Immunohistochemical detection of changes in tumor hypoxia.

Abstract

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. 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. 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. HIF1alpha can be compared with either CAIX or a previously administered nitroimidazole to provide an estimate of reoxygenation.