Name: coculture assays to study macrophage and microglia stimulation of glioblastoma invasion
Uploaded: 2016-10-20T12:30:00
Description: Watch this Scientific Journal Video about Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion at JoVE.com

We would like to thank Dr. Konstantin Dobrenis for providing murine microglia for these studies.

1.细胞荧光标记注：标签胶质母细胞瘤细胞系和小胶质细胞用荧光染料9。可替换地，产生如在14中所述组成型表达的荧光蛋白如GFP / RFP的细胞系。 <ol><li>在6孔板板细胞，他们将70 - 对染色天80％融合。对于鼠恶性胶质瘤细胞系GL261和人成胶质细胞瘤细胞系U87，板1×10 6和1.5×10 6个细胞，分别在染色前一个6cm皿24小时。 </li><li>在DMSO准备荧光细胞染色染料溶液，?...

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高级别星形细胞瘤和胶质母细胞瘤的高度创伤性使这些脑癌非常致命的。因此至关重要它是理解胶质母细胞瘤侵袭的分子和细胞机制。很多人都了解胶质母细胞瘤入侵已经17的过程。使用本文详述的测定形式，我们的实验室已表明在小鼠和人模型肿瘤相关巨噬细胞可以通过5刺激神经胶质瘤细胞侵袭 - 10倍。这个共培养模型忠实复制成胶质细胞瘤细胞的物理上与巨噬细胞/小胶质细胞相互作?...

