Blautia coccoides and its metabolic products enhance the efficacy of bladder cancer immunotherapy by promoting CD8+ T cell infiltration

Abstract

Background: Immune checkpoint inhibitors (ICIs) have emerged as a novel and effective treatment strategy, yet their effectiveness is limited to a subset of patients. The gut microbiota, recognized as a promising anticancer adjuvant, is being increasingly suggested to augment the efficacy of ICIs. Despite this, the causal link between the gut microbiota and the success of immunotherapy is not well understood. This gap in knowledge has driven us to identify beneficial microbiota and explore the underlying molecular mechanisms.

Methods: Through 16S rDNA sequencing, we identified distinct gut microbiota in patients undergoing treatment with ICIs. Following this, we assessed the impact of probiotics on anti-PD-1 therapy in bladder cancer using mouse models, employing a multi-omics strategy. Subsequently, we uncovered the mechanisms through which Blautia-produced metabolites enhance antitumor immunity, utilizing untargeted metabolomics and a range of molecular biology techniques.

Results: In our research, the LEfSe analysis revealed a significant enrichment of the Blautia genus in the gut microbiota of patients who responded to immunotherapy. We discovered that the external addition of Blautia coccoides hampers tumor growth in a bladder cancer mouse model by enhancing the infiltration of CD8⁺ T cells within the tumor microenvironment (TME). Further investigations through untargeted metabolomics and molecular biology experiments showed that oral administration of Blautia coccoides elevated trigonelline levels. This, in turn, suppresses the β-catenin expression both in vitro and in vivo, thereby augmenting the cancer-killing activity of CD8⁺ T cells.

Conclusions: This research provided valuable insights into enhancing the efficacy of PD-1 inhibitors in clinical settings. It was suggested that applying Blautia coccoides and its metabolic product, trigonelline, could serve as a synergistic treatment method with PD-1 inhibitors in clinical applications.

Keywords: Blautia coccoides; Anti-PD-1 immunotherapy; Gut microbiota; Trigonelline.

Fig. 1

Marker gene sequence profiling of the gut microbiota in patients with immunotherapy. A The flowchart of patient recruitment, treatment and grouping details. B The unweighted-UniFrac distance of β-diversity. C Species composition of gut microbiota at the phylum and genus level in both groups of patients. D Results of LEfSe analysis at the genus level of gut microbiota. E The ROC curve showing the predictive efficacy of the Blautia genus. F Distribution of Blautia in fecal samples. RCC: renal cell carcinoma; NSCLC: non-small cell lung cancer; SCLC: small cell lung cancer; CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease

Fig. 2

Blautia enhanced immunotherapy efficacy by increasing CD8⁺ T cell infiltration. A Experimental grouping and treatment flowchart for mice. B In vivo bioluminescence monitoring pictures of tumor growth in mice from different. C The images of bladder tumors, tumor volume growth curves and statistical charts of tumor weight for mice (n = 6). D Proportion of CD8⁺ T cells in mouse spleen tissue. E Proportion of IFN-γ and Granzyme B in mouse spleen tissue. F Proportion of CD8⁺ T cells in mouse tumor tissue. G Immunohistochemical staining to detect the expression levels of CD8⁺T cells in tumor tissues (200 ×, scale bar: 100 μm). H Proportion of IFN-γ and Granzyme B in mouse tumor tissue. (n = 4) Significant differences were evaluated by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001). Data were presented as Mean ± SEM. NC: negative control; BL: Blautia coccoides; B + P: Blautia coccoides and anti-PD-1; ns: no significant difference

Fig. 3

Microbial metabolite trigonelline promoted anti-PD-1 immunotherapy efficacy through CD8⁺T cell antitumor immunity. A LC–MS analysis of trigonelline content in mice serum samples from the anti-PD-1 group and the B + P combination treatment group. B Experimental grouping and treatment flowchart for mice. C The final subcutaneous tumor images in mice, growth curves of tumor volume and statistical chart of tumor weight(n = 6). D Proportion of CD8⁺ T cells in mouse tumor tissue. E Immunohistochemical staining to detect the expression levels of CD8⁺T cells in tumor tissues (200 ×, scale bar: 100 μm). F Proportion of IFN-γ and Granzyme B in mouse tumor tissue (n = 4). Significant differences were evaluated by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001). Data were presented as Mean ± SEM. NC: negative control; Tri: trigonelline; T + P: trigonelline and anti-PD-1; ns: no significant difference

Fig. 4

Blautia coccoides-derived trigonelline inhibited β-catenin expression. A Immunohistochemical staining to detect the expression levels of β-catenin in tumor tissues (200 ×, scale bar: 100 μm, n = 3). B Immunofluorescence staining of β-catenin expression (red represents β-catenin, blue represents DAPI, 400 ×, scale bar: 20 μm, n = 3). C Statistical analysis graph of β-catenin protein immunohistochemical results; D Statistical analysis graph of β-catenin protein immunofluorescence staining results(n = 3). E The qPCR to detect the expression levels of β-catenin in tumor tissue (n = 6). Significant differences were evaluated by one-way ANOVA (*p < 0.05, ***p < 0.001, ns: no statistical difference).Data were presented as Mean ± SEM. NC: negative control; Tri: trigonelline; T + P: trigonelline and anti-PD-1; ns: no significant difference

Fig. 5

The trigonelline-pretreated CD8⁺T cells increased cytotoxicity against tumors. A The 2D and 3D structure of trigonelline. B The CCK-8 assay was used to evaluate the proliferative capacity of various bladder cancer cell lines treated with trigonelline (n = 3). C The cell scratch assay assesses the impact of different concentrations of trigonelline treatment on the migration ability of T24 cells (n = 3, scale bar: 500 μm). D The cell scratch assay assesses the impact of different concentrations of trigonelline treatment on the migration ability of BIU cells. E The cell scratch assay assesses the impact of different concentrations of trigonelline treatment on the migration ability of 5637 cells. Significant differences were evaluated by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ns, no statistical difference). Data were presented as Mean ± SEM. NC: negative control; ns: no significant difference

Fig. 6

The effect of co-culture trigonelline and CD8⁺ T cells on 5637 tumor cells. A Annexin V/PI double staining flow cytometry to determine the apoptosis ratio in 5637 cells. The apoptosis ratio is the sum of early apoptosis (Q3) and late apoptosis (Q2). (n = 3). B Immunofluorescence images and statistical analysis of β-catenin expression in 5637 cells treated with different concentrations of trigonelline. (Red representing β-catenin, blue representing DAPI, 400 ×, scale bar: 20 μm). Significant differences were evaluated by one-way ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001). Data were presented as Mean ± SEM. NC: negative control

Fig. 7

Schematic diagram showed the mechanism by which Bl. coccoides enhances the anti-tumor effect of PD-1 inhibitor therapy. The gut microbiota Bl. coccoides, along with metabolite trigonelline, played a pivotal role in amplifying PD-1 inhibitor therapy effectiveness. It achieved this by boosting the infiltration of CD8⁺T cells and suppressing the β-catenin expression, consequently hindering the advancement of bladder cancer