Table_1_Residual OXPHOS is required to drive primary and metastatic lung tumours in an orthotopic breast cancer model.xlsx

BackgroundFast adaptation of glycolytic and mitochondrial energy pathways to changes in the tumour microenvironment is a hallmark of cancer. Purely glycolytic ρ0 tumour cells do not form primary tumours unless they acquire healthy mitochondria from their micro-environment. Here we explored the effects of severely compromised respiration on the metastatic capability of 4T1 mouse breast cancer cells.
Methods4T1 cell lines with different levels of respiratory capacity were generated; the Seahorse extracellular flux analyser was used to evaluate oxygen consumption rates, fluorescent confocal microscopy to assess the number of SYBR gold-stained mitochondrial DNA nucleoids, and the presence of the ATP5B protein in the cytoplasm and fluorescent in situ nuclear hybridization was used to establish ploidy. MinION nanopore RNA sequence analysis was used to compare mitochondrial DNA transcription between cell lines. Orthotopic injection was used to determine the ability of cells to metastasize to the lungs of female Balb/c mice.
ResultsOXPHOS-deficient ATP5B-KO3.1 cells did not generate primary tumours. Severely OXPHOS compromised ρ0D5 cells generated both primary tumours and lung metastases. Cells generated from lung metastasis of both OXPHOS-competent and OXPHOS-compromised cells formed primary tumours but no metastases when re-injected into mice. OXPHOS-compromised cells significantly increased their mtDNA content, but this did not result in increased OXPHOS capacity, which was not due to decreased mtDNA transcription. Gene set enrichment analysis suggests that certain cells derived from lung metastases downregulate their epithelial-to-mesenchymal related pathways.
ConclusionIn summary, OXPHOS is required for tumorigenesis in this orthotopic mouse breast cancer model but even very low levels of OXPHOS are sufficient to generate both primary tumours and lung metastases.

背景：糖酵解与线粒体能量通路可快速适应肿瘤微环境变化，这是癌症的标志性特征。仅依赖糖酵解的ρ0肿瘤细胞无法形成原发肿瘤，除非从微环境中获取健康线粒体。本研究探讨了呼吸功能严重受损对4T1小鼠乳腺癌细胞转移能力的影响。
方法：构建了具有不同呼吸功能水平的4T1细胞系；采用Seahorse细胞外通量分析仪评估氧消耗速率，通过荧光共聚焦显微镜计数SYBR金染色的线粒体DNA类核数量、检测细胞质中ATP5B蛋白的表达情况，并利用荧光原位核杂交确定细胞倍性。采用MinION纳米孔RNA测序分析比较不同细胞系的线粒体DNA转录水平。通过向雌性BALB/c小鼠实施原位注射，检测细胞向肺部转移的能力。
结果：氧化磷酸化（OXPHOS）缺陷的ATP5B-KO3.1细胞无法形成原发肿瘤。呼吸功能严重受损的ρ0D5细胞可同时形成原发肿瘤与肺部转移灶。从氧化磷酸化功能正常及受损的细胞的肺部转移灶中分离得到的细胞，再次注射入小鼠体内后仅能形成原发肿瘤，无法发生转移。氧化磷酸化功能受损的细胞其线粒体DNA（mtDNA）含量显著升高，但该变化并未提升其氧化磷酸化能力，且这一现象并非由线粒体DNA转录水平降低所导致。基因集富集分析结果显示，部分源自肺部转移灶的细胞下调了上皮-间充质转化相关通路的表达。
结论：综上，在本原位小鼠乳腺癌模型中，氧化磷酸化功能是肿瘤发生所必需的，但即便仅维持极低水平的氧化磷酸化活性，也足以支持原发肿瘤形成与肺部转移。