Name: semi-automatic pd-l1 characterization and enumeration of circulating tumor cells from non-small cell lung cancer patients by immunofluorescence
Uploaded: 2019-08-14T14:30:00
Description: Watch this Scientific Journal Video about Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence at JoVE.com

这项工作得到了阿斯利康(英国伦敦)、生物研究所(新加坡)和癌症联盟(法国萨奥内和卢瓦尔)的研究资助。作者感谢阿斯利康和生物科技公司的财政支持。

在患者书面同意后,在里昂大学医院的CIRCAN("CIRCsatCANcer")队列框架内,对样本进行了前瞻性采集。这项研究被纳入了CIRCAN_ALL队列。该研究 CIRCAN_ALL 在参考 L15-188 下被 CPP 东南 IV 于 2015 年 4 月 4 日确认为不干预。2016年9月20日,根据参考L16-160,修订版被确认为不干预。CIRCAN_ALL 研究于 2015 年 1 月 12 日根据参考文献 15-131 向里昂市公民协会 IT 和自由通讯员宣布。当医生观察到肿瘤进展的最早迹象时,进行血液...

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本研究提出了两个主要观点,第一点涉及将其转移到临床应用的工作流程的性能,第二点关于分析所获得的荧光图像的主观性降低。
在细胞富集后,通过CTC无标签微流体系统(螺旋微流体装置),使用可定制的IF测定,初步确定了CTC枚举的可执行和优化的工作流程。利用这一工作流程,一项试验性研究证实,来自转移性NSCLC患者的所有样本都含有非典型细胞,这些细胞都是CD45(-)。它们也可?...

肿瘤抗原特异性细胞毒性T淋巴细胞(CTLs)在癌症反应中扮演着关键角色,这个过程被称为癌症"免疫监测"。免疫检查点封锁抗体(如CTLA-4抑制剂和PD-1/PD-L1抑制剂)增强了其抗肿瘤功能。在非小细胞肺癌(NSCLC)中,抗PD-1/PD-L1疗法在PD-L1阴性肿瘤患者中导致反应率在0%-17%到36%-100%。在黑色素瘤和NSCLC中观察到的对PD-1/PD-L1封锁的有力反应通过总体反应率(RR)、持久临床益处和无进展生存(PFS)的证据显示。目前,抗PD1治疗是二线NSCLC治疗的护理标准,无论PD-L1表达如何,对表达PD-L1±1的患者中,使用二线NSCLC治疗均与尼沃鲁马布和pmbrolizumab为标准。在一线治疗中,在NSCLC表达PD-L1+50%的患者中,护理标准是pmbrolizumab,并且可以通过化疗(根据组织学亚型的platin和双药)1、2来增强。
然而,这种治疗患者的方法是有争议的3,因为PD-L1在肿瘤细胞中通过免疫组织化学(IHC)表达可能不是最理想的伴生生物标志物。其他如肿瘤突变负担4(TMB),微卫星不稳定性(MSI)和/或微生物群可能在这个环境中有趣,单独或组合。NSCLC 已知是异构肿瘤,无论是在空间上(从肿瘤部位到另一个)还是从时间上(从诊断到复发)。NSCLC患者通常很脆弱,反复侵入性组织活检可能是一个问题。事实上,第一次进展的复检率从46%-84%不等,取决于系列,而成功的复检(指组织学和全分子分析)范围为33%-75%。这意味着25%-67%的患者在第一次进展5、6、7、8期间不能接受全面的再活检分析。
因此,"液体活检"的出现在这个特定环境中引起了相当大的热情,因为它通过检查从循环中衍生的循环自由DNA(cfDNA),能够对疾病进展期间的分子变化进行关键的重新评估肿瘤细胞(CTCs)。这些活细胞从肿瘤释放到血液中,在那里自由循环。虽然不经常使用,但CTC的分析在肺癌的分子和表型表征、预后和预测意义(通过DNAAq、RNAeq、miRNA和蛋白质分析)方面似乎很有希望。事实上,CTC可能含有活性疾病的型板特征,而不是最初的标记物(在诊断时在组织活检上检测到)。此外,CTCs绕过了肿瘤组织的空间异质性问题,这可能是小活检中的一个关键问题。因此,在CTC上的PD-L1表达可能会揭示其作为肿瘤组织的预测性生物标志物所产生的差异。
最近,PD-L1表达在NSCLC的TC中进行了测试。几乎所有测试的患者9是PD-L1阳性,使结果的解释及其临床使用复杂化。总体而言,在平均4.5个细胞/mL10的样本中,69.4%的样本中检测到PD-L1阳性的CTC。放射治疗开始后,PD-L1阳性CTC的比例显著增加,表明PD-L1表达对辐射11的反应上升。因此,PD-L1 CTCs 分析可用于监测肿瘤的动态变化和免疫反应,这可能反映对化疗、放疗和可能的免疫治疗 (IT) 治疗的反应。
迄今为止,CTC隔离和PD-L1表征依赖于各种方法,如抗EpCAM涂层磁珠的CTC捕获、富集基测定和基于尺寸的12、13CTC捕获测定。然而,在45%-65%的转移NSCLC患者中只检测到了CTCs,从而限制了他们为超过一半的转移性NSCLC患者提供任何信息的能力。此外,在大多数使用基于尺寸的方法10的研究中,CTC计数较低。此外,这种方法还导致在健康献血者血液中检测CD45(-)/DAPI(+)细胞具有"恶性细胞形态"的差异。这些关切突出表明,需要使用NSCLC中额外的癌症生物标志物(即TTF1、维门丁、EpCAM和CD44)从健康全血中分离非典型CD45(-)细胞进行免疫-phenotment收集,从而获得高度敏感的CTC收集方法。
因此,我们评估了一种螺旋微流体装置,该装置使用惯性和Dean阻力,通过微流体芯片根据大小和可塑性分离细胞。微流体芯片中存在的Dean涡流的形成导致沿内壁的较大CT和沿着芯片外壁的较小免疫细胞。浓缩过程通过将较大的细胞作为浓缩的CTC部分吸入收集出口完成。这种方法特别敏感和具体(检测全血约1CTC/mL)14,可与定制的免疫荧光(IF)分析相关。这些工具将启用临床解释的正阈值。因此,描述了一个工作流程,使生物学家能够分离和免疫类三氯苯甲酸碳酸盐具有高回收率和特异性。该协议描述了螺旋微流体器件收集CTC的最佳用途,根据癌症类型可定制的优化IF检测,以及使用免费的开源软件测量和分析细胞图像,以执行半自动根据荧光染色对细胞进行计数。此外,显微镜复用可以根据可用的荧光过滤器/标记的数量进行。

