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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.<\/jats:p>","DOI":"10.3390\/microorganisms14092067","type":"journal-article","created":{"date-parts":[[2026,9,16]],"date-time":"2026-09-16T13:50:27Z","timestamp":1789566627000},"page":"2067","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Functional Heterogeneity of Fusobacterium nucleatum Subspecies in the Immune Microenvironment of Oral Squamous Cell Carcinoma"],"prefix":"10.3390","volume":"14","author":[{"given":"Megumu","family":"Yano","sequence":"first","affiliation":[{"name":"Department of Pathophysiology, Graduate School of Medicine, Osaka Metropolitan University, Osaka 545-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"validation","vocabulary":"credit"},{"role":"investigation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-6970-9512","authenticated-orcid":false,"given":"Tomonori","family":"Kamiya","sequence":"additional","affiliation":[{"name":"Department of Pathophysiology, Graduate School of Medicine, Osaka Metropolitan University, Osaka 545-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"investigation","vocabulary":"credit"},{"role":"data-curation","vocabulary":"credit"},{"role":"formal-analysis","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"validation","vocabulary":"credit"}]},{"given":"Yuki","family":"Yamamoto","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology and Head & Neck Surgery, Graduate School of Medicine, Osaka Metropolitan University, Osaka 545-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"resources","vocabulary":"credit"}]},{"given":"Teppei","family":"Kouga","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology and Head & Neck Surgery, Graduate School of Medicine, Osaka Metropolitan University, Osaka 545-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"resources","vocabulary":"credit"}]},{"given":"Masahiro","family":"Oishi","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology and Head & Neck Surgery, Graduate School of Medicine, Osaka Metropolitan University, Osaka 545-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"resources","vocabulary":"credit"}]},{"given":"Miki","family":"Nishio","sequence":"additional","affiliation":[{"name":"Division of Molecular and Cellular Biology, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"resources","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-5950-8808","authenticated-orcid":false,"given":"Akira","family":"Suzuki","sequence":"additional","affiliation":[{"name":"Division of Molecular and Cellular Biology, Graduate School of Medicine, Kobe University, 7-5-1 Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"resources","vocabulary":"credit"}]},{"given":"Kishiko","family":"Sunami","sequence":"additional","affiliation":[{"name":"Department of Otolaryngology and Head & Neck Surgery, Graduate School of Medicine, Osaka Metropolitan University, Osaka 545-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"resources","vocabulary":"credit"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8934-0797","authenticated-orcid":false,"given":"Naoko","family":"Ohtani","sequence":"additional","affiliation":[{"name":"Department of Pathophysiology, Graduate School of Medicine, Osaka Metropolitan University, Osaka 545-8585, Japan"}],"role":[{"vocabulary":"crossref","role":"author"},{"role":"conceptualization","vocabulary":"credit"},{"role":"writing-original-draft","vocabulary":"credit"},{"role":"writing-review-editing","vocabulary":"credit"},{"role":"funding-acquisition","vocabulary":"credit"},{"role":"supervision","vocabulary":"credit"},{"role":"project-administration","vocabulary":"credit"}]}],"member":"1968","published-online":{"date-parts":[[2026,9,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"5002","DOI":"10.1128\/JB.00542-10","article-title":"The Human Oral Microbiome","volume":"192","author":"Dewhirst","year":"2010","journal-title":"J. Bacteriol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"292","DOI":"10.1101\/gr.126573.111","article-title":"Genomic analysis identifies association of Fusobacterium with colorectal carcinoma","volume":"22","author":"Kostic","year":"2012","journal-title":"Genome Res."