Detection of hypoxic cells in a C3H mouse mammary carcinoma using the comet assay

Abstract

Summary The comet assay was used to estimate radiobiological hypoxic fraction across a full range of tumour oxygenations in C3H mammary tumours implanted into the feet of female CDF1 mice. Tumours were either clamped before irradiation or mice were allowed to breath air, 100% oxygen, carbogen or carbon monoxide for 5-35 min before and during exposure to 15 Gy. For the alkaline comet assay, tumours were excised after irradiation and individual tumour cells were analysed for DNA single-strand breaks. Hypoxic cells were defined as those cells with approximately three times fewer single-strand breaks than aerobic cells. Radiobiological hypoxic fraction was calculated by fitting DNA damage histograms to two normal distributions, representing the response of the aerobic and hypoxic populations. The percentage of hypoxic cells estimated using the comet assay was then compared with hypoxic fraction measured using a clamped tumour control assay.Carbogen and oxygen breathing reduced the normal hypoxic fraction from 14% to 2-3% in this tumour, whereas 75-660 p.p.m. carbon monoxide progressively increased the hypoxic fraction from 18% to 82%. The slope of the line comparing the two methods was 1.23 with 95% confidence limits of 1.12-1.33 (r2 = 0.994). In the SCCVII squamous cell carcinoma growing subcutaneously in C3H mice, a similar correlation was observed between hypoxic fraction measured using the comet assay and hypoxic fraction measured in the same tumour cells using the paired survival curve assay (slope = 1.20 with 95% confidence limits of 1.03-1.37). These results confirm the ability of the comet assay to provide an accurate estimate of radiobiological hypoxic fraction over a wide range of tumour oxygenations and between two tumour types.

Vascular endothelial growth factor expression is independent of hypoxia in human malignant glioma spheroids and tumours

“…A similar correlative study would be possible in patients, but would require in situ hypoxia marker labelling prior to biopsy (Urtasun et al, 1986). The hypoxic fraction values obtained with the M006 xenografts for the comet assay are close to those of the C 3 H murine mammary tumour under air-breathing conditions (Olive et al, 1997) and are consistent with our 3 H-misonidazole data suggesting that most but not all M006 xenografts contain substantial radiobiological hypoxia.The oxygen dependence of misonidazole binding is slightly different from the hypoxic marker EF5 . EF5 shows less inhibition of binding at moderate oxygen tensions, whereas misonidazole binding is inhibited by oxygen in a somewhat more complex fashion down to levels of hypoxia which would be considered significant from the point of view of induction of radiation resistance (Chapman, 1984).…”

“…In this report we have examined the oxygen dependence of VEGF mRNA expression in the M006X line undergoing mild hypoxic stress (6% oxygen (O 2 )), and we have also examined VEGF expression in spheroids of both M006X and M006XLo lines and in xenograft tumours of the M059K, M006 and M010b lines. We also sought to independently validate the presence of radiobiologically hypoxic cells in situ in M006 tumours using the comet assay of in-situ DNA damage (Olive et al, 1997(Olive et al, , 1998.Finally, we have performed preliminary experiments to assess the presence of tumour-cell VEGF receptor (Vaisman et al, 1990) expression which has previously been seen in melanoma and ovarian carcinoma lines (Boocock et al, 1995;Liu et al, 1995), suggesting autocrine/paracrine growth stimulation. …”

“…In this report we have examined the oxygen dependence of VEGF mRNA expression in the M006X line undergoing mild hypoxic stress (6% oxygen (O 2 )), and we have also examined VEGF expression in spheroids of both M006X and M006XLo lines and in xenograft tumours of the M059K, M006 and M010b lines. We also sought to independently validate the presence of radiobiologically hypoxic cells in situ in M006 tumours using the comet assay of in-situ DNA damage (Olive et al, 1997(Olive et al, , 1998.…”

“…DNA was stained with propidium iodide and individual cell 'comets' were viewed using a Zeiss epifluorescence microscope attached to an intensified solid state charge device camera and image analysis system (Olive et al, 1997). The hypoxic fraction was estimated by iterative fitting of histograms of 'tail moment' (product of the percentage of DNA in the comet tail multiplied by the distance between the means of the head and tail distributions) with two normal distributions, representing the aerobic and hypoxic populations as described previously (Olive et al, 1997). …”

The Comet Assay in Clinical Practice

“…Unlike other techniques, the comet assay is specific for whole nucleated cells and is thus largely unaffected by necrosis, and it measures radiobiologic hypoxia during irradiation. In murine tumours, the proportion of cells that are hypoxic by this assay predicts for tumour cure by radiation (15). However, there is an important limitation in using the comet assay for detection of hypoxic cells.…”