Establishment and characterization of a highly metastatic hepatocellular carcinoma cell line

The prevalence of alcohol-related hepatocellular carcinoma (HCC) has been increasing during the last decade. Cancer research requires cell lines suitable for both <i>in vitro</i> and <i>in vivo</i> assays. However, there is a lack of cell lines with a high <i>in vivo</i> metastatic capacity for this HCC subtype. Herein, a new HCC cell line was established, named HCC-ZJ, using cells from a patient diagnosed with alcohol-related HCC. The karyotype of HCC-ZJ was 46, XY, del (p11.2). Whole-exome sequencing identified several genetic variations in HCC-Z that occur frequently in alcohol-associated HCC, such as mutations in <i>TERT</i>, <i>CTNNB1</i>, <i>ARID1A</i>, <i>CDKN2A</i>, <i>SMARCA2</i>, and <i>HGF</i>. Cell counting kit-8 assays, colony formation assays, and Transwell assays were performed to evaluate the proliferation, migration, and sensitivity to sorafenib and lenvatinib of HCC-Z <i>in vitro</i>. HCC-ZJ showed a robust proliferation rate, a weak foci-forming ability, a strong migration capacity, and a moderate invasion tendency <i>in vitro</i>. Finally, the tumorigenicity and metastatic capacity of HCC-Z were evaluated using a subcutaneous xenograft model, an orthotopic xenograft model, and a tail-veil injection model. HCCZJ exhibited strong tumorigenicity in the subcutaneous xenograft and orthotopic tumor models. Moreover, HCC-ZJ spontaneously formed pulmonary metastases in the orthotopic tumor model. In summary, a new HCC cell line derived from a patient with alcohol-related HCC was established, which showed a high metastatic capacity and could be applied for <i>in vitro</i> and <i>in vivo</i> experiments during pre-clinical research. <b>Highlights</b> • An alcohol-related HCC cell line, HCC-ZJ, was established • HCC-ZJ was applicable for <i>in vitro</i> functional experiment and gene editing • HCC-ZJ was applicable for <i>in vivo</i> tumor growth and spontaneous metastasis models

近十年来，酒精相关性肝细胞癌（hepatocellular carcinoma, HCC）的患病率持续攀升。癌症研究亟需适用于体外（in vitro）与体内（in vivo）检测的细胞系，但目前针对该亚型肝细胞癌，仍缺乏具备高体内转移能力的细胞系。
本研究从一名确诊为酒精相关性肝细胞癌的患者体内分离细胞，成功构建了一株全新的肝细胞癌细胞系，命名为HCC-ZJ。该细胞系的核型为46, XY, del (p11.2)。全外显子测序结果显示，HCC-ZJ中存在多种在酒精相关性肝细胞癌中高频出现的遗传变异，包括TERT、CTNNB1、ARID1A、CDKN2A、SMARCA2及HGF的突变。
本研究采用细胞计数试剂盒-8（Cell counting kit-8）实验、集落形成实验及Transwell实验，体外评估了HCC-ZJ的增殖能力、迁移能力以及对索拉非尼（sorafenib）和仑伐替尼（lenvatinib）的敏感性。结果表明，HCC-ZJ体外增殖能力旺盛，集落形成能力较弱，迁移能力较强，侵袭倾向中等。
最后，通过皮下移植瘤模型、原位移植瘤模型及尾静脉注射模型，评估了HCC-ZJ的成瘤性与转移能力。实验结果显示，HCC-ZJ在皮下移植瘤及原位移植瘤模型中均表现出较强的成瘤能力；此外，该细胞系在原位肿瘤模型中可自发形成肺转移。
综上，本研究成功构建了一株源自酒精相关性肝细胞癌患者的全新肝细胞癌细胞系HCC-ZJ，该细胞系具备高转移能力，可用于临床前研究中的体外及体内实验。
**研究亮点**
• 成功构建了一株酒精相关性肝细胞癌细胞系HCC-ZJ
• HCC-ZJ可用于体外功能实验及基因编辑研究
• HCC-ZJ可应用于体内肿瘤生长及自发转移模型研究