这项工作是由癌症研究协会安大略省（＃08NOV230），加拿大创新基金会（＃13199），前列腺癌加拿大，扬森肿瘤，在伦敦地区癌症计划，并从约翰和唐娜布里斯托尔通过捐助者的支持赠款支持伦敦健康科学基金会（以ALA）。 LEL是由弗雷德里克班廷和查尔斯最佳加拿大研究生奖学金博士奖的支持。 ALA是由CIHR新研究者奖，并从研究和创新的安大略省的早期研究者奖支持。

在这个手稿中描述的所有的人进行了研究，根据批准的西方大学的人类研究伦理委员会的协议。所有动物实验均按照加拿大理事会关于动物保护的建议进行的，根据批准的西方大学动物使用小组委员会的协议。 1。标准四氯化碳枚举从病人的血液样本使用的CSS 1。人体血液样品采集和加工准备的制备仪器<ol><li>使用标准无菌采血技术，绘制一个最小为8.0毫升人类血液到10ml CellSave管（以下简称为CTC防腐剂管），其中包含乙二胺四乙酸（EDTA）和专有的蜂窝防腐剂。逆转管5倍，以防止血液凝固。样本可立即被处理或在室温下保存长达96小时。 </li><li>祛瘀从冰箱里éCSS的试剂，并允许他们使用前加热到室温。 </li><li>使用一次性装置10毫升吸管和自动移液器，收集7.5毫升血液从四氯化碳防腐管，慢慢地血液分配到一个适当标记的制备仪器加工管。 </li><li>加6.5毫升的稀释缓冲液对每个样品。颠倒样品5倍混合。在800×g离心10分钟，在“关”位置的制动离心样品。请按照准备仪器的屏幕上的说明来加载所有患者样本在系统上进行处理。样品必须在1小时内准备处理。 </li></ol> 2。控制加工配制的制备仪器<ol><li>然后振荡控制小瓶和反转5X混合。 </li><li>小心地控制瓶取下瓶盖并放置一个倒置的准备仪器加工管上不封顶的小瓶的顶部。在一个迅速的动作，反转管制瓶，倒内容到处理管。而倒，轻轻一抖控制小瓶的两侧，以排出残留的内容。 </li><li>小心地从处理管取出反转控制小瓶中，确保没有液体丢失，并放置在一边。使用1,000微升移液器，收集小瓶和盖任何剩余的内容，轻轻地吸取到处理管。 </li><li>按照关于编制仪器屏幕上的指示来控制加载到系统中进行处理。 </li></ol> 3。对分析仪器的样品扫描<ol><li>请按照准备仪器的屏幕上的说明卸载所有样品从系统中。松散封顶每个磁性装置盒，用双手或实验室工作台上释放被粘在盒的边缘任何气泡挖掘磁性器件。一旦所有的泡沫已被删除，坚决帽筒，奠定了磁性器件平坦，而我ncubate在黑暗中为至少20分钟，在室温下进行。样品必须在准备24小时扫描。 </li><li>打开分析仪和初始化灯泡。一旦回暖（约15分钟），加载系统验证匣放入分析仪器并选择QC测试选项卡。按照屏幕上的说明进行必要的质量控制措施。 </li><li>样品加载到分析仪器并选择病人测试标签。从准备仪器内的所有保存的信息将被显示。单击开始初始化样本扫描。该系统将磁器件盒上进行粗调焦和边缘检测。 </li><li>调整所有的边按需要使用方向键。选择接受 。该系统将进行微调对焦，并开始样品扫描。 </li><li>下面的控制扫描的结果，应使用尖高（CK + DA对细胞的定义的标准进行验证PI + CD45 - APC +）和低（CK + DAPI + CD45 - FITC +）的浓度。下列患者样本扫描结果应使用四氯化碳定义的标准（CK + DAPI + CD45 - ）捕获的CTC进行审查。 </li></ol> 2。 CTC表征使用CSS的用户自定义标记 1。用户自定义的标记和仪器初始化准备<ol><li>稀释的使用下面的公式，其中，工作浓度是抗体除了样品和库存浓缩后的浓度使用债券初级抗体稀释液中的标记试剂杯的期望浓度感兴趣的抗体是抗体的浓度试剂杯。对于多个样品，调整为表1中所述的抗体量。将标记试剂杯到试剂盒和L在位置1OAD墨盒上的CSS。 股票集中度=（工作浓度X 850微升）/ 150微升 </li><li>收集血液，制备样品，并载入准备仪器如上面的标准四氯化碳枚举从病人的血液样本使用了CSS协议描述。要启用自定义标记此外，选择用户定义检测时的准备仪器提示。输入标记名称，然后选择保存 。由于样品装载到机器上，操作员将被提示来指示应根据需要选择是或否接收自定义标记。 </li></ol> 2。对分析仪器用户定义的标记样品扫描<ol><li>打开分析仪，初始化灯，并作为标准四氯化碳枚举的第3.2节从病人的血液样本使用CSS的描述进行质量控制和系统验证</sTRONG&gt;的协议。 </li><li>样品加载到分析仪器，然后选择设置选项卡。要初始化FITC通道，选择CellSearch四氯化碳作为下测试协议部分套件的ID。从这个菜单中，选择四氯化碳研究 ，单击编辑按钮，并根据需要设置曝光时间。建议在1.0秒的曝光时间不使用的CSS四氯化碳组件时，因为这可以提高渗色成用于反恐委员会确定的其他荧光通道超标。 </li></ol> 3。标准CSSProtocol适应于临床前小鼠模型的使用 *从Veridex的鼠标/鼠CellCapture套件改编（不再市售） 1。小鼠血液收集和存储<ol><li>前采血，跑〜30微升0.5M的EDTA通过22号针头来回，留下少量的EDTA的枢纽。 </li><li>收集至少50微升小鼠血液从小鼠通过原位，尾静脉或心内途径与人类肿瘤细胞预先注入。收集血液从大隐静脉（串行CTC分析）或通过心脏穿刺（终端CTC分析）。卸下针和血液分配到1毫升EDTA的微量血液收集管。通过倒置或轻轻一抖管混匀，以防止血液凝结。长达48小时，在加入CytoChex蜂窝防腐剂等体积的血液可以被立即处理或在室温下保存。 </li></ol> 2。 CTC富集<ol><li>从冰箱中取出的CSS试剂，并允许他们使用前加热到室温。 </li><li>转移的50微升全血的相当于12毫米×75毫米流式细胞管。加入500微升的稀释缓冲液中向每个样品，洗下残留在管的两侧任何血液。如果需要的话，短离心机旋可用于收集任何残留的血液。 </li><li>然后振荡的抗-EpCAM的铁磁流体，并添加25μl的各样品通过将移液管的尖端直接进样。加入25微升捕获增强试剂和旋涡轻轻混匀。孵育的样品在室温下放置15分钟。 </li><li>放置样品管放置在磁体并孵育10分钟。而试样管仍然在磁体中，使用玻璃吸管小心吸出残留的液体不接触所述管的壁旁的磁铁并丢弃。 </li></ol> 3。 CTC法<ol><li>从磁体，重悬在50μl核酸染料，加入50μl染色试剂，1.5微升抗小鼠CD45-APC，5.0微升的抗人HLA-的Alexa Fluor 488，和100微升的透化的的取出样品管试剂。对于多个样品，这些试剂可预混和206.5微升的混合物可以被加入到每个管中。涡轻轻混匀孵育20分钟，在室温下在黑暗中。 </li><li>加入500微升的稀释缓冲液，轻轻涡旋，这里的样品管插入磁铁，并孵育10分钟。而试样管仍然在磁体中，使用玻璃吸管小心吸出残留的液体不接触所述管的壁旁的磁铁并丢弃。从磁体取出样品管，并重新悬浮于350微升的稀释缓冲液。轻轻旋涡混匀。 </li></ol> 4。磁性设备装载<ol><li>使用凝胶加载提示，小心地从样品管的整个体积转移到磁性装置中的墨盒。开始在盒的底部，并慢慢抽出尖的样品分配。 </li><li>一旦整个样品已转移，松松盖住磁性装置盒，用双手或实验室工作台上释放在SECTIO描述了被粘在盒的边缘任何气泡挖掘磁性器件N 从使用的CSS病人的血液样本标准四氯化碳枚举的3.1。 </li><li>用无菌22号针头的斜面和卡盒的边缘之间将它们俘获弹出任何气泡。一旦所有的泡沫已被删除，坚决帽筒，奠定了磁性器件平坦，孵育在黑暗中至少10分钟。样品必须在准备24小时扫描。 </li></ol> 5。对分析仪器手工分离样品扫描<ol><li>打开分析仪，初始化灯，并作为标准四氯化碳枚举的使用CSS的协议病人的血液样本 3.2节所述的方法进行质量控制和系统验证。 </li><li>样品加载到分析仪器，然后选择设置选项卡。通过单击格式示例按钮清除磁器件的数据按钮的任何现有数据。使FITC通道次的曝光时间设置为1.0秒如四氯化碳表征使用CSS用户定义的标记第2.2节所述。 </li><li>点击病人测试选项卡上，选择编辑输入样品信息。选择CellSearch四氯化碳作为工具包的ID和CTC作为研究的测试协议 。输入的星号所指示的其余必要的信息。 保存样品信息，并单击开始 。 </li></ol>

