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，20％FBS，5毫克/毫升肝素，100μg/ ml的内皮细胞生长因子，和L-谷氨酰胺）在microslide孵育20分钟，在室温。在灌注，准备HUVEC细胞悬液。 </li><li>冲洗脐静脉内皮细胞用PBS加0.05％胰蛋白酶-EDTA在室温1-2分钟。离心机在内皮细胞在180×g离心5分钟2毫升生长培养基。 </li><li>使用neuba测量细胞浓度UER血球并准备10 7个细胞HUVEC微升生长培养基。然后，小心地从用200微升枪头microslide的入口取出介质。 </li><li>使microslide到眼睛的高度，并用1毫升的鲁尔锁注射器，轻轻灌注200μl的脐静脉内皮细胞的制备的浓度的进入通道。注意，需要在这一步，以防止泡沫的形成。如果出现气泡，保持灌流一个稍长的时间，直到气泡进入出口通道。 </li><li>放HUVEC媒体等体积的（约80微升）在这两个入口和microslide的插座。这可以防止细胞在两个方向上的流动。 </li><li>覆盖滑动，并保持它在培养箱（37℃）为1.5小时。 </li></ol> 2，准备流室组件的隔夜培养的HUVEC在Microslide <ol><li>放置一个无菌的20毫升注射器，女性和男性的鲁尔连接器，管道和注射器泵在培养箱中培养15分钟Ñ​​。 </li><li>对于最小的死体积，使用与0.04英寸内径的管道。较小的管径可以防止泡沫的形成。 </li><li>用温水（37℃）介质内皮细胞（12毫升）填充20毫升注射器。安装与连接器的油管到注射器。除去气泡。这个组件连接到microslide。 </li><li>完全充满HUVEC媒体microslide的入口。使填充20毫升的注射器连接到连接器旁边的microslide。轻轻将连接器连接到microslide。 </li><li>使集上放入培养箱，并将其连接到注射器泵中，设置在10微升/分的剪切速率。离开细胞O / N在孵化器在37°C </li><li>第二天，通过去除附着在microslide的入口连接器拆卸的设置。 </li><li>上调内皮细胞上的E-选择素的表达，制备含IL-1β在10在4毫升媒体纳克/毫升的新鲜生长培养基。吸媒体在10毫升注射器。除去叔他从microslide的入口媒体和注射器连接到幻灯片中。 </li><li>设定注射器泵以10微升/分的剪切速率为在孵化4小时。 </li></ol> 3，抗PSMA的制备（J591-488）标记的前列腺癌细胞<ol><li>在内皮细胞中IL-1β孵育，制备抗PSMA J591-ALEXA488标记前列腺癌细胞。添加0.05％胰蛋白酶-EDTA对MDA细胞1分钟。请勿将细胞胰蛋白酶更长的时间，因为它可以影响存在于细胞表面的糖肽。酶无细胞解离试剂，也可用于代替胰蛋白酶。离心机在200×g离心5分钟。 </li><li> MDA重悬细胞沉淀于1毫升的H / H缓冲液（Hanks平衡盐solution/0.1％HSA/10毫米肝素钠/ 1毫米氯化钙2）。添加抗PSMA J591-ALEXA488抗体在黑暗的地方20微克/毫升在室温30分钟。在温育过程中重悬细胞。 </li><li> 30分钟后，离心分离，在800×g离心5分钟，将细胞溶液。 ASPI率和悬浮颗粒在1ml H / H缓冲液中。计数标记的MDA的细胞和使终浓度为1×10 6细胞/ ml。填补5毫升注射器J591-488标记的MDA细胞及消除气泡。 </li></ol> 4，抗PSMA（J591-488）标记的CTC富集的前列腺癌患者的制备<ol><li>收集7.5毫升血液前列腺癌患者在一个蓝色的帽筒（含柠檬酸钠）。轻轻地稀释血液1:1 0.1％BSA / 1毫摩尔EDTA / PBS中。 </li><li>加5.3毫升的Ficoll-Paque上加在50ml的锥形管中。层稀释血液的菲可帕克的顶部。离心机在400×g离心30分钟。 </li><li>预涂所有的管子或提示来在接触的CTC用2％FBS/RPMI-1640 / 1毫米氯化钙2/4毫摩尔MgCl 2（R / S缓冲液），以防止的CTC的非特异性结合到表面上。维护媒体和缓冲区来与接触的CTC 4°C的温度。 </li><li>收集的外周血单核细胞（PBMCs）中馏分含的CTC从另一个50毫升锥形管中装有30ml的R / S缓冲器的接口。离心机在400×g离心8分钟。 </li><li>重悬沉淀，并在400×g离心8分钟的R / S缓冲洗一遍。离心后，沉淀重悬在H / H缓冲液中。添加抗PSMA J591-488抗体在黑暗的地方20微克/毫升在室温30分钟。 </li><li> 30分钟后，离心分离含J591-488标记的CTC，在800×g离心5分钟的PBMC。重悬在H / H缓冲。填补1-ml注射器（预涂覆的R / S缓冲液中）与样本。 </li></ol> 5，准备显微镜，注射泵和流动室组件<ol><li>打开倒置显微镜和设置柯勒照明在10X物镜。使注射器泵以相同的水平显微镜的试样台上，并将其设置为1达因/厘米2的剪切应力（〜10微升/分钟）。 </li><li>打开蔡司的AxioVision软件。选择计算机屏幕上的目标。创建一个新的在该软件中的工具选项文件夹。 </li><li>在的AxioVision打开智能实验选项，并调整设置为30分钟记录短短30秒的视频。 </li><li>对于实时荧光的视频，设置图像选项 - 12毫秒曝光，2×2箱，5增益。这些参数，可以在实现视频接近视频帧速率（〜每秒23帧）与蔡司MRM相机。 </li><li>可视化的流道的整个宽度在计算机屏幕上10 X目标，使用C型安装转接器（0.63 xf/60 mm接口）。 </li><li>开关上的水银灯。 </li><li>将注射器和含有细胞的连接器上的注射器泵。 </li><li>带来的IL-1β刺激的HUVECs从孵化器。请将此接头连接到含有内皮细胞的microslide的填充进口通道。 </li><li>连接的出口通道与连接到管道的连接器。把管子插入一盘菜或15毫升锥形管通过收集流量。 </li><li>启动infusi上通过microslide以10μl/分钟。观察内皮细胞和MDA标记细胞或下488纳米过滤器是来自于患者的落射荧光显微镜标记的CTC之间的相互作用。 </li><li>开始记录实验的30秒短片。在播放过程中分析，测量滚动速度。轧制速度除以细胞随时间移动的距离测量。 </li></ol>该Microslide 6。免疫染色<ol><li>对IL-1β刺激血管内皮细胞MDA灌注细胞10分钟后，从microslide的入口和出口中取出介质。 </li><li>用200微升尖，放温水（37℃）PBS中含有钙和镁成microslide 5分钟的入口。利用其作为盖在板凳上的一个道具倾斜microslide。保持microslide在一个倾斜的位置为免疫染色在所有的温育。 </li><li>修复血管内皮细胞由暖灌注2％的甲醛进入入口</li><lI&gt;孵育20分钟，在RT。用PBS漂洗两次，每次5分钟。 </li><li>添加曲拉通X-100（0.1％）在PBS中5％BSA于室温10分钟。 </li><li>用PBS漂洗两次，每次5分钟。用在PBS中的5％BSA阻断30分钟。 </li><li>孵育一抗山羊抗VE-钙粘蛋白（1:100）在2.5％BSA的O / N在4℃下</li><li>第二天，用PBS洗两次，每次5分钟。添加辅助驴抗山羊647抗体45分钟。用PBS，每次5分钟洗涤两次。 </li><li>在室温下孵育以人源化J591-ALEXA488缀合的抗体1小时。 </li><li>用PBS，每次5分钟洗涤两次。加DAPI 5分钟。用蒸馏水5分钟洗净。 </li><li>添加PBS（或MOWIOL对于长期贮存）。可视化的共聚焦显微镜下microslide。 </li></ol>

