Characterization of hepatic stellate cells (HSC) genes in the normal liver or diverse liver fibrotic models by scRNAseq and bulk RNAseq.

Hepatocellular carcinoma (HCC), the fourth leading cause of cancer mortality, develops almost exclusively in patients with chronic liver disease (CLD) and advanced fibrosis. Here we interrogated functions of hepatic stellate cells (HSC), the main source of liver fibroblasts, during hepatocarcinogenPesis. Genetic depletion, activation or inhibition established HSC as tumour-promoting in mouse models of HCC. HSC were enriched in the preneoplastic environment, where they closely interacted with hepatocytes and modulated hepatocarcinogenesis by regulating hepatocyte proliferation and death. Analysis of mouse and human HSC subpopulations and their associated mediators by single cell RNA-sequencing in conjunction with genetic ablation revealed dual functions of HSC in hepatocarcinogenesis. Hepatocyte growth factor, enriched in quiescent and cytokine-producing HSC (cyHSC), protected from hepatocyte death and HCC development. In contrast, type I collagen, enriched in activated myofibroblastic HSC (myHSC), promoted proliferation and tumour development via increased stiffness and TAZ activation in pretumoural hepatocytes and via activation of discoidin domain receptor 1 in established tumours. An increasing HSC dysbalance between cyHSC and myHSC during liver disease progression was associated with elevated HCC risk in patients. In summary, the dynamic shift of HSC subpopulations and their mediators during CLD is associated with a switch from HCC protection to HCC promotion. - Single Cell RNAseq of isolated HSC from fibrotic livers induced by CCl4 (RS039, n=1), TAZ+FPC-NASH diet (RS042, n=1) and HF-CDAA diet (RS043, n=1) - Bulk RNA sequencing of isolated HSC from FPC-treated mice (n=5), or by CCl4 (n=4) - Bulk RNA-sequencing from normal (n=8), adjacent (n=10) and HCC (n=10) mouse livers with or without HSC inhibition induced by YAP deletion in HSC during hepatocarcinogenesis induced by DEN+CCl4

肝细胞癌（Hepatocellular carcinoma, HCC）是第四大癌症致死病因，其发生几乎仅见于慢性肝病（chronic liver disease, CLD）及晚期纤维化患者。本研究探究了肝星状细胞（hepatic stellate cells, HSC，即肝脏成纤维细胞的主要来源）在肝致癌过程中的功能。通过基因敲除、激活或抑制实验，证实HSC在肝细胞癌小鼠模型中具有促瘤作用。HSC富集于癌前微环境中，在此处与肝细胞紧密相互作用，并通过调控肝细胞增殖与死亡来调节肝致癌进程。通过单细胞RNA测序（single cell RNA-sequencing）结合基因消融技术，对小鼠及人类HSC亚群及其相关介导因子进行分析，揭示了HSC在肝致癌过程中的双重功能：富集于静止型与细胞因子分泌型HSC（cytokine-producing HSC, cyHSC）中的肝细胞生长因子，可抑制肝细胞死亡及肝细胞癌的发生；与之相反，富集于活化肌成纤维样HSC（myofibroblastic HSC, myHSC）中的I型胶原，可通过增加癌前肝细胞的基质硬度并激活TAZ，以及在已形成的肿瘤中激活盘状结构域受体1（discoidin domain receptor 1, DDR1），促进肝细胞增殖与肿瘤发展。在肝病进展过程中，cyHSC与myHSC之间的HSC失衡不断加剧，与患者的肝细胞癌风险升高相关。综上，慢性肝病过程中HSC亚群及其介导因子的动态变化，与肝细胞癌的保护作用向促瘤作用的转变密切相关。
- 四氯化碳（CCl₄）诱导的纤维化肝脏中分离的HSC的单细胞RNA测序（single cell RNA-sequencing，样本标识RS039，n=1）、TAZ+FPC-NASH饮食诱导的纤维化肝脏中分离的HSC的单细胞RNA测序（single cell RNA-sequencing，样本标识RS042，n=1），以及HF-CDAA饮食诱导的纤维化肝脏中分离的HSC的单细胞RNA测序（single cell RNA-sequencing，样本标识RS043，n=1）
- 经FPC处理的小鼠中分离的HSC的批量RNA测序（bulk RNA sequencing，n=5），以及经CCl₄处理的小鼠中分离的HSC的批量RNA测序（bulk RNA sequencing，n=4）
- 正常（n=8）、癌旁（n=10）及肝细胞癌（n=10）小鼠肝脏的批量RNA测序（bulk RNA-sequencing），这些小鼠均处于二乙基亚硝胺（diethylnitrosamine, DEN）+CCl₄诱导的肝致癌过程中，部分小鼠通过在HSC中敲除Yes相关蛋白（Yes-associated protein, YAP）实现了HSC抑制，其余未进行该处理