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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+epithelial-mesenchymal+transition-inducing+transcription+factors+in+primary+breast+cancer:+The+effect+of+neoadjuvant+therapy.">Expression of epithelial-mesenchymal transition-inducing transcription factors in primary breast cancer: The effect of neoadjuvant therapy.</a> Int. J. Cancer. 130 (4), 808-816 (2012).</li><li>Yu, M., Bardia, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+breast+tumor+cells+exhibit+dynamic+changes+in+epithelial+and+mesenchymal+composition.">Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition.</a> Science. 339 (6119), 580-584 (2013).</li><li>Armstrong, A. J., Marengo, M. 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笔者（ALA）收到由扬森肿瘤，其母公司强生公司还拥有扬森诊断有限责任公司，主要生产在这篇文章中使用的试剂和仪器提供的资金。

尽管自2004年实行了CSS的许多新的反恐技术的发展，这种技术仍然是唯一在市场上临床批准的技术今天，因此它被认为是目前的黄金标准CTC检测和计数。这个手稿表明，虽然CSS有严格的质量控制标准也可以是有待诠释偏见和四氯化碳识别患者样本是从识别太大的不同加标样品中。确定了六大类常用物品不符，可能会导致四氯化碳错误分类的发生。这些差异的项目突出了需要对每一个病人样品多个审...

