Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer.

Abstract

It is becoming recognized that screening of oncology drugs on a platform using two-dimensionally (2D)-cultured cell lines is unable to precisely select clinically active drugs; therefore three-dimensional (3D)-culture systems are emerging and show potential for better simulating the invivo tumor microenvironment. The purpose of this study was to reveal the differential effects of chemotherapeutic drugs between 2D- and 3D-cultures and to explore their underlying mechanisms. We evaluated differences between 2D- and 3D-cultured breast cancer cell lines by assessing drug sensitivity, oxygen status and expression of Ki-67 and caspases. Three cell lines (BT-549, BT-474 and T-47D) developed dense multicellular spheroids (MCSs) in 3D-culture, and showed greater resistance to paclitaxel and doxorubicin compared to the 2D-cultured cells. An additional three cell lines (MCF-7, HCC-1954, and MDA-MB‑231) developed only loose MCSs in 3D, and showed drug sensitivities similar to those found in the 2D-culture. Treatment with paclitaxel resulted in greater increases in cleaved-PARP expression in the 2D-culture compared with the 3D-culture, but only in cell lines forming dense 3D-MCSs, suggesting that MCS formation protected the cells from paclitaxel-induced apoptosis. Hypoxia was observed only in the dense 3D-MCSs. BT-549 had fewer cells positive for Ki-67 in 3D- than in 2D-culture, suggesting that the greater G0-dormant subpopulation was responsible for its drug resistance in the 3D-culture. BT-474 had a lower level of caspase-3 in the 3D- than in the 2D-culture, suggesting that the 3D-environment was anti-apoptotic. Finally, we compared staining for Ki-67 and caspases in the 2D- and 3D-primary‑cultured cells originating from a patient-derived xenograft (PDX), fresh PDX tumor, and the patient's original tumor; 2D-cultured cells showed greater proportions of Ki-67-positive and caspase-3-positive cells, in agreement with the view that 3D-primary culture better represents characteristics of tumors invivo. In conclusion, 3D-cultured cells forming dense MCSs may be better than 2D-cultured cells in simulating important tumor characteristics invivo, namely hypoxia, dormancy, anti-apoptotic features and their resulting drug resistance.