Fig 2: OV-aCD47-G1 is effective for locoregional delivery of the anti-CD47 antibody, while systemic administration of aCD47-G1 is not effective.a Experimental timeline for in vivo studies using an orthotopic model of human GBM. Experiment details are provided in the main text. Group 1, saline; Group 2, OV-Q1; Group 3, a combination of OV-Q1 plus i.p. administration of aCD47-G1; and Group 4, OV-aCD47-G1. After tumor implantation, Groups 2, 3 and 4 received intracranial injection of oHSV (OV-Q1 or OV-aCD47-G1) at a dose of 2 × 105 PFU per mouse on day 21. Group 1 received saline as control. On day 22, Group 3 received i.p. injection of purified aCD47-G1 at the dose of 150 µg per mouse. Groups 1, 2, and 4 received i.p. injection of saline as control. All mice were euthanized on day 23 for blood and brain harvesting. b The concentration of aCD47-G1 in plasma measured by ELISA in mice from different treatments. One-way ANOVA with P values corrected for multiple comparisons by the Bonferroni test (n = 3 animals). c, d Slides from the brain tissues isolated from experimental mice were subjected to H&E and immunohistochemical staining, the latter with anti-HSV, anti-human Fc, which identifies IgG, anti-CD11b or anti-NKp46 antibodies. Images with high and low magnifications are shown in (c) and (d), respectively. The boxed images in (d) are shown in higher power in (c). Data presented are representative of one (c, d) or three (b) mice of at least three mice in total with similar data. Data in (b) are presented as mean values ± SD.

Fig 3: Characterization and identification of designed EVs. (A) Schematic of CD47-overexpressing lentiviral construct. (B) Cytosolic EGFP fluorescence observed under inverted fluorescence microscopy. Scale bar = 100 μm. (C&D) Flow cytometric histograms and quantification for CD47 expression (n = 5). (E) Schematic diagram of EV extraction. (F) EV morphology and size distribution were examined using transmission electron microscopy (TEM) and nanoparticle tracking analysis (NTA), respectively. Scale bar = 50 nm. (G-I) Size distribution, zeta potential, and polydispersity index (PDI) were determined by dynamic light scattering. Statistical evaluations were conducted using a two-tailed unpaired Student’s t-test. Results were presented as mean ± SEM (*** P < 0.001)

Fig 4: Expression of CD47 and common markers on EV Surfaces. (A) Schematic diagram depicting ExoView 200-based EV biological characterization. (B) Representative images demonstrating co-localization of CD81 and CD9 on individual EVs. An enlarged image is displayed in the top right corner to highlight details. Scale bar = 20 μm. (C&D) Quantification of CD9+ and CD81+ EVs from CD81 or CD9 capture spots. (E-J) Aldehyde/sulfate latex beads-based flow cytometric histograms detail CD81, CD63, and CD47 expression on individual EVs (n = 5). Statistical evaluations were conducted using one-way ANOVA. Results were presented as mean ± SEM (*** P < 0.001)

Fig 5: Blocking the SIRPα-CD47 axis enhances macrophage phagocytosis of obese vWAT-Exos. A-C Representative confocal microscopy images showing BMDMs (F4/80+ cells, green) phagocytosis of PKH26-labeled vWAT-Exos (panel a, left), iWAT-Exos (panel b, left) and BAT-Exos (panel c, left) derived from HFD-fed and LFD-fed mice, with quantitation shown in right. The cells nuclei were stained with DAPI (blue). n = 50 cells per group. Scale bar, 50 μm. D Normalized expression of inflammatory cytokine and chemokine genes in BMDMs treated with vWAT-Exos derived from HFD-fed and LFD-fed mice. n = 6 per group. E and F Flow cytometry analysis and quantitation of CD47 expression in PKH26+ AT-Exos (panel e) and CD63+ AT-Exos (panel f) derived from HFD-fed and LFD-fed mice. n = 4 per group. G Representative confocal microscopy images showing macrophage (F4/80+ cells, green) derived from HFD-fed SIRPαΔMac and SIRPαloxp/loxp mice phagocytosis for PKH26-labeled vWAT-Exos (left), with quantitation shown in right. The cells nuclei were stained with DAPI (blue). n = 50 cells per group. Scale bar, 50 μm. H Flow cytometry analysis (left) and quantitation (right) of macrophage (F4/80+ cells) derived from HFD-fed SIRPαΔMac and SIRPαloxp/loxp mice phagocytosis of PKH26.+ vWAT-Exos. n = 6 mice per group. All data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001