Extracellular Vesicle Antibody Microarray for Multiplexed Inner and Outer Protein Analysis

Abstract

Proteins are found both outside and inside of extracellular vesicles (EVs) and govern the properties and functions of EVs, while also constituting a signature of the cell of origin and of biological function and disease. Outer proteins on EVs can be directly bound by antibodies to either enrich EVs, or probe the expression of a protein on EVs, including in a combinatorial manner. However, co-profiling of inner proteins remains challenging. Here, we present the high-throughput, multiplexed analysis of extracellular vesicle inner and outer proteins (EVPio). We describe the optimization of fixation and heat-induced protein epitope retrieval for EVs, along with oligo-barcoded antibodies and branched DNA signal amplification for sensitive, multiplexed and high-throughput assays. We captured 4 subpopulations of EVs from colorectal cancer cell lines HT29 and SW403 based on EpCAM, CD9, CD63 and CD81 expression, and quantified the co-expression of 8 outer (integrins and tetraspanins) and 4 inner (heat shock, endosomal and inner leaflet) proteins. The differences in co-expression patterns were consistent with the literature and known biological function. In conclusion, EVPio analysis can simultaneously detect multiple inner and outer proteins in EVs immobilized on a surface, opening the way to extensive combinatorial protein profiles for both discovery and clinical translation.

Effect of Sample Preprocessing and Size-Based Extraction Methods on the Physical and Molecular Profiles of Extracellular Vesicles

“…The conclusions drawn from both studies emphasize the need for additional information to assist scientists in constructing their own extraction platforms by understanding the advantages and disadvantages of each method. Moreover, while previous studies 29−31 have delved into the diversity of extraction methods and provided tentative comparisons, our approach in this study involves correlating the information obtained from standardized instruments with the data generated by an in-house microarray immunoassay 32 to study the protein profile of EVs. We share here a global view incorporating not only the impact of each method of extraction but also the influence of the selected preprocessing protocols.…”