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

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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.

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Most cited references 73

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STAR: ultrafast universal RNA-seq aligner.

Alexander Dobin, Carrie A. Davis, Felix Schlesinger … (2013)

Accurate alignment of high-throughput RNA-seq data is a challenging and yet unsolved problem because of the non-contiguous transcript structure, relatively short read lengths and constantly increasing throughput of the sequencing technologies. Currently available RNA-seq aligners suffer from high mapping error rates, low mapping speed, read length limitation and mapping biases. To align our large (>80 billon reads) ENCODE Transcriptome RNA-seq dataset, we developed the Spliced Transcripts Alignment to a Reference (STAR) software based on a previously undescribed RNA-seq alignment algorithm that uses sequential maximum mappable seed search in uncompressed suffix arrays followed by seed clustering and stitching procedure. STAR outperforms other aligners by a factor of >50 in mapping speed, aligning to the human genome 550 million 2 × 76 bp paired-end reads per hour on a modest 12-core server, while at the same time improving alignment sensitivity and precision. In addition to unbiased de novo detection of canonical junctions, STAR can discover non-canonical splices and chimeric (fusion) transcripts, and is also capable of mapping full-length RNA sequences. Using Roche 454 sequencing of reverse transcription polymerase chain reaction amplicons, we experimentally validated 1960 novel intergenic splice junctions with an 80-90% success rate, corroborating the high precision of the STAR mapping strategy. STAR is implemented as a standalone C++ code. STAR is free open source software distributed under GPLv3 license and can be downloaded from http://code.google.com/p/rna-star/.

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fastp: an ultra-fast all-in-one FASTQ preprocessor

Shifu Chen, Yanqing Zhou, Yaru Chen … (2018)

Abstract Motivation Quality control and preprocessing of FASTQ files are essential to providing clean data for downstream analysis. Traditionally, a different tool is used for each operation, such as quality control, adapter trimming and quality filtering. These tools are often insufficiently fast as most are developed using high-level programming languages (e.g. Python and Java) and provide limited multi-threading support. Reading and loading data multiple times also renders preprocessing slow and I/O inefficient. Results We developed fastp as an ultra-fast FASTQ preprocessor with useful quality control and data-filtering features. It can perform quality control, adapter trimming, quality filtering, per-read quality pruning and many other operations with a single scan of the FASTQ data. This tool is developed in C++ and has multi-threading support. Based on our evaluation, fastp is 2–5 times faster than other FASTQ preprocessing tools such as Trimmomatic or Cutadapt despite performing far more operations than similar tools. Availability and implementation The open-source code and corresponding instructions are available at https://github.com/OpenGene/fastp.

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Comprehensive Integration of Single-Cell Data

Tim Stuart, Andrew Butler, Paul Hoffman … (2019)

Single-cell transcriptomics has transformed our ability to characterize cell states, but deep biological understanding requires more than a taxonomic listing of clusters. As new methods arise to measure distinct cellular modalities, a key analytical challenge is to integrate these datasets to better understand cellular identity and function. Here, we develop a strategy to "anchor" diverse datasets together, enabling us to integrate single-cell measurements not only across scRNA-seq technologies, but also across different modalities. After demonstrating improvement over existing methods for integrating scRNA-seq data, we anchor scRNA-seq experiments with scATAC-seq to explore chromatin differences in closely related interneuron subsets and project protein expression measurements onto a bone marrow atlas to characterize lymphocyte populations. Lastly, we harmonize in situ gene expression and scRNA-seq datasets, allowing transcriptome-wide imputation of spatial gene expression patterns. Our work presents a strategy for the assembly of harmonized references and transfer of information across datasets.

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Author and article information

Journal

Journal ID (nlm-ta): Gut

Journal ID (iso-abbrev): Gut

Journal ID (hwp): gutjnl

Journal ID (publisher-id): gut

Title: Gut

Publisher: BMJ Publishing Group (BMA House, Tavistock Square, London, WC1H 9JR )

ISSN (Print): 0017-5749

ISSN (Electronic): 1468-3288

Publication date (Print): May 2023

Publication date (Electronic): 10 June 2022

Volume: 72

Issue: 5

Pages: 958-971

Affiliations

[1 ] departmentDepartment of General Surgery, Pancreatic Disease Center , Ruijin Hospital, Shanghai Jiaotong University School of Medicine , Shanghai, People's Republic of China

[2 ] departmentResearch 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 ] departmentState Key Laboratory of Oncogenes and Related Genes , Institute of Translational Medicine, Shanghai Jiaotong University , Shanghai, People's Republic of China

[4 ] departmentShanghai 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 ] departmentDepartment of Pathology , Ruijin Hospital, Shanghai Jiaotong University School of Medicine , Shanghai, People's Republic of China

[6 ] departmentCenter for Biomedical Big Data , the First Affiliated Hospital, School of Medicine, Zhejiang University , Hangzhou, Zhejiang Province, People's Republic of China

[7 ] departmentBiomedical Pioneering Innovation Center (BIOPIC), School of Life Sciences , Peking University , Beijing, People's Republic of China

[8 ] departmentSino-French Research Center for Life Sciences and Genomics , Ruijin Hospital, Shanghai Jiao Tong University School of Medicine , Shanghai, People's Republic of China

Author notes

[Correspondence to ] Dr Baiyong Shen, Department of General Surgery, Shanghai Jiao Tong University Medical School Affiliated Ruijin Hospital, Shanghai 200025, China; shenby@ 123456shsmu.edu.cn ; Dr Lingxi Jiang; jlx12120@ 123456rjh.com.cn ; Saijuan Chen; sjchen@ 123456stn.sh.cn

Author information

Baiyong Shen http://orcid.org/0000-0002-3994-248X

Article

Publisher ID: gutjnl-2021-326070

DOI: 10.1136/gutjnl-2021-326070

PMC ID: 10086491

PubMed ID: 35688610

SO-VID: f12c211c-15e8-467a-a0d5-ebaede80bea0

License:

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

History

Date received : 08 September 2021

Date accepted : 27 May 2022

Funding

Funded by: FundRef http://dx.doi.org/10.13039/501100001809, National Natural Science Foundation of China;

Award ID: 81802316

Award ID: 81871906

Award ID: 82002460

Award ID: 82073326

Funded by: FundRef http://dx.doi.org/10.13039/501100002858, China Postdoctoral Science Foundation;

Award ID: 2019M661552

Funded by: Shanghai Pilot Program for Basic Research-Shanghai Jiao Tong University;

Award ID: 21TQ1400205

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ScienceOpen disciplines: Gastroenterology & Hepatology

Keywords: pancreatic cancer,immunotherapy,glucose metabolism

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ScienceOpen disciplines: Gastroenterology & Hepatology

Keywords: pancreatic cancer, immunotherapy, glucose metabolism

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

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Abstract

Objective

Design

Results

Conclusion

Related collections

Karger: Gastroenterology

Most cited references 73

STAR: ultrafast universal RNA-seq aligner.

fastp: an ultra-fast all-in-one FASTQ preprocessor

Comprehensive Integration of Single-Cell Data

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Cited by 38

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