Application of a fluorescence resonance energy transfer (FRET)-based biosensor for detection of drug-induced apoptosis in a 3D breast tumor model
Application of a fluorescence resonance energy transfer (FRET)-based biosensor for detection of drug-induced apoptosis in a 3D breast tumor model
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DOI：
10.1002/bit.25572
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发表时间：
2015-08-01
影响因子：
3.8
通讯作者：
Luo, Kathy Q.
中科院分区：
文献类型：
--
作者：
Anand, Padmaja;Fu, Afu;Luo, Kathy Q.
二维（2D）培养通常用于测试药物作用，主要是因为它们易于维护。但它们并不代表肿瘤内细胞的空间相互作用。三维（3D）培养可以克服这些限制，从而模仿实体瘤的结构。然而，长期在3D培养中评价药物效果并不容易。这就需要开发3D平台的实时和纵向分析。在这项研究中，我们将编码基于荧光共振能量转移（FRET）的生物传感器的质粒DNA转染到人乳腺癌细胞中，并产生两种细胞系MCF7-C3和MDA-MB-231-C3（231-C3）细胞。我们用它们来确定caspase-3的激活，通过FRET成像，健康细胞显示为绿色，凋亡细胞显示为蓝色。由于半胱天冬酶传感器可以在细胞内不断产生，并迅速响应半胱天冬酶激活，我们假设这些传感器细胞将允许纵向检测细胞凋亡。产生MCF7-C3和231-C3球状体并进行组织学检查、基因表达研究、药物处理和FRET分析。我们的结果表明，MCF7-C3细胞形成紧密的3D球体，并模仿体内肿瘤结构。在3D培养的细胞中，致瘤标志物如MMP-9、SOX 2和OCT 4A的mRNA水平比2D培养的细胞高得多。最后，在用紫杉醇处理后，FRET效应在MCF7-C3球状体的边缘以剂量和时间依赖性方式降低，证明这些传感器细胞可用于在3D设置中确定药物诱导的细胞凋亡。这项研究支持开发基于生物传感器的体外3D乳腺肿瘤模型的可能性，用于以非侵入性方式确定抗癌药物在长时间内的渗透。Biotechnol. Bioeng. 2015; 112：1673 - 1682. (c)2015 Wiley Periodicals，Inc.
Two-dimensional (2D) cultures are commonly used for testing drug effects largely because of their easy maintenance. But they do not represent the spatial interactions of the cells within a tumor. Three-dimensional (3D) cultures can overcome those limitations thus mimicking the architecture of solid tumor. However, it is not easy to evaluate drug effects in 3D culture for a long time. This necessitates the development of a real-time and longitudinal analysis of 3D platforms. In this study, we transfected the plasmid DNA encoding the fluorescence resonance energy transfer (FRET)-based biosensor into human breast cancer cells and generated two cell lines of MCF7-C3 and MDA-MB-231-C3 (231-C3) cells. We used them to determine the activation of caspase-3, whereby healthy cells appear green and apoptotic cells appear blue by FRET imaging. As the caspase sensors can be constantly produced within the cells and quickly respond to caspase activation, we hypothesized that these sensor cells will allow longitudinal detection of apoptosis. MCF7-C3 and 231-C3 spheroids were generated and subjected to histological examination, gene expression studies, drug treatment, and FRET analyses. Our results demonstrated that MCF7-C3 cells formed tight 3D spheroids, and mimicked in vivo tumor architecture. The mRNA level of tumorigenic markers such as MMP-9, SOX2, and OCT4A were much higher in cells cultured in 3D than in 2D. Finally, upon treatment with paclitaxel, the FRET effect was reduced at the rim of MCF7-C3 spheroids in a dose and time-dependent manner demonstrating these sensor cells can be used to determine drug-induced apoptosis in a 3D set up. This study supports the possibility of developing a biosensor-based in vitro 3D breast tumor model for determination of anti-cancer drug penetration over a long course of time in a non-invasive manner. Biotechnol. Bioeng. 2015;112: 1673-1682. (c) 2015 Wiley Periodicals, Inc.