Cisplatin plus pemetrexed combination therapy is considered the standard treatment for patients with advanced, non-squamous, non-small-cell lung cancer (NSCLC). However, advanced NSCLC has a 5-year survival rate of below 10%, which is mainly due to therapy resistance. We previously showed that the NSCLC cell line A549 harbors different subpopulations including a mesenchymal-like subpopulation characterized by increased chemo- and radiotherapy resistance. Recently, therapy resistance in hematological and solid tumors has been associated with increased mitochondrial activity. Thus, the aim of this study was to investigate the role of the mitochondrial activity in NSCLC chemotherapy resistance. Based on MitoTracker staining, subpopulations characterized by the highest 10% (Mito-High) or lowest 10% (Mito-Low) mitochondrial mass content were sorted by FACS (Fluorescence-Activated Cell Sorting) from paraclonal cultures of the NSCLC A549 cell line . Mitochondrial DNA copy numbers were quantified by real-time PCR whereas basal cellular respiration was measured by high-resolution respirometry. Cisplatin and pemetrexed response were quantified by proliferation and colony formation assay. Pemetrexed treatment of parental A549 cells increased mitochondrial mass over time. FACS-sorted paraclonal Mito-High cells featured increased mitochondrial mass and mitochondrial DNA copy number compared to the Mito-Low cells. Paraclonal Mito-High cells featured an increased proliferation rate and were significantly more resistant to cisplatin treatment than Mito-Low cells. Interestingly, cisplatin-resistant, paraclonal Mito-High cells were significantly more sensitive to pemetrexed treatment than Mito-Low cells. We provide a working model explaining the molecular mechanism underlying the increased cisplatin- and decreased pemetrexed resistance of a distinct subpopulation characterized by high mitochondrial mass. This study revealed that cisplatin resistant A549 lung cancer cells can be identified by their increased levels of mitochondrial mass. However, Mito-High cells feature an increased sensitivity to pemetrexed treatment. Thus, pemetrexed and cisplatin target reciprocal lung cancer subpopulations, which could explain the increased efficacy of the combination therapy in the clinical setting.

中文翻译：

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顺铂耐药的 A549 非小细胞肺癌细胞可以通过增加的线粒体质量来识别，并且对培美曲塞治疗敏感

顺铂加培美曲塞联合治疗被认为是晚期非鳞状非小细胞肺癌（NSCLC）患者的标准治疗。然而，晚期非小细胞肺癌的5年生存率低于10%，这主要是由于治疗耐药。我们之前表明，NSCLC 细胞系 A549 具有不同的亚群，包括以增加的化疗和放疗抗性为特征的间充质样亚群。最近，血液和实体瘤的治疗抗性与线粒体活性增加有关。因此，本研究的目的是研究线粒体活性在 NSCLC 化疗耐药中的作用。基于 MitoTracker 染色，以最高 10% (Mito-High) 或最低 10% (Mito-Low) 线粒体质量含量为特征的亚群通过 FACS（荧光激活细胞分选）从 NSCLC A549 细胞系的旁克隆培养物中分选。线粒体 DNA 拷贝数通过实时 PCR 定量，而基础细胞呼吸通过高分辨率呼​​吸测量法测量。通过增殖和集落形成测定来量化顺铂和培美曲塞的反应。培美曲塞治疗亲代 A549 细胞随时间增加线粒体质量。与 Mito-Low 细胞相比，FACS 分选的旁克隆 Mito-High 细胞具有增加的线粒体质量和线粒体 DNA 拷贝数。旁克隆 Mito-High 细胞的增殖率增加，并且比 Mito-Low 细胞对顺铂治疗的抵抗力明显更强。有趣的是，顺铂耐药的旁克隆 Mito-High 细胞对培美曲塞治疗的敏感性明显高于 Mito-Low 细胞。我们提供了一个工作模型，解释了以高线粒体质量为特征的不同亚群增加顺铂和降低培美曲塞耐药性的分子机制。这项研究表明，顺铂耐药的 A549 肺癌细胞可以通过线粒体质量水平的增加来识别。然而，Mito-High 细胞对培美曲塞治疗的敏感性增加。因此，培美曲塞和顺铂靶向互惠肺癌亚群，这可以解释联合治疗在临床环境中的疗效增加。我们提供了一个工作模型，解释了以高线粒体质量为特征的不同亚群增加顺铂和降低培美曲塞耐药性的分子机制。这项研究表明，顺铂耐药的 A549 肺癌细胞可以通过线粒体质量水平的增加来识别。然而，Mito-High 细胞对培美曲塞治疗的敏感性增加。因此，培美曲塞和顺铂靶向互惠肺癌亚群，这可以解释联合治疗在临床环境中的疗效增加。我们提供了一个工作模型，解释了以高线粒体质量为特征的不同亚群增加顺铂和降低培美曲塞耐药性的分子机制。这项研究表明，顺铂耐药的 A549 肺癌细胞可以通过线粒体质量水平的增加来识别。然而，Mito-High 细胞对培美曲塞治疗的敏感性增加。因此，培美曲塞和顺铂靶向互惠肺癌亚群，这可以解释联合治疗在临床环境中的疗效增加。这项研究表明，顺铂耐药的 A549 肺癌细胞可以通过线粒体质量水平的增加来识别。然而，Mito-High 细胞对培美曲塞治疗的敏感性增加。因此，培美曲塞和顺铂靶向互惠肺癌亚群，这可以解释联合治疗在临床环境中的疗效增加。这项研究表明，顺铂耐药的 A549 肺癌细胞可以通过线粒体质量水平的增加来识别。然而，Mito-High 细胞对培美曲塞治疗的敏感性增加。因此，培美曲塞和顺铂靶向互惠肺癌亚群，这可以解释联合治疗在临床环境中的疗效增加。