DETECTION OF HYPOXIC CELLS IN A MURINE TUMOR WITH THE USE OF THE COMET ASSAY

Background: Hypoxic cells within solid tumors are likely to limit tumor curability by radiation therapy and some chemotherapeutic agents. Purpose: To quantify a hypoxic fraction in solid tumors, we developed a method which measures radiation-induced DNA single-strand breaks in individual tumor cells and makes use of the fact that three times more strand breaks are produced in aerobic than in hypoxic cells. Methods: Immediately after irradiation with doses of 4-20 Gy, SCCVII squamous cell carcinomas growing in C3H mice were removed and cooled, and a single-cell suspension was prepared. These cells were then embedded in agarose, lysed in an alkaline solution, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye. The amount and migration distance of damaged DNA from individual cells were scored by using a fluorescence image processing system, where differentially radiosensitive aerobic and hypoxic cell populations resulted in bimodal damage distributions. Curve-fitting routines provided quantitative estimates of the fraction of hypoxic cells. Results: After the mice were exposed to 10-20 Gy, the SCCVII tumors (450-600 mg) were shown to have a hypoxic fraction of 18.5% +/- 10.6% (mean +/- SD for 11 tumors), which compares well with the value of 11.6% observed using the paired survival curve method. Conclusions: Our results indicate that this method, which requires only a few thousand cells, is a rapid and sensitive way to detect hypoxic cells in solid animal tumors. Implications: Estimating hypoxia in accessible human tumors undergoing radiotherapy may be possible if the sensitivity of the method can be improved to allow detection of hypoxic cells after a dose of 2 Gy.