膜 RRM2 阳性细胞代表了肝内胆管癌中具有癌干细胞特征的恶性细胞群。

IF 11.4 1区 医学 Q1 ONCOLOGY Journal of Experimental & Clinical Cancer Research Pub Date : 2024-09-06 DOI:10.1186/s13046-024-03174-w
Yongzhi Zhao, Shuting Xue, Danduo Wei, Jianjuan Zhang, Nachuan Zhang, Liping Mao, Niya Liu, Lei Zhao, Jianing Yan, Yifan Wang, Xiujun Cai, Saiyong Zhu, Stephanie Roessler, Junfang Ji
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引用次数: 0

摘要

背景:肝内胆管癌(iCCA)是最致命的恶性肿瘤之一,具有高度异质性。因此,我们旨在鉴定具有严重恶性特征的 iCCA 细胞亚群并确定其特征:我们使用了来自三个独立 iCCA 队列(iCCA 队列 1-3,n = 382)的转录组数据集和来自 iCCA 队列 4(n = 31)的福尔马林固定和石蜡包埋组织。建立了无偏见的整体筛选策略,包括对iCCA 1-3队列中活化的恶性/干性(MS)特征进行转录组分析,以及对分选的干性报告阳性iCCA细胞进行质谱分析。为了研究候选者的功能作用,还进行了一组细胞实验和皮下肿瘤异种移植实验。对 iCCA 队列 4 进行了免疫组化,以检测候选者的表达和定位。分子和生化试验用于评估候选者的膜定位和功能蛋白域。进行了细胞分选,并利用相应的细胞分子测定来检查分选细胞的癌症干细胞特征:无偏见的全局筛选确定RRM2为首选候选蛋白,在具有MS特征激活的iCCA患者和干性报告阳性的iCCA细胞中,RRM2的水平明显较高。同样,沉默RRM2能显著抑制体外和体内的iCCA恶性表型。此外,iCCA 患者肿瘤组织的免疫组化显示,RRM2 的细胞膜定位与通常的细胞质定位不同。随后,在iCCA细胞中通过免疫荧光、细胞膜通透或不通透、细胞分馏试验和细胞表面生物素化试验证实了RRM2的细胞膜定位。同时,实验还发现了RRM2的非经典信号肽和跨膜结构域。它们对于RRM2通过传统的内质网(ER)-高尔基体分泌途径转运至细胞膜至关重要。此外,还成功分选了膜RRM2阳性的iCCA细胞。这些细胞具有明显的癌症干细胞恶性特征,包括细胞分化能力、自我更新能力、肿瘤诱发能力和干性/恶性基因特征。膜RRM2阳性iCCA细胞的患者预后较差:结论:RRM2具有另一种细胞膜定位方式。结论:RRM2具有另一种细胞膜定位方式,膜RRM2阳性iCCA细胞代表了一种具有癌症干细胞特征的恶性亚群。
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Membrane RRM2-positive cells represent a malignant population with cancer stem cell features in intrahepatic cholangiocarcinoma.

Background: Intrahepatic cholangiocarcinoma (iCCA) is one of the most lethal malignancies and highly heterogeneous. We thus aimed to identify and characterize iCCA cell subpopulations with severe malignant features.

Methods: Transcriptomic datasets from three independent iCCA cohorts (iCCA cohorts 1-3, n = 382) and formalin-fixed and paraffin-embedded tissues from iCCA cohort 4 (n = 31) were used. An unbiased global screening strategy was established, including the transcriptome analysis with the activated malignancy/stemness (MS) signature in iCCA cohorts 1-3 and the mass spectrometry analysis of the sorted stemness reporter-positive iCCA cells. A group of cellular assays and subcutaneous tumor xenograft assay were performed to investigate functional roles of the candidate. Immunohistochemistry was performed in iCCA cohort 4 to examine the expression and localization of the candidate. Molecular and biochemical assays were used to evaluate the membrane localization and functional protein domains of the candidate. Cell sorting was performed and the corresponding cellular molecular assays were utilized to examine cancer stem cell features of the sorted cells.

Results: The unbiased global screening identified RRM2 as the top candidate, with a significantly higher level in iCCA patients with the MS signature activation and in iCCA cells positive for the stemness reporter. Consistently, silencing RRM2 significantly suppressed iCCA malignancy phenotypes both in vitro and in vivo. Moreover, immunohistochemistry in tumor tissues of iCCA patients revealed an unreported cell membrane localization of RRM2, in contrast to its usual cytoplasmic localization. RRM2 cell membrane localization was then confirmed in iCCA cells via immunofluorescence with or without cell membrane permeabilization, cell fractionation assay and cell surface biotinylation assay. Meanwhile, an unclassical signal peptide and a transmembrane domain of RRM2 were revealed experimentally. They were essential for RRM2 trafficking to cell membrane via the conventional endoplasmic reticulum (ER)-Golgi secretory pathway. Furthermore, the membrane RRM2-positive iCCA cells were successfully sorted. These cells possessed significant cancer stem cell malignant features including cell differentiation ability, self-renewal ability, tumor initiation ability, and stemness/malignancy gene signatures. Patients with membrane RRM2-positive iCCA cells had poor prognosis.

Conclusions: RRM2 had an alternative cell membrane localization. The membrane RRM2-positive iCCA cells represented a malignant subpopulation with cancer stem cell features.

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1.80%
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期刊介绍: The Journal of Experimental & Clinical Cancer Research is an esteemed peer-reviewed publication that focuses on cancer research, encompassing everything from fundamental discoveries to practical applications. We welcome submissions that showcase groundbreaking advancements in the field of cancer research, especially those that bridge the gap between laboratory findings and clinical implementation. Our goal is to foster a deeper understanding of cancer, improve prevention and detection strategies, facilitate accurate diagnosis, and enhance treatment options. We are particularly interested in manuscripts that shed light on the mechanisms behind the development and progression of cancer, including metastasis. Additionally, we encourage submissions that explore molecular alterations or biomarkers that can help predict the efficacy of different treatments or identify drug resistance. Translational research related to targeted therapies, personalized medicine, tumor immunotherapy, and innovative approaches applicable to clinical investigations are also of great interest to us. We provide a platform for the dissemination of large-scale molecular characterizations of human tumors and encourage researchers to share their insights, discoveries, and methodologies with the wider scientific community. By publishing high-quality research articles, reviews, and commentaries, the Journal of Experimental & Clinical Cancer Research strives to contribute to the continuous improvement of cancer care and make a meaningful impact on patients' lives.
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