Rate of Death of Hypoxic Cells in Multicell Spheroids

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Abstract
The rate of death of hypoxic cells was measured in multicell spheroids, which are considered to model in vitro the microenvironments surrounding such cells within solid tumors. Two types of experiments were performed: (i) All of the cells in spheroids were made hypoxic (less than 100 ppm O2) and the total number of viable cells was determined by clonogenic assay at later times up to 7 days. The rate of death of cells appeared biphasic. At least 15% of the cells died within the first 6 hr. The rate of development of histological changes in the spheroids suggested that the innermost cells were most sensitive. The more peripheral layers of cells died much more slowly so that there was still about 5% survival and a significant number of histologically normal cells at 6 days. Changes in the glucose concentration in the medium (from one-third to three times normal) during exposure to hypoxia had little effect on the survival time of these outer cells. (ii) The cells in the inner half of spheroids grown in 20% O2 were made hypoxic by equilibrating the growth medium with 5% O2, and the number of resistant hypoxic cells at different times later was determined after a radiation dose of 3500 rad. The number of surviving clonogenic cells after this dose of radiation decreased with a half-time for cell death of 3 hr. These results indicate that the rate of death of hypoxic cells in the central regions of spheroids is much more rapid than has been reported for monolayer cultures although the resistance of the outer cells is similar to or greater than monolayers. Since spheroids may model the necrosis and other microenvironments near chronically hypoxic cells in tumors, the relatively rapid rate of death of hypoxic cells demonstrated here must be considered in evaluating their contribution to the size of the radiation-resistant hypoxic fraction and possible mechanisms which might contribute to the phenomenon of reoxygenation.