Name: testing the vascular invasive ability of cancer cells in zebrafish (danio rerio)
Uploaded: 2016-11-03T15:00:00
Description: Watch this Scientific Journal Video about Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio at JoVE.com

We thank Peter Johnson of the Georgetown University Microscopy Core for assistance with imaging the zebrafish embryos. The Microscopy &amp; Imaging Shared Resource and the Zebrafish Shared Resource are partially supported by NIH/NCI grant P30-CA051008. This work was also supported by NIH/NCI CA71508 (AW) and CA177466 (AW).

伦理学声明：根据经批准的协议IACUC产生了斑马鱼的胚胎。这些实验是在遵守进行与由乔治城大学动物护理和使用委员会的建议。 1.组织胚胎注射和创建库存解决方案<ol><li>生成必要的斑马鱼来评估癌细胞血管浸润。 <ol><li>设置成对或组交叉交配与TG（kdrl：grcfp）的Zn1; mitfa b692; ednrb1 B140鱼。 注：我们产生的Tg（kdrl：grcfp）的Zn1;...

<ol>
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这种技术利用了斑马鱼模型来有效地测试癌症细胞（参见图1）的血管侵袭能力。在这里，我们采用的技术的乳腺癌细胞系的组，以提供一个基线到其上其他研究者然后可以建立他们自己的研究（ 见表1; 图2 - 3）。该MDA-MB-231细胞很容易地侵入到斑马鱼胚胎的尾部区域观察会使该细胞系适用于测试可能抑制血管浸润剂。相对较少创伤的细胞系，像HCC1806或MDA-MB-468...

转移性疾病是癌症死亡率和许多机制，使癌细胞传播有待发现1的一个主要原因。为了使癌细胞成功转移，它首先必须通过围绕一个原发肿瘤中，输入（intravasate）进入循环系统基质侵入，生存在从循环中转，出口（渗出），最后建立一个可行的菌落在远处器官的网站2。血管内和外渗因此在转移级联关键步骤，但每一个癌细胞是不是破坏，并通过内皮连接3迁移本身娴熟。事实上，有一系列的周围癌细胞的血管侵袭和方法可以通过多种4内源性和外源性因素的进一步影响独特的选择压力。由于这些原因，该探头的晚期癌症的攻击行为技术通常专注上的vascular浸润能力作为一种手段来预测转移扩散。 各种模型系统中，以方便癌细胞血管浸润的体外研究。 在体外测定法中最常用的涉及任一的transwell系统通过内皮屏障5或电动细胞-基底阻抗传感（ECIS）技术评估癌症细胞迁移并监控由癌细胞6完整内皮单层的实时中断。这些测定通常缺乏流体动力学和基质因素，否则将影响癌细胞附着到内皮壁上。这个问题在某种程度上被从内皮细胞的三维培养与出现支持基质细胞perfusable血管网络规避，而这些3D微流体系统现在代表目前在体外选项7,8前列。尽管如此，这些方法省略官能循环系统的鲁棒微因此只在体内模型中部分替代品。 最广泛使用的血管浸润的体内模型是鼠标，其中因为它们发生在相对短的时间尺度，并且一般指示转移能力9实验性转移测定法，通常进行。这些分析包括直接注入肿瘤细胞进入流通，因此转移，器官即外渗和癌细胞定植结束阶段模型。实验转移测定不同根据肿瘤细胞注射的部位和器官，最终分析。在第一测定类型，癌症的细胞注射到小鼠和癌细胞播种在肺部尾静脉监视10,11。第二测定涉及执行腔内注射直接朝向骨微环境12-14转移性播种，而且脑15。在第三个实验中，癌症Ç厄尔注入脾脏以允许肝脏16的定植而第四传送路径进入颈动脉进行癌细胞到大脑17,18。无论癌细胞交货方式，器官殖民化是接受实验的端点，一般是通过发光，组织学，或基于PCR的技术来确定。尽管小鼠主机中进行实验测定转移的生理优势，这些实验仍然需要几个星期到几个月才能完成和分析。 斑马鱼（ 斑马鱼 ）模型最近出现的一个新的系统来研究癌症进展19,20，并允许癌细胞血管浸润过短得多的时间尺度的功能循环系统内的评估时，小鼠相比，21-24。该方法利用有其内皮标记有一个绿色的珊瑚暗礁FLUO透明斑马鱼品系rescent蛋白报告由kdrl启动子，血管内皮生长因子25的斑马鱼受体驱动。在测定中，癌细胞被标记有红色荧光标记物，并注入2日龄胚胎precardiac窦。间48到96小时的任何地方的注射后，即已经侵入了脉管的和成胚胎的尾部区域癌细胞可以有效地在荧光显微镜评分。在这里，我们采用的技术中常用的人乳腺癌细胞系组成的小组，证明他们的血管侵袭能力明显的差异。此外，我们证明，改变注射部位的胚胎卵黄囊允许异质细胞相互作用的研究，如癌症的细胞群可以用荧光染料进行差异标记，并注射入缺乏荧光脉管斑马鱼的胚胎。在后一种测定法中，已经侵入蛋黄和癌细胞intravasated入vasculaturE分别打进尾区域24注射后48小时。由于该模型的有效性和方便性，斑马鱼日益用于快速测试下一个生理设置癌细胞的血管侵袭能力。

