福尔马林固定癌组织标本免疫肿瘤学自动复合免疫荧光小组研究
Summary
对6个标记多重免疫荧光面板的详细协议进行了优化和执行, 使用自动着色器实现更一致的结果和更短的手术时间。这种方法可以直接由任何实验室用于免疫肿瘤学研究。
Abstract
免疫肿瘤学的持续发展需要对癌症免疫学机制有更多的了解。从福尔马林固定、石蜡包埋 (ffpe) 活检中对组织样本进行免疫分析, 已成为了解肿瘤免疫学复杂性和发现癌症免疫治疗新的预测生物标志物的关键工具。组织免疫分析需要评估肿瘤微环境中的组合标志物, 包括炎性细胞亚群和免疫检查点。新型多重免疫组织化学方法的出现使得单组织切片的多参数分析比标准单次免疫组织化学 (ihc) 更有效。一种商业上可用的多重免疫荧光 (if) 方法基于钟形信号放大, 并结合多谱显微分析, 可以更好地分离组织中的各种标记物的信号。这种方法与使用未共轭的原生抗体是兼容的, 这些抗体在 ffpe 组织样本上已经为标准 ihc 进行了优化。在此, 我们详细介绍了一种自动化协议, 该协议允许用 pd-1、pd-1、cd68、cd8、ki-67 和 aeeenae3 细胞角蛋白的6个标记抗体面板对肿瘤组织样本进行多重 if 标记, 其中含有 4 ‘、6-二胺-2-苯二胺作为核细胞的反染色多面板协议在自动化 ihc 染色机中进行了优化, 染色时间比手动协议短, 任何实验室调查员都可以直接应用和调整多路面板协议, 以便对人体 ffpe 组织样本进行免疫肿瘤学研究。还介绍了几种控制和工具, 包括一种用于新的多重 if 面板的精细质量控制的下拉控制方法, 这些方法对于优化和验证该技术非常有用。
Introduction
ffpe 肿瘤组织样本的免疫分析已成为免疫肿瘤学研究的重要组成部分, 特别是在临床试验背景下发现和验证癌症免疫治疗的新预测生物标志物1 ,2。使用二胺苯并定等化学色素的致变色剂, 仍然是诊断活检组织3免疫标记的标准技术.标准 ihc 还可用于肿瘤组织免疫分析, 包括定量肿瘤相关淋巴细胞的亚群, 以及评估免疫检查点的表达水平, 如程序性细胞死亡配体 1 (pd-l1)4 ,5。标准 ihc 是有限的, 但是, 因为每个组织部分只能标记一种抗原。由于免疫分析研究通常需要分析几个标记的组合表达, 使用标准 ihc 将需要对多个组织切片进行染色, 每个部分都用一个标记染色, 因此,极大地限制了小组织样品的分析, 如芯针活检。标准 ihc 方法对于评估由不同细胞群体共同表达的标记也有限, 这与免疫检查标志物 (如 pd-l1) 很常见, 后者由肿瘤相关的巨噬细胞和癌细胞表达。例如, 病理学家使用标准单效 ihc 对不同类型的细胞6所表达的 ihc 标记进行定量分析时就报告了这一限制。在同一组织切片上采用不同颜色的 ihc 技术的发展代表了比标准 ihc 单次方法的进步,7尽管它们仍然受到少数人免疫标记的限制标记, 也是一个重要的技术挑战, 以正确评估标记表达在相同的亚细胞隔间相同的细胞群。
上述关于临床样本组织可用性的警告, 以及多重显色 ic 技术的局限性, 导致有必要开发改进的多重方法, 用于免疫肿瘤学研究。荧光标记与成像系统相结合, 可以有效地将多个荧光体的信号与同一幻灯片分离。其中一项技术是基于 tylab 信号放大 (tsa) 与多光谱显微镜成像相结合,以实现高效的分色8。一种市售的基于 tsa 的试剂盒采用了针对多光谱成像8进行优化的荧光 (见材料表)。该系统的一个关键优势是它与相同的未标记的原生抗体的兼容性, 这些抗体已经为标准显色 ihc9、10、11进行了验证和优化。这不仅可以实现更快的优化, 还可以灵活地进行包含新目标的优化和面板修改。此外, 多重免疫荧光 (mif) tsa 方法可以针对市售的自动化 ihc 污渍系统进行优化, 从而实现从单剂显色原 ihc 到 mif 的直接转移。
在这里, 我们提出了一个用于免疫肿瘤学研究的 mif 面板的协议, 该程序基于自动 mif tsa 染色, 并使用多光谱扫描仪进行成像。任何能够访问所述仪器和试剂的实验室用户都可以对该协议进行调整和修改。该协议包括一个由六种主要抗体组成的小组, 用于肿瘤的免疫分析: pd-l1、pd-1、cd68 (作为一种泛巨噬细胞标记)、cd8 (t-细胞毒性细胞)、ki-67 和 ae半 e3 (泛细胞角蛋白, 用作识别的上皮标记物癌细胞)。最近的一项研究描述了利用显色 ihc 作为标准参考来验证多重染色12的手动 tsa mif 协议的优化。此处介绍的更新方法是通过使用商用七色 tsa 套件在自动着色器中进行优化而开发的, 将染色时间从3-5天大幅缩短到 14小时, 同时还提高了染色的一致性。除了这里介绍的详细的主要协议外,补充材料部分还包括 “下拉控制” 方法、评估新 mif 面板的附加质量控制过程以及优化的技术说明,故障排除, 并开发新的多路面板, 以帮助实验室用户建立和优化 mif tsa 方法的定制 mif 面板。
Protocol
Representative Results
Discussion
正在进行的癌症免疫治疗革命为癌症患者开辟了新的、有希望的治疗选择。免疫肿瘤学领域的进展将需要增加对炎症性肿瘤微环境的了解, 这不仅是为了了解致癌过程中免疫机制的生物学, 也是为了寻找新的预测生物标志物。免疫治疗 1,2。由于癌症免疫学的复杂性, 通常需要对肿瘤组织样本进行审讯, 以形成越来越多的免疫标记物, 这些…
Disclosures
The authors have nothing to disclose.
Acknowledgements
medimmune 的 deborah s人提供了编辑支持。
Materials
