Name: adaptation of semiautomated circulating tumor cell (ctc) assays for clinical and preclinical research applications
Uploaded: 2014-02-28T14:45:00
Description: Watch this Scientific Journal Video about Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications at JoVE.com

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?...

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S., 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+tumor+cells+from+patients+with+advanced+prostate+and+breast+cancer+display+both+epithelial+and+mesenchymal+markers.">Circulating tumor cells from patients with advanced prostate and breast cancer display both epithelial and mesenchymal markers.</a> Mol. Cancer Res. 9 (8), 997-1007 (2011).</li></ol>

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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</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 ...

Research

JoVE Journal

Medicine

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

Generation of Alginate Microspheres for Biomedical Applications

生物医学应用海藻酸钠微球的一代

Alginate-based materials have received considerable attention for biomedical applications because of their hydrophilic nature, biocompatibility, and ...

科研

医学

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

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 ...

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

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

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 ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI for Cell Tracking

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

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

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

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

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

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

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

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, ...

Percutaneous Contrast Echocardiography-guided Intramyocardial Injection and Cell Delivery in a Large Preclinical Model

经皮超声心动图引导心肌注射液与大鼠前体细胞的临床研究

Cell and gene therapy are exciting and promising strategies for the purpose of cardiac regeneration in the setting of heart failure with reduced ejection ...