Highly Multiplexed Immunofluorescence PhenoCycler Panel for Murine Formalin-Fixed Paraffin-Embedded Tissues Yields Insight Into Tumor Microenvironment Immunoengineering.

IF 5.1 2区 医学 Q1 MEDICINE, RESEARCH & EXPERIMENTAL Laboratory Investigation Pub Date : 2024-10-26 DOI:10.1016/j.labinv.2024.102165
Sachin S Surwase, Xin Ming M Zhou, Kathryn M Luly, Qingfeng Zhu, Robert A Anders, Jordan J Green, Stephany Y Tzeng, Joel C Sunshine
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Abstract

Spatial proteomics profiling is an emerging set of technologies that has the potential to elucidate the cell types, interactions, and molecular signatures that make up complex tissue microenvironments, with applications in the study of cancer, immunity, and much more. An emerging technique in the field is CoDetection by indEXing, recently renamed as the PhenoCycler system. This is a highly multiplexed immunofluorescence imaging technology that relies on oligonucleotide-barcoded antibodies and cyclic immunofluorescence to visualize many antibody markers in a single specimen while preserving tissue architecture. Existing PhenoCycler panels are primarily designed for fresh frozen tissues. Formalin-fixed paraffin-embedded blocks offer several advantages in preclinical research, but few antibody clones have been identified in this setting for PhenoCycler imaging. Here, we present a novel PhenoCycler panel of 28 validated antibodies for murine formalin-fixed paraffin-embedded tissues. We describe our workflow for selecting and validating clones, barcoding antibodies, designing our panel, and performing multiplex imaging. We further detail our analysis pipeline for comparing marker expressions, clustering and phenotyping single-cell proteomics data, and quantifying spatial relationships. We then apply our panel and analysis protocol to profile the effects of 3 gene delivery nanoparticle formulations, in combination with systemic anti-PD1, on the murine melanoma tumor immune microenvironment. Intralesional delivery of genes expressing the costimulatory molecule 4-1BBL and the cytokine IL-12 led to a shift toward intratumoral M1 macrophage polarization and promoted closer associations between intratumoral CD8 T cells and macrophages. Delivery of interferon gamma, in addition to 4-1BBL and IL-12, not only further increased markers of antigen presentation on tumor cells and intratumoral antigen-presenting cells but also promoted greater expression of checkpoint marker PD-L1 and closer associations between intratumoral CD8 T cells and PD-L1-expressing tumor cells. These findings help explain the benefits of 4-1BBL and IL-12 delivery while offering additional mechanistic insights into the limitations of interferon gamma therapeutic efficacy.

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用于小鼠 FFPE 的高复用免疫荧光 PhenoCycler 面板可深入了解肿瘤微环境免疫工程。
空间蛋白质组学分析是一套新兴技术,有可能阐明构成复杂组织微环境的细胞类型、相互作用和分子特征,可应用于癌症、免疫等方面的研究。该领域的一项新兴技术是 "共沉淀检测(CODEX)",最近更名为 "PhenoCycler 系统"。这是一种高度复用的免疫荧光成像技术,依靠寡核苷酸条形码抗体和循环免疫荧光,在保留组织结构的同时,对单个标本中的多种抗体标记进行可视化。现有的 PhenoCycler 面板主要是为新鲜冷冻组织设计的。福尔马林固定石蜡包埋(FFPE)组织块在临床前研究中具有多种优势,但很少有抗体克隆能在这种情况下用于 PhenoCycler 成像。在这里,我们展示了一个由 28 种经过验证的抗体组成的新型 PhenoCycler 面板,适用于小鼠 FFPE 组织。我们介绍了选择和验证克隆、对抗体进行条形码编码、设计面板和进行多重成像的工作流程。我们进一步详细介绍了比较标记物表达、聚类和表型单细胞蛋白质组学数据以及量化空间关系的分析流程。然后,我们应用我们的面板和分析方案来分析三种基因递送纳米粒子制剂与全身抗 PD1 联用对小鼠黑色素瘤肿瘤免疫微环境的影响。瘤内递送表达激动分子4-1BBL和细胞因子IL-12的基因会导致瘤内M1巨噬细胞极化,并促进瘤内CD8 T细胞和巨噬细胞之间更紧密的联系。除4-1BBL和IL-12外,IFNγ的递送进一步增加了肿瘤细胞和瘤内抗原递呈细胞上的抗原递呈标记物,但也促进了检查点标记物PD-L1的更高表达,并使瘤内CD8 T细胞与表达PD-L1的肿瘤细胞之间的联系更加紧密。这些发现有助于解释4-1BBL和IL-12递送的益处,同时为IFNγ疗效的局限性提供了更多的机理启示。
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来源期刊
Laboratory Investigation
Laboratory Investigation 医学-病理学
CiteScore
8.30
自引率
0.00%
发文量
125
审稿时长
2 months
期刊介绍: Laboratory Investigation is an international journal owned by the United States and Canadian Academy of Pathology. Laboratory Investigation offers prompt publication of high-quality original research in all biomedical disciplines relating to the understanding of human disease and the application of new methods to the diagnosis of disease. Both human and experimental studies are welcome.
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