Characterizing the Tumor Immune Microenvironment with Tyramide-Based Multiplex Immunofluorescence.
Characterizing the Tumor Immune Microenvironment with Tyramide-Based Multiplex Immunofluorescence.
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DOI：
10.1007/s10911-021-09479-2
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发表时间：
2020-12
影响因子：
2.5
通讯作者：
Campbell MJ
中科院分区：
文献类型：
--
作者：
Mori H;Bolen J;Schuetter L;Massion P;Hoyt CC;VandenBerg S;Esserman L;Borowsky AD;Campbell MJ
多重免疫荧光（mIF）允许在单个组织切片上同时基于抗体检测多种标记物和核复染。最近的研究表明，mIF正在成为肿瘤微环境免疫分析的重要工具，进一步推进了我们对癌症和免疫系统之间相互作用的理解，并确定了对免疫治疗反应的预测性生物标志物。加快mIF的发现正在导致改进的诊断面板，而重要的是，mIF协议的标准化，以促进其过渡到临床使用。手动处理用于mIF的切片非常耗时，并且是众多样本之间变异性的潜在来源。为了提高重现性和通量，我们演示了使用自动载玻片染色机进行包含酪胺信号放大（TSA）的mIF。我们描述了两个小组，旨在表征肿瘤免疫微环境。第1组包括CD 3、CD 20、CD 117、FOXP 3、Ki 67、泛细胞角蛋白（CK）和DAPI，第2组包括CD 3、CD 8、CD 68、PD-1、PD-L1、CK和DAPI。首先通过标准免疫组织化学和单重IF测试一抗，然后开发多重面板，并使用Vectra 3.0多光谱成像系统获得图像。概述了各种图像分析方法（识别细胞类型，确定细胞密度，表征细胞-细胞关联）。这些mIF方案将是肿瘤微环境免疫分析的宝贵工具。
Multiplex immunofluorescence (mIF) allows simultaneous antibody-based detection of multiple markers with a nuclear counterstain on a single tissue section. Recent studies have demonstrated that mIF is becoming an important tool for immune profiling the tumor microenvironment, further advancing our understanding of the interplay between cancer and the immune system, and identifying predictive biomarkers of response to immunotherapy. Expediting mIF discoveries is leading to improved diagnostic panels, whereas it is important that mIF protocols be standardized to facilitate their transition into clinical use. Manual processing of sections for mIF is time consuming and a potential source of variability across numerous samples. To increase reproducibility and throughput we demonstrate the use of an automated slide stainer for mIF incorporating tyramide signal amplification (TSA). We describe two panels aimed at characterizing the tumor immune microenvironment. Panel 1 included CD3, CD20, CD117, FOXP3, Ki67, pancytokeratins (CK), and DAPI, and Panel 2 included CD3, CD8, CD68, PD-1, PD-L1, CK, and DAPI. Primary antibodies were first tested by standard immunohistochemistry and single-plex IF, then multiplex panels were developed and images were obtained using a Vectra 3.0 multispectral imaging system. Various methods for image analysis (identifying cell types, determining cell densities, characterizing cell-cell associations) are outlined. These mIF protocols will be invaluable tools for immune profiling the tumor microenvironment.
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DOI：
10.1083/jcb.201004104
发表时间：
2010-05-31
期刊：
The Journal of cell biology
影响因子：
--
作者：
Linkert M;Rueden CT;Allan C;Burel JM;Moore W;Patterson A;Loranger B;Moore J;Neves C;Macdonald D;Tarkowska A;Sticco C;Hill E;Rossner M;Eliceiri KW;Swedlow JR
通讯作者：
Swedlow JR
影响因子：
4.6
作者：
Bankhead P;Loughrey MB;Fernández JA;Dombrowski Y;McArt DG;Dunne PD;McQuaid S;Gray RT;Murray LJ;Coleman HG;James JA;Salto-Tellez M;Hamilton PW
通讯作者：
Hamilton PW
影响因子：
64.5
作者：
Goltsev Y;Samusik N;Kennedy-Darling J;Bhate S;Hale M;Vazquez G;Black S;Nolan GP
通讯作者：
Nolan GP
影响因子：
46.9
作者：
Merritt, Christopher R.;Ong, Giang T.;Beechem, Joseph M.
通讯作者：
Beechem, Joseph M.
影响因子：
2.5
作者：
Levenson, Richard M.;Lynch, David T.;Backer, Marina V.
通讯作者：
Backer, Marina V.