| "InForm 2.4.2" Software for Spectral Unmixing and Image Analysis | PerkinElmer | CLS151066 | Called "spectral unmixing software" in text |
| "Phenochart 1.0.9" QPTIFF Software for Selection of MSI and Overall Slide Scan Viewing | PerkinElmer | CLS151067 | Called "QPTIFF software" in text |
| #1.5 Coverslips | Sigma Aldrich | 2975246 | |
| 200 Proof Ethanol | Koptec | V1001 | |
| 20x Tris-Buffered Saline | VWR | J640-4L | |
| Antibody Diluent | DAKO | S2203 | |
| Anti-CD68 Mouse Monoclonal | DAKO | M087601-2 | Clone PG-M1 |
| Anti-CD8 Rabbit Monoclonal | Ventana | M5392 | Clone SP239 |
| Anti-CK Mouse Monoclonal | DAKO | M351501-2 | Clone AE1/AE3 |
| Anti-ki67 Mouse Monoclonal | DAKO | M724001-2 | Clone MIB-1 |
| Anti-PD-1 Rabbit Monoclonal | Cell Signaling | #86163 | Clone D4W2J |
| Anti-PD-L1 Rabbit Monoclonal | Ventana | 790-4905 | Clone SP263 |
| Bond Dewax Solution | Leica | AR9222 | Called "dewax solution" in text |
| Bond Epitope Retrieval Solution 1 | Leica | AR9961 | Called "ER1" in text |
| Bond Epitope Retrieval Solution 2 | Leica | AR9640 | Called "ER2" in text |
| Bond Open Containers, 30 mL | Leica | OP309700 | Called "30 mL open containers" in text |
| Bond Open Containers, 7 mL | Leica | OP79193 | Called "7 mL open containers" in text |
| Bond Polymer Refine Detection | Leica | DS9800 | Called "chromogenic detection kit" in text |
| Bond Research Detection Kit | Leica | DS9455 | Called "research detection kit" in text |
| Bond Titration Kit | Leica | OPT9049 | Called "titration kit" in text |
| Bond Universal Covertile Novocastra | Leica | S21.2001 | Called "covertiles" in text |
| Bond Wash Solution 10X Concentrate | Leica | AR9590 | Called "10x wash solution" in text |
| BondRX Autostainer | Leica | Called "automated stainer" in text | |
| BondRX Software Version 5.2.1.204 | Leica | Called "automated stainer software" in text | |
| Opal 7-Color Automation IHC Kit | PerkinElmer | NEL801001KT | Called "multispectral staining kit" in text |
| Peroxidase Block | Leica | RE7101 | |
| ProLong Diamond Antifade Mountant | Thermo | P36965 | Called "slide mountant" in text |
| Starfrost Slides | Fisher | 15-183-51 | |
| Vectra Polaris Multispectral Microscope with "Vectra 3.0.5" Software for Multispectral Microscope Control | PerkinElmer | CLS143455 | Called "microscope control software" in text |
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