胶质母细胞瘤是一种积极的人脑肿瘤与来自诊断1,2的时间的大约12个月的中位生存期。胶质母细胞瘤是最致命和临床具有挑战性的癌症之一，因为它是难治标准化疗和手术切除。胶质母细胞瘤的扩散性质，使肿瘤细胞在整个大脑的正常传播使晚期肿瘤几乎不可能完全手术切除。这种高度侵入性的方面是胶质母细胞瘤等先进星形细胞瘤的标志特征。因此，许多研究的重点是胶质母细胞瘤细胞侵袭的分子机制。胶质母细胞瘤肿瘤微环境在建立恶性肿瘤3-6至关重要的作用。肿瘤相关巨噬细胞/被证明小胶质细胞负责促进胶质母细胞瘤侵袭7,8。大部分这些研究然而用测量的巨噬细胞/小胶质细胞的作用测定其物理上胶质瘤细胞将它们分开。我们的实验室已着手产生提高检测这让我们能够研究胶质母细胞瘤入侵是如何依赖于共同培养巨噬细胞/小胶质细胞和入侵期间使我们能够像它们之间的物理相互作用。 经典实验测定细胞侵袭包括“标准”Boyden小室趋化和化学侵袭格式。这里待研究的细胞铺在含有对具有指定尺寸（一般为0.4和8微米的直径）的孔的底部的聚碳酸酯过滤器的塑料室中。细胞侵袭的方法，包括一个物理屏障，通常由细胞外基质蛋白质。在化学侵袭测定中，所使用的优选的基质是Matrigel中（以下简称为“矩阵”），通过Engelbreth-Holm的-群（EHS）分泌的胞外基质蛋白混合物小鼠肉瘤细胞和大部分由山口的拉根Ⅳ型和层粘连蛋白。然后将腔室放置在包含有或没有被怀疑刺激侵袭生长因子的细胞培养基的组织培养孔。具有较高侵袭能力通过细胞外基质涂覆的过滤器在较高的频率会侵入并附着到过滤器的下侧的细胞。我们已在以评估小胶质细胞和肿瘤相关巨噬细胞对成胶质细胞瘤细胞侵袭的作用改性该测定。 我们已经能够确定使用该纸张胶质可由5刺激两成胶质细胞瘤细胞系的侵袭中描述的共培养测定法- 10倍9,10。这反映了在胶质母细胞瘤的动物模型中观察到。此外，我们开发了一种三维侵袭测定，其中成胶质细胞瘤细胞和巨噬细胞之间的相互作用/小胶质细胞，可以更直接地检测。胶质母细胞瘤细胞浸润由macropha刺激的程度在3D试验GES /小胶质细胞可媲美什么是使用矩阵涂层的腔体的方式看到。类似的分析是以前开发的入侵在11-13研究与巨噬细胞乳腺癌的相互作用。本文中所描述这两种方法应在解剖胶质母细胞瘤细胞的巨噬细胞/小胶质细胞刺激的入侵的分子机制（S）的能力帮助。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Corning BioCoat Matrigel Invasion Chamber: With BD Matrigel Matrix</td>
			<td>Corning/Fisher Scientific</td>
			<td>Cat: 354481</td>
			<td></td>
		</tr>
		<tr>
			<td>Macrophage Serum Free Media (MSFM) (500 ml)</td>
			<td>Life Technologies</td>
			<td>12065-074</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Red CMTPX Dye</td>
			<td>Life Technologies/Molecular Probes</td>
			<td>C34552</td>
			<td></td>
		</tr>
		<tr>
			<td>CellTracker Green CMFDA Dye</td>
			<td>Life Technologies/Molecular Probes</td>
			<td>C2925</td>
			<td></td>
		</tr>
		<tr>
			<td>GL261 cell line</td>
			<td>National Cancer Institute (NCI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>U87 cell line</td>
			<td>American Tissue Type Culture Collection</td>
			<td>HTB-14</td>
			<td></td>
		</tr>
		<tr>
			<td>THP-1 cell line</td>
			<td>American Tissue Type Culture Collection</td>
			<td>ATCC TIB-202</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium (500 ml)</td>
			<td>Life Technologies/Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde solution</td>
			<td>Sigma Aldrich</td>
			<td>F1635</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Transwell polycarbonate membrane cell culture inserts (8 &#181;M pore) 48 per pack.</td>
			<td>Corning</td>
			<td>CLS3422</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex 3-D Culture Matrix Reduced Growth Factor Basement Membrane Extract, PathClear</td>
			<td>Trevigen</td>
			<td>3445-005-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Calf Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>Cat: 10500064</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin, Fraction V, Heat Shock Treated</td>
			<td>Fisherscientific</td>
			<td>BP1600-100</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5M EDTA</td>
			<td>ThermoFisher Scientific</td>
			<td>15575-020</td>
			<td></td>
		</tr>
		<tr>
			<td>phorbol 12-myristate 13-acetate (PMA)</td>
			<td>Sigma Aldrich</td>
			<td>P8139-1MG</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculture assays to study macrophage and microglia stimulation of glioblastoma invasion

胶质母细胞瘤（IV级胶质瘤）是一个非常积极的人类癌症1年后诊断中位生存期。尽管引起胶质母细胞瘤的分子事件的了解增多，这种癌症仍然是非常难治常规治疗。高档脑肿瘤的手术切除是很少完整由于胶质瘤细胞的浸润性高的性质。因此，这削弱胶质母细胞瘤细胞侵袭的治疗方法是一个有吸引力的选择。我们的实验室和其他人已经表明，肿瘤相关巨噬细胞和小胶质细胞（脑居民巨噬细胞）强烈刺激胶质母细胞瘤入侵。本文中所描述的协议被用来模拟使用体外培养测定胶质母细胞瘤，巨噬细胞/小胶质细胞的相互作用。这种方法可以极大地方便，使这个恶性behav的扰乱与巨噬细胞的沟通药物的开发和/或发现IOR。我们已经建立了两个强大的共培养入侵检测，其中的小胶质细胞/巨噬细胞刺激5胶质瘤细胞侵袭 - 10倍。不具有和具有巨噬细胞/小胶质细胞接种于涂覆基质 - 聚碳酸酯室插入标记有荧光标记物或组成型表达的荧光蛋白胶质母细胞瘤细胞或嵌入在三维基质。细胞的侵袭是通过使用荧光显微镜图像和计数仅侵袭细胞在过滤器的下侧进行评估。使用这些测定，若干药理学抑制剂（JNJ-28312141，PLX3397，吉非替尼，和塞马莫德），已经鉴定阻断巨噬细胞/小胶质细胞刺激胶质母细胞瘤侵袭。

