Abstract
BackgroundTetraspanin expression of extracellular vesicles (EVs) is often used as a surrogate for their detection and classification, a practice that typically assumes their consistent expression across EV sources.ResultsHere we demonstrate that there are distinct patterns in colocalization of tetraspanin expression of EVs enriched from a variety of in vitro and in vivo sources. We report an optimized method for the use of single particle antibody-capture and fluorescence detection to identify subpopulations according to tetraspanin expression and compare our findings with nanoscale flow cytometry. We found that tetraspanin profile is consistent from a given EV source regardless of isolation method, but that tetraspanin profiles are distinct across various sources. Tetraspanin profiles measured by flow cytometry do not totally agree, suggesting that limitations in subpopulation detection significantly impact apparent protein expression. We further analyzed tetraspanin expression of single EVs captured non-specifically, revealing that tetraspanin capture can bias the apparent multiplexed tetraspanin profile. Finally, we demonstrate that this bias can have significant impact on diagnostic sensitivity for tumor-associated EV surface markers.ConclusionOur findings may reveal key insights into protein expression heterogeneity of EVs that better inform EV capture and detection platforms for diagnostic or other downstream use.Graphical abstract
Highlights
The promise of extracellular vesicles (EVs) for unbiased diagnostic and therapeutic application requires identification of ubiquitous markers that can distinguish EVs from the contaminating background of free protein aggregates, microparticles, lipoprotein, and numerous other nanoparticulate assemblies found in human biofluids
We explore the impact of heterogeneous tetraspanin colocalization as a form of bias that may impact sensitivity to specific disease markers, especially for platforms where CD9, CD63, or CD81 capture is the common first step to enrich EVs for downstream biomarking
We developed a custom assay on the ExoView platform utilizing non-specific biotin-based EV capture to examine the bias of direct tetraspanin capture in selecting specific subpopulations
Summary
The promise of extracellular vesicles (EVs) for unbiased diagnostic and therapeutic application requires identification of ubiquitous markers that can distinguish EVs from the contaminating background of free protein aggregates, microparticles, lipoprotein, and numerous other nanoparticulate assemblies found in human biofluids. There is a lack of techniques that can assess multiplexed expression of membrane proteins at single vesicle resolution across the size range of EVs, with the majority of EVs being between ~ 30 and 100 nm in diameter [15]. Commercial flow cytometers can assay protein expression at single EV resolution [20, 21] but only above a critical nominal size of ~ 80 nm, since detection is limited by particle scattering, is dependent on particle diameter, particle refractive index, the angle of collection, and the intensity and wavelength of the laser [20]. Some studies have reduced this size limit by detecting particles using fluorescence instead of scattering, yet are still limited in sensitivity to EVs that highly express a single protein [22]. Tetraspanin expression of extracellular vesicles (EVs) is often used as a surrogate for their detection and classification, a practice that typically assumes their consistent expression across EV sources
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