在2013年估计580,350人死于癌症，这种疾病是1660290新病例被诊断在美国就有1。其中大多数死亡发生转移性疾病2的后续发展。目前尚缺乏有效的治疗方法在治疗转移灶和转移级联的了解有限，使这一阶段的疾病死亡率很高。血液中的循环肿瘤细胞（CTCs的）的存在已被证实与转移性疾病3。这些细胞是极为罕见的，他们的检测是反映整体生存的转移性乳腺癌4，前列腺癌5和6大肠癌癌。在这些患者中，存在≥时相比，这些患者具有更少或在SAM中没有可检测到的CTC 5（乳腺癌和前列腺癌）或≥3（大肠癌），7.5毫升血液的CTC指示预后较差的Ë血容量。此外，期间或之后的治疗干预中的四氯化碳数的变化已被证明是有用的治疗响应的预测，通常早于目前所使用的技术7-10。 据估计，在转移性癌症患者中，发生的CTC在约1 CTC每10 5 -10 7血单核细胞的频率和患者的局部疾病，这个频率可以是什至更低（〜1 10 8）。这些细胞的罕见的性质可以使其难以精确地和可靠地检测和分析的CTC 11。有几种方法（先前12-14审阅）已用于通过利用从周围血液成分区分它们的属性来丰富和检测这些细胞。在一般情况下，四氯化碳枚举是一个两部分的过程，既需要一个浓缩步骤和检测步骤。传统上，富集步骤依赖于物理差异车辆检验中心（细胞大小，密度，变形）或蛋白质标志物的表达（ 即上皮细胞粘附分子[EpCAM的]，细胞角蛋白[CK]）的iCal属性。以下浓缩，CTC检测可以以许多不同的方式，其中最常见的是基于核酸的测定法和/或流式细胞术的方法来进行。这些策略都是独一无二的，具有不同的优点和缺点，但他们都缺乏标准化;一个必要进入临床设置。因此CellSearch系统（CSS）的开发是为了提供一个标准化的方法，用荧光显微镜和基于抗体的技术4-6在人体血液中罕见的CTC的检测和计数。该平台目前正在考虑在CTC枚举的金标准，是在临床使用15通过了美国食品和药物管理局（FDA）唯一技术。 这个CSS是一种双组份平台consistin克，（1）本CellTracks AutoPrep系统（以下简称为制备仪），它能够自动人体血液样品的制备方法，和（2）的CellTracks分析器II（以下简称为分析仪），该扫描这些下面的样品制备。从污染的白细胞准备仪器分辨的CTC采用介导的抗体，铁磁流体基磁性分离法与差分荧光染色。最初，该系统采用共轭铁纳米颗粒抗EpCAM抗体标记的CTC。然后将样品孵育在磁场中，并且所有未标记的细胞被吸出。选择的肿瘤细胞重新悬浮，并孵育在差分荧光染色，包括荧光标记的抗体和核染色试剂。最后，将样品转移到一个磁盒，称为MagNest（以下简称为磁性装置），以及SCanned使用分析仪器。 该分析仪被用来使用不同的荧光过滤器，每个优化，以适当的荧光颗粒，使用10X物镜扫描准备样品。的CTC被确定为是由抗EpCAM，抗泛CK-藻红蛋白（PE）结合的细胞（CK8，18，和19），以及核染色剂4&#39;，6 - 二脒基-2 - 苯基吲哚（DAPI）。相反，白细胞污染被确定为是由抗CD45-别藻蓝蛋白（APC）和DAPI结合的细胞。下面扫描，计算机定义的潜在的肿瘤细胞被呈现给用户。从这些图像时，用户必须使用定义的参数和差分染色上面所讨论的，以确定哪些事件的CTC采用定性分析。 除了四氯化碳枚举提供了一种标准化的方法，该CSS允许的CTC的基于感兴趣的蛋白标志物分子特征。这个审讯Ç一个可以在单细胞水平进行，采用不需要四氯化碳识别16异硫氰酸荧光素（FITC）荧光通道。虽然这个平台提供了分子鉴定的能力，协议开发和优化的详细过程没有明确界定。三种市售的标记物已被开发由生产用于与CSS的使用，包括表皮生长因子受体（EGFR），人表皮生长因子受体2（HER2），胰岛素样生长因子1受体（IGF-1R）。 HER2分析，与CSS结合，已动用了几组，以说明四氯化碳表征的潜力，告知临床决策，并有可能改变现有的治疗指南。例如，Fehm 等 17证实，乳腺癌患者的HER-2原发肿瘤大约有三分之一HER2阳性的CTC。此外，刘等人。18近日报道，患者的HER +转移性乳腺癌高达50％的人没有HER2阳性的CTC。赫赛汀，一个HER2受体干扰单克隆抗体证明大大有利于患者的肿瘤表达HER2的足够的水平，是一个为患者常用的治疗HER2阳性的原发性肿瘤19-21。然而，这些研究表明，曲妥珠单抗可以是亚最优利用和四氯化碳表征可以在预测治疗反应助剂。最终，CTC表征可能要提高个性化服务的潜力。 CTC的研究是独一无二的，它已经在很大程度上利用了床边到台式办法。这种方法，不像台式到床边的研究，而这往往需要几年时间才能影响病人护理，已经允许的CTC快速进入临床。然而，医生正在犹豫中病人的治疗决策麦从使用四氯化碳分析结果ING由于缺乏它们的基础生物学的理解。因此，转移和互补CTC分析技术，适当的临床前小鼠模型必须被利用，以调查这些悬而未决的问题。一般而言，有两种类型的临床前模型中的用于学习的转移级联，（1）自发转移模型，它允许在转移级联中的所有步骤的研究，和（2）实验性转移模型，只允许该研究在转移过程的后续步骤，如外渗和继发性肿瘤的形成22。自发转移模型，涉及肿瘤细胞注射到适当的原位位置（ 如注射前列腺癌细胞进入前列腺前列腺癌的研究）。然后细胞被给定的时间，形成原发肿瘤和转移自发到辅助站点，如骨，肺和淋巴结肿大。在相比之下，实验性转移模型涉及直接注射肿瘤细胞进入血液（ 经尾静脉或心腔内注射，例如对靶细胞的特定位置），因此跳过血管内和传播的最初步骤，二级机关22。到目前为止，大部分在体内模型系统CTC分析一直采用流式细胞仪要么基于23或改装以人为本的CTC技术（ 如 AdnaTest）24执行。虽然有用，没有这些技术使用金标准的CSS充分反映四氯化碳枚举。根据临床审批，规范的性质，和CSS的广泛使用， 在体内建模四氯化碳捕获和检测技术，利用等效的样品制备，加工，鉴定标准的发展将是有利的，因为结果将是媲美从患者样本获得的。然而，由于该卷的REQ制备仪器uirements不可能处理的血液使用该自动化平台小卷。此外，以前的工作由ELIANE 等人 25表明，与小鼠上皮细胞样品（其也符合标准CTC定义[EpCAM的+ CK + DAPI + CD45 - ]）的污染可导致小鼠鳞状上皮细胞误判为车辆检验中心。解决这些问题的一个适应的技术，使CSS的四氯化碳试剂盒试剂结合的手动分离过程的利用率被开发。另外一个FITC标记的人白细胞抗原（HLA）抗体的测定法允许人肿瘤细胞是从小鼠鳞状上皮细胞区别开来。 这份手稿描述了标准，商业开发和优化的CSS协议处理病人的血液样本和常见的陷阱可能遇到的，包括discrepan吨的项目，可导致四氯化碳误分。此外，CSS试验，以检查捕获的CTC和可比适于CSS技术，其允许的CTC的由小体积的血液中转移的临床前小鼠模型的富集和检测的用户定义的蛋白质的特性进行自定义进行说明。

