Expression profiling of glioma initiating cells (GICs) in the sphere and differentiation conditions. Homo sapiens

Glioma initiating cells (GICs) are considered responsible for the therapeutic resistance and recurrence of malignant glioma. To clarify the molecular mechanism of GIC maintenance/differentiation, we established GIC clones from GBM patient tumors having the potential to differentiate into malignant gliomas in mouse intracranial xenograft, and established an in vitro glioma induction system by using serum stimulation. Upon the serum stimulation, the GIC spheres showed increased cellular proliferation, motility, filopodia/lameripodia formation and adhesion to the culture dishes. Simultaneously, the NSC marker proteins such as CD133 and Sox2 were down-regulated, and the astrocyte/glioma marker GFAP and the malignancy marker CD44 dramatically up-regulated. To identify genes/proteins whose expression changes dynamically during the differentiation of GICs into glioma cells, these GICs were subjected to DNA microarray/iTRAQ based integrated proteomics. Overall design: Within 4 hours of tumor removal from GBM patients, tissues were subjected to GIC preparation. After successive cloning, total RNA from GIC clones (GIC03A and GIC03U) on day 2 or 7 of subculture in NSC medium with or without 10% FCS was subjected to the analysis with Affymetrix microarrays. Simultaneously, the proteins extracted from the same set of cells were subjected to LC-shot gun analyses using the 8-plex iTRAQ method. We sought to obtain the information of the common molecules that were up- or down-regulated during the GSC differentiation process, and functional targets for the early onset of GIC-associated glioma.

胶质瘤起始细胞（Glioma Initiating Cells, GICs）被认为是恶性胶质瘤治疗抵抗与复发的关键诱因。为阐明GIC维持与分化的分子机制，我们从胶质母细胞瘤（Glioblastoma Multiforme, GBM）患者肿瘤样本中分离获得具备在小鼠颅内异种移植模型中分化为恶性胶质瘤能力的GIC克隆，并通过血清刺激构建了体外胶质瘤诱导体系。
血清刺激处理后，GIC神经球的细胞增殖能力、迁移能力、丝状伪足/板层伪足形成能力以及对培养皿的黏附能力均显著提升。与此同时，CD133、Sox2等神经干细胞（Neural Stem Cell, NSC）标记蛋白的表达水平显著下调，而星形胶质细胞/胶质瘤标记物GFAP以及恶性标志物CD44的表达则明显上调。
为筛选GIC向胶质瘤细胞分化过程中表达动态变化的基因与蛋白，我们对上述GIC开展了基于DNA微阵列与同位素标记相对和绝对定量（isobaric Tags for Relative and Absolute Quantitation, iTRAQ）的整合蛋白质组学分析。
实验整体设计：在胶质母细胞瘤患者肿瘤切除后4小时内，即刻对组织样本进行GIC分离培养。经过连续克隆筛选后，我们分别收集在添加10%胎牛血清（Fetal Calf Serum, FCS）或不含血清的神经干细胞培养基中继代培养第2天和第7天的GIC克隆（GIC03A与GIC03U）的总RNA，采用Affymetrix微阵列芯片进行转录组分析；同时，提取同一批细胞的蛋白质，利用8-plex iTRAQ标记法开展液相色谱-鸟枪法（LC-shot gun）蛋白质组学分析。本研究旨在筛选GSC分化过程中共同上调或下调的分子，以及与GIC相关胶质瘤早期发生相关的功能性靶标。