Surwase Sachin S, Zhou Xin Ming M, Luly Kathryn M, Zhu Qingfeng, Anders Robert A, Green Jordan J, Tzeng Stephany Y, Sunshine Joel C
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland; Translational Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland; Johns Hopkins Translational ImmunoEngineering Center, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Lab Invest. 2025 Jan;105(1):102165. doi: 10.1016/j.labinv.2024.102165. Epub 2024 Oct 26.
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.
空间蛋白质组学分析是一组新兴技术，有潜力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，在癌症、免疫等研究领域有广泛应用。该领域的一项新兴技术是索引共检测技术，最近更名为PhenoCycler系统。这是一种高度多重免疫荧光成像技术，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，在单个样本中可视化多种抗体标记物。现有的PhenoCycler检测板主要针对新鲜冷冻组织设计。福尔马林固定石蜡包埋块在临床前研究中有诸多优势，但在此背景下，用于PhenoCycler成像的抗体克隆很少被鉴定出来。在此，我们展示了一个针对小鼠福尔马林固定石蜡包埋组织的由28种经过验证的抗体组成的新型PhenoCycler检测板。我们描述了选择和验证克隆、给抗体加条形码、设计检测板以及进行多重成像的工作流程。我们进一步详细介绍了用于比较标记物表达、对单细胞蛋白质组学数据进行聚类和表型分析以及量化空间关系的分析流程。然后，我们应用我们的检测板和分析方案，来分析3种基因递送纳米颗粒制剂与全身性抗PD1联合联合联合联合使用对小鼠黑色素瘤肿瘤免疫微环境的影响。瘤内递送表达共刺激分子4-1BBL和细胞因子IL-12的基因，导致肿瘤内M1巨噬细胞极化转变，并促进肿瘤内CD8 T细胞与巨噬细胞之间的紧密关联。除了4-1BBL和IL-12之外，递送干扰素γ不仅进一步增加了肿瘤细胞和肿瘤内抗原呈递细胞上抗原呈递的标记物，还促进了检查点标记物PD-L1的更高表达以及肿瘤内CD8 T细胞与表达PD-L1的肿瘤细胞之间的更紧密关联。这些发现有助于解释递送4-1BBL和IL-12的益处，同时为干扰素γ治疗效果的局限性提供了额外的机制性见解。
