Multiparametric analysis of circulating exosomes and other small extracellular vesicles by advanced imaging flow cytometry

Extracellular vesicles (EVs) are responsible for a multitude of physiological functions, including immunomodulation. A heterogenous mixture of small EV (sEV) subsets, including putative exosomes, is derived when commonly used “exosome” isolation techniques are employed. Subset diversity relates in part to their different intracellular origins, and can be associated with distinct functional properties. Recent progress in the EV field has enabled the categorization of such subsets based on their surface composition.

For the first time, researchers at King’s College London combine such emerging subset-specific markers with advanced imaging flow cytometry (iFCM) to perform high-throughput, multiparametric, vesicle-by-vesicle characterization, and functional assessment of specific small EV subsets, and exosomes in particular. The approach allows researchers to address three important applications. First, it is known that different isolation techniques result in the divergent recovery of particular vesicle subsets. Taking three commonly used “exosome” isolation techniques as test cases (ultracentrifugation, size-exclusion chromatography, and polymer-based precipitation), the capacity for convenient and accurate isolate compositional analysis by iFCM is demonstrated. The approach was able to corroborate and to quantify the known skewing of subtype recovery among different isolation approaches. Second, exosomes are a particularly widely studied EV subset. Applying exosome-specific markers to samples collected from an optimal clinical transplantation model, the researchers verify the capacity for iFCM to detect exosomes in circulation, to establish their tissue of origin, and to provide insights as to their functional immunological potential. Finally, they describe a technique for establishing whether the transfer of a molecule of interest to a target cell is exosomally mediated. In so doing, they highlight the approach’s utility in assessing the functional impact of circulating exosomes and in identifying their targets.

Profiling of circulating exosomes for tissue-specific biomarker discovery and functional analysis

exosomes

(A) Small EVs (sEVs) from an HLA-B27-positive, HLA-B8-negative recipient were gated in G1 before and after receiving a liver allograft from an HLA-B8-positive, HLA-B27-negative donor. Bona fide CD63-positive exosomes were analyzed for their expression of donor and recipient HLA. Donor HLA-bearing exosomes become detectable in circulation after liver transplantation. (B) PD-L1 expression was analyzed in exosomes bearing either donor or recipient HLA and found higher in the former group post-transplantation. (C) Percentage of HLA-A3 positive sEVs were compared between genotypically confirmed HLA-A3+ (n = 3) and HLA-A3 negative (n = 3) individuals. (D) Percentage of donor-HLA + sEVs observed pre- and post-liver transplantation, in genotypically confirmed HLA-mismatched liver transplant recipients (n3). Data represented as mean ± SD. *P < 0.05.

These researchers set out a new methodological approach by which small extracellular vesicle subsets, exosomes in particular, can be conveniently and comprehensively investigated, thereby offering novel phenotypic and functional insights.

Mastoridis S, Bertolino GM, Whitehouse G, Dazzi F, Sanchez-Fueyo A, Martinez-Llordella M. (2018) Multiparametric Analysis of Circulating Exosomes and Other Small Extracellular Vesicles by Advanced Imaging Flow Cytometry. Front Immunol 9:1583. [article]

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