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			<td height="20" style="height:20px;width:376px;">4&#39;,6-diamidino-2-ph&#233;nylindole (DAPI)</td>
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			<td style="width:375px;">Storage conditions: +4&#176;C</td>
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			<td height="34" style="height:34px;width:376px;">BD Facs Clean &#8211; 5L</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:105px;">340345</td>
			<td style="width:375px;">Bleach-based cleaning agent. Storage conditions: Room temperature</td>
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			<td height="20" style="height:20px;width:376px;">Bleach 1% Cleaning Solution 100 mL</td>
			<td style="width:148px;">Biolidics</td>
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			<td height="20" style="height:20px;width:376px;">Bovine Serum Albumin (BSA) 7.5%</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:105px;">A8412</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
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			<td height="20" style="height:20px;width:376px;">CD45 monoclonal antibody (clone HI30) Alexa Fluor 647</td>
			<td style="width:148px;">BioLegend</td>
			<td style="width:105px;">BLE304020</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
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			<td height="20" style="height:20px;width:376px;">Centrifuge device</td>
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			<td style="width:375px;">Storage conditions: Room temperature</td>
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			<td height="20" style="height:20px;width:376px;">Centrifuge tube 50 mL</td>
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			<td style="width:375px;">Storage conditions: Room temperature</td>
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			<td height="20" style="height:20px;width:376px;">Centrifuge Tube 15 mL</td>
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			<td style="width:375px;">Storage conditions: Room temperature</td>
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			<td style="width:148px;">Biolidics</td>
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			<td style="width:375px;">Spiral microfluidic device. Storage conditions: Room temperature</td>
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			<td height="34" style="height:34px;width:376px;">Coulter Clenz Cleaning Agent &#8211; 5L</td>
			<td style="width:148px;">Beckman Coulter</td>
			<td style="width:105px;">8448222</td>
			<td style="width:375px;">All-purpose cleaning reagent. Storage conditions: Room temperature</td>
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			<td height="20" style="height:20px;width:376px;">CTChip FR1S</td>
			<td style="width:148px;">Biolidics</td>
			<td style="width:105px;">CBB-FR001002</td>
			<td style="width:375px;">Microfluidic chip. Storage conditions: Room temperature</td>
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			<td height="20" style="height:20px;width:376px;">Cytospin 4</td>
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			<td style="width:105px;">A78300003</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
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			<td height="20" style="height:20px;width:376px;">Diluent Additive Reagent &#8211; 20 mL</td>
			<td style="width:148px;">Biolidics</td>
			<td style="width:105px;">CBB-F016009</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
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			<td height="34" style="height:34px;width:376px;">EZ Cytofunnels</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:105px;">A78710003</td>
			<td style="width:375px;">Sample chamber with cotton. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">FcR blocking Agent</td>
			<td style="width:148px;">Miltenyi Biotec</td>
			<td style="width:105px;">130-059-901</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Fetal Calf Serum (FCS)</td>
			<td style="width:148px;">Gibco</td>
			<td style="width:105px;">10270-106</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Fluoromount</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:105px;">F4680</td>
			<td style="width:375px;">Mounting solution. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Fungizone - 50 mg</td>
