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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Oncol. 26 (19), 3213-3221 (2008).</li><li>Allard, W. J., Matera, J., 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=Tumor+cells+circulate+in+the+peripheral+blood+of+all+major+carcinomas+but+not+in+healthy+subjects+or+patients+with+nonmalignant+diseases.">Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases.</a> Clin. Cancer Res. 10 (20), 6897-6904 (2004).</li><li>Thiery, J. P., Acloque, H., Huang, R. Y. J., Nieto, M. 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Cell. 14 (6), 818-829 (2008).</li><li>Gradilone, A., Raimondi, C., 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+tumour+cells+lacking+cytokeratin+in+breast+cancer:+the+importance+of+being+mesenchymal.">Circulating tumour cells lacking cytokeratin in breast cancer: the importance of being mesenchymal.</a> J. Cell. Mol. 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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。

<table><tbody><tr><td>0.5 M EDTA</td><td></td><td></td><td></td></tr><tr><td>Anti-human CD44-FITC</td><td>BD Pharmigen</td><td>555478</td><td></td></tr><tr><td>Anti-human CD44-PE</td><td>BD Pharmigen</td><td>555479</td><td></td></tr><tr><td>Anti-human HLA-Alexa Fluor 488</td><td>BioLegend</td><td>311415</td><td></td></tr><tr><td>Anti-mouse CD45-APC</td><td>eBioscience</td><td>17-0451-82</td><td></td></tr><tr><td>Bond Primary Antibody Diluent</td><td>Leica</td><td>AR9352</td><td></td></tr><tr><td>CellSave Preservative Tubes</td><td>Veridex</td><td>952820 (20 pack) 79100005 (100 pack)</td><td></td></tr><tr><td>CellSearch CTC Control Kit</td><td>Veridex</td><td>7900003</td><td></td></tr><tr><td>CellSearch CTC Kit</td><td>Veridex</td><td>7900001</td><td></td></tr><tr><td>CellSearch CXC Control Kit</td><td>Veridex</td><td>7900018RUO</td><td></td></tr><tr><td>CellSearch CXC Kit</td><td>Veridex</td><td>7900017RUO</td><td></td></tr><tr><td>Instrument Buffer</td><td>Veridex</td><td>7901003</td><td></td></tr><tr><td>Streck Cell Preservative (aka CytoChex)</td><td>Streck</td><td>213350</td><td></td></tr><tr><td>1 ml Syringe</td><td></td><td></td><td></td></tr><tr><td>10 ml Serological pipette</td><td></td><td></td><td></td></tr><tr><td>1,000 &amp;micro;l Pipette</td><td></td><td></td><td></td></tr><tr><td>1,000 &amp;micro;l Pipette tips</td><td></td><td></td><td></td></tr><tr><td>12 mm x 75 mm Flow tubes</td><td></td><td></td><td></td></tr><tr><td>200 &amp;micro;l Gel loading tips</td><td></td><td></td><td></td></tr><tr><td>200 &amp;micro;l Pipette</td><td></td><td></td><td></td></tr><tr><td>22 G Needle</td><td></td><td></td><td></td></tr><tr><td>5 3/4 in Disposible Pasteur pipet</td><td>VWR</td><td>14672-200</td><td></td></tr><tr><td>5 ml Serological pipette</td><td></td><td></td><td></td></tr><tr><td>Automated pipettor</td><td></td><td></td><td></td></tr><tr><td>Capillary Blood Collection Tube (EDTA)</td><td>BD Microtainer</td><td>365974</td><td></td></tr><tr><td>CellSearch Analyzer II</td><td>Veridex</td><td>9555</td><td>Includes magnests and verification cartirdges</td></tr><tr><td>CellSearch AutoPrep System</td><td>Veridex</td><td>9541</td><td></td></tr><tr><td>Centrifuge</td><td></td><td></td><td></td></tr><tr><td>MagCellect Magnet&amp;nbsp;</td><td>R&amp;amp;D Systems</td><td>MAG997</td><td></td></tr><tr><td>Small Latex Bulb</td><td>VWR</td><td>82024-550</td><td></td></tr><tr><td>Vortex</td><td></td><td></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 ...

