A novel multiplex bead‐based platform highlights the diversity of extracellular vesicles

Abstract

The surface protein composition of extracellular vesicles (EVs) is related to the originating cell and may play a role in vesicle function. Knowledge of the protein content of individual EVs is still limited because of the technical challenges to analyse small vesicles. Here, we introduce a novel multiplex bead-based platform to investigate up to 39 different surface markers in one sample. The combination of capture antibody beads with fluorescently labelled detection antibodies allows the analysis of EVs that carry surface markers recognized by both antibodies. This new method enables an easy screening of surface markers on populations of EVs. By combining different capture and detection antibodies, additional information on relative expression levels and potential vesicle subpopulations is gained. We also established a protocol to visualize individual EVs by stimulated emission depletion (STED) microscopy. Thereby, markers on single EVs can be detected by fluorophore-conjugated antibodies. We used the multiplex platform and STED microscopy to show for the first time that NK cell–derived EVs and platelet-derived EVs are devoid of CD9 or CD81, respectively, and that EVs isolated from activated B cells comprise different EV subpopulations. We speculate that, according to our STED data, tetraspanins might not be homogenously distributed but may mostly appear as clusters on EV subpopulations. Finally, we demonstrate that EV mixtures can be separated by magnetic beads and analysed subsequently with the multiplex platform. Both the multiplex bead-based platform and STED microscopy revealed subpopulations of EVs that have been indistinguishable by most analysis tools used so far. We expect that an in-depth view on EV heterogeneity will contribute to our understanding of different EVs and functions.

Evaluation of serum extracellular vesicle isolation methods for profiling miRNAs by next‐generation sequencing

“…Similar findings were recently presented in a publication by Stranska et al, which demonstrated the absence of CD81 and TSG101 in plasma EVs isolated by membrane affinity [69]. Additionally, recent advances in the field have demonstrated that so-called exosome markers can also be present on other classes of EVs and that EV isolates are a heterogeneous mixture of various subpopulations with specific protein profiles [81,82]. It is therefore conceivable that isolation methods are biased towards only partially overlapping EV populations, resulting in different protein profiles.…”

Extracellular vesicles: the growth as diagnostics and therapeutics; a survey

“…In a similar vein, the 10 major device companies, skilled in flow technologies for cell analyses, have explored adaptations to EV studies towards “liquid biopsy” and longitudinal monitoring of personalized therapies. EVs are analysed using NTA, TRPS, SPR, atomic force microscopy, cryo-electron microscopy and others [26], and device manufacturers are providing flow cytometry approaches for quantitative particle-by-particle multiplex analyses of EVs [27]. Flow-cytometry resolution can approach 20 nm, and reagent EV and antibody standards are emerging to enable replication studies.…”

Therapeutic and diagnostic applications of extracellular vesicles

“…For example, MHC II is found on EVs secreted by antigen presenting cells (APC) [35,47]. As another example, CD2, CD8 and CD56 were found in EVs derived from natural killer (NK) cells and not in EVs derived from platelets where the opposite holds true for CD41b, CD42a and CD61 [48].…”