Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids
Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids
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DOI：
10.3791/53486
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发表时间：
2015-12-01
影响因子：
1.2
通讯作者：
Haass, Nikolas K.
中科院分区：
文献类型：
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作者：
Beaumont, Kimberley A.;Anfosso, Andrea;Haass, Nikolas K.
与传统的二维（2D）细胞培养相比，三维（3D）肿瘤球体在癌症研究中被用作更准确的体内肿瘤微环境模型。球体模型能够模拟细胞间相互作用、缺氧和营养剥夺以及药物渗透的影响。该模型的一个特征是坏死核心的发育，被G1细胞包围，球状体的外层有增殖细胞。在癌症领域，人们感兴趣的是球体的不同区域对药物治疗以及遗传或环境操纵的反应。我们在这里描述了使用荧光泛素化细胞周期指示器（FUCCI）系统以及使用商业软件的细胞术和图像分析来表征细胞周期状态及其在黑色素瘤球体内的位置。这些方法可用于跟踪肿瘤球体不同亚区随时间和不同条件下细胞周期状态、基因/蛋白表达或细胞活力的变化。
Three-dimensional (3D) tumor spheroids are utilized in cancer research as a more accurate model of the in vivo tumor microenvironment, compared to traditional two-dimensional (2D) cell culture. The spheroid model is able to mimic the effects of cell-cell interaction, hypoxia and nutrient deprivation, and drug penetration. One characteristic of this model is the development of a necrotic core, surrounded by a ring of G1 arrested cells, with proliferating cells on the outer layers of the spheroid. Of interest in the cancer field is how different regions of the spheroid respond to drug therapies as well as genetic or environmental manipulation. We describe here the use of the fluorescence ubiquitination cell cycle indicator (FUCCI) system along with cytometry and image analysis using commercial software to characterize the cell cycle status of cells with respect to their position inside melanoma spheroids. These methods may be used to track changes in cell cycle status, gene/protein expression or cell viability in different sub-regions of tumor spheroids over time and under different conditions.