DETECTION OF HYPOXIC FRACTIONS IN MURINE TUMORS BY COMET ASSAY - COMPARISON WITH OTHER TECHNIQUES

The alkaline comet assay was used to detect the hypoxic fractions of murine tumors, A total of four tumor types were tested using needle aspiration biopsies taken immediately after a radiation dose of 15 Gy, Initial studies confirmed that the normalized tail moment, a parameter reflecting single-strand DNA breaks induced by the radiation, was linearly related to radiation dose. Further, it was shown that for a mixed population (1:1) of cells irradiated under air-breathing or hypoxic conditions, the histogram of normal tail moment values obtained from analyzing 400 cells in the population had a double peak which, when fitted with two Gaussian distributions, gave a good estimate of the proportion of the two subpopulations. For the four tumor types, the means of the calculated hypoxic fractions from four or five individual tumors were 0.15 +/- 0.04 for B16F1, 0.08 +/- 0.04 for KHT-LP1, 0.17 +/- 0.04 for RIF-1 and 0.14 +/- 0.01 for SCCVII. Analysis of variance showed that the hypoxic fraction in KHT-LP1 tumors is significantly lower than those of the other three tumors (P = 0.026) but that there is no significant difference in hypoxic fraction between B16F1, RIF-1 and SCCVII tumors (P = 0.574), Results from multiple samples taken from each of five RIF-I tumors showed that the intertumor heterogeneity of hypoxic fractions was greater than that within the same tumor. The mean hypoxic fraction obtained using the comet assay for the four tumor types was compared with the hypoxic fraction determined by the clonogenic assay, or median pO(2) values, or [H-3]misonidazole binding in the same tumor types. With the tumors studied, the values of hypoxic fraction obtained with the comet assay were two to four times lower than those measured by the paired survival method. Preliminary results obtained with a dose of 5 Gy were consistent with those obtained using 15 Gy. These results suggest the further development of the comet assay for clinical studies. (C) 1995 by Radiation Research Society