Noninvasive Molecular Imaging of Hypoxia in Human Xenografts: Comparing Hypoxia-Induced Gene Expression with Endogenous and Exogenous Hypoxia Markers

Tumor hypoxia is important in the development and treatment of human cancers. We have developed a novel xenograft model for studying and imaging of hypoxia-induced gene expression. A hypoxia-inducible dual reporter herpes simplex virus type I thymidine kinase and enhanced green fluorescence protein (HSV1-TKeGFP), under the control of hypoxia response element (9HRE), was stably transfected into human colorectal HT29 cancer cells. Selected clones were further enriched by repeated live cell sorting gated for hypoxia-induced eGFP expression. Fluorescent microscopy, fluorescence-activated cell sorting, and radioactive substrate trapping assays showed strong hypoxia-induced expression of eGFP and HSV1-tk enzyme in the HT29-9HRE cells in vitro. Sequential micropositron emission tomography (PET) imaging of tumor-bearing animals, using the hypoxic cell tracer F-18-FMISO and the reporter substrate 124 I-FIAU, yielded similar tumor hypoxia images for the HT29-9HRE xenograft but not in the parental HT29 tumor. Using autoradiography and IHC, detailed spatial distributions in tumor sections were obtained and compared for the following hypoxia-associated biomarkers in the HT29-9HRE xenograft: I-124-FIAU, is F-18-FMISO, Hoechst (perfusion), lectin-TRITC (functional blood vessels), eGFP, pimonidazole, EF5, and CA9. Intratumoral distributions of I-124-FIAU and F-18-FMISO were similar, and eGFP, pimonidazole, EF5, and CA9 colocalized in the same areas but not in well-perfused regions that were positive for Hoechst and lectin-TRITC. In enabling the detection of hypoxia-induced molecular events and mapping their distribution in vivo with serial noninvasive positron emission tomography imaging, and multiple variable analysis with immunohistochemistry and fluorescence microscopy, this human xenograft model provides a valuable tool for studying tumor hypoxia and in validating existing and future exogenous markers for tumor hypoxia. [Cancer Res 2008;68(20):8597-606]