Fusobacterium nucleatum enhances the efficacy of PD-L1 blockade in colorectal cancer

Abstract

Given that only a subset of patients with colorectal cancer (CRC) benefit from immune checkpoint therapy, efforts are ongoing to identify markers that predict immunotherapeutic response. Increasing evidence suggests that microbes influence the efficacy of cancer therapies. Fusobacterium nucleatum induces different immune responses in CRC with different microsatellite-instability (MSI) statuses. Here, we investigated the effect of F. nucleatum on anti-PD-L1 therapy in CRC. We found that high F. nucleatum levels correlate with improved therapeutic responses to PD-1 blockade in patients with CRC. Additionally, F. nucleatum enhanced the antitumor effects of PD-L1 blockade on CRC in mice and prolonged survival. Combining F. nucleatum supplementation with immunotherapy rescued the therapeutic effects of PD-L1 blockade. Furthermore, F. nucleatum induced PD-L1 expression by activating STING signaling and increased the accumulation of interferon-gamma (IFN-γ)⁺ CD8⁺ tumor-infiltrating lymphocytes (TILs) during treatment with PD-L1 blockade, thereby augmenting tumor sensitivity to PD-L1 blockade. Finally, patient-derived organoid models demonstrated that increased F. nucleatum levels correlated with an improved therapeutic response to PD-L1 blockade. These findings suggest that F. nucleatum may modulate immune checkpoint therapy for CRC.

Fig. 1

Patients with high levels of F. nucleatum were more responsive to PD-1 blockade than those with low levels of F. nucleatum. a F. nucleatum was detected by a fluorescence in situ hybridization (FISH) assay in CRC tumor tissues and adjacent tissues. A FISH assay showed that F. nucleatum (red) was present in the tumor tissues of mice. The nuclei (blue) of cells in tumor tissue samples were stained with DAPI. The white arrows indicate positive staining (red) for F. nucleatum. b The F. nucleatum levels in tumor tissues were measured by RT-PCR. The progression-free survival of patients with CRC (n = 38) stratified by their F. nucleatum levels in tumor tissues. Fn+ and Fn– represent the patients with F. nucleatum-positive and negative tumor tissues, respectively. Log-rank test. c Correlation analysis between patient outcomes and F. nucleatum levels in CRC tumor tissues (n = 24). Patients showing no sign of progression within 6 months after PD-1 blockade treatment were defined as responders, while those who progressed within 6 months after PD-1 blockade treatment were defined as non-responder. Chi-square test (one-sided). d The progression-free survival of patients with CRC (n = 27). The F. nucleatum levels in feces were measured by RT-PCR. Patients were divided into high F. nucleatum groups and low F. nucleatum groups according to the median. Log-rank test. e Representative CT images of a patient with lung metastasis before and after treatment of PD-1 blockade. The red arrows indicate lung metastases nucleatum were more responsive to PD-1 blockade than those with low levels of F. nucleatum

Fig. 2

F. nucleatum treatment augments the antitumor effects of PD-L1 blockade on CRC. a–d CT26.WT cells were subcutaneously injected into BALB/c mice. Tumor-bearing mice were intratumorally injected with PBS (control) or F. nucleatum and intraperitoneally injected treated with an anti-PD-L1 mAb or an isotype control mAb. a CT26.WT tumors from BALB/c mice in different groups at the end of the experiment are shown. Growth curves display tumor volumes b and relative tumor volumes c over time and the bar graph shows tumor weights at the end of the experiment d (One-way analysis of variance [ANOVA] and Bonferroni’s multiple comparison test). e Representative image of a colon of AOM/DSS-treated C57BL/6 mice with or without F nucleatum treatment and PD-L1 blockade. f The tumor numbers (diameter > 1 mm) of the different groups (n = 8 per group, One-way ANOVA and Bonferroni’s multiple comparison test). g Survival curves of AOM/DSS-treated C57BL/6 mice receiving different treatments (n = 10 per group) with Log-rank test. h Tumor-bearing mice were first injected intraperitoneally with an anti-PD-L1 mAb or an isotype control mAb and subsequently injected intratumorally with PBS or F. nucleatum and continued to be treated with an anti-PD-L1 mAb. i A picture of tumors from mice in different groups. j–l Tumor volume growth, relative tumor volume growth and tumor weights at the end of the experiment are shown. One-way ANOVA and Bonferroni’s multiple comparison test. Data are expressed as the mean + s.d. *P < 0.05, **P < 0.01. NS, no statistical difference. CRC, colorectal cancer. Fn, F. nucleatum

