Hernandez Sharia, Rojas Frank, Laberiano Caddie, Lazcano Rossana, Wistuba Ignacio, Parra Edwin Roger
Department of Translational Molecular Pathology, The University of Texas MD Anderson Cancer Center, Houston, TX, United States.
Front Mol Biosci. 2021 Apr 29;8:667067. doi: 10.3389/fmolb.2021.667067. eCollection 2021.
Every day, more evidence is revealed regarding the importance of the relationship between the response to cancer immunotherapy and the cancer immune microenvironment. It is well established that a profound characterization of the immune microenvironment is needed to identify prognostic and predictive immune biomarkers. To this end, we find phenotyping cells by multiplex immunofluorescence (mIF) a powerful and useful tool to identify cell types in biopsy specimens. Here, we describe the use of mIF tyramide signal amplification for labeling up to eight markers on a single slide of formalin-fixed, paraffin-embedded tumor tissue to phenotype immune cells in tumor tissues. Different panels show different markers, and the different panels can be used to characterize immune cells and relevant checkpoint proteins. The panel design depends on the research hypothesis, the cell population of interest, or the treatment under investigation. To phenotype the cells, image analysis software is used to identify individual marker expression or specific co-expression markers, which can differentiate already selected phenotypes. The individual-markers approach identifies a broad number of cell phenotypes, including rare cells, which may be helpful in a tumor microenvironment study. To accurately interpret results, it is important to recognize which receptors are expressed on different cell types and their typical location (i.e., nuclear, membrane, and/or cytoplasm). Furthermore, the amplification system of mIF may allow us to see weak marker signals, such as programmed cell death ligand 1, more easily than they are seen with single-marker immunohistochemistry (IHC) labeling. Finally, mIF technologies are promising resources for discovery of novel cancer immunotherapies and related biomarkers. In contrast with conventional IHC, which permits only the labeling of one single marker per tissue sample, mIF can detect multiple markers from a single tissue sample, and at the same time, deliver extensive information about the cell phenotypes composition and their spatial localization. In this matter, the phenotyping process is critical and must be done accurately by a highly trained personal with knowledge of immune cell protein expression and tumor pathology.
每天，越来越多的证据表明癌症免疫治疗反应与癌症免疫微环境之间关系的重要性。众所周知，为了识别预后和预测性免疫生物标志物，需要对免疫微环境进行深入表征。为此，我们发现通过多重免疫荧光（mIF）对细胞进行表型分析是一种在活检标本中识别细胞类型的强大且有用的工具。在此，我们描述了使用mIF酪胺信号放大技术在福尔马林固定、石蜡包埋的肿瘤组织的单个载玻片上标记多达八个标志物，以对肿瘤组织中的免疫细胞进行表型分析。不同的 panel 显示不同的标志物，不同的 panel 可用于表征免疫细胞和相关的检查点蛋白。panel 的设计取决于研究假设、感兴趣的细胞群体或所研究的治疗方法。为了对细胞进行表型分析，使用图像分析软件来识别单个标志物表达或特定的共表达标志物，这些标志物可以区分已经选定的表型。单标志物方法可识别大量细胞表型，包括罕见细胞，这在肿瘤微环境研究中可能会有所帮助。为了准确解释结果，重要的是要识别不同细胞类型上表达的哪些受体及其典型位置（即细胞核、细胞膜和/或细胞质）。此外，mIF 的放大系统可能使我们比单标志物免疫组织化学（IHC）标记更容易看到弱标志物信号，如程序性细胞死亡配体 1。最后，mIF 技术是发现新型癌症免疫疗法和相关生物标志物的有前景的资源。与传统 IHC 不同，传统 IHC 每个组织样本仅允许标记一个单一标志物，mIF 可以从单个组织样本中检测多个标志物，同时提供有关细胞表型组成及其空间定位的广泛信息。在这方面，表型分析过程至关重要，必须由具有免疫细胞蛋白表达和肿瘤病理学知识的训练有素的人员准确完成。
