Figure 2.

Oral administration of bilberry anthocyanins enhances anti-PD-L1 efficiency and changes the mouse gut microbiome structure. (A) C57BL/6 mice were inoculated subcutaneously with 5 × 10⁵ MC38-OVA cells and treated with 200 μg of the immunoglobulin G2b (IgG2b) isotype or the α-PD-L1 mAb on days 7 and 10. CT, the IgG2b isotype control; PD, the α-PD-L1 group; PE, the α-PD-L1/LCP–chitosan group; BA, the α-PD-L1/anthocyanin group; BP, the α-PD-L1/anthocyanin combo group. Repeated measures ANOVA (time × tumor volume) and Sidak’s multiple comparisons test were used to test mouse tumor growth between groups. Data indicates mean ± SEM, n = 6 per group, * p < 0.05. (B) Beta-diversity analysis of microbial communities by using principal component analysis (PCA) based on OTUs. (C) The associated contribution plot illustrating the key OTUs that impact the PCA clustering. (D) Significant increase of Muribaculaceae in the α-PD-L1/LCP–chitosan group. (E) For alpha diversity, richness, chao1, Shannon, Simpson, and ace indices were calculated using random subsamples of 35,043 sequences per sample, Data indicates mean ± SEM. * p < 0.05, ** p < 0.01 (Student’s t-test). (F) OTU richness under different thresholds of relative abundance.