Wataru Ariyoshi

Cancer stem cells (CSCs) are considered to be responsible for recurrence, metastasis, and resistance to treatment in many types of cancers; therefore, new treatment strategies targeting CSCs are attracting attention. In this study, we fabricated a polyethylene glycol-tagged microwell device that enabled spheroid formation from human oral squamous carcinoma cells. HSC-3 and Ca9-22 cells cultured in the microwell device aggregated and generated a single spheroid per well within 24–48 h. The circular shape and smooth surface of spheroids were maintained for up to five days, and most cells comprising the spheroids were Calcein AM-positive viable cells. Interestingly, the mRNA expression of CSC markers (Cd44, Oct4, Nanog, and Sox2) were significantly higher in the spheroids than in the monolayer cultures. CSC marker-positive cells were observed throughout the spheroids. Moreover, resistance to cisplatin was enhanced in spheroid-cultured cells compared to that in the monolayer-cultured cells. Furthermore, some CSC marker genes were upregulated in HSC-3 and Ca9-22 cells that were outgrown from spheroids. In xenograft model, the tumor growth in the spheroid implantation group was comparable to that in the monolayer culture group. These results suggest that our spheroid culture system may be a high-throughput tool for producing uniform CSCs in large numbers from oral cancer cells.

The effect of hydrogen peroxide, an antiseptic dental treatment, on Aggregatibacter actinomycetemcomitans, the main causative agent of localized invasive periodontitis, was investigated. Hydrogen peroxide treatment (0.06%, 4× minimum inhibitory concentration) resulted in the persistence and survival of approximately 0.5% of the bacterial population. The surviving bacteria did not genetically acquire hydrogen peroxide resistance but exhibited a known persister behavior. Sterilization with mitomycin C significantly reduced the number of A. actinomycetemcomitans persister survivors. RNA sequencing of hydrogen peroxide-treated A. actinomycetemcomitans showed elevated expression of Lsr family members, suggesting a strong involvement of autoinducer uptake. In this study, we found a risk of A. actinomycetemcomitans persister residual from hydrogen peroxide treatment and hypothesized associated genetic mechanisms of persister from RNA sequencing.