Digital Profiling of Tumor Extracellular Vesicle-associated RNAs Directly from Unprocessed Blood Plasma

Tumor-derived extracellular vesicle (tEV)-associated RNAs hold promise as diagnostic biomarkers, but their clinical use is hindered by the rarity of tEVs among non-tumor EVs. Here, we present EV-CLIP, a highly sensitive droplet-based digital method for profiling EV RNA. EV-CLIP utilizes the fusion of EVs with charged liposomes (CLIPs) in a microfluidic chip. Optimized CLIP surface charge enables exceptional sensitivity and selectivity for EV-derived miRNAs and mRNAs. This approach streamlines detection with minimal plasma volume (20 μL) and eliminates the need for prior EV isolation or RNA preparation, preventing loss of EVs or RNA. In testing with 83 patient samples, EV-CLIP detected EGFR L858R and T790M mutations with high AUC values of 1.0000 and 0.9784, respectively. Its success in serial monitoring during chemotherapy highlights its potential for precise quantification of rare EV subpopulations, facilitating the exploration of single EV RNA content and enhancing understanding of diverse EV populations in various disease states. ### Competing Interest Statement EMC, SK, YKC are named inventors of a patent pending for the method described in this study. The remaining authors have no conflicts of interest to declare.