Metabolically labeled exosomes for biogenesis and functional studies

Exosomes are nanovesicles containing bioactive molecules, including proteins and nucleic acids, which make them a potent means of cellular communication. In our laboratory, we developed a powerful method to metabolically label fluorescent exosome (C16 exo) that allowed us to study some aspects of the biogenesis of exosomes through the use of a fluorescent lipid (Bodipy FL C16) which, once internalized by cells, is transformed into phospholipids which will form part of the lipid bilayer of the exosomal vesicles. Bodipy C16 is readily internalized by cells showing an initial perinuclear diffused fluorescence distribution to later concentrate in more discrete compartments. Confocal microscopy showed colocalization with lipid transformation sites such as ER and mitochondria, and with specific markers of late endosomes or other organelles (tetraspanins, Golgi markers). C16 exo by melanoma cells are purified by differential ultracentrifugations, quantified by Flow Citometry (FC) and Nanoparticle Tracking Analysis (NTA) and sorted by Fluorescence Activated Cell Sorting (FACS). Electron microscopy analysis of purified and sorted C16 exo showed that C16 exo have the typical shape and size of exosomal vesicles. The secretion kinetic of fluorescent exosomes showed an early release into the extracellular medium that reaches a plateau at about 6 hours. Colocalization studies of C16 exo with tetraspanins fluorescent antibody showed colocalization with CD63 and CD81. If we label with CFSE vesicle pellets derived from unlabelled cells and we compare these CFSE-vesicles with C16 exo on a density gradient we observed a single peak at density 1,08-1,09 g/ml for C16 exo and an additional peak at higher density for CFSE-vesicles. Fractions were analysed by Western Blot for the presence of typical exosome markers and ESCRT components. Taken together these results show an effective labelling of a discrete exosome population that can be further exploited for biogenesis and functional studies