Fig. 4

CircDLG1 promotes tumor progression by increasing the infiltration of MDSCs. a C57BL/6 mice were injected with MFC-sh-NC or MFC-sh-circDLG1 cells, and the tumor volume and tumor metastasis are shown. The mice were sacrificed at 4 weeks after injection. Left: the tumor size was measured, and the tumor volume was calculated every 4 days (n = 10 mice per group), **P < 0.05, two-way ANOVA. Right: the lungs of the mice were removed and paraffin embedded, consecutive sections (4 μm) were made and stained with hematoxylin-eosin, and the micrometastases in the lungs were examined and counted under a dissecting microscope (n = 10 mice per group), **P < 0.05, Student’s t-test. All experiments were repeated three times. Scale bar, left: 5 mm; right: 100 μm. b BALB/c nude mice were injected with MFC-sh-NC or MFC-sh-circDLG1 cells. The mice were sacrificed at 4 weeks after injection. Left: the tumor size was measured, and the tumor volume was calculated every 4 days (n = 10 mice per group). ns, no significance, two-way ANOVA. Right: The lungs of the mice were removed and paraffin embedded. Consecutive sections (4 μm) were made and stained with hematoxylin-eosin, and the micrometastases in the lungs were examined and counted under a dissecting microscope (n = 10 mice per group). ns, no significance, Student’s t-test. All experiments were repeated three times. Scale bar, left: 5 mm; right: 100 μm. c IHC analysis was performed using subcutaneous tumors for CD4, CD8, Gr-1, and CD11b (Gr-1 and CD11b are mouse MDSC markers). HPF, high-power visual field. ns, no significance; **P < 0.05. Student’s t-test. d Flow cytometric analysis of immune cells in subcutaneous tumors. **P < 0.05, Student’s t-test; the experiment was repeated three times. e The expression of CD33 (a marker of human MDSCs) was detected by IHC in human gastric cancer tissues. Scale bar, 50 μm. f The correlation between circDLG1 expression measured by qRT–PCR and CD33⁺ cells. n = 30, *P < 0.05, Pearson correlation analysis