Intracellular Fusobacterium nucleatum infection attenuates antitumor immunity in esophageal squamous cell carcinoma

Currently, the influence of the tumor microbiome on the effectiveness of immunotherapy remains largely unknown. Intratumoural Fusobacterium nucleatum (Fn) functions as an oncogenic bacterium and can promote tumor progression in esophageal squamous cell carcinoma (ESCC). Our previous study revealed that Fn is a facultative intracellular bacterium and that its virulence factor Fn-Dps facilitates the intracellular survival of Fn. In this study, we find that Fn DNA is enriched in the nonresponder (NR) group among ESCC patients receiving PD-1 inhibitor and that the serum antibody level of Fn is significantly higher in the NR group than in the responder (R) group. In addition, Fn infection has an opposite impact on the efficacy of αPD-L1 treatment in animals. Mechanistically, we confirm that Fn can inhibit the proliferation and cytokine secretion of T cells and that Fn-Dps binds to the PD-L1 gene promoter activating transcription factor-3 (ATF3) to transcriptionally upregulate PD-L1 expression. Our results suggest that it may be an important therapeutic strategy to eradicate intratumoral Fn infection before initiating ESCC immunotherapies.

(i) Summary of spleen weight data of C57BL/6 xenografts (the experiment was done once; n = 6 mice in each group; n = 1 experiment; n = 6 mice in each group).
The statistical significance of results in figure d, h, i and l was determined by a one-way ANOVA analysis.(e-f) NSG mice (the experiment was done once; n = 5 mice in each group) were implanted with 5 × 10 6 E109 cells and Fn infection (i.v., 10 7 CFU/mouse) three times.Then the mice were tail vein injected with 2 × 10 6 human CD3 + T cells.αPD-L1 (i.p., 100 μg/mouse) once every three days.

Figure S10 Human CD8 + T cells were obtained from PBMCs
Human CD3 + T cells were obtained and purified from human peripheral blood mononuclear cells (PBMCs) using CD3 microbeads according to the manufacturer's instructions.

Fig. S1
Fig.S1 Representative images of immunohistochemistry staining of PD-L1 expression in the NR

Figure
Figure S1 Representative images of immunohistochemistry staining of PD-L1 expression in

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Figure S2 Representative images of immunohistochemistry staining of PD-L1 expression in

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Figure S3 Immunohistochemistry positive rates of PD-L1 in ESCC paraffin sections

Figure S5 F
Figure S5 F. nucleatum infection decreases the effectiveness of αPD-L1 in female

Figure S6 F
Figure S6 F. nucleatum infection decreases the effectiveness of αPD-L1 in larger

Figure S7 F
Figure S7 F. nucleatum infection attenuated T-cell activation in vivo

Figure S8 F
Figure S8 F. nucleatum protects ESCC cells against direct cytotoxicity from splenocytes in

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Figure S9 The effect of F. nucleatum infection on PBMCs, human CD8 + T cells and Jurkat

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Figure S11 The effect of lipopolysaccharide (LPS) in Jurkat cells

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Figure S12 Only live F. nucleatum could survive and upregulate the expression of PD-L1 in

Figure 13
Figure 13 Dichromatic IF staining of PD-L1 in ESCC cells

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Figure S15 IF staining of PD-L1 or ATF3 in tumor tissues from C57BL/6 xenografts.

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Figure S16 Validation of Fn and Fn-Dps antibodies specificity.

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Figure S17 Validation of Fn primer specificity.

Table S1 :
ESCC patients details for ELISA

Table S2 :
ESCC patients details for PD-L1 and Fn detection

Table S3 :
All antibodies used in flow cytometry analysis

Table S4 :
All antibodies used in histology and immunofluorescence

Table S5 :
The sequences of primers for the qPCR analysis

Table S6 :
All antibodies used in western blotting and Co-IP

Table S7 :
Table summary of differentially expressed genes, related to Figure 6E