Background/Objectives: Cancer cells rely on metabolic reprogramming that is supported by altered mitochondrial redox status and an increased demand for NAD+. Over expression of Nampt, the rate-limiting enzyme of the NAD+biosynthesis salvage pathway, is common in breast cancer cells, and more so in triple negative breast cancer (TNBC) cells. Targeting the salvage pathway has been pursued for cancer therapy. However, TNBC cells have heterogeneous responses to Nampt inhibition, which contributes to the diverse outcomes. There is a lack of imaging biomarkers to differentiate among TNBC cells under metabolic stress and identify which are responsive. We aimed to characterize and differentiate among a panel of TNBC cell lines under NAD-deficient stress and identify which subtypes are more dependent on the NAD salvage pathway.Methods: Optical redox imaging (ORI), a label-free live cell imaging microscopy technique was utilized to acquire intrinsic fluorescence intensities of NADH and FAD-containing flavoproteins (Fp) thus the mitochondrial redox ratio Fp/(NADH + Fp) in a panel of TNBC cell lines. Various fluorescence probes were then added to the cultures to image the mitochondrial ROS, mitochondrial membrane potential, mitochondrial mass, and cell number.Results: Various TNBC subtypes are sensitive to Nampt inhibition in a dose- and time-dependent manner, they have differential mitochondrial redox responses; furthermore, the mitochondrial redox indices linearly correlated with mitochondrial ROS induced by various doses of a Nampt inhibitor. Moreover, the changes in the redox indices correlated with growth inhibition. Additionally, the redox state was found fully recovered after removing the Nampt inhibitor.Conclusions: This study supports the utility of ORI in rapid metabolic phenotyping of TNBC cells under NAD-deficient stress to identify responsive cells and biomarkers of treatment response, facilitating combination therapy strategies.
背景/目的：癌细胞依赖于代谢重编程，这一过程由线粒体氧化还原状态改变和NAD+需求增加所支持。NAD+生物合成补救途径的限速酶Nampt在乳腺癌细胞中普遍过表达，在三阴性乳腺癌（TNBC）细胞中尤为显著。靶向补救途径已成为癌症治疗的研究方向。然而，TNBC细胞对Nampt抑制存在异质性反应，这导致了治疗结果的多样性。目前缺乏能够在代谢应激下区分TNBC细胞并识别其反应性的影像学生物标志物。本研究旨在表征并区分一组在NAD缺乏应激下的TNBC细胞系，并确定哪些亚型更依赖于NAD补救途径。 方法：采用光学氧化还原成像技术（一种无标记活细胞成像显微镜技术），获取一组TNBC细胞系中NADH和含FAD黄素蛋白（Fp）的内源性荧光强度，从而计算线粒体氧化还原比值Fp/(NADH+Fp)。随后在培养体系中添加多种荧光探针，分别对线粒体活性氧、线粒体膜电位、线粒体质量和细胞数量进行成像分析。 结果：不同TNBC亚型对Nampt抑制表现出剂量和时间依赖性敏感，其线粒体氧化还原反应存在差异；此外，线粒体氧化还原指数与不同剂量Nampt抑制剂诱导的线粒体活性氧呈线性相关。氧化还原指数的变化与生长抑制程度相关。值得注意的是，在撤除Nampt抑制剂后，细胞氧化还原状态可完全恢复。 结论：本研究证实了ORI技术在NAD缺乏应激下快速代谢分型TNBC细胞的实用性，能够识别反应性细胞和治疗反应的生物标志物，为联合治疗策略的制定提供支持。
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