Hepatocellular carcinoma (HCC) is the most common type of canine primary liver tumor; however, most chemotherapies against HCC are ineffective due to resistance to anticancer agents. Sphere-forming cells are considered to act as cancer stem cells for various types of solid tumors and have been established for many canine tumor cell lines, yet no studies have reported sphere-forming cells for canine HCC. In this study, we established sphere-forming cells from a canine HCC cell line (AZACH). These cells displayed increased stem cell marker mRNA expression (Nanog, Sox2, c-Myc, and Klf4), aldehyde dehydrogenase activity, and chemoresistance against mitoxantrone, gemcitabine, and doxorubicin. In addition, sphere-forming cells exhibited higher xCT expression and lower intracellular reactive oxygen species levels than adherent cells. Treatment with sulfasalazine, a xCT-specific inhibitor, reduced sphere formation efficiency. Together, these results indicate that sphere-forming cells derived from a canine HCC cell line have similar characteristics to cancer stem cells and that their increased xCT expression and associated resistance to oxidative stress contribute toward sphere formation.

肝细胞癌 (HCC) 是最常见的犬原发性肝肿瘤类型。然而，由于对抗癌药物的耐药性，大多数针对 HCC 的化学疗法是无效的。球形成细胞被认为是各种类型实体瘤的癌症干细胞，并且已经在许多犬肿瘤细胞系中建立起来，但没有研究报道犬 HCC 的球形成细胞。在这项研究中，我们从犬 HCC 细胞系 (AZACH) 中建立了球体形成细胞。这些细胞显示出增加的干细胞标志物 mRNA 表达（Nanog、Sox2、c-Myc 和 Klf4）、醛脱氢酶活性以及对米托蒽醌、吉西他滨和阿霉素的化学抗性。此外，与贴壁细胞相比，球体形成细胞表现出更高的 xCT 表达和更低的细胞内活性氧水平。用柳氮磺吡啶（一种 xCT 特异性抑制剂）处理会降低球体形成效率。总之，这些结果表明源自犬 HCC 细胞系的球形成细胞具有与癌症干细胞相似的特征，并且它们增加的 xCT 表达和相关的对氧化应激的抵抗力有助于球形成。