Liu Dan, Li Hong, Dong Hui, Qu Mincheng, Yang Liguang, Chen Lina, Li Yixue, Wang Hongyang, He Yufei
Model Animal Research Center, Medical School, Nanjing University, Nanjing, China.
Molecular Pathology Laboratory, National Center for Liver Cancer, Eastern Hepatobiliary Surgery Hospital, Shanghai, China.
Front Cell Dev Biol. 2022 Feb 9;10:810687. doi: 10.3389/fcell.2022.810687. eCollection 2022.
Cancer stem cells (CSCs) usually account for a very small tumor cell population but play pivotal roles in human cancer development and recurrence. A fundamental question in cancer biology is what genetic and epigenetic changes occur in CSCs. Here we show that the global levels of DNA cytosine modifications, including 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC), are similar between liver cancer stem-like (LCSL) cells and paratumor liver cells of liver cancer patients. We then developed a robust method combining immunohistochemistry, laser capture microdissection and genome sequencing with ultra-low-input cells (CIL-seq) to study the detailed genetic and DNA methylation changes in human LCSL cells. We first used clinical samples of mixed hepatocellular carcinoma-cholangiocarcinoma (HCC-CCA) with stem cell features to investigate human LCSL cells. The CIL-seq analysis of HCC-CCA and HCC patients showed that LCSL cells had strong spatial genetic and epigenetic heterogeneity. More interestingly, although the LCSL cells had some potential key changes in their genome, they had substantially fewer somatic single nucleotide variants (SNVs), copy number alterations (CNAs) and differentially methylated regions than other tumor parenchymal cells. The cluster analysis of SNVs, CNAs, DNA methylation patterns and spatial transcriptomes all clearly showed that the LCSL cells were clustered with the paratumor liver cells. Thus, spatial multiomics analysis showed that LCSL cells had only minor genetic and epigenetic changes compared with other tumor parenchymal cells. Targeting key changes in CSCs, not just changes in bulk tumor cells, should be more effective for human cancer therapy.
癌症干细胞（CSCs）通常仅占肿瘤细胞群体的很小一部分，但在人类癌症的发展和复发中起着关键作用。癌症生物学中的一个基本问题是CSCs中发生了哪些基因和表观遗传变化。在此，我们表明，肝癌干细胞样（LCSL）细胞与肝癌患者的癌旁肝细胞之间，包括5-甲基胞嘧啶（5mC）、5-羟甲基胞嘧啶（5hmC）和5-甲酰基胞嘧啶（5fC）在内的DNA胞嘧啶修饰的整体水平相似。然后，我们开发了一种强大的方法，将免疫组织化学、激光捕获显微切割和基因组测序与超低输入细胞相结合（CIL-seq），以研究人类LCSL细胞中详细的基因和DNA甲基化变化。我们首先使用具有干细胞特征的混合肝细胞癌-胆管癌（HCC-CCA）临床样本，来研究人类LCSL细胞。对HCC-CCA和HCC患者的CIL-seq分析表明，LCSL细胞具有很强的空间基因和表观遗传异质性。更有趣的是，尽管LCSL细胞在其基因组中存在一些潜在的关键变化，但与其他肿瘤实质细胞相比，它们的体细胞单核苷酸变异（SNVs）、拷贝数改变（CNAs）和差异甲基化区域要少得多。SNVs、CNAs、DNA甲基化模式和空间转录组的聚类分析均清楚地表明，LCSL细胞与癌旁肝细胞聚集在一起。因此，空间多组学分析表明，与其他肿瘤实质细胞相比，LCSL细胞仅发生了微小的基因和表观遗传变化。针对CSCs中的关键变化，而不仅仅是大块肿瘤细胞的变化，对人类癌症治疗应该更有效。
