Resistance of V79 Multicell Spheroids to Mitoxantrone: Drug Uptake and Cytotoxicity

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Abstract
We have previously shown that V79 multicell spheroids are resistant to the anthracenedione mitoxantrone (1). In this paper we demonstrate that this resistance is not solely due to restricted drug penetration into the spheroid, but also to an altered intrinsic resistance of the cells when grown as a three-dimensional structure. We have studied the uptake and toxicity of mitoxantrone in V79-0CF4 monolayers, 100 μm spheroids, 650 μm spheroids, and outer and inner cells of 650 μm spheroids. The LD90 for cells exposed as monolayers to mitoxantrone for two hours was 0.016 μg/ml, 0.055 μg/ml for 100 μm spheroids, 1.5 μg/ml for outer spheroid cells and 6.2 μg/ml for inner spheroid cells. Uptake of [14C]mitoxantrone was linear for all populations with no plateau up to the highest doses used. The uptake of drug required to kill 90% of the cells in a population (UP90) of, monolayers was 3.7 ng/106 cells, 10.7 ng/106 cells for 100 μm spheroids, 169 ng/106 cells for outer spheroid cells, and 146 ng/106 cells for inner spheroid cells. The relative resistance of spheroids compared to monolayers, based on drug concentration in the medium, was 3.4 for small spheroids, 92 for outer cells, and 390 for inner cells. When cell survival was normalized to drug uptake, the relative resistance of spheroids to monolayers was 2.9 for small spheroids, 46 for outer cells and 40 for inner cells of large spheroids. The data demonstrate that the resistance of multicell spheroids to mitoxantrone is not solely due to restricted drug penetration into the cell aggregate but is also due, to a spheroid-induced altered intrinsic resistance of the V79 cells.