Abstract：
BACKGROUND: Tumour-induced skeletal muscle wasting in the context of cancer cachexia is a condition with profound implications for patient survival. The loss of muscle mass is a significant clinical obstacle and is linked to reduced tolerance to chemotherapy and increased frailty. Understanding the molecular mechanisms driving muscle atrophy is crucial for the design of new therapeutics.
METHODS: Lewis lung carcinoma tumours were utilized to induce cachexia and muscle atrophy in mice. Single-nucleus libraries of the tibialis anterior (TA) muscle from tumour-bearing mice and their non-tumour-bearing controls were constructed using 10X Genomics applications following the manufacturer\'s guidelines. RNA sequencing results were analysed with Cell Ranger software and the Seurat R package. Oxygen consumption of mitochondria isolated from TA muscle was measured using an Oroboros O2k-FluoRespirometer. Mouse primary myotubes were treated with a recombinant ectodysplasin A2 (EDA-A2) protein to activate EDA-A2 receptor (EDA2R) signalling and study changes in gene expression and oxygen consumption.
RESULTS: Tumour-bearing mice were sacrificed while exhibiting moderate cachexia. Average TA muscle weight was reduced by 11% (P = 0.0207) in these mice. A total of 12 335 nuclei, comprising 6422 nuclei from the control group and 5892 nuclei from atrophying muscles, were studied. The analysis of single-nucleus transcriptomes identified distinct myonuclear gene signatures and a shift towards type IIb myonuclei. Muscle atrophy-related genes, including Atrogin1, MuRF1 and Eda2r, were upregulated in these myonuclei, emphasizing their crucial roles in muscle wasting. Gene set enrichment analysis demonstrated that EDA2R activation and tumour inoculation led to similar expression patterns in muscle cells, including the stimulation of nuclear factor-kappa B, Janus kinase-signal transducer and activator of transcription and transforming growth factor-beta pathways and the suppression of myogenesis and oxidative phosphorylation. Muscle oxidative metabolism was suppressed by both tumours and EDA2R activation.
CONCLUSIONS: This study identified tumour-induced transcriptional changes in muscle tissue at single-nucleus resolution and highlighted the negative impact of tumours on oxidative metabolism. These findings contribute to a deeper understanding of the molecular mechanisms underlying muscle wasting.
METHODS: Lewis lung carcinoma tumours were utilized to induce cachexia and muscle atrophy in mice. Single-nucleus libraries of the tibialis anterior (TA) muscle from tumour-bearing mice and their non-tumour-bearing controls were constructed using 10X Genomics applications following the manufacturer\'s guidelines. RNA sequencing results were analysed with Cell Ranger software and the Seurat R package. Oxygen consumption of mitochondria isolated from TA muscle was measured using an Oroboros O2k-FluoRespirometer. Mouse primary myotubes were treated with a recombinant ectodysplasin A2 (EDA-A2) protein to activate EDA-A2 receptor (EDA2R) signalling and study changes in gene expression and oxygen consumption.
RESULTS: Tumour-bearing mice were sacrificed while exhibiting moderate cachexia. Average TA muscle weight was reduced by 11% (P = 0.0207) in these mice. A total of 12 335 nuclei, comprising 6422 nuclei from the control group and 5892 nuclei from atrophying muscles, were studied. The analysis of single-nucleus transcriptomes identified distinct myonuclear gene signatures and a shift towards type IIb myonuclei. Muscle atrophy-related genes, including Atrogin1, MuRF1 and Eda2r, were upregulated in these myonuclei, emphasizing their crucial roles in muscle wasting. Gene set enrichment analysis demonstrated that EDA2R activation and tumour inoculation led to similar expression patterns in muscle cells, including the stimulation of nuclear factor-kappa B, Janus kinase-signal transducer and activator of transcription and transforming growth factor-beta pathways and the suppression of myogenesis and oxidative phosphorylation. Muscle oxidative metabolism was suppressed by both tumours and EDA2R activation.
