Variable presence of hypoxia in M006 human glioma spheroids and in spheroids and xenografts of clonally derived sublines.

Recently we reported the variable presence of hypoxia adjacent to necrosis in human glioma lines grown as subcutaneous tumours in severe combined immunodeficient (SCID) mice. To assess the basis for this observation, we examined the pattern of oxygenation in M006 and M006XLo glioma spheroids. We found a wide range of binding of [3H]misonidazole to cells adjacent to the necrotic core, analogous to the patterns seen in xenografts, indicating substantial differences in the central oxygen tension of the spheroids. Clonal selection was used to isolate single cell-derived sublines of the M006XLo line. Some sublines gave spheroids that showed narrow distributions of [3H]misonidazole binding to the cells adjacent to necrosis, whereas other sublines showed a range of binding similar to that seen in spheroids of the parent line. After additional passages in monolayer culture, clonal sublines occasionally gave rise to spheroids in which the mean oxygen tension of cells adjacent to necrosis differed substantially from that of the initial spheroids. No relationship was evident between the thickness of the rim of viable cells and the presence or absence of central hypoxia, over a wide range of rim thickness. These results indicate that different oxygenation characteristics of glioma spheroids and tumour microregions are unlikely to arise from stable genetic variants coexisting in the parent line.

Anomalous patterns of nitroimidazole binding adjacent to necrosis in human glioma xenografts: possible role of decreased oxygen consumption.

In contrast to reports of extensive hypoxia in human gliomas in situ measured by pO2 histography, non-invasive methods of assessing glioma oxygenation, including nitroimidazole binding, have yielded surprisingly contradictory results. In order to investigate the relationship of necrosis, hypoxia, nitroreductase activity and cellular respiration in human gliomas, subcutaneous models using the human glioma cell lines M059K, M006 and M010b were developed in the murine SCID host. Intracranial growth of the M006 line was achieved in nude rats. The nitroreductive capacity of glioma cell lines was assessed and found to be similar to transplanted tumours previously reported in the literature. This suggests that if substantial numbers of viable hypoxic cells were present in situ in gliomas, then nitroimidazole-binding techniques should be capable of identifying them. Inter-tumour variability in the amount of necrosis was seen. M006 xenografts growing in subcutaneous and intracranial sites revealed extensive necrotic regions histologically, some of which were surrounded by cells labelled heavily for [3H]misonidazole, while other areas were lightly labelled. Similar binding patterns were seen for subcutaneous M059K tumours, while subcutaneous M010b tumours display necroses of which almost all were surrounded by heavily labelled cells. The oxygen consumption rates of tumour cell lines grown in vivo, in which venous pO2 concentrations are of the order of 2-5%, were two to sevenfold less than those of the same lines grown as monolayers in vitro under oxygen concentrations of 18%. We postulate that glioma cell lines behave as 'oxygen conformers', in that their rate of oxygen consumption appears to vary with the availability of oxygen. Together with the misonidazole-binding data, the results in this glioma tumour model are consistent with coordinate inhibition or down-regulation of respiration under moderate hypoxia.