Punt Simone, Baatenburg de Jong Robert J, Jordanova Ekaterina S
Department of Pathology, Leiden University Medical Center;
Department of Otorhinolaryngology and Head and Neck Surgery, Erasmus University Medical Center.
J Vis Exp. 2017 Jul 29(125):55589. doi: 10.3791/55589.
The four-color fluorescence immunohistochemistry (IHC) technique is a method to quantify cell populations of interest while taking into account their relative distribution and their localization in the tissue. This technique has been extensively applied to study the immune infiltrate in various tumor types. The tumor microenvironment is infiltrated by immune cells that are attracted to the tumor site. Different immune cell populations have been found to play different roles in the tumor microenvironment and to have a different impact on the outcome of disease. This manuscript describes the use of multiparameter fluorescence IHC on oropharyngeal squamous cell carcinoma (OPSCC) as an example. This technique can be extended to other tissue samples and cell types of interest. In the presented study, we analyzed the intraepithelial and stromal compartment of a large OPSCC cohort (n = 162). We focused on total T lymphocytes (CD3), immunosuppressive regulatory T cells (Tregs; i.e., FoxP3), and T helper 17 (Th17) cells (i.e., IL-17CD3) using a nuclear counterstain to distinguish tumor epithelium from stroma. A high number of T cells was found to be correlated with improved disease-free survival in patients with a low number of intratumoral IL-17 non-T cells. This suggests that IL-17 non-T cells may be correlated with a poor immune response in OPSCC, which is in agreement with the correlation described between IL-17 and poor survival in cancer patients. Currently, novel multiparameter fluorescence IHC techniques are being developed using up to 7 different fluorochromes and will enable the more precise characterization and localization of immune cells in the tumor microenvironment.
四色荧光免疫组织化学（IHC）技术是一种在考虑感兴趣的细胞群体在组织中的相对分布及其定位的同时对其进行定量的方法。该技术已被广泛应用于研究各种肿瘤类型中的免疫浸润情况。肿瘤微环境中浸润着被吸引到肿瘤部位的免疫细胞。已发现不同的免疫细胞群体在肿瘤微环境中发挥不同作用，并对疾病结局产生不同影响。本手稿以口咽鳞状细胞癌（OPSCC）为例描述了多参数荧光IHC的应用。该技术可扩展到其他感兴趣的组织样本和细胞类型。在本研究中，我们分析了一个大型OPSCC队列（n = 162）的上皮内和基质部分。我们使用核复染来区分肿瘤上皮和基质，重点关注总T淋巴细胞（CD3）、免疫抑制调节性T细胞（Tregs；即FoxP3）和辅助性T细胞17（Th17）细胞（即IL-17⁺CD3）。在肿瘤内IL-17⁻非T细胞数量较少的患者中，发现大量T细胞与无病生存期的改善相关。这表明IL-17⁻非T细胞可能与OPSCC中不良的免疫反应相关，这与癌症患者中IL-17与不良生存之间的相关性描述一致。目前，正在开发使用多达7种不同荧光染料的新型多参数荧光IHC技术，这将能够更精确地表征和定位肿瘤微环境中的免疫细胞。
