With the explosion of immuno-oncology and the approval of many immune checkpoint therapies by regulatory agencies in the last few years, understanding the tumor microenvironment (TME) in the context of patients' immune status has become essential. Among available immune profiling techniques, multiplex immunofluorescence (mIF) assays offer the unique advantage of preserving the architectural features of the tumor and revealing the spatial relationships between tumor cells and immune cells. A number of mIF and image analysis assays have been described for solid tumors but most are not sufficiently suitable in lymphoma, where the lack of clear tumor-stromal boundaries and high tumor density present significant challenges. Here we describe the development and optimization of a reliable workflow using Akoya Opal staining kits to label and analyze 6 markers per slide in diffuse large B-cell lymphoma (DLBCL) tissue sections. Five panels totaling 30 markers were developed to characterize infiltrating immune cells and relevant check-point proteins such as PD1, PD-L1, ICOS, SIRP-alpha and Lag3 on 70 DLBCL sections. Multiplexed sections were scanned using an Akoya multispectral scanner. An image analysis workflow using InForm and Matlab was developed to overcome challenges inherent to the DLBCL environment. Using the assays and workflows detailed here, we were able to quantify cell densities of subsets of infiltrating immune cells and observe their spatial patterns within the tumors. We highlight heterogeneous distribution of cytotoxic T cells across tumors with similar T cell density to underscores the importance of considering spatial context when studying the effects of immunological therapies in DLBCL.

中文翻译：

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弥漫性大B细胞淋巴瘤的多重免疫荧光染色和图像分析测定。

近年来，随着免疫肿瘤学的爆炸式增长以及监管机构对许多免疫检查点疗法的认可，了解患者免疫状况下的肿瘤微环境（TME）变得至关重要。在可用的免疫谱分析技术中，多重免疫荧光（mIF）分析提供了保留肿瘤的结构特征并揭示肿瘤细胞与免疫细胞之间空间关系的独特优势。已经描述了许多针对实体瘤的mIF和图像分析方法，但大多数方法不足以用于淋巴瘤，因为缺乏清晰的肿瘤基质边界和高肿瘤密度是淋巴瘤的一大挑战。在这里，我们描述了使用Akoya蛋白石染色试剂盒来标记和分析弥漫大B细胞淋巴瘤（DLBCL）组织切片中每张幻灯片6个标记的可靠工作流程的开发和优化。开发了五个面板，共30个标记，以在70个DLBCL切片上表征浸润的免疫细胞和相关的检查点蛋白，例如PD1，PD-L1，ICOS，SIRP-alpha和Lag3。使用Akoya多光谱扫描仪扫描多路切片。开发了使用InForm和Matlab的图像分析工作流，以克服DLBCL环境固有的挑战。使用此处详述的测定和工作流程，我们能够量化浸润免疫细胞子集的细胞密度，并观察其在肿瘤内的空间格局。