PO.TB10.18 · 肿瘤生物学
用于在治疗条件下评估巨噬细胞极化及功能的CyTOF抗体组合的开发
Development of a CyTOF panel for macrophage polarization and functional assessment under treatment conditions
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
目的：肿瘤相关巨噬细胞（TAM）在肉瘤肿瘤微环境中广泛存在，常常促进免疫抑制、血管生成和肿瘤进展。TAM表现出一系列极化状态，从抗肿瘤（M1）到促肿瘤（M2）功能不等。质谱流式（CyTOF）能够进行高维单细胞分析，非常适合剖析肿瘤内各种巨噬细胞表型的存在及丰度。本研究旨在设计并验证一个靶向巨噬细胞及相关免疫标志物的CyTOF抗体组合，以表征巨噬细胞极化状态并评估治疗诱导的巨噬细胞亚型变化。
方法：开发了一个以小鼠巨噬细胞为重点的CyTOF抗体组合，包含40种标志物，涵盖谱系标志物、活化标志物以及与M1/M2表型和其他免疫细胞群相关的细胞因子相关蛋白。为优化抗体滴定和染色条件，使用市售的小鼠来源巨噬细胞系RAW 264.7进行体外巨噬细胞极化。在标准条件下诱导巨噬细胞极化状态：M1：LPS + IFN-gamma；M2a：IL-4 + IL-13；M2b：OVA、抗OVA IgG；M2c：IL-10；M2d：NECA刺激。验证后，将该组合应用于经一种新型化合物处理的原位植入骨肉瘤细胞的小鼠模型，以量化TAM极化及相关免疫网络。将肿瘤组织样本解离为单细胞悬液，使用CyTOF组合染色，并采用降维（UMAP）和聚类算法进行分析。
结果：该40标志物CyTOF组合成功用于在体外及小鼠异种移植瘤中识别和表征巨噬细胞亚型群体。小鼠RAW264.7细胞的体外极化显示出对应M1和M2亚型的不同表型聚类。这些结果也实现了有效的抗体滴定。与溶媒对照相比，用该新型化合物处理原位植入的骨肉瘤肿瘤后，M1标志物表达增加，M2相关标志物减少，表明该化合物诱导了巨噬细胞极化的转变。
结论：所开发的CyTOF组合为巨噬细胞极化及治疗诱导的表型变化的高分辨率分析提供了稳健的平台，可对免疫调节策略进行更详尽的研究。该组合可作为未来针对巨噬细胞亚群及其功能作用研究的工具，并将有助于进一步剖析肿瘤免疫微环境，推进对治疗反应中免疫学变异性的理解。
查看英文原文 English abstract
Purpose: Tumor associated macrophages (TAMs) are prevalent in the sarcoma tumor microenvironment, and often contribute to immune suppression, angiogenesis, and tumor progression. TAMs exhibit a spectrum of polarization states, ranging from anti-tumor (M1) to tumor-supportive (M2) functions. Mass cytometry (CyTOF) enables high-dimensional single-cell profiling, making it ideal for dissecting the presence and abundance of various macrophage phenotypes within a tumor. This study aimed to design and validate a CyTOF panel targeting macrophages and associated immune markers, to characterize macrophage polarization states and evaluate treatment induced changes in macrophage subtypes.
Methods: A mouse macrophage-focused CyTOF panel was developed comprised of 40 markers, including lineage markers, activation markers, and cytokine-associated proteins relevant to M1/M2 phenotypes and other immune cell populations. For optimization of antibody titration and staining conditions, macrophage polarization was performed in vitro using the commercially available mouse-derived macrophage cell line RAW 264.7. Macrophage polarization states were induced under standard conditions: M1: LPS + IFN-gamma and M2a: IL-4 + IL-13, M2b- OVA, Anti-OVA IgG, M2c- IL-10, and M2d-NECA stimuli. Following validation, the panel was applied to a mouse model of orthotopically implanted osteosarcoma cells treated with a novel compound to quantify the TAM polarization and associated immune networks. Tumor tissue samples were dissociated into single cell suspensions, stained using the CyTOF panel, and analyzed using dimensionality reduction (UMAP) and clustering algorithms.
Results: The 40-marker CyTOF panel was successfully used to identify and characterize macrophage subtype populations in vitro as well as in mouse xenograft tumors. In vitro polarization of mouse RAW264.7 cells showed distinct phenotypic clusters corresponding to M1 and M2 subtypes. These results also enabled effective antibody titration. Treatment of orthotopically implanted osteosarcoma tumors with the novel compound resulted in increased expression of M1 markers and a reduction in M2-associated markers compared to vehicle control, indicating a shift in macrophage polarization induced by this compound.
Conclusion: The developed CyTOF panel provides a robust platform for high-resolution analysis of macrophage polarization and treatment-induced phenotypic changes, offering more detailed investigation of immune modulation strategies. This panel could be applied as a tool for future investigations focused on macrophage subpopulations and their functional roles and will allow for further dissection of the tumor immune microenvironment, advancing the understanding of immunological variability in response to treatment.
利益披露 Disclosure
R. Weil, None..
K. Q. McKinney, None..
D. M. Loeb, None.