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. 2022 Feb;41(9):1269-1280.
doi: 10.1038/s41388-021-02137-1. Epub 2022 Jan 28.

A dysbiotic microbiome promotes head and neck squamous cell carcinoma

Daniel N Frank  1 , Yue Qiu  2   3 , Yu Cao  2   4 , Shuguang Zhang  2 , Ling Lu  2 , Jennifer M Kofonow  5 , Charles E Robertson  5 , Yanqiu Liu  2 , Haibo Wang  2 , Cassandra L Levens  6 , Kristine A Kuhn  6 , John Song  2 , Vijay R Ramakrishnan  2 , Shi-Long Lu  7
Affiliations

A dysbiotic microbiome promotes head and neck squamous cell carcinoma

Daniel N Frank et al. Oncogene. 2022 Feb.

Abstract

Recent studies have reported dysbiotic oral microbiota and tumor-resident bacteria in human head and neck squamous cell carcinoma (HNSCC). We aimed to identify and validate oral microbial signatures in treatment-naïve HNSCC patients compared with healthy control subjects. We confirm earlier reports that the relative abundances of Lactobacillus spp. and Neisseria spp. are elevated and diminished, respectively, in human HNSCC. In parallel, we examined the disease-modifying effects of microbiota in HNSCC, through both antibiotic depletion of microbiota in an induced HNSCC mouse model (4-Nitroquinoline 1-oxide, 4NQO) and reconstitution of tumor-associated microbiota in a germ-free orthotopic mouse model. We demonstrate that depletion of microbiota delays oral tumorigenesis, while microbiota transfer from mice with oral cancer accelerates tumorigenesis. Enrichment of Lactobacillus spp. was also observed in murine HNSCC, and activation of the aryl-hydrocarbon receptor was documented in both murine and human tumors. Together, our findings support the hypothesis that dysbiosis promotes HNSCC development.

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Conflict of interest statement

