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="http://ecsource.jove.com/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 border="0" cellpadding="0" cellspacing="0" width="603">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="106">
			<td height="106" style="height:107px;width:216px;">Slot Filter</td>
			<td style="width:101px;">Circulogix Inc.</td>
			<td style="width:119px;">MSF-01</td>
			<td style="width:167px;">Different size filters available based for filtration for CTC from blood or urine (www.circulogixinc.com)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">poly(N-iso-propylacrylamide) (PIPAAm)&#160;</td>
			<td style="width:101px;">Ploysciences Inc.</td>
			<td style="width:119px;">21458</td>
			<td style="width:167px;">Non-Hazardous. Store at room temp.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1-Butanol</td>
			<td style="width:101px;">Sigma Aldrich</td>
			<td style="width:119px;">B7906</td>
			<td style="width:167px;">Use in well ventilated area</td>
		</tr>
		<tr height="118">
			<td height="118" style="height:119px;width:216px;">Plastic Microscope Slides</td>
			<td style="width:101px;">Cole-Parmer</td>
			<td style="width:119px;">48510-30</td>
			<td style="width:167px;">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 height="59">
			<td height="59" style="height:59px;width:216px;">Spin Coater</td>
			<td style="width:101px;">Specialty Coating Systems</td>
			<td style="width:119px;">SCS G3 Spin Coater</td>
			<td>Instrument</td>
		</tr>
		<tr height="62">
			<td height="62" style="height:63px;width:216px;">Polyimide Tape</td>
			<td style="width:101px;">Uline</td>
			<td style="width:119px;">S-7595</td>
			<td style="width:167px;">Polyimide is the generic name for Kapton Tape which can be purchased form multiple vendors (Amazon, Kaptontape.com)</td>
		</tr>
		<tr height="47">
			<td height="47" style="height:47px;width:216px;">HBSS- Hank&#39;s Balanced Salt Solution</td>
			<td style="width:101px;">Gibco</td>
			<td style="width:119px;">14025-092</td>
			<td></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:216px;">1XPBS</td>
			<td style="width:101px;">Gibco</td>
			<td style="width:119px;">10010-023</td>
			<td></td>
		</tr>
		<tr height="189">
			<td height="189" style="height:189px;width:216px;">McCoy&#39;s</td>
			<td style="width:101px;">Gibco</td>
			<td style="width:119px;">16600-082</td>
			<td style="width:167px;">Warm in 37 &#8304;C water bath before use. McCoys was used for SKBr3 cells, if you use different cell lines or patient blood, please use media that would be optimal for that particular case</td>
		</tr>
		<tr height="74">
			<td height="74" style="height:74px;width:216px;">Falcon Petri dishes 35x10 mm</td>
			<td style="width:101px;">VWR</td>
			<td style="width:119px;">25373-041</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="87">
			<td height="87" style="height:87px;width:216px;">Microfilter Cassette</td>
			<td style="width:101px;">Circulogix Inc.</td>
			<td style="width:119px;">FC-01</td>
			<td style="width:167px;">Custom catridges are avilable based on filtration for CTC from blood or urine&#160;</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Syringe 20mL</td>
			<td style="width:101px;">BD Scientific</td>
			<td style="width:119px;">302830</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Syringe Pump</td>
			<td style="width:101px;">KD scientific&#160;</td>
			<td style="width:119px;">78-0100V</td>
			<td style="width:167px;">Any syringe pump capable of holding a 25mL syringe may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cellstar 50mL Centrifuge tube</td>
			<td style="width:101px;">VWR</td>
			<td style="width:119px;">82050-322</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Greiner Bio One 6 well plate</td>
			<td style="width:101px;">VWR</td>
			<td style="width:119px;">89131-688</td>
			<td style="width:167px;">Any brand can be used, as long as the surface is compatiable for cell adesion and not repellant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">SKBR3 Cells</td>
			<td style="width:101px;">ATCC</td>
			<td style="width:119px;">HTB-30</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Live Dead Assay</td>
			<td style="width:101px;">Life Technologies</td>
			<td style="width:119px;">L3224</td>
			<td style="width:167px;">Any assay that can provide a reasonable analysis to evaluate live cells will work</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Cell Culture Incubator</td>
			<td style="width:101px;">VWR</td>
			<td style="width:119px;">98000-368</td>
			<td style="width:167px;">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 de Cartagena UDEC

