Time-Resolved Quantification of Single-Cell Extracellular Vesicles Secretion for Drug Resistance Evaluation

Extracellular vesicles (EVs) play a crucial role in mediating cell–cell communication and shaping cellular functions. However, the relationship between EVs secretion and cellular drug sensitivity, as well as the role of EVs in drug resistance development, remains unclear. Therefore, characterizing rare drug-resistant cells within cell populations and obtaining time-resolved information on single-cell EVs secretion are essential for understanding EVs’ role in drug resistance. Herein, we utilize a single-cell EVs secretion quantification chip (SCQ-chip) to compartmentalize individual cells and longitudinally monitor their secretion during drug treatment. The results show that EVs secretion is closely related to cellular drug sensitivity and strongly depends on the drug dose. Low-dose drugs induce increased levels of EVs secretion as a protective mechanism, whereas high-dose drugs reduce both cell viability and EVs secretion. Notably, stem-like cells exhibit a unique slow-fluctuating EVs secretion pattern. Furthermore, coculturing drug-sensitive cells with EVs from drug-resistant cells revealed that EVs facilitate drug resistance transfer. Proteomic analysis indicates that this process is primarily driven by metabolism-related pathways, confirming EVs as mediators of drug resistance dissemination. Our results proposed that EVs secretion is a promising indicator for drug resistance analysis, which will inspire us to develop new drugs targeting EVs secretion as a strategy to overcome drug resistance.