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1.
Figure 1.. Oral microbiotas differ between HNSCC cases and non-HNSCC controls.
Panel A. Percent relative abundance (%RA) of genus-level taxa, stratified by HNSCC occurrence and tumor location. Taxa with %RA less than 1% were collapsed into the “Other” category. Results of PERMANOVA tests are summarized above each plot for tests across all groups (red lines/symbols) and pairwise tests (blue lines/symbols). Panel B. Principal coordinates analysis. Individual subjects are indicated by smaller symbols (circles/squares/diamonds/triangles), with group affiliations designated by symbol shapes and color-coding. Mean PC values for each group along the x- and y-axes are indicated by larger shapes, with 95% confidence intervals about the means marked by whiskers.
Figure 2.
Figure 2.. Alpha-diversity indices differ between HNSCC cases and non-HNSCC controls.
Boxplots show distributions of alpha-diversity indices by HNSCC occurrence (top panels) and tumor location (bottom panels). Results of ANOVA tests are summarized above each plot for tests across all groups (red lines/symbols) and pairwise tests (blue lines/symbols).
Figure 3.
Figure 3.. Differentially abundant taxa between HNSCC cases and non-HNSCC controls.
Panel A. Volcano plot of fold-change (FC; Log2 transformed) vs FDR-corrected p-values (-Log10 transformed) ascertained by ALDEx2 analysis Vertical and horizontal dashed lines represent cutoffs of FC ≥2 and FDR-corrected p-value ≤0.05, respectively. Panel B. ALDEx2-calculated effect sizes of taxa meeting FC and p-value cutoffs. Panel C. Overlap in influential taxa identified through multiple analytic methods. In all three panels, taxa enriched in cases compared to controls are highlighted in red with FC and effect sizes greater than zero, while taxa enriched in controls are highlighted in blue with FC and effect sizes less than zero.
Figure 4.
Figure 4.. Overlap in taxa differentially abundant between non-HNSCC controls and different tumor locations.
Taxa that were differentially abundant between each of the three tumor locations (i.e., OSCC, OPSCC, LSCC) and controls were identified through ALDEx2 analysis. The Venn diagram shows the overlap in taxa identified in the three analyses, including four taxa that were found in common (identified in the box). “Neisseriaceae other” represents taxa that could not be classified to the genus-level. Taxa enriched in cases compared to controls are colored red, while those enriched in controls are colored blue.
Figure 5.
Figure 5.. Differential abundance of Lactobacillus and Neisseria species in HNSCC cases and controls.
Panel A. Volcano plot of fold-change (FC; Log2 transformed) vs FDR-corrected p-values (-Log10 transformed) ascertained by ALDEx2 analysis Horizontal and vertical dashed lines represent cutoffs of FC ≥2 and FDR-corrected p-value ≤0.05, respectively. Panel B. ALDEx2-calculated effect sizes of taxa meeting FC and p-value cutoffs. In both panels, the control group served as the reference, such that taxa enriched in controls are assigned FC and effect sizes <0 and are displayed to the left and colored blue. Taxa enriched in cases have FC and effect sizes >0 and are displayed to the right and colored red.
Fig. 6.
Fig. 6.. Reduced tongue tumor formation and size upon antibiotic (Abx) treatment.
Panel A. Timeline of 4NQO-OSCC pathogenesis and Abx treatment. Panel B. 16S bacterial qPCR, Abx: antibiotics. Panels C and D. Ablation of microbiota by antibiotics (Abx) in the 4NQO model diminishes the size of tongue tumors. Dashed lines denote tumor margins. Scale bar: 10mm. **: p<0.01 for Student’s t-test. Panel E. H&E staining of tongue tumors without (i, ii) or with (iii, iv) Abx reveals decreased size and depth of invasion in Abx-treated mice. Scale bar: 50 μm. Panels F, G, H. Enrichment of lactic acid bacteria in saliva of 4NQO-OSCC mice compared to controls. F. Representative MRS agar plates showing more colonies in the OSCC saliva. G. Quantification of colonies (N=3). **: p<0.01 for Student’s t-test. Plot summarizes mean±s.d. values for each group. H. Representative figure of MRS colony validation using Lactobacillus PCR.
Fig. 7.
Fig. 7.. Aryl-hydrocarbon Receptor (AhR) expression in human and murine HNSCC.
Immunohistochemical staining of AhR in human HNSCC (Panel A) and murine 4NQO-OSCC (Panel B) tumor tissues. Nuclear staining (dark brown) denotes AhR activation. Dashed lines delineate epithelial and stromal boundaries. Scale bar: 50 μm. Panel C. Summary of AhR staining results. Ahr positivity represents the percentage of tumors (human or murine) with detectable AhR staining. The number of specimens analyzed for each group is shown below the barchart. *: p<0.05 for Chi-squared test.
Fig. 8.
Fig. 8.. Effects of microbiome on tongue tumor development in germ-free and reconstitution conditions.
Panel A. Kaplan-Meier survival curve of SPF and GF mice receiving tongue injection of the murine Cu110 HNSCC cell line (n = 8 for each group). SPF: specific pathogen free, GF: germ free. P-value determined by Mantel-Cox test. Panel B. Gross pathology of tongue tumors in syngeneic orthotopic mouse model (sOMM) hosted in SPF and GF conditions (n = 10 for each group). Dotted circles and arrows indicate visible tongue tumors. Scale bar: 10 mm. Panel C. Quantification of tongue tumor volumes from the experiment in panel B. *: p<0.05 for Student’s t-test in comparison to SPF group. Panel D. H&E staining of tongue tumor development in GF mice reconstituted from tumor-bearing donor mice (upper row of tissues, n = 4 donor/recipient pairs) compared with reconstitution from tumor-free donor mice (middle row of tissues, n = 4 donor/recipient pairs) or non-reconstituted GF mice (lower panel of tissues, n = 4). Tumors are indicated by dashed oval lines. Scale bar: 5 mm. Panel E. Quantification of tongue tumor volumes from the experiment in panel D. *: p<0.05 for Student’s t-test comparisons to (+)Tumor donor group.
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