After reaching the tumor site, neutrophils deploy a series of immune defense strategies. Initially, they initiate receptor-mediated endocytosis, known as phagocytosis. Studies have shown that when target cells are identified as abnormal through antibodies or complement systems, phagocytic cells are primed for optimal function (46, 55). The CR3 and Fc receptors carried by neutrophils mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cytotoxicity (ADCC), respectively (56). The onset of phagocytosis depends on proximity between tumor cells and CD11b/CD18 expressed on neutrophils (57). Opsonized tumor cells contact with CR3 or FcRs and adhere to neutrophils through receptors on the surface of neutrophils (58), ultimately initiating phagocytosis after the formation of immune synapses. The engulfment of tumor cells by neutrophils to form phagosomes requires the involvement of calcium ions (Ca²⁺) and a series of functional proteins (58). After this, neutrophils use various methods to eliminate tumor cells, including degranulation, NETosis, and the release of ROS ( Figure 1 ).

Neutrophils internalize fragmented cancer cell plasma membranes and cytoplasmic components through successive bites, eventually precipitating cell necrosis (59, 60). Moreover, it has been observed that C-type lectin receptors (CLRs) on neutrophils can recognize Nidogen-1 on the tumor cell surface, thereby enhancing neutrophil-mediated destruction of these cells (61).

The activation of neutrophils is caused by signals emitted by inflammation or infectious lesions, leading to the release of cytotoxins and activation of phagocytosis. Throughout this cascade reaction, neutrophils increased the expression of CD11b, CD18, and CD66b, which are stored within granules (40, 62). In certain circumstances, CD177 and proteinase 3 (PRTN3) are also appreciably upregulated (63). Unlike the conventionally activated neutrophils found in the peripheral blood, those neutrophils that infiltrate into tumors exhibit a distinct activated phenotype, characterized by the increased expression of CD54 (40). After activation, neutrophils predominantly fulfill their function through the mechanism of phagocytosis, and this process is primarily mediated via several pathways ( Figure 2 ): 1) Upon the capture the microbe by the pseudopodia of neutrophils and the subsequent formation of phagosomes, a segment of the plasma membrane invaginates, allowing the combination of cytochrome b558, one of the subunits of NADPH oxidase embedded in the plasma membrane, with another oxidase subunit resident in the cytoplasm. This juxtaposition culminates in the activation of NADPH oxidase, thereby initiating an oxygen-dependent cytotoxic process; 2) Phagosomes detach from the plasma membrane and enter the cytoplasm where they fuse with lysosomes that store cytotoxic proteins, peptides, and enzymes. This connection can cause degranulation and trigger non-oxidative damage.

Cytoplasmic granules, which constitute an arsenal of cytotoxic agents, are a hallmark of neutrophils. According to the composition of matrix and membrane proteins, these particles can be divided into four different subgroups ( Table 1 ): primary (azurophilic) granules, secondary (specific) granules, tertiary (gelatinase) granules, and secretory vesicles (SVs) (2, 21). Primary granules contain antimicrobial substances, including neutrophil elastase (ELANE), cathepsin G, and myeloperoxidase (MPO). Secondary granules contain phagocytic receptors (such as Fc receptors and complement receptors), NADPH oxidase complexes, and lactoferrin. And tertiary granules contain receptors and gelatinase (56, 59, 64). The composition of neutrophil granules is related to various diseases, including rheumatoid arthritis, bullous pemphigoid, and cancer (53). For example, CD18, primarily stored in tertiary granules with some present in secondary granules (57), is associated with liver metastasis in colorectal cancer (65). NE and MMP-9 contribute to tumor metastasis by degrading the basement membrane and releasing vascular endothelial growth factor (VEGF) to promote angiogenesis (2, 53, 57, 66). ELANE is a neutrophil-specific serine protease stored in the azurophil granules (53, 57, 67). Studies have found that neutrophils exert anti-cancer effects by releasing ELANE, which selectively triggers cancer cell apoptosis and supports CD8⁺ T cell-mediated destruction of distant metastasis (68). ELANE selectively kills different types of tumor cells with minimal toxicity to non-cancer cells, which increases the possibility of developing it as a broad anti-cancer therapy. Degranulation, also referred to as exocytosis in neutrophils, relates to the release of pre-formed mediators from granules. The critical step in neutrophil degranulation is the fusion of granules with the plasma membrane, a process mediated by soluble N-ethylmaleimide-sensitive factor activating protein receptors (SNARE) (2, 67). Subsequently, the concentration of Ca²⁺ in neutrophils increases, triggering the degranulation of neutrophils (53, 69).

