Zebrafish larvae as a model for studying the impact of oral bacterial vesicles on tumor cell growth and metastasis.

Oral bacteria naturally secrete extracellular vesicles (EVs), which have attracted attention for their promising biomedical applications including cancer therapeutics. However, our understanding of EV impact on tumor progression is hampered by limited in vivo models. In this study, we propose a facile in vivo platform for assessing the effect of EVs isolated from different bacterial strains on oral cancer growth and dissemination using the larval zebrafish model. EVs were isolated from: wild-type Aggregatibacter actinomycetemcomitans and its mutant strains lacking the cytolethal distending toxin (CDT) or lipopolysaccharide (LPS) O-antigen; and wild-type Porphyromonas gingivalis. Cancer cells pretreated with EVs were xenotransplanted into zebrafish larvae, wherein tumor growth and metastasis were screened. We further assessed the preferential sites for the metastatic foci development. Interestingly, EVs from the CDT-lacking A. actinomycetemcomitans resulted in an increased tumor growth, whereas EVs lacking the lipopolysaccharide O-antigen reduced the metastasis rate. P. gingivalis-derived EVs showed no significant effects. Cancer cells pretreated with EVs from the mutant A. actinomycetemcomitans strains tended to metastasize less often to the head and tail compared to the controls. In sum, the proposed approach provided cost- and labor-effective yet efficient model for studying bacterial EVs in oral carcinogenesis, which can be easily extended for other cancer types. Furthermore, our results support the notion that these nanosized particles may represent promising targets in cancer therapeutics.