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			<td height="20" style="height:20px;width:267px;">0.5M EDTA</td>
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			<td height="20" style="height:20px;width:267px;">Anti-human CD44-FITC</td>
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			<td style="width:161px;">555478</td>
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			<td height="20" style="height:20px;width:267px;">Anti-human HLA-AlexaFluor488</td>
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			<td height="20" style="height:20px;width:267px;">Anti-mouse CD45-APC</td>
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			<td style="width:161px;">17-0451-82</td>
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			<td style="width:161px;">952820 (20 pack)&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 79100005 (100 pack)</td>
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			<td height="20" style="height:20px;width:267px;">CellSearch CTC Control Kit</td>
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			<td style="width:161px;">7900003</td>
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			<td height="20" style="height:20px;width:267px;">CellSearch CTC Kit</td>
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			<td height="20" style="height:20px;width:267px;">CellSearch CXC Control Kit</td>
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			<td height="20" style="height:20px;width:267px;">CellSearch CXC Kit</td>
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			<td style="width:161px;">7900017RUO</td>
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			<td height="20" style="height:20px;width:267px;">Instrument Buffer</td>
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			<td height="40" style="height:40px;width:267px;">Streck Cell Preservative (aka CytoChex)</td>
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			<td>213350</td>
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			<td height="20" style="height:20px;width:267px;">1 ml syringe</td>
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			<td height="20" style="height:20px;width:267px;">10 ml serological pipette</td>
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			<td height="21" style="height:21px;width:267px;">1000&#181;l pipette</td>
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			<td height="20" style="height:20px;width:267px;">1000&#181;l pipette tips</td>
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		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">12 x 75mm flow tubes</td>
			<td style="width:175px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">200&#181;l gel loading tips</td>
			<td style="width:175px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">200&#181;l pipette</td>
			<td style="width:175px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">22 gauge needle</td>
			<td style="width:175px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">5 3/4&#34; disposible pasteur pipet</td>
			<td style="width:175px;">VWR</td>
			<td style="width:161px;">14672-200</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">5 ml serological pipette</td>
			<td style="width:175px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">Automated pipettor</td>
			<td style="width:175px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:267px;">Capillary Blood Collection Tube (EDTA)</td>
			<td style="width:175px;">BD Microtainer</td>
			<td style="width:161px;">365974</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">CellSearch Analyzer II</td>
			<td style="width:175px;">Veridex</td>
			<td style="width:161px;">9555</td>
			<td rowspan="2" style="width:188px;">Includes magnests and verification cartirdges</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">CellSearch AutoPrep System</td>
			<td style="width:175px;">Veridex</td>
			<td style="width:161px;">9541</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Centrifuge</td>
			<td style="width:175px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:267px;">MagCellect Magnet&#160;</td>
			<td style="width:175px;">R&#38;D Systems</td>
			<td style="width:161px;">MAG997</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Small Latex Bulb</td>
			<td style="width:175px;">VWR</td>
			<td>82024-550</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:267px;">Vortex</td>
			<td style="width:175px;"></td>
			<td style="width:161px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

