Name: a cancer cell spheroid assay to assess invasion in a 3d setting
Uploaded: 2015-11-20T15:00:00
Description: Watch this Scientific Journal Video about A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting at JoVE.com

由美国国立卫生研究院资助P30 CA051008和T32 CA009686（ATR）的支持。

1.球体产生的<ol><li>制备单细胞悬浮液通过分离用的PBS 70％汇合的贴壁〜癌细胞培养物悬滴培养洗涤随后暴露于0.05％的胰蛋白酶-EDTA溶液。 <ol><li>中和与细胞培养基的胰蛋白酶溶液和计数用细胞悬浮液的等分试样的细胞。注意：特定细胞培养基将取决于细胞系进行测试。遵循ATCC建议媒体，其中该细胞培养基是通常的DMEM + 10％FBS。更多信息可在材料表中找到。 </li><li>执行稀释，以允许每细胞培养?...

<ol>
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本研究评估癌细胞系面板的性能在一个球体侵袭测定（ 表1）。一般地，我们发现，球体的形成增强了更多上皮前瞻性的细胞系，其中细胞 - 细胞连接的存在促进了球体状结构的形成。已知已经历上皮间质转变的细胞系，如MDA-MB-231细胞不形成在最有可能的悬滴培养球状体由于它们的减小E-，N-，和P-cadherin表达16。 一些在球体侵袭测定的步骤需要密切遵守的...

经确定癌症细胞运动是预测转移潜能的，因为这样的行为通过基底膜和进入循环系统1促进侵袭。大多数研究蠕动集中于细胞如何表现在二维（2D）的设置，虽然它变得普遍认识到，细胞在三维（3D）矩阵的运动是更具代表性如何这些相同的细胞将实际的表现在体内 2。三维培养系统正越来越多地用于研究的细胞行为，范围从细胞形态，对生长动力学和药物敏感性3。到3D环境的上下文中监测癌细胞侵袭的愿望已经导致了通过悬滴培养法4，接着通过嵌入这些球体成3D胞涉及三维细胞聚集体（球状体）的产生先前建立的技术合成矩阵（ECM）的胶原蛋白和基底膜材料5组成。该方法旨在通过提供可以很容易地利用在各种实验条件下，比较入侵一个简化的方法来改善这些先前建立的技术。 更传统的方式来评估在体外细胞运动是划痕缠绕法和Transwell小实验6。前者测定描绘细胞运动在二维设置，因此独立的多种功能用于体内侵入关键的， 如蛋白酶活性7。 Transwell小测定可以更好的模型细胞入侵时井刀片涂覆有细胞外基质底物，但是，只有一个参数， 即细胞在相对 ​​膜表面的外观被测量，和细胞侵袭的许多细微差别，因此不是很容易观察到。相对于这些技术中，癌细胞球体侵袭测定（图1 &lt;/强&gt;）允许细胞侵入中的设置，不仅生理学相关的实时监控，而且还允许重要细胞系特有的功能而被可视化，如个人与集体细胞迁移8。这种方法也得到了标准的3D文化生长测定的优点。通过悬滴法细胞聚集的产生最初约束细胞运动，从而使细胞将被诱因侵入后该约束被提升。此外，一旦该约束被抬起，细胞出口将进行在一致的方向，然后可以方便地定量。 用于癌症细胞球体测定中最流行的ECM材料是基底膜和I型胶原，其中每个组件具有在影响转移行为重要和独特的作用。基质胶是由Engelbreth-Holm的群小鼠肉瘤细胞产生的蛋白质的分泌的混合物中，并富集basement的膜蛋白诸如层粘连蛋白，巢蛋白和IV型胶原9。出于这个原因基质胶是以下称为“基膜材料。”这些基底膜材料提供了所需的除了该施加的范围内的效果对细胞行为11许多其它蛋白质癌细胞侵袭10期间整合附着力必需配体。相比较而言，I型胶原，通常从肌腱等致密的胶原结构12的酸消化制备，是在作为结缔组织和间质支持组织的身体和器官的一个主要的结构元件更简单的基质材料。它已被证明胶原的物理特性可以调节的一些细胞运动的特征;例如，胶原纤维在肿瘤基质界面的取向允许癌细胞沿着这些原纤维侵入到基质13时，随后迁移。在这里介绍的试验中，两个I型胶原和基底膜材料被用作工具来研究三维癌细胞 - 基质相互作用。 抑制控制侵入可监控的细胞已经嵌入在三维矩阵后途径的或刺激的效果。细胞可以生长在挂滴剂或经转移到三维培养，这取决于一个漫长的处理是否需要调节入侵期间进行预处理。对于更短的处理，建议该药物可以与采集后的球体悬浮液，以及将包围3D培养，以促进适当的药物暴露于细胞的培养基混合。接着，正常或肿瘤相关的基质细胞可以混合与基质材料，以评估其在调节肿瘤细胞侵袭的作用，或以确定如何旁分泌和自分泌信号影响细胞的行为。这个想法是在一项研究显示，其中山坳共培养对癌细胞和内皮细胞在悬滴导致球状体14内的血管网。最后，将ECM成分也可以改变，如癌细胞的侵袭是通过不同的基板15的影响。下面提出的方法将由此提供用于评估在各种条件癌细胞侵袭的框架。一般来说我们发现，并非所有的细胞系将在悬滴创建球状体和上皮前瞻性细胞系通常形成定期球体。

<table>
	<tbody>
		<tr>
			<td>Matrigel Growth Factor Reduced</td>
			<td>Corning</td>
			<td>CB-40234</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I, Rat Tail, 100 mg</td>
			<td>Millipore</td>
			<td>08-115</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Life Technologies</td>
			<td>11995-065</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640 Medium&#160;</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Heat Inactivated</td>
			<td>Omega Scientific</td>
			<td>FB-12</td>
			<td></td>
		</tr>
		<tr>
			<td>100mm TC-treated Dishes</td>
			<td>Corning Incorporated</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well TC-treated Plates</td>
			<td>NEST Biotechnlology</td>
			<td>702001</td>
			<td></td>
		</tr>
		<tr>
			<td>Olympus IX-71 Inverted Microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4">Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and LNCaP cells, which were mantained in RPMI + 10% FBS.</td>
		</tr>
	</tbody>
</table>

a cancer cell spheroid assay to assess invasion in a 3d setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

使用球体侵袭测定（图1），肿瘤细胞系的组为他们以形成球状体，以及用于小区出口的在3D基质植入后表现出组成的基底膜材料和I型胶原蛋白的量的能力进行了测试（ 表1）。这些结果表明，并不是每一个细胞系将产生良好的球状体，其中细胞系具有体外上皮形态倾向于产生定期和光滑聚集体。不同的细胞系在测定侵入的能力也是可变的。已经建立的作为是侵入性...

Watch this Scientific Journal Video about A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting at JoVE.com

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

癌细胞球体检测，以评估入侵的3D设置

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

Research

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Modeling and Imaging 3-Dimensional Collective Cell Invasion

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Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

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It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

一种改进<em&gt;在体外</em&gt;入侵检测，以确定激素，在调节肿瘤细胞侵袭的细胞因子和/或生长因子的潜在作用

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

在实时的间皮细胞卵巢癌球体附件和入侵评估

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

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