title: In vivo identification and specificity assessment of mRNA markers of hypoxia in human and mouse tumors.
abstract: BACKGROUND
  Tumor hypoxia is linked to poor prognosis, but identification and quantification of tissue hypoxia remains a challenge. The hypoxia-specificity of HIF-1α target genes in vivo has been questioned due to the confounding influence of other microenvironmental abnormalities known to affect gene expression (e.g., low pH). Here we describe a new technique that by exploiting intratumoral oxygenation heterogeneity allows us to identify and objectively rank the most robust mRNA hypoxia biomarkers.
  
  
  METHODS
  Mice carrying human (FaDudd) or murine (SCCVII) tumors were injected with the PET hypoxia tracer FAZA. Four hours post-injection tumors were removed, frozen, and crushed into milligram-sized fragments, which were transferred individually to pre-weighed tubes containing RNAlater and then weighed. For each fragment radioactivity per tissue mass and expression patterns of selected mRNA biomarkers were analyzed and compared.
  
  
  RESULTS
  In both tumour models, fragmentation into pieces weighing 10 to 60 mg resulted in tissue fragments with highly variable relative content of hypoxic cells as evidenced by an up to 13-fold variation in FAZA radioactivity per mass of tissue. Linear regression analysis comparing FAZA retention with patterns of gene expression in individual tissue fragments revealed that CA9, GLUT1 and LOX mRNA levels were equally and strongly correlated to hypoxic extent in FaDudd. The same link between hypoxia and gene expression profile was observed for CA9 and GLUT1, but not LOX, in SCCVII tumors. Apparent in vivo hypoxia-specificity for other putative molecular markers of tissue hypoxia was considerably weaker.
  
  
  CONCLUSIONS
  The portrayed technique allows multiple pairwise measurements of mRNA transcript levels and extent of hypoxia in individual tumors at a smallest possible volumetric scale which (by limiting averaging effects inherent to whole-tumor analysis) strengthen the conclusiveness on true hypoxia-specificity of candidate genes while limiting the required number of tumors. Among tested genes, our study identified CA9, GLUT1 and possibly LOX as highly specific biomarkers of tumor hypoxia in vivo.
authors.affiliation: Department of Experimental Clinical Oncology, Aarhus University Hospital, Aarhus, Denmark. morten@oncology.dk
authors.forename: Morten
authors.initials: M
authors.lastname: Busk
authors.forename: Kasper
authors.initials: K
authors.lastname: Toustrup
authors.forename: Brita S
authors.initials: BS
authors.lastname: Sørensen
authors.forename: Jan
authors.initials: J
authors.lastname: Alsner
authors.forename: Michael R
authors.initials: MR
authors.lastname: Horsman
authors.forename: Steen
authors.initials: S
authors.lastname: Jakobsen
authors.forename: Jens
authors.initials: J
authors.lastname: Overgaard
mesh_terms: D000818:Animals; D014408:Biomarkers, Tumor / Q000235:genetics*; D002256:Carbonic Anhydrases / Q000235:genetics; D045744:Cell Line, Tumor; D005260:Female; D020869:Gene Expression Profiling; D015972:Gene Expression Regulation, Neoplastic; D051272:Glucose Transporter Type 1 / Q000235:genetics; D006801:Humans; D000860:Hypoxia / Q000235:genetics*; D051379:Mice; D008809:Mice, Inbred C3H; D008815:Mice, Inbred Strains; D008819:Mice, Nude; D009374:Neoplasms, Experimental / Q000000981:diagnostic imaging / Q000235:genetics* / Q000473:pathology; D009593:Nitroimidazoles; D049268:Positron-Emission Tomography / Q000379:methods; D008249:Protein-Lysine 6-Oxidase / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014183:Transplantation, Heterologous
chemical_list: D014408:Biomarkers, Tumor; D051272:Glucose Transporter Type 1; D009593:Nitroimidazoles; D012333:RNA, Messenger; C498052:fluoroazomycin arabinoside; D008249:Protein-Lysine 6-Oxidase; D002256:Carbonic Anhydrases