A Systematic Approach to Identify Markers of Distinctly Activated Human Macrophages

Polarization has been a useful concept for describing activated macrophage phenotypes and gene expression profiles. However, macrophage activation status within tumors and other settings are often inferred based on only a few markers. Complicating matters for relevance to human biology, many of the best studied macrophage activation markers have been best characterized in mice and sometimes are not similarly regulated in human macrophages. To identify novel markers of activated human macrophages, gene expression profiles for human macrophages of a single donor subjected to 33 distinct activating conditions were obtained and a set of putative activation markers were subsequently evaluated in macrophages from multiple donors using integrated fluidic circuit (IFC)-based RT-PCR. Using unsupervised hierarchical clustering of the microarray screen, highly-altered transcripts (>4-fold change in expression) sorted the macrophage transcription profiles into two major and 13 minor clusters. Among the 1874 highly-altered transcripts, over 100 were uniquely altered in one major or two related minor clusters. IFC PCR-derived data confirmed the microarray results and to show the kinetics of expression of potential macrophage activation markers. Transcripts encoding chemokines, cytokines, and cell surface were prominent in our analyses. The activation markers identified by this study could be used to better characterize tumor-associated macrophages from biopsies as well as other macrophage populations collected from human clinical samples. 36 samples total from primary human monocyte-derived macrophages (3 technical replicates for untreated controls and 33 individual samples of uniquely activated macrophage types)

极化（Polarization）是描述活化巨噬细胞表型与基因表达谱的经典研究概念。然而，肿瘤及其他微环境中的巨噬细胞活化状态，往往仅通过少量标志物即可推断。而制约该类标志物向人体生物学研究转化的难点在于：多数被广泛研究的巨噬细胞活化标志物均以小鼠为模型完成系统表征，但其在人类巨噬细胞中的调控模式往往并不保守。为鉴定活化人巨噬细胞的新型标志物，本研究首先获取了单一名供体的人类巨噬细胞在33种不同活化条件下的基因表达谱；随后，依托集成流体回路（IFC）的反转录聚合酶链式反应（RT-PCR）技术，对多供体来源巨噬细胞中的一系列候选活化标志物进行了验证评估。通过对基因芯片（microarray）筛选结果开展无监督层次聚类分析，表达量变化倍数超过4倍的差异转录本将巨噬细胞转录谱划分为2个主要簇与13个次要簇。在1874个差异转录本中，超过100个仅在单个主要簇或两个相关联的次要簇中发生特异性表达改变。IFC PCR所得数据验证了基因芯片的分析结果，并阐明了潜在巨噬细胞活化标志物的表达动力学特征。本研究的分析结果显示，编码趋化因子、细胞因子与细胞表面分子的转录本占据显著比例。本研究鉴定得到的活化标志物，可用于更精准地表征活检样本中的肿瘤相关巨噬细胞，以及其他从人类临床样本中分离得到的巨噬细胞群。本研究共纳入36份原代人单核细胞衍生巨噬细胞样本：其中未处理对照组设置3份技术重复，剩余33份分别对应33种独特活化状态的巨噬细胞样本。