Name: capture and release of viable circulating tumor cells from blood
Uploaded: 2016-10-28T14:00:00.000+00:00
Duration: 8 min 10 s
Description: Watch this Scientific Journal Video about Capture and Release of Viable Circulating Tumor Cells from Blood at JoVE.com

We thank all the patients who have donated blood samples to support this work. We thank Drs. Guiseppe Giaconne, Ritesh Parajuli, and Marc E. Lippman for their assistance in clinical sample acquirement, and Drs. Carmen Gomez, Ralf Landgraf, Stephan Z&#252;chner, Toumy Guettouche, Diana Lopez for their insightful discussions. Zheng Ao thanks partial support and assistance from the Sheila and David Fuente Graduate Program in Cancer Biology, Sylvester Comprehensive Cancer Center.

道德声明:为了保护人类受试者的权益，根据如下迈阿密机构审查委员会的IRB 20150020下的大学批准的方案知情同意书，获得血液样本。 注:血液要过滤四氯化碳采集应以EDTA管收集，以防血液凝结。 1.涂层用聚（N-异丙丙基丙烯酰胺）（PIPAAm）的精密的过滤<ol><li>称出PIPAAm以制备丁醇10％w / v的溶液。混合使用涡旋直至溶液澄清。 </li><li>切胶显微镜滑入大致12毫米×12平方毫米或者用剪刀或锋利的刀刃的。或者使用断头台。 注意:这些方块将作为在旋涂过程中的过滤器保持器。 </li><li>用锋利的直边剪，切槽孔微过滤片成8毫米×8平方毫米。 </li><li>用聚酰亚胺薄膜胶带固定的截止滤光片到塑料SQUA水库先前切割步骤1.3。应用该膜只对边缘和过滤器的角，使得过滤面积在小于7毫米×7 mm表示未包括的胶带。 </li></ol><img alt="图2" src="/files/ftp_upload/54435/54435fig2.jpg" /> 图2:在精密的过滤设置为PIPAAm被膜 8毫米×8毫米微滤器的代表性示意图被定位在由塑料显微镜载片12毫米×12毫米塑料方形切割。聚酰亚胺胶带是用来固定微过滤到位，旋涂的PIPAAm.Reproduced与基准15的许可。 <a href="https://ecsource-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54435/54435fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="5"><li>放置被固定在塑料方形，上旋涂机的真空吸盘的微过滤。 </li><li>编程旋涂器，以下列配方:开始，500rpm下10秒，6000ř分60秒，500转10秒，停。 </li><li>分配足够的10％w / v的PIPAAm溶液（在1.1中制备）使用一个标准的塑料移液管完全覆盖微过滤表面并启动旋涂机。 </li><li>从旋涂器中取出PIPAAm涂覆微滤器完成包衣过程后。