Sequencing-free, joint single-EV profiling of DNA and protein cargos enables accurate cancer detection at early stages

Abstract

Accurate diagnosis at early stages is crucial for improved survival in cancer patients. Analysis of cell-free DNA represents a transformative liquid biopsy technology, yet high-throughput sequencing is expensive and time-consuming. Here, we report a facile and affordable strategy for the identification of specific extracellular vesicle (EV) subpopulations towards early cancer detection with high accuracy. A hydrogel-based method was developed for profiling the distributions of DNA and protein cargos concurrently among single EVs with near-single-molecule sensitivity. In a retrospective cohort study, we found that merely the proportion of EVs carrying both DNA and carbohydrate antigen 125 (CA125), with no need to decipher the DNA sequences, can serve as an effective biomarker for ovarian cancer (OC) detection. Remarkably, combined evaluation of the DNA and CA125 abundances in plasma EVs could resolve healthy, benign, early-stage malignant, and late-stage malignant subjects, with an overall accuracy of 95.0%. More importantly, distinguishing early-stage OC from benign diseases, which is challenging in clinical practice, was achieved. Single-EV analysis not only addresses EV heterogeneity for mechanism elucidation, but also reveals rare EV subpopulations for development of personalized treatment strategies.