Department of Dermatology, University Medical Center of the Johannes Gutenberg University Mainz, 55131 Mainz, Germany.
Zellkraftwerk GmbH, 04103 Leipzig, Germany.
Int J Mol Sci. 2021 Jan 20;22(3):1011. doi: 10.3390/ijms22031011.
The presence and interaction of immune cells in the tumor microenvironment is of significant importance and has a great impact on disease progression and response to therapy. Hence, their identification is of high interest for prognosis and treatment decisions. Besides detailed phenotypic analyses of immune, as well as tumor cells, spatial analyses is an important parameter in the complex interplay of neoplastic and immune cells-especially when moving into focus efforts to develop and validate new therapeutic strategies. Ex vivo analysis of tumor samples by immunohistochemistry staining methods conserves spatial information is restricted to single markers, while flow cytometry (disrupting tissue into single cell suspensions) provides access to markers in larger numbers. Nevertheless, this comes at the cost of scarifying morphological information regarding tissue localization and cell-cell contacts. Further detrimental effects incurred by, for example, tissue digestion include staining artifacts. Consequently, ongoing efforts are directed towards methods that preserve, completely or in part, spatial information, while increasing the number of markers that can potentially be interrogated to the level of conventional flow cytometric methods. Progression in multiplex immunohistochemistry in the last ten years overcame the limitation to 1-2 markers in classical staining methods using DAB with counter stains or even pure chemical staining methods. In this study, we compared the multiplex method Chipcytometry to flow cytometry and classical IHCP using DAB and hematoxylin. Chipcytometry uses frozen or paraffin-embedded tissue sections stained with readily available commercial fluorophore-labeled antibodies in repetitive cycles of staining and bleaching. The iterative staining approach enables sequential analysis of a virtually unlimited number of markers on the same sample, thereby identifying immune cell subpopulations in the tumor microenvironment in the present study in a humanized mouse melanoma model.
免疫细胞在肿瘤微环境中的存在和相互作用具有重要意义，对疾病的进展和治疗反应有很大影响。因此，它们的鉴定对于预后和治疗决策具有重要意义。除了对免疫细胞和肿瘤细胞进行详细的表型分析外，空间分析也是肿瘤和免疫细胞复杂相互作用的一个重要参数，尤其是当研究重点转移到开发和验证新的治疗策略时。免疫组织化学染色方法对肿瘤样本进行的离体分析保留了空间信息，但仅限于单个标志物，而流式细胞术（将组织分解成单细胞悬液）可以获得更多数量的标志物。然而，这是以牺牲组织定位和细胞间接触的形态学信息为代价的。进一步的不利影响包括，例如，组织消化导致的染色伪影。因此，人们正在努力开发方法，这些方法可以在保留空间信息的同时，增加可以通过传统流式细胞术方法进行检测的标记物的数量。在过去十年中，多重免疫组化技术的进展克服了使用 DAB 进行传统染色方法时只能检测 1-2 个标记物的限制，甚至可以使用纯化学染色方法。在这项研究中，我们将 Chipcytometry 与流式细胞术和使用 DAB 和苏木精的经典 IHCP 进行了比较。Chipcytometry 使用冷冻或石蜡包埋的组织切片，使用现成的商业荧光标记抗体进行重复的染色和漂白循环。迭代染色方法能够在同一样本上对几乎无限数量的标记物进行顺序分析，从而在本研究中在人源化黑色素瘤模型中鉴定肿瘤微环境中的免疫细胞亚群。
