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3D cell culture for compound de-risking
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Cell-based anticancer drug screening generally utilizes rapidly proliferating tumour cells grown as monolayer cultures. Hit compounds from such screens are not necessarily effective on hypoxic and slowly proliferating cells in 3-D tumour tissue. The aim of this study was to examine the potential usefulness of 3-D cultured tumour cells for anticancer drug screening. We used colon carcinoma multicellular spheroids containing hypoxic and quiescent cells in core areas for this purpose. Three libraries (∼11 000 compounds) were screened using antiproliferative activity and/or apoptosis as end-points. Screening of monolayer and spheroid cultures was found to identify different sets of hit compounds. Spheroid screening enriched for hydrophobic compounds: median XLogP values of 4.3 and 4.4 were observed for the hits in two independent screening campaigns. Mechanistic analysis revealed that the majority of spheroid screening hits were microtubuli inhibitors. One of these inhibitors was examined in detail and found to be effective against non-dividing cells in the hypoxic centres of spheroids. Spheroid screening represents a conceptually new strategy for anticancer drug discovery. Our findings have implications for drug library design and hit selection in projects aimed to develop drugs for the treatment of solid tumours.
Spheroids of EMT-6 mammary tumor cells were markedly more resistant to different exposure doses of Adriamycin (ADR) than monolayer cells in exponential or plateau growth phases. For example, after 1 hr exposures to 0.5 μ/ml, surviving fractions determined by colony formation assay were approximately 0.3 for cells from spheroids treated intact and 0.001 for single exponential phase cells. To evaluate whether this resistance was related to poor drug uptake the distribution of the natural fluorescence of ADR equivalents was determined fluorimetrically and by direct microscopic observations. A concentration gradient of fluorescence was observed from the outside to the centers of spheroids even after high concentrations (10 μ/ml) and long exposure times (2 hrs). Cells from dissociated spheroids took up more drug than intact spheroids further indicating the existence of a significant diffusion barrier. When the surviving fraction of cells was plotted versus absorbed ADR equivalents the cells in intact spheroids were still more resistant and both curves were bicomponent with the most resistant fraction comprising about 20% of the cells. By using a selective disaggregation technique after intact spheroids had been exposed to the drug it was possible to show directly that the inner spheroid cells were most resistant (D0 = 0.25 μ/106 cells). This resistance was not due to differences in the cell cycle state of these inner cells since separate experiments showed that both exponential and plateau phase monolayer cells were about equally sensitive when the surviving fraction was plotted vs absorbed drug (D0, = 0.04 μ/106 cells). Thus, other factors related to the metabolic state of the cells, the microenvironment, or the formation of different drug products must account for the observed resistance. Pretreatment of spheroids with misonidazole before ADR effectively reduced this resistant population of cells.