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.
Nelson
Daniel W
DW
Department of Radiation Oncology, Stanford University, Stanford, California 94305-5847, USA.
Cao
Hongbin
H
Zhu
Yonghua
Y
Sunar-Reeder
Bulbin
B
Choi
Clara Y H
CY
Faix
James D
JD
Brown
J Martin
JM
Koong
Albert C
AC
Giaccia
Amato J
AJ
Le
Quynh-Thu
QT
D000818:Animals; D002294:Carcinoma, Squamous Cell / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000652:urine; D015687:Cell Hypoxia / Q000502:physiology; D045744:Cell Line, Tumor; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000235:genetics* / Q000652:urine; D015179:Colorectal Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000652:urine; D004268:DNA-Binding Proteins / Q000235:genetics; D005419:Flavonoids / Q000494:pharmacology; D017930:Genes, Reporter / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D006258:Head and Neck Neoplasms / Q000188:drug therapy / Q000235:genetics / Q000378:metabolism* / Q000652:urine; D006801:Humans; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D051379:Mice; D008807:Mice, Inbred BALB C; D018632:Mice, Inbred SENCAR; D009368:Neoplasm Transplantation; D009687:Nuclear Proteins / Q000235:genetics; D010880:Piperidines / Q000494:pharmacology; D019772:Topotecan / Q000494:pharmacology; D014157:Transcription Factors / Q000235:genetics; D014162:Transfection; D014183:Transplantation, Heterologous
D018997:Chorionic Gonadotropin, beta Subunit, Human; D004268:DNA-Binding Proteins; D005419:Flavonoids; C497442:HIF1A protein, human; C497443:Hif1a protein, mouse; D051793:Hypoxia-Inducible Factor 1; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009687:Nuclear Proteins; D010880:Piperidines; D014157:Transcription Factors; C077990:alvocidib; D019772:Topotecan