Exploiting Manipulated Small Extracellular Vesicles to Subvert Immunosuppression at the Tumor Microenvironment through Mannose Receptor/CD206 Targeting

Immunosuppression at tumor microenvironment (TME) is one of the major obstacles to be overcome for an effective therapeutic intervention against solid tumors. Tumor-associated macrophages (TAMs) comprise a sub-population that plays multiple pro-tumoral roles in tumor development including general immunosuppression, which can be identified in terms of high expression of mannose receptor (MR or CD206). Immunosuppressive TAMs, like other macrophage sub-populations, display functional plasticity that allows them to be re-programmed to inflammatory macrophages. In order to mitigate immunosuppression at the TME, several efforts are ongoing to effectively re-educate pro-tumoral TAMs. Extracellular vesicles (EVs), released by both normal and tumor cells types, are emerging as key mediators of the cell to cell communication and have been shown to have a role in the modulation of immune responses in the TME. Recent studies demonstrated the enrichment of high mannose glycans on the surface of small EVs (sEVs), a subtype of EVs of endosomal origin of 30–150 nm in diameter. This characteristic renders sEVs an ideal tool for the delivery of therapeutic molecules into MR/CD206-expressing TAMs. In this review, we report the most recent literature data highlighting the critical role of TAMs in tumor development, as well as the experimental evidences that has emerged from the biochemical characterization of sEV membranes. In addition, we propose an original way to target immunosuppressive TAMs at the TME by endogenously engineered sEVs for a new therapeutic approach against solid tumors

Cell Propagation of Cholera Toxin CTA ADP-Ribosylating Factor by Exosome Mediated Transfer

In this study, we report how the cholera toxin (CT) A subunit (CTA), the enzyme moiety responsible for signaling alteration in host cells, enters the exosomal pathway, secretes extracellularly, transmits itself to a cell population. The first evidence for long-term transmission of CT's toxic effect via extracellular vesicles was obtained in Chinese hamster ovary (CHO) cells. To follow the CT intracellular route towards exosome secretion, we used a novel strategy for generating metabolically-labeled fluorescent exosomes that can be counted by flow cytometry assay (FACS) and characterized. Our results clearly show the association of CT with exosomes, together with the heat shock protein 90 (HSP90) and Protein Disulfide Isomerase (PDI) molecules, proteins required for translocation of CTA across the ER membrane into the cytoplasm. Confocal microscopy showed direct internalization of CT containing fluorescent exo into CHO cells coupled with morphological changes in the recipient cells that are characteristic of CT action. Moreover, Me665 cells treated with CT-containing exosomes showed an increase in Adenosine 3',5'-Cyclic Monophosphate (cAMP) level, reaching levels comparable to those seen in cells exposed directly to CT. Our results prompt the idea that CT can exploit an exosome-mediated cell communication pathway to extend its pathophysiological action beyond an initial host cell, into a multitude of cells. This finding could have implications for cholera disease pathogenesis and epidemiology

Generation, quantification, and tracing of metabolically labeled fluorescent exosomes

Over the last 10 years, the constant progression in exosome (Exo)-related studies highlighted the importance of these cell-derived nano-sized vesicles in cell biology and pathophysiology. Functional studies on Exo uptake and intracellular trafficking require accurate quantification to assess sufficient and/or necessary Exo particles quantum able to elicit measurable effects on target cells. We used commercially available BODIPY® fatty acid analogues to label a primary melanoma cell line (Me501) that highly and spontaneously secrete nanovesicles. Upon addition to cell culture, BODIPY fatty acids are rapidly incorporated into major phospholipid classes ultimately producing fluorescent Exo as direct result of biogenesis. Our metabolic labeling protocol produced bright fluorescent Exo that can be examined and quantified with conventional non-customized flow cytometry (FC) instruments by exploiting their fluorescent emission rather than light-scattering detection. Furthermore, our methodology permits the measurement of single Exo-associated fluorescence transfer to cells making quantitative the correlation between Exo uptake and activation of cellular processes. Thus the protocol presented here appears as an appropriate tool to who wants to investigate mechanisms of Exo functions in that it allows for direct and rapid characterization and quantification of fluorescent Exo number, intensity, size, and eventually evaluation of their kinetic of uptake/secretion in target cells

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

Genetic and metabolic labelling of Extracellular Vescicles: a new tool for biogenesis studies

It has been described that the Human Immunodeficiency Virus (HIV)-1 Nef protein associates with exosomes through anchoring its N-terminal myristoylation to lipid raft microdomains at the endosome membranes. To study Nef association with exosomes, we used a Nef protein mutant defective for all anti-cellular Nef functions that presents a N-terminal palmitoylation, fused at C-terminal with Green Fluorescent Protein (GFP). To demonstrate that Nefmut-GFP goes to compartments like late endosomes or other organelles involved in exosomes biogenesis, 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 Nefmut-GFP vector and pulsing them with C12 we could purify exosomes containing Nefmut-GFP (Nef-GFP exo) and/or C12 (C12 exo). Fluorescent exosomes were characterized for typical exosomes markers and were analyzed by density gradients showing that Nef-GFP exo could be separated in two distinct peaks whereas C12 exo displayed only one fluorescent peak. Furthermore, they could be sorted by Fluorescence-activated Cell Sorting (FACS) that can be further characterized. In conclusion Nefmut-GFP efficiently incorporates into exosomes that display typical exosome markers (CD63, CD81, CD9, Alix, TSG101). NanoSight analysis showed that vesicles have a mean diameter of 124 nm, compatible with the dimensions of the exosomes reported in literature. Analysis pre-sorting by FACS of double labelled Nefmut-GFP/C12 exosomes showed that the majority of exosomes had incorporated the Nefmut-GFP protein, (92,4% ), of which 40% of the total labeled exosomes is double labeled with Nefmut-GFP and C12 fluorescent lipid. Density gradient separation of double labelled Nefmut-GFP/C12 exosomes showed two distinct peaks for Nefmut-GFP exo whereas C12 exo displayed only one fluorescent peak. Distribution analysis of exo markers by western blot suggested we may have unraveled two different populations of exosomes that can be further characterized