Using single-vesicle technologies to unravel the heterogeneity of extracellular vesicles

Abstract

Extracellular vesicles (EVs) are heterogeneous lipid containers with a complex molecular cargo comprising several populations with unique roles in biological processes. These vesicles are closely associated with specific physiological features, which makes them invaluable in the detection and monitoring of various diseases. EVs play a key role in pathophysiological processes by actively triggering genetic or metabolic responses. However, the heterogeneity of their structure and composition hinders their application in medical diagnosis and therapies. This diversity makes it difficult to establish their exact physiological roles, and the functions and composition of different EV (sub)populations. Ensemble averaging approaches currently employed for EV characterization, such as western blotting or 'omics' technologies, tend to obscure rather than reveal these heterogeneities. Recent developments in single-vesicle analysis have made it possible to overcome these limitations and have facilitated the development of practical clinical applications. In this review, we discuss the benefits and challenges inherent to the current methods for the analysis of single vesicles and review the contribution of these approaches to the understanding of EV biology. We describe the contributions of these recent technological advances to the characterization and phenotyping of EVs, examination of the role of EVs in cell-to-cell communication pathways and the identification and validation of EVs as disease biomarkers. Finally, we discuss the potential of innovative single-vesicle imaging and analysis methodologies using microfluidic devices, which promise to deliver rapid and effective basic and practical applications for minimally invasive prognosis systems.

exoRBase 2.0: an atlas of mRNA, lncRNA and circRNA in extracellular vesicles from human biofluids

“…Extracellular vesicles (EVs) are nano- to micrometer-sized lipid membrane vesicles (mainly including exosomes and microvesicles) secreted by virtually all cell types ( 1 , 2 ). These vesicles are highly abundant in blood-derived plasma/serum, urine, cerebrospinal fluid (CSF), bile, saliva and other human biofluids ( 3 , 4 ).…”

Small extracellular vesicles from malignant ascites of patients with advanced ovarian cancer provide insights into the dynamics of the extracellular matrix

“…Among the primary finding of our study, by applying a recently developed high‐throughput AFM‐based mechanical characterization method, which analyses the contact angle (a parameter related to the deformation extent) of a vesicle once adsorbed on a substrate, we demonstrated that small‐EVs derived from the two sources, bulk fluid ascites and tumor cells short‐term cultures, showed a markedly different stiffness. Compared to classical AFM‐based Force Spectroscopy (AFM‐FS), which represents one of the most widely used techniques for the mechanical characterization of lipid nanovesicles [ 49 ], the herein employed mechanical characterization allows obtaining information on vesicle mechanics just exploiting AFM imaging; this results in larger number of vesicles being sampled at the same time, together with a less challenging data analysis [ 50 , 51 , 52 ]. Moreover, in the original work, Ridolfi et al .…”

Extracellular Vesicles Mediate Communication between Endothelial and Vascular Smooth Muscle Cells

“…Numerous reports have shown that cells secrete a heterogeneous population of EVs with different sizes and composition [ 30 , 31 ]. For example, EVs positive for CD63, a widely used exosome marker, represent only a fraction of EVs purified from dendritic or Hela cells [ 32 ].…”