistic studies using agents that induced lysosomal acidification or lysosomal alkalinization supported that both β-Gal activity and elevated lysosomal pH are required for fluorescence activation. ECD-Gal enabled confocal imaging, flow-cytometric quantification, in vivo visualization of doxorubicin or bleomycin-induced senescence, and its fluorescence readout correlated with SA-β-Gal staining, senescence marker expression, and fibrosis-associated pathological remodeling. This dual-locked sensing strategy provides a robust molecular tool for monitoring therapy-induced senescence and evaluating senolytic interventions in living systems.