肝内胆管癌（iCCA）是一种高度异质性和侵袭性的肝癌，治疗选择有限。精准分类和免疫治疗是改善治疗的前景。我们报道了 septin 9 在顶端基底极性和上皮间质转化 (EMT) 中的作用。在这里，我们旨在阐明它在 iCCA 中的作用。我们根据表型和细胞状态分析了人类 iCCA 肿瘤细胞的单细胞转录组。使用小干扰 RNA (siRNA) 敲低 septin 9 基因 (SEPT9)；使用不同的CCA细胞进行干扰素-γ（IFN-γ）刺激；通过逆转录和实时PCR分析（RT-qPCR）分析基因表达；并进行免疫荧光、免疫印迹和流式细胞术来评估蛋白质的表达。 SEPT9 和波形蛋白 (VIM) 基因表达的差异分布使我们能够定义恶性细胞的特定细胞轨迹，从而识别不同的 iCCA 细胞簇。其中一个簇富含 VIM 和细胞外基质 (ECM) 重塑分子，另一个簇则高表达 SEPT9 和参与免疫逃逸的“别吃我”信号基因。 SEPT9和VIM之间的这种拮抗作用通过体外实验得到证实。值得注意的是，SEPT9 和“别吃我”基因表达与波形蛋白和 EMT 标记物呈负相关。 SEPT9 表达被 IFN-γ 上调，而 SEPT9 敲除降低了“别吃我”信号基因的表达，并增加了间充质标记物的表达。癌细胞系百科全书 (CCLE) 转录组数据库分析证实，富含 septin 9 的 iCCA 细胞表现出上皮样特征。这项研究揭示了 septin 9 是 iCCA 上皮样细胞的细胞骨架元件和免疫系统反应的调节剂。它还为 EMT 和免疫反应之间的神秘关系带来了新的见解。值得注意的是，我们解码了一种可能使患者对免疫疗法敏感的潜在机制。© 2024 作者。约翰·威利出版的《分子肿瘤学》

Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous and aggressive liver cancer with limited therapeutic options. Precise classification and immunotherapy are perspectives to improve the treatments. We reported the role of septin 9 in apico-basal polarity and epithelial-to-mesenchymal transition (EMT). Here, we aim to elucidate its role in iCCA. We analyzed single-cell transcriptomes from human iCCA tumor cells based on phenotype and cell state. Knockdown of the septin 9 gene (SEPT9) was done using small interfering RNA (siRNA); interferon-γ (IFN-γ) stimulation was performed using different CCA cells; gene expressions were analyzed by reverse transcription and real-time PCR analysis (RT-qPCR); and immunofluorescence, immunoblotting, and flow cytometry were performed to assess the expression of proteins. The differential distributions of SEPT9 and vimentin (VIM) gene expressions allowed us to define specific cellular trajectories of malignant cells and thus identified distinct clusters of iCCA cells. One cluster was enriched in VIM and extracellular-matrix (ECM) remodeling molecules, and another had high expression of SEPT9 and genes from the 'don't eat me' signal involved in immune escape. This antagonism between SEPT9 and VIM was confirmed by in vitro experiments. Notably, SEPT9 and 'don't eat me' gene expressions were inversely correlated to those of vimentin and the EMT markers. SEPT9 expression was upregulated by IFN-γ and SEPT9 knockdown decreased expression of 'don't eat me' signal genes and increased expression of mesenchymal markers. Cancer Cell Line Encyclopedia (CCLE) transcriptome database analyses confirmed that iCCA cells enriched in septin 9 exhibit epithelial-like features. This study revealed septin 9 as a cytoskeleton element of iCCA epithelial-like cells and a regulator of the immune system response. It also brings new insights into the enigmatic relationship between EMT and immune response. Notably, we decoded a potential mechanism that could sensitize patients to immunotherapies.© 2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.