A Preclinical Model For Imaging Hypoxia-Driven Gene Expression In Human Nasopharyngeal Carcinoma Xenograft

Tumor hypoxia has been associated with malignant phenotype and resistance to chemo- and radio-therapy. The purpose is to research tumor hypoxia by establishing a preclinical model of nasopharyngeal cancer (NPC) with hypoxia-driven gene expression, comparing the biodistribution between exogenous hypoxia tracer and endogenous hypoxia gene expressions and investigating effect of synthetic lethality by irradiation combined with chemotherapeutic agent or PI3K/mTOR inhibitor. SUNE1 cells, originated from an NPC patient were transfected with plasmid containing a hypoxia-inducible dual reporter herpes simplex virus type 1 thymidine kinase and enhanced green fluorescence protein (HSV1-TKeGFP) under control of 9 repeats of hypoxia response element (9x HRE) from human EGFR. Single cloned cells were obtained by repetitive selection with G418 and repeated live cell FACS sorting gated for hypoxia-induced eGFP expression and in vitro radioactive substrate trapping assays with 14C-FIAU. MicroPET imaging study, immunofluorescence on tumor hypoxia and immunohistochemical (IHC) analysis of xenografts were made for comparison of exogenous hypoxia tracer and endogenous hypoxia gene expressions. This hypoxia research model was used in vitro and in vivo to research the effect of synthetic lethality by applying cisplatin, inhibitor targeted on PI3K/mTOR in combination with irradiation including foci formation, clonogenic survival formation and tumor growth delay. We have established and characterized a novel model for studying and imaging of hypoxia-induced gene expression. A hypoxia-inducible dual reporter HSV1-TKeGFP, under the control of 9xHRE, was stably transfected into SUNE1 cells. Selected clones were further enriched by repeated live cell sorting gated for hypoxia-induced eGFP expression. Fluorescent microscopy, and radioactive substrate trapping assays showed strong hypoxia-induced expression of eGFP and HSV1-tk enzyme in SUNE1/9xHRE cells. Detailed spatial distributions in tumor sections were obtained and compared for following hypoxia-associated biomarkers in SUNE1/9xHRE xenograft: Hoechst (blood perfusion), pimonidazole (exogenous biomarker), eGFP and CA9 (endogenous biomarkers). Intratumoral distributions of eGFP, pimonidazole, and CA9 colocalized in the same areas but not in well-perfused regions that were positive for Hoechst and anti-CD31 (blood vessels). The effect of synthetic lethality was observed when SUNE1/9xHRE-TKeGFP cells or xenografts are exposed to cisplatin or PI3K/mTOR inhibitor and irradiation. In enabling the detection of hypoxia-induced molecular events and mapping their distribution in vivo including serial noninvasive PET imaging, and multiple variable analysis with immunohistochemistry assay, this human xenograft model provides a valuable tool for investigating tumor hypoxia and exploring therapeutic efficacy of different treatment modalities to overcome hypoxia.