Response of spheroids implanted in the peritoneal cavity of mice exposed to cyclophosphamide and ionizing radiation

Abstract

Chinese hamster V79-171B spheroids implanted in the peritoneal cavity of C3H mice were characterized as a model for evaluating the toxicity of drugs requiring metabolic activation in vivo. After 24 hours in the peritoneal cavity, spheroid cellularity and plating efficiency were not significantly decreased, and host cell infiltration was estimated between 5 and 10%. The oxygenation of spheroids in the peritoneal cavity was assessed using their response to ionizing radiation. Spheroids were recovered after irradiation, incubated for 20 minutes in vitro with the slowly penetrating fluorescent dye, Hoechst 33342, and reduced to single cells with trypsin. Cells were analysed for clonogenicity as a function of position within the spheroid by using fluorescence-activated cell sorting in conjunction with the Hoechst diffusion gradient. When spheroids were first placed in the peritoneal cavity, the hypoxic fraction was close to 100%, but after 24 hours, cell oxygenation increased, probably due to a decrease in cell respiration rate. However, the location of a spheroid within the peritoneal cavity did not influence the radiosensitivity of individual spheroids or the amount of Hoechst 33342 delivered to the spheroid when Hoechst was given intravenously; individual spheroids recovered from mice given an intravenous injection of Hoechst showed no greater heterogeneity in binding than that observed when spheroids were incubated with Hoechst in vitro. Mice implanted with spheroids were also exposed to cyclophosphamide; the external cells of 0.6 mm diameter spheroids were about 30% more sensitive than the internal cells to the toxic effects of both cyclophosphamide and X-rays, and the combination of the two agents was additive at all depths within the spheroid.