Fig. 3: HGF NPs inhibited exosomal PD-L1 and triggered robust immunostimulation.

a Western blot analysis of exosome marker CD63 and PD-L1 via collecting the exosomes from the cell-free media of B16F10 cells with various treatments. GAPDH served as a loading control. Images were representative of the three experiments. b Scheme of in vivo experimental design for (c–m). C57BL/6 mice were subcutaneously inoculated with 1 × 10⁶ B16F10 cells. When tumor volumes reached ~ 100 mm³, mice were treated with PBS, HACA-Fe, HACA-GW, or HGF on day 0, day 2, and day 4. Tumor and tumor-draining lymph node (TDLN) were collected on day 7 for further analysis. c Western blot analysis of exosome marker CD63 and PD-L1 in tumor tissues after treatment. Images were representative of the three experiments. d–f Flow cytometric plots (d) and quantification of CD8⁺ (e) and CD4⁺ (f) T cells, respectively, gated by CD3⁺ T cells in the TDLN (n = 5) in groups of PBS (G1), HACA-Fe (G2), HACA-GW (G3), HGF (G4), and HGF + liproxstatin (G5). g–m Flow cytometric analysis of granzyme B⁺ (GzmB⁺, g-i), Ki67⁺ (j and k), and Tim3⁺ (l and m) in CD8⁺ and CD4⁺ T cells. n = 5 biologically independent animals per group. Data were presented as mean ± SD. Statistically significant differences between groups were identified by one-way ANOVA with Tukey’s post hoc test. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.