College of Life Science and Bioengineering, Faculty of Environmental and Life Sciences, Beijing University of Technology, Beijing 100124, China; Academy of Military Medical Sciences (AMMS), Academy of Military Sciences, Beijing 100071, China; State Key Laboratory of Experimental Hematology, Fifth Medical Center of Chinese PLA General Hospital, Beijing 100071, China.
College of Life Science and Bioengineering, Faculty of Environmental and Life Sciences, Beijing University of Technology, Beijing 100124, China; Stem Cell and Regenerative Medicine Lab, Institute of Health Service and Transfusion Medicine, AMMS, Beijing 100850, China; South China Research Center for Stem Cell & Regenerative Medicine, SCIB, Guangzhou, Guangdong 510005, China.
J Hepatol. 2020 Nov;73(5):1118-1130. doi: 10.1016/j.jhep.2020.05.039. Epub 2020 Jun 5.
BACKGROUND & AIMS: Intrahepatic cholangiocarcinoma (ICC) is the second most common liver malignancy. ICC typically features remarkable cellular heterogeneity and a dense stromal reaction. Therefore, a comprehensive understanding of cellular diversity and the interplay between malignant cells and niche cells is essential to elucidate the mechanisms driving ICC progression and to develop therapeutic approaches.
Herein, we performed single-cell RNA sequencing (scRNA-seq) analysis on unselected viable cells from 8 human ICCs and adjacent samples to elucidate the comprehensive transcriptomic landscape and intercellular communication network. Additionally, we applied a negative selection strategy to enrich fibroblast populations in 2 other ICC samples to investigate fibroblast diversity. The results of the analyses were validated using multiplex immunofluorescence staining, bulk transcriptomic datasets, and functional in vitro and in vivo experiments.
We sequenced a total of 56,871 single cells derived from human ICC and adjacent tissues and identified diverse tumor, immune, and stromal cells. Malignant cells displayed a high degree of inter-tumor heterogeneity. Moreover, tumor-infiltrating CD4 regulatory T cells exhibited highly immunosuppressive characteristics. We identified 6 distinct fibroblast subsets, of which the majority were CD146-positive vascular cancer-associated fibroblasts (vCAFs), with highly expressed microvasculature signatures and high levels of interleukin (IL)-6. Functional assays indicated that IL-6 secreted by vCAFs induced significant epigenetic alterations in ICC cells, particularly upregulating enhancer of zeste homolog 2 (EZH2) and thereby enhancing malignancy. Furthermore, ICC cell-derived exosomal miR-9-5p elicited high expression of IL-6 in vCAFs to promote tumor progression.
Our single-cell transcriptomic dataset delineates the inter-tumor heterogeneity of human ICCs, underlining the importance of intercellular crosstalk between ICC cells and vCAFs, and revealing potential therapeutic targets.
Intrahepatic cholangiocarcinoma is an aggressive and chemoresistant malignancy. Better understanding the complex transcriptional architecture and intercellular crosstalk of these tumors will help in the development of more effective therapies. Herein, we have identified important interactions between cancer cells and cancer-associated fibroblasts in the tumor stroma, which could have therapeutic implications.
肝内胆管癌（ICC）是第二大常见的肝脏恶性肿瘤。ICC 通常具有显著的细胞异质性和密集的基质反应。因此，全面了解细胞多样性以及恶性细胞与生态位细胞之间的相互作用对于阐明驱动 ICC 进展的机制以及开发治疗方法至关重要。
本研究对 8 例人 ICC 及其相邻样本中的未经选择的存活细胞进行了单细胞 RNA 测序（scRNA-seq）分析，以阐明全面的转录组景观和细胞间通讯网络。此外，我们应用负选择策略富集了另外 2 例 ICC 样本中的成纤维细胞群体，以研究成纤维细胞的多样性。通过多重免疫荧光染色、批量转录组数据集以及体外和体内功能实验对分析结果进行了验证。
我们总共对源自人 ICC 和相邻组织的 56871 个单细胞进行了测序，鉴定出了多种肿瘤、免疫和基质细胞。恶性细胞表现出高度的肿瘤间异质性。此外，肿瘤浸润性 CD4 调节性 T 细胞表现出高度的免疫抑制特征。我们鉴定出 6 种不同的成纤维细胞亚群，其中大多数为 CD146 阳性血管癌相关成纤维细胞（vCAFs），具有高度表达的微血管特征和高水平的白细胞介素（IL）-6。功能测定表明，vCAFs 分泌的 IL-6 可诱导 ICC 细胞发生显著的表观遗传学改变，特别是上调增强子结合蛋白 2（EZH2），从而增强恶性程度。此外，ICC 细胞衍生的外泌体 miR-9-5p 可诱导 vCAFs 中 IL-6 的高表达，从而促进肿瘤进展。
我们的单细胞转录组数据集描绘了人 ICC 的肿瘤间异质性，强调了 ICC 细胞与 vCAFs 之间细胞间相互作用的重要性，并揭示了潜在的治疗靶点。
肝内胆管癌是一种侵袭性和化疗耐药性的恶性肿瘤。更好地了解这些肿瘤的复杂转录结构和细胞间相互作用将有助于开发更有效的治疗方法。在此，我们鉴定了肿瘤基质中癌细胞与癌相关成纤维细胞之间的重要相互作用，这可能具有治疗意义。