adaptation-semiautomated-circulating-tumor-cell-ctc-assays-for

Universidad de Cartagena UDEC

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

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip for downstream analysis and/or culture. The strategy employs the use of a novel Parylene C membrane slot pore microfilter to capture CTC and a coating of poly (N-iso-propylacrylamide) (PIPAAm) for thermoresponsive viable release of the captured CTC. The capture of live cells is enabled by leveraging the design of a slot pore geometry with specific dimensions to reduce the shear stress typically associated with the filtration process. While the microfilter exhibits a high capture efficiency, the release of these cells is non-trivial. Typically, only a small percentage of cells are released when techniques such as reverse flow or cell scraping are used. The strong adhesion of these epithelial cancer cells to the Parylene C membrane is attributable to non-specific electrostatic interaction. To counteract this effect, we employed the use of PIPAAm coating and exploited its thermal responsive interfacial properties to release the cells from the filter. Blood is first filtered at room temperature. Below 32 &#176;C, PIPAAm is hydrophilic. Thereafter, the filter is placed in either culture media or a buffer maintained at 37 &#176;C, which results in the PIPAAm turning hydrophobic, and subsequently releasing the electrostatically bound cells.

A protocol to utilize a poly(N-iso-propylacrylamide) (PIPAAm) coated microfilter for effective capture and thermoresponsive release of viable circulating tumor cells (CTC) is presented. This method allows capture of CTC from patients&#39; blood and subsequent release of viable CTC for downstream off-chip culture, analyses and characterization.

捕获和血液中的活循环肿瘤细胞释放

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

科研

癌症研究

Rapid Isolation of Viable Circulating Tumor Cells from Patient Blood Samples

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary tumors at distant sites. CTC count can be used to follow disease progression based on the correlation between CTC concentration in blood and disease severity1. As a treatment tool, CTC could be studied in the laboratory to develop personalized therapies. To this end, CTC isolation must cause no cellular damage, and contamination by other cell types, particularly leukocytes, must be avoided as much as possible2. Many of the current techniques, including the sole FDA-approved device for CTC enumeration, destroy CTC as part of the isolation process (for more information see Ref. 2). A microfluidic device to capture viable CTC is described, consisting of a surface functionalized with E-selectin glycoprotein in addition to antibodies against epithelial markers3. To enhance device performance a nanoparticle coating was applied consisting of halloysite nanotubes, an aluminosilicate nanoparticle harvested from clay4. The E-selectin molecules provide a means to capture fast moving CTC that are pumped through the device, lending an advantage over alternative microfluidic devices wherein longer processing times are necessary to provide target cells with sufficient time to interact with a surface. The antibodies to epithelial targets provide CTC-specificity to the device, as well as provide a readily adjustable parameter to tune isolation. Finally, the halloysite nanotube coating allows significantly enhanced isolation compared to other techniques by helping to capture fast moving cells, providing increased surface area for protein adsorption, and repelling contaminating leukocytes3,4. This device is produced by a straightforward technique using off-the-shelf materials, and has been successfully used to capture cancer cells from the blood of metastatic cancer patients. Captured cells are maintained for up to 15 days in culture following isolation, and these samples typically consist of &gt;50% viable primary cancer cells from each patient. This device has been used to capture viable CTC from both diluted whole blood and buffy coat samples. Ultimately, we present a technique with functionality in a clinical setting to develop personalized cancer therapies.

Circulating tumor cells are isolated from the blood of cancer patients without inflicting cellular damage. Isolation of tumor cells is accomplished using a bimolecular surface of E-selectin in addition to antibodies against epithelial markers. A nanotube coating specifically promotes cancer cell adhesion resulting in high capture purities.

从病人的血液样本可行的循环肿瘤细胞的快速分离

Circulating tumor cells (CTC) are cells that disseminate from a primary tumor throughout the circulatory system and that can ultimately form secondary ...