使用此处所述的方法中，我们已经表明，小胶质细胞和巨噬细胞可基本上刺激成胶质细胞瘤细胞的侵袭。两种不同的侵袭测定法采用与图1中描绘在图2中，组成型表达的荧光蛋白mCherry GL261细胞涂布在有和没有小胶质细胞48小时预涂布腔室。然而，当培养与小胶质细胞的侵袭能力增加了大约10倍，GL261细胞在自己的微创。 <p class="jove_content" fo:keep-to...

Watch this Scientific Journal Video about Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion at JoVE.com

Coculture Assays to Study Macrophage and Microglia Stimulation of Glioblastoma Invasion

Understanding the malignant behavior of cancer requires creating accurate models of how tumor cells interact with components of the tumor microenvironment, such as macrophages. Here we describe two methods to study glioblastoma cell interaction with tumor associated macrophages and microglia where the effect on glioblastoma invasion is assessed.

共培养化验，以研究胶质母细胞瘤入侵的巨噬细胞和小胶质细胞刺激

Glioblastoma multiforme (grade IV glioma) is a very aggressive human cancer with a median survival of 1 year post diagnosis. Despite the increased ...

Research

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Cancer Research

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

PET 与 MRI 引导下微器对大鼠胶质瘤模型的照射

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

科研

癌症研究

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

Fluorescence Molecular Tomography for In Vivo Imaging of Glioblastoma Xenografts

胶质瘤移植中体内成像的荧光分子层析成像研究

Tumorigenicity is the capability of cancer cells to form a tumor mass. A widely used approach to determine if the cells are tumorigenic is by injecting ...

Live-Cell Imaging Assays to Study Glioblastoma Brain Tumor Stem Cell Migration and Invasion

活体细胞成像技术研究胶质瘤脑干细胞的迁移和侵袭

Glioblastoma (GBM) is an aggressive brain tumor that is poorly controlled with the currently available treatment options. Key features of GBMs include ...

The Clinical Application of Tumor Treating Fields Therapy in Glioblastoma

肿瘤治疗胶质母细胞瘤的临床应用

Glioblastoma is the most common and lethal form of brain cancer, with a median survival of 15 months after diagnosis and a 5 year survival rate of only 5% ...

Yeast As a Chassis for Developing Functional Assays to Study Human P53

酵母作为开发功能分析研究人类P53的机箱

The finding that the well-known mammalian P53 protein can act as a transcription factor (TF) in the yeast S. cerevisiae has allowed for the development of ...

In Vitro Assay to Study Tumor-macrophage Interaction

体外测定研究肿瘤-巨噬菌体相互作用

Tumor associated macrophages (TAMs) account for a large percentage of cells in the tumor mass for different types of cancers. Glioblastoma (GBM), a ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

多维共同培养系统模拟肺鳞癌进展

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

基于阻抗的癌细胞迁移和入侵的实时测量

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

A 3D Spheroid Model for Glioblastoma

胶质母细胞瘤的3D球形模型

Two-dimensional (2D) cell cultures do not mimic in vivo tumor growth satisfactorily. Therefore, three-dimensional (3D) culture spheroid models were ...

Co-culture of Glioblastoma Stem-like Cells on Patterned Neurons to Study Migration and Cellular Interactions

胶质母细胞瘤干细胞在模式神经元上共同培养，以研究迁移和细胞相互作用

Glioblastomas (GBMs), grade IV malignant gliomas, are one of the deadliest types of human cancer because of their aggressive characteristics. Despite ...