Growing multicellular prostate tumor spheroids develop quiescent cell subpopulations in central regions with features of intrinsic multicell-mediated drug resistance. Doxorubicin (dox) uptake was significantly reduced in large spheroids (diameter 400 ؎ 70 m), which consist predominantly of quiescent cells, as compared to small spheroids (diameter 100 ؎ 50 m), which consist entirely of proliferating cells. After removal of dox from the incubation medium, dox fluorescence declined more efficiently in large spheroids, which led to a decreased dox toxicity as revealed by colonyforming assays. Verapamil significantly increased dox retention in large spheroids and, consequently, augmented dox toxicity. At a depth 80 m from the spheroid periphery, a significantly decreased dox fluorescence was observed in the deep, quiescent cell layers of large spheroids. The Pglycoprotein-mediated multidrug resistance (MDR)-reversing agents verapamil, cyclosporin A, quinidine, sodium orthovanadate and tamoxifen significantly increased dox fluorescence at this depth, whereas genistein, indomethacin, probenecid and brefeldin A, which reverse multidrugresistance-associated protein (MRP) function, exerted no effect. Anti-P-glycoprotein immunohistochemistry of multicellular tumor spheroids revealed an increase of P-glycoprotein expression in large speroids as compared to small spheroids, which was most prominent in the Ki-67-negative, quiescent cell layers 60 to 100 m distant from the periphery of the spheroid, indicating that the MDR phenotype is related to cell quiescence. This was corroborated by whole-cell patchclamp experiments, where the C219 antibody, which is directed against the ATP-binding site of P-glycoprotein, significantly inhibited P-glycoprotein-associated, volume-activated chloride currents in quiescent, but not proliferating cells from multicellular tumor spheroids. Int.