离开储存期间附连到塑料正方形的过滤器。 注:涂布的过滤器，可以在室温下储存长达3个月。 </li></ol> 2.过滤盒的组装与PIPAAm涂层微尘<ol><li>通过将仍然固定在塑料平方成培养皿的微滤器再水合室温PIPAAm涂覆微过滤。加1×PBS中，直到微滤器被完全淹没，静置5分钟。 </li><li>水合步骤后，通过切割使用一对剪刀的聚酰亚胺薄膜带释放从塑料正方形的过滤器。 注:请注意其中的过滤器侧面有在PIPAAm涂层。 </li><li>组装微滤器进入过滤盒中，通过与2聚二甲基硅氧烷（PDMS）个用作垫圈/密封沿夹着的顶部和底部的丙烯酸片之间的微过滤。钳夹子来固定过滤器，并提供了一个密封的压克力盒。 注:PIPAAm涂层应朝上，从而将样品过滤通过时，细胞将在过滤器上（ 图1）的PIPAAm涂覆的表面捕获。 </li></ol> 3.血液样本过滤从微尘捕获和CTC释放<ol><li>因为每个注射泵品牌都有其自己的用户界面，参阅泵的用户手册和编程注射泵在75毫升/小时的速度流动并定容至20ml。 </li><li>暖3毫升的McCoy的（可商购）细胞培养基至37℃（通过添加10％胎牛血清和1％青霉素和链霉素的方法制备）。 <li>添加7.5毫升市售Hank氏平衡盐溶液（HBSS）中来的7.5毫升血液样品和吸此稀释血液进入25毫升注射器。 注:1用HBSS 例如，如果使用的10ml血，然后用等体积的10毫升的HBSS以达到1混合:1稀释，以使血液样品与等体积的1稀释，调整的HBSS的音量。 </li><li>接合用注射器过滤盒，并将其放置到注射泵。放置在50ml的管中，在底部收集通过流动和启动泵。 注:过滤应采取大约10 12分钟所有的过滤步骤是进行出在正常室温下（20-25℃），包括3.8的步骤</li><li>过滤完成后，脱开过滤盒，注意到这一侧是具有抓PIPAAm涂覆表面上的细胞的顶部。吸1ml所温暖的培养基到一个新的注射器。 </li><li>再接合与包含媒体的注射器过滤盒，但此时，以使滤波器的PIPAAm涂覆表面朝向从注射器远离接合盒的底端。 注意:这将是对释放可以通过施加平缓逆流被嵌入到槽孔隙任何细胞。 </li><li>放置注射器返回到注射泵和右边的过滤盒下保持一小培养皿或6孔板中。设置流速为100毫升/小时，并启动泵。 </li><li>通过所有媒体通过过滤器，并收集在培养皿流通。等待所有的媒体通过，然后停泵，取下注射器从泵浦Nd过滤盒。 </li><li>脱离注射器过滤盒和打开它来检索从盒的过滤器。放置与PIPAAm表面朝下到用于释放从孔中的细胞时，收集逆流相同的培养皿的过滤器。添加温介质的其余部分，培养皿放入设定在37℃的培养箱中。 </li><li> 30分钟后，所有的细胞将来自过滤器被释放。然而，离开过滤器在培养皿长达24小时，以确保所有的细胞分离。 注意:可替换地，释放的细胞可以被收集到一个离心管中，以进行进一步的下游分析，如PCR，ELISA，Western印迹，仅举几个例子。 </li><li>在培养24小时，使用镊子小心地取出微型过滤器。 注:该过滤器可以在此时被丢弃的细胞已经从它释放。 </li><li>轻轻吸取培养基上下用1000＆＃181:L移液管，以重新悬浮的红细胞和外周血单核细胞（PBMC），该被捕获在过滤器上，并释放到与四氯化碳的板。小心，轻轻地执行此操作，以避免拆卸松散连接的癌细胞。小心地取出含有再悬浮红细胞和PBMC同时留下附在癌细胞，并以新鲜培养基预先加热到37℃，后面整个介质。 注意:如果过多的红细胞是一次洗涤后，目前，该步骤可重复一次。 </li></ol> 4. CTC生存能力评估<ol><li>用活/死测定8评价CTC培养生存能力。 注:众多活/死测定法是市售的。按照制造商的协议是高度宜。请参考材料/试剂列表用于此实验商用测定试剂盒。 </li></ol>