During the respiratory burst, superoxide anions catalyze the formation of ROS (70), including hydrogen peroxide (H₂O₂), hydroxyl radicals (OH·), and alkoxyl (RO·). Enormous evidence suggests that these oxidants play a crucial role in the clearance of tumor cells. The sudden increase of ROS can cause cell lysis necrosis, apoptosis, or other cell death pathways, so many studies emphasize the role of using ROS to promote oxidative death of cancer cells (71, 72). Traditional antitumor methods such as radiotherapy and chemotherapy, as well as emerging methods such as photodynamic therapy, are all aimed at increasing ROS levels to selectively eradicate malignant cells. Neutrophils can also inhibit lung metastasis and colonization by generating H₂O₂ (73).

ROS can modulate the protein conformation within tumor cells, thereby indirectly affecting the function of crucial signal transduction enzymes, such as kinases and phosphatases. This intricate interplay between ROS and signal transduction proteins forms the cornerstone of tumor therapeutic mechanisms. ROS can activate the intracellular signaling pathways of tumor cells, leading to the opening of non-selective cation channels transient receptor potential of melastatin 2 (TRPM2), which in turn causes lethal calcium influx into the cells (10). ROS can also disrupt the Ras-PI3K Akt signaling cascade by oxidizing specific cysteine residues in Ras and PI3K, thereby inhibiting their interaction and subsequently activating signaling pathways that drive tumor proliferation (74). The impact of ROS is not limited to the PI3K Akt signaling axis; It can also regulate the activity of its upstream signaling molecules, including receptor tyrosine kinase (RTK), ultimately leading to the death of tumor cells (75). In breast cancer cells, ROS can undermine the protein-protein interactions between Bcl-2 and apoptosis-promoting members of the Bcl-2 family, such as Bax and Bak (76), thereby fostering the production of apoptotic pores within mitochondrial membranes. This results in the release of Cytochrome c and the initiation of caspase cascade reactions, ultimately leading to the apoptosis of tumor cells (74).

Nevertheless, the levels of ROS are not static and can fluctuate drastically throughout the various stages of cancer progression. For example, in the early stages of cancer, an increase in ROS levels may accelerate cancer cell death, whereas in the advanced stages, targeting the antioxidant defense systems that permit cancer cells to prosper under elevated ROS conditions may be more effective. This dynamic change indicates that targeting ROS requires different strategies tailored to specific stages of cancer.

Activated neutrophils can manufacture extracellular structures called NETs, which play an important role in capturing and eliminating microorganisms. These NETs are constituted of DNA, originating from either the nucleus or mitochondria (66), and a variety of proteins. Mass spectrometry has delineated 24 proteins associated with NETs, predominantly characterized by their cationic nature, among which are NE, citrullinated H3 histones, defensins, PRTN3, cathepsin G, and MPO (22). The implications of NETs in the context of cancer remain a subject of debate. Research demonstrated that tumor cells can stimulate neutrophils to form NETs through suicidal NETosis (77), and use it to facilitate the tumor metastasis (78). Recent studies also elucidated that NETs can directly attract tumor cells through the cell surface DNA sensor CCDC25 (79). ITGαvβ1, a component of NETs, can cause an epithelial mesenchymal transition in cancer cells, thereby promoting tumor cell chemoresistance (80). NETs can impede the interaction between immune cells and tumor cells by encapsulating the latter, thereby shielding them from the cytotoxic effects mediated by CD8⁺ T cells and natural killer (NK) cells (22). On the contrary, our previous research indicated that in patients with non-small cell lung cancer, NETs have the potential to hinder the migration of tumor cells and lead to their destruction by neighboring neutrophils (19). Recent studies have clarified that activated neutrophils in pancreatic ductal adenocarcinoma patients release NETs, where arginase 1 (ARG1) interacts with cathepsin S to obtain enzyme activity, thereby exerting anticancer effects (81).

Evidence from rodent cancer models has elucidated the potential benefits of neutrophil-targeted depletion therapies (45, 98), with the use of anti-Ly6G monoclonal antibodies (mAbs) demonstrating a selective consumption of neutrophils.

