Fig. 6

Modified exosomes loaded with miR-21 inhibitor improved PD-1 blockade therapy

(A) Schematic diagram of DNA plasmid construction and the process used to produce modified exosomes loaded with miR-21 inhibitor. (B) DC were transfected with the GNSTM-ANG-Lamp2b-HA plasmid, and the expression of HA in DC and DC-derived exosomes (Dex) was measured by western blotting. (C) ANG-modified Dex was labeled with PKH26 (red) and injected intravenously into mice implanted with GL261 and GFP-labeled MSCs (green) in situ. The uptake of Dex by MSCs was observed using a fluorescence microscope. Scale bar = 50 μm. (D) Relative expression of miR-21 inhibitor in Dex without electroporation, with electroporation, without electroporation and with RNase treatment, or with electroporation and RNase A treatment. (E) Cy3-labeled miR-21 inhibitor (red) were loaded into PKH67-labeled ANG-modified Dex (green) by electroporation and used to treat GA-MSCs. Confocal microscopy showed the uptake of Dex by MSCs. Scale bar = 20 μm. (F, G) ANG-modified Dex containing miR-21 inhibitor were injected intravenously into GL261-bearing mice. The percentage of Gr-1⁺CD11b⁺ MDSCs and the expression of CD73 on MDSCs infiltrating in glioma tissues in mice were measured by flow cytometry. (H) Glioma-infiltrating MDSCs were cocultured with CFSE-labeled splenocytes from normal C57BL/6 mice, and CD8⁺ T-cell proliferation was measured using flow cytometry. (I) Schematic diagram of the schedule for glioma implantation and drug treatment. ANG-modified Dex (30 µg/mouse/time) containing miR-21 inhibitor were injected intravenously three times a week for 2 weeks. Anti-PD-1 antibodies were intraperitoneally injected into the mice (250 µg/mouse/time) on days 7, 10, 13, 16 and 19 after glioma implantation. (J) Tumor volume was evaluated using bioluminescence imaging, and the luminescence quantification is shown. (K) The survival curves of glioma-bearing mice are shown. Statistical significance was determined by the log-rank test. (L) Proposed working model of the miR-21/SP1/DNMT1 positive feedback loop in GA-MSCs induced by glioma exosomal CD44. The data are presented as the mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001