Cargo and cell‐specific differences in extracellular vesicle populations identified by multiplexed immunofluorescent analysis

Abstract

Extracellular vesicles (EVs) have been 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 the 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 a 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 and/or to interrogate small subsets of EVs of interest within larger EV populations in biological samples.

Metabolic Syndrome Is Associated With Altered mRNA and miRNA Content in Human Circulating Extracellular Vesicles

“…Circulating EVs are released from various tissues and organs, reflect the status of their parental cells, and may also mediate important processes in target cells (5). We found that several biological protein functions, including protein complex biogenesis and transport are targeted by dysregulated miRNAs within those EVs.…”

“…Besides apoptotic bodies, the two main types of EVs are exosomes and microvesicles, which are the most studied EVs populations. Known as carriers for circulating miRNAs, plasma EVs also contain other nucleotides, lipids, and proteins (5). EVs function as mediators of intracellular communication by shuttling bioactive cargo between the parent cells in which they originate and neighboring or distant recipient cells (6).…”

Efficient Neutrophil Activation Requires Two Simultaneous Activating Stimuli

“…Although we did not observe clear differences in the mean buoyant density between these two EV types, the light scattering signals do show differences between the 10 kg and 100 kg EVs, which could indicate differences in size and/or composition. Since molecular cargo selection of EVs is not a random process, but rather regulated via distinct and regulated processes that are dependent on the type and state of the cell, the type of stimuli that were received, and the subcellular origin of EVs [ 48 , 49 ]. For mast cells for example, we previously observed clear differences in the buoyant density, light scattering, and CD9 and CD63 content of EVs released from unstimulated and activated cells [ 50 ].…”

Unsupervised 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 the separately‐labeled and pooled EV samples a very low number of EV with multiple labels was observed using SBA.…”