<table border="0" cellpadding="0" cellspacing="0" width="1253">
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			<td height="21" style="height:21px;width:491px;">0.05% Trypsin-EDTA</td>
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			<td height="21" style="height:21px;">100mm Dishes</td>
			<td>Corning Incorporated</td>
			<td>3160-100</td>
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			<td height="21" style="height:21px;">5 3/4&#34; Disposable Pastur Pipets, borosilicate Glass</td>
			<td>Fisher Brand</td>
			<td>13-678-20B</td>
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			<td height="21" style="height:21px;">60 mm Dish</td>
			<td>Corning Incorporated</td>
			<td>3160-60</td>
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			<td height="21" style="height:21px;">Agarose, Low Melting</td>
			<td>Fisher</td>
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			<td height="21" style="height:21px;">Agarose, Molecular Grade</td>
			<td>Bioline</td>
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			<td height="21" style="height:21px;">Capillary Glass, Standard, 1.2MM x 0.68MM, 4&#34;</td>
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			<td>Omega Scientific</td>
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			<td height="21" style="height:21px;">Fisherbrand Transfer Pipettes</td>
			<td>ThermoFisher Scientific</td>
			<td>13-711-7M</td>
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			<td height="21" style="height:21px;">Latex Rubber Bulbs, 2mL, Pack of 72</td>
			<td>Heathrow Scientific</td>
			<td>HS20622B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SP8 Confocal Microscope</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulator</td>
			<td>Narishige</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eclipse E600</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Life Technologies</td>
			<td>10010-023</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-G Potassium</td>
			<td>Fisher Biotech</td>
			<td>BP914-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Plates, 100mm x 15mm</td>
			<td>Fisher Brand</td>
			<td>&#160;FB0875713</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Picospritzer II</td>
			<td>General Valve Corporation</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium&#160;</td>
			<td>Life Technologies</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin Sulfate</td>
			<td>Fisher Biotech</td>
			<td>BP910-50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vybrant DiI</td>
			<td>ThermoFisher Scientific</td>
			<td>V22885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vybrant DiO</td>
			<td>ThermoFisher Scientific</td>
			<td>V22886</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Zebrafish&#160;</td>
			<td>Georgetown Zebrafish Shared Resources</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td colspan="3" height="21" style="height:21px;">Cell lines were maintained in DMEM + 10% FBS, with the expection of BT-474 and HCC18-6 cells, which were mantained in RPMI + 10% FBS.</td>
			<td></td>
		</tr>
	</tbody>
</table>

testing the vascular invasive ability of cancer cells in zebrafish (danio rerio)

