F11R is a novel monocyte prognostic biomarker for malignant glioma [Seq]

Objective: Brain tumors (gliomas) contain large populations of infiltrating macrophages and recruited microglia, which in experimental murine glioma models promote tumor formation and progression. Among the barriers to understanding the contributions of these stromal elements to high-grade glioma (glioblastoma; GBM) biology is the relative paucity of tools to characterize infiltrating macrophages and resident microglia. In this study, we leveraged multiple RNA analysis platforms to identify new monocyte markers relevant to GBM patient outcome. Methods: High-confidence lists of mouse resident microglia- and bone marrow-derived macrophage-specific transcripts were generated using converging RNA-seq and microarray technologies and validated using qRT-PCR and flow cytometry. Expression of select cell surface markers was analyzed in brain-infiltrating macrophages and resident microglia in an induced GBM mouse model, while allogeneic bone marrow transplantation was performed to trace the origins of infiltrating and resident macrophages. Glioma tissue microarrays were examined by immunohistochemistry, and the Gene Expression Omnibus (GEO) database was queried to determine the prognostic value of identified microglia biomarkers in human GBM. Results: We generated a unique catalog of differentially-expressed bone marrow-derived monocyte and resident microglia transcripts, and demonstrated that brain-infiltrating macrophages acquire F11R expression in GBM and following bone-marrow transplantation. Moreover, mononuclear cell F11R expression positively correlates with human high-grade glioma and additionally serves as a biomarker for GBM patient survival, regardless of GBM molecular subtype. Significance: These studies establish F11R as a novel monocyte prognostic marker for GBM critical for defining a subpopulation of stromal cells for future potential therapeutic intervention. Total RNA was isolated from three independently-generated sets of flow sorted bone marrow monocytes (CD11b+ CD45high CD115+ Ly6G- cells) and brainstem microglia (CD11b+ CD45low CD115low Ly6G- cells) for Illumina RNA-Seq, and two additional pools were subsequently generated and submitted for Affymetrix Mouse Exon 1.0ST microarray. Two of the RNA-Seq samples were additionally analyzed by the microarray, for a total of 6 samples (3 monocyte, 3 microglia) in each platform. Data outputs were analyzed by two analysis methods each (RNA-Seq data: ALEXA-Seq and Cufflinks; microarray data: Partek and Aroma). All four lists were merged into a new high-confidence gene list of transcripts that were significantly differentially expressed (DE) in three out of the four analyses. In this dataset, we include RNA-Seq data obtained from flow sorted mouse bone marrow monocytes and brainstem microglia.研究目标：脑肿瘤（胶质瘤）中存在大量浸润性巨噬细胞与募集的小胶质细胞，在实验性小鼠胶质瘤模型中，这类细胞可促进肿瘤形成与进展。目前阻碍我们解析这些基质成分对高级别胶质瘤（胶质母细胞瘤，GBM）生物学特性影响的核心瓶颈之一，是缺乏可有效表征浸润性巨噬细胞与常驻小胶质细胞的研究工具。本研究依托多种RNA分析平台，筛选出与胶质母细胞瘤患者预后相关的新型单核细胞标志物。 研究方法：本研究通过整合RNA测序（RNA-seq）与基因芯片（microarray）技术，构建了小鼠常驻小胶质细胞与骨髓来源巨噬细胞的高置信度特异性转录本列表，并通过实时定量聚合酶链反应（qRT-PCR）与流式细胞术（flow cytometry）对该列表进行验证。在诱导型胶质母细胞瘤小鼠模型中，我们分析了特定细胞表面标志物在脑浸润性巨噬细胞与常驻小胶质细胞中的表达情况；同时通过异基因骨髓移植追踪浸润性与常驻巨噬细胞的起源。我们采用免疫组化检测胶质瘤组织微阵列，并检索基因表达综合数据库（Gene Expression Omnibus, GEO），以明确筛选出的小胶质细胞标志物在人类胶质母细胞瘤中的预后价值。 研究结果：本研究构建了一套独特的差异表达骨髓来源单核细胞与常驻小胶质细胞转录本目录，并证实胶质母细胞瘤状态下以及骨髓移植后，脑浸润性巨噬细胞会表达F11R。此外，单核细胞F11R的表达水平与人类高级别胶质瘤呈正相关，且可作为胶质母细胞瘤患者生存预后的生物标志物，不受胶质母细胞瘤分子亚型影响。 研究意义：本研究证实F11R可作为胶质母细胞瘤的新型单核细胞预后标志物，为精准定义基质细胞亚群以开展未来潜在的治疗干预提供了关键依据。 本研究从3组独立流式分选的骨髓单核细胞（CD11b+ CD45high CD115+ Ly6G-细胞）与脑干小胶质细胞（CD11b+ CD45low CD115low Ly6G-细胞）中提取总RNA，用于Illumina RNA测序；此外额外构建了2个混合样本，提交至Affymetrix Mouse Exon 1.0ST基因芯片进行检测。其中2份RNA测序样本同时接受了芯片检测，因此每个平台共包含6份样本（3份单核细胞样本、3份小胶质细胞样本）。我们分别采用两种分析方法对各平台的数据进行处理（RNA测序数据采用ALEXA-Seq与Cufflinks；基因芯片数据采用Partek与Aroma），最终将4组分析结果合并，得到一套高置信度基因列表：即4次分析中有3次均显著差异表达的转录本。本数据集包含上述从流式分选的小鼠骨髓单核细胞与脑干小胶质细胞中获取的RNA测序数据。