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班德尔博士是分配给康奈尔大学研究基金会（“CRF”）为本文中所使用的J591抗体专利的发明者。班德尔博士是一名顾问，并拥有股票BZL生物制剂，公司以该专利已经授权由CRF的进一步研究和开发。

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。

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			<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

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。

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

Metastasis is a process in which tumor cells shed from the primary tumor intravasate blood vascular and lymphatic system, thereby, gaining access to ...

in-vitro-method-to-observe-e-selectin-mediated-interactions-between

Research

JoVE Journal

Medicine

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

9.0K 次观看.  Weill Cornell Medical College. 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。 

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

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

人牙髓干细胞的分离，鉴定和比较性恒牙使用两种不同的方法，得出

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

科研

医学

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

癌症干细胞从人类前列腺癌样本隔离

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Generation of Induced Pluripotent Stem Cells from Muscular Dystrophy Patients: Efficient Integration-free Reprogramming of Urine Derived Cells

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with muscular dystrophy is an invaluable cellular source for in&#160;vitro disease model systems and can be used for drug screening studies. Patient-derived urine cells have been used in successful reprogramming into induced pluripotent stem cells in order to model dystrophic cardiomyopathy1. Addressing the safety concerns of integrating vector systems, we present a protocol using a non-integrating Sendai virus vector for transduction of Yamanaka factors into urine cells collected from patients with muscular dystrophy. This protocol generates fully reprogrammed clones within 2&#8211;3 weeks. The pluripotent cells are vector-free by passage-13. These dystrophic iPSCs can be differentiated into cardiomyocytes and used either to study disease mechanisms or for drug screening.

This protocol entails detailed procedures for isolation of urine derived cells from muscular dystrophy patients; their efficient and rapid reprogramming through Sendai virus transduction.

从生成性肌营养不良患者诱导多能干细胞的：尿源性细胞的有效整合，免费重编程

Dystrophic cardiomyopathy is a poorly understood consequence of muscular dystrophy. Generating induced Pluripotent Stem Cells (iPSCs) from patients with ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

在卵巢癌肿瘤起始细胞体外富集

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

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

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

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

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

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

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

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

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 patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

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 ...