An agonist antibody against TRAIL-R2, DS-8273a, has been evaluated in patients with advanced cancers, showing a reduction in MDSCs in the peripheral blood of half the patients without affecting the maturation of myeloid cells and lymphocytes (45). Furthermore, the engagement of CD300ld, a biomarker of PMN-MDSCs, has been shown to modulate the cellular composition of the TME. Knockout and competitive blockade of the extracellular domain (ECD) of CD300ld can reverse the protumor effect of PMN-MDSCs (104). Ruxolitinib is a JAK inhibitor that has been shown in phase I clinical trials to significantly reduce the number of immature neutrophils without affecting the production of normal neutrophils, thereby achieving anti-tumor effects (105). However, in humans, depletion of neutrophils increases the risk of invasive infections, which may lead to increased treatment costs, antibiotic use, prolonged hospitalization, reduced or delayed use of chemical drugs, and in severe cases, life-threatening complications such as septic shock and sepsis syndrome, and even patient death. In clinical practice, G-CSF is often used preventively in cancer patients to prevent adverse consequences caused by excessive reduction of neutrophils (106).

The cytotoxic potential of neutrophils is regulated by a delicate balance of signals transmitted through immune checkpoints and activating receptors. Neutrophils also express a suite of lymphocyte checkpoint ligands, including V-domain Ig suppressor of T cell activation (VISTA), PD-L1, CD86, 4-1BB ligand (4-1BBL), and OX40L (22), signaling a new frontier in cancer immunotherapy through targeted neutrophil checkpoints.

Recent findings suggest that targeting NAMPT can inhibit SIRT1 signaling and neutrophil angiogenic gene transcription, offering a potential mechanism for tumor growth inhibition (12). ATG-019, which inhibits both PAK4 and NAMPT, has shown improved efficacy in combination with anti-PD-1 therapy in murine tumor models compared to anti-PD-1 alone, representing a novel treatment strategy for advanced solid tumors and non-Hodgkin’s lymphoma (107, 108). Additionally, the concurrent administration of TNF and anti-CD40 mAb boosts neutrophil activation and toxicity, further promoting tumor cell apoptosis and clearance by enhancing oxidative damage and N1 neutrophil recruitment in the TME (13, 101). Additionally, the anti-CD40 therapy increased the Sell^hi neutrophils accumulation and maturation, thus eliciting the neutrophils antitumor response (109).

Compared to traditional radiotherapy and chemotherapy, immune checkpoint inhibitors typically have a lower incidence of adverse reactions. Even if patients stop using immune checkpoint inhibitors, they can often still maintain the treatment benefits (110). However, immunotherapy also faces the challenge of drug resistance. Evidence suggests that the binding affinity of anti-PD-1 receptors decreases within 2-3 months after the last dose of medication (111). In addition, multiple types of tumors and most patients have limited response to single immune checkpoint therapy, and combining other antitumor therapies may be the future path for immune checkpoint inhibitors.

Traditional strategies typically involve depleting the original tumor neutrophils or disrupting their migratory ability, rather than eliminating the entire neutrophil population. As previously noted, CXCR2 and CXCR1 are indispensable for the recruitment of neutrophils into the TME (21). The inhibition of neutrophil recruitment by blocking CXCL8, CXCR1, and CXCR2 has progressed to the clinical evaluation stage (22). The spleen serves as a reservoir for the precursors of TANs, from which TANs are recruited into the tumor stroma by CXCL8 (48, 49). Under normal physiological conditions, CXCL8 is barely detectable; however, within the TME, it rapidly accumulates to effectively regulate the protumor effects of N2 neutrophils, including the promotion of angiogenesis, dedifferentiation, and metastasis of tumor cells (50). The lipid metabolism-related gene enoyl-CoA δ-isomerase 2 (ECI2) can reduce CXCL8 to decrease neutrophil infiltration and NETs formation in the TME, thereby inhibiting colorectal cancer progression (112). Similarly, active PRSS35 can inactivate and degrade CXCL2 by cleaving it, ultimately inhibiting the development of HCC through a similar pathway (113). Inhibiting CXCL8 signal transduction can reduce the recruitment of N2 neutrophils. HuMax-IL8 (BMS-986253), an inhibitor of CXCL8, has been demonstrated to be well-tolerated in patients with advanced cancers and is currently being evaluated for its safety and efficacy in combination with nivolumab (NCT03400332) (50, 114). ABX-IL8, functioning as a neutralizing antibody against CXCL8, can significantly inhibit the promoter activity and collagenase activity of MMP2 in melanoma cells, thereby diminishing tumor metastasis, reducing tumor angiogenesis, and augmenting tumor apoptosis (50).

