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 in vivo 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 in vivo. In conclusion, 3D-cultured cells forming dense MCSs may be better than 2D-cultured cells in simulating important tumor characteristics in vivo, namely hypoxia, dormancy, anti-apoptotic features and their resulting drug resistance.

Concise Review: 3D cell culture systems for anticancer drug screening

“…This may be a primary reason as to why results of anticancer drug assessments using MCTS are more predictive of clinical efficacy than 2D cell assessments (Carver et al, 2014). Many antineoplastic agents have been reported to be less effective for cancer cells cultured in 3D than 2D (Frankel et al, 2000;Imamura et al, 2015;Karlsson et al, 2012). The architectural structure of MCTS is the main reason for this difference.…”

“…Secondly, the tightly adhered cells and ECM in MCTS can limit drug penetration (Frankel et al, 2000). Moreover, the hypoxic core generates a G0-dormant cell population which is highly resistant to chemotherapy (Imamura et al, 2015). Gene expression of cells cultured in 3D systems differs from that of cells in 2D monolayer; for instance, expression of genes related to chemoresistance has been found to vary from 3D versus 2D systems (Lin and Chang, 2008).…”

Alginate foam-based three-dimensional culture to investigate drug sensitivity in primary leukaemia cells

“…Foam-based 3D culture similar to fiber scaffolds [16] increased the resistance of the leukaemia cell lines to inhibitory effects of imatinib and to the maintenance of AML cells [29,30] and also selects bortezomib-resistant CML stem cells [31]. It has been shown that 3D-cultures are better than 2D-cultures in simulating important tumor characteristics in vivo such as hypoxia and resulting drug resistant phenotype [32]. The mechanism of resistance might also be due to poor access of drugs to the cells hidden in the niche like spaces in the scaffold.…”

Formation of cell spheroids using Standing Surface Acoustic Wave (SSAW)

“…Retention in 3D structure, establishment of cell-cell contacts, and presence of extracellular matrix (ECM) are important reasons for spheroidal aggregation [16][17][18] . Currently, cell spheroids are used extensively in the study of tissue anatomy, drug screening [19,20] , toxicology [21] , and cell proliferation and differentiation [22,23] because they represent more similar in vivo biological behaviors. Therefore, cell spheroids could be an alternative format of the bioink.…”