Behavior Analysis of Oral Cancer Cells in an Acidic Microenvironment

Objectives: The objective of this study is to improve our knowledge on the effects of the tumor microenvironment acidosis on the behavior of oral cancer cells.
Methods: The oral squamous cell carcinoma cells SCC-4 were cultured in regular complete DMEM medium or supplemented with HCL acid to a pH6.8. Cells cultured in neutral (pH7.4) were compared with cells cultured at pH6.8 after different time points. The collective or individual migration of the cells was compared by the wound healing and by the time lapse assay, respectively. The fraction of cells with cancer stem phenotype was assessed by the orospheres assay and by gene expression of Bmi-I and Nanog. Cell cycle analysis was assessed with propidium iodide. Moreover, the effect of acidosis on the survival to Cisplatin was analyzed by the sulphorodamine B cytotoxic assay. All experiments were performed in triplicates and assessed by a blind observer. The groups were compared by the t test or ANOVA followed by Tukey test.
Results: Cells exposed to pH6.8 for 7 and 21 days showed increased collective (p=0.0211 ANOVA) and individual (p=0.0039 ANOVA) migration ability when compared to cells kept in neutral pH for the same period. After 21 days under pH6.8, a cancer stem cell phenotype was induced because SCC-4 cells were able to form more than twice of orospheres (p=0.0104 ANOVA) and presented increased expression of Bmi-I. Cells under acidosis became more resistant (p<0,01) to Cisplatin at different doses (1-100μM). No significant alterations on cell cycle distribution were observed.
Conclusions: Acidosis increases aggressiveness of cancer cells by inducing a cancer stem-cell phenotype, chemoresistance and boosting cell migration. The tumor pH may have a major impact in disease progression and prognosis, therefore to inhibit the acidification of tumor microenvironment may represent an important therapeutic tool.