Figure 4.

Epigenetic therapy with the combination of 5-aza-dC and TSA promotes reprogramming of M2-type TAMs into the M1-like phenotype in the tumor microenvironment. (A, B) qRT-PCR analysis of the relative mRNA levels of M2- (A) and M1-type (B) cytokines and markers in tumor tissues after treatment of 4T1 tumor-bearing mice with 5-aza-dC (referred as A) and TSA (referred as T), alone or in combination (A+T). (C–F) Flow cytometric analysis of the population of CD206⁺ M2 macrophages and CD86⁺ M1 macrophages (C), CD11b⁺Gr1⁺ MDSCs (D), CD4⁺ T cells (E), and CD8⁺ T cells (F) in the tumor tissues after treatments. (G, H) Tumor volumes after treatments in the absence or presence of CD8⁺ T cell depletion (G) and macrophage depletion using clodronate (H). Control (Ctrl) liposomes were used as control for clodronate. Data represent the mean ± SD of three separate experiments performed in triplicates (A–F) and three mice (n=3) per group (G, H). *, P<0.05; **, P<0.01; ***, P<0.001; n.s., not significant, as assessed using one-way ANOVA followed by Tukey’s multiple comparison post-hoc test.