Cargo and Cell Specific Differences in Extracellular Vesicle Populations Identified by Multiplexed Immunofluorescent Analysis

Abstract

Extracellular vesicles (EVs) are implicated in a wide variety of biological activities, have been implicated in the pathogenesis of numerous diseases, and have been proposed to serve as potential biomarkers of disease in human patients and animal models. However, characterization of EV populations is often performed using methods that do not account for heterogeneity of EV populations and require comparatively large sample sizes to facilitate analysis. Here, we describe an imaging-based method that allows for the multiplexed characterization of EV populations at the single EV level following centrifugation of EV populations directly onto cover slips, allowing comprehensive analysis of EV populations with relatively small samples. We observe that canonical EV markers are present on subsets of EVs which differ substantially in a producer cell and cargo specific fashion, including differences in EVs containing different HIV-1 proteins previously reported to be incorporated into pathogenic EVs. We also describe a lectin binding assay to interrogate EVs based on their glycan content, which we observe to change in response to pharmacological modulation of secretory autophagy pathways. These studies collectively reveal that multiplexed analysis of EV populations using fluorescent microscopy can reveal differences in specific EV populations that may be used to understand the biogenesis of specific EV populations or to interrogate small subsets of EVs of interest within larger EV populations.

Urinary extracellular vesicles: A position paper by the Urine Task Force of the International Society for Extracellular Vesicles

“…EV flow cytometry uses specific antibodies and/or ligands to either enrich uEVs or exploit the signal of a fluorescent tag linked to the antibody. An example is the use of anti-tetraspanin coated magnetic beads when analysing EVs with conventional flow cytometry, which offers combined isolation and analysis of uEVs (Campos-Silva et al, 2019;Welsh et al, 2020). Further developments of flow cytometric based analysis of EVs include use of imaging flow cytometry (Musante et al, 2020) and nano-flow cytometry (Tian et al, 2020) for direct uEV analysis in cell-free urine.…”

“…Microfluidic devices such as nanoscale lateral displacement arrays on a chip (Nano‐DLD arrays), double filtration microfluidic system on a microchip, microfluidic nanowires followed by in situ RNA extraction, centrifugal lab‐on‐a‐disc nanofilters, and nanoparticle‐based time resolved fluorescence immunoassay (NP‐TRFIA) are prototypes showing the feasibility of isolating and analysing uEVs directly from cell‐free urine (Duijvesz et al., 2015; Islam et al., 2019; Liang et al., 2017; Smith et al., 2018; Woo et al., 2017; Yasui et al., 2017). Important developments allow multiplexing and enable the detection of combinations of markers on the EV surface (Burbidge et al., 2020). In line with this, a single particle interferometric reflectance imaging sensor platform (SP‐IRIS) is now commercially available (Daaboul et al., 2016).…”

Machine learning-based clustering of nanosized fluorescent extracellular vesicles

“…These low amounts of multiple labeled EV are similar to those observed in other studies. 27–30 Again, our new SBA based approach was capable of specifically detecting multiple markers on a single EV allowing to sort single EV into different subgroups. In control experiments, using separately labeled and pooled EV samples a very low number of multiple labeled EV were observed using SBA.…”