Figure 6. The combination of epigenetic modulators and anti-PD-1 increases CXCR3 ligand expression and converts non-responders to responders.

(A) Expression of CXCL9 and CXCL10 in gated CD11c⁺ MHCII⁺ cells within the indicated solid tumors of REX3 transgenic mice. Bar graph represents the mean values of the indicated data points, and the error bars represent SEM. (B) Schematic of experimental design for the treatment schedule for the epigenetic modulators and anti-PD-1. Orthotopic injection of AT-3 tumor cells (1×10⁶) into the mammary fat pad of mice was performed on day 0. Following tumor inoculation, tumor-bearing mice were treated with four different treatment schemes on day 8, 11 and 14: (1) control; (2) DZNeP (5mg/kg) + 5-AZA-Dc (0.2mg/kg); (3) anti-PD-1 (200μg); and (4) DZNeP (5mg/kg) + 5-AZA-Dc (0.2mg/kg) + anti-PD-1 (200μg). (C) Tumors were excised from REX3 transgenic mice on day 15 of the schedule presented in Figure 6B, followed by analysis of CXCL9 and CXCL10 expression in CD11c⁺ MHCII⁺ cells from AT-3 tumors of REX3 transgenic mice treated with control, epigenetic modulators, anti-PD-1 or a combination of epigenetic modulators and anti-PD-1 antibody. Bar graph represent the mean values of the indicated data points, and the error bars represent SEM; ***p<0.001, with statistical significance determined by Student’s t-test. Representative data are shown from two independent experiments. (D) AT-3 tumor growth in WT mice that were treated with control, epigenetic modulators, anti-PD-1 or a combination of epigenetic modulators and anti-PD-1 (n=6 mice per group). Data represent the mean ± SEM; ***p<0.001, with statistical significance determined by two-way ANOVA. Survival curves show the percentage of mice whose tumor sizes were <100mm² at each time point. Statistical differences in survival were assessed by a Mantel-Cox log-rank test (**p<0.01). Data are representative of two independent experiments. (E) 1×10⁶ AT3 tumor cells were injected into the mammary fat pad of chimeric mice, which were generated using the protocol presented in Figure 5D. Following tumor inoculation, administration of DT (20μg/kg) started one day before control or a combination of epigenetic modulators and anti-PD-1 treatment, and continued until day 19. Tumor growth was monitored until the experimental endpoints (n=5–8 mice per group). Data are given as the mean ± SEM; ***p<0.001, with statistical significance determined by two- way ANOVA.