Inhibiting TGFβ-1 pathway reduces the aggressiveness of intrahepatic CCA HuCCT1 CD90 positive cells

Molecular mechanisms responsible for the poor prognosis in patients with intrahepatic cholangiocarcinoma (CCA) are still unknown, however, the stem cell marker cluster differentiation 90 (CD90), has been reported to be associated with a more aggressive cancer phenotype. In this scenario, TGFβ1 signaling pathway likely has a role as master gene regulator. Aim of the study is to investigate the role of CD90 in iCCA aggressiveness. The molecular profile of HuCCT1/CD90+ and HuCCT1/CD90- cells was obtained through transcriptomic analysis (NGS). Bioinformatic data were confirmed in both cell lines by qRT-PCR and western blot. Cells were treated with Gemcitabine in monotherapy or in combination with Galunisertib, a selective TGFβRI inhibitor in 2D and 3D models. HuCCT1 CD90 positive cells are more proliferative, less migratory, and resistant to Gemcitabine treatment. Next, HuCCT1/CD90+ express lower levels of TGFβ1 compared to /CD90- cell lines. Finally, HuCCT1/CD90+ are resistant to Gemcitabine, while the combination of both Gemcitabine and Galunisertib displays a synergistic effect on HuCCT1/CD90+ cell proliferation. These results underline that CD90 inducing Gemcitabine resistance can be overcome by adding a TGFβ1 inhibitor such as Galunisertib, thereby moving further toward a precision medicine approach in patients with iCCA. We used RNA sequencing technology to understand the molecular changes of CD90 increase in iCCA cell line

肝内胆管癌（CCA）患者预后不良的分子机制迄今尚未明确，不过已有研究表明干细胞标志物簇分化抗原90（CD90）与更具侵袭性的癌症表型密切相关。在此背景下，转化生长因子β1（TGFβ1）信号通路可能充当主基因调控因子。本研究旨在探讨CD90在肝内胆管癌侵袭性中的作用。研究人员通过转录组分析（NGS，即二代测序）获取了HuCCT1/CD90+与HuCCT1/CD90-细胞的分子表达谱，并通过实时定量逆转录聚合酶链反应（qRT-PCR）与蛋白质印迹法（western blot）在两种细胞系中验证了生物信息学分析所得的数据。在二维（2D）与三维（3D）细胞培养模型中，分别采用单药吉西他滨（Gemcitabine），或联合选择性转化生长因子βI型受体（TGFβRI）抑制剂加鲁尼替尼（Galunisertib）处理细胞。实验结果显示：HuCCT1/CD90+细胞的增殖能力更强、迁移能力更弱，且对吉西他滨治疗产生耐药性；其次，相较于HuCCT1/CD90-细胞系，HuCCT1/CD90+细胞的TGFβ1表达水平更低；最后，HuCCT1/CD90+细胞对吉西他滨具有耐药性，而吉西他滨与加鲁尼替尼联合使用可对HuCCT1/CD90+细胞的增殖产生协同抑制效应。上述研究结果证实，CD90介导的吉西他滨耐药性可通过加入转化生长因子β1抑制剂（如加鲁尼替尼）加以克服，进而为肝内胆管癌患者的精准治疗方案提供了新的研究方向。本研究采用RNA测序技术，旨在解析肝内胆管癌细胞系中CD90表达上调所引发的分子改变。