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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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?…
Declarações
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 4',6-diamidino-2-phénylindole (DAPI) | Ozyme | BLE 422801 | Storage conditions: +4°C |
| BD Facs Clean – 5L | BD Biosciences | 340345 | Bleach-based cleaning agent. Storage conditions: Room temperature |
| Bleach 1% Cleaning Solution 100 mL | Biolidics | CBB-F016012 | Bleach. Storage conditions: Room temperature |
| Bovine Serum Albumin (BSA) 7.5% | Sigma | A8412 | Storage conditions: +4°C |
| CD45 monoclonal antibody (clone HI30) Alexa Fluor 647 | BioLegend | BLE304020 | Storage conditions: +4°C |
| CellProfiler Software | Broad Institute | Image Analysis Software | |
| Centrifuge device | Hettich | 4706 | Storage conditions: Room temperature |
| Centrifuge tube 50 mL | Corning | 430-829 | Storage conditions: Room temperature |
| Centrifuge Tube 15 mL | Biolidics | CBB-F001004-25 | Storage conditions: Room temperature |
| ClearCell FX-1 System | Biolidics | CBB-F011002 | Spiral microfluidic device. Storage conditions: Room temperature |
| Coulter Clenz Cleaning Agent – 5L | Beckman Coulter | 8448222 | All-purpose cleaning reagent. Storage conditions: Room temperature |
| CTChip FR1S | Biolidics | CBB-FR001002 | Microfluidic chip. Storage conditions: Room temperature |
| Cytospin 4 | ThermoFisher | A78300003 | Storage conditions: Room temperature |
| Diluent Additive Reagent – 20 mL | Biolidics | CBB-F016009 | Storage conditions: +4°C |
| EZ Cytofunnels | ThermoFisher | A78710003 | Sample chamber with cotton. Storage conditions: Room temperature |
| FcR blocking Agent | Miltenyi Biotec | 130-059-901 | Storage conditions: +4°C |
| Fetal Calf Serum (FCS) | Gibco | 10270-106 | Storage conditions: +4°C |
| Fluoromount | Sigma | F4680 | Mounting solution. Storage conditions: Room temperature |
| Fungizone – 50 mg | Bristol-Myers-Squibb | 90129TB29 | Anti-fungal reagent. Storage conditions: +4°C |
| FX1 Input Straw with lock cap | Biolidics | CBB-F013005 | Straw. Storage conditions: Room temperature |
| KovaSlide | Dutscher | 50126 | Chambered slide. Storage conditions: Room temperature |
| PanCK monoclonal antibody (clone AE1/AE3) Alexa Fluor 488 | ThermoFisher | 53-9003-80 | Storage conditions: +4°C |
| Paraformaldehyde 16% | ThermoFisher | 11490570 | Fixation solution. Storage conditions: +4°C |
| PD-L1 monoclonal antibody (clone 29E2A3) – Phycoerythrin | BioLegend | BLE329706 | Storage conditions: +4°C |
| Petri Dish | Dutscher | 632180 | Storage conditions: Room temperature |
| Phosphate Buffered Saline (PBS) | Ozyme | BE17-512F | Storage conditions: +4°C |
| Phosphate Buffered Saline Ultra Pure Grade 1X – 1L | 1st Base Laboratory | BUF-2040-1X1L | Storage conditions: Room temperature |
| Pluronic F-68 10% | Gibco | 24040-032 | Anti-binding solution. Storage conditions: Room temperature |
| Polylysine slides | ThermoFisher | J2800AMNZ | Storage conditions: Room temperature |
| Polypropylene Conical Tube 50 mL | Falcon | 352098 | Storage conditions: Room temperature |
| RBC Lysis Buffer – 100 mL | G Biosciences | 786-649 | Storage conditions: +4°C |
| RBC Lysis Buffer – 250 mL | G Biosciences | 786-650 | Storage conditions: +4°C |
| Resuspension Buffer (RSB) | Biolidics | CBB-F016003 | Storage conditions: +4°C |
| Shandon Cytopsin4 centrifuge | ThermoFisher | A78300003 | Dedicated centrifuge. Storage conditions: Room temperature |
| Silicon Isolator | Grace bio-Labs | 664270 | Storage conditions: Room temperature |
| Sterile Deionized Water – 100 mL | 1st Base Laboratory | CUS-4100-100ml | Storage conditions: Room temperature |
| Straight Fluorescent microscope Axio Imager D1 | Zeiss | Storage conditions: Room temperature | |
| Surgical Sterile Bag | SPS Laboratoires | 98ULT01240 | Storage conditions: Room temperature |
| Syringe BD Discardit II 20 mL sterile | BD Biosciences | 300296 | Storage conditions: Room temperature |
| Syringe Filter 0.22 µm 33 mm sterile | ClearLine | 51732 | Storage conditions: Room temperature |
| Zen lite 2.3 Lite Software | Zeiss | Microscope associated software |
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