Comparison between the comet assay and the oxygen microelectrode for measurement of tumor hypoxia.

BACKGROUND AND PURPOSE: Hypoxic cells are present in some solid tumours and are known to limit radiocurability. To compare two measures of tumour hypoxia, 25 patients with locally advanced disease and accessible tumours or metastatic nodes were examined using an oxygen microelectrode and the alkaline comet assay. MEASUREMENTS AND METHODS: For the comet assay, fine needle aspirate biopsies were taken immediately following a dose of 5-10 Gy. Single cells were examined for radiation-induced DNA strand breaks, and the percentage of radio-resistant hypoxic cells within the population was calculated from DNA damage histograms. For oxygen tension (pO2) measurements, multiple tracks were made using an Eppendorf oxygen microelectrode. The possibility that application of the first method might influence hypoxic fraction measurement by the second method was examined in a more controlled system by creating four tracks in murine SCC-VII tumours using an oxygen electrode, and measuring hypoxic fraction at subsequent times. RESULTS: For 28 tumours from 25 patients, hypoxic fraction measured by comet assay correlated with the percentage of PO2 values < 5 mmHg (r2 = 0.46, P < 0.001). The mean comet hypoxic fraction was 0.36 for five tumours with a median PO2 < 10 mmHg. For the remaining 23 tumours with a median PO2 > 10 mmHg, the mean hypoxic fraction was 0.09. Advancement of an oxygen electrode through SCCVII tumours had no significant effect on hypoxic fraction measured 5 min to 24 h later using the alkaline comet assay. CONCLUSIONS: Tumours defined as hypoxic based on a median pO2 < 10 mmHg appear to contain more than 20% radio-biologically hypoxic cells as estimated by the comet assay. In an animal tumour model, puncture of the tumour with an oxygen electrode did not influence hypoxic fraction measured using the comet assay, in agreement with the clinical data that the order in which the two methods were performed was not important.

Increase in the fraction of necrotic, not apoptotic, cells in SiHa xenograft tumours shortly after irradiation.

BACKGROUND AND PURPOSE: Approximately 18% of the cells recovered by rapid mechanical dissociation of SiHa xenograft tumours contain large numbers of DNA strand breaks. The number of damaged cells increases to 30-40% 4-6 h after exposure to 5 or 15 Gy, returning to normal levels by 12 h. This observation is reminiscent of the rate of production of apoptotic cells in other murine and human xenograft tumours. The nature of this damage, rate of development and relation to cell proliferation rate were therefore examined in detail. MATERIALS AND METHODS: SiHa human cervical carcinoma cells were grown as xenograft tumours in SCID mice. Single-cell suspensions were prepared as a function of time after irradiation of the mouse and examined for DNA damage using the alkaline comet assay. Cell cycle progression was measured by flow cytometry evaluation of anti-bromodeoxyuridine-labelled tumour cells. RESULTS: Significant numbers of apoptotic cells could not be detected in irradiated SiHa tumours using an end-labelling assay, electron microscopy, or histological examination of thin sections. Instead, xenograft cells exhibiting extensive DNA damage in the comet assay were predominantly necrotic cells. The increase in the proportion of heavily damaged cells 4-6 h after irradiation could be the result of an interplay between several factors including loss of viable cells and change in production or loss of necrotic cells. Analysis of the progression of BrdUrd-labelled cells confirmed that while 35% of cells from untreated SiHa tumours had divided and entered G1 phase by 6 h after BrdUrd injection, none of the labelled cells from tumours exposed to 5 or 15 Gy had progressed to G1. CONCLUSIONS: The increase in the percentage of SiHa tumour cells with extensive DNA damage 4-6 h after irradiation is attributable to necrosis, not apoptosis. Cell cycle progression and cell loss are likely to influence the kinetics of appearance of both apoptotic and necrotic cells in irradiated tumours.

Use of the comet assay to identify cells sensitive to tirapazamine in multicell spheroids and tumors in mice.

Tirapazamine, a bioreductive drug preferentially toxic to hypoxic cells, produces significant numbers of DNA single-strand breaks that can be detected using the alkaline comet assay. Our goal was to determine whether single-strand breaks measured using this assay could act as a surrogate end point for cell killing in multicell spheroids and solid tumors in mice. In spheroids composed of Chinese hamster V79 cells, WiDr human colon carcinoma cells, or SiHa human cervical carcinoma cells, histograms of tail moments (indicators of DNA damage in the comet assay) could be used to identify the percentage of cells that sustained sufficient DNA damage to cause cell death after treatment with tirapazamine. The proportion of comets with tail moments

Hypoxic fractions measured in murine tumors and normal tissues using the comet assay.

PURPOSE: To apply the alkaline comet assay to the detection of radiobiologically hypoxic cells in solid tumors and normal tissues of mice, and to examine the influence of strand break repair on the oxygen enhancement ratio measured using the alkaline comet assay. METHODS AND MATERIALS: In previous studies, we found that hypoxic fraction in squamous cell carcinomas growing in C3H mice could be reliably and easily measured using the alkaline comet assay. The comet assay applies fluorescence microscopy and image analysis to examine patterns of migration of deoxyribonucleic acid from individual cells embedded in agarose and exposed to an electric field. This method has sufficient resolution to detect subpopulations of hypoxic cells which show about 3 x fewer strand breaks than aerobic cells after irradiation. RESULTS: Fast rejoining kinetics in vitro are comparable to those measured in vivo, and rejoining of strand breaks in hypoxic tumor cells occurs at a similar rate as rejoining in aerobic cells. Little residual damage was detectable using the comet assay in tumors 4-24 h following 15 Gy, allowing repeat measurements to be performed. Bone marrow and testis, but not liver, spleen, or jejunum contained a small fraction of hypoxic cells when mice breathed 10% oxygen during irradiation. CONCLUSION: The comet assay confirms that some normal tissues may border on hypoxia. Rejoining of strand breaks occurs rapidly in both oxic and hypoxic cells so that the oxygen enhancement ratio remains relatively constant with time after irradiation. Interestingly, a smaller oxygen enhancement ratio was observed in tumors than was expected, probably as a result of the presence of acutely hypoxic cells.