Targeting neutrophil extracellular traps in cancer progression and metastasis

Abstract

Neutrophils serve as pivotal effectors and regulators of the intricate immune system. Their contributions are indispensable, encompassing the obliteration of pathogens and a significant role in both cancer initiation and progression. Conversely, malignancies profoundly affect neutrophil activity, maturation, and lifespans. Cancer cells manipulate their biology to enhance or suppress the key functions of neutrophils. This manipulation is one of the most remarkable defensive mechanisms used by neutrophils, including the formation of neutrophil extracellular traps (NETs). NETs are filamentous structures comprising DNA, histones, and proteins derived from cytotoxic granules. In this review, we discuss the bidirectional interplay in which cancer elicits NET formation, and NETs concurrently facilitate cancer progression. Here, we discuss how vascular dysfunction and thrombosis induced by neutrophils and NETs contribute to an elevated risk of mortality from cardiovascular complications in patients with cancer. Ultimately, we propose a series of therapeutic strategies that hold promise for effectively targeting NETs in clinical settings.

Keywords: cancer progression; metastasis; neutrophil extracellular traps; therapeutic targets; tumor microenvironment.

Figure 1

Research history of NETs. This timeline highlights significant milestones in the research and understanding of Neutrophil Extracellular Traps (NETs) from 2004 to 2024. The timeline illustrates the evolution of key discoveries and their impact on various domains, particularly in the field of oncology and immunology. *Created with BioRender.com.

Figure 2

Composition of NETs. Neutrophil extracellular traps (NETs) are composed of a DNA scaffold released by neutrophils and a variety of functional proteins that play critical roles in tumor biology, immune modulation, and microenvironment remodeling. The core components and their functions include: (1) Neutrophil Elastase: Facilitates angiogenesis by degrading the extracellular matrix (ECM) and modulates endothelial adhesion, inducing metabolic changes that impact immune cells, ECM, and endothelial cell functions. (2) Histones and Antimicrobial Peptides: Directly cause endothelial cell damage and exhibit antimicrobial activity against pathogens. (3) Programmed Death-Ligand 1 (PD-L1): Suppresses immune responses by modulating adaptive immune cells. (4) Matrix Metalloproteinases (MMPs): Promote endothelial damage, angiogenesis, and ECM remodeling through degradation, influencing immune cells, ECM, and endothelial cell dynamics. (5) Chemokines and Cytokines: Recruit immune cells and platelets by inducing chemotaxis and regulating vascular adhesion, thereby orchestrating inflammation and tumor progression. (6) Adhesion Molecules: Act as scaffolds or physical barriers for immune cells, promoting cancer cell proliferation, migration, and immune evasion. (7) Cathepsins: Contribute to angiogenesis, ECM remodeling, and cytokine cleavage, modulating immune responses and ECM architecture. (8) DNA Scaffold: Provides structural support and physical entrapment for immune and tumor cells, while generating chemotactic signals that influence tumor migration and differentiation. *Created with BioRender.com.

Figure 3

Formation of NETs. The formation of NETs includes suicide NETosis and vital NETosis. Suicidal NETosis (left) is initiated by stimuli such as PMA, LPS, immune complexes, cholesterol crystals, or IL-8. These extracellular signals mainly activate the NOX complex via several pathways, such as the Raf-MEK-ERK signaling pathways. IL-8 also activates NF-κB via PI3K-AKT pathways. The influx of extracellular calcium ions can activate mitochondria. These signaling pathways subsequently generate ROS in the cytoplasm, resulting in the release of NE and MPO from azurophilic granules, activation of PAD4, and their translocation into the cell nucleus. Subsequently, activated PAD4 catalyzes the citrullination of histones and chromatin decondensation with the aid of NE and MPO. Additionally, ROS can both activate RIPK3-MLKL, leading to membrane perforation, and cause membrane rupture through NE release. Moreover, intracellular bacterial LPS forms GSDMD pores via caspases 4/5 or 11. When the nuclear membrane breaks down, the decondensed chromatin enters the cytoplasm, mixes with granular proteins, and forms NETs. Finally, NETs are released following the membrane and the mechanical action of swollen chromatin. Vital NET formation (right) is initiated by stimuli such as S. aureus, DAMPs, LPS, activated platelets, and bacterial derivatives. One process is mainly mediated by Ca²⁺ but is independent of the NOX complex. Activated PAD4, NE, and MPO also translocate into the nucleus to promote chromatin decondensation. Mitochondria participate in another pathway by releasing mtDNA and generating mtROS. Lastly, NETs, which may include nuclear DNA and mtDNA, are stored within vesicles budding from nuclei and released by neutrophils without membrane rupture. Abbreviations: LPS, lipopolysaccharide; PMA, phorbol-12-myristate-13-acetate; RAGE, Receptor for Advanced Glycation End-products; FcR, Fc receptor; IL-8, interleukin-8; DAMPs, damage-associated molecular patterns; TLR, toll-like receptor; MPO, myeloperoxidase; NE, neutrophil elastase; NOX, NADPH oxidase; PAD4, protein-arginine deiminase 4; ROS, reactive oxygen species; GSDMD, gasdermin D; mtDNA, mitochondrial DNA; MLKL, mixed lineage kinase domain-like protein.*Created with BioRender.com.

