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

Abstract

Summary 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 pattem of oxygenation in M006 and M006XLo glioma spheroids. We found a wide range of binding of [3H] aExduding two spheroids with a mixture of high and low grains ( Figure 3B).completely at each feeding. The number of spheroids w-as reduced at each feeding to stabilize the rate of consumption of nutrients. The first spheroids reached diameters of 0.8-1.2 mm. at x-hich they w-ere eliaible for experiments. w ithin 4-5 eeks. M006XLo sublineIn an effort to denrve a subline that w-as adapted to grow-th at lowambient oxy gen. the M006XLo subline x as established from small M006 spheroids that had been exposed continuously to 0.6%7 oxygen for 13 days. beainning 1 week after initiation in air. The spheroids were disaggregated wxith 0.25%c trypsin. and the cells w ere groxx n in monolayer culture for four passages (5 w eeks). then injected subcutaneously in SCID mice. A tumour x-as disaggregated to yield a new line designated M006XLo. w hich w as propagated in monolayer culture. Cells from the third passage were stored in liquid nitrogen. and each line x-as discarded after 3 months in culture and replaced from frozen stock.Clonally derived sublines (Franko et al. 1987). The desired oxygen level was obtained by a series of partial evacuations. each of which was followed by refilling of the chambers with 95% nitroaenlr5% carbon dioxide. Metabolic actixation of misonidazole during the degrassing procedure w-as minimized by precooling the medium and the chambers to 0°C and maintainingr this temperature during, the 30-to 40-min degassing process. The incubation period was 3 h for the spinner flask. and an additional 30 min for the aluminum chambers. w-hich is the time required for the medium in the dishes to return to 37' in the cabinet used for incubation. The oxygen tension in each chamber was measured at the end of the incubation period. as described previously (Franko et al. 1987). AutoradiographyThe spheroids were fixed in formalin and embedded in wax. and 4-gm serial sections w-ere taken throughout the spheroids. Slides w-ith sections near the centres of the majority of the spheroids were

Evidence that involucrin, a marker for differentiation, is oxygen regulated in human squamous cell carcinomas

“…Conversely, shallow gradients would increase the distance over which divergent K m s are traversed allowing for involucrin induction in advance of pimonidazole binding as appears to be the case in Figure 4A and B. Variations in the slope of oxygen gradients have been proposed to account for the lack of nitroimidazole binding around areas of necrosis in a subset of glioma xenografts (Parliament et al, 1997;Franko et al, 1998;Turcotte et al, 2002), but it remains to be seen whether this occurs among subsets of squamous cell carcinomas.…”

Variation in mitochondrial function in hypoxia-sensitive and hypoxia-tolerant human glioma cells

“…In vivo measurement of hypoxia in a variety of human tumours by SPECT imaging of iodoazomycin arabinoside showed an unusual pattern of heterogeneity in GBM tumours specifically (Urtasun et al, 1996). Using cell lines derived from human GBM biopsy material and human GBM xenografts, we have shown that regional heterogeneity of oxygen consumption exists in GBMs and that modulation of cellular respiration is an important component of the hypoxia defence mechanisms utilized by GBMs (Parliament et al, 1997;Franko et al, 1998;AllalunisTurner et al, 1999). However, individual tumours and cell lines derived from them vary considerably in their ability to modulate cellular respiration, with some being more or less proficient than others.…”

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

“…Lack of correlation between hypoxia marker binding and VEGF expression in xenograft tumours is somewhat complex in that intratumoural drug distribution may be suboptimal because of the aberrant tumoural vasculature. However, glioma spheroids (Franko et al, 1998), in which nitroimidazole drug penetration is not impaired, also reveal substantial variation in hypoxia marker binding in the viable cells adjacent to necrosis, a pattern similar to that seen in xenograft tumours.From the Northern analysis it is apparent that severe, pathophysiological levels of hypoxia are not necessary for the up-regulation of VEGF messenger RNA and protein expression; indeed substantial upregulation is evident at modest levels of O 2 , which would be similar to the physiological hypoxic conditions noted in many mammalian organ systems. Our results are in agreement with those of Leith and Michelson (1995) who showed that modest hypoxia (i.e.…”

“…It is becoming apparent that primary brain tumours of astrocytic lineage, especially glioblastoma multiforme, exhibit hypoxia-induced neoangiogenesis in vivo through induction of the endothelial cell-specific mitogen, vascular endothelial growth factor (VEGF) (Shweiki et al, 1992;Plate et al, 1992Plate et al, , 1994. A strong correlation was found between intense VEGF expression and severe hypoxia in situ in HT29 and EMT-6 spheroids (Waleh et al, 1995).Recently we have observed variable presence of hypoxia adjacent to necrosis in some xenografted glioma lines and spheroids (Parliament et al, 1997;Franko et al, 1998). The results were interpreted in terms of regional variations in oxygen consumption, based on the concept of modulation of oxygen consumption in regions of oxygen and nutrient deprivation in solid tumours (Hochachka et al, 1996).…”

“…One tumour was disaggregated to yield a new line designated M006XLo, which was propagated in monolayer culture (Parliament et al, 1997). Details of the initiation and growth of spheroids were as described in Franko et al (1998). M006XLo, M006X, M059K and M010b tumours were raised s.c. in the flanks of NOD/SCID mice.…”