CONCLUSIONS: This study identified tumour-induced transcriptional changes in muscle tissue at single-nucleus resolution and highlighted the negative impact of tumours on oxidative metabolism. These findings contribute to a deeper understanding of the molecular mechanisms underlying muscle wasting.
摘要：
背景：癌症恶病质背景下肿瘤诱导的骨骼肌萎缩是一种对患者生存具有深远意义的疾病。肌肉质量的损失是重要的临床障碍,并且与对化疗的耐受性降低和虚弱增加有关。了解驱动肌肉萎缩的分子机制对于设计新疗法至关重要。
方法：利用Lewis肺癌肿瘤诱导小鼠恶病质和肌肉萎缩。根据制造商的指南，使用10倍基因组学应用构建了荷瘤小鼠及其非荷瘤对照的胫骨前（TA）肌肉的单核文库。用CellRanger软件和SeuratR包分析RNA测序结果。使用OroborosO2k-荧光计测量从TA肌肉分离的线粒体的氧消耗。小鼠原代肌管用重组胞外异型增生蛋白A2(EDA-A2)蛋白处理,以激活EDA-A2受体(EDA2R)信号并研究基因表达和耗氧量的变化。
结果:处死荷瘤小鼠,同时表现出中度恶病质。在这些小鼠中,平均TA肌肉重量减少11%(P=0.0207)。总共12.335个原子核，包括来自对照组的6422个细胞核和来自萎缩肌肉的5892个细胞核，被研究过。单核转录组的分析确定了不同的肌核基因特征，并向IIb型肌核转移。肌肉萎缩相关基因，包括Atrogin1，MuRF1和Eda2r，在这些肌核中上调，强调它们在肌肉萎缩中的关键作用。基因集富集分析表明，EDA2R激活和肿瘤接种导致肌肉细胞中相似的表达模式，包括核因子-κB的刺激,Janus激酶-信号转导和转录和转化生长因子-β途径的激活因子以及抑制肌生成和氧化磷酸化。肿瘤和EDA2R激活均抑制了肌肉氧化代谢。
结论：这项研究确定了肿瘤诱导的单核分辨率肌肉组织的转录变化，并强调了肿瘤对氧化代谢的负面影响。这些发现有助于更深入地了解肌肉萎缩的分子机制。
方法：利用Lewis肺癌肿瘤诱导小鼠恶病质和肌肉萎缩。根据制造商的指南，使用10倍基因组学应用构建了荷瘤小鼠及其非荷瘤对照的胫骨前（TA）肌肉的单核文库。用CellRanger软件和SeuratR包分析RNA测序结果。使用OroborosO2k-荧光计测量从TA肌肉分离的线粒体的氧消耗。小鼠原代肌管用重组胞外异型增生蛋白A2(EDA-A2)蛋白处理,以激活EDA-A2受体(EDA2R)信号并研究基因表达和耗氧量的变化。
结果:处死荷瘤小鼠,同时表现出中度恶病质。在这些小鼠中,平均TA肌肉重量减少11%(P=0.0207)。总共12.335个原子核，包括来自对照组的6422个细胞核和来自萎缩肌肉的5892个细胞核，被研究过。单核转录组的分析确定了不同的肌核基因特征，并向IIb型肌核转移。肌肉萎缩相关基因，包括Atrogin1，MuRF1和Eda2r，在这些肌核中上调，强调它们在肌肉萎缩中的关键作用。基因集富集分析表明，EDA2R激活和肿瘤接种导致肌肉细胞中相似的表达模式，包括核因子-κB的刺激,Janus激酶-信号转导和转录和转化生长因子-β途径的激活因子以及抑制肌生成和氧化磷酸化。肿瘤和EDA2R激活均抑制了肌肉氧化代谢。
结论：这项研究确定了肿瘤诱导的单核分辨率肌肉组织的转录变化，并强调了肿瘤对氧化代谢的负面影响。这些发现有助于更深入地了解肌肉萎缩的分子机制。
