Abstract：
Multiple myeloma (MM) is an incurable plasma cell malignancy primarily localized within the bone marrow (BM). Myeloma plasma cells, like many other cancer cells, change their metabolism in response to internal and external stimuli. The main metabolic alterations of MM cells include deregulated glycolysis (commonly associated with enhanced uptake and utilization of glucose), lipid metabolism dysregulation, as well as deregulated mitochondrial respiration (commonly associated with the deregulated formation of reactive oxygen species). Over the past decade, the discovery of novel methodologies and the commercialization of sophisticated instrumentation and reagents have facilitated the detection of real-time changes in cellular bioenergetics. Of those, the Seahorse™ extracellular flux (XF) analyzer has been widely used to evaluate the glycolytic flux and mitochondrial respiration in many cell types. While adherent cell lines are easy to use with this technology, non-adherent suspension cells are more difficult to handle especially when their metabolic activities are being investigated in response to drug treatment. Here, we provide an integrated protocol that allows the detection of extracellular acidification rate (ECAR) of live myeloma plasma cells in response to chemotherapeutic drugs. Our optimized protocol consists of treating myeloma cells with cytotoxic drug of interest in a standard culture plate prior to the real-time analysis in the XF analyzer. Furthermore, we provide results of experiments in which the metabolic activities of myeloma cells in response to cytotoxic treatment were compared between the manufacturer\'s basic procedure and our optimized protocol. Our observations suggest that our integrated protocol can be used to achieve consistent, well-standardized results and thus it may have broad applications in studies focusing on the characterization of metabolic events in non-adherent suspension cells.
摘要：
多发性骨髓瘤（MM）是一种无法治愈的浆细胞恶性肿瘤，主要位于骨髓（BM）内。骨髓瘤浆细胞，像许多其他癌细胞一样，改变它们的新陈代谢以响应内部和外部刺激。MM细胞的主要代谢改变包括糖酵解失调（通常与葡萄糖的摄取和利用增强有关）。脂质代谢失调，以及线粒体呼吸失调（通常与活性氧的失调形成有关）。在过去的十年里，新方法的发现以及复杂仪器和试剂的商业化促进了细胞生物能量学实时变化的检测。其中,Seahorse™细胞外通量（XF）分析仪已广泛用于评估许多细胞类型的糖酵解通量和线粒体呼吸。虽然贴壁细胞系易于使用这种技术，非贴壁悬浮细胞更难处理，尤其是当它们的代谢活性正在研究响应于药物治疗时。这里,我们提供了一个综合方案,该方案允许检测骨髓瘤活浆细胞对化疗药物的反应的细胞外酸化率(ECAR).我们的优化方案包括在XF分析仪中进行实时分析之前，在标准培养板中用感兴趣的细胞毒性药物处理骨髓瘤细胞。此外，我们提供了实验结果，其中在制造商的基本程序和我们的优化方案之间比较了骨髓瘤细胞对细胞毒性治疗的反应的代谢活性。我们的观察表明，我们的集成协议可以用来实现一致，因此，它可能在专注于表征非粘附悬浮细胞中代谢事件的研究中具有广泛的应用。