Fig. 3

F. nucleatum increases the accumulation of CD8⁺ IFN-γ⁺ TILs during treatment with an anti-PD-L1 mAb. a, b CT26.WT cells were subcutaneously injected into BALB/c mice. Tumor-bearing mice were treated with PBS or F. nucleatum by intratumoral injection and intraperitoneally injected with an anti-PD-L1 mAb or an isotype control mAb. a, c Flow cytometry was used to detect CD8⁺ cells in tumor tissues from mice and the proportion of IFN-γ⁺ cells in CD8⁺ cells. One-way ANOVA and Bonferroni’s multiple comparison test. b–d CT26.WT cells were subcutaneously injected into BALB/c mice. Tumor-bearing mice were treated with PBS or F. nucleatum by garvage and intraperitoneally injected with an anti-PD-L1 mAb or an isotype control mAb. c, d Flow cytometry was used to detect CD8⁺ cells in tumor tissues from mice and the proportion of IFN-γ⁺ cells in CD8⁺ cells is shown. One-way ANOVA and Bonferroni’s multiple comparison test. e Tumor-bearing mice were intraperitoneally injected with an anti-CD8 mAb or an isotype control mAb, then mice were treated with PBS or F. nucleatum by intratumoral injection and intraperitoneally injected with an anti-PD-L1 mAb or an isotype control mAb. An image of tumors collected at the end of the experiment is shown. f, g Tumor volumes and relative tumor volumes at various time points are shown. One-way ANOVA and Bonferroni’s multiple comparison test. Bars, s.d. *P < 0.05; **P < 0.01. Fn, F. nucleatum

Fig. 4

F. nucleatum induces the expression of PD-1 and PD-L1. a Flow cytometry was used to detect the expression of PD-1 in tumor tissues from mice treated with F. nucleatum and/or an anti-PD-L1 mAb. One-way ANOVA and Bonferroni’s multiple comparison test. b The protein levels of PD-L1 were detected by IHC, and F. nucleatum was detected by FISH in tumor tissue samples from mice. Brown staining in IHC and red staining in FISH (white arrows) indicate positive staining. c, d DLD1 and Caco-2 cells were treated with Fn (1:1000) for different time course, and the mRNA and protein levels of PD-L1 were detected by RT-PCR and Western blotting, respectively. Student’s t-test. e DLD1 cells were treated with different dilutions of different Fn isolates obtained from CRC patients. f DLD1 cells were treated with Fn (1:1000) for different time course. g DLD1 cells were treated with Fn (1:1000) and/or 5 μM BAY 11–7082 for 24 h. The expression of the indicated proteins was detected by Western blotting. Actin was used as a loading control. Bars represent s.d. of at least three experiments. *P < 0.05; **P < 0.01. Fn, F. nucleatum. NS, no significant difference

Fig. 5

F. nucleatum enhanced the therapeutic effect of PD-L1 blockade by activating STING signaling. a–d The indicated proteins were detected by Western blotting. Actin was used as a loading control. a DLD1 cells were treated with Fn (1:1000) for different time course. b DLD1 cells were pre-treated with H151 for 2 h, and then treated with Fn (1:1000) for 12 h. c CT26.WT cells were treated with Fn (1:1000) for different time course. d CT26.WT cells were pre-treated with C176 for 2 h, and then treated with Fn (1:1000) for different time course. e–h CT26.WT cells were subcutaneously injected into BALB/c mice (n = 7 for each group). Tumor-bearing mice were pre-treated with C176 or vehicle, then intratumorally injected with F. nucleatum or PBS and intraperitoneally injected treated with an anti-PD-L1 mAb or an isotype control mAb every three days until the end of the experiment. Tumor volumes were measured. e An image of tumors collected at the end of the experiment is shown. f, g Tumor volumes and relative tumor volumes at various time points are shown. One-way ANOVA and Bonferroni’s multiple comparison test. h Flow cytometry was used to detect the proportion of IFN-γ⁺ cells in CD8⁺ TILs from mice. One-way ANOVA and Bonferroni’s multiple comparison test. *P < 0.05; **P < 0.01. Fn, F. nucleatum

Fig. 6

CRC Organoids treated with F. nucleatum were more responsive to PD-L1 blockade than those not treated with F. nucleatum. CRC organoids were mixed with T lymphocytes (10⁵/well) and F. nucleatum (10⁸ CFU) and treated with an anti-PD-L1 mAb or an isotype control mAb for one week. a CRC organoids in different groups. Organoid morphology was examined by staining for E-cadherin (red) and with DAPI (blue). Ki-67 expression was detected by IHC (brown staining). Cell apoptosis was detected by TUNEL staining (green). b The proportions of proliferating cells were detected by Ki-67 staining. c The proportions of apoptotic cells were detected by TUNEL staining. One-way ANOVA and Bonferroni’s multiple comparison test. d, e Flow cytometry was used to detect the proportion of CD8⁺ cells in CRC organoids and the proportion of IFN-γ⁺ cells in CD8⁺ cells in different groups. One-way ANOVA and Bonferroni’s multiple comparison test. f CRC organoids were treated with F. nucleatum for different time course. The indicated proteins were detected by Western blotting. One-way ANOVA and Bonferroni’s multiple comparison test. *P < 0.05; **P < 0.01. Fn, F. nucleatum