Metabolically labeled exosomes for biogenesis and functional studies

Introduction: Exosomes are small Extracellular Vesicles (sEV) formed by an endosomal route by inward budding of the lateendosome/multivesicular body (MVB) membrane. Despite in recent years much progress has been made to better define sEVcomposition and biogenesis pathways, their small size and heterogeneity pose challenges to find new reliable labelling strategies toidentify specific exosome populations. We developed an innovative methodology to metabolically label fluorescent sEV throughthe use of a fluorescent lipid (BODIPY C16) that is readily internalized by cells and is transformed into phospholipids which willform part of the lipid bilayer of the secreted vesicles.Methods: Fluorescent sEV secreted in the conditioned media of melanoma cells pulsed with BODIPY FL C16 were purifiedby differential ultracentrifugation, quantified by Flow Cytometry (FC) and Nanoparticle Tracking Analysis (NTA), sorted byFluorescence Activated Cell Sorting (FACS) and further characterized by density gradient separation and Western Blot analysisfor typical sEVs markers. Colocalization studies were performed by confocal microscopy and electron microscopy.Results: Confocal images showed colocalization of BODIPY lipids with lipid transformation sites such as ER and mitochondriaand with specific markers of late endosomes/MVB or other organelles (tetraspanins, Golgi markers, lysosomes) but not with the  of ABSTRACTplasma membrane. Secretion of fluorescent sEV (Bodipy sEV) was followed over time showing an early release of Bodipy sEVinto the extracellular medium with a constant ratio of Bodipy sEV/total EVs, as determined by NTA, up to 6 hours. Bodipy sEVsecreted in the conditioned media purified by differential ultracentrifugation were separated by density gradient fractionation.Fractions analysed by FC displayed a single low density peak at 1,08-1,09 g/ml that is detergent sensitive demonstrating thatfluorescent particles are indeed lipid vesicles and contain tetraspanins (CD63, CD81 and CD9), syntenin and ESCRT componentswhen analysed by Western Blot. Electron microscopy analysis of ultracentrifuged and sorted Bodipy sEV showed that BodipysEVhavethetypicalshapeandsize(about80nm)ofasubpopulationofsEVoftenreferredtoassmallexosomes(Exo-S).Finally,colocalization studies of single Bodipy sEV with tetraspanins fluorescent antibodies showed colocalization of Bodipy sEV withCD63, CD81 and CD9.Summary/Conclusion: Taken together these results show a very specific and effective labelling of a discrete sEV subpopulationthat can be further exploited for biogenesis, internalization and functional studies. This work was supported by the Italian Ministry of Health (grant RF-2019-12369719)