Fig 2: Effect of skeletal muscle myoblast differentiation into myotubes on the expression of proteins related to EV subtypes. (A) Representative immunoblots showing equal amounts (20 µg) of myoblast (MB) or myotube (MT) EP protein lysates were subjected to 12% SDS–PAGE. EPs were isolated using Total Exosome Isolation Reagent (ThermoFisher, Waltham, MA, USA). Coomassie blue gel staining was used as a loading control. (B) Quantification of immunoblot analysis showing the expression of different markers of small and medium/large EVs and non-EV co-isolates. Expression of small EV marker proteins TSG101, ALIX and tetraspanins CD63 and CD81 were enriched by several orders of magnitude in myoblast-EP preparations compared with myotube-EPs (* p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, n = 7–12). Conversely, levels of ARF6, a microvesicle marker, and APO-A1, a lipoprotein non-EV co-isolate, were only expressed in myotube-EPs when compared with myoblast-EPs (* p < 0.05, ** p < 0.01, n = 4–12). Cytochrome c and beta-actin were barely expressed in myotube-EPs compared to myoblast-EPs (** p < 0.01, n = 6–9). Data were analyzed using an unpaired Student’s t test and are expressed as scatter plots showing the mean.

Fig 3: Characterization of murine MSCs and MSC exosomes. (A) MSCs were characterized by flow cytometry analysis for surface markers (anti-CD105–PE, clone MJ7/18 [562759], anti-CD106–PE, clone 429 [561613], anti-CD44–APC, clone IM-7 [561862]; all BD Biosciences; anti-CD45–PE, clone 30-F11 [12-0451-82], anti-CD29–PECy7, clone HMb1-1 [25-0291-80], anti-stem cells antigen-1–APC, clone D7 [17-5981-81]; all eBioscience, Vienna, Austria) using the LSRII flow cytometer (BD Biosciences) and analyzed with FlowJo software (Tree Star, Inc, Ashland, OR). (B) MSC differentiation staining for adipocytes (lipid droplets) and osteoblasts (calcium deposit and alkaline phosphatase activity).5 (C) Western blot for exosomal markers flottilin-1 and alix. Western blot analysis was performed using 25 µg protein loaded on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis8 and transferred to polyvinylidene difluoride membranes (0.45-µm pore size, 10401196; Whatman, Maidstone, UK). Ponceau staining (3504; Sigma-Aldrich) was used to confirm equal loading. Blots were incubated with primary antibodies to rabbit anti-alix, clone 3A9 (MCA2493; Bio-Rad Laboratories, Hercules, CA) and mouse anti–flotillin-1, clone EPR6041 (1333497; Abcam, Cambridge, UK). (D) Transmission electron microscopy images of exosomes isolated from MSCs. From a purified exosome suspension, 3 µL was put on a copper electron microscopy grid that was glow-discharged in air. By using an electron microscopy grid plunger freezer (Leica), excess suspension was removed by blotting for 1 second using filter paper (Whatman) at 95% humidity at room temperature and the grid was plunged into liquid ethane at -183°C. Samples were transferred to a Tecnai F20 Transmission Electron Microscope (ThermoFisher Scientific) using a Gatan cryo holder and images were recorded at 15,000× magnification on a Gatan 2kx2k CCD camera (Gatan, Pleasanton, CA) behind a 2001 energy filter operating at 20 electron-volts slit width. MW, molecular weight.

Fig 4: Representative Western Blot images of EVs isolated via precipitation, precipitation + SEC and SEC. The expression of CD63, HSP90 and Alix confirmed the presence of EVs in the samples. Calnexin and albumin were used as a negative control. N=3, all the western blots were repeated at least 2 independent times.

Fig 5: Quantification of TAM-EVs and Validation of the TAM-EV Protein Signature by IP of CD11b+ Tumor-Derived EVs(A) Procedure to isolate EVs from MC38 tumors grown in LysM.Cre/ROSAmT/mG mice.(B) WB analysis of EVs isolated from cultured MC38 cells or MC38 tumors grown in LysM.Cre/ROSAmT/mG mice, after IP with anti-CD9 or control-coated magnetic beads. One representative EV preparation per condition is shown.(C and D) Flow cytometry analysis of tumor-EVs isolated from wild-type (WT) or LysM.Cre/ROSAmT/mG mice. Data show representative dot plots of GFP and tdTomato (C) and quantitative values (mean ± SD; n = 3 independent experiments; D). Statistics by two-way ANOVA, using Sidak’s multiple comparison test.(E) LC-MS/MS analysis of EVs showing quantitative values (mean ± SEM; n = 4 or 3 independent preparations) for CD11b, ALIX, and CD81, normalized to CD9. Statistics by two-way ANOVA, using Tukey’s multiple comparison test.(F) IP procedure for capturing CD11b+ EVs.(G) Flow cytometry analysis of PKH67-labeled EVs bound to anti-CD9, anti-CD11b or isotype-control beads. Data show percentage values (mean ± SEM; n = 4 and 5 preparations for CD11b and CD9 IP, respectively).(H) WB analysis of tumor-derived EVs after IP. One representative EV preparation per condition is shown.(I) Venn diagrams comparing proteins enriched in IgG-EVs versus anti-CSF1R-EVs (FC > 1.7; see Table S2) with those detected in CD11b+ IgG-EVs after IP (FC versus isotype control beads > 2; see Table S4). IP was performed on the same IgG-EV preparation shown in (H).(J) Radial table reporting the 62 proteins of the MC38 TAM-EV signature and their association with biological pathways. The graphical representation limits connection of each protein to two pathways (see also Table S5).(K) The data in (I) are shown after removing potential protein contaminants identified according to the CRAPome database.See also Figures S3, S4, S5, and S6.