Intracellular pathways involved in exosome biogenesis: a study of their roles and mechanisms

Rationale/background Exosomes are small extracellular vesicles (sEV) formed within late endocytic compartments/multivesicular bodies (MVB) by invagination of the limiting membrane into the lumen. They play a crucial role in intercellular communication under both physiological and pathological conditions by delivering proteins, metabolites, and nucleic acids to recipient cells. The molecular composition of exosomes is determined by the cell type of origin and by the intracellular pathways involved in their biogenesis and release into the extracellular space. Various molecular machineries have been implicated in the regulation of exosome biogenesis, including the ESCRT machinery, the syntenin–alix pathway, tetraspanins and lipids, such as ceramide. However, several aspects of these regulatory processes remain incompletely understood. General objectives In our laboratory we developed a methodology to obtain fluorescent exosomes (Bodipy exo) of endosomal origin by using Bodipy FL C16, a fluorescent palmitic acid. Upon internalization by cells, C16 is converted into phospholipids that are incorporated into the bilayer of secreted vesicles. Bodipy exo can be precisely quantified by flow cytometry (FC) and further characterized. The objective of this project is to gain insight into the different mechanisms participating in exosome biogenesis, combining this labeling technique with the use of a panel of known inhibitors of intracellular pathways and investigating the release and characterization of fluorescent exosomes. Experimental design and methods Bodipy exo secreted by melanoma cells pulsed with BODIPY FL C16 and treated with inhibitors of cellular pathways were isolated by differential ultracentrifugation and quantified by flow cytometry and Nanoparticle Tracking Analysis (NTA). Colocalization between Bodipy exo and anti-tetraspanins fluorescent antibodies was determined by FC. Characterization of sEV protein content was performed by Western Blot. Transmission Electron Microscopy (TEM) analysis of cells treated with inhibitors was also performed. Results Significant differences in Bodipy exo secretion were observed by flow cytometry following treatment of melanoma cells with selected intracellular pathway inhibitors. Compounds targeting cholesterol and lysobisphosphatidic acid (LBPA) metabolism, ceramide synthesis, endolysosomal acidification, and intracellular trafficking were evaluated. The strongest increase in fluorescent exosome release was seen with U18666A in contrast with Thioperamide, suggesting that cholesterol, rather than LBPA, is the main driver of this effect. Ceramide metabolism inhibitors GW4869 and Desipramine, which target neutral and acid sphingomyelinases (SMase), respectively, showed opposing effects, highlighting distinct roles for SMase isoforms in exosome regulation. Notably, GW4869 selectively reduced Bodipy exo secretion without affecting total sEV release. Inhibitors of endolysosomal acidification had variable outcomes: Bafilomycin A1, a V-ATPase inhibitor, increased Bodipy exo secretion, whereas Monensin, acting via a different mechanism, had no effect, supporting a specific role for V-ATPase in exosome biogenesis. Across most conditions, the percentage of Bodipy exo positive for tetraspanins (CD63, CD81, CD9) remained stable, suggesting minimal effect on tetraspanin loading. However, Western blot analysis revealed marked differences in sEV protein composition, even when secretion levels were unchanged, indicating that inhibitors can selectively affect cargo sorting. Finally, TEM analysis showed notable variations in the number of multivesicular bodies (MVBs) and intraluminal vesicles (ILVs), reflecting structural changes linked to the modulation of intracellular pathways involved in exosome biogenesis. Conclusions The use of selected inhibitors targeting intracellular pathways affects not only the secretion of sEV, but also their protein composition. These findings suggests that alterations in cellular trafficking pathways can influence protein sorting, thereby modulating the exosome biogenesis process. In conclusion, this experimental approach provides valuable insights into the molecular mechanisms underlying exosome formation and offers a foundation for further exploration of the regulatory pathways involved.