Fig 2: GC-EVs Loaded with miR-675-3p Increase PD-L1 Expression to Stimulate the Immune Escape in Mice Bearing Transplanted Tumors In Vivo(A) Process diagram of EV treatment during tumor formation. (B) Quantitative analysis of differences in body weight of mice in each group. (C) Quantitative analysis of tumor volume of mice. (D) Representative images of tumors from mice in each group. (E) Quantitative analysis of tumor weight of mice. (F) IHC assay to detect CD3+ expression in T cells and tumors of mice in each group. (G) Flow cytometry to determine the proportion of IFN-?+ in CD3+ cells in TILs of tumors. (H) CXXC4 protein level in tumor tissues normalized to GAPDH determined by western blot analysis. *p < 0.05 compared with mice treated with EV-mimic-NC. NC, negative control. The measurement data are expressed as mean ± standard deviation. An unpaired t test was used for comparison between the two groups. Data at different time points were analyzed using repeated-measures ANOVA followed by a Bonferroni’s post hoc test. n = 8 in each group.

Fig 3: SNHG12 depletion suppresses immune escape in vivo. (A) Weight of mice recorded every 3 d. (B) Growth curve of transplanted tumor recorded every 5 d. (C) image of transplanted tumors (Scale = 1 mm). (D) CD3+ cells in transplanted tumors determined by IHC. (E) the rate of IFN-? positive cells in transplanted tumors determined by flow cytometry. * p < 0.05 vs. SKOV3 + T cells. Statistical comparisons were performed using unpaired t test when only two groups were compared or by Tukey’s test-corrected one-way ANOVA with when more than two groups were compared. Variables were analyzed at different time points using Bonferroni-corrected repeated measures ANOVA.

Fig 4: Up-regulated CXXC4 exerts inhibitory effects on the proliferation of gastric cancer cells and activation of T cells by inhibiting the expression of MIR100HG. A, The expression of CXXC4 and MIR100HG measured by RT-qPCR, n = 3. B, The proliferation of SGC7901 cells detected by CCK-8 assay, n = 3. C, The proliferative ability of CD3+ T cell and the proportion of activated IFN-?+ T cells detected by flow cytometry, n = 3. D, The IFN-? cytokine secreted by T cells measured by ELISA, n = 3, *P < .05 oe-CXXC4 + oe-NC vs oe-NC, # P < .05 oe-CXXC4 + oe-MIR100HG vs oe-CXXC4 + oe-NC. Measurement data were expressed as mean ± SD. Comparisons between two groups in panels A, C and D were analysed by unpaired t test. Comparisons among multiple groups in panels A, C and D were analysed using one-way ANOVA, followed by Bonferroni's post hoc test. The data at different time points in panel B were analysed by the repeated measures ANOVA followed by Bonferroni's post hoc test. The experiment was repeated three times independently

Fig 5: HOTTIP enhances PD-L1 expression in neutrophils, thereby contributing to immune escape of OC cells. The mice (5 in each group) were intraperitoneally injected respectively with 100 µL PBS, neutrophils + T cells (5 × 106 cells for either;, 1: 1, a total of 100 µL), SKOV3-neutrophils + T cells (5 × 106 cells for either; 1: 1, a total of 100 µL), SKOV3-neutrophils overexpressing HOTTIP + T cells (5 × 106 cells for either; 1: 1, a total of 100 µL), or PD-L1 antibody (20 µg/mL)-treated SKOV3-neutrophils overexpressing HOTTIP and T cells (5 × 106 cells for either; 1: 1, a total of 100 µL). a The body weight of mice after treatment. b The tumor volume of mice after different treatment. c Immunohistochemical staining of CD3+ T cell infiltration (400 ×). d The expression of IFN-?+ in CD3 T+ cells as detected by flow cytometry. * p < 0.05 vs. the SKOV3-neutrophils + T cells. The measurement data were summarized as mean ± standard deviation. The one-way analysis of variance was adopted to compare the data among multiple groups, followed by Tukey’s post hoc test. Repeated measures analysis of variance was applied to compare the data at different time points, followed by Bonferroni post hoc test. n = 5. The experiment was repeated 3 times independently