Fig 1: BC can attenuate the ability of tumor cells to bind PD-1, enhance the cytotoxicity of T cells, and mediate a T cell-dependent antitumor effect(A) PD-L1/PD-1 binding assay in RKO cells treated with BC (10 μM, 24 h). The nuclei were stained with Hoechst. Scale bar, 100 μm.(B) Bound PD-1 was calculated according to the intensity of green fluorescence (n = 3, t test). ∗∗∗p < 0.001 compared with the DMSO group.(C) PD-L1/PD-1 blockade assay performed with RKO cells treated with 1, 5, and 10 μM BC for 12 h. Jurkat NFAT-luciferase reporter cells (10,000 cells/well) were added, and the cells were cocultured for 4 h. Data are presented as the fold induction over the untreated control (n = 3, t test). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with the DMSO group.(D‒G) T cells were cocultured with RKO and HCT116 cells in 12-well plates for 24 days in the presence of BC, and the surviving tumor cells were visualized by crystal violet staining. Relative fold ratios of surviving cell intensity are shown (n = 3, one-way ANOVA). ##p < 0.01 compared with the RKO and HCT116 DMSO groups; ∗∗p < 0.01 and ∗∗∗p < 0.001 compared with the RKO + T cell or HCT116 + T cell groups.(H) MC38 cells were injected into C57BL/6 mice on day −5, and BC was administered as indicated.(I) Ex vivo observation of the tumors from the treated mice (0, 5, 25, and 50 mg/kg).(J) C57BL/6 mice with MC38 tumors were treated intraperitoneally (i.p.) with PBS or BC, and tumor growth was monitored.(K) Comparison of the weight of the tumors from the mice treated with PBS or BC.(L) The body weight curves of the mice measured every 2 days.(M) Expression of PD-L1 in the tumors of mice treated with PBS or BC.(N) Nude mice bearing MC38 tumors received daily i.p. injections of PBS or BC (50 mg/kg) for 12 days(O–Q) (O) Ex vivo observation of the tumors from the treated mice (0 and 50 mg/kg), and (P) tumor growth and (Q) tumor weight were measured.(R) Representative immunohistochemistry (IHC) staining results for PD-L1, CD3, cleaved caspase 3, and FOXP3 in PBS- or BC (50 mg/kg)-treated C57BL/6 mice.Representative positive expressions are indicated by arrows. Scale bar, 10 μm.(S) Quantification of IHC staining. The data shown are the mean value ± SEM (one-way ANOVA). ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 compared with the PBS group.