Name: in vitro method to observe e-selectin-mediated interactions between prostate circulating tumor cells derived from patients and human endothelial cells
Uploaded: 2014-05-15T13:00:00
Description: Watch this Scientific Journal Video about In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells at JoVE

这项工作是由国防部，前列腺癌研究计划（W81XWH-12-1-0124）的资金支持，由美国国家癌症研究所和罗伯特·麦柯奕泌尿生殖系统肿瘤研究基金U54CA143876。我们要感谢Annarita洛伦佐博士（病理科）提供VE-钙粘蛋白抗体，和Marco Seandel博士（外科），以脐静脉内皮细胞。

1，培养内皮细胞上Microslides观察四氯化碳内皮的相互作用<ol><li>根据组织文化引擎盖，第一冲洗microslide，1毫米，用PBS通道宽度。轻轻外套用200μl的50微克/毫升用1毫升鲁尔锁注射器纤连蛋白（溶于PBS中）的microslide。 </li><li>盖上盖子的microslide，并保持它的组织培养罩内30分钟。在microslide液体的慢分配可防止气泡形成的通道。 </li><li>灌注200μl的温水（37℃）的HUVEC的生长培养基（M199媒体，1M HEPES，...

<ol>
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由于CTCs的血细胞中的数量低，这是难以分离的CTC作为信元的纯群体。为了研究四氯化碳/ EC相互作用，车辆检验中心的稀有和不纯的人口带来了两大挑战：1）血细胞中车辆检验中心鉴定的;二）四氯化碳/ EC相互作用观察。 为了克服识别血细胞中前列腺车辆检验中心的一个限制，我们注意到，几乎所有的前列腺肿瘤细胞表达PSMA 6的事实。单克隆抗体...

转移是一个复杂的多步骤的过程，仍然知之甚少。该E-selectin/selectin配体轴已被证明通过促 ​​进血管内皮细胞和癌细胞1,2之间主要粘附相互作用在肿瘤转移中发挥重要作用。内皮（E）-选择素是由活化的内皮细胞上表达的跨膜蛋白，而不同的E-选择素配体（s）是由肿瘤细胞表达3。许多体外方法已被成功地应用到模型的肿瘤细胞和内皮细胞之间E-selectin/selectin配体相互作用（ECS）1。为了研究这些相互作​​用，不同的流室系统被用来模拟血液血管系统。之间流动室组件，平行平板流动腔室（PPFC）与内皮细胞一起被常规用作体外模型模拟体内剪切应力条件。在这种方法，内皮细胞生长在35毫米培养皿，并实现了单层后，内皮细胞附着到PPFC和剪切应力为基础的实验中进行。 然而，PPFC和其它现有系统存在许多局限性，研究循环从患者和内皮细胞衍生的肿瘤细胞（CTCs的）之间的粘合相互作用主要是因为CTCs的是一种罕见的细胞群，从原发肿瘤脱落，其中数百万的血细胞循环（1 CTC每10 9血细胞）4。因此，无限量供应的培养细胞系的不同，低CTC计数导致很少和罕见的四氯化碳/ EC相互作用，需要适当的流道宽度来记录回放分析的相互作用。此外，由于来自患者的车辆检验中心是不纯的人口，因此识别标记是需要在特定的跟踪检验中心。为了解决这个问题，我们开发了一种新的方法，以确定前列腺癌（PCa）的CTCS通过利用几乎所有这些的CTC的表达前列腺特异性膜抗原（PSMA）在其细胞表面5,6的事实的优点。在这份报告中，我们使用了前列腺癌细胞株，MDA PCa2b（MDA）含量，以展示我们的新系统来研究前列腺四氯化碳相互作用与内皮细胞的潜在效用，最终以了解有无转移的机制。 我们的方法可以应用于各种基于剪切实验模拟体内血管系统7-9。除了检查前列腺四氯化碳/ EC相互作用，电流流室系统可以很容易地适应用于分析的外周血单核细胞或肿瘤细胞“与内皮的相互作用。拆卸和流动室的重新组装的简易性，一个microslideⅢ（0.1）（以下简称microslide），使培养内皮下灌注和刺激内皮细胞有不同的细胞因子以诱导PRotein表达。此外，培养的内皮细胞，重组蛋白，如E-和P-选择素，可涂覆在microslide和相互作用与肿瘤细胞可以层流条件10下被观察到。

