Imaging of hypoxia-driven gene expression in an orthotopic liver tumor model

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The purpose of this study was to monitor hypoxia in an orthotopic liver tumor model using a hypoxia-sensitive reporter imaging system and to image enhanced gene expression after clamping the hepatic artery. C6 and RH7777 Morris hepatoma cells were transduced with a triple reporter gene (HSV1-tk/green fluorescent protein/firefly luciferase-triple fusion), placed under the control of a HIF-1-inducible hypoxia responsive element (HRE). The cells showed inducible luciferase activity and green fluorescent protein expression in vitro. Isolated reporter-transduced Morris hepatoma cells were used to produce tumors in livers of nude rats, and the effect of hepatic artery clamping was evaluated. Tumor hypoxia was shown by immunofluorescence microscopy with the hypoxia marker EF5 [2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl acetamide)] and the fluorescent perfusion marker Hoechst 33342, and by pO(2) electrode measurements. For tumor hypoxia imaging with the HRE-responsive reporter, both luciferase bioluminescence and [(18)F]2'-fluoro-2'-deoxyarabinofuranosyl-5-ethyluracil positron emission tomography was done, and the presence of hypoxia in Morris hepatoma tumors were successfully imaged by both techniques. Transient clamping of the hepatic artery caused cessation of tumor perfusion and severe hypoxia in liver tumors, but not in adjacent liver tissue. These results show that the orthotopic reporter-transduced RH7777 Morris hepatomas are natively hypoxic and poorly perfused in this animal model, and that the magnitude of hypoxia can be monitored using a HRE-responsive reporter system for both bioluminescence and positron emission tomography imaging. However, the severity of tumor ischemia after permanent ligation of the hepatic artery limits our ability to image severe hypoxia in this animal model.