Quantitative Proteomic Profiling Studies of Pancreatic Cancer Stem Cells

Analyzing subpopulations of tumor cells in tissue is a challenging subject in proteomic studies. Pancreatic cancer stem cells (CSCs) are such a group of cells that only constitute 0.2−0.8% of the total tumor cells but have been found to be the origin of pancreatic cancer carcinogenesis and metastasis. Global proteome profiling of pancreatic CSCs from xenograft tumors in mice is a promising way to unveil the molecular machinery underlying the signaling pathways. However, the extremely low availability of pancreatic tissue CSCs (around 10 000 cells per xenograft tumor or patient sample) has limited the utilization of currently standard proteomic approaches which do not work effectively with such a small amount of material. Herein, we describe the profiling of the proteome of pancreatic CSCs using a capillary scale shotgun technique by coupling offline capillary isoelectric focusing(cIEF) with nano reversed phase liquid chromatography(RPLC) followed by spectral counting peptide quantification. A whole cell lysate from 10 000 cells which corresponds to ∼1 μg of protein material is equally divided for three repeated cIEF separations where around 300 ng of peptide material is used in each run. In comparison with a nontumorigenic tumor cell sample, among 1159 distinct proteins identified with FDR less than 0.2%, 169 differentially expressed proteins are identified after multiple testing corrections where 24% of the proteins are upregulated in the CSCs group. Ingenuity Pathway analysis of these differential expression signatures further suggests significant involvement of signaling pathways related to apoptosis, cell proliferation, inflammation, and metastasis.

组织内肿瘤细胞亚群的分析是蛋白质组学研究中的一大挑战。胰腺肿瘤干细胞（Cancer Stem Cells, CSCs）仅占肿瘤细胞总数的0.2%~0.8%，却被证实是胰腺癌发生与转移的根源。对小鼠异种移植瘤来源的胰腺CSCs开展全局蛋白质组分析，是揭示其信号通路下游分子机制的极具前景的研究路径。然而，胰腺CSCs的获取量极低（每枚异种移植瘤或患者样本仅约10000个细胞），这极大限制了当前主流蛋白质组学方法的应用——此类方法无法对如此微量的样本实现有效分析。为此，本研究采用毛细管级鸟枪法蛋白质组技术，将离线毛细管等电聚焦（Capillary Isoelectric Focusing, cIEF）与纳米反相液相色谱（Nano Reversed Phase Liquid Chromatography, RPLC）联用，并结合光谱计数肽段定量法，对胰腺CSCs的蛋白质组进行了表征。本研究将10000个细胞制备的全细胞裂解液（对应约1μg蛋白质总量）均分为三份，用于三次重复的cIEF分离，每次分离使用约300ng肽段样品。与非致瘤性肿瘤细胞样本相比，在错误发现率（False Discovery Rate, FDR）小于0.2%的条件下鉴定得到的1159种独特蛋白质中，经多重检验校正后共鉴定出169种差异表达蛋白质，其中24%的蛋白质在CSCs组中呈上调表达。对上述差异表达特征进行Ingenuity通路分析后进一步揭示，细胞凋亡、细胞增殖、炎症反应及转移相关的信号通路均显著富集。