Surace Michael, DaCosta Karma, Huntley Anna, Zhao Weiguang, Bagnall Christopher, Brown Charles, Wang Chichung, Roman Kristin, Cann Jennifer, Lewis Arthur, Steele Keith, Rebelatto Marlon, Parra Edwin R, Hoyt Clifford C, Rodriguez-Canales Jaime
Laboratory of Pathology, MedImmune;
Laboratory of Pathology, MedImmune.
J Vis Exp. 2019 Jan 21(143). doi: 10.3791/58390.
Continued developments in immuno-oncology require an increased understanding of the mechanisms of cancer immunology. The immunoprofiling analysis of tissue samples from formalin-fixed, paraffin-embedded (FFPE) biopsies has become a key tool for understanding the complexity of tumor immunology and discovering novel predictive biomarkers for cancer immunotherapy. Immunoprofiling analysis of tissues requires the evaluation of combined markers, including inflammatory cell subpopulations and immune checkpoints, in the tumor microenvironment. The advent of novel multiplex immunohistochemical methods allows for a more efficient multiparametric analysis of single tissue sections than does standard monoplex immunohistochemistry (IHC). One commercially available multiplex immunofluorescence (IF) method is based on tyramide-signal amplification and, combined with multispectral microscopic analysis, allows for a better signal separation of diverse markers in tissue. This methodology is compatible with the use of unconjugated primary antibodies that have been optimized for standard IHC on FFPE tissue samples. Herein we describe in detail an automated protocol that allows multiplex IF labeling of carcinoma tissue samples with a six-marker multiplex antibody panel comprising PD-L1, PD-1, CD68, CD8, Ki-67, and AE1/AE3 cytokeratins with 4',6-diamidino-2-phenylindole as a nuclear cell counterstain. The multiplex panel protocol is optimized in an automated IHC stainer for a staining time that is shorter than that of the manual protocol and can be directly applied and adapted by any laboratory investigator for immuno-oncology studies on human FFPE tissue samples. Also described are several controls and tools, including a drop-control method for fine quality control of a new multiplex IF panel, that are useful for the optimization and validation of the technique.
免疫肿瘤学的持续发展需要我们对癌症免疫学机制有更深入的了解。对福尔马林固定、石蜡包埋（FFPE）活检组织样本进行免疫谱分析，已成为理解肿瘤免疫学复杂性以及发现癌症免疫治疗新预测生物标志物的关键工具。组织的免疫谱分析需要评估肿瘤微环境中的多种标志物，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，相比标准的单重免疫组织化学（IHC），能够对单个组织切片进行更高效的多参数分析。一种市售的多重免疫荧光（IF）方法基于酪胺信号放大，并结合多光谱显微镜分析，能够更好地分离组织中不同标志物的信号。这种方法与未偶联的一抗兼容，这些一抗已针对FFPE组织样本上的标准IHC进行了优化。在此，我们详细描述了一种自动化方案，该方案允许使用包含PD-L1、PD-1、CD68、CD8、Ki-67和AE1/AE3细胞角蛋白的六标志物多重抗体面板对癌组织样本进行多重IF标记，并以4',6-二脒基-2-苯基吲哚作为细胞核复染剂。该多重面板方案在自动化IHC染色仪中进行了优化，染色时间比手动方案短，任何实验室研究人员都可直接应用并适用于对人类FFPE组织样本进行的免疫肿瘤学研究。文中还介绍了几种对照和工具，包括用于新多重IF面板精细质量控制的点滴对照法，这些对照和工具对该技术的优化和验证很有用。
