Immunohistochemical detection of changes in tumor hypoxia.
OBJECTIVE
Although hypoxia is a known prognostic factor, its effect will be modified by the rate of reoxygenation and the extent to which the cells are acutely hypoxic. We tested the ability of exogenous and endogenous markers to detect reoxygenation in a xenograft model. Our technique might be applicable to stored patient samples.


METHODS
The human colorectal carcinoma line, HT29, was grown in nude mice. Changes in tumor hypoxia were examined by injection of pimonidazole, followed 24 hours later by EF5. Cryosections were stained for these markers and for carbonic anhydrase IX (CAIX) and hypoxia-inducible factor 1alpha (HIF1alpha). Tumor hypoxia was artificially manipulated by carbogen exposure.


RESULTS
In unstressed tumors, all four markers showed very similar spatial distributions. After carbogen treatment, pimonidazole and EF5 could detect decreased hypoxia. HIF1alpha staining was also decreased relative to CAIX, although the effect was less pronounced than for EF5. Control tumors displayed small regions that had undergone spontaneous changes in tumor hypoxia, as judged by pimonidazole relative to EF5; most of these changes were reflected by CAIX and HIF1alpha.


CONCLUSIONS
HIF1alpha can be compared with either CAIX or a previously administered nitroimidazole to provide an estimate of reoxygenation.
Russell
James
J
Department of Medical Physics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA. russellj@mskcc.org
Carlin
Sean
S
Burke
Sean A
SA
Wen
Bixiu
B
Yang
Kwang Mo
KM
Ling
C Clifton
CC
D000818:Animals; D000951:Antigens, Neoplasm / Q000378:metabolism*; D014408:Biomarkers, Tumor / Q000378:metabolism; D002245:Carbon Dioxide / Q000494:pharmacology; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000378:metabolism*; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology*; D017341:Etanidazole / Q000031:analogs & derivatives* / Q000378:metabolism; D005260:Female; D019073:HT29 Cells; D006801:Humans; D006845:Hydrocarbons, Fluorinated / Q000378:metabolism*; D051795:Hypoxia-Inducible Factor 1, alpha Subunit / Q000378:metabolism*; D007150:Immunohistochemistry; D051379:Mice; D008819:Mice, Nude; D009593:Nitroimidazoles / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism* / Q000494:pharmacology; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D005466:Fluorocarbons
D000951:Antigens, Neoplasm; D014408:Biomarkers, Tumor; D002245:Carbon Dioxide; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases; D017341:Etanidazole; D006845:Hydrocarbons, Fluorinated; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D009593:Nitroimidazoles; D010100:Oxygen; D011838:Radiation-Sensitizing Agents; C096011:2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-pentafluoropropyl)acetamide; D005466:Fluorocarbons; C033815:pimonidazole; C011700:carbogen; C089226:CA9 protein, human