Detection of Hypoxic cells in a Murine Tumour With the Use of the Comet Assay

• Published in: JNCI : Journal of the National Cancer Institute 1992-05, Vol.84 (9), p.707-711
• Main Authors: Olive, Peggy L., Durand, Ralph E.
• Format: Article
• Language: English
• Subjects: Animals; Carcinoma, Squamous Cell - metabolism; Carcinoma, Squamous Cell - pathology; DNA Damage; DNA, Neoplasm - chemistry; DNA, Neoplasm - genetics; DNA, Neoplasm - radiation effects; DNA, Single-Stranded; Hydrogen-Ion Concentration; Hypoxia; Male; Mice; Mice, Inbred C3H; Neoplasms, Experimental - metabolism; Neoplasms, Experimental - pathology; X-Rays
• ISSN: 0027-8874; 1460-2105
• DOI: 10.1093/jnci/84.9.707

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 insolid tumors, we developed a method which measures radiationinduced DNA singlestrand 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 singlecell suspension was prepared. These cells were then embedded in agarose, lysed in an alkaline solution, subjected to electrophoresis, and stained with a fluorescent DNAbindirg 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. Curvefitting 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. [J NatI Cancer Inst 84:707–711, 1992]