Imamura Yoshinori, Mukohara Toru, Shimono Yohei, Funakoshi Yohei, Chayahara Naoko, Toyoda Masanori, Kiyota Naomi, Takao Shintaro, Kono Seishi, Nakatsura Tetsuya, Minami Hironobu
Division of Medical Oncology/Hematology, Department of Medicine, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan.
Division of Breast and Endocrine Surgery, Department of Surgery, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan.
Oncol Rep. 2015 Apr;33(4):1837-43. doi: 10.3892/or.2015.3767. Epub 2015 Jan 29.
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.
人们逐渐认识到，在使用二维（2D）培养细胞系的平台上筛选肿瘤药物无法精确选择具有临床活性的药物；因此，三维（3D）培养系统正在兴起，并显示出更好模拟体内肿瘤微环境的潜力。本研究的目的是揭示化疗药物在2D和3D培养之间的差异效应，并探索其潜在机制。我们通过评估药物敏感性、氧状态以及Ki-67和半胱天冬酶的表达来评估2D和3D培养的乳腺癌细胞系之间的差异。三种细胞系（BT-549、BT-474和T-47D）在3D培养中形成了致密的多细胞球体（MCS），与2D培养的细胞相比，对紫杉醇和阿霉素表现出更强的耐药性。另外三种细胞系（MCF-7、HCC-1954和MDA-MB‑231）在3D培养中仅形成松散的MCS，并且显示出与2D培养相似的药物敏感性。与3D培养相比，紫杉醇处理导致2D培养中裂解的PARP表达增加更多，但仅在形成致密3D-MCS的细胞系中如此，这表明MCS的形成保护细胞免受紫杉醇诱导的凋亡。仅在致密的3D-MCS中观察到缺氧。BT-549在3D培养中Ki-67阳性细胞比2D培养中少，这表明更大的G0休眠亚群是其在3D培养中耐药的原因。BT-474在3D培养中的半胱天冬酶-3水平低于2D培养，这表明3D环境具有抗凋亡作用。最后，我们比较了源自患者来源异种移植（PDX）、新鲜PDX肿瘤和患者原发肿瘤的2D和3D原代培养细胞中Ki-67和半胱天冬酶的染色情况；2D培养的细胞显示出更高比例的Ki-67阳性和半胱天冬酶-3阳性细胞，这与3D原代培养更好地代表体内肿瘤特征的观点一致。总之，表示体内肿瘤重要特征，即缺氧、休眠、抗凋亡特征及其导致的耐药性方面，形成致密MCS的3D培养细胞可能比2D培养细胞更好。