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1101\/gr.126516.111","article-title":"Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma","volume":"22","author":"Castellarin","year":"2012","journal-title":"Genome Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"973","DOI":"10.1126\/science.aay9189","article-title":"The human tumor microbiome is composed of tumor type-specific intracellular bacteria","volume":"368","author":"Nejman","year":"2020","journal-title":"Science"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"1156","DOI":"10.1126\/science.aah5043","article-title":"Potential role of intratumor bacteria in mediating tumor resistance to the chemotherapeutic drug gemcitabine","volume":"357","author":"Geller","year":"2017","journal-title":"Science"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"1356","DOI":"10.1016\/j.cell.2022.02.027","article-title":"Tumor-resident intracellular microbiota promotes metastatic colonization in breast cancer","volume":"185","author":"Fu","year":"2022","journal-title":"Cell"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1016\/j.chom.2013.07.007","article-title":"Fusobacterium nucleatum Potentiates Intestinal Tumorigenesis and Modulates the Tumor-Immune Microenvironment","volume":"14","author":"Kostic","year":"2013","journal-title":"Cell Host Microbe"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1799","DOI":"10.1158\/1078-0432.CCR-25-4420","article-title":"Association of Intratumoral Microbiota with Prognosis in Head and Neck Squamous Cell Carcinoma","volume":"32","author":"Yin","year":"2026","journal-title":"Clin. Cancer Res."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1016\/j.chom.2013.07.012","article-title":"Fusobacterium nucleatum Promotes Colorectal Carcinogenesis by Modulating E-Cadherin\/\u03b2-Catenin Signaling via its FadA Adhesin","volume":"14","author":"Rubinstein","year":"2013","journal-title":"Cell Host Microbe"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"215","DOI":"10.1016\/j.chom.2016.07.006","article-title":"Fap2 Mediates Fusobacterium nucleatum Colorectal Adenocarcinoma Enrichment by Binding to Tumor-Expressed Gal-GalNAc","volume":"20","author":"Abed","year":"2016","journal-title":"Cell Host Microbe"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"344","DOI":"10.1016\/j.immuni.2015.01.010","article-title":"Binding of the Fap2 Protein of Fusobacterium nucleatum to Human Inhibitory Receptor TIGIT Protects Tumors from Immune Cell Attack","volume":"42","author":"Gur","year":"2015","journal-title":"Immunity"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"398","DOI":"10.1158\/1940-6207.CAPR-16-0178","article-title":"Fusobacterium Nucleatum Subspecies Animalis Influences Proinflammatory Cytokine Expression and Monocyte Activation in Human Colorectal Tumors","volume":"10","author":"Ye","year":"2017","journal-title":"Cancer Prev. Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"424","DOI":"10.1038\/s41586-024-07182-w","article-title":"A distinct Fusobacterium nucleatum clade dominates the colorectal cancer niche","volume":"628","author":"Minot","year":"2024","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"7870","DOI":"10.1038\/s41598-021-86816-9","article-title":"Prognostic value of intratumoral Fusobacterium nucleatum and association with immune-related gene expression in oral squamous cell carcinoma patients","volume":"11","author":"Neuzillet","year":"2021","journal-title":"Sci. Rep."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"e04596-22","DOI":"10.1128\/spectrum.04596-22","article-title":"Intratumoral Microbiota Changes with Tumor Stage and Influences the Immune Signature of Oral Squamous Cell Carcinoma","volume":"11","author":"Singh","year":"2023","journal-title":"Microbiol. Spectr."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1038\/s41392-023-01693-0","article-title":"Intratumoural microbiota: A new frontier in cancer development and therapy","volume":"9","author":"Cao","year":"2024","journal-title":"Signal Transduct. Target. Ther."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Li, C., Dong, X., and Li, B. (2024). Tumor microenvironment in oral squamous cell carcinoma. Front. Immunol., 15.","DOI":"10.3389\/fimmu.2024.1485174"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"942","DOI":"10.1038\/nm1093","article-title":"Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival","volume":"10","author":"Curiel","year":"2004","journal-title":"Nat. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1002\/cre2.477","article-title":"Tumor-infiltrating FoxP3+ T cells are associated with poor prognosis in oral squamous cell carcinoma","volume":"8","author":"Hayashi","year":"2022","journal-title":"Clin. Exp. Dent. Res."