The Intrinsic Resistance Of V79 Multicell Spheroids To Mitoxantrone, Adriamycin And Menogaril

Cancer chemotherapy usually fails to completely cure solid tumors due to surviving cells often repopulating the tumor or metastasizing to other sites. It is not clear whether tumor cells are apparently resistant due to a restricted drug penetration into the tumor mass, or whether the resistance is intrinsic being due to the tumor microenvironment.;We have used an in vitro model of tumor nodules, the multicell spheroid, to determine the apparent and intrinsic resistances of cells growing in contact in a tumor-like environment to two anthracyclines: adriamycin, and menogaril, and the anthracenedione mitoxantrone. A combination of HPLC, radiolabel, and flow cytometric techniques were used to measure the uptake of the three drugs by monolayers, 100 {dollar}\mu{dollar}m multicell spheroids, and 650 {dollar}\mu{dollar}m multicell spheroids. The 650 {dollar}\mu{dollar}m spheroids were dissociated into two fractions comprising an outer cell layer approximately 50 {dollar}\mu{dollar}m thick, which contained mostly oxygenated proliferating cells, and an inner cell layer approximately 100 {dollar}\mu{dollar}m thick, which contained a subpopulation of quiescent hypoxic cells. The toxicity of each of the three drugs, on the V79 cells grown in the various culture conditions, was normalized to drug accumulation within the cells. The results demonstrate a restriction to drug uptake of approximately 5 fold in 650 {dollar}\mu{dollar}m spheroids compared to monolayers. The LD{dollar}\sb{lcub}90{rcub}{dollar} of 650 {dollar}\mu{dollar}m spheroids exposed to the cytotoxic compounds was between 6 and 250 fold higher than in monolayers. However, once normalized for drug uptake, the LD{dollar}\sb{lcub}90{rcub}{dollar} was approximately 4-40 fold greater for spheroids compared to monolayers. Adriamycin is more toxic to outer cells than inner cells by a factor of five, while outer cells and inner cells are equisensitive to mitoxantrone and menogaril. The data suggest that restricted drug penetration, or reduced accumulation by cells in multicell spheroids, only partially explains their resistance to three drugs. Hence, normalizing survival to drug uptake removes the apparent resistance factor from survival studies allowing the calculation of intrinsic resistance due to the spheroid microenvironment