			<td style="width:148px;">Bristol-Myers-Squibb</td>
			<td style="width:105px;">90129TB29</td>
			<td style="width:375px;">Anti-fungal reagent. Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">FX1 Input Straw with lock cap</td>
			<td style="width:148px;">Biolidics</td>
			<td style="width:105px;">CBB-F013005</td>
			<td style="width:375px;">Straw. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">KovaSlide</td>
			<td style="width:148px;">Dutscher</td>
			<td style="width:105px;">50126</td>
			<td style="width:375px;">Chambered slide. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">PanCK monoclonal antibody (clone AE1/AE3) Alexa Fluor 488</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:105px;">53-9003-80</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Paraformaldehyde 16%</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:105px;">11490570</td>
			<td style="width:375px;">Fixation solution. Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">PD-L1 monoclonal antibody (clone 29E2A3) - Phycoerythrin</td>
			<td style="width:148px;">BioLegend</td>
			<td style="width:105px;">BLE329706</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Petri Dish</td>
			<td style="width:148px;">Dutscher</td>
			<td style="width:105px;">632180</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:148px;">Ozyme</td>
			<td style="width:105px;">BE17-512F</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Phosphate Buffered Saline Ultra Pure Grade 1X &#8211; 1L</td>
			<td style="width:148px;">1st Base Laboratory</td>
			<td style="width:105px;">BUF-2040-1X1L</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Pluronic F-68 10%</td>
			<td style="width:148px;">Gibco</td>
			<td style="width:105px;">24040-032</td>
			<td style="width:375px;">Anti-binding solution. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Polylysine slides</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:105px;">J2800AMNZ</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Polypropylene Conical Tube 50 mL</td>
			<td style="width:148px;">Falcon</td>
			<td style="width:105px;">352098</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">RBC Lysis Buffer &#8211; 100 mL</td>
			<td style="width:148px;">G Biosciences</td>
			<td style="width:105px;">786-649</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">RBC Lysis Buffer &#8211; 250 mL</td>
			<td style="width:148px;">G Biosciences</td>
			<td style="width:105px;">786-650</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Resuspension Buffer (RSB)</td>
			<td style="width:148px;">Biolidics</td>
			<td style="width:105px;">CBB-F016003</td>
			<td style="width:375px;">Storage conditions: +4&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Shandon Cytopsin4 centrifuge</td>
			<td style="width:148px;">ThermoFisher</td>
			<td style="width:105px;">A78300003</td>
			<td style="width:375px;">Dedicated centrifuge. Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Silicon Isolator</td>
			<td style="width:148px;">Grace bio-Labs</td>
			<td style="width:105px;">664270</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Sterile Deionized Water &#8211; 100 mL</td>
			<td style="width:148px;">1st Base Laboratory</td>
			<td style="width:105px;">CUS-4100-100ml</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Straight Fluorescent microscope Axio Imager D1</td>
			<td style="width:148px;">Zeiss</td>
			<td></td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Surgical Sterile Bag</td>
			<td style="width:148px;">SPS Laboratoires</td>
			<td style="width:105px;">98ULT01240</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Syringe BD Discardit II 20 mL sterile</td>
			<td style="width:148px;">BD Biosciences</td>
			<td style="width:105px;">300296</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:376px;">Syringe Filter 0.22 &#181;m 33 mm sterile</td>
			<td style="width:148px;">ClearLine</td>
			<td style="width:105px;">51732</td>
			<td style="width:375px;">Storage conditions: Room temperature</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:376px;">Zen lite 2.3 Lite Software</td>
			<td style="width:148px;">Zeiss</td>
			<td style="width:105px;"></td>
			<td style="width:375px;">Microscope associated software</td>
		</tr>
	</tbody>
</table>