adaptation of semiautomated circulating tumor cell (ctc) assays for clinical and preclinical research applications

多数癌症相关死亡的发生转移性疾病的后续发展。这种高度致命性疾病分期与循环肿瘤细胞（CTCs的）的存在有关。这些罕见的细胞已被证明是在转移性乳腺癌，前列腺癌和大肠癌的临床意义。在临床CTC检测和计数目前的黄金标准是FDA批准CellSearch系统（CSS）。这个手稿概述了该平台所使用的标准协议，以及描述用户自定义标记优化患者CTCs的蛋白质特性和极低的血量可比协议CTC捕捉的详细过程，使用标准的两个附加议定书改编CSS的试剂， 在转移的体内临床前小鼠模型研究。另外，在健康供者血液的CTC质量差异掺入细胞从组织培养物与患者血液SAMP文件被高亮显示。最后，一​​些可能导致四氯化碳误分常用物品不符的概述。两者合计，这些协议将提供一个有用的资源为这个平台感兴趣的有关转移和CTCs的临床前和临床研究的用户。

标准CTC枚举含量在CSS的敏感性和特异性得到了很好的记载在文献中。然而，要验证等效四氯化碳回收，尖刺（1,000 LNCaP人前列腺癌细胞）和来自健康志愿者供体无稀释剂的人类血液的样品用标准的CSSCTC协议上的CSS处理。正如预期的那样，未加标的样品是免费的CTC的，0.00±0.00％，和CTC恢复被证明是86.9±4.71％，加标样品（图1A）。从加标样品获得的CSS画廊图像进行的最?...

Watch this Scientific Journal Video about Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications at JoVE.com

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

循环肿瘤细胞（CTCs的）是影响预后的几个转移性癌症。此手稿描述的金标准CellSearch系统（CSS）四氯化碳枚举平台和亮点常见的误分。此外，两个适配协议被描述为的CTC和CTC枚举在转移的使用这种技术的临床前小鼠模型中的用户定义的标记物的表征。

