Development of an intrinsic P‐glycoprotein‐mediated doxorubicin resistance in quiescent cell layers of large, multicellular prostate tumor spheroids

Abstract

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.

Identification of genomic classifiers that distinguish induction failure in T-lineage acute lymphoblastic leukemia: a report from the Children's Oncology Group

“…Several recent studies have suggested that cellular quiescence may be mechanistically linked with intrinsic drug resistance to multiagent therapy for both solid and liquid tumor cells. 66,67 In support of this change in paradigm, we found that T lymphoblasts in patients with IF may have a genetic profile that suggests they resist multiagent therapy though cellular quiescence. A number of genes that control the G1/S transition in cell cycle progression, specifically TGF␤1, LYN, and LATS2, were upregulated (Figure 3).…”

Regulation of the multidrug resistance transporter P‐glycoprotein in multicellular prostate tumor spheroids by hyperthermia and reactive oxygen species

“…48 The data of our study clearly demonstrate that hyperthermia treatment resulted in a robust increase in P-glycoprotein expression in small drug-sensitive tumor spheroids on the transcriptional as well as on the translational level, whereas only moderate increase was observed in large tumor spheroids that are characterized by high intrinsic levels of P-glycoprotein and a MDR phenotype. 16,18,49 The hyperthermia-induced increase in P-glycoprotein expression in small tumor spheroids was approximately 50% of the value of intrinsic P-glycoprotein levels in untreated large tumor spheroids that have been previously analyzed by us. 16 Apparently the hyperthermia-induced induction of P-glycorprotein was not associated to hsp90, which has been previously shown to copre-FIGURE 7 -RT-PCR analysis of mdr-1 gene expression after hyperthermia treatment, and effects of the NADPH-oxidase inhibitors AEBSF and DPI as well as the free radical scavenger vitamin E. HPRT mRNA expression was used as a house-keeping gene to control gel loading.…”

The Use of 3-D Cultures for High-Throughput Screening: The Multicellular Spheroid Model

“…Bearing in mind, for example, that the heterogeneity in the binding of monoclonal antibodies depends on both cell and antibody type, as well as on cell cycle and metabolic status of the cells, 88,98,[117][118][119][120] spheroid mono-and cocultures should be considered as a screening tool for local penetration, distribution, and efficacy of new drug candidates.…”