Three-Dimensional Lung Tumor Microenvironment Modulates Therapeutic Compound Responsiveness In Vitro - Implication for Drug Development
Three-Dimensional Lung Tumor Microenvironment Modulates Therapeutic Compound Responsiveness In Vitro - Implication for Drug Development
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DOI：
10.1371/journal.pone.0092248
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发表时间：
2014-03-17
期刊：
影响因子：
3.7
通讯作者：
Colter, David C.
中科院分区：
文献类型：
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作者：
Ekert, Jason E.;Johnson, Kjell;Colter, David C.
三维(3D)细胞培养正在被接受，以回应对更好地模拟生理组织的细胞模型的需求。球体就是这样一种3D模型，在这种模型中，成团的细胞将进行自我组装，形成可存活的3D肿瘤样结构。然而，到目前为止，人们对椭圆形生物学与更传统和更广泛使用的2D单层培养的比较知之甚少。因此，这项研究的目的是描述2D单层培养的肺肿瘤细胞与3D球状培养的肺癌细胞在表型和功能上的差异。以8株表达不同水平的表皮生长因子受体(EGFR)和cMET蛋白的肺癌细胞株为材料，利用低附着U-Bottom板建立三维球形细胞培养模型。用流式细胞仪检测EGFR和cMET受体的表达，用MSD法检测EGFR和cMET的磷酸化，以及3)细胞对表皮生长因子(EGF)和肝细胞生长因子(HGF)的刺激。此外，通过测量细胞增殖和迁移的程度来评估药物对EGFR和cMET抑制剂(Erlotinib、Crizotinib、Cetuximab[Erbitux]和Onartuzumab[MetMab])的敏感性。数据显示，在未经处理的球体培养的第四天，与单层相比，EGFR和cMET的表达减少。与单层培养相比，球状细胞中EGFR和cMET的基础磷酸化水平更高。与2D培养相比，当配体刺激时，球体显示EGFR和cMET的磷酸化水平降低。与单层培养相比，球形细胞对HGF以及EGFR和cMET抑制剂的细胞增殖反应发生了改变。最后，球体培养在细胞迁移分析中显示出特殊的用途。总体而言，3D球体培养改变了细胞对药物和生长因子的反应，可能更准确地模拟自然的肿瘤微环境。
Three-dimensional (3D) cell culture is gaining acceptance in response to the need for cellular models that better mimic physiologic tissues. Spheroids are one such 3D model where clusters of cells will undergo self-assembly to form viable, 3D tumor-like structures. However, to date little is known about how spheroid biology compares to that of the more traditional and widely utilized 2D monolayer cultures. Therefore, the goal of this study was to characterize the phenotypic and functional differences between lung tumor cells grown as 2D monolayer cultures, versus cells grown as 3D spheroids. Eight lung tumor cell lines, displaying varying levels of epidermal growth factor receptor (EGFR) and cMET protein expression, were used to develop a 3D spheroid cell culture model using low attachment U-bottom plates. The 3D spheroids were compared with cells grown in monolayer for 1) EGFR and cMET receptor expression, as determined by flow cytometry, 2) EGFR and cMET phosphorylation by MSD assay, and 3) cell proliferation in response to epidermal growth factor (EGF) and hepatocyte growth factor (HGF). In addition, drug responsiveness to EGFR and cMET inhibitors (Erlotinib, Crizotinib, Cetuximab [Erbitux] and Onartuzumab [MetMab]) was evaluated by measuring the extent of cell proliferation and migration. Data showed that EGFR and cMET expression is reduced at day four of untreated spheroid culture compared to monolayer. Basal phosphorylation of EGFR and cMET was higher in spheroids compared to monolayer cultures. Spheroids showed reduced EGFR and cMET phosphorylation when stimulated with ligand compared to 2D cultures. Spheroids showed an altered cell proliferation response to HGF, as well as to EGFR and cMET inhibitors, compared to monolayer cultures. Finally, spheroid cultures showed exceptional utility in a cell migration assay. Overall, the 3D spheroid culture changed the cellular response to drugs and growth factors and may more accurately mimic the natural tumor microenvironment.