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

Abstract

ObjectiveInnate 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.DesignWe 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.ResultsExploration 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.ConclusionThe dynamic properties of TANs revealed by this study will be helpful in advancing PDAC therapy targeting innate immunity.

Prognostic value of glycolysis markers in pancreatic cancer: A systematic review and meta-analysis

“…It has been well demonstrated that glycolytic by-product lactic acid not only blunts tumor immunosurveillance by T and NK cells but also enhances immunosuppressive phenotype of Tregs, TAMs and MDSCs ( 49 – 52 ). A recent study further indicated that a terminally differentiated subpopulation of tumor-associated neutrophils exhibited hyperactivated glycolytic activity in PDAC TME promotes pro-tumor and immunosuppression functions of neutrophils ( 53 ). Therefore, targeting enhanced glycolysis of multiple components in the TME, not just tumor cells, may contribute to the development of novel therapeutic strategies against PDAC.…”

Single Cell RNA Sequencing: A New Frontier in Pancreatic Ductal Adenocarcinoma

“…Other stromal components have also been studied using scRNA-seq. Wang et al highlighted the differences in tumor-associated polymorphonuclear cells using an scRNA-seq analysis [ 55 ]. Their work delineated four distinct tumor-associated neutrophil lineages with different metabolic and immune behaviors and identified a key regulator driving this differentiation.…”

Glycolysis in tumor microenvironment as a target to improve cancer immunotherapy

“…The intrinsic heterogeneity of tumor-associated neutrophils (TANs) and cancer contexts determine these myeloid cells' proor anti-tumor effects. In pancreatic ductal adenocarcinoma (PDAC), the multi-omics approach exhibits that TANs undergoing glycolytic switch mediated by LDHA upregulation showed tumor-promoting phenotype (Wang et al, 2022b). Triple-negative breast cancer (TNBC) cells with accelerated glycolysis highly express granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) under the regulation of liver-enriched activator Frontiers in Cell and Developmental Biology frontiersin.org protein and AMP-activated protein kinase (AMPK)-serine/ threonine-protein kinase (ULK1) pathway, supporting myeloid-derived suppressor cells (MDSCs) development and facilitating CD8 + T cell inhibition and cancer progression (Li et al, 2018).…”