生物工程

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

循环肿瘤细胞的微操作, 用于下游分子分析和转移电位评估

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL of blood) warrant a poor prognosis and are correlated with shorter overall survival in several cancers (e.g., breast, prostate and colorectal). Currently, the anti-EpCAM-coated magnetic bead-based CTC capturing system is the gold standard test approved by the U.S. Food and Drug Administration (FDA) for enumerating CTCs in the bloodstream. This test is based on the use of magnetic beads coated with anti-EpCAM markers, which specifically target epithelial cancer cells. Many studies have illustrated that EpCAM is not the optimal marker for CTC detection. Indeed, CTCs are a heterogeneous subpopulation of cancer cells and are able to undergo an epithelial-to-mesenchymal transition (EMT) associated with metastatic proliferation and invasion. These CTCs are able to reduce the expression of cell surface epithelial marker EpCAM, while increasing mesenchymal markers such as vimentin. To address this technical hurdle, other isolation methods based on physical properties of CTCs have been developed. Microfluidic technologies enable a label-free approach to CTC enrichment from whole blood samples. The spiral microfluidic technology uses the inertial and Dean drag forces with continuous flow in curved channels generated within a spiral microfluidic chip. The cells are separated based on the differences in size and plasticity between normal blood cells and tumoral cells. This protocol details the different steps to characterize the programmed death-ligand 1 (PD-L1) expression of CTCs, combining a spiral microfluidic device with customizable immunofluorescence (IF) marker set.

The characterization of circulating tumor cells (CTCs) is a popular topic in translational research. This protocol describes a semi-automatic immunofluorescence (IF) assay for PD-L1 characterization and enumeration of CTCs in non-small cell lung cancer (NSCLC) patient samples.

免疫荧光对非小细胞肺癌患者循环肿瘤细胞的半自动PD-L1特征化及枚举

Circulating tumor cells (CTCs) derived from the primary tumor are shed into the bloodstream or lymphatic system. These rare cells (1&#8722;10 cells per mL ...

Quantitative High-throughput Single-cell Cytotoxicity Assay For T Cells

Cancer immunotherapy can harness the specificity of immune response to target and eliminate tumors. Adoptive cell therapy (ACT) based on the adoptive transfer of T cells genetically modified to express a chimeric antigen receptor (CAR) has shown considerable promise in clinical trials1-4. There are several advantages to using CAR+ T cells for the treatment of cancers including the ability to target non-MHC restricted antigens and to functionalize the T cells for optimal survival, homing and persistence within the host; and finally to induce apoptosis of CAR+ T cells in the event of host toxicity5.
Delineating the optimal functions of CAR+ T cells associated with clinical benefit is essential for designing the next generation of clinical trials. Recent advances in live animal imaging like multiphoton microscopy have revolutionized the study of immune cell function in vivo6,7. While these studies have advanced our understanding of T-cell functions in vivo, T-cell based ACT in clinical trials requires the need to link molecular and functional features of T-cell preparations pre-infusion with clinical efficacy post-infusion, by utilizing in vitro assays monitoring T-cell functions like, cytotoxicity and cytokine secretion. Standard flow-cytometry based assays have been developed that determine the overall functioning of populations of T cells at the single-cell level but these are not suitable for monitoring conjugate formation and lifetimes or the ability of the same cell to kill multiple targets8.
Microfabricated arrays designed in biocompatible polymers like polydimethylsiloxane (PDMS) are a particularly attractive method to spatially confine effectors and targets in small volumes9. In combination with automated time-lapse fluorescence microscopy, thousands of effector-target interactions can be monitored simultaneously by imaging individual wells of a nanowell array. We present here a high-throughput methodology for monitoring T-cell mediated cytotoxicity at the single-cell level that can be broadly applied to studying the cytolytic functionality of T cells.

We describe a single-cell high-throughput assay to measure cytotoxicity of T cells when incubated with tumor target cells. This method employs a dense, elastomeric array of sub-nanoliter wells (~100,000 wells/array) to spatially confine the T cells and target cells at defined ratios and is coupled to fluorescence microscopy to monitor effector-target conjugation and subsequent apoptosis.

