Section of Pharmaceutical Chemistry, Department of Pharmacy, School of Health Sciences, National and Kapodistrian University of Athens (NKUA), 15701 Athens, Greece.
Section of Cell Biology and Biophysics, Department of Biology, School of Science, National and Kapodistrian University of Athens (NKUA), 15701 Athens, Greece.
Int J Mol Sci. 2020 Mar 31;21(7):2436. doi: 10.3390/ijms21072436.
Melanoma is the most aggressive type of skin cancer, leading to metabolic rewiring and enhancement of metastatic transformation. Efforts to improve its early and accurate diagnosis are largely based on preclinical models and especially cell lines. Hence, we herein present a combinational Nuclear Magnetic Resonance (NMR)- and Ultra High Performance Liquid Chromatography-High-Resolution Tandem Mass Spectrometry (UHPLC-HRMS/MS)-mediated untargeted metabolomic profiling of melanoma cells, to landscape metabolic alterations likely controlling metastasis. The cell lines WM115 and WM2664, which belong to the same patient, were examined, with WM115 being derived from a primary, pre-metastatic, tumor and WM2664 clonally expanded from lymph-node metastases. Metabolite samples were analyzed using NMR and UHPLC-HRMS. Multivariate statistical analysis of high resolution NMR and MS (positive and negative ionization) results was performed by Principal Component Analysis (PCA), Partial Least Squares-Discriminant Analysis (PLS-DA) and Orthogonal Partial Least Squares-Discriminant Analysis (OPLS-DA), while metastasis-related biomarkers were determined on the basis of VIP lists, S-plots and Student's t-tests. Receiver Operating Characteristic (ROC) curves of NMR and MS data revealed significantly differentiated metabolite profiles for each cell line, with WM115 being mainly characterized by upregulated levels of phosphocholine, choline, guanosine and inosine. Interestingly, WM2664 showed notably increased contents of hypoxanthine, myo-inositol, glutamic acid, organic acids, purines, pyrimidines, AMP, ADP, ATP and UDP(s), thus indicating the critical roles of purine, pyrimidine and amino acid metabolism during human melanoma metastasis.
黑色素瘤是最具侵袭性的皮肤癌，导致代谢重排和转移转化增强。改善其早期和准确诊断的努力主要基于临床前模型，特别是细胞系。因此，我们在此提出了一种组合的核磁共振（NMR）和超高效液相色谱-高分辨率串联质谱（UHPLC-HRMS/MS）介导的黑色素瘤细胞非靶向代谢组学分析，以描绘可能控制转移的代谢改变。检查了属于同一患者的 WM115 和 WM2664 细胞系，其中 WM115 源自原发、前转移肿瘤，WM2664 则从淋巴结转移中克隆扩增而来。使用 NMR 和 UHPLC-HRMS 分析代谢物样本。通过主成分分析（PCA）、偏最小二乘判别分析（PLS-DA）和正交偏最小二乘判别分析（OPLS-DA）对高分辨率 NMR 和 MS（正离子和负离子）结果进行多变量统计分析，同时基于 VIP 列表、S-图和学生 t 检验确定与转移相关的生物标志物。NMR 和 MS 数据的接收器操作特征（ROC）曲线显示每个细胞系的代谢物谱明显不同，WM115 主要表现为磷酸胆碱、胆碱、鸟苷和肌苷水平上调。有趣的是，WM2664 显示出显著增加的次黄嘌呤、肌醇、谷氨酸、有机酸、嘌呤、嘧啶、AMP、ADP、ATP 和 UDP(s)含量，这表明嘌呤、嘧啶和氨基酸代谢在人类黑色素瘤转移过程中起着关键作用。
