Application of Hypoxia-Driven Dual-Reporter Model to Explore the Effect of Hypoxia on Radiosensitivity of Human Nasopharyngeal Carcinoma Cells.

Abstract

PURPOSE/OBJECTIVE(S): hypoxia has been commonly observed in human nasopharyngeal carcinoma (NPC) which is associated with resistance to radiation and malignant phenotypes. The purpose of the study was to apply our build-in hypoxia-driven dual reporter model to explore the effect of hypoxia on radiosensitivity in vitro and in vivo.\n\n\nMATERIALS/METHODS\nThe modalities to research the effect of hypoxia in vitro includes clonogenic survival assay and γH2AX foci formation. Tumor growth delay with radiation and/or cisplatin, imaging study on spatial biodistribution between blood perfusion (Hoechst 33342), endogenous (eGFP and CA9) and exogenous (pimonidazole) hypoxia markers were used to investigate the effect of hypoxia in vivo.\n\n\nRESULTS\nThe constructed hypoxia-driven dual-reporter model contains 9 repeats of hypoxia responsive element and fusion gene of 9xHRE-HSV1-TKeGFP in NPC cells (abbreviated as SUNE-1/HRE cells) in which hypoxia-induced molecular events can be visualized by fluorescence or nuclear imaging techniques. There is no difference in radiosensitivity between SUNE1/Parental and SUNE1/HRE cells, while cisplatin significantly increases radiosensitivity in both cell lines. γH2AX serves as a surrogate for unrepaired DSB. When the cells are exposed to 0.5% O2, eGFP is observed only in SUNE1/HRE cells. DSB repair rate under hypoxia is similar in SUNE1/Parental and SUNE1/HRE cells. The number of γH2AX foci is dramatically higher in the presence of cisplatin compared to that given irradiation alone under the condition of normoxia or hypoxia. It indicates that the addition of cisplatin enhances radiosensitivity by compromising the ability of cells to repair DSB. Cisplatin alone shows modest whereas irradiation moderate effect on delayed tumor growth. Combined treatment of irradiation and cisplatin further enhances the effect on tumor growth delay. High expression of eGFP is observed in the nonperfused regions. The spatial biodistribution of hypoxia-induced expression of eGFP reporter gene is similar to other endogenous or exogenous hypoxia biomarkers, i.e., CA9 and pimonidazole.\n\n\nCONCLUSION\nThe build-in hypoxia-driven dual reporter 9HRE-HSV1-TKeGFP hypoxia research model does not modify intrinsic radiosensitivity of SUNE1 cells under the condition of normoxia or hypoxia, suggesting it be used as preclinical model to explore the therapeutic efficacy of various treatment modalities to overcome hypoxia by fluorescence or nuclear imaging techniques.