A High-Throughput-Compatible 3D Microtissue Co-Culture System for Phenotypic RNAi Screening Applications
A High-Throughput-Compatible 3D Microtissue Co-Culture System for Phenotypic RNAi Screening Applications
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DOI：
10.1177/1087057113499071
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发表时间：
2013-12-01
影响因子：
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通讯作者：
Kelm, Jens M.
中科院分区：
文献类型：
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作者：
Thoma, Claudio R.;Stroebel, Simon;Kelm, Jens M.
肿瘤细胞在体内受到交互三维微环境的协调影响。然而，药物靶点的鉴定和初始靶点验证通常在二维细胞培养系统中进行。设计3D共培养模型的机会，至少在一定程度上反映了这些异型相互作用，当与RNA干扰相结合时，将能够在更接近于体内肿瘤生长的模型中研究基因功能的表型影响。在这里，我们描述了一种高通量兼容的方法，用于在由人DLD1结肠癌细胞和小鼠成纤维细胞组成的共培养3D肿瘤微组织模型系统中发现癌症基因功能。引人注目的是，该模型中的DLD1细胞在sirna介导的Kif11/Eg5（有丝分裂运动蛋白家族的一员）缺失后未能扩增。相比之下，当这些癌细胞作为2D单层生长时，这些癌细胞被证明对Kif11/Eg5耗尽更具抵抗力。这些结果表明，某些癌细胞在3D和2D环境下的生长可以揭示其生存对特定基因的差异依赖。此外，他们表示，高通量兼容，基于悬挂滴技术的3D共培养模型将能够发现，表征和验证关键生物和病理过程中的基因功能。
Cancer cells in vivo are coordinately influenced by an interactive 3D microenvironment. However, identification of drug targets and initial target validations are usually performed in 2D cell culture systems. The opportunity to design 3D co-culture models that reflect, at least in part, these heterotypic interactions, when coupled with RNA interference, would enable investigations on the phenotypic impact of gene function in a model that more closely resembles tumor growth in vivo. Here we describe a high-throughput-compatible method to discover cancer gene functions in a co-culture 3D tumor microtissue model system composed of human DLD1 colon cancer cells together with murine fibroblasts. Strikingly, DLD1 cells in this model failed to expand upon siRNA-mediated depletion of Kif11/Eg5, a member of the mitotic kinesin-like motor protein family. In contrast, these cancer cells proved to be more resistant to Kif11/Eg5 depletion when grown as a 2D monolayer. These results suggest that growth of certain cancer cells in 3D versus 2D can unveil differential dependencies on specific genes for their survival. Moreover, they denote that the high-throughput-compatible, hanging drop technology-based 3D co-culture model will enable the discovery, characterization, and validation of gene functions in key biological and pathological processes.