Single-cell RNA-seq analysis reveals BHLHE40-driven pro-tumour neutrophils with hyperactivated glycolysis in pancreatic tumour microenvironment

Liwen Wang; Yihao Liu; Yuting Dai; Xiaomei Tang; Tong Yin; Chaofu Wang; Ting Wang; Lei Dong; Minmin Shi; Jiejie Qin; Meilin Xue; Yizhi Cao; Jia Liu; Pengyi Liu; Jinyan Huang; Chenlei Wen; Jun Zhang; Zhiwei Xu; Fan Bai; Xiaxing Deng; Chenghong Peng; Hao Chen; Lingxi Jiang; Saijuan Chen; Baiyong Shen

doi:10.1136/gutjnl-2021-326070

Single-cell RNA-seq analysis reveals BHLHE40-driven pro-tumour neutrophils with hyperactivated glycolysis in pancreatic tumour microenvironment

Gut. 2023 May;72(5):958-971. doi: 10.1136/gutjnl-2021-326070. Epub 2022 Jun 10.

Authors

Liwen Wang^#^{1

2

3}, Yihao Liu^#^{1

2

3}, Yuting Dai^#⁴, Xiaomei Tang^#^{1

2

3}, Tong Yin⁴, Chaofu Wang⁵, Ting Wang⁵, Lei Dong⁵, Minmin Shi^{1

2

3}, Jiejie Qin^{1

2

3}, Meilin Xue^{1

2

3}, Yizhi Cao^{1

2

3}, Jia Liu^{1

2

3}, Pengyi Liu^{1

2

3}, Jinyan Huang^{4

6}, Chenlei Wen^{1

2

3}, Jun Zhang^{1

2

3}, Zhiwei Xu^{1

2

3}, Fan Bai⁷, Xiaxing Deng^{1

2

3}, Chenghong Peng^{1

2

3}, Hao Chen^{1

2

3}, Lingxi Jiang^{8

2

3}, Saijuan Chen^{9

10}, Baiyong Shen^{8

2

3}

Affiliations

¹ Department of General Surgery, Pancreatic Disease Center, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China.
² Research Institute of Pancreatic Diseases, Shanghai Key Laboratory of Translational Research for Pancreatic Neoplasms, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China.
³ State Key Laboratory of Oncogenes and Related Genes, Institute of Translational Medicine, Shanghai Jiaotong University, Shanghai, People's Republic of China.
⁴ Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.
⁵ Department of Pathology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China.
⁶ Center for Biomedical Big Data, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, People's Republic of China.
⁷ Biomedical Pioneering Innovation Center (BIOPIC), School of Life Sciences, Peking University, Beijing, People's Republic of China.
⁸ Department of General Surgery, Pancreatic Disease Center, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, People's Republic of China shenby@shsmu.edu.cn jlx12120@rjh.com.cn sjchen@stn.sh.cn.
⁹ Shanghai Institute of Hematology, State Key Laboratory of Medical Genomics, National Research Center for Translational Medicine at Shanghai, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China shenby@shsmu.edu.cn jlx12120@rjh.com.cn sjchen@stn.sh.cn.
¹⁰ Sino-French Research Center for Life Sciences and Genomics, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, People's Republic of China.

^# Contributed equally.

Abstract

Objective: Innate immunity plays important roles in pancreatic ductal adenocarcinoma (PDAC), as non-T-cell-enriched tumour. Neutrophils are major players in innate immune system. Here, we aimed to explore the heterogeneity and pro-tumour mechanisms of neutrophils in PDAC.

Design: We analysed single-cell transcriptomes of peripheral blood polymorphonuclear leucocytes (PMNs) and tumour-infiltrating immune cells from five patients with PDAC, and performed immunofluorescence/immunohistochemistry staining, multi-omics analysis and in vitro experiments to validate the discoveries of bioinformatics analysis.

Results: Exploration of the heterogeneity of tumour-associated neutrophils (TANs) revealed a terminally differentiated pro-tumour subpopulation (TAN-1) associated with poor prognosis, an inflammatory subpopulation (TAN-2), a population of transitional stage that have just migrated to tumour microenvironment (TAN-3) and a subpopulation preferentially expressing interferon-stimulated genes (TAN-4). Glycolysis signature was upregulated along neutrophil transition trajectory, and TAN-1 was featured with hyperactivated glycolytic activity. The glycolytic switch of TANs was validated by integrative multi-omics approach of transcriptomics, proteomics and metabolomics analysis. Activation of glycolytic activity by LDHA overexpression induced immunosuppression and pro-tumour functions in neutrophil-like differentiated HL-60 (dHL-60) cells. Mechanistic studies revealed BHLHE40, downstream to hypoxia and endoplasmic reticulum stress, was a key regulator in polarisation of neutrophils towards TAN-1 phenotype, and direct transcriptional regulation of BHLHE40 on TAN-1 marker genes was demonstrated by chromatin immunoprecipitation assay. Pro-tumour and immunosuppression functions were observed in dHL-60 cells overexpressing BHLHE40. Importantly, immunohistochemistry analysis of PDAC tissues revealed the unfavourable prognostic value of BHLHE40⁺ neutrophils.

Conclusion: The dynamic properties of TANs revealed by this study will be helpful in advancing PDAC therapy targeting innate immunity.

Keywords: glucose metabolism; immunotherapy; pancreatic cancer.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Basic Helix-Loop-Helix Transcription Factors / genetics
Carcinoma, Pancreatic Ductal* / pathology
Homeodomain Proteins / genetics
Humans
Neutrophils
Pancreatic Neoplasms* / pathology
Single-Cell Gene Expression Analysis
Tumor Microenvironment

Substances

BHLHE40 protein, human
Homeodomain Proteins
Basic Helix-Loop-Helix Transcription Factors