Abstract 319: Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer
Authors: Imamura Yoshinori , Mukohara Toru , Shimono Yohei , Funakoshi Yohei , Chayahara Naoko , Toyoda Masanori , Kiyota Naomi , Takao Shintaro , Kono Seishi , Nakatsura Tetsuya , Minami Hironobu 
Published: August 2015

While 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, three-dimensional (3D)-culture systems are emerging and show potential for better simulating the in vivo tumor microenvironment. The aim of this study was to reveal differential effects of chemotherapeutic drugs between 2D- and 3D-cultures and to explore their underlying mechanisms. We first 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 multi-cellular spheroids (MCSs) in 3D-culture, and tended to show greater resistance to paclitaxel and doxorubicin than in 2D-culture. An additional three cell lines (MCF-7, HCC-1954, and MDA-MB-231) developed only loose MCSs in 3D-culture, and showed drug sensitivities similar to those in 2D-culture. Treatment with paclitaxel resulted in greater increases in cleaved PARP expression in 2D- than in 3D-culture, but only in cell lines forming dense 3D-MCSs, suggesting that MCS formation protected cells from paclitaxel-induced apoptosis. Hypoxia, as measured with phosphorescent LOX-1, was observed only in dense 3D-MCSs. BT-549 had considerably fewer cells positive for Ki-67 in 3D- than in 2D-culture, suggesting that the greater G0 dormant sub-population may be responsible for its drug resistance in 3D-culture. In addition, BT-474 had a lower level of caspase-3 in 3D- than in 2D-culture, suggesting that the 3D-environment was anti-apoptotic. We next examined whether these findings were reproduced in primary cells utilizing a breast cancer patient-derived xenograft (PDX). The fresh disaggregated PDX tissue developed dense MCSs in 3D-culture. We compared staining for Ki-67, and caspase-3 and -8 in 2D- and 3D-cultured primary cells obtained from the PDX, and the original patient tumor (Ki-67 only): 2D-cultured cells showed much greater proportions of Ki-67-positive and caspase-3-positive cells than the others. 3D-cultured cells tended to show greater resistance to paclitaxel and doxorubicin compared to the others. Residual spheroids after exposure to paclitaxel did not grow after removal of the drug but majority of cells in the spheroids appeared alive based on their excluding trypan blue dye. In the residual spheroids, greater population of cells showed phosphorylated histone H2AX expression than those in the spheroids unexposed to paclitaxel, suggesting that chemotherapy-induced senescence associated with drug resistance. In conclusion, 3D-cultured cells forming dense MCSs may be better than 2D-cultured cells in simulating important characteristics of tumor grown in vivo, namely hypoxia, dormancy, anti-apoptotic features, and their resulting drug resistance. 3D primary culture will be a model for the study of chemotherapy-induced senescence.

Citation Format: Yoshinori Imamura, Toru Mukohara, Yohei Shimono, Yohei Funakoshi, Naoko Chayahara, Masanori Toyoda, Naomi Kiyota, Shintaro Takao, Seishi Kono, Tetsuya Nakatsura, Hironobu Minami. Comparison of 2D- and 3D-culture models as drug-testing platforms in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 319. doi:10.1158/1538-7445.AM2015-319