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Part of this work has been protected under U.S. Provisional Patent Application No. 62/219,808. Siddarth Rawal is a shareholder of Circulogix Inc. that commercially produces the filters and filter cassettes/cartridges used in this Article.

从全血中捕获可行四氯化碳和从微滤器释放他们的方法是相对简单;然而，几个关键点值得一提。这是必要的，因为与无菌条件在整个过程中保持所有细胞培养。涂布PIPAAm过滤器的初始步骤是重要的，因为用于释放从过滤器单元的技术的基础是基于利用PIPAAm的温度响应界面性质。为了确保过滤器已被有效地涂覆到涂覆之前测量在显微镜下在过滤器上的孔（狭缝孔的宽度和长度）的大小，然后再次测...

转移性疾病是负责大多数癌症死亡。为转移预后开发和伴随诊断的生物标记物是癌症管理和治疗的关键。循环肿瘤细胞（CTC）在肿瘤传播和转移中心作用。此外，作为一个"液体活检"生物标记方便，CTC肿瘤患者的外周血中一直在上升为"癌症生物标志物研究的温床"。 CTC已经被证实为各种癌症的设置，包括乳腺癌，前列腺癌和直肠癌1预后标志物- 3。然而，最近在CTC领域的进展已经表明，这些稀有细胞的单纯列举具有有限的临床效用，如图介入临床试验4。因此，存在对技术，使四氯化碳的分子和功能表征的新兴需求。目前，只有一些技术存在允许FOR无抗原偏差，可行的捕获和CTC的释放，使下游强劲分子和功能分析5,6。这些微加工器件的大多数被耦合到微流体平台，因此具有在可处理的血液量，这从2-4毫升7范围的限制因素- 10。四氯化碳是在抽血（7.5毫升）的单管罕见的事件，因此，进一步降低了可处理血液量，极大地妨碍了捕获和隔离感兴趣的这些细胞的机会。 我们已经开发出两种类型的C型帕利灵膜微过滤装置的四氯化碳的其中采用更大的肿瘤细胞和正常的小血细胞11,12之间的大小差异捕获。我们以前曾报道对圆孔径枚举过滤并用FDA批准的平台，其中该微滤器被证明是在CTC捕获效率优于比较它对于癌症患者的血液样本13,14。然而，圆形过滤器的一个限制是在过滤之前使用基于甲醛固定液的必要性。这个过程会保留细胞形态，同时使它们能够承受在过滤过程的剪切应力和压力。而枚举和分子研究可以片上13来执行，在固定剂削弱以执行功能特性的能力。为了解决此限制，我们开发了一个狭槽的孔的过滤器 ，否定的必要性来修复之前过滤细胞（ 图1）。槽孔隙几何形状（6微米宽度x 40微米长度槽的孔）允许而仅部分闭塞的细孔，从而仍允许用于其它血细胞的分类通道和减轻压力的上升，这将导致细胞损伤要捕获的肿瘤细胞和最终破裂15,16槽孔隙盒由2丙烯酸系片其三明治的顶部和底部件与聚二甲基硅氧烷（PDMS）作为垫圈之间的槽中微孔过滤以提供防漏密封14,15（ 图1）。 而槽细孔过滤器的捕获效率是高的，（ 表1），所捕获的CTC由强的非特异性静电相互作用，而不是介导的粘附15外基质（ECM）结合于C型帕利灵膜。方法，例如逆流或使用细胞刮刀不能有效地从过滤器释放的细胞，或导致细胞损伤和细胞死亡。我们探讨的一个非常规的使用PIPAAm来制定发布策略15。 PIPAAm是经历在液温32℃17的可逆低临界溶液温度（LCST）相转变的聚合物。传统上，PIPAAm的这一特性已被广泛地探讨了组织工程应用。通常，细胞是在PIPAAm培养涂层表面在37℃时PIPAAm是疏水性。当培养温度低于32°C，其中的PIPAAm涂层表面变得水合17,18转移到细胞可以被分离成片状。我们通过在室温（低于32℃）进行过滤处理，然后通过将保持在37℃的培养介质中的过滤器使得细胞释放利用此热性能。在此温度下，PIPAAm聚合物层成为疏水性的，从而释放静电结合的细胞15（ 图1）。 虽然温度响应的方法以及其它方法已成功地实施，以实现可行四氯化碳捕获和释放19 -由这些技术报告共享21，一键潜在的缺点是，它们都采用四氯化碳捕获的抗原依赖性原理。基于抗原捕获CTC，如所示的Previously，可能导致偏CTC分析11,14。例如，许多基于亲和力的技术采用结合EpCAM的四氯化碳捕获抗体。然而，四氯化碳已经显示出表达的EpCAM的各级，通过这些技术导致的EpCAM低和EpCAM的负四氯化碳遗漏。此外，当CTC非上皮起源是在黑色素瘤和肉瘤设置兴趣爱好，如CTC可能发生的局限性。因此，这种技术允许可行CTC捕获和释放无基于抗原捕获引入潜在的偏见是非常可取的。 重要的是，基于微滤器捕获设备纯粹的尺寸和释放的策略是不可知的某些表面标记的存在。我们认为，PIPAAm涂层微孔过滤器的工作将有助于扩大我们的转移过程的理解，通过提供有效且高效地吸收和释放CTC为下游分析的能力。今年5月烈性ially露出的量新型靶向全身治疗可能被定向以及提供一种能够很容易地被监测，并在癌症患者管理助剂的生物标志物的新分子。

