Glioma-Associated Microglia/Macrophages Display an Expression Profile Different from M1 and M2 Polarization and Highly Express <i>Gpnmb</i> and <i>Spp1</i>

Malignant glioma belong to the most aggressive neoplasms in humans with no successful treatment available. Patients suffering from glioblastoma multiforme (GBM), the highest-grade glioma, have an average survival time of only around one year after diagnosis. Both microglia and peripheral macrophages/monocytes accumulate within and around glioma, but fail to exert effective anti-tumor activity and even support tumor growth. Here we use microarray analysis to compare the expression profiles of glioma-associated microglia/macrophages and naive control cells. Samples were generated from CD11b+ MACS-isolated cells from naïve and GL261-implanted C57BL/6 mouse brains. Around 1000 genes were more than 2-fold up- or downregulated in glioma-associated microglia/macrophages when compared to control cells. A comparison with published data sets of M1, M2a,b,c-polarized macrophages revealed a gene expression pattern that has only partial overlap with any of the M1 or M2 gene expression patterns. Samples for the qRT-PCR validation of selected M1 and M2a,b,c-specific genes were generated from two different glioma mouse models and isolated by flow cytometry to distinguish between resident microglia and invading macrophages. We confirmed in both models the unique glioma-associated microglia/macrophage phenotype including a mixture of M1 and M2a,b,c-specific genes. To validate the expression of these genes in human we MACS-isolated CD11b+ microglia/macrophages from GBM, lower grade brain tumors and control specimens. Apart from the M1/M2 gene analysis, we demonstrate that the expression of Gpnmb and Spp1 is highly upregulated in both murine and human glioma-associated microglia/macrophages. High expression of these genes has been associated with poor prognosis in human GBM, as indicated by patient survival data linked to gene expression data. We also show that microglia/macrophages are the predominant source of these transcripts in murine and human GBM. Our findings provide new potential targets for future anti-glioma therapy.恶性胶质瘤是人类恶性程度最高的实体肿瘤之一，目前尚无获批的有效治疗方案。多形性胶质母细胞瘤（glioblastoma multiforme, GBM）作为最高级别胶质瘤，确诊患者的平均生存期仅约1年。小胶质细胞与外周巨噬细胞/单核细胞均会在胶质瘤组织及其周边区域聚集，但无法发挥有效的抗肿瘤免疫活性，甚至会促进肿瘤生长。本研究采用微阵列分析技术，对比胶质瘤相关小胶质细胞/巨噬细胞与未致敏对照细胞的基因表达谱。实验样本取自未致敏及接种GL261细胞的C57BL/6小鼠脑组织中经磁性激活细胞分选术（MACS）分离得到的CD11b阳性细胞。与对照细胞相比，胶质瘤相关小胶质细胞/巨噬细胞中约有1000个基因的表达量出现2倍以上的上调或下调。将该表达谱与已发表的M1型、M2a/b/c型极化巨噬细胞数据集进行比对后发现，胶质瘤相关小胶质细胞/巨噬细胞的基因表达模式仅与上述M1或M2型巨噬细胞的表达模式存在部分重叠。为验证筛选出的M1型及M2a/b/c型特异性基因的表达情况，本研究从两种不同的胶质瘤小鼠模型中获取经流式细胞术分选常驻小胶质细胞与浸润性巨噬细胞制备的样本，用于实时定量逆转录聚合酶链反应（qRT-PCR）验证。我们在两种小鼠模型中均证实，胶质瘤相关小胶质细胞/巨噬细胞具有独特的表型：其基因表达谱同时混合了M1型与M2a/b/c型巨噬细胞的特异性基因。为验证上述基因在人体中的表达情况，本研究从多形性胶质母细胞瘤（GBM）、低级别脑肿瘤患者及正常对照样本中，通过MACS分选出CD11b阳性小胶质细胞/巨噬细胞。除M1/M2型基因分析外，本研究还证实，Gpnmb与Spp1基因在小鼠及人类的胶质瘤相关小胶质细胞/巨噬细胞中均呈现显著上调表达。结合患者生存数据与基因表达数据的分析结果显示，这两个基因的高表达与人类GBM患者的不良预后密切相关。此外，本研究证实，在小鼠及人类GBM组织中，小胶质细胞/巨噬细胞是这两种转录本的主要来源。本研究的发现为未来抗胶质瘤治疗提供了全新的潜在靶点。