semi-automatic pd-l1 characterization and enumeration of circulating tumor cells from non-small cell lung cancer patients by immunofluorescence

从原发性肿瘤衍生的循环肿瘤细胞(CTC)被流入血液或淋巴系统。这些稀有细胞(每mL血液1~10个细胞)预后不佳,与几种癌症(如乳腺癌、前列腺癌和结肠直肠)的整体存活率较短有关。目前,抗EpCAM涂层磁珠的CTC捕获系统是美国食品和药物管理局(FDA)批准的用于在血液中列举CTC的黄金标准测试。此测试基于使用涂有抗 EpCAM 标记的磁珠,该标记专门针对上皮癌细胞。许多研究表明,EpCAM不是四氯化委员会检测的最佳标记。事实上,CTCs是癌细胞的异质亚群,能够经历与转移性增殖和入侵相关的上皮到中位转移(EMT)。这些CC能够减少细胞表面上皮标记EpCAM的表达,同时增加等分体标记,如维门他命。为了解决这一技术障碍,开发了基于CTC物理特性的其他隔离方法。微流体技术使全血样本的CTC富集方法无标签。螺旋微流体技术使用惯性和Dean阻力力,在螺旋微流体芯片内产生的曲线通道中连续流动。根据正常血细胞和肿瘤细胞的大小和可塑性的差异,分离细胞。该协议详细介绍了将螺旋微流体装置与可自定义的免疫荧光 (IF) 标记集相结合,以表征 CC 的编程死亡配体 1 (PD-L1) 表达的不同步骤。

第一个先决条件是获取未受污染的(无传染性代理)的CTC集合,用于组织培养,并避免生成IF背景。净化协议能够清洁所有管道和泵,并收集了具有良好回收率的四氯苯二苯C,没有细菌污染。将富集样品与螺旋微流体装置的去污方案工作流程进行比较。为了验证去污方案,A549细胞系在没有全血的情况下使用,并直接使用螺旋微流体装置进行浓缩。如果没有优化的去污方案,仅24小时后,在富集A549细胞系的组织?...

Watch this Scientific Journal Video about Semi-automatic PD-L1 Characterization and Enumeration of Circulating Tumor Cells from Non-small Cell Lung Cancer Patients by Immunofluorescence at JoVE.com

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

循环肿瘤细胞(CTC)的表征是转化研究的一个热门话题。该协议描述了一种半自动免疫荧光(IF)测定,用于PD-L1表征和非小细胞肺癌(NSCLC)患者样本中CTC的枚举。

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 ...