<table><tbody><tr><td>Slot Filter</td><td>Circulogix Inc.</td><td>MSF-01</td><td>Different size filters available based for filtration for CTC from blood or urine (www.circulogixinc.com)</td></tr><tr><td>poly(&lt;em&gt;N&lt;/em&gt;-iso-propylacrylamide) (PIPAAm)&amp;nbsp;</td><td>Ploysciences Inc.</td><td>21458</td><td>Non-Hazardous. Store at room temp.</td></tr><tr><td>1-Butanol</td><td>Sigma Aldrich</td><td>B7906</td><td>Use in well ventilated area</td></tr><tr><td>Plastic Microscope Slides</td><td>Cole-Parmer</td><td>48510-30</td><td>Any plastic slides or alternatively any sort of square (Metal, Acrylic etc.) can be used if it will be bale to hold the 8mmx8mm filter square</td></tr><tr><td>Spin Coater</td><td>Specialty Coating Systems</td><td>SCS G3 Spin Coater</td><td>Instrument</td></tr><tr><td>Polyimide Tape</td><td>Uline</td><td>S-7595</td><td>Polyimide is the generic name for Kapton Tape which can be purchased form multiple vendors (Amazon, Kaptontape.com)</td></tr><tr><td>HBSS- Hank&#039;s Balanced Salt Solution</td><td>Gibco</td><td>14025-092</td><td></td></tr><tr><td>1x PBS</td><td>Gibco</td><td>10010-023</td><td></td></tr><tr><td>Falcon Petri dishes 35 x 10 mm</td><td>VWR</td><td>25373-041</td><td></td></tr><tr><td>Microfilter Cassette</td><td>Circulogix Inc.</td><td>FC-01</td><td>Custom catridges are avilable based on filtration for CTC from blood or urine&amp;nbsp;</td></tr><tr><td>Syringe 20 ml</td><td>BD Scientific</td><td>302830</td><td></td></tr><tr><td>Syringe Pump</td><td>KD scientific&amp;nbsp;</td><td>78-0100V</td><td>Any syringe pump capable of holding a 25 ml syringe may be used</td></tr><tr><td>Cellstar 50 ml Centrifuge tube</td><td>VWR</td><td>82050-322</td><td></td></tr><tr><td>Greiner Bio One 6 well plate</td><td>VWR</td><td>89131-688</td><td>Any brand can be used, as long as the surface is compatiable for cell adesion and not repellant</td></tr><tr><td>SKBR3 Cells</td><td>ATCC</td><td>HTB-30</td><td></td></tr><tr><td>Live Dead Assay</td><td>Life Technologies</td><td>L3224</td><td>Any assay that can provide a reasonable analysis to evaluate live cells will work</td></tr><tr><td>Cell Culture Incubator</td><td>VWR</td><td>98000-368</td><td>Any incubator that can be used for cell culture will suffice</td></tr></tbody></table>

capture and release of viable circulating tumor cells from blood

我们证明了从全血存活的循环肿瘤细胞（CTC）的大小基于捕获的方法，用这些细胞从芯片释放用于下游分析和/或培养沿。该战略采用采用了新型的C型帕利灵膜孔槽的微过滤器捕获CTC和聚对捕获的CTC的温敏可行的释放涂层（N -异丙基丙烯酰胺）（PIPAAm）。活细胞的捕获是通过利用与特定尺寸的槽孔隙几何形状的设计，以减少通常与过滤过程相关的剪切应力使能。而微滤器表现出高的捕集效率，这些细胞的释放是不平凡的。典型地，使用的技术，如逆流或细胞刮时只有细胞的一小部分被释放。这些上皮癌细胞的C型帕利灵膜的附着力强的原因是非特异性静电相互作用。为了抵消日是影响，我们采用的使用PIPAAm涂层和利用它的热响应界面性能从过滤器释放的细胞。血液是首先在室温下过滤。低于32°C，PIPAAm是亲水性的。此后，将过滤器放置在任一培养基或维持在37℃的缓冲液，这导致在PIPAAm转动疏水性的，并且随后释放静电结合的细胞。

用健康捐献者的血液（迈阿密IRB 20150020的大学批准的方案如下知情同意下而得到的）培养的癌细胞，可行的循环肿瘤细胞（CTC），实现了采集，发布和检索效率的释放温敏技术飙升分别为94％±9％，82％±5％和77％±5％（ 表1）15。通过比较，未涂覆的过滤器的释放和检索效率显著降低（7％±1％的释放效率和6％±1％检索效率）为（ 表1）15。</su...