<table border="0" cellpadding="0" cellspacing="0" width="744">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Microslide</td>
			<td style="width:192px;">Ibidi</td>
			<td style="width:119px;">80331</td>
			<td style="width:189px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Fibronectin</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">FC010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Plastic tubing</td>
			<td style="width:192px;">Tygon</td>
			<td style="width:119px;">AAQ04103</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Male luer adapter</td>
			<td style="width:192px;">GlycoTech</td>
			<td style="width:119px;">31-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Female luer adapter</td>
			<td style="width:192px;">GlycoTech</td>
			<td style="width:119px;">31-001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Syringe pump</td>
			<td style="width:192px;">Chemyx Inc</td>
			<td style="width:119px;">Fusion 100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Luer-lock syringe</td>
			<td style="width:192px;">BD Biosciences</td>
			<td style="width:119px;">309628</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">M199 medium</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">M7653</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Endothelial Mitogen</td>
			<td style="width:192px;">Biomedical Technologies</td>
			<td style="width:119px;">BT-203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">HBSS</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">H9269</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">Anti-PSMA J591-488</td>
			<td style="width:192px;">Weill Cornell Medical College-Lab of Urologic Oncology</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Interleukin-1 beta</td>
			<td style="width:192px;">Peprotech</td>
			<td style="width:119px;">200-01B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Trypsin</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">SM-2002-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Heparin</td>
			<td style="width:192px;">Sigma</td>
			<td style="width:119px;">H-3149</td>
			<td></td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:244px;">HUVECs</td>
			<td style="width:192px;">Weill Cornell Medical College-Department of Surgery</td>
			<td style="width:119px;"></td>
			<td>provided by Marco Seandel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">VE-Cadherin</td>
			<td style="width:192px;">Santa Cruz</td>
			<td style="width:119px;">sc-5648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">10x objective</td>
			<td style="width:192px;">Zeiss Plan Neofluar</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:244px;">Enzyme free cell dissociation reagent</td>
			<td style="width:192px;">Millipore</td>
			<td style="width:119px;">S-004-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">RPMI-1640</td>
			<td style="width:192px;">Lonza</td>
			<td style="width:119px;">12-702-F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Ficoll-paque plus</td>
			<td style="width:192px;">GE healthcare</td>
			<td style="width:119px;">17-1440-02</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vitro method to observe e-selectin-mediated interactions between prostate circulating tumor cells derived from patients and human endothelial cells

转移是一个过程，其中肿瘤细胞从原发肿瘤intravasate血液血管和淋巴系统散出，从而接触到外渗形成一个次级定位。肿瘤细胞从血液中血管系统的渗漏可以通过从不同的细胞系获得的内皮细胞（EC）和肿瘤细胞进行研究。最初的研究是使用静态条件下进行，但也得到了很好的记载，内皮细胞的生理流动条件下表现不同。因此，不同的流动腔室组件当前被用于研究内皮细胞与癌细胞相互作用。电流流室组件使用提供了在不同的剪切应力条件下两种不同的细胞系或液体可重复的结果。然而，观察和研究稀有细胞如循环肿瘤细胞（CTCs的）的相互作用，还需要一定的变化作出常规的流室组件。车辆检验中心是在数以百万的血细胞的罕见细胞群。其结果是，难以得到的CTC的纯群体。使用目前富集或耗损技术的CTC的污染与不同类型的细胞中循环通常存在是不可避免的。在本报告中，我们描述了一种独特的方法，以荧光标记的循环前列腺癌细胞，并研究在自组装的流动腔室系统它们与内皮细胞的相互作用。这种技术还可以进一步应用到观察前列腺的CTC和任何感兴趣的蛋白质之间的相互作用。

图1显示了内皮细胞上的microslide单层的O / N培养。 图1A上的定标显示，microslide 100％是可见用5X物镜，而70％是可见的用10倍的目标（ 图1B）。对于E-选择素介导的相互作用，细胞滚动的边缘不认为这使得microslide的70％以上，可用于视频录制和回放分析。根据我们的经验，最初这个设置程序集需要一些练习来培养内皮细胞的剪切应力作用下单层。在microslide建立EC?...

Watch this Scientific Journal Video about In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells at JoVE

In vitro Method to Observe E-selectin-mediated Interactions Between Prostate Circulating Tumor Cells Derived From Patients and Human Endothelial Cells

在体外的方法来观察派生病人和人内皮细胞前列腺循环肿瘤细胞之间的E-选择素介导的相互作用

我们的报告描述了一种独特的方法来可视化和分析生理流动条件下，在前列腺癌CTC / EC相互作用。

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Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

前列腺癌患者产生派生异种移植模型从循环肿瘤细胞

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

从人类肿瘤异种移植的小鼠细胞的消耗显着改善靶细胞下游分析

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...