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Size matters in nanoscale communication

Exosomes are heterogeneous, nanoscale vesicles that mediate cellular communication. A study now leverages a size separation strategy to identify sub-classes of nanoparticles, revealing a subtype without an encapsulating membrane and variation in vesicle cargo, suggesting that size is not the only driver of heterogeneity.

Extracellular vesicles, of which exosomes represent one nanoscale subpopulation1, have been recognized as bona fide promoters of cancer progression because they function as mediators of communication between the cell of origin and adjacent cells, as well as distant tissues1,2. The underpinnings of this mechanism of communication and the nature of what is being communicated have been under intense investigation over the past five years. An active and specialized role for cancer-derived extracellular vesicles in tumour progression has been confirmed, which includes communication with and transfer of active biomolecules to the tumour microenvironment3. In addition, a functional role for extracellular-vesicle cargo sorting in disease progression has been demonstrated and the mechanism revealed4. The inhibition of exosome secretion or biogenesis also leads to defective tumour cell migration5, suggesting that the tumour-promoting function of extracellular vesicles can be therapeutically targeted3,6.

In this issue of Nature Cell Biology, Zhang et al.7 report the implementation of asymmetric flow-field fractionation (AF4) of nanoscale particles released by cells to classify these components of the cellular communication milieu according to size. In doing so, this work confirmed the presence of at least two previously reported exosome subclasses8, named small and large exosomes, and also identified a previously unappreciated nanoparticle, termed exomere. This is a discrete and abnormally small nanoparticle of about 35 nm in diameter that exhibits a distinct protein, lipid, RNA and DNA profile compared to exosomes.

Zhang et al. performed an in-depth molecular analysis to compare the cargo of exomeres with two different exosome populations (large exosomes, Exo-L, and small exosomes, Exo-S), purified from the same cell type. Their findings indicate that both exosome subpopulations have distinct biophysical and molecular properties, which differ from those of exomeres. More specifically, all three nanosized particles displayed diverse proteomic, lipidic, RNA and DNA profiles, and N-glycosylation patterns, suggesting that they might originate from different biogenesis mechanisms. Moreover, once injected into the bloodstream of animals, the three particles also exhibited different biodistribution. The authors therefore propose that exosomes and exomeres are likely to carry out different functions in intercellular communication.

Possibly the most compelling question in the extracellular vesicle field is the mechanism by which heterogeneity of nanoscale particles is accomplished. Size and cargo heterogeneity among extracellular vesicles is now commonly appreciated, with substantial evidence in the literature8,9,10. In addition, micro-sized vesicles such as exophers, migrasomes and large oncosomes, have been discovered by several laboratories11,12,13 (Fig. 1). It is generally presumed that this diversity is the result of distinct biogenesis mechanisms14. However, only the biogenesis of exosomes has been revealed in some detail to date15.

Fig. 1: Schematic representation of vesicle subpopulations that comprise the cellular communication milieu.
figure1

Stratified extracellular vesicles, including recently identified exomeres, listed according to size ranges. Whereas the biogenesis of exosomes is relatively well defined, biogenesis of larger vesicles and exomeres is currently unknown. Extracellular vesicles contain cargo material from the cytoplasm, mitochondria, Golgi, plasma membrane, endoplasmic reticulum and nucleus. EV, extracellular vesicle; MVB, multivesicular bodies.

Zhang et al. further found that exomeres were enriched in proteins involved in metabolism, especially glycolysis and mTORC1 metabolic pathways. These results suggest that exomere biogenesis involves subcellular organelles or activity associated with the cell’s metabolism, such as mitochondrial function. Similarly, they speculate that the receiving cells are likely to interpret this exomere-delivered signal in the form of a metabolic response. The authors demonstrated that exomeres were also enriched in proteins associated with the endoplasmic reticulum, mitochondria and microtubules, proposing a potential role for these proteins in exomere biogenesis and secretion. However, these classes of proteins have been previously attributed to exosomes. Another interesting observation is that even though exomeres did not have a lipid bilayer, they were enriched in certain types of lipids, which differ from those found in Exo-S and Exo-L. In addition, DNA packaging in exomeres and exosomes varied by tumour-type whereas RNA was packaged in Exo-S and Exo-L independently of the tumour cells they originated from.

The analysis of nanoparticle cargo revealed differences not only between each size subclass, but also within each class. Previously, expression of CD9, CD63, CD81, Tsg101 and Alix1 has been considered as specific markers of exosomes. However, in this work they were distributed very differently between Exo-L and Exo-S, suggesting that more than one type of exosome populations exists. These data are in line with a previous study reporting that extracellular vesicle populations are heterogeneous8. The detailed, in-depth analysis of particle composition performed by the authors demonstrated that each particle subclass had a notable enrichment of specific cargo, possibly indicating different functions and properties. Compared to Exo-S and Exo-L, exomeres were also enriched in enzymes, suggesting potential roles in influencing the metabolic program in target organ cells, as well as in proteins associated with coagulation (for instance, factors VIII and X) and hypoxia. Conversely, Exo-S predominantly contained proteins associated with endosomes, multivesicular bodies, vacuoles and phagocytic vesicles, whereas Exo-L were specifically enriched in proteins that make up the plasma membrane, cell–cell contacts or junctions, the late-endosome, and the trans-Golgi network. These findings suggest that Exo-S are most likely bona fide, canonical exosomes, for example derived from intraluminal vesicles of endosomal compartments. By contrast, Exo-L may represent non-canonical exosomes or extracellular vesicles of different sub-cellular origin, for instance derived from plasma membrane budding.

Although enrichment of certain cargos within a subclass indicates commonality in biogenesis and function, the authors also revealed substantial intra-class heterogeneity evident in cargo variability between cell lines. By comparing several cancer cell lines, including breast cancer, pancreatic cancer and melanoma in parallel, Zhang et al. confirmed that even though each cell line contained the three nanoparticle subclasses, the composition of their cargo was very much dependent on the cell line from which they are derived. Although this result is not unexpected, it additionally corroborates that heterogeneity among nanoparticle populations is driven not only by size but also by cargo composition. Considering that the cargo will dictate, in large part, the response of the recipient cell to the particle, the mechanisms that control cargo composition in different types of particles will be an interesting matter to focus on in future studies.

One of the most important implications of this study is that it shows that extracellular vesicle heterogeneity is not only defined by variation in size, but also by variation in cargo between and within each size class. Although the possibility that exomeres are simply aggregates of molecules rather than extracellular vesicles was not ruled out, the finding that exomeres contain a complex array of macromolecules corroborates the hypothesis that exomeres are indeed a unique class of nanoparticle with, as of yet, unknown function. If true, then exomeres add another dimension to the heterogeneity of the nanoparticle communication milieu, which, if better understood, might allow improved insights into cellular communication mechanisms and also provide a previously overlooked biomarker. The discovery of exomeres and stratification of exosomes using AF4 emphasizes the effectiveness of this technique and the potential to reveal additional populations of particles. One limitation of this study is the lack of functional experiments. However, the difference in cargo, and especially in N-glycosylation, which has been rarely investigated in extracellular vesicles, is highly suggestive of functional differences that can be investigated in the future. Collectively, this comprehensive investigation will certainly open new avenues for translational studies of extracellular vesicles and particles in diagnostic, prognostic and therapeutic applications.

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Correspondence to Dolores Di Vizio.

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Zijlstra, A., Di Vizio, D. Size matters in nanoscale communication. Nat Cell Biol 20, 228–230 (2018). https://doi.org/10.1038/s41556-018-0049-8

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