Significance: Deranged metabolism and dysregulated growth factor signaling are closely associated with abnormal levels of proliferation, a recognized hallmark in tumorigenesis. Fluorescence lifetime imaging microscopy (FLIM) of endogenous nicotinamide adenine dinucleotide (NADH), a key metabolic coenzyme, offers a non-invasive, diagnostic indicator of disease progression, and treatment response. The model-independent phasor analysis approach leverages FLIM to rapidly evaluate cancer metabolism in response to targeted therapy. Aim: We combined lifetime and phasor FLIM analysis to evaluate the influence of human epidermal growth factor receptor 2 (HER2) inhibition, a prevalent cancer biomarker, on both nuclear and cytoplasmic NAD(P)H of two squamous cell carcinoma (SCC) cultures. While better established, the standard lifetime analysis approach is relatively slow and potentially subject to intrinsic fitting errors and model assumptions. Phasor FLIM analysis offers a rapid, model-independent alternative, but the sensitivity of the bound NAD(P)H fraction to growth factor signaling must also be firmly established. Approach: Two SCC cultures with low- and high-HER2 expression, were imaged using multiphoton-excited NAD(P)H FLIM, with and without treatment of the HER2 inhibitor AG825. Cells were challenged with mitochondrial inhibition and uncoupling to investigate AG825’s impact on the overall metabolic capacity. Phasor FLIM and lifetime fitting analyses were compared within nuclear and cytoplasmic compartments to investigate epigenetic and metabolic impacts of HER2 inhibition. Results: NAD(P)H fluorescence lifetime and bound fraction consistently decreased following HER2 inhibition in both cell lines. High-HER2 SCC74B cells displayed a more significant response than low-HER2 SCC74A in both techniques. HER2 inhibition induced greater changes in nuclear than cytoplasmic compartments, leading to an increase in NAD(P)H intensity and concentration. Conclusions: The use of both, complementary FLIM analysis techniques together with quantitative fluorescence intensity revealed consistent, quantitative changes in NAD(P)H metabolism associated with inhibition of growth factor signaling in SCC cell lines. HER2 inhibition promoted increased reliance on oxidative phosphorylation in both cell lines.

中文翻译：

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基于相量的 FLIM 分析的开发和表征，以评估 HER2 抑制对鳞状细胞癌培养物的代谢和表观遗传影响

意义：紊乱的新陈代谢和失调的生长因子信号与异常水平的增殖密切相关，这是肿瘤发生的公认标志。内源性烟酰胺腺嘌呤二核苷酸 (NADH)（一种关键的代谢辅酶）的荧光寿命成像显微镜 (FLIM) 提供了疾病进展和治疗反应的非侵入性诊断指标。独立于模型的相量分析方法利用 FLIM 来快速评估癌症代谢以响应靶向治疗。目的：我们结合寿命和相量 FLIM 分析来评估人表皮生长因子受体 2 (HER2) 抑制（一种流行的癌症生物标志物）对两种鳞状细胞癌 (SCC) 培养物的核和细胞质 NAD(P)H 的影响。虽然建立得更好，标准寿命分析方法相对较慢，并且可能受到固有拟合误差和模型假设的影响。Phasor FLIM 分析提供了一种快速、独立于模型的替代方法，但还必须牢固地建立结合的 NAD(P)H 分数对生长因子信号的敏感性。方法：使用多光子激发的 NAD(P)H FLIM 对具有和不具有 HER2 抑制剂 AG825 处理的两种具有低和高 HER2 表达的 SCC 培养物进行成像。细胞受到线粒体抑制和解偶联的挑战，以研究 AG825 对整体代谢能力的影响。在细胞核和细胞质区室中比较了 Phasor FLIM 和寿命拟合分析，以研究 HER2 抑制的表观遗传和代谢影响。结果：在两种细胞系中抑制 HER2 后，NAD(P)H 荧光寿命和结合分数持续下降。在这两种技术中，高 HER2 SCC74B 细胞显示出比低 HER2 SCC74A 更显着的反应。HER2 抑制诱导的细胞核变化大于细胞质区室，导致 NAD(P)H 强度和浓度增加。结论：两种互补的 FLIM 分析技术与定量荧光强度的结合使用揭示了与 SCC 细胞系中生长因子信号传导抑制相关的 NAD(P)H 代谢的一致定量变化。HER2 抑制促进了两种细胞系对氧化磷酸化的依赖性增加。在这两种技术中，高 HER2 SCC74B 细胞显示出比低 HER2 SCC74A 更显着的反应。HER2 抑制诱导的细胞核变化大于细胞质区室，导致 NAD(P)H 强度和浓度增加。结论：两种互补的 FLIM 分析技术与定量荧光强度的结合使用揭示了与 SCC 细胞系中生长因子信号传导抑制相关的 NAD(P)H 代谢的一致定量变化。HER2 抑制促进了两种细胞系对氧化磷酸化的依赖性增加。在这两种技术中，高 HER2 SCC74B 细胞显示出比低 HER2 SCC74A 更显着的反应。HER2 抑制诱导的细胞核变化大于细胞质区室，导致 NAD(P)H 强度和浓度增加。结论：两种互补的 FLIM 分析技术与定量荧光强度的结合使用揭示了与 SCC 细胞系中生长因子信号传导抑制相关的 NAD(P)H 代谢的一致定量变化。HER2 抑制促进了两种细胞系对氧化磷酸化的依赖性增加。互补的 FLIM 分析技术与定量荧光强度一起揭示了与 SCC 细胞系中生长因子信号传导抑制相关的 NAD(P)H 代谢的一致定量变化。HER2 抑制促进了两种细胞系对氧化磷酸化的依赖性增加。互补的 FLIM 分析技术与定量荧光强度一起揭示了与 SCC 细胞系中生长因子信号传导抑制相关的 NAD(P)H 代谢的一致定量变化。HER2 抑制促进了两种细胞系对氧化磷酸化的依赖性增加。