Development of a novel and economical agar-based non-adherent three-dimensional culture method for enrichment of cancer stem-like cells.
Development of a novel and economical agar-based non-adherent three-dimensional culture method for enrichment of cancer stem-like cells.
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DOI：
10.1186/s13287-018-0987-x
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发表时间：
2018-09-26
影响因子：
7.5
通讯作者：
Chan FL
中科院分区：
文献类型：
--
作者：
Gao W;Wu D;Wang Y;Wang Z;Zou C;Dai Y;Ng CF;Teoh JY;Chan FL
非贴壁或超低贴壁三维(3D)培养，也称为球体形成实验，已被广泛用于评估转化细胞或癌细胞的恶性表型和干细胞潜能。这种方法也被广泛用于分离癌症干细胞(CSCs)或肿瘤启动细胞，因为它们具有独特的锚定非依赖性生长或抗失巢凋亡能力。不同的非粘附性包衣剂，如聚-甲基丙烯酸羟乙酯(Poly-HEMA)和合成水凝胶，已被用于这种非粘附性3D培养。然而，非粘附性文化器皿的制备是劳动密集型和技术要求高的，而且用各种涂层剂制备的商业非粘附性文化器皿的成本相对较高，并且不能重复使用。在本研究中，我们开发了一种基于琼脂涂层的非贴壁3D培养方法，用于在非贴壁和无血清的条件下生长来自各种癌细胞系和前列腺癌组织的肿瘤球体。通过逆转录定量聚合酶链式反应分析CSC相关标志物的表达谱，荧光激活细胞分选(CD133+/CD44+细胞分选)和对OCT4和SOX2反应的CSC可视化报告系统(SORE6)分析CSC的存在和相对比例，以及体内致瘤性。对于连续传代的肿瘤球体，重复使用琼脂涂层平板也进行了评估。结果表明，用该方法培养的多细胞肿瘤微球富含CSCs，表现为CSC标志物表达增强，CD133+/CD44+或SORE6+细胞的存在，自我更新能力增强，体内致瘤性增强，提示其在CSCs分离和富集性方面有一定的应用价值。琼脂涂层平板可以在连续传代的肿瘤球体中多次使用。与其他方法相比，所描述的基于琼脂的3D培养方法在分离CSC方面具有几个优点，包括其简单、低成本和重复使用琼脂涂层平板来连续传代CSC富集球。本文的在线版本(10.1186/s13287-0180987-x)包含补充材料，可供授权用户使用。
Non-adherent or ultra-low attachment three-dimensional (3D) culture, also called sphere formation assay, has been widely used to assess the malignant phenotype and stemness potential of transformed or cancer cells. This method is also popularly used to isolate the cancer stem-like cells (CSCs) or tumor-initiating cells based on their unique anchorage-independent growth or anoikis-resistant capacity. Different non-adhesive coating agents, such as poly-2-hydroxyethyl methacrylate (poly-HEMA) and synthetic hydrogels, have been used in this non-adherent 3D culture. However, preparation of non-adherent culture-ware is labor-intensive and technically demanding, and also costs of commercial non-adherent culture-ware prepared with various coating agents are relatively expensive and the culture-ware cannot be used repeatedly. In this study, we developed a non-adherent 3D culture method based on agar coating for growing tumor spheres derived from various cancer cell lines and primary prostate cancer tissues under a non-adherent and serum-free condition. The tumor spheres generated by this 3D culture method were analyzed on their expression profiles of CSC-associated markers by reverse transcription quantitative polymerase chain reaction, presence and relative proportion of CSCs by fluorescence-activated cell sorting (CD133+/CD44+ cell sorting) and also a CSC-visualizing reporter system responsive to OCT4 and SOX2 (SORE6), and in vivo tumorigenicity. The repeated use of agar-coated plates for serial passages of tumor spheres was also evaluated. Our results validated that the multicellular tumor spheres generated by this culture method were enriched of CSCs, as evidenced by their enhanced expression profiles of CSC markers, presence of CD133+/CD44+ or SORE6+ cells, enhanced self-renewal capacity, and in vivo tumorigenicity, indicating its usefulness in isolation and enrichment of CSCs. The agar-coated plates could be used multiple times in serial passages of tumor spheres. The described agar-based 3D culture method offers several advantages as compared with other methods in isolation of CSCs, including its simplicity and low-cost and repeated use of agar-coated plates for continuous passages of CSC-enriched spheres. The online version of this article (10.1186/s13287-018-0987-x) contains supplementary material, which is available to authorized users.
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影响因子：
10.5
作者：
Dontu, G;Abdallah, WM;Wicha, MS
通讯作者：
Wicha, MS
影响因子：
11.2
作者：
Huang EH;Hynes MJ;Zhang T;Ginestier C;Dontu G;Appelman H;Fields JZ;Wicha MS;Boman BM
通讯作者：
Boman BM
CD44+ CD24（ - ）前列腺细胞是早期的癌症祖/干细胞，为预后不良的患者提供了模型。
影响因子：
8.8
作者：
Hurt, E. M.;Kawasaki, B. T.;Klarmann, G. J.;Thomas, S. B.;Farrar, W. L.
通讯作者：
Farrar, W. L.
影响因子：
--
作者：
Ivascu, Andrea;Kubbies, Manfred
通讯作者：
Kubbies, Manfred
影响因子：
3.7
作者：
Deng S;Yang X;Lassus H;Liang S;Kaur S;Ye Q;Li C;Wang LP;Roby KF;Orsulic S;Connolly DC;Zhang Y;Montone K;Bützow R;Coukos G;Zhang L
通讯作者：
Zhang L