基于微流体技术，从正常血细胞分离和表征CTC不需要使用特定的CTC生物标志物，并且与细胞培养相容。该协议通过评估恶性细胞形态特征，促进高CTC提取率，并提供广泛的细胞学分析。CTC的PD-L1表达在肺癌管理中具有预测价值。利用此测试改进其检测可促进长期更好的患者管理。该协议的其他应用包括使用 FISH 检测遗传畸变，或对 CTC 进行转录分析，以及隔离母亲体内胎儿起源的细胞。演示这个程序的将是朱莉·巴兰迪尔，一个来自我们实验室的技术人员。对于预分析循环肿瘤细胞富集，首先将7.5毫升血液收集到EDTA钾管中，并保持样品在温和搅拌下，以避免细胞沉淀和凝固。在采集的6小时内，使用微生物安全柜下的血清移液器，将高达7.5毫升全血转移到新的50毫升离心管中，并在室温下以1600倍G离心10分钟。在离心结束时，使用转移移液器收集血浆分数，而不会干扰薄毛涂层，并使用相当于 PBS 的量稀释等离子体，高达 7.5 毫升。接下来，在血液样本中小心添加红细胞解液缓冲液，最终体积为30毫升，轻轻反转血液采集管三次，然后将导管置于室温下10分钟。在孵化结束时，通过离心收集细胞，并小心地去除除最后四到五毫升的上经剂。使用过滤的微移液来去除剩余的上清液，并使用带过滤尖端的 P1000 微移子在管壁上添加一毫升的再增液缓冲液。为了避免引入气泡，用温和的移液重新暂停细胞颗粒，直到样品是均匀的。当颗粒重新分配时，在管壁上再加三毫升的再增缓冲液，而不产生气泡，然后再次轻轻地混合细胞。对于循环肿瘤细胞富集的螺旋微流体装置，首先将一个新的螺旋微流体芯片加载到装置上，然后将一个空的50毫升离心管加载到每个输入和输出端口中。单击素数以对螺旋微流体设备进行三分钟的准备。在循环结束时拆下输入和输出管。将重新暂停的血液样本加载到输入端口中，然后将一个 15 毫升的透明锥形管加载到输出端口中。然后单击运行，并选择程序三启动31分钟循环肿瘤细胞富集程序。在循环结束时，将输出管转移到离心机，并使用五毫升血清移液器去除除最后两毫升的上流液器的所有部分。然后使用微管去除除最后100微升的上一升剂。对于免疫荧光染色，在血细胞计中计算细胞，将富集的样品稀释至每100微升0.2%抗结合溶液浓度的10至5个细胞的1倍。接下来，使用用 50 微升防结合溶液浸泡的棉签来滋润样品室的轮廓，并在样品室中放置多晶硅玻璃滑梯。关闭腔室，上下移液器三次，用结合溶液涂覆微管尖端，然后再将样品重新在上流液的最后 100 微升中重新制备。将细胞溶液转移到样品室，并在专用离心机中以每分钟 400 次旋转的速度将样品固定，以低加速度进行 4 分钟。离心结束时，在沉积区域周围放置一个硅胶隔离器，让滑轨在微生物安全柜下干燥两分钟，然后在室温下用100微升4%半甲醛将细胞孢子样品固定10分钟，然后用每次洗涤的200微升PBS进行三次两分钟洗涤。上次洗涤后，在室温下用30分钟的孵化阻断试剂，阻断任何非特异性结合，然后用100微升的抗体溶液进行标记。将标有的幻灯片放在 100 到 15 毫升的培养皿中，放在用两毫升无菌水润湿的吸水纸上，并在四摄氏度的夜间将盘子关闭，防止光线。第二天早上，如所示，用PBS清洗样品三次，用10微升适当的安装溶液和玻璃盖滑将样品安装。然后用透明指甲油密封盖滑。要对细胞孢子样本进行成像，请用 X Y 电动平台将幻灯片加载到直荧光显微镜上，然后根据用于抗体标记的荧光团选择 20 倍的靶点和适当的通道。打开汞灯，使显微镜和相关软件适应半自动拍摄。当灯准备就绪时，在采集菜单中定义四个通道，设置曝光时间，并定义要扫描的磁贴。单击磁贴和高级实验，并定义要扫描的区域。然后调整屏幕上的焦点，然后单击"开始实验"。在实验结束时，导出每个通道的 TIF 文件，并特别命名该文件以包括样本、时间、染料和子切片数。对于图像分析，从广院网站打开图像分析软件，然后单击文件、管道文件和分析四个通道CTC。将文件放入文件列表中，并更新元数据以按磁贴对文件进行分组。然后单击分析图像以打开与测量强度参数对应的电子表格文件。如果没有优化的去污方案，仅在24小时后，在富集A549细胞系的组织培养中观察到高细菌污染，导致真核细胞死亡和细胞形态变化。相比之下，在清洁方案之后，活的 A549 细胞在组织培养和培养中去除 10 小时后，在 3D 培养条件下和在患者样本中获得 2D 培养。使用液体免疫荧光染色测定或多晶硅涂层幻灯片上的免疫荧光染色测定，可以观察到两种测定之间列举的核数。如果没有细胞素步骤，很难区分白血球和肿瘤细胞，因为非细胞素细胞准备的模糊轮廓。在这里，可以从不同的患者样本中观察到不同的代表性结果，特别是白血球的残留计数被确定为强变量，并取决于整个血液样本。在获得循环肿瘤细胞子种群中也观察到高变异性。细胞针上的每个细胞由图像分析软件识别，并能够跟踪细胞并手动确认结果。事实上，试验性分析表明，手动枚举和图像分析软件枚举之间是一致性的。内部为蛋白质抗体杂交设计的湿培养皿的设置至关重要，因为在整个孵化过程中，该装置仍然足够潮湿。

Research

JoVE Journal

Cancer Research

Lung Tumor Cell Recruitment Assay

肺肿瘤细胞招募分析

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

科研

癌症研究

Capture and Release of Viable Circulating Tumor Cells from Blood

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 ...

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

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 ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

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

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

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

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 ...

Using 22C3 Anti-PD-L1 Antibody Concentrate on Biopsy and Cytology Samples from Non-small Cell Lung Cancer Patients

22C3 Anti-PD-L1 抗体精矿在非小细胞肺癌活检及细胞学标本中的应用

Pembrolizumab monotherapy has been approved for the first- and second-line treatment of patients with PD-L1-expressing advanced non-small cell lung cancer ...

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

合成肺腺癌细胞原位移植研究 pd-l1 表达

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

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

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 ...