title: Combining Optical Reporter Proteins with Different Half-lives to Detect Temporal Evolution of Hypoxia and Reoxygenation in Tumors.
abstract: Here we have developed a hypoxia response element driven imaging strategy that combined the hypoxia-driven expression of two optical reporters with different half-lives to detect temporal changes in hypoxia and hypoxia inducible factor (HIF) activity. For this purpose, human prostate cancer PC3 cells were transfected with the luciferase gene fused with an oxygen-dependent degradation domain (ODD-luc) and a variant of the enhanced green fluorescent protein (EGFP). Both ODD-luciferase and EGFP were under the promotion of a poly-hypoxia-response element sequence (5xHRE). The cells constitutively expressed tdTomato red fluorescent protein. For validating the imaging strategy, cells were incubated under hypoxia (1% O2) for 48 hours and then reoxygenated. The luciferase activity of PC3-HRE-EGFP/HRE-ODD-luc/tdtomato cells detected by bioluminescent imaging rapidly decreased after reoxygenation, whereas EGFP levels in these cells remained stable for several hours. After in vitro validation, PC3-HRE-EGFP/HRE-ODD-luc/tdtomato tumors were implanted subcutaneously and orthotopically in nude male mice and imaged in vivo and ex vivo using optical imaging in proof-of-principle studies to demonstrate differences in optical patterns between EGFP expression and bioluminescence. This novel "timer" imaging strategy of combining the short-lived ODD-luciferase and the long-lived EGFP can provide a time frame of HRE activation in PC3 prostate cancer cells and will be useful to understand the temporal changes in hypoxia and HIF activity during cancer progression and following treatments including HIF targeting strategies.
authors.affiliation: Division of Cancer Imaging Research, The Johns Hopkins University In Vivo Cellular and Molecular Imaging Center, The Russell H. Morgan Department of Radiology and Radiological Science, Baltimore, MD, USA.
authors.forename: Pierre
authors.initials: P
authors.lastname: Danhier
authors.affiliation: Division of Cancer Imaging Research, The Johns Hopkins University In Vivo Cellular and Molecular Imaging Center, The Russell H. Morgan Department of Radiology and Radiological Science, Baltimore, MD, USA.
authors.forename: Balaji
authors.initials: B
authors.lastname: Krishnamachary
authors.affiliation: Division of Cancer Imaging Research, The Johns Hopkins University In Vivo Cellular and Molecular Imaging Center, The Russell H. Morgan Department of Radiology and Radiological Science, Baltimore, MD, USA.
authors.forename: Santosh
authors.initials: S
authors.lastname: Bharti
authors.affiliation: Division of Cancer Imaging Research, The Johns Hopkins University In Vivo Cellular and Molecular Imaging Center, The Russell H. Morgan Department of Radiology and Radiological Science, Baltimore, MD, USA.
authors.forename: Samata
authors.initials: S
authors.lastname: Kakkad
authors.affiliation: Division of Cancer Imaging Research, The Johns Hopkins University In Vivo Cellular and Molecular Imaging Center, The Russell H. Morgan Department of Radiology and Radiological Science, Baltimore, MD, USA.
authors.forename: Yelena
authors.initials: Y
authors.lastname: Mironchik
authors.affiliation: Division of Cancer Imaging Research, The Johns Hopkins University In Vivo Cellular and Molecular Imaging Center, The Russell H. Morgan Department of Radiology and Radiological Science, Baltimore, MD, USA; Sidney Kimmel Comprehensive Cancer Center, The Johns Hopkins University School of Medicine, Baltimore, MD, USA. Electronic address: zaver@mri.jhu.edu.
authors.forename: Zaver M
authors.initials: ZM
authors.lastname: Bhujwalla
mesh_terms: D000818:Animals; D015153:Blotting, Western; D015687:Cell Hypoxia; D045744:Cell Line, Tumor; D003513:Cycloheximide / Q000494:pharmacology; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000860:Hypoxia; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000235:genetics / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008297:Male; D008819:Mice, Nude; D008856:Microscopy, Fluorescence; D010100:Oxygen / Q000378:metabolism / Q000494:pharmacology*; D011471:Prostatic Neoplasms / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020218:Response Elements / Q000235:genetics*; D059008:Time-Lapse Imaging / Q000379:methods; D014183:Transplantation, Heterologous; D000097573:Red Fluorescent Protein
chemical_list: D003513:Cycloheximide; D049452:Green Fluorescent Proteins; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D008156:Luciferases; D008164:Luminescent Proteins; D010100:Oxygen; D011500:Protein Synthesis Inhibitors; D000097573:Red Fluorescent Protein; C485184:enhanced green fluorescent protein