Yijian Zhang1, Chunman Zuo2, Liguo Liu3, Yunping Hu1, Bo Yang4, Shimei Qiu1, Yang Li3, Dongyan Cao5, Zheng Ju5, Jing Ge2, Qiu Wang2, Ting Wang1, Lu Bai1, Yang Yang3, Guoqiang Li3, Ziyu Shao1, Yuan Gao3, Yongsheng Li3, Rui Bian3, Huijie Miao3, Lin Li3, Xuechuan Li3, Chengkai Jiang3, Siyuan Yan3, Ziyi Wang3, Zeyu Wang3, Xuya Cui3, Wen Huang1, Dongxi Xiang3, Congjun Wang6, Qiyun Li7, Xiangsong Wu8, Wei Gong8, Yun Liu3, Rong Shao9, Fatao Liu10, Maolan Li11, Luonan Chen12, Yingbin Liu13. 1. Department of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, China; Shanghai Key Laboratory of Biliary Tract Disease Research, Shanghai, 200092, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai, 200127, China; Shanghai Research Center of Biliary Tract Disease, Shanghai, 200092, China. 2. State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China. 3. Department of Biliary-Pancreatic Surgery, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, China; Shanghai Key Laboratory of Biliary Tract Disease Research, Shanghai, 200092, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai, 200127, China; Shanghai Research Center of Biliary Tract Disease, Shanghai, 200092, China. 4. Key Laboratory of Diagnosis and Treatment of Severe Hepato-Pancreatic Diseases of Zhejiang Province, Department of Surgery, First Affiliated Hospital of Wenzhou Medical University, Wenzhou, 325000, China. 5. Novogene Bioinformatics Institute, Beijing, 100015, China. 6. Department of Gastroenterology Surgery, Songjiang Central Hospital Affiliated to The First People's Hospital of Shanghai Jiao Tong University, Shanghai, 201600, China. 7. Department of Abdominal Surgery, Jiangxi Provincial Cancer Hospital, Nanchang, 330029, China. 8. Department of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, China; Shanghai Key Laboratory of Biliary Tract Disease Research, Shanghai, 200092, China; Shanghai Research Center of Biliary Tract Disease, Shanghai, 200092, China. 9. Department of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, China; Shanghai Key Laboratory of Biliary Tract Disease Research, Shanghai, 200092, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai, 200127, China; Shanghai Research Center of Biliary Tract Disease, Shanghai, 200092, China; Department of Pharmacology, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China. Electronic address: rongshao@sjtu.edu.cn. 10. Department of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, China; Shanghai Key Laboratory of Biliary Tract Disease Research, Shanghai, 200092, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai, 200127, China; Shanghai Research Center of Biliary Tract Disease, Shanghai, 200092, China. Electronic address: liufatao@xinhuamed.com.cn. 11. Department of General Surgery, Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, China; Shanghai Key Laboratory of Biliary Tract Disease Research, Shanghai, 200092, China; Shanghai Research Center of Biliary Tract Disease, Shanghai, 200092, China. Electronic address: limaolan@xinhuamed.com.cn. 12. State Key Laboratory of Cell Biology, Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences, Shanghai, 200031, China; Key Laboratory of Systems Health Science of Zhejiang Province, Hangzhou Institute for Advanced Study, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Hangzhou, 310024, China; Center for Excellence in Animal Evolution and Genetics, Chinese Academy of Sciences, Kunming, 650223, China; School of Life Science and Technology, ShanghaiTech University, Shanghai, 201210, China. Electronic address: lnchen@sibs.ac.cn. 13. Department of Biliary-Pancreatic Surgery, Renji Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, China; Shanghai Key Laboratory of Biliary Tract Disease Research, Shanghai, 200092, China; State Key Laboratory of Oncogenes and Related Genes, Shanghai, 200127, China; Shanghai Research Center of Biliary Tract Disease, Shanghai, 200092, China. Electronic address: laoniulyb@shsmu.edu.cn.
