Gorris Mark A J, Halilovic Altuna, Rabold Katrin, van Duffelen Anne, Wickramasinghe Iresha N, Verweij Dagmar, Wortel Inge M N, Textor Johannes C, de Vries I Jolanda M, Figdor Carl G
Department of Tumor Immunology, Radboud Institute for Molecular Life Sciences, Radboud university medical center, 6525 GA Nijmegen, the Netherlands.
Department of Pathology, Radboud university medical center, 6525 GA Nijmegen, the Netherlands; and.
J Immunol. 2018 Jan 1;200(1):347-354. doi: 10.4049/jimmunol.1701262. Epub 2017 Nov 15.
Therapies targeting immune checkpoint molecules CTLA-4 and PD-1/PD-L1 have advanced the field of cancer immunotherapy. New mAbs targeting different immune checkpoint molecules, such as TIM3, CD27, and OX40, are being developed and tested in clinical trials. To make educated decisions and design new combination treatment strategies, it is vital to learn more about coexpression of both inhibitory and stimulatory immune checkpoints on individual cells within the tumor microenvironment. Recent advances in multiple immunolabeling and multispectral imaging have enabled simultaneous analysis of more than three markers within a single formalin-fixed paraffin-embedded tissue section, with accurate cell discrimination and spatial information. However, multiplex immunohistochemistry with a maximized number of markers presents multiple difficulties. These include the primary Ab concentrations and order within the multiplex panel, which are of major importance for the staining result. In this article, we report on the development, optimization, and application of an eight-color multiplex immunohistochemistry panel, consisting of PD-1, PD-L1, OX40, CD27, TIM3, CD3, a tumor marker, and DAPI. This multiplex panel allows for simultaneous quantification of five different immune checkpoint molecules on individual cells within different tumor types. This analysis revealed major differences in the immune checkpoint expression patterns across tumor types and individual tumor samples. This method could ultimately, by characterizing the tumor microenvironment of patients who have been treated with different immune checkpoint modulators, form the rationale for the design of immune checkpoint-based immunotherapy in the future.
针对免疫检查点分子CTLA-4和PD-1/PD-L1的疗法推动了癌症免疫治疗领域的发展。针对不同免疫检查点分子（如TIM3、CD27和OX40）的新型单克隆抗体正在临床试验中研发和测试。为了做出明智的决策并设计新的联合治疗策略，深入了解肿瘤微环境中单个细胞上抑制性和刺激性免疫检查点的共表达情况至关重要。多重免疫标记和多光谱成像的最新进展使得能够在单个福尔马林固定石蜡包埋组织切片中同时分析三种以上的标志物，并能准确区分细胞和获取空间信息。然而，使用最大数量标志物的多重免疫组化存在诸多困难。这些困难包括多重检测组中一抗的浓度和顺序，它们对染色结果至关重要。在本文中，我们报告了一种八色多重免疫组化检测组的开发、优化及应用，该检测组由PD-1、PD-L1、OX40、CD27、TIM3、CD3、一种肿瘤标志物和DAPI组成。这个多重检测组能够同时对不同肿瘤类型中单个细胞上的五种不同免疫检查点分子进行定量分析。该分析揭示了不同肿瘤类型和单个肿瘤样本在免疫检查点表达模式上的主要差异。通过表征接受不同免疫检查点调节剂治疗患者的肿瘤微环境，这种方法最终可为未来基于免疫检查点的免疫治疗设计提供理论依据。
