Profiling of CD63 and EpCAM Membrane Proteins of Extracellular Vesicles on Tannic Acid-Coated Magnetic Beads Using Conventional Flow Cytometry.

Extracellular vesicles (EVs) are considered to be a promising tool in disease diagnosis. However, the clinical translation of EV-based liquid biopsy faces significant challenges due to the lack of inexpensive, rapid, and high-throughput methods of EV analysis. Bead-based platforms, combined with conventional flow cytometry, allow for the simultaneous capture and immunolabeling of EVs. In this study, we present a new approach based on the label-free isolation of EVs by tannic acid-coated superparamagnetic beads (TASPMB) combined with immunofluorescence detection of EV membrane proteins using flow cytometry. First, we tested the molecular profiling capabilities of the approach using EVs derived from human breast and colorectal cancer cell lines and from plasma of colorectal cancer patients to recognize the tetraspanin protein CD63 and the epithelial cell adhesion molecule (EpCAM). Subsequently, the developed approach was validated to identify proteins on EVs enriched with TASPMB from the conditioned media of SKBR3 and HT29 cell cultures without preliminary purification by a size-exclusion chromatography (SEC) column. The developed approach demonstrates a high capacity for isolating EVs and subsequently profiling of their membrane proteins, with a total assay time of approximately 2 h. The approach presented here can be a promising tool for rapid detection of EV membrane proteins using conventional instruments, such as flow cytometry.