Engineered Extracellular Vesicles for biogenesis and immunomodulation studies

Small Extracellular Vesicles (sEV), as naturally occurring vesicles, have a low intrinsic immunogenic profile, thus showing great therapeutic potential. Over the past few years, several engineering strategies have been devised to manipulate tumor-derived sEVs in order to induce cellular and innate immunity. In our study we use a mutant Human Immunodeficiency Virus (HIV)-1 Nef protein that presents a N-terminal palmitoylation (NefG3C), which increase the specificity of the protein for sEV association and a mutated NefG3C with two additional mutations (Nefmut) to render the protein biologically inactive. Both Nef mutants were fused at C-terminus with Green Fluorescent Protein (GFP) (NefG3C/Nefmut-GFP). To follow the biogenesis of these engineered sEV we used a novel methodology developed in our laboratory to metabolically label exosomes by incubating cells with a red fluorescent fatty acid BODIPY 558/568 C12 (C12). The lipid is readily taken up by cells and transformed into phospholipids that will ultimately form the exosome lipid bilayer. By transfecting HEK293 cells with the NefG3C-GFP or Nefmut-GFP vectors and pulsing them with C12 we could purify exosomes containing NefG3C/mut-GFP and/or C12 (C12 exo). Results show that the number of cell secreted sEV greatly differs for the two constructs probably due to a different association with sEV. Fluorescent sEV were also characterized for typical exosomes markers and analyzed in iodixanol density gradients. NefG3C/Nefmut-GFP/C12 exo could be separated in two distinct peaks whereas C12 exo displayed only one fluorescent peak. Further analysis will show if these two different populations of sEV display different behaviour in terms of efficiency of transfer to recipient cells and ultimately stimulation of the immune system

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