High sensitivity detection of extracellular vesicles immune-captured from urine by conventional flow cytometry

Abstract

Extracellular vesicles (EVs) provide an invaluable tool to analyse physiological processes because they transport, in biological fluids, biomolecules secreted from diverse tissues of an individual. EV biomarker detection requires highly sensitive techniques able to identify individual molecules. However, the lack of widespread, affordable methodologies for high-throughput EV analyses means that studies on biomarkers have not been done in large patient cohorts. To develop tools for EV analysis in biological samples, we evaluated here the critical parameters to optimise an assay based on immunocapture of EVs followed by flow cytometry. We describe a straightforward method for EV detection using general EV markers like the tetraspanins CD9, CD63 and CD81, that allowed highly sensitive detection of urinary EVs without prior enrichment. In proof-of-concept experiments, an epithelial marker enriched in carcinoma cells, EpCAM, was identified in EVs from cell lines and directly in urine samples. However, whereas EVs isolated from 5–10 ml of urine were required for western blot detection of EpCAM, only 500 μl of urine were sufficient to visualise EpCAM expression by flow cytometry. This method has the potential to allow any laboratory with access to conventional flow cytometry to identify surface markers on EVs, even non-abundant proteins, using minimally processed biological samples.

Extracellular Vesicle Isolation and Characterization from Periprosthetic Joint Synovial Fluid in Revision Total Joint Arthroplasty

“…Another matter under debate is the variety of technologies for EV isolation and analysis, which have classically been strenuous and hampering EV biomarker research in larger patient cohorts [12]. However, bead-based EV capture followed by flow cytometrical analysis has been shown to be a reliable, sensitive and reproducible surface marker detection method and has become readily available [23].…”

“…Given this, the present pilot study aimed to employ reproducible and clinically feasible methodology using a standardized bead-based cytometry assay to analyze EVs from periprosthetic joint aspirates. Forthcoming studies may be able to isolate and examine EV surface signatures with little preparation from biological samples [12]. In the future these developments could encourage wider utilization of this tool.…”

“…Since the first description of exosomes in the 1980s as a vehicle to dispose cellular waste, scientific convergence has evolved to attribute enormous diagnostic potential to nanoscale vesicles as multimodal reflectors of a cell's (patho-)physiological state [9,10]. These properties have dubbed EVs "liquid biopsies", which are released into the extracellular space by multicellular organisms with enclosed proteins, lipids and nucleic acids as their cargo to act on distant receiver cells in form of exosomes (30-100 nm) and microvesicles (50-1000 nm) or, in case of cell death, apoptotic bodies (1000-5000 nm) [11,12]. Lipid-bilayer nanovesicles have been isolated from various biological fluids including synovial fluid of patients with rheumatoid-and osteoarthritis [13][14][15][16].…”

Regulation of MT1-MMP Activity through Its Association with ERMs

“…A modification of our bead-assisted flow cytometry assay [64,65], the ExoStep kit (Immunostep), was used to quantitate MT1-MMP incorporation into EVs. This assay is based on the capture of EVs on magnetic beads coated with an anti-CD63 antibody and staining with anti-CD9 antibody, since both CD63 and CD9 tetraspanins are highly enriched on the surface of EVs from most cell types.…”

Optimized Protocol for Isolation of Small Extracellular Vesicles from Human and Murine Lymphoid Tissues

“…Extracellular vesicles (EVs) are lipid bilayer-enveloped nanovesicles secreted by both eukaryotic and prokaryotic cells and carrying cargos of proteins, lipids, and nucleic acids [1,2]. EVs contain both surface and luminal factors which can be used as markers for specific EV populations representing the different biogenesis pathways [3,4]. Although the definition of EVs is continuously being refined, currently three main subtypes of eukaryotic cell-derived EVs can be distinguished based on their size, composition, and cellular origin-small EVs (sEVs or exosomes, 30-150 nm), microvesicles (MVs, 100 nm −1 µm), and apoptotic bodies (1-5 µm) [5,6].…”