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

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. 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.

垂体腺瘤可引发显著的内分泌功能紊乱及占位相关并发症，目前学界对垂体肿瘤发生发展的分子机制尚知之甚少。本研究首先在垂体肿瘤中搜寻侧群细胞（side population, SP）——这类细胞具备高效外排药物的特性，理论上可富集肿瘤干细胞（cancer/tumor stem cells, CSC/TSC）。无论激素表型如何，人类垂体腺瘤中均存在SP组分。肿瘤来源的SP组分，以及经进一步纯化、去除内皮与免疫细胞后的纯化侧群细胞（purified SP, pSP），均可富集表达“肿瘤干性”标志物及相关信号通路分子的细胞，其中包括上皮间质转化（epithelial-mesenchymal transition, EMT）相关调控因子。研究发现垂体腺瘤中存在可自我更新的成球细胞，而这正是肿瘤干细胞的典型特征之一，且成球起始细胞可在pSP组分中被分离获取。
由于良性垂体腺瘤无法在体外培养增殖，且无法在免疫缺陷小鼠体内成瘤扩增，本研究后续采用了垂体肿瘤细胞系AtT20进行实验。我们在该细胞系中同样检测到了SP组分，且发现其致瘤能力显著高于主群细胞（main population, MP）。在本研究检测的两条EMT调控通路中，抑制Cxcr4信号通路可在体外降低EMT相关的细胞迁移能力，同时抑制异种移植瘤的生长；而激活TGFβ信号通路则未产生此类效应。肿瘤来源的pSP组分中，垂体干细胞标志物SOX2的表达显著上调。携带催乳素瘤的多巴胺D2受体敲除（Drd2-/-）小鼠的垂体组织，相较于野生型小鼠垂体，其pSP组分、SOX2阳性细胞以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SP组分，并鉴定出其携带的肿瘤干细胞相关特征。本研究有助于深入阐明垂体肿瘤的发生机制，同时可为开发新型治疗靶点提供理论依据。本研究对9例人类垂体腺瘤样本进行了基因表达谱分析（其中5例为无功能垂体腺瘤（non-functioning adenoma, NF-A），4例为生长激素型垂体腺瘤（growth hormone adenoma, GH-A））。所有样本均经经蝶窦手术切除后立即获取（手术由鲁汶大学医院神经外科的van Loon医师主刀），所有患者均签署了知情同意书。样本的激素表型由鲁汶大学医院影像与病理科完成鉴定。针对每一例样本，我们均通过荧光激活细胞分选术（fluorescence-activated cell sorting, FACS）分离获取总SP与MP组分，并分别对两个组分开展后续实验。本研究最终共呈现18组微阵列芯片检测数据。