Watch this Scientific Journal Video about Capture and Release of Viable Circulating Tumor Cells from Blood at JoVE.com

Capture and Release of Viable Circulating Tumor Cells from Blood

的协议，利用聚（N -异丙基丙烯酰胺）（PIPAAm）有效捕获和可行的循环肿瘤细胞（CTC）的温敏缓释包衣微型过滤器呈现。这种方法可以从患者的血液和可行的CTC随后释放CTC的捕获下游片文化，分析和鉴定。

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 ...

capture-and-release-of-viable-circulating-tumor-cells-from-blood

Universidad San Sebastian

Research

JoVE Journal

Cancer Research

8.4K 次观看.  University of Miami. 的协议，利用聚（N -异丙基丙烯酰胺）（PIPAAm）有效捕获和可行的循环肿瘤细胞（CTC）的温敏缓释包衣微型过滤器呈现。这种方法可以从患者的血液和可行的CTC随后释放CTC的捕获下游片文化，分析和鉴定。 

视频: Capture and Release of Viable Circulating Tumor Cells from Blood

Characterization of Tumor Cells Using a Medical Wire for Capturing Circulating Tumor Cells: A 3D Approach Based on Immunofluorescence and DNA FISH

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a better understanding of tumor heterogeneity and thus facilitate the selection of patients for personalized treatment. However, this is hampered because of the rarity of CTCs. We present an innovative approach for sampling a high volume of the patient blood and obtaining information about presence, phenotype, and gene translocation of CTCs. The method combines immunofluorescence staining and DNA fluorescent-in-situ-hybridization (DNA FISH) and is based on a functionalized medical wire. This wire is an innovative device that permits the in vivo isolation of CTCs from a large volume of peripheral blood. The blood volume screened by a 30-min administration of the wire is approximately 1.5-3 L. To demonstrate the feasibility of this approach, epithelial cell adhesion molecule (EpCAM) expression and the chromosomal translocation of the ALK gene were determined in non-small-cell lung cancer (NSCLC) cell lines captured by the functionalized wire and stained with an immuno-DNA FISH approach. Our main challenge was to perform the assay on a 3D structure, the functionalized wire, and to determine immuno-phenotype and FISH signals on this support using a conventional fluorescence microscope. The results obtained indicate that catching CTCs and analyzing their phenotype and chromosomal rearrangement could potentially represent a new companion diagnostic approach and provide an innovative strategy for improving personalized cancer treatments.

We present a new approach to characterize tumor cells. We combined immunofluorescence with DNA fluorescent-in-situ-hybridization to evaluate cells captured by a functionalized medical wire capable of in vivo enriching CTCs directly from patient blood.

用医用金属丝捕获循环肿瘤细胞的肿瘤细胞: 基于免疫荧光和 DNA 的3D 方法

Circulating tumor cells (CTCs) are associated with poor survival in metastatic cancer. Their identification, phenotyping, and genotyping could lead to a ...

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 ...

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 ...

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

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

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

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

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 ...

医学

Automatic Separation and Collection of Cancer-Related Substances from Clinical Samples

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, and nucleic acids originating from normal tissues, but some of these substances also originate from the diseased area. The investigation and analysis of these substances in the body fluids play a pivotal role in the diagnosis of various diseases. Therefore, it is important to accurately separate the required substances, and several techniques are developed to be used for this purpose.
We have developed a lab-on-a-disc type of device and platform named CD-PRIME. This device is automated and has good results for sample contamination and sample stability. Moreover, it has advantages of a good acquisition yield, a short operation time, and high reproducibility. In addition, depending on the type of disc to be mounted, plasma containing cell-free DNA, circulating tumor cells, peripheral blood mononuclear cells, or buffy coats can be separated. Thus, the acquisition of a variety of materials present in the body fluids can be done for a variety of downstream applications, including the study of omics.

This paper describes the application of automated equipment to easily and efficiently separate and collect substances, such as cell-free DNA and circulating tumor cells, from whole blood.

从临床样本中自动分离和收集癌症相关物质

Recently, liquid biopsies have been used to diagnose various diseases, including cancer. Body fluids contain many substances, including cells, proteins, ...

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 ...

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 ...

CTC Immunofluorescence Assay : A Technique to  Characterize Circulating Tumor Cells

Source: Garcia, J., et al. <a href="https://www-jove-com.bdigitaluss.remotexs.co/v/59873/semi-automatic-pd-l1-characterization-enumeration-circulating-tumor">Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence.</a>&#160;J. Vis. Exp.&#160;(2019)
This video describes the Circulating Tumor Cells (CTC) Immunofluorescence assay, a technique to characterize the biomarkers on circulating tumor cells in non-small cell lung cancer patient samples. This assay could be used to monitor dynamic changes of the tumor and immune response, which may reflect the response to chemotherapy, radiation, and possible immunotherapy treatments.

CTC 免疫荧光检测:一种表征循环肿瘤细胞的技术

Source: Garcia, J., et al. Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by ...