Cytotoxicity of Hypoxic Cell Sensitizers in Multicell Spheroids

Multicell tumor spheroids of EMT6/Ro cells were used to evaluate the cytotoxicity of several hypoxic cell sensitizers. The number of clonogenic cells per spheroid was determined after different exposure periods and concentrations of misonidazole, Ro-05-9963, and SR 2508 which have similar electron affinities. The kinetics of cytotoxicity were similar for each drug and the clonogenic fraction was reduced by about 0.5 to a plateau level after 24 hours at 3.0 mM for spheroids grown in 20% O2. Extended exposure periods caused additional cytotoxicity for both Ro-05-9963 and SR 2508. Lower concentrations (0.5 mM) of these sensitizers were not cytotoxic even in spheroids grown in low concentrations of oxygen (2.5% O2) to increase the hypoxic fraction. However, at cytotoxic concentrations (3.0 mM) spheroids grown in this low oxygen concentration exhibited almost twice as much cytotoxicity. No cytotoxicity was produced at 3.0 mM misonidazole in small spheroids without necrotic centers and hypoxic cells. In addition to being cytotoxic, continuous exposure of spheroids to high concentrations (3.0 mM) of misonidazole were cytostatic. The spheroid experiments were predictive of the relative effectiveness of the different sensitizers for EMT6/Ro tumors in vivo but the rate and extent of cytotoxicity was greater in tumors especially at low concentrations. In both spheroids and tumors there was little change in growth rate after 24 hours or single intraperitoneal exposures respectively, even with concentrations which reduced the clonogenic fraction by 0.9. This was related to a rapid repopulation of cells and increase in growth fraction after misonidazole cytotoxicity. Addition of adriamycin immediately after misonidazole cytotoxicity resulted in an apparent supra-additive overall response. The significance of these results for interpretation of tumor properties and responses in vivo and for tumor therapy were discussed.