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression. Traditional in vitro models of cancer cell invasion of endothelia typically lack the fluid dynamics that invading cells are otherwise exposed to in vivo. However, in vivo systems such as mouse models, though more physiologically relevant, require longer experimental timescales and present unique challenges associated with monitoring and data analysis. Here we describe a zebrafish assay that seeks to bridge this technical gap by allowing for the rapid assessment of cancer cell vascular invasion and extravasation. The approach involves injecting fluorescent cancer cells into the precardiac sinus of transparent 2-day old zebrafish embryos whose vasculature is marked by a contrasting fluorescent reporter. Following injection, the cancer cells must survive in circulation and subsequently extravasate from vessels into tissues in the caudal region of the embryo. Extravasated cancer cells are efficiently identified and scored in live embryos via fluorescence imaging at a fixed timepoint. This technique can be modified to study intravasation and/or competition amongst a heterogeneous mixture of cancer cells by changing the injection site to the yolk sac. Together, these methods can evaluate a hallmark behavior of cancer cells and help uncover mechanisms indicative of malignant progression to the metastatic phenotype.

在这里，我们测试了在斑马鱼胚胎模型常用乳腺癌细胞系（ 图1）的血管侵袭能力。严格的标准在得分外渗被用于这些不同的细胞系，其中，如果癌细胞已经明确外渗积极事件只算的，这之中主要是做以限制可从得分细胞碎片出现的任何误报。 我们的分析显示，该线之间明显的差异时侵入被注入precardiac窦（ ...

Watch this Scientific Journal Video about Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio at JoVE.com

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish Danio Rerio

这种方法利用斑马鱼的胚胎以有效地测试癌细胞的血管侵袭能力。荧光癌细胞注射到发育中的胚胎的precardiac窦或卵黄囊。癌细胞血管浸润和渗出通过尾部区域的荧光显微镜24至96小时后评估。

测试癌细胞的斑马鱼的血管侵袭能力（<em&gt;斑马鱼</em&gt;）

Cancer cell vascular invasion and extravasation is a hallmark of metastatic progression.  Traditional in vitro models of cancer cell invasion of ...

Research

JoVE Journal

Cancer Research

Testing the Vascular Invasive Ability of Cancer Cells in Zebrafish (Danio Rerio)

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

科研

癌症研究

Invasive Behavior of Human Breast Cancer Cells in Embryonic Zebrafish

在斑马鱼胚胎人乳腺癌细胞的侵袭行为

In many cases, cancer patients do not die of a primary tumor, but rather because of metastasis. Although numerous rodent models are available for studying ...

A Time-lapse, Label-free, Quantitative Phase Imaging Study of Dormant and Active Human Cancer Cells

休眠和活性人肿瘤细胞的时间推移、无标签、定量相成像研究

The acquisition of the angiogenic phenotype is an essential component of the escape from tumor dormancy. Although several classic in vitro assays (e.g., ...

In Vivo Targeting of Xenografted Human Cancer Cells with Functionalized Fluorescent Silica Nanoparticles in Zebrafish

斑马鱼中具有功能化荧光硅纳米粒子的异种移植人类癌细胞的Vivo靶向

Developing nanoparticles capable of detecting, targeting, and destroying cancer cells is of great interest in the field of nanomedicine. In vivo animal ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

斑马鱼拉瓦尔异种草和肿瘤行为分析的一代

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

肝细胞癌原位小鼠模型中的无创PET/MR成像

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

基于同步成像和流式细胞术检测治疗诱导的衰老癌细胞中的多种荧光衰老标志物

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

通过颈内动脉注射癌细胞模拟脑转移

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Zebrafish Larvae as a Model to Evaluate Potential Radiosensitizers or Protectors

斑马鱼幼虫作为评估潜在放射增敏剂或保护剂的模型

Zebrafish are extensively used in several kinds of research because they are one of the easily maintained vertebrate models and exhibit several features ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

小鼠患者来源的癌细胞原位植入概括了晚期结直肠癌

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...