A noninvasive approach for assessing tumor hypoxia in xenografts: developing a urinary marker for hypoxia

Tumor hypoxia modifies the efficacy of conventional anticancer therapy and promotes malignant tumor progression. Human chorionic gonadotropin (hCG) is a glycoprotein secreted during pregnancy that has been used to monitor tumor burden in xenografts engineered to express this marker. We adapted this approach to use urinary beta-hCG as a secreted reporter protein for tumor hypoxia. We used a hypoxia-inducible promoter containing five tandem repeats of the hypoxia-response element (HRE) ligated upstream of the beta-hCG gene. This construct was stably integrated into two different cancer cell lines, FaDu, a human head and neck squamous cell carcinoma, and RKO, a human colorectal cancer cell line. In vitro studies showed that tumor cells stably transfected with this plasmid construct secrete beta-hCG in response to hypoxia or hypoxia-inducible factor 1alpha (HIF-1alpha) stabilizing agents. The hypoxia responsiveness of this construct can be blocked by treatment with agents that affect the HIF-1alpha pathways, including topotecan, 1-benzyl-3-(5'-hydroxymethyl-2'-furyl)indazole (YC-1), and flavopiridol. Immunofluorescent analysis of tumor sections and quantitative assessment with flow cytometry indicate colocalization between beta-hCG and 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide (EF5) and beta-hCG and pimonidazole, two extrinsic markers for tumor hypoxia. Secretion of beta-hCG from xenografts that contain these stable constructs is directly responsive to changes in tumor oxygenation, including exposure of the animals to 10% O2 and tumor bed irradiation. Similarly, urinary beta-hCG levels decline after treatment with flavopiridol, an inhibitor of HIF-1 transactivation. This effect was observed only in tumor cells expressing a HRE-regulated reporter gene and not in tumor cells expressing a cytomegalovirus-regulated reporter gene. The 5HRE beta-hCG reporter system described here enables serial, noninvasive monitoring of tumor hypoxia in a mouse model by measuring a urinary reporter protein.