Fig. 5: TKD restores immune microenvironment and improves ICB therapy.

a HCT116 cells were treated with the indicated amount of TKD for 24 h and levels of IRF2, PD-L1, KRAS and β-tubulin were determined by western blotting. b HCT116 cells were treated with TKD for 24 h and the expression of PD-L1 on cell surface was determined by flow cytometry. c KRAS and β-tubulin were determined by western blotting assay and the cell growth was evaluated by CCK-8 assay in TKD-treated MC38K cells. d MC38K cecal transplantation CRC mice were treated with vehicle or TKD (50 mg/kg) per 3 days, then tumors were collected and dispersed into single cells for flow cytometry analysis, the amount of cytotoxic T cells (CD45⁺CD3⁺CD8⁺) and MDSCs (CD45⁺CD11b⁺Gr-1⁺) were analyzed. n = 3. e MC38K-luc cecal transplantation CRC mice were treated with vehicle, PD-1 antibody (200 μg), TKD (50 mg/kg), or the combination of TKD and PD-1 antibody every 3 days and tumor growth was recorded according to fluorescence intensity. n = 5. f Ceca, along with the cecal tumors were collected and weighed after 7 administrations as indicated. n = 5. Scale bar = 1 cm. Arrow, tumor site. g The synergistic effect of PD-1 antibody and TKD was analyzed by CI statistics. CI < 1 indicates synergistic effect. h IF staining for CD8⁺ T cells (CD8) and MDSCs (Gr-1) in TKD, PD-1 antibody or a combination of these two agents treated tumors. n = 3. Scale bar = 100 μm. Data are representative of three independent experiments. Statistical analyses were performed using unpaired t-test in c and d, Kruskal–Wallis test in f, and ordinary one-way ANOVA in h; Error bars, SD; n.s. not significant; *P < 0.05; **P < 0.01; ****P < 0.0001.