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Weed, D.T., Walker, G., De La Fuente, A.C., Nazarian, R., Vella, J.L., Gomez-Fernandez, C.R., and Serafini, P. (2013). FOXP3 Subcellular Localization Predicts Recurrence in Oral Squamous Cell Carcinoma. PLoS ONE, 8.","DOI":"10.1371\/journal.pone.0071908"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"446","DOI":"10.1038\/nature12721","article-title":"Commensal microbe-derived butyrate induces the differentiation of colonic regulatory T cells","volume":"504","author":"Furusawa","year":"2013","journal-title":"Nature"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"451","DOI":"10.1038\/nature12726","article-title":"Metabolites produced by commensal bacteria promote peripheral regulatory T-cell generation","volume":"504","author":"Arpaia","year":"2013","journal-title":"Nature"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1126\/science.1241165","article-title":"The Microbial Metabolites, SCFAs, Regulate Colonic Treg Cell Homeostasis","volume":"341","author":"Smith","year":"2013","journal-title":"Science"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"337","DOI":"10.1126\/science.1198469","article-title":"Induction of Colonic Regulatory T Cells by Indigenous Clostridium Species","volume":"331","author":"Atarashi","year":"2011","journal-title":"Science"},{"key":"ref_25","doi-asserted-by":"crossref","unstructured":"Yamamoto, Y., Kamiya, T., Yano, M., Huyen, V.T., Oishi, M., Nishio, M., Suzuki, A., Sunami, K., and Ohtani, N. (2023). Oral Microbial Profile Analysis in Patients with Oral and Pharyngeal Cancer Reveals That Tumoral Fusobacterium nucleatum Promotes Oral Cancer Progression by Activating YAP. Microorganisms, 11.","DOI":"10.3390\/microorganisms11122957"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"1919","DOI":"10.1128\/aem.56.6.1919-1925.1990","article-title":"Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations","volume":"56","author":"Amann","year":"1990","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"eaay3324","DOI":"10.1126\/sciadv.aay3324","article-title":"YAP1 is a potent driver of the onset and progression of oral squamous cell carcinoma","volume":"6","author":"Omori","year":"2020","journal-title":"Sci. Adv."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1016\/j.ccell.2023.02.014","article-title":"Tumor-infiltrating regulatory T cells as targets of cancer immunotherapy","volume":"41","author":"Tay","year":"2023","journal-title":"Cancer Cell"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"635","DOI":"10.1111\/jop.12445","article-title":"Regulatory T cells in oral squamous cell carcinoma","volume":"45","author":"Liu","year":"2016","journal-title":"J. Oral Pathol. Med."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"4729","DOI":"10.1128\/IAI.66.10.4729-4732.1998","article-title":"Role of Fusobacterium nucleatum and Coaggregation in Anaerobe Survival in Planktonic and Biofilm Oral Microbial Communities during Aeration","volume":"66","author":"Bradshaw","year":"1998","journal-title":"Infect. Immun."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"138","DOI":"10.1038\/ijos.2016.27","article-title":"Characterization of Fusobacterium nucleatum ATCC 23726 adhesins involved in strain-specific attachment to Porphyromonas gingivalis","volume":"8","author":"Park","year":"2016","journal-title":"Int. J. Oral Sci."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"e0518422","DOI":"10.1128\/spectrum.05184-22","article-title":"Pangenomic Study of Fusobacterium nucleatum Reveals the Distribution of Pathogenic Genes and Functional Clusters at the Subspecies and Strain Levels","volume":"11","author":"Ma","year":"2023","journal-title":"Microbiol. Spectr."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"5674","DOI":"10.1038\/s41467-021-25965-x","article-title":"Gut bacteria identified in colorectal cancer patients promote tumourigenesis via butyrate secretion","volume":"12","author":"Okumura","year":"2021","journal-title":"Nat. Commun."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"645","DOI":"10.1038\/s41586-020-03045-2","article-title":"Metabolic support of tumour-infiltrating regulatory T cells by lactic acid","volume":"591","author":"Watson","year":"2021","journal-title":"Nature"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"201","DOI":"10.1016\/j.ccell.2022.01.001","article-title":"Lactic acid promotes PD-1 expression in regulatory T cells in highly glycolytic tumor microenvironments","volume":"40","author":"Kumagai","year":"2022","journal-title":"Cancer Cell"}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/9\/2067\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,18]],"date-time":"2026-09-18T04:26:27Z","timestamp":1789705587000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/14\/9\/2067"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,9,16]]},"references-count":35,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["microorganisms14092067"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms14092067","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,9,16]]}}}