Low-Intensity Ultrasound as a Method to Improve the Effect of Oncolytic Virotherapy on Oral Cancer

Statement of the problem: Oncolytic virotherapy is based on the ability of an attenuated virus to destroy infected tumor cells. This therapy with herpes simplex virus type-1 (HSV-1) is now progressing to its clinical application for the treatment of cancer because the results obtained for the efficiency of oncolytic HSV-1 in advanced head and neck cancer in phase I/II studies are promising [1]. Since the most reliable method by which to deliver oncolytic HSV-1 to solid tumors is direct inoculation, the efficient and rapid entry of the inoculated virus into tumor cells is required to achieve a significant antitumor effect by oncolytic HSV-1. Low-intensity ultrasound is a useful method to introduce materials into cells due to the transient formation of micropores, called sonoporation, on the cell membrane. The effect of ultrasound on oncolytic HSV-1 infection in oral squamous cell carcinoma (SCC) cells was examined.

Materials and methods: Human SCC cell line SAS and HSV-1 RH2, which exhibits a fusogenic ability in human SCC cells, was deficient in the gamma 34.5 gene and had mutations in gB were used [2]. Cells were grown in multi-well plates and exposed to ultrasound in the presence or absence of lipid-shelled microbubbles filled with perfluorocarbon gas. An ultrasound machine Sonitron 2000V was used. Cell viability was evaluated using MTT assay.

Methods of data analysis: Results are reported as means±SD. Comparisons of mean plaque numbers were achieved using a one-way ANOVA, followed by Tukey’s honest significant difference (HSD) test. A value of P<0.05 was considered to be significant.

Results: Cell viability at 1 W/cm²was approximately 80% that of the untreated control during an exposure time of 60 sec. When cells were inoculated with HSV-1, incubated for 30 min, and exposed to ultrasound, a maximal effect was observed after an exposure of 10 sec and the plaque number was 3.6-fold that of the control. Plaques were produced even if SAS cells were exposed to ultrasound and immediately infected with RH2 without an adsorption time. This effect was abolished when the interval from ultrasound exposure to virus inoculation was prolonged. Scanning electron microscopy revealed pore formation on the cell surface after exposure to ultrasound. HSV enters cells by fusion of the viral envelope with either the plasma membrane or endosomal membrane. Based on experiments with lysosomotrophic agents, which elevate the normally low pH of endosomes, acidic pH has been implicated in the endocytotic entry of HSV to several cell types. Monensin is a carboxylic ionophore that blocks endosomal acidification and has been used to evaluate the role of low pH in HSV-1 replication. Monensin inhibited the replication of RH2 that entered into cells by ultrasound.

Conclusion: These results demonstrated that the entry of oncolytic HSV-1 RH2 into SAS cells was enhanced by ultrasound due to the formation of pores and endocytosis. The application of this method to oncolytic virotherapy for oral SCC with HSV-1 may facilitate the efficiency of viral infection at the site of inoculation.

References: [1] Harrington KJ, Hingorani M, Tanay MA, Hickey J, Bhide SA, Clarke PM, Renouf LC, Thway K, Sibtain A,  McNeish IA, Newbold KL,  Goldsweig H, Coffin R, Nutting CM: Phase I/II study of oncolytic HSV^GM-CSF in combination with radiotherapy and cisplatin in untreated stage III/IV squamous cell cancer of the head and neck. Clin Cancer Res 2010; 16: 4005-4015. [2] Takahashi G, Meshii N, Hamada M, Iwai S, Yura Y: Sequence of a fusogenic herpes simplex virus RH2 for oncolytic virotherapy. J Gen Virol 2013; 94:726-737.