Different sensitivity to cytotoxic agents of internal and external cells of spheroids composed of thioguanine-resistant and sensitive cells.

When thioguanine-resistant V79 cells are introduced into spinner flasks containing V79 multicell spheroids, the mutant cells attach to the surface of the spheroids. The composite spheroids thus formed consist of external, thioguanine-resistant (TGr) and internal, thioguanine-sensitive cells. Cell sorting with a Becton Dickinson FACS II was used to determine the relative position of TGr cells and sensitivity to fluorescent drugs. After 2-4 days, the TGr cells are found internally as well as externally. The initial percentage of TGr cells varies from 1 to 50%, depending on the size of the single-cell inoculum, size of spheroids and frequency of addition of cells during a 24th period. Differential effects of drugs or radiation on external (cycling, oxic) vs internal (non-cycling, hypoxic) cells of composite spheroids can be assayed by simply plating cells trypsinized from spheroids into standard medium or medium with 2 mu g/ml 6-thioguanine, which allows growth of only TGr cells. For example, Adriamycin, which penetrates poorly into spheroids, is preferentially toxic to external cells, and causes a decrease in the percentage of TGr survivors. Irradiation of composite spheroids also causes preferential killing of external (oxic) TGr cells. However, AF-2, a nitrofuran which preferentially kills hypoxic cells, caused an increase in the 0/0 of TGr cells. This technique should prove useful in the evaluation of the preferential cytotoxicity of chemotherapeutic agents, alone or in combination.