A Noninvasive Approach for Assessing Tumor Hypoxia in Xenografts: Developing a Urinary Marker for Hypoxia Daniel W. Nelson, 1 Hongbin Cao, 1 Yonghua Zhu, 1 Bulbin Sunar-Reeder, 1 Clara Y.H. Choi, 1 James D. Faix, 2 J. Martin Brown, 1 Albert C. Koong, 1 Amato J. Giaccia, 1 and Quynh-Thu Le 1 1 Departments of Radiation Oncology and 2 Pathology, Stanford University, Stanford, California Abstract Tumor hypoxia modifies the efficacy of conventional antican- cer 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 B-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 b-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 B-hCG in response to hypoxia or hypoxia-inducible factor 1A (HIF-1A) stabilizing agents. The hypoxia responsiveness of this con- struct can be blocked by treatment with agents that affect the HIF-1A pathways, including topotecan, 1-benzyl-3-(5V -hydrox- ymethyl-2V -furyl)indazole (YC-1), and flavopiridol. Immuno- fluorescent analysis of tumor sections and quantitative assessment with flow cytometry indicate colocalization between B-hCG and 2-(2-nitro-1H -imidazol-1-yl)-N -(2,2,3,3,3- pentafluoropropyl)acetamide (EF5) and B-hCG and pimoni- dazole, two extrinsic markers for tumor hypoxia. Secretion of B-hCG from xenografts that contain these stable constructs is directly responsive to changes in tumor oxygenation, including exposure of the animals to 10% O 2 and tumor bed irradiation. Similarly, urinary B-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 cytomega- lovirus-regulated reporter gene. The 5HRE B-hCG reporter system described here enables serial, noninvasive monitoring of tumor hypoxia in a mouse model by measuring a urinary reporter protein. (Cancer Res 2005; 65(14): 6151-8) Introduction Tumor hypoxia is a major determinant of the malignant progression of transformed cells and their response to therapy. It has been shown to promote tumor progression through the selection of tumor cells with diminished apoptotic potential, stimulate proangiogenic gene expression, and increase metastatic potential (1). Clinical studies have shown a strong correlation between pretreatment tumor pO 2 and tumor control and survival in patients with head and neck cancers (2, 3) and cervical cancers (4–6). Clinical and preclinical studies have also indicated that hypoxia increases tumor invasiveness and dissemination in several human solid tumor types (7–10). At the molecular level, hypoxia-inducible factor 1 (HIF-1) is an important transcription factor that regulates gene expression under hypoxic conditions (11). Over three dozen HIF-1–regulated genes have been identified to date (12, 13). The protein products of these genes play key roles in angiogenesis, vascular remodeling, glucose metabolism, cell proliferation, and cell survival. Recent studies suggest that HIF-1 is a novel target for anticancer therapy and many research groups are actively developing small molecular inhibitors of HIF-1 (14–16). Therefore, a noninvasive and cost- effective system to monitor tumor hypoxia in vivo would aid in determining the efficacy of targeted therapy to HIF-1 or other hypoxia-regulated genes or proteins. Various methods have been developed for assessing tumor hypoxia in xenograft tumor models and in patients (17–21). Most approaches only allow assessment of tumor hypoxia only at a single time point, as it is often necessary to remove the tumor or to sacrifice the animal at the time of hypoxia measurement. Only few methods can be used for serial measurements of tumor hypoxia. These include various oxygen sensing devices and hypoxia imaging techniques (22–24). The oxygen sensing devices, such as needle electrodes, can only access superficial tumors, are highly depen- dent on the technical skill of the user, and often fail to distinguish viable hypoxic cells from necrosis (25, 26). Imaging studies have also been developed to monitor hypoxia-induced stability of reporter proteins, such as luciferase (27), or HIF-1 reporter gene activity, such as positron emission tomography (PET) imaging of the 2V -[ 18 F]flouro-2V deoxy-1h-D-arabionofuranosyl-5-ethyl-uracil (FEAU) product of a HIF-1–driven herpes simplex virus type 1 thymidine kinase/green fluorescent protein fusion (23, 24), but these approaches often require expensive dedicated animal imaging facilities and local imaging expertise for optimal results. Our goal was therefore to develop a noninvasive, rapid, and inexpensive system for dynamic monitoring of tumor hypoxia in living animals. Human chorionic gonadotropin (hCG) is a glycoprotein normally secreted by syncytiotrophoblasts during pregnancy and has long been used as a sensitive and specific marker for pregnancy. h-hCG is readily measured in both serum and urine and has been used for the diagnosis and monitoring of pregnancy. Shih et al. (28) have previously developed a system using h-hCG engineered to be constitutively secreted from implanted tumor xenografts to monitor Note: D.W. Nelson and H. Cao contributed equally to this work. Presented at the 94th Annual Meeting of the AACR, July 11-14, 2003, Washington, DC. Requests for reprints: Quynh-Thu Le, Department of Radiation Oncology, 875 Blake Wilbur Dr, MC 5847, Stanford, CA 94305-5847. Phone: 650-498-5032; Fax: 650- 725-8231; E-mail: qle@stanford.edu. I2005 American Association for Cancer Research. www.aacrjournals.org 6151 Cancer Res 2005; 65: (14). July 15, 2005 Research Article