Fusobacterium is enriched in oral cancer and promotes induction of programmed death-ligand 1 (PD-L1)

Abstract

Recently, increased number of studies have demonstrated a relationship between the oral microbiome and development of head and neck cancer, however, there are few studies to investigate the role of oral bacteria in the context of the tumor microenvironment in a single head and neck subsite. Here, paired tumor and adjacent normal tissues from thirty-seven oral tongue squamous cell carcinoma (SCC) patients were subjected to 16S rRNA gene sequencing and whole exome sequencing (WES), in addition to RNA sequencing for tumor samples. We observed that Fusobacterium was significantly enriched in oral tongue cancer and that Rothia and Streptococcus were enriched in adjacent normal tissues. A decrease in alpha diversity was found in tumor when compared to adjacent normal tissues. While increased Fusobacterium in tumor samples was not associated with changes in immune cell infiltration, it was associated with increased PD-L1 mRNA expression. Therefore, we examined the effects of Fusobacterium on PD-L1 expression in head and neck SCC cell lines. We demonstrated that infection with Fusobacterium species can increase both PD-L1 mRNA and surface PD-L1 protein expression on head and neck cancer cell lines. The correlation between Fusobacterium and PD-L1 expression in oral tongue SCC, in conjunction with the ability of the bacterium to induce PD-L1 expression in vitro suggests a potential role for Fusobacterium on modulation of the tumor immune microenvironment in head and neck cancer.

Keywords: Fusobacterium; Head and neck cancer; Microbiome; Oral cancer; PD-L1; Periodontal bacteria.

Fig. 1

Bacterial Profiling. (A) Individual relative abundance of top 5 phyla in adjacent normal (n = 36) and (B) tumor (n = 33) tissues. (C) Average relative abundance of phyla, (D) genera, and (E) species. In total, 15 phyla, 252 genera, and 492 species were found in all samples.

Fig. 2

Bacterial differences in tumor and adjacent normal tissues. (A) Comparison of alpha diversity scores in adjacent normal (n = 36) and tumor (n = 33) using the Chao 1 (p = 0.02), the Shannon (p = 0.16), and Simpson (p = 0.18) indices by Mann-Whitney U (MW) test. (B) Beta-diversity using weighted UniFrac distances (left) and Bray-Curtis dissimilarity (right). (C) Association networks (Anets) of samples in terms of pair-wise similarity of shared species richness profiles. The isolated samples (bottom left) have no unique information. Nodes of the network represent samples connected by edges if they have significant pair-wise association of the shared richness profile (Pearson correlation R>0.70). (D) Cladogram with linear discriminant analysis (LDA) effect size (LEfSe) method to show the phylogenetic distribution of bacteria which were significantly enriched in the tumor (green) or normal (red) samples. LDA scores computed for differentially abundant taxa and the length indicates effect size associated with a taxon. p = 0.05 for the Kruskal-Wallis H statistic; LDA score > 2.0. (E) Differentially enriched bacteria at genus level and alpha-diversity indices in tumor, the Chao 1, the Shannon, and the Simpson.

Fig. 2

Bacterial differences in tumor and adjacent normal tissues. (A) Comparison of alpha diversity scores in adjacent normal (n = 36) and tumor (n = 33) using the Chao 1 (p = 0.02), the Shannon (p = 0.16), and Simpson (p = 0.18) indices by Mann-Whitney U (MW) test. (B) Beta-diversity using weighted UniFrac distances (left) and Bray-Curtis dissimilarity (right). (C) Association networks (Anets) of samples in terms of pair-wise similarity of shared species richness profiles. The isolated samples (bottom left) have no unique information. Nodes of the network represent samples connected by edges if they have significant pair-wise association of the shared richness profile (Pearson correlation R>0.70). (D) Cladogram with linear discriminant analysis (LDA) effect size (LEfSe) method to show the phylogenetic distribution of bacteria which were significantly enriched in the tumor (green) or normal (red) samples. LDA scores computed for differentially abundant taxa and the length indicates effect size associated with a taxon. p = 0.05 for the Kruskal-Wallis H statistic; LDA score > 2.0. (E) Differentially enriched bacteria at genus level and alpha-diversity indices in tumor, the Chao 1, the Shannon, and the Simpson.

Fig. 3

Association with TP53 status and alpha-diversity in tumor. Mut, mutation; WT, wild-type.

Fig. 4

Differentially enriched bacteria and PD-L1 (CD274) expression.

Fig. 5

mRNA expression of PD-L1 and associated genes in head and neck SCC cell lines after Fusobacterium infection. (A) PD-L1 mRNA expression with T. denticola and T. forsythia in OQ01 cell lines and with F. periodonticum and F. Vincentii in OQ01 and RPMI 2650 cell lines. (B) mRNA expression of PD-L1 pathway associated genes with the in OQ01 (left) and RPMI 2650 cells (right). Data are shown as the log2 fold change of gene expression relative to uninfected control. All bar graph results are presented as mean ± SEM based on 3 independent experiments (* p < 0.05; ** p < 0.01, *** p < 0.001). Solid bars represent infection with F. periodonticum (FP) and bars with dots represent infection with F. vincentii (FV).

Fig. 6

PD-L1 surface protein expression in head and neck SCC cell lines after Fusobacterium infection. (A)% PD-L1+ cells as determined by flow cytometry after infection with Fusobacterium in OQ01 and RPMI 2650 cell lines. (B)Representative flow histograms for the respective cell lines. Bar graph results are presented as mean ± SEM based on 3 independent infection experiments for each cell line (* p < 0.05; ** p < 0.01).