Identification of distinct nanoparticles and subsets of extracellular vesicles by asymmetric flow field-flow fractionation

Abstract

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution, and functions. By employing asymmetric-flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90-120 nm; small exosome vesicles, Exo-S, 60-80 nm) and discovered an abundant population of non-membranous nanoparticles termed “exomeres” (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins and specific pathways, such as glycolysis and mTOR signaling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signaling pathways, respectively. Exo-S, Exo-L, and exomeres each had unique N-glycosylation, protein, lipid, and DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating and addressing the complexities of heterogeneous nanoparticle subpopulations.

Extracellular Vesicles from Neurosurgical Aspirates Identifies Chaperonin Containing TCP1 Subunit 6A as a Potential Glioblastoma Biomarker with Prognostic Significance

“…The EV research field is rapidly expanding and the existence of diverse EV subtypes with distinct biophysical and biochemical properties is becoming evident. It is important to note that the methods employed here do not separate subtypes of small EVs or other structurally and functionally distinct non‐membranous particles, e.g., exomeres …”

Exosomes in cancer development and clinical applications

“…The heterogeneity of exosomes in association with exosome‐mediated cell functions, distinct molecular cargo profiles, and the resultant biophysical characteristics have been investigated according to their distinct morphology and/or sizes. The latter has been used to discriminate large exosome vesicles (90‐120 nm), small exosome vesicles (60‐80 nm), or nonmembranous nanoparticles (also called exomeres, ~35 nm) . Intrinsically, distinct molecular signatures in exomeres and exosomes were further seen in proteomic, lipidomic, and glycomic analyses.…”

“…Based on proteomic profiling, enzymes related to metabolism and hypoxia as well as microtubule and coagulation proteins are more abundantly expressed in exomeres compared to large or small membranous exosomal vesicles. In contrast, large and small exosome vesicles show enrichment in mitotic spindle and interleukin (IL)‐2/STAT5 signaling, and proteins related to endosomal secretion, respectively . Moreover, the difference in sialylated glycoproteins (ie, galectin‐3‐binding protein) between exosomes and exomeres is organ distribution .…”

Proteomic Analysis of Extracellular Vesicles for Cancer Diagnostics

“…EVs are generally categorized into subtypes based on their cellular origin and size. While exomeres are suggested to be non‐membranous nanoparticles, they are illustrated here for comparison purposes. Inlet, an EV consists of the outer and inner membrane leaflets with the cytosol in the center.…”