Lactate constitutes an effector molecule that not only contributes to inflammation but also plays a crucial role in the onset and progression of cancer (115). Consequently, lactate dehydrogenase inhibitors may represent effective targets for inhibiting tumor progression. Such inhibitors have been proven to alleviate the harmful neutrophil mobilization associated with TAN in malignant tumors (115).

While NETosis plays an important role in microbial defense, it may be adeptly subverted by tumors to serve their own benefit (59). NETs not only inhibit T cell function but also facilitate the metastasis of tumor cells. By forming a physical barrier on the tumor’s surface, NETs shield tumor cells from CD8⁺ T cell-mediated assaults (116). Moreover, NETs can induce CD4⁺ T cells to differentiate into regulatory T cells (Treg cells), thereby establishing a tumor tolerant environment (39). As an emerging target for tumor therapy, the employment of deoxyribonuclease I (DNase I) to clear NETs or the use of related metabolic inhibitors can effectively repress NETosis (21). DNase I has been shown to have the ability to prevent the invasion and migration of lung and colon cancer cells, as well as significantly diminish lung metastasis in tumor models (22, 53). In addition, metformin targeting the mitochondrial DNA (mtDNA) of NETs can alleviate metastasis-abetting inflammatory state, thereby weakening the metastatic potential of hepatocellular carcinoma. The NET inhibitor chloroquine can alleviate hypercoagulability by diminishing NET-dependent thrombosis in cancer patients (39).

The citrullination of histones by peptidylarginine deiminase 4 (PAD4) is considered the main event in the formation of NETs in vivo (21, 46, 59). PDA4 inhibitors can enhance the clinical prognosis for tumor patients by diminishing NETosis (117). Consequently, PAD4 is expected to become an apt target for therapeutic intervention (66). The currently used PAD4 inhibitor in clinical practice is Cl-amidine (21). Moreover, other PAD4 inhibitors, such as BMS-P5, JBI-589, resveratrol (RES), and Simvastatin, have also exhibited therapeutic efficacy in animal models (118).

The formation of NETs is contingent upon H₂O₂ produced by NADPH oxidase and further metabolized by MPO (2). Therefore, ROS are amongst the most significant inducers of NETs (46, 53, 59), and any substance capable of inhibiting ROS production holds the potential to inhibit NETosis. Previous studies revealed that vitamin C, flavonoids, 5-aminosalicylic acid (5-ASA), N-acetyl-L-cysteine (NAC), PF-1355, or diphenyliodonium (DPI) can inhibit ROS production, thereby suppressing NETosis (21, 118). However, this aspect remains unexplored in the context of tumor treatment.

Although numerous studies have demonstrated that they have a pro-tumor effect, there are also studies suggesting that NETs can inhibit tumor growth by capturing and killing cancer cells (19, 81). In clinical practice, it is crucial to identify the role that NET plays in tumor progression.

The immunomodulatory characteristics of neutrophils within TME are closely related to metabolic remodeling. In response to the increased energy demand caused by rapid proliferation of tumor tissue, neutrophils are adept at utilizing glucose, amino acids, and lipids as key energy substrates within the TME (119). Notably, TANs predominantly obtains energy from glycolytic metabolism, allowing them to function effectively in hypoxic conditions (42, 59). The hypoxic-glycolytic niche in tumors can reprogram mature and immature neutrophils into long-lived and terminally-differentiated subsets, thereby promoting angiogenesis and tumor growth (120). HIF-1α, a pivotal transcriptional regulator, control the expression of enzymes related to glycolysis and facilitates a metabolic shift from oxidative phosphorylation (OXPHOS) to glycolysis in PMN-MDSCs (48). In vitro experimentation with PX-478, an inhibitor of HIF-1α, has demonstrated synergistic enhancement of tumor cell apoptosis when used in conjunction with immune checkpoint inhibitors (121). Despite being highly dependent on glycolysis, neutrophils have the metabolic versatility to eschew glycolysis and upregulate alternative metabolic pathways, including glutaminolysis, OXPHOS, and fatty acid oxidation (FAO), when necessary (22, 60, 119). This metabolic adaptability highlights the challenge of targeting TAN metabolism within TME. Findings have elucidated that immunosuppressive neutrophils present within the TME elevate the expression of lipid transport-related proteins, including CD36 and fatty acid transporter protein 2 (FATP2) (22). The increase in CD36 expression promotes the metabolic transition from glycolysis to FAO, making FAO the main energy source for PMN-MDSC (95). Meanwhile, FATP2 promotes tumorigenesis and immune evasion by regulating the accumulation of arachidonic acid in PMN-MDSC and subsequent synthesis of prostaglandin E₂ (PGE₂) (48, 122). Consequently, FATP2 and CD36 have emerged as viable targets for selective inhibition to enhance the therapeutic efficacy of cancer treatments (95, 122). The metabolic profiling of TANs critically influences their behavior during tumorigenesis. The metabolic remodeling undergone by neutrophils within neoplastic tissue may provide novel avenues for research and development within future cancer treatment strategies.

