Neutrophils in Cancer immunotherapy: friends or foes?

1Neutrophils, the most abundant population of white blood cells in circulation, play critical roles in host defense against infection. Traditionally recognized as inflammatory immune cells, neutrophils eliminate pathogens through phagocytosis, degranulation, and neutrophil extracellular trap (NET) formation. Under normal conditions, neutrophils have a brief lifespan, and their activation and mobilization are tightly controlled by the body to prevent potential harm to normal tissues from the highly toxic nature of neutrophil responses. However, in pathological settings, the continuous infiltration of neutrophils can lead to chronic inflammation and tissue damage, which often contribute to tumorigenesis. Recent research has elucidated the multifaceted nature of neutrophils, revealing that they not only serve as effective antibacterial cells but also exhibit phenotypic and functional heterogeneity in pathological contexts such as cancer. Consequently, the impact of neutrophils on tumors has emerged as a prominent area of investigation.

2In this work, we present a comprehensive overview of recent research advancements aimed at unraveling the dual mechanisms of neutrophils in tumor pathogenesis and their potential in immunotherapy. We specifically focus on various factors influencing the phenotypic alterations of tumor-associated neutrophils and predict their clinical utility as diagnostic tools and vital targets for antitumor interventions. Additionally, we explore the existing opportunities and challenges in neutrophil research. This work enhances our understanding of the role of neutrophils in cancer and offers systematic insights for the development of cancer immunotherapy strategies targeting neutrophils.

3Neutrophils are derived from hematopoietic stem cells (HSCs) located in the bone marrow. The process begins with the differentiation of HSCs into common myeloid progenitors (CMPs), which further develop into granulocyte monocyte progenitors (GMPs) and eventually give rise to mature segmented neutrophils. During carcinogenesis, the hematopoietic system may tend to produce more neutrophils and monocytes. This shift in differentiation is driven by the upregulation of granulocyte colony-stimulating factor (G-CSF) and myeloid-related protein S100 calcium-binding protein A9 (S100A9), inhibiting dendritic cell differentiation while promoting the accumulation of bone marrow-derived suppressor cells. This skewed differentiation process further facilitates immune evasion by tumors, thereby promoting tumor progression and treatment resistance (Fig. 1). Of particular intrigue, polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs), exhibit protumor activity akin to that of immunosuppressive neutrophils. Under the stimulation of granulocyte colony-stimulating factor (G-CSF), GM-CSF, pro-inflammatory cytokines interleukin-6 (IL-6), IL-8, TGF-β, and leukotriene B4, immature PMN-MDSCs undergo massive expansion from their precursors in the bone marrow and venture into the peripheral blood and tumor microenvironment. While distinguishing PMN-MDSCs from regular neutrophils in mice remains challenging, distinct markers have been identified for human PMN-MDSCs. The biomarkers/features related to classic neutrophils and PMN MDSCs are shown in Table 1.

4Factors like G-CSF and GM-CSF regulate neutrophil development, prompting the release of mature neutrophils from the bone marrow. However, sustained chronic inflammation, autoimmune ailments, and the provocation of cancer may result in neutrophil depletion, setting off an occurrence known as “emergency granulopoiesis,” rapidly facilitating the influx of mature or even immature neutrophils into the circulation. As neutrophils age within the body, they depart from their target sites and are subsequently cleared by macrophages in the bone marrow, spleen, liver, and lungs. Interestingly, both intrinsic and extrinsic factors influence neutrophil aging. Brain and muscle Arnt-like 1 (BMAL1) drives cell aging by inducing the production of CXCL2 and subsequent CXCR2 signaling, while CXCR4 antagonizes this aging effect. Moreover, the microbiome influences neutrophil senescence through a signaling pathway mediated by toll-like receptors and myeloid differentiation factor 88 (MYD88), representing an external mode of regulation. During transendothelial cell migration, the interaction between integrin α9β1 on neutrophils and vascular cell adhesion molecule 1 (VCAM-1) on endothelial cells triggers the release of GM-CSF, thereby prolonging neutrophil survival. Neutrophils also undergo a distinct form of cell death known as NETosis, wherein they release their nuclear DNA and lytic proteins into the extracellular space in the form of NETs. Neutrophils have considerable plasticity and complexity in the tumor microenvironment. The transforming growth factor-β (TGF-β) and type I interferons (IFN 1) respectively induce the polarization of neutrophils towards the N2 and N1 phenotypes (Fig. 1). In the circulation of cancer patients and mice, high-density neutrophils (HDNs) are a uniform group of mature (segmented) neutrophils with anti-tumor properties, while low-density neutrophils (LDNs) are a mix of immaturce (banded) and mature neutrophils with immunosuppressive properties.

5Therefore, tumor cells have significant regulatory effects on neutrophil recruitment, clearance, and polarization. There is confusion in naming cells with similar or overlapping mechanisms of action, such as PMN-MDSC and N2 neutrophils. Hence, it is necessary to further explore a more appropriate and comprehensive nomenclature.

6Neutrophils secrete various toxic factors or directly contact tumor cells to exert anti-tumor activity. In a mouse model of uterine cancer, the response to low oxygen levels attracted neutrophils to the tumor area. These neutrophils then induced the separation of tumor cells from the basement membrane, thereby limiting the growth of early tumors. Recent research has illuminated a captivating phenomenon: although reducing the hypoxic tumor environment may lead to fewer neutrophils being recruited, their ability to kill cells is exceptional. This remarkable observation can be attributed to the secretion of reactive oxygen species (ROS) and matrix metalloproteinases (MMP-9) by activated neutrophils, leading to the degradation of the epithelial basement membrane. Additionally, neutrophils treated with β-glucan rely on high ROS levels to fight tumors, a process linked to the development of neutrophil precursors’ memory in the bone marrow of trained mice. Neutrophils secrete hydrogen peroxide (H2O2), which induces apoptosis in tumor cells through the influx of calcium ions (Ca2+) mediated by the transient receptor potential cation channel, subfamily M, member 2 (TRPM2). Notably, neutrophil elastase (NE) secreted by neutrophils can hydrolyze and release the CD95 death domain (DD), selectively killing cancer cells and minimizing toxicity to noncancer cells. NE also induces distant effects mediated by CD8+ T cells. Inflammatory modulators such as TNF