Binding of 14C-misonidazole to hypoxic cells in V79 spheroids.

A J Franko,J D Chapman

doi:10.1038/bjc.1982.110

Abstract

The metabolism-induced binding of 14C-labelled misonidazole (MISO) to hypoxic V79 cells in multicell spheroids has been quantitated using autoradiography. Hypoxia was shown to be the major determinant of the rate of binding. Maximally hypoxic cells bound MISO several times more rapidly than necrotic material in the centre of the spheroids, and up to 50 times more rapidly than well oxygenated cells. The rate of binding to chronically hypoxic cells at the edge of the necrotic centre was 20 times less than to similar cells in other spheroids made maximally hypoxic with N2. This difference is consistent with the greater radio-sensitivity of the chronically hypoxic cells, which is a consequence of their intermediate level of oxygenation. The results indicated that the ability to bind MISO might have considerable potential as a marker for hypoxic cells in tumours. However, some binding patterns cannot be explained by the simplest model of O2 diffusion. It may be necessary to invoke more complex models of O2 diffusion or metabolic gradients within the spheroid which affect the rate of binding.

Highlights

Whole medium was outer 100,um of the necrotic centre, and prepared with 50DuM labelled MISO and squares with equivalent positions in these degassed for 1.5 h at 37°C
[02] in the regions of the viable rim, while reduced labelling is seen over the necrotic centre and the outer half of the viable rim
FIG. 3.-Grain densities over ARGs of central sections of spheroids grown in air and incubated with 14C-MISO in air or 50% 02The sections were stained with Feulgen before the application of the emulsion

Summary

Thus it was not possible to pool the data

171b spheroids have been described (Suther- squares which were the same distance from land & Durand, 1976), as have the techniques the spheroid surface, as beyond 150 ,um for altering [02] in the medium The the spheroids were averaged, each rim was purity was checked by thin-layer chromato- divided into 3 regions, the outer 100 ,um, the graphy (silica gel G (Canlab), ethyl acetate) innermost 100 ,um of the viable rim and the and found to be >95%. Whole medium was outer 100 ,um of the necrotic centre, and prepared with 50DuM labelled MISO and squares with equivalent positions in these degassed for 1.5 h at 37°C. This meant that in added in a small volume of medium and spheroids with viable rims > 200 ,um, a few incubated for 3 h with continuous gas flow. Squares at the centres of the viable rims. The [02] in the effluent gas was measured were ignored, while in spheroids with viable with a modified Clarke electrode During the final 2 h of incubation for the flask which received 97% N2-3% CO2

RESULTS

DISCUSSION

The gradual rise in the binding rate