The administration of Coley’s Toxin and the BCG vaccine represents one of the pioneering instances of immunotherapy in modern medical practice. The therapeutic essence of both Coley’s Toxin and the BCG vaccine lies in their capacity to stimulate neutrophils and other components of the immune system through the introduction of inactivated or attenuated bacterial agents (123, 124). Coley’s Toxin can trigger the release of a series of immune regulatory mediators, including cytokines, lysosomal enzymes, and ROS, which play an important role in directly killing tumor cells and activating other immune effectors (such as NK cells and T lymphocytes) to participate in synergistic antitumor responses (123). Similarly, early investigations have revealed that neutrophils activated by BCG immunotherapy in the context of bladder cancer undergo polarization into an antitumoral phenotype (14). The cytokines released by neutrophils activated by BCG vaccine indirectly enhance the chemotaxis of T cells, thereby enhancing the efficacy of BCG immunotherapy (125).

Applying microbial bioparticles to tumor growth not only activates the killing ability of neutrophils, but also causes significant changes in their transcriptional landscape, migration patterns, and functional properties, ultimately inhibiting tumor proliferation in a neutrophil-dependent manner. The intratumoral delivery of microbial bioparticles has also been observed to enhance the expression of chemokines recruiting NK cells and CD8⁺ T cells in neutrophils, thereby enhancing the antitumor immune response (126).

Previous studies have shown that tumor cells can release various types of extracellular vesicles, one of which is called tumor microparticles (T-MP) with sizes ranging from 100-1000 nm (127). T-MPs have been shown to affect various cancer-related cells and immune cells ( Figure 3 ). As a carrier for chemotherapeutic drug delivery, MP has the advantages of biological safety, simple preparation, and stable properties. The MPs deliver the chemotherapeutic drugs to the nucleus and kill the tumor repopulation cell (128). In addition, the MP contains the tumor common antigen, which activated the DC by cGas-STING pathway, then promotes the tumor-specific CD8⁺ T cells antitumor response (129). Our previous studies have clarified that methotrexate-loaded tumor cell microparticles (MTX-MPs) activated neutrophils and demonstrated a high level of efficacy and safety in a clinical trial involving patients with MPE or cholangiocarcinoma (18, 19). Recently, we found that the MTX-MP-activated neutrophils promote the T cell antitumor response by increasing the PD-1 degradation in the lysosome pathway in neutrophils (16). Moreover, the MPs without drug promote tumor metastasis by promoting M2 macrophage differentiation, while the MTX-MP promotes M2 polarized toward M1 by activating lysosomal cytochrome P450 and nuclear hnRNPA2B1, thereby recruiting neutrophils to kill tumor cells (17, 129, 130).

For a long time, people have believed that immune memory is a unique feature of adaptive immune response. Professor Mihai Netea from Nijmegen University first proposed that the innate immune system can also exhibit adaptive features, known as trained immunity (131). One of the most typical examples is that the BCG can induce the well-trained immunity of neutrophils through the genome-wide epigenetic modifications in trimethylation at histone 3 lysine 4 (132). β-glucan can train the innate immune system through type I interferon signaling, reprogram GMP, and reprogram neutrophils into antitumor phenotypes, ultimately inhibiting the occurrence and development of tumors (133). Mulder et al. designed a bone marrow-targeted nanobiological agent that can induce training immunity, thus enhance proliferation and metabolism of neutrophils to inhibit tumor growth (134).