Analysis of Exosome Release as a Cellular Response to MAPK Pathway Inhibition

Abstract

Exosome size distributions and numbers of exosomes released per cell are measured by asymmetric flow-field flow fractionation/multi-angle light scattering (A4F/MALS) for three thyroid cancer cell lines as a function of a treatment that inhibits MAPK signaling pathways in the cells. We show that these cell lines release exosomes with well-defined morphological features and size distributions that reflect a common biological process for their formation and release into the extracellular environment. We find that those cell lines with constitutive activation of the MAPK signaling pathway display MEK-dependent exosome release characterized by increased numbers of exosomes released per cell. Analysis of the measured exosome size distributions based on a generalized extreme value distribution model for exosome formation in intracellular multivesicular bodies highlights the importance of this experimental observable for delineating different mechanisms of vesicle formation and predicting how changes in exosome release can be modified by pathway inhibitors in a cell context-dependent manner.

Dynamic Scaling of Exosome Sizes

“…44 The Fréchet EVD was identified previously by us as the EVD function that best represents TPC1 cell-derived exosome size distributions when fitting the measured distributions to the probability density function for the generalized EVD. 16 We note that fitting exosome size distributions using the Weibull distribution can give a negative and therefore unphysical minimum vesicle diameter for the distribution. 34 …”

“…As a consequence of the lower resolution of A4F/MALS for smaller vesicles, the A4F/MALS distributions are shifted to larger vesicle diameters relative to the cryo-TEM distributions. 16 Equation 8 suggests that the difference in resolution between the two measurements can be taken into account by normalizing the exosome diameters by 〈 D 〉. The extended tails of the two distributions plotted using this normalization are in good agreement, indicating that the scaling exponent is insensitive to the difference in the minimum detectable vesicle size for the two methods.…”

“…Probability distribution, p ( D ), of vesicle diameters from cryo-TEM images (circles) and independently from A4F/MALS measurements (squares) for exosomes released from untreated TPC1 thyroid cancer cells 16 plotted as a function of reduced diameter, D /〈 D 〉, where 〈 D 〉 = 46.6 nm (cryo-TEM, n = 1387) and 〈 D 〉 = 61.9 nm (A4F/MALS). The line corresponding to the extended tail of the distributions is provided to guide the eye.…”

“…5–7 However, much broader size distributions have been reported, 14,15 and spherical vesicles with the morphological properties of exosomes and diameters up to ~200 nm have been observed directly in cryogenic transmission electron microscopy (cryo-TEM) images. 13,14,16 Moreover, exosome size distributions are notably right-skewed to larger vesicles, a characteristic that is not captured by specifying either an average vesicle diameter or a range of diameters as a defining criterion for the exosome subpopulation of EVs.…”

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

“…The state-of-the-art technology, asymmetric-flow field-flow fractionation (AF4) 8 , exhibits unique capability to separate nanoparticles and has been widely utilized to characterize nanoparticles and polymers in the pharmaceutical industry and to examine various biological macromolecules, protein complexes and viruses 8, 9 , but rarely tested for extracellular vesicle (EV) analysis 10-14 . Using AF4, nanoparticles are separated based on their density and hydrodynamic properties by two perpendicular flows, i.e., the forward laminar channel flow and the variable crossflow.…”