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EV-Ident: Identifying Tumor-Specific Extracellular Vesicles by Size Fractionation and Single-Vesicle Analysis

Single-EV analysis using EV-Ident provides a practical way to understand EV heterogeneity and to successfully identify potent subpopulation of EVs for breast and prostate cancer

 

Identifying EVs and their biomarkers within a sample can predict their cellular origin with high accuracy. Such evidence shows that there is potential for the use of EVs as diagnostic markers for liquid biopsies. Understanding the heterogeneous physical and biochemical properties of EVs originating from multiple complex biogenesis pathways remains a major challenge. There is an urgent need for more efficient and reliable EV isolation methods so that the cellular and molecular mechanisms of the biogenesis and function of specific EV subtypes can be readily studied. Standard bulk biochemical assays provide limited information for the characterization of heterogeneous populations of EVs. To overcome this challenges, Professor Cho’s group present a novel technique, called EV-Ident, to isolate EVs into three size fractions and characterize each individual EV. The size fractions are prepared by serial filtration through three differently sized nanoporous membranes in a centrifugal microfluidic device. Because the blood plasma contains not only EVs but also many lipoprotein particles of similar size, we enriched “plasma-derived nanoparticles” (PNPs), including EVs from blood plasma.

  

Three size fractions of nanoparticles for analysis of a size specific single extracellular vesicle (EV)

 

EV-Ident was designed to analyze EV subpopulations by considering both the physical (size) and biochemical (biomarker) features of EVs. This was carried out by enriching EVs into three different size fractions, followed by biochemical marker expression characterization at the single-EV level. Single-EV analysis in each EV size fraction uncovered distinct characteristics of EVs in different subpopulations. Individual EV analysis in EV populations of different sizes successfully removed the ambiguity that results from averaging in conventional EV analysis. As a proof of-concept group has performed two demonstrations to identify the specific type of breast or prostate cancer-positive EVs or PNPs from conditioned medium or blood plasma

 

The study has been supported through the Korean Health Technology R&D Project of the Ministry of Health & Welfare, the IBS Center for Soft and Living Matter and Bio & Medical Technology Development Program of the National Research Foundation. This work is detailed in a paper published in the Analytical Chemistry (Dongyoung Kim et al., EV-Ident: Identifying Tumor-Specific Extracellular Vesicles by Size Fractionation and Single-Vesicle Analysis, Analytical Chemistry), DOI: 10.1021/acs.analchem.0c00285.