title: Imaging of HIF-1-active tumor hypoxia using a protein effectively delivered to and specifically stabilized in HIF-1-active tumor cells.
abstract: Hypoxia-inducible factor-1 (HIF-1) plays an important role in malignant tumor progression and in the development of resistance to radiotherapy. We designed a novel fusion protein (PTD-ODD-SAV [POS]) consisting of a protein transduction domain (PTD), streptavidin (SAV), and a portion of the oxygen-dependent degradation domain (ODD) of HIF-1alpha that confers the same oxygen-dependent regulation as HIF-1alpha on POS. (3-(123/125)I-iodobenzoyl)norbiotinamide ((123/125)I-IBB) was conjugated to the SAV moiety of POS to synthesize (123/125)I-IBB-labeled POS ((123/125)I-IPOS). The purpose of this study was to evaluate the feasibility of (123)I-IPOS as an imaging probe for HIF-1-active tumor hypoxia.
  
  
  METHODS
  After a 24-h incubation of (125)I-IPOS with various tumor cell lines under either normoxic (20% O(2)) or hypoxic (0.1% O(2)) conditions, the intracellular radioactivity was investigated. Then, the biodistribution of (123/125)I-IPOS was examined with tumor-implanted mice, and an in vivo imaging study was performed. The tumoral accumulation of (125)I-IPOS was compared with HIF-1 activity using the mice carrying tumors with the HIF-1-dependent luciferase reporter gene. Furthermore, the intratumoral localization of (125)I-IPOS was examined by the autoradiographic study, and then the same slide was subjected to immunostaining for pimonidazole, which is the hypoxic marker.
  
  
  RESULTS
  The ratios of radioactivity in hypoxic cells to that in normoxic cells were more than 2. These results indicate incorporation of (125)I-IPOS into these cells and degradation of (125)I-IPOS by normoxic tumor cells. In the biodistribution study, (125)I-IPOS accumulated in the tumor (1.4 +/- 0.3 percentage injected dose per gram) 24 h after administration. At that time, (125)I-IPOS showed high tumor-to-blood and tumor-to-muscle ratios (5.1 +/- 0.3 and 14.0 +/- 3.9, respectively). The tumors were clearly visualized by in vivo imaging 24 h after (123)I-IPOS injection (tumor-to-muscle ratio was 9.6). The tumoral accumulation of (125)I-IPOS correlated with HIF-1 activity (R = 0.71, P < 0.05), and its intratumoral distribution coincided with the hypoxic regions.
  
  
  CONCLUSIONS
  (123)I-IPOS is a potential probe for the imaging of HIF-1 activity in tumors. Given the role of HIF-1 in tumor biology, its detection may be considered an indicator of aggressive cancer phenotypes.
authors.affiliation: Department of Patho-Functional Bioanalysis, Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Kudo
authors.forename: Masashi
authors.initials: M
authors.lastname: Ueda
authors.forename: Yuji
authors.initials: Y
authors.lastname: Kuge
authors.forename: Takahiro
authors.initials: T
authors.lastname: Mukai
authors.forename: Shotaro
authors.initials: S
authors.lastname: Tanaka
authors.forename: Maki
authors.initials: M
authors.lastname: Masutani
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Kiyono
authors.forename: Shinae
authors.initials: S
authors.lastname: Kizaka-Kondoh
authors.forename: Masahiro
authors.initials: M
authors.lastname: Hiraoka
authors.forename: Hideo
authors.initials: H
authors.lastname: Saji
mesh_terms: D000818:Animals; D001710:Biotin / Q000031:analogs & derivatives; D015687:Cell Hypoxia; D002478:Cells, Cultured; D005260:Female; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000032:analysis*; D007457:Iodine Radioisotopes; D051379:Mice; D008809:Mice, Inbred C3H; D009369:Neoplasms / Q000737:chemistry*; D019275:Radiopharmaceuticals; D011993:Recombinant Fusion Proteins; D014018:Tissue Distribution
chemical_list: C106664:(3-iodobenzoyl)norbiotinamide; D051793:Hypoxia-Inducible Factor 1; D007457:Iodine Radioisotopes; D019275:Radiopharmaceuticals; D011993:Recombinant Fusion Proteins; D001710:Biotin