Abstract
BACKGROUND & AIMS: Our previous genomic whole-exome sequencing (WES) data identified the key ErbB pathway mutations that play an essential role in regulating the malignancy of gallbladder cancer (GBC). Herein, we tested the hypothesis that individual cellular components of the tumor microenvironment (TME) in GBC function differentially to participate in ErbB pathway mutation-dependent tumor progression. METHODS: We engaged single-cell RNA-sequencing to reveal transcriptomic heterogeneity and intercellular crosstalk from 13 human GBCs and adjacent normal tissues. In addition, we performed WES analysis to reveal the genomic variations related to tumor malignancy. A variety of bulk RNA-sequencing, immunohistochemical staining, immunofluorescence staining and functional experiments were employed to study the difference between tissues with or without ErbB pathway mutations. RESULTS: We identified 16 cell types from a total of 114,927 cells, in which epithelial cells, M2 macrophages, and regulatory T cells were predominant in tumors with ErbB pathway mutations. Furthermore, epithelial cell subtype 1, 2 and 3 were mainly found in adenocarcinoma and subtype 4 was present in adenosquamous carcinoma. The tumors with ErbB pathway mutations harbored larger populations of epithelial cell subtype 1 and 2, and expressed higher levels of secreted midkine (MDK) than tumors without ErbB pathway mutations. Increased MDK resulted in an interaction with its receptor LRP1, which is expressed by tumor-infiltrating macrophages, and promoted immunosuppressive macrophage differentiation. Moreover, the crosstalk between macrophage-secreted CXCL10 and its receptor CXCR3 on regulatory T cells was induced in GBC with ErbB pathway mutations. Elevated MDK was correlated with poor overall survival in patients with GBC. CONCLUSIONS: This study has provided valuable insights into transcriptomic heterogeneity and the global cellular network in the TME, which coordinately functions to promote the progression of GBC with ErbB pathway mutations; thus, unveiling novel cellular and molecular targets for cancer therapy. LAY SUMMARY: We employed single-cell RNA-sequencing and functional assays to uncover the transcriptomic heterogeneity and intercellular crosstalk present in gallbladder cancer. We found that ErbB pathway mutations reduced anti-cancer immunity and led to cancer development. ErbB pathway mutations resulted in immunosuppressive macrophage differentiation and regulatory T cell activation, explaining the reduced anti-cancer immunity and worse overall survival observed in patients with these mutations.
BACKGROUND & AIMS: Our previous genomic whole-exome sequencing (WES) data identified the key ErbB pathway mutations that play an essential role in regulating the malignancy of gallbladder cancer (GBC). Herein, we tested the hypothesis that individual cellular components of the tumor microenvironment (TME) in GBC function differentially to participate in ErbB pathway mutation-dependent tumor progression. METHODS: We engaged single-cell RNA-sequencing to reveal transcriptomic heterogeneity and intercellular crosstalk from 13 human GBCs and adjacent normal tissues. In addition, we performed WES analysis to reveal the genomic variations related to tumor malignancy. A variety of bulk RNA-sequencing, immunohistochemical staining, immunofluorescence staining and functional experiments were employed to study the difference between tissues with or without ErbB pathway mutations. RESULTS: We identified 16 cell types from a total of 114,927 cells, in which epithelial cells, M2 macrophages, and regulatory T cells were predominant in tumors with ErbB pathway mutations. Furthermore, epithelial cell subtype 1, 2 and 3 were mainly found in adenocarcinoma and subtype 4 was present in adenosquamous carcinoma. The tumors with ErbB pathway mutations harbored larger populations of epithelial cell subtype 1 and 2, and expressed higher levels of secreted midkine (MDK) than tumors without ErbB pathway mutations. Increased MDK resulted in an interaction with its receptor LRP1, which is expressed by tumor-infiltrating macrophages, and promoted immunosuppressive macrophage differentiation. Moreover, the crosstalk between macrophage-secreted CXCL10 and its receptor CXCR3 on regulatory T cells was induced in GBC with ErbB pathway mutations. Elevated MDK was correlated with poor overall survival in patients with GBC. CONCLUSIONS: This study has provided valuable insights into transcriptomic heterogeneity and the global cellular network in the TME, which coordinately functions to promote the progression of GBC with ErbB pathway mutations; thus, unveiling novel cellular and molecular targets for cancer therapy. LAY SUMMARY: We employed single-cell RNA-sequencing and functional assays to uncover the transcriptomic heterogeneity and intercellular crosstalk present in gallbladder cancer. We found that ErbB pathway mutations reduced anti-cancer immunity and led to cancer development. ErbB pathway mutations resulted in immunosuppressive macrophage differentiation and regulatory T cell activation, explaining the reduced anti-cancer immunity and worse overall survival observed in patients with these mutations.