The incidence of oral cancer has recently been increasing worldwide, particularly among young individuals and women. The primary risk factors for head and neck cancers, including oral and pharyngeal cancers, are smoking, alcohol consumption, poor oral hygiene, and repeated exposure to mechanical stimuli. However, approximately one-third of the patients with oral and pharyngeal cancers are neither smokers nor drinkers, which points to the existence of other mechanisms. Recently, human microbes have been linked to various diseases, including cancer. Oral pathogens, especially periodontal pathobionts, are reported to play a role in the development of colon and other types of cancer. In this study, we employed a series of bioinformatics analyses to pinpoint Fusobacterium nucleatum as the predominant oral bacterial species in oral and pharyngeal cancer tissue samples. We successfully isolated Fn. polymorphum from the saliva of patients with oral cancer and demonstrated that Fn. polymorphum indeed promoted oral squamous cell carcinoma development by activating YAP in a mouse tongue cancer model. Our research offers scientific evidence for the role of the oral microbiome in oral cancer progression and provides insights that would help in devising preventative strategies against oral cancer, potentially by altering oral bacterial profiles.

Oral squamous cell carcinoma (OSCC) develops through multiple pathogenic and environmental factors. Increasing evidence suggests that the oral microbiota, particularly intratumoral bacteria, is also associated with OSCC progression; however, the relationship between individual bacterial subspecies and the tumor immune microenvironment remains poorly understood. We previously demonstrated that Fusobacterium nucleatum is enriched in OSCC tissues. In this study, we investigated the biological and immunological properties of F. nucleatum subspecies, with a particular focus on regulatory T-cell (Treg) induction. We reanalyzed our 16S rRNA gene sequencing dataset from 12 oral and pharyngeal squamous cell carcinoma specimens at the amplicon sequence variant (ASV) level and examined archived human OSCC tissues by immunofluorescence and fluorescence in situ hybridization (FISH). Treg differentiation and bacterial metabolite production were examined in vitro, and selected F. nucleatum subspecies were evaluated in a mouse tongue carcinogenesis model (n = 6/group). ASVs most closely matching F. nucleatum subsp. animalis were the most abundant among the detected F. nucleatum-associated ASVs. In three human OSCC specimens with detectable F. nucleatum, FOXP3-positive cells preferentially accumulated in the peritumoral stroma. In vitro, culture supernatants from F. nucleatum subsp. animalis, but not subsp. polymorphum, promoted Treg differentiation in two independent experiments, and butyrate production may be associated with the Treg-promoting strains. In vivo, subsp. animalis significantly increased Ki67-positive, nuclear YAP1-positive, and Foxp3-positive cell numbers, whereas subsp. polymorphum significantly increased only nuclear YAP1-positive cell numbers. Furthermore, FISH demonstrated spatial proximity between F. nucleatum and FOXP3-positive cells in a human OSCC case. These findings indicate functional heterogeneity among F. nucleatum subspecies and suggest that their effects on epithelial and immune parameters differ among subspecies. Our findings support possible subspecies-specific immunomodulatory effects of F. nucleatum in OSCC and highlight the importance of subspecies- and strain-level analyses for understanding the biological significance of the intratumoral microbiota.