Figure 4

Role of NETs in the initial progression of cancer. The impact of Neutrophil Extracellular Traps (NETs) on the initial progression of cancer can be divided into four categories: (1) Anti-tumor Effect: NETs release DNA and associated proteins that are processed and presented by MHC molecules on tumor cells. Tumor cells recruit CD4+ T cells and downregulate their activation threshold through ZAP70. NETs interact with tumor cells and T cells through integrin-mediated adhesion, promoting proximity for effective immune cell-tumor cell interactions. Bacillus Calmette-Guérin (BCG), as an immunotherapeutic agent, augments this response by enhancing the anti-tumor properties of neutrophils. (2) Tumors and Tumor-Associated Neutrophils (TANs) Positive Feedback Loop: Tumors stimulate TANs to secrete IL-8 through exosomes and cellular molecules (G-CSF, CXCL-1, HMGB1, and Cathepsin C), which in turn leads to the formation of NETs. NETs activate tumors via substances such as HMGB1, promoting their differentiation and metastasis. This positive feedback loop forms a malignant cycle, facilitated by molecules such as HMGB1 that activate TANs via TLR and RAGE signaling pathways, inducing further NETosis and secretion of proinflammatory cytokines (IL-1β, IL-6, IL-8), enhancing the recruitment and activation of additional TANs. (3) NETs' Impact on the Tumor Microenvironment: NETs damage epithelial cells and capture circulating tumor cells (CTCs), providing potential support for tumor colonization. They promote tumor migration through the secretion of HMGB1 and cytokines via epithelial-mesenchymal transition (EMT). Additionally, NETs inhibit the cytotoxic activity of T cells through soluble PD-L1 (sPD-L1). (4) Components and Signaling Pathways of NETs in Tumor Cells: NET-derived components, including NET-DNA, HMGB1, neutrophil elastase (NE), and matrix metalloproteinase-9 (MMP-9), interact with tumor cells (Ts) through multiple pathways. CCDC25 and TLR9 recognize NET-DNA, activating the ILK/β-parvin pathway to promote tumor cell motility. TLR4 engagement triggers NF-κB and MAPK signaling cascades. Additionally, reconstituted laminin binds to integrins α3β1, activating the FAK/ERK/MLCR/YAP signaling axis to enhance tumor cell proliferation and IL-6 production. Abbreviations: NETs, Neutrophil Extracellular Traps; Ts, Tumor cells; BCG, Bacillus Calmette-Guérin; MHC, Major Histocompatibility Complex; TANs, Tumor-Associated Neutrophils; G-CSF, Granulocyte Colony-Stimulating Factor; CXCL-1, C-X-C Motif Chemokine Ligand 1; HMGB1, High Mobility Group Box 1; RAGE, Receptor for Advanced Glycation End-products; IL-8, Interleukin 8; TLR, Toll-Like Receptor; CTCs, Circulating Tumor Cells; NE, Neutrophil Elastase; MMP-9, Matrix Metallopeptidase 9; CCDC25, Coiled-Coil Domain Containing 25. *Created with BioRender.com.

