Min Zhang1, Hui Yang2, Lingfei Wan2, Zhaohai Wang3, Haiyang Wang4, Chen Ge2, Yunhui Liu2, Yajing Hao5, Dongdong Zhang5, Gaona Shi6, Yandong Gong7, Yanli Ni8, Chaojie Wang9, Yuan Zhang10, Jiafei Xi11, Sen Wang3, Lei Shi3, Lina Zhang12, Wen Yue13, Xuetao Pei14, Bing Liu15, Xinlong Yan16. 1. 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. 2. 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. 3. Department of Hepatobiliary Surgery, Fifth Medical Center of Chinese PLA General Hospital, Beijing 100039, China. 4. 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. 5. Key Laboratory of RNA Biology, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China. 6. State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100050, China. 7. Academy of Military Medical Sciences (AMMS), Academy of Military Sciences, Beijing 100071, China. 8. State Key Laboratory of Experimental Hematology, Fifth Medical Center of Chinese PLA General Hospital, Beijing 100071, China. 9. Department of Pathology and Pathophysiology, Medical College, Jinan University, Guangzhou 510632, China. 10. Department of Integrated Traditional Chinese and Western Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. 11. Academy of Military Medical Sciences (AMMS), Academy of Military Sciences, Beijing 100071, 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. 12. College of Life Science and Bioengineering, Faculty of Environmental and Life Sciences, Beijing University of Technology, Beijing 100124, China. 13. Academy of Military Medical Sciences (AMMS), Academy of Military Sciences, Beijing 100071, 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. Electronic address: yuewen0206@126.com. 14. Academy of Military Medical Sciences (AMMS), Academy of Military Sciences, Beijing 100071, 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. Electronic address: Peixt@nic.bmi.ac.cn. 15. State Key Laboratory of Experimental Hematology, Fifth Medical Center of Chinese PLA General Hospital, Beijing 100071, China. Electronic address: bingliu17@yahoo.com. 16. College of Life Science and Bioengineering, Faculty of Environmental and Life Sciences, Beijing University of Technology, Beijing 100124, China. Electronic address: yxlong2000@bjut.edu.cn.
Abstract
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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. LAY SUMMARY: 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.
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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. LAY SUMMARY: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.
Authors: Isabella Lurje; Zoltan Czigany; Jan Bednarsch; Nadine Therese Gaisa; Edgar Dahl; Ruth Knüchel; Hannah Miller; Tom Florian Ulmer; Pavel Strnad; Christian Trautwein; Frank Tacke; Ulf Peter Neumann; Georg Lurje Journal: Liver Cancer Date: 2022-01-25 Impact factor: 12.430