Pituitary tumors contain a side population with 'tumor stem cell'-associated characteristics [set 1]. Homo sapiens

Pituitary adenomas cause significant endocrine and mass-related morbidity. Not much is known about mechanisms underlying pituitary tumor pathogenesis.We searched for a side population (SP) in pituitary tumor, representing cells with high efflux capacity and potentially enriching for cancer/tumor stem cells (CSC/TSC). Human pituitary adenomas contain a SP irrespective of hormonal phenotype. This adenoma SP, as well as the further purified SP (pSP) depleted from endothelial and immune cells, enriches for cells that express ‘tumor stemness’ markers and signaling pathways, including epithelial-mesenchymal transition (EMT)-linked factors. The adenomas were found to contain self-renewing sphere-forming cells, considered a property of TSC. The sphere-initiating cells were retrieved in the pSP.Because benign pituitary adenomas do not grow in vitro and failed to expand in immunodeficient mice, the pituitary tumor cell line AtT20 was further used. We identified a SP in this cell line and found it more tumorigenic than the main population (MP). Of the two EMT-regulatory pathways tested, inhibition of Cxcr4 signaling reduced EMT-linked cell motility in vitro as well as xenograft tumor growth, whereas activation of TGFβ had no effect.The tumor pSP showed upregulated expression of the pituitary stem cell marker SOX2. Pituitaries from dopamine receptor D2 knockout (Drd2-/-) mice bearing prolactinomas, contain more pSP, Sox2+ and colony-forming cells than wildtype glands.In conclusion, we detected a SP in pituitary tumors and identified TSC-associated characteristics. Our study may contribute to a better understanding of pituitary tumor pathogenesis and lead to new therapeutic targets. Overall design: We determined gene expression profiles for 9 human pituitary adenoma samples (5 x NF-A; 4 x GH-A). The samples were obtained immediately after transsphenoidal resection (by the neurosurgeon Dr. van Loon, Division Neurosurgery, University Hospitals Leuven). Informed consent was received from the patients. Hormonal phenotype was determined by the Department of Imaging and Pathology (University Hospitals Leuven). For each sample, total SP and MP cells were sorted by FACS. Separate assays were run subsequently for the two fractions. As such we present data from 18 microarrays.

垂体腺瘤可引发严重的内分泌紊乱及占位性并发症，目前学界对垂体肿瘤发生发展的潜在分子机制仍知之甚少。本研究在垂体腺瘤中筛选得到侧群细胞（side population, SP）——这类细胞具备较高的药物外排能力，可富集癌症/肿瘤干细胞（cancer/tumor stem cells, CSC/TSC）。无论激素表型如何，人垂体腺瘤中均存在侧群细胞。该腺瘤侧群细胞，以及进一步剔除内皮细胞与免疫细胞后的纯化侧群细胞（pSP），均富集表达肿瘤干性标志物及相关信号通路分子，包括上皮间质转化（epithelial-mesenchymal transition, EMT）相关调控因子。研究发现，垂体腺瘤中存在自我更新的成球细胞，这被认为是肿瘤干细胞的核心特性之一，而成球起始细胞可在纯化侧群细胞中被分离得到。

由于良性垂体腺瘤无法在体外培养增殖，且在免疫缺陷小鼠体内无法形成移植瘤并扩增，因此本研究进一步采用垂体肿瘤细胞系AtT20开展实验。我们在该细胞系中检测到侧群细胞，且发现其致瘤能力显著强于主群细胞（main population, MP）。在测试的两条上皮间质转化调控通路中，抑制Cxcr4信号通路可在体外降低上皮间质转化相关的细胞迁移能力，并显著抑制异种移植瘤的生长，而激活转化生长因子β（transforming growth factor β, TGFβ）则无此类效果。肿瘤纯化侧群细胞中，垂体干细胞标志物SOX2的表达水平显著上调。相较于野生型小鼠垂体组织，携带催乳素瘤的多巴胺D2受体敲除（Drd2-/-）小鼠的垂体中，纯化侧群细胞、Sox2阳性细胞及集落形成细胞的数量均显著增多。

综上，本研究在垂体肿瘤中成功检测到侧群细胞，并鉴定出其具备肿瘤干细胞相关的特性。本研究有助于加深对垂体肿瘤发生发展机制的理解，同时可为新型治疗靶点的开发提供理论依据。

整体实验设计：本研究对9例人垂体腺瘤样本（5例无功能垂体腺瘤（non-functioning adenoma, NF-A）、4例生长激素型垂体腺瘤（growth hormone adenoma, GH-A））进行了基因表达谱分析。所有样本均由鲁汶大学医院神经外科分部的神经外科医生van Loon博士经蝶窦切除术后立即获取，且本研究已获得所有患者的知情同意。激素表型由鲁汶大学医院影像与病理科完成鉴定。针对每个样本，我们通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）分选出总侧群细胞与主群细胞，随后针对两个细胞组分分别开展独立实验，最终本研究共得到18组微阵列检测数据。