Figure 4.

Enhanced antitumor immunity caused by KDM4C inhibition is mediated by CXCL10. (A) C57BL/6 mice were injected subcutaneously with 1×10⁶ Lewis cells. When the tumor volume reached an average of 100 mm³, the mice were given a vehicle either alone or in combination with SD70 and/or anti-CXCL10. Tumor volume was measured every 3 days (mean±SEM; n=8; ***p<0.001; two-tailed t-test). (B) Tumor wet weights at 22 days’ postinoculation. n=8, **p<0.01. (C, D) Flow cytometry analyses showing the changes in the percentages of CD8⁺ T cells (C) and IFN-γ⁺ CD8⁺ T cells (D) in each group. n=8/group, *p<0.05, ***p<0.001. (E) Changes in Ki67⁺ CD8⁺ T cells with treatment. n=3, **p<0.01, ***p<0.001. (F) CFSE staining of proliferating CD8⁺ T cells measured by flow cytometry. Each experiment was repeated three times independently. (G) Changes in the in vitro migration of CD8⁺ T cells. n=3, ***p<0.001. (H) Schematic illustration of the in vivo migration experiment. (I, J) IHC was used to analyze the infiltration of CD8⁺ T cells in subcutaneously transplanted tumors for each group. Scale bar: 100 µm; n=8/group; ***p<0.001. (K, L) Flow cytometry was used to detect the expression of cytotoxicity markers (IFN-γ, GZMB, Perforin, and CD107a) and exhaustion markers (PD-1 and CD39) in CD8⁺ T cells in each group. n=3; *p<0.05, **p<0.01, ***p<0.001. (M) Lewis cells in 24-well plates were cocultured with pretreated CD8⁺ T cells in the absence or presence of an anti-CXCL10 neutralizing antibody or control antibody for 24 hours. Crystal violet staining identified the surviving tumor cells. Each group of experiments was carried out three times.