半自动适应循环肿瘤细胞（CTC）分析临床和临床前研究中的应用

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

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Research

JoVE Journal

Medicine

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

15.8K 次观看.  London Health Sciences Centre. 循环肿瘤细胞（CTCs的）是影响预后的几个转移性癌症。此手稿描述的金标准CellSearch系统（CSS）四氯化碳枚举平台和亮点常见的误分。此外，两个适配协议被描述为的CTC和CTC枚举在转移的使用这种技术的临床前小鼠模型中的用户定义的标记物的表征。 

视频: Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

加工的主要脑肿瘤组织的干细胞检测和流量排序

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

科研

医学

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

在体内 19女性 MRI对细胞跟踪

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone resorption. Osteoclasts can be generated in vitro from monocyte/macrophage precursor cells in the presence of certain cytokines, which promote survival and differentiation. Here, both in vivo and in vitro techniques are demonstrated, which allow scientists to study different cytokine contributions towards osteoclast differentiation, signaling, and activation. The minicircle DNA delivery gene transfer system provides an alternative method to establish an osteoporosis-related model is particularly useful to study the efficacy of various pharmacological inhibitors in vivo. Similarly, in vitro culturing protocols for producing osteoclasts from human precursor cells in the presence of specific cytokines enables scientists to study osteoclastogenesis in human cells for translational applications. Combined, these techniques have the potential to accelerate drug discovery efforts for osteoclast-specific targeted therapeutics, which may benefit millions of osteoporosis and arthritis patients worldwide.

A Novel in vivo Gene Transfer Technique and in vitro Cell Based Assays for the Study of Bone Loss in Musculoskeletal Disorders

Differentiation of precursor cells into osteoclasts is regulated by cytokines and growth factors. Here, a novel gene transfer technique for differentiation of osteoclasts in vivo and cell culture protocols for differentiating precursor cells into osteoclasts in vitro as a method to study the effects of cytokines on osteoclastogenesis are described.

一种新型的<em&gt;在体内</em&gt;基因转移技术和<em&gt;在体外</em&gt;细胞分析为肌肉骨骼疾病骨质流失的研究

Differentiation and activation of osteoclasts play a key role in the development of musculoskeletal diseases as these cells are primarily involved in bone ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

原发性肿瘤和/或转移相关的临床前和治疗的临床开发的离体处理响应

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

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 ...

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, ...

Proton Therapy Delivery and Its Clinical Application in Select Solid Tumor Malignancies

Radiation therapy is a frequently used modality for the treatment of solid cancers. Although the mechanisms of cell kill are similar for all forms of radiation, the in vivo properties of photon and proton beams differ greatly and maybe exploited to optimize clinical outcomes. In particular, proton particles lose energy in a predictable manner as they pass through the body. This property is used clinically to control the depth at which the proton beam is terminated, and to limit radiation dose beyond the target region. This strategy can allow for substantial reductions in radiation dose to normal tissues located just beyond a tumor target. However, the degradation of proton energy in the body remains highly sensitive to tissue density. As a consequence, any changes in tissue density during the course of treatment may significantly alter proton dosimetry. Such changes may occur through alterations in body weight, respiration, or bowel filling/gas, and may result in unfavorable dose deposition. In this manuscript, we provide a detailed method for the delivery of proton therapy using both passive scatter and pencil beam scanning techniques for prostate cancer. Although the described procedure directly pertains to prostate cancer patients, the method may be adapted and applied for the treatment of virtually all solid tumors. Our aim is to equip readers with a better understanding of proton therapy delivery and outcomes in order to facilitate the appropriate integration of this modality during cancer therapy.

The fundamentals of radiation planning and delivery for proton therapy using prostate cancer as a model are presented. The application of these principles to other selected disease sites highlights how proton radiotherapy may enhance clinical outcomes for cancer patients.

质子治疗在实体肿瘤恶性肿瘤选择中的临床应用

Radiation therapy is a frequently used modality for the treatment of solid cancers. Although the mechanisms of cell kill are similar for all forms of ...

