Role of extracellular vesicles from periodontopathogens in serum resistance and oral cancer

Oral squamous cell carcinoma (OSCC) is the most common cancer encountered in the head and neck area. Oral dysbiosis and periodontopathogens induce periodontitis, an inflammatory disease of the teeth-supporting tissues. Recent evidence supports that the oral microbiota and dysbiosis are linked to several cancers, including OSCC. Both anti- and pro-tumorigenic effects of the oral microbiota in cancer are reported. Periodontopathogens secrete nanoparticles, extracellular vesicles (EVs), that transport virulence factors and modulate immune responses. Although EVs contribute to inflammation, they are also potential therapeutic agents. New diagnostic and therapeutic strategies are needed since the OSCC 5-year survival rate is rather low and has not significantly improved recently. The results of Study I indicated that EVs from the bacterium Aggregatibacter actinomycetemcomitans increased its survival in the host and protected the bacterium from the human innate immune system in vitro. The effect was dose-dependent on the inflammation-stimulating molecule lipopolysaccharide (LPS) expressed on the EV-surface. Such an effect on serum resistance could promote the survival of A. actinomycetemcomitans and other periodontopathogens and contribute to periodontitis. Understanding EV-host interactions may advance the development of EV-based therapies. In Study II, we conducted a systematic review, where we confirmed the association between oral bacteria and head and neck squamous cell carcinoma (HNSCC), including OSCC. We analyzed 34 studies comprising 2469 patients. Importantly, many periodontopathogens, such as Porphyromonas gingivalis and Fusobacterium nucleatum, were associated with HNSCC. Furthermore, we summarized the association between the expression profiles of different bacterial species and their effect on tumor progression and prognosis. For example, F. nucleatum was linked to a better survival rate among OSCC patients. In Study III, we investigated how EVs from A. actinomycetemcomitans, P. gingivalis, F. nucleatum, and Parvimonas micra influenced primary and metastatic OSCC cell behavior in vitro. We showed that A. actinomycetemcomitans-derived EVs inhibited pro-cancerous features of metastatic OSCC cells, such as cancer cell proliferation, invasion, and tube formation potential. The effect on cell proliferation was cytolethal distending toxin (CDT)-dependent, while the expression of both CDT and LPS influenced cell invasion and tube formation. EVs from F. nucleatum decreased cancer cell proliferation and increased apoptotic cell death, whereas P. gingivalis-derived EVs induced their invasion. All tested EVs increased the motility of OSCC cells. Finally, in Study IV, we proposed zebrafish larvae as a novel model in bacterial EV research. In this study, we designed a protocol for studying the effect of EVs on the tumor area and the metastasis rate of OSCC cells in zebrafish larvae. The highly metastatic OSCC cells were first treated with EVs and then injected into zebrafish. Interestingly, cells challenged with EVs from the CDT-lacking A. actinomycetemcomitans strain formed larger tumors, while cells challenged with EVs from the LPS O-antigen-lacking strain showed reduced metastatic potential. Collectively, these studies support the conclusion that bacterial EVs are potential contributors of periodontitis through mediating serum protection. Regarding OSCC, EVs showed both pro- and anti-cancer effects by influencing cancer cell behavior in vitro and in vivo. Oral bacteria in OSCC patients have the potential to provide both diagnostic and prognostic utilities; however, standardized techniques and validation are required for sampling and the analysis to be employed in the clinical context.