Cells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. While a wide range of putative biological functions have been attributed to exosomes, they are assumed to represent a homogenous population of EVs.
A team led by researchers at the University of Oxford hypothesized the existence of subpopulations of exosomes with defined molecular compositions and biological properties. Density gradient centrifugation of isolated exosomes revealed the presence of two distinct subpopulations, differing in biophysical properties and their proteomic and RNA repertoires. Interestingly, the subpopulations mediated differential effects on the gene expression programmes in recipient cells.
B16F10 melanoma cells release distinct subpopulations of exosomes
(A) Schematic overview of the isolation protocol used to obtain different exosome populations. (B,C) P110 was loaded on top (B) or at the bottom (C) of a sucrose density gradient and subjected to ultracentrifugation for 16 h. The resulting fractions (1–10) with increasing density were analyzed for particle number by NTA (upper panels) and the presence of exosome marker proteins ALIX and TSG101 by Western blotting (lower panels). For Western blots, an equal volume of each sample was analyzed. Fractions 3–5 (LD-Exo) and 8–9 (HD-Exo) were pooled for further analysis. (D) Exosomes or MVs were negatively stained with uranyl acetate and visualized by transmission electron microscopy. Scale bars represent 100 nm. (E) Size distribution profiles as determined by NTA. Data shown are representative of three independent experiments. (F,G) Exosomes from B16F10 melanoma cells cultured for 48h in the absence or presence of 7.5 μM GW4869 were isolated as in Fig. 1A. P110 was loaded at the bottom of a sucrose gradient and subjected to ultracentrifugation for 16 h. The resulting fractions (1–10) with increasing density were analyzed for particle number by NTA (F) and the presence of exosome marker proteins ALIX and TSG101 by Western blotting (G). A control sample (Ctrl) was included to show consistent analysis across the membranes. For Western blots, an equal volume of each sample was analyzed. Data shown are representative of two independent experiments.