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity is disturbed in gastrointestinal, cardiovascular and metabolic diseases, which may result in easier access of biologically active compounds, such as gut bacterial metabolites, to the bloodstream. Thus, the permeability of the GBB may be a marker of both intestinal and extraintestinal diseases. Furthermore, the increased penetration of bacterial metabolites may affect the functioning of the entire organism.
Commonly used methods for studying the GBB permeability are performed ex vivo. The accuracy of those methods is limited, because the functioning of the GBB depends on intestinal blood flow. On the other hand, commonly used in vivo methods may be biased by liver and kidney performance, as those methods are based on evaluation of urine or/and peripheral blood concentrations of exogenous markers. Here, we present a direct measurement of GBB permeability in rats using an in vivo method based on portal blood sampling, which preserves intestinal blood flow and is virtually not affected by the liver and kidney function. 
Polyurethane catheters are inserted into the portal vein and inferior vena cava just above the hepatic veins confluence. Blood is sampled at baseline and after administration of a selected marker into a desired part of the gastrointestinal tract. Here, we present several applications of the method including (1) evaluation of the colon permeability to TMA, a gut bacterial metabolite, (2) evaluation of liver clearance of TMA, and (3) evaluation of a gut-portal blood-liver-peripheral blood pathway of gut bacteria-derived short-chain fatty acids. Furthermore, the protocol may also be used for tracking intestinal absorption and liver metabolism of drugs or for measurements of portal blood pressure.

An In Vivo Method for Evaluating the Gut-Blood Barrier and Liver Metabolism of Microbiota Products

The access of nutrients, microbiota metabolites and medicines to the circulation is controlled by the gut-blood barrier (GBB). We describe a direct method for measuring the GBB permeability in vivo, which, in contrast to commonly used indirect methods, is virtually not affected by liver and kidney functions.

微生物肠道血屏障及肝脏代谢的体内评价方法

The gut-blood barrier (GBB) controls the passage of nutrients, bacterial metabolites and drugs from intestinal lumen to the bloodstream. The GBB integrity ...

An Ex Vivo Tissue Culture Model for Fibrovascular Complications in Proliferative Diabetic Retinopathy

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No current animal models of diabetes and oxygen-induced retinopathy develop the full-range progressive changes manifested in human proliferative diabetic retinopathy (PDR). Therefore, understanding of the disease pathogenesis and pathophysiology has relied largely on the use of histological sections and vitreous samples in approaches that only provide steady-state information on the involved pathogenic factors. Increasing evidence indicates that dynamic cell-cell and cell-extracellular matrix (ECM) interactions in the context of three-dimensional (3D) microenvironments are essential for the mechanistic and functional studies towards the development of new treatment strategies. Therefore, we hypothesized that the pathological fibrovascular tissue surgically excised from eyes with PDR could be utilized to reliably unravel the cellular and molecular mechanisms of this devastating disease and to test the potential for novel clinical interventions. Towards this end, we developed a novel method for 3D ex vivo culture of surgically-excised patient-derived fibrovascular tissue (FT), which will serve as a relevant model of human PDR pathophysiology. The FTs are dissected into explants and embedded in fibrin matrix for ex vivo culture and 3D characterization. Whole-mount immunofluorescence of the native FTs and end-point cultures allows thorough investigation of tissue composition and multicellular processes, highlighting the importance of 3D tissue-level characterization for uncovering relevant features of PDR pathophysiology. This model will allow the simultaneous assessment of molecular mechanisms, cellular/tissue processes and treatment responses in the complex context of dynamic biochemical and physical interactions within the PDR tissue architecture and microenvironment. Since this model recapitulates PDR pathophysiology, it will also be amenable for testing or developing new treatments.

Here, we present a protocol to study the pathophysiology of proliferative diabetic retinopathy by using patient-derived, surgically-excised, fibrovascular tissues for three-dimensional native tissue characterization and ex vivo culture. This ex vivo culture model is also amenable for testing or developing new treatments.

增殖性糖尿病视网膜病变纤维血管并发症的体外组织培养模型

Diabetic retinopathy (DR) is the most common microvascular complication of diabetes and one of the leading causes of blindness in working-age adults. No ...