Advantage of reduced oxygen tension in growth of human melanomas in semi-solid cultures: Quantitative analysis

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Abstract
A systematic study was undertaken to compare the growth characteristics of human melanomas in liquid monolayer cultures at ambient oxygen tension, and in semi-solid cultures at ambient or reduced oxygen tension. Physically dispersed single cell suspensions from 200 freshly-excised melanomas (66 primary, 134 secondary) from 169 patients were cultured in monolayers, or plated in semi-solid cultures maintained either in 5% CO2 in room air (20% O2) or in 5% CO2, 5% O2 and 90% N2, to assay tumour colony-forming units (T-CFU). Aliquots were taken at each passage of the monolayer cultures for T-CFU assay in semi-solid culture at ambient and reduced O2 concentrations. Of 200 melanomas tested, 153 (77%) grew in monolayer culture, 94 (47%) in semi-solid culture at 5% O2, and only 48 (24%) in semi-solid culture at 20% O2. The mean number (+/-s.e.) of colonies in the 94 tumours which grew in semi-solid culture at 5% O2 (29 +/- 4 per 5 x 10(5) cells plated) was significantly greater than the mean in the same tumours in semi-solid culture at 20% O2 (11 +/- 2 per 5 x 10(5) cells). Furthermore, hypoxic colonies showed a morphologically different growth pattern. There was a significant correlation (r = 0.749, P less than 0.001) between the number of colonies growing at 5% O2 and the number at 20% O2; hypoxia appeared to act both by recruiting additional T-CFU and by increasing the proliferative activity of those already present. Short-term monolayer cultured cell lines showing evidence of persistent tumour cell characteristics were successfully established from 74 tumours, and the proportions of T-CFU assayed at each passage. In 63% of cultures the proportion of T-CFU increased initially and then declined, while in the remainder it declined progressively throughout. Although monolayer cultures were successfully maintained for up to 15 passages, T-CFU became undetectable by the eighth passage and remained so thereafter.