软琼脂集落形成分析
Summary
The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.
Abstract
Anchorage-independent growth is the ability of transformed cells to grow independently of a solid surface, and is a hallmark of carcinogenesis. The soft agar colony formation assay is a well-established method for characterizing this capability in vitro and is considered to be one of the most stringent tests for malignant transformation in cells. This assay also allows for semi-quantitative evaluation of this capability in response to various treatment conditions. Here, we will demonstrate the soft agar colony formation assay using a murine lung carcinoma cell line, CMT167, to demonstrate the tumor suppressive effects of two members of the Wnt signaling pathway, Wnt7A and Frizzled-9 (Fzd-9). Concurrent overexpression of Wnt7a and Fzd-9 caused an inhibition of colony formation in CMT167 cells. This shows that expression of Wnt7a ligand and its Frizzled-9 receptor is sufficient to suppress tumor growth in a murine lung carcinoma model.
Introduction
软琼脂集落形成测定是广泛用于评估体外细胞转化的技术。在历史上，另一个试验中，集落形成试验，通过冰球等人在1956年描述。用于评价细胞形成集落1的能力。在该技术中，将细胞分散到培养板中，在“进料器”细胞或条件培养基的存在下，以提供必要的生长因子生长。这种技术的局限性是，它仅对于集落形成提供信息。正常细胞是从锚定非依赖性生长抑制，由于特定类型的凋亡性死亡，称为失巢2。然而，转化细胞有能力生长和分裂，而不结合到基片上。利用这一概念，研究人员开发出了软琼脂克隆形成实验。软琼脂集落形成测定法进行了修改，在最近几年，为解决藻ecific需求。一个变化涉及到荧光染料的掺入，允许高通量菌落计数。另一种变型涉及使用专门的琼脂溶液中，以允许集落形成后检索的活细胞时，需要的蛋白质或DNA样本。
在传统的软琼脂集落形成测定法，细胞生长在一层软琼脂与搁置在软琼脂中，还混有细胞培养基的另外一个层细胞培养基混合的，但含有琼脂的浓度较高。这可以防止细胞粘附到培养板上，还允许转化的细胞形成可见的菌落。该技术背后的基本原理是，正常细胞依赖于细胞外基质接触，以便能够生长和分裂。相反，转化细胞具有生长和分化而不论其周围环境的能力。因此，细胞能够形成菌落在锚地无关的方式为Considered待转化和致癌性。该方法的总体目标是测量在一个半定量的和严格的方式在细胞中这种能力。
Wnt信号通路是在胚胎发育的关键和经常去调节肿瘤发生3-6。有与Wnt信号相关联的多种途径。规范的途径，通过对转录共激活因子的β-catenin的影响涉及Wnt信号和下游基因的转录调控。 Wnts还通过几个非经典途径的信号，例如，在平面细胞极性通路，它调节参与细胞骨架结构7的元素，并且所述的Wnt-钙通路，调节钙的释放从内质网8。的Wnt配体通过结合型卷曲受体发挥其活性。虽然几个Wnts已被证明在肺癌中上调，WNT7A已显示被下调非SMaL公司通过启动子甲基化9升细胞肺癌。 WNT7A结合Fzd9并作为通过非经典途径中的肿瘤抑制基因。的Wnt-7a和FZD-9的恢复抑制非小细胞肺癌细胞10的生长。 WNT7A / Fzd9的作用是通过ERK-5，这反过来，激活过氧化物酶体增殖物激活受体γ（PPARγ）11,12的激活介导的。在这里，我们表明WNT7A和Fzd9的过度表达导致的小鼠肺癌细胞株的锚定非依赖性生长的抑制。鼠CMT167细胞来自于C57BL / lcrf小鼠13个肺腺癌和稳定转染WNT7A和Fzd9。 WNT7A和Fzd9的过表达通过定量PCR（Q-PCR）和WNT7A和Fzd9过表达的功能性被证实是通过PPARγ的下游激活确认。
Protocol
Representative Results
Discussion
中的信号蛋白的肿瘤抑制功能的体外确认是困难的。一个可用来调查该属性的最严格的测定法是软琼脂集落形成测定。这里，我们已经说明了利用鼠肺癌细胞稳定过表达WNT7A和Fzd9相比其亲CMT167细胞系的软琼脂集落形成测定。
有考虑有关软琼脂法的几个要点。在此测定法中最关键的步骤是电镀的细胞。细胞计数必须是准确的，并将琼脂溶液不能是太热了。如果没?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This study was supported by a Merit Award from the U.S. Department of Veterans Affairs, and an NIH grant R01CA1385282522717 to RW.
Materials
| Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
| Cancer Cell Line of Interest | Sigma-Aldrich | 10032302 | CMT-167 Cells |
| Powdered RPMI 1640 Medium | Gibco | 31800-089 | Used to prepare 2X cell culture medium. |
| Liquid RPMI 1650 Medium | Cellgro | 10-040-CV | Referred to as 1X cell culture medium. |
| Fetal Bovine Serum | HyClone | SH30910.03 | Used to supplement cell culture medium. |
| Penicillin/Streptomycin | CellGro | 30-002-Cl | Used in cell culture medium. |
| Difco Noble Agar | BD Biosciences | 214230 | Used to prepare 1.0% and 0.6% Agar. |
| Sodium Bicarbonate | Fisher | BP-328-1 | Used in 2X cell culture medium. |
| Trypsin | Cellgro | 25-050-Cl | |
| Sterile Bottle-Top Filters | Fisher | 09-761-126 | Used to sterile filter 2X medium. |
| Lipofectamine Reagent | Invitrogen | 18324-020 | Used in PPAR-RE luciferase assay. |
| 6-well Plates Tissue-culture Treated | |||
| 37 degree C/5% CO2 Incubator | |||
| Chemi-Doc Imager | Bio-Rad | Used to take pictures of colonies. | |
| Quantity One Software | Bio-Rad | Used to count cell colonies. | |
| 15mL Conical Tubes | |||
| 50mL Conical Tubes | |||
| 250mL Erlenmeyer Flasks | |||
| Microwave | |||
| 5mL Serological Pipettes | |||
| Pipette Aid | |||
| Micropipette | |||
| Hemacytometer w/ cover slip | |||
| Pipette Tips | |||
| Inverted Light Microscope | |||
| Centrifuge | |||
| Heat-Resistant Gloves | |||
| Saran Wrap | |||
| Ice Bucket |
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