In vitro analysis of Fusobacterium Polymorphum from oral leukoplakia patients identifies “high-risk” isolates

Introduction Mounting evidence links F. nucleatum , particularly subsp. polymorphum (now classified as F. polymorphum ) to oral squamous cell carcinoma (OSCC). However, not all studies support this association, and its ubiquitous presence in health complicates its role as a driver of disease, raising a fundamental question: Can some isolates be pathogenic “high risk” while others are not? Here, we investigated the carcinogenic potential of clinical isolates of F. polymorphum from oral dysplastic lesions. Methods Sixteen, fully sequenced F. polymorphum isolates from healthy subjects (6 isolates) or patients with mild epithelial dysplasia (3 isolates), moderate dysplasia (3 isolates) or severe dysplasia/OSCC (4 isolates) were included. The isolates were assessed for their effect on the proliferation, migration, invasion, transcriptome and cytokinome of dysplastic oral keratinocytes (DOK). The isolates were also subject to RNA-seq and secreted amyloid FadA analyses. Results Although genetically indistinguishable, the isolates differed markedly in their effects on dysplastic oral keratinocytes (DOK) with isolates from dysplastic lesions demonstrating enhanced proliferation, migration, and invasion of DOK in proportion to the dysplasia severity of their clinical origin. Strikingly, amyloid-like FadA levels were also significantly higher in dysplasia-associated “high-risk” isolates and correlated with their pro-carcinogenic effects. RNA-seq further showed upregulation of heme acquisition genes in high risk isolates. Transcriptome and cytokine profiling of DOK revealed a uniform pro-inflammatory response across all isolates, independent of origin, but genes and pathways related to proliferation correlated with dysplasia severity. Supporting this, 45% of the most severity-correlated host genes identified in vitro were also differentially expressed in the same direction in tumors versus normal tissues in the TCGA OSCC dataset. Conclusion These results show, for the first time, that clinical oral isolates of F. polymorphum vary in their carcinogenic properties, establishing the novel concept of “high-risk” versus “low-risk” isolates in oral carcinogenesis, potentially driven by genome-independent regulation.