Purpose: Development of a double hypoxic cell marker assay, using the bioreductive nitroimidazole derivatives CCI-103F and pimonidazole, to study changes in tumor hypoxia after treatments that modify tumor oxygenation.
Methods and materials: Both hypoxic markers were visualized by immunohistochemical techniques to detect changes in hypoxic fraction induced by carbogen breathing (95% O(2) and 5% CO(2)) or hydralazine injection. The protocol was tested in a human laryngeal squamous cell carcinoma xenograft line. Quantitative measurements were derived from consecutive tissue sections that were analyzed by a semiautomatic image analysis system. Qualitative analysis was obtained by double staining of the two hypoxic markers on the same tissue section.
Results: A significant correlation between the hypoxic fractions for the two markers, CCI-103F and pimonidazole, was found in air breathing animals. After carbogen breathing, the hypoxic fraction decreased significantly from 0.07 to 0.03, and after hydralazine treatment, the hypoxic fraction increased significantly. Reduction of hypoxia after carbogen breathing was most pronounced close to well-perfused tumor regions.
Conclusions: With this method, employing two consecutively injected bioreductive markers, changes in tumor hypoxia can be studied. A significant reduction in hypoxia after carbogen breathing and a significant increase in hypoxia after hydralazine administration was demonstrated.