A Cell Culture Model to Study Tobacco Associated Oral Cancer Progression and Treatment Resistance

Head and Neck Squamous cell carcinoma is the 6th most common cancer in the world. Risk factors for HNSCC include alcohol and tobacco consumption, infection with Human Papilloma Virus, and exposure to environmental toxins. Unlike many other cancers, the incidence of HNSCC is growing, yet the treatment options and outcomes have not been significantly improved for decades. An increased understanding of the molecular mechanisms of these cancers is needed for improvement in early diagnosis, enhanced treatment effectiveness, and cancer prevention. 4-nitroquinoline 1-oxide (4NQO) is a well-tested, tobacco mimicking, carcinogen. 4NQO produces similar histological and chemical changes found in human oral carcinomas. Programmed cell death resistance and anchorage-independency are acquired abilities that allow tumor cells to invade adjacent tissues and disseminate through the body. Spheroids are aggregates of cells formed in three-dimensional culture that can mimic the structural character of microtumors. In this study, mouse oral mucosal epithelial cells were treated with 4NQO to induce neoplastic transformation. Spheroid formation was achieved with the use of non-attachment cell culture plates. Anchorage independent growth capability was assessed by measuring the average spheroid diameter for 4NQO treated and 4NQO Non-treated cell lines. Cisplatin, a chemotherapy drug, was also tested for its effect on spheroid formation. Western blot analysis was performed to analyze the molecular events of the DNA damage signaling and repair, in 4NQO treated and untreated cells. As the results of our studies, we derived three cell lines post 4NQO exposure. Two of these cell lines exhibited significantly increased spheroid formation in 3D culture. These two cell lines were also more resistant to cisplatin treatment, measured by spheroid growth in the presence of 10µM Cisplatin. Along with these observations, our Western blot analysis detected some levels of heterogenetic responses to DNA damage in these cell lines, compared to the parental epithelial cell line. These results suggested potential transformative events in oral epithelial cells, induced by 4NQO. The connections between these events and altered DNA damage responses, genomic instability, tumorigenesis, and treatment resistance will be investigated in future studies