定量高通量单细胞的细胞毒性T细胞的测定

Cancer immunotherapy can harness the specificity of  immune response to target and eliminate tumors. Adoptive cell therapy (ACT)  based on the adoptive ...

免疫与感染

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.
Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.
In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

循环肿瘤细胞系:基础和转化研究的创新工具

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

实时的细胞毒性分析在人全血

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...

医学

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 ...

Analysis of Human T Cell Activity in an Allogeneic Co-Culture Setting of Pre-Treated Tumor Cells

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and activity of cytotoxic T cells within tumors and their tumor microenvironment (TME) are now well-established prognostic and predictive biomarkers for numerous tumor types. However, it is well-known that various tumor treatment modalities, including radiotherapy, chemotherapy, immunotherapy, and targeted therapy, modulate not only the immunogenicity of the tumor but also the immune system itself. Consequently, the interaction between tumor cells and T cells requires more intensive study in different therapeutic contexts to fully understand the complex role of T cells during tumor therapy. To address this need, a protocol was developed to analyze the activity and proliferative capacity of human cytotoxic (CD8+) T cells in co-culture with pre-treated tumor cells. Specifically, CD8+ T cells from healthy donors are stained with the non-toxic proliferation marker carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated using CD3/CD28-coated plates. Subsequently, T cells are co-cultured with pre-treated tumor cells. As a readout, T cell proliferation is quantified by measuring CFSE signal distribution and assessing the expression of surface activation markers via flow cytometry. This can be further complemented by quantifying cytokine release using enzyme-linked immunosorbent assay (ELISA). This method facilitates the evaluation of treatment-induced changes in the interaction between tumor cells and T cells, providing a foundation for more detailed analyses of tumor treatment modalities and their immunogenicity in a human ex vivo setting. Additionally, it contributes to the reduction of pre-clinical in vivo analyses.

The present protocol describes an experimental workflow that allows for the ex vivo analysis of human T cell stimulation in an allogenic co-culture system with pre-treated tumor cells.

在预处理肿瘤细胞的同种异体共培养环境中分析人 T 细胞活性

Cytotoxic T cells play a key role in the elimination of tumor cells and are, therefore, intensively studied in cancer immunology. The frequency and ...

Clinical Microfluidic Chip Platform for the Isolation of Versatile Circulating Tumor Cells

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient disease since CTCs are rare and heterogeneous. CTCs are detached from the primary tumor, enter the blood circulation system, and potentially grow at distant sites, thus metastasizing the tumor. Since CTCs carry similar information to the primary tumor, CTC isolation and subsequent characterization can be critical in monitoring and diagnosing cancer. The enumeration, affinity modification, and clinical immunofluorescence staining of rare CTCs are powerful methods for CTC isolation because they provide the necessary elements with high sensitivity. Microfluidic chips offer a liquid biopsy method that is free of any pain for the patients. In this work, we present a list of protocols for clinical microfluidic chips, a versatile CTC isolating platform, that incorporate a set of functionalities and services required for CTC separation, analysis, and early diagnosis, thus facilitating biomolecular analysis and cancer treatment. The program includes rare tumor cell counting, clinical patient blood preprocessing, which includes red blood cell lysis, and the isolation and recognition of CTCs in situ on microfluidic chips. The program allows the precise enumeration of tumor cells or CTCs. Additionally, the program includes a tool that incorporates CTC isolation with versatile microfluidic chips and immunofluorescence identification in situ on the chips, followed by biomolecular analysis.

The clinical microfluidic chip is an important biomedical analysis technique that simplifies clinical patient blood sample preprocessing and immunofluorescently stains circulating tumor cells (CTCs) in situ on the chip, allowing the rapid detection and identification of a single CTC.

用于分离多功能循环肿瘤细胞的临床微流控芯片平台

Circulating tumor cells (CTCs) are significant in cancer prognosis, diagnosis, and anti-cancer therapy. CTC enumeration is vital in determining patient ...