Comparison between the comet assay and pimonidazole binding for measuring tumour hypoxia

Pimonidazole is finding increasing use in histochemical analyses of hypoxia in tumours. Whether it can identify every hypoxic cell in a tumour, and wh ether the usual subjective criteria used to define 'positive' cells are opt imal, are less certain. Therefore, our aim was to develop an objective flow cytometry procedure for quantifying pimonidazole binding in tumours, and t o validate this method by using a more direct indicator of radiobiologic hy poxia, the comet assay. SCCVII rumours in C3H mice were analysed for pimoni dazole binding using flow cytometry and an iterative curve-fitting procedur e, and the results were compared to the comet assay for the same cell suspe nsions. On average, cells defined as anoxic by flow analysis (n = 43 tumour s) bound 10.8 +/- 0.95 times more antibody than aerobic cells. In samples c ontaining known mixtures of aerobic and anoxic cells, hypoxic fractions as low as 0.5% could easily be detected. To assess the flow cytometry assay un der a wider range of tumour oxygen contents, mice were injected with hydral azine to reduce tumour blood flow, or allowed to breathe various gas mixtur es during the 90 min exposure to pimonidazole. Hypoxic fraction estimated b y the pimonidazole binding method agreed well with the hypoxic fraction mea sured using the comet assay in SCCVII rumours (r2 = 0.87, slope = 0.98), wi th similar results in human U87 glioma cells and SiHa cervical carcinoma xe nografts. We therefore conclude that this objective analysis of pimonidazol e labelling by flow cytometry gives a convenient and accurate estimate of r adiobiological hypoxia. Preliminary analyses of biopsies from 3 patients gi ven 0.5 g m-2 pimonidazole also suggest the suitability of this approach fo r human tumours. (C) 2000 Cancer Research Campaign http://www.bjcancer.com.