Figure 5

NET-mediated metastasis in various tumor types. NETs facilitate tumor metastasis through distinct organ-specific pathways: In the nervous system, GBM metastasis is promoted via HMGB1/RAGE and IL-8/PI3K/AKT/ROS signaling. In OSCC, NETs facilitate cancer progression through the induction of epithelial-mesenchymal transition (EMT), enhancing tumor cell motility and invasiveness. NETs contribute to lung metastasis by supporting polarization of neutrophils and EMT, influenced by cytokines such as IL-4 and IL-13, and pathways including STAT3 and NAMPT/SIRT1. Additionally, lung cancer cells can promote NET formation through the release of mitochondrial DNA. Colorectal cancer metastasis features mutant KRAS-driven NET formation and mast cell interactions. Omental metastasis occurs through NET-mediated CTC entrapment. Breast cancer metastasis involves NET-induced P53/IL-1β/NF-κB signaling, promoting CD44high/CD24low phenotype transition, EMT, and angiogenesis. Liver metastasis is facilitated through multiple NET-dependent pathways: DNA webs activating RAGE/TLR9, cholesterol-mediated mechanisms, and integrin α3β1-dependent ECM remodeling. Gastric cancer metastasis is driven by TGF-β/ANGPT2/Tie2 axis activation. In pancreatic cancer, NETs promote metastasis through inflammatory pathways (IL-1β/IL-17) and collagen-mediated EMT. *Created with BioRender.com.

Figure 6

NETs promote coagulation and cancer-associated thrombosis. Platelets increase intracellular calcium due to histone activation, which triggers neutrophil activation through P-selectin signaling. Tumor cells (Ts) recruit tumor-associated neutrophils (TANs) and induce their transformation into NETs. Activated endothelial cells secrete IL-8, promoting NET formation. Depending on the microenvironment, NET components can activate or induce apoptosis in endothelial cells. Erythrocytes infected with Phasmodium species and free heme participate directly or indirectly in the conversion of neutrophils into NETs. NETs contribute to thrombosis through several mechanisms. Initially, they promote platelet adhesion, activation, and aggregation through interactions with von Willebrand factor (VWF), fibrinogen, and intermediate filament 1(IF1). Both exogenous and endogenous pathways involving histones, oxidative stress markers, phosphatidylserine, and tissue factor (TF) pathway inhibitors facilitate NET-mediated fibrin formation. Additionally, a positive feedback loop involving neutrophils exists: complement activation through C3a receptor (C3aR), signal STAT4/ROS signaling promotes NET formation. Concurrently, C3b and C5a mediate further neutrophil recruitment and activation. These processes collectively contribute to erythrocyte lysis and aggregation, crucial for thrombus formation in cancer-related contexts. Abbreviations: TANs, tumor-associated neutrophils; Ts, Tumor cells; VWF, von Willebrand factor; IF1, intermediate filament 1; TF, tissue factor; C3aR, C3a receptor; ROS, reactive oxygen species. *Created with BioRender.com.

Figure 7

NETs regulate the functions of immune cells in the tumor microenvironment. NETs modulate multiple immune cell populations through distinct mechanisms: In CD4+ T cells, NETs trigger TLR4-mediated oxidative phosphorylation gene regulation, affecting both regulatory T cells (Treg) and Th17 differentiation. CD8+ T cells respond to NETs through multiple pathways: forming physical barriers that inhibit infiltration, suppressing cytokine production (IFN-γ, TNF-α, IL-2), inducing functional proteins (Tim-3, LAG3, PD-1), and causing T cell depletion via TMO6/TCR signaling. NETs promote Th17 cell differentiation through TLR2/STAT pathway activation. In the NK cell compartment, NETs create physical barriers preventing infiltration, while inducing apoptosis and NK cell dysfunction through Angiopoietin-2 and inflammatory cytokines (IL-2, IL-15). NETs-derived cathepsin G and MMP-9 regulate NK cell receptor expression (e.g., NKp46) affecting IFN-γ production and function. Dendritic cells (DCs) respond to NETs through two opposite pathways: activation of DCs and mitochondrial damage-induced apoptosis. Macrophages exposed to NETs undergo polarization toward an M2 phenotype. Abbreviations: Treg; regulatory T cells; NK, natural killer cells; DC, dendritic cells. *Created with BioRender.com.