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

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

在直肠癌原位小鼠模型中循环肿瘤细胞的分离

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

科研

癌症研究

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

肺定植法在肺部组织循环肿瘤细胞可视化中的建立

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

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 ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been shown to be responsible for tumor initiation, metastasis, and drug resistance. Therefore, it is important to develop specific TIC-targeting therapy in addition to current chemotherapy strategies, which mostly focus on the bulk of non-TICs. In order to further understand the mechanism behind the malignancy of TICs, we describe a method to isolate and to characterize TICs in human sarcomas. Herein, we show a detailed protocol to generate patient-derived xenografts (PDXs) of human sarcomas and to isolate TICs by fluorescence-activated cell sorting (FACS) using human leukocyte antigen class I (HLA-1) as a negative marker. Also, we describe how to functionally characterize these TICs, including a sphere formation assay and a tumor formation assay, and to induce differentiation along mesenchymal pathways. The isolation and characterization of PDX TICs provide clues for the discovery of potential targeting therapy reagents. Moreover, increasing evidence suggests that this protocol may be further extended to isolate and characterize TICs from other types of human cancers.

We describe a detailed protocol for the isolation of tumor-initiating cells from human sarcoma patient-derived xenografts by fluorescence-activated cell sorting, using human leukocyte antigen-1 (HLA-1) as a negative marker, and for the further validation and characterization of these HLA-1-negative tumor-initiating cells.

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

Investigation of Genetic Dependencies Using CRISPR-Cas9-based Competition Assays

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene disruption, many of these studies have made use of complex gene knockout models. While these studies with knockout mice offer an elegant and time-tested system for investigating genotype-to-phenotype relationships, a rapid and scalable method for assessing candidate genes that play a role in AML cell proliferation or survival in AML models will help accelerate the parallel interrogation of multiple candidate genes. Recent advances in genome-editing technologies have dramatically enhanced our ability to perform genetic perturbations at an unprecedented scale. One such system of genome editing is the CRISPR-Cas9-based method that can be used to make rapid and efficacious alterations in the target cell genome. The ease and scalability of CRISPR/Cas9-mediated gene-deletion makes it one of the most attractive techniques for the interrogation of a large number of genes in phenotypic assays. Here, we present a simple assay using CRISPR/Cas9 mediated gene-disruption combined with high-throughput flow-cytometry-based competition assays to investigate the role of genes that may play an important role in the proliferation or survival of human and murine AML cell lines.

This manuscript describes a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR-Cas9-based method for simple and expeditious investigation of the role of multiple candidate genes in Acute Myeloid Leukemia (AML) cell proliferation in parallel. This technique is scalable and can be applied in other cancer cell lines as well.

基于 crispr-cas9 的竞争检测对遗传依赖性的调查

Gene perturbation studies have been extensively used to investigate the role of individual genes in AML pathogenesis. For achieving complete gene ...

Mesenchymal Stem Cell Isolation from Pulp Tissue and Co-Culture with Cancer Cells to Study Their Interactions

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor environment and cell-cell interactions. Identification of cancer and stem cell interactions, including changes in extracellular environment, physical interactions, and secreted factors, might enable the discovery of new therapy options. We combine known co-culture techniques to create a model system for mesenchymal stem cells (MSCs) and cancer cell interactions. In the current study, dental pulp stem cells (DPSCs) and PC-3 prostate cancer cell interactions were examined by direct and indirect co-culture techniques. Condition medium (CM) obtained from DPSCs and 0.4 &#181;m pore sized trans-well membranes were used to study paracrine activity. Co-culture of different cell types together was performed to study direct cell-cell interaction. The results revealed that CM increased cell proliferation and decreased apoptosis in prostate cancer cell cultures. Both CM and trans-well system increased cell migration capacity of PC-3 cells. Cells stained with different membrane dyes were seeded into the same culture vessels, and DPSCs participated in a self-organized structure with PC-3 cells under this direct co-culture condition. Overall, the results indicated that co-culture techniques could be useful for cancer and MSC interactions as a model system.

We provide protocols for evaluation of mesenchymal stem cells isolated from dental pulp and prostate cancer cell interactions based on direct and indirect co-culture methods. Condition medium and trans-well membranes are suitable to analyze indirect paracrine activity. Seeding differentially stained cells together is an appropriate model for direct cell-cell interaction.

骨髓间充质干细胞在浆组织中的分离及与癌细胞联合培养研究其相互作用

Cancer as a multistep process and complicated disease is not only regulated by individual cell proliferation and growth but also controlled by tumor ...

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

木尿造血干细胞和祖细胞和MLL-AF9驱动白血病线粒体反应氧物种的流细胞学分析

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

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 ...

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者生物流体中肿瘤相关循环DNA的检测与监测

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

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 ...