冷冻延迟时间对用于代谢组学研究的组织样本的影响。
Impact of Freezing Delay Time on Tissue Samples for Metabolomic Studies.
作者信息
Haukaas Tonje H, Moestue Siver A, Vettukattil Riyas, Sitter Beathe, Lamichhane Santosh, Segura Remedios, Giskeødegård Guro F, Bathen Tone F
机构信息
Department of Circulation and Medical Imaging, Norwegian University of Science and Technology, Trondheim, Norway; Faculty of Medicine, K. G. Jebsen Center for Breast Cancer Research, Institute of Clinical Medicine, University of Oslo, Oslo, Norway.
Department of Circulation and Medical Imaging, Norwegian University of Science and Technology, Trondheim, Norway; St. Olavs Hospital, Trondheim University Hospital, Trondheim, Norway.
出版信息
Front Oncol. 2016 Jan 28;6:17. doi: 10.3389/fonc.2016.00017. eCollection 2016.
INTRODUCTION
Metabolic profiling of intact tumor tissue by high-resolution magic angle spinning (HR MAS) MR spectroscopy (MRS) provides important biological information possibly useful for clinical diagnosis and development of novel treatment strategies. However, generation of high-quality data requires that sample handling from surgical resection until analysis is performed using systematically validated procedures. In this study, we investigated the effect of postsurgical freezing delay time on global metabolic profiles and stability of individual metabolites in intact tumor tissue.
MATERIALS AND METHODS
Tumor tissue samples collected from two patient-derived breast cancer xenograft models (n = 3 for each model) were divided into pieces that were snap-frozen in liquid nitrogen at 0, 15, 30, 60, 90, and 120 min after surgical removal. In addition, one sample was analyzed immediately, representing the metabolic profile of fresh tissue exposed neither to liquid nitrogen nor to room temperature. We also evaluated the metabolic effect of prolonged spinning during the HR MAS experiments in biopsies from breast cancer patients (n = 14). All samples were analyzed by proton HR MAS MRS on a Bruker Avance DRX600 spectrometer, and changes in metabolic profiles were evaluated using multivariate analysis and linear mixed modeling.
RESULTS
Multivariate analysis showed that the metabolic differences between the two breast cancer models were more prominent than variation caused by freezing delay time. No significant changes in levels of individual metabolites were observed in samples frozen within 30 min of resection. After this time point, levels of choline increased, whereas ascorbate, creatine, and glutathione (GS) levels decreased. Freezing had a significant effect on several metabolites but is an essential procedure for research and biobank purposes. Furthermore, four metabolites (glucose, glycine, glycerophosphocholine, and choline) were affected by prolonged HR MAS experiment time possibly caused by physical release of metabolites caused by spinning or due to structural degradation processes.
CONCLUSION
The MR metabolic profiles of tumor samples are reproducible and robust to variation in postsurgical freezing delay up to 30 min.
引言
通过高分辨率魔角旋转（HR MAS）磁共振波谱（MRS）对完整肿瘤组织进行代谢谱分析可提供重要的生物学信息，可能有助于临床诊断和新治疗策略的开发。然而，要生成高质量的数据，需要从手术切除到分析的整个样本处理过程都使用经过系统验证的程序。在本研究中，我们调查了术后冷冻延迟时间对完整肿瘤组织整体代谢谱和单个代谢物稳定性的影响。
材料与方法
从两个患者来源的乳腺癌异种移植模型收集的肿瘤组织样本（每个模型n = 3）在手术切除后0、15、30、60、90和120分钟被切成小块并在液氮中速冻。此外，立即分析一个样本，代表既未接触液氮也未接触室温的新鲜组织的代谢谱。我们还评估了乳腺癌患者活检样本（n = 14）在HR MAS实验中延长旋转时间的代谢影响。所有样本均在Bruker Avance DRX600光谱仪上通过质子HR MAS MRS进行分析，并使用多变量分析和线性混合模型评估代谢谱的变化。
结果
多变量分析表明，两个乳腺癌模型之间的代谢差异比冷冻延迟时间引起的变化更为显著。在切除后30分钟内冷冻的样本中，未观察到单个代谢物水平的显著变化。在此时间点之后，胆碱水平升高，而抗坏血酸、肌酸和谷胱甘肽（GS）水平降低。冷冻对几种代谢物有显著影响，但对于研究和生物样本库目的而言是必不可少的程序。此外，四种代谢物（葡萄糖、甘氨酸、甘油磷酸胆碱和胆碱）受HR MAS实验时间延长的影响，这可能是由于旋转导致代谢物物理释放或由于结构降解过程引起的。
结论
肿瘤样本的磁共振代谢谱具有可重复性，并且对术后冷冻延迟长达30分钟的变化具有稳健性。
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