Background and Aims: Somatic mutations in mitochondrial DNA (mtDNA) are abundant in HCC and directly affect metabolic homeostasis and tumor progression. The mixed population of mutant and wild-type mtDNA alleles within a cell, termed heteroplasmy, can vary from cell-to-cell and orchestrate tumorigenesis. However, the systematic evolutionary dynamics of somatic mtDNA mutations in HCC tissues remain to be delineated at single-cell resolution. Approach and Results: We established the single-cell capture-based mtDNA sequencing approach for accurately detecting somatic mtDNA mutations at single-cell resolution. Based on single-cell capture-based mtDNA sequencing, the single-cell somatic mtDNA mutational landscape, intratumor heterogeneity (ITH), and spatiotemporal clonal evolution were systematically investigated in 1641 single cells from 11 patients with HCC and 528 single cells from 2 patient-derived xenografts mouse models. Our data revealed the presence of 2 distinct categories of mtDNA mutation at single-cell resolution, including stable mutations exhibiting similar heteroplasmy levels and unstable mutations exhibiting remarkable cell-to-cell variability of heteroplasmy levels. Furthermore, the proportion of unstable mtDNA mutations was positively associated with the ITH of patients with HCC, with high ITH reflecting the proliferative and aggressive clinicopathological features of HCC cells. In addition, reconstruction of the evolutionary history classified HCC evolution patterns as linear or branched. Notably, spatiotemporal lineage tracing in patient-derived xenograft mouse models and multifocal lesions revealed bidirectional evolution of unstable mtDNA mutations during HCC progression. Conclusions: Our study unravels the landscape of single-cell somatic mtDNA mutations in HCC tissues and reveals the bidirectional evolution of unstable mtDNA mutations, with potential implications for HCC stratification and therapeutic intervention.

中文翻译：

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在单细胞分辨率下揭示肝细胞癌不稳定线粒体 DNA 突变的双向进化


背景和目的： 线粒体 DNA （mtDNA） 中的体细胞突变在 HCC 中丰富，直接影响代谢稳态和肿瘤进展。细胞内突变型和野生型 mtDNA 等位基因的混合种群称为异质性，可能因细胞而异并协调肿瘤发生。然而，HCC 组织中体细胞 mtDNA 突变的系统进化动力学仍有待在单细胞分辨率下描述。方法和结果： 我们建立了基于单细胞捕获的 mtDNA 测序方法，用于在单细胞分辨率下准确检测体细胞 mtDNA 突变。基于基于单细胞捕获的 mtDNA 测序，系统研究了 11 例 HCC 患者的 1641 个单细胞和 2 个患者来源的异种移植小鼠模型的 528 个单细胞的单细胞体细胞 mtDNA 突变景观、肿瘤内异质性 （ITH） 和时空克隆进化。我们的数据揭示了在单细胞分辨率下存在 2 种不同类别的 mtDNA 突变，包括表现出相似异质水平的稳定突变和表现出异质水平显着细胞间变异性的不稳定突变。此外，不稳定 mtDNA 突变的比例与 HCC 患者的 ITH 呈正相关，高 ITH 反映了 HCC 细胞的增殖和侵袭性临床病理特征。此外，进化历史的重建将 HCC 进化模式分类为线性或分支。值得注意的是，患者来源的异种移植小鼠模型和多灶性病变中的时空谱系追踪揭示了 HCC 进展过程中不稳定 mtDNA 突变的双向进化。 结论： 我们的研究揭示了 HCC 组织中单细胞体细胞 mtDNA 突变的前景，并揭示了不稳定 mtDNA 突变的双向进化，对 HCC 分层和治疗干预具有潜在意义。