Department of Molecular Biology and Genetics, Bilkent University, 06800, Ankara, Turkey.
Izmir International Biomedicine and Genome Institute (iBG-izmir), Dokuz Eylul University, 35340, Izmir, Turkey.
Sci Rep. 2018 Jan 25;8(1):1570. doi: 10.1038/s41598-018-19817-w.
Acetylcholinesterase (AChE), an enzyme responsible for degradation of acetylcholine, has been identified as a prognostic marker in liver cancer. Although in vivo Ache tumorigenicity assays in mouse are present, no established liver cancer xenograft model in zebrafish using an ache mutant background exists. Herein, we developed an embryonic zebrafish xenograft model using epithelial (Hep3B) and mesenchymal (SKHep1) liver cancer cell lines in wild-type and ache sibling mutant larvae after characterization of cholinesterase expression and activity in cell lines and zebrafish larvae. The comparison of fluorescent signal reflecting tumor size at 3-days post-injection (dpi) revealed an enhanced tumorigenic potential and a reduced migration capacity in cancer cells injected into homozygous ache mutants when compared with the wild-type. Increased tumor load was confirmed using an ALU based tumor DNA quantification method modified for use in genotyped xenotransplanted zebrafish embryos. Confocal microscopy using the Huh7 cells stably expressing GFP helped identify the distribution of tumor cells in larvae. Our results imply that acetylcholine accumulation in the microenvironment directly or indirectly supports tumor growth in liver cancer. Use of this model system for drug screening studies holds potential in discovering new cholinergic targets for treatment of liver cancers.
乙酰胆碱酯酶（AChE）是一种负责降解乙酰胆碱的酶，已被确定为肝癌的预后标志物。尽管在小鼠体内存在 Ache 肿瘤发生测定法，但在使用 ache 突变背景的斑马鱼中尚未建立成熟的肝癌异种移植模型。在此，我们在野生型和 ache 同胞突变幼虫中，使用上皮（Hep3B）和间充质（SKHep1）肝癌细胞系，开发了一种胚胎斑马鱼异种移植模型。在细胞系和斑马鱼幼虫中对胆碱酯酶表达和活性进行了表征后，比较了反映注射后 3 天（dpi）肿瘤大小的荧光信号。与野生型相比，注射到纯合 ache 突变体中的癌细胞具有更高的致瘤潜力和更低的迁移能力。使用针对基因分型异种移植斑马鱼胚胎修改的基于 ALU 的肿瘤 DNA 定量方法，证实了肿瘤负荷的增加。使用稳定表达 GFP 的 Huh7 细胞进行共聚焦显微镜检查有助于确定肿瘤细胞在幼虫中的分布。我们的结果表明，微环境中的乙酰胆碱积累直接或间接地支持肝癌中的肿瘤生长。该模型系统在药物筛选研究中的应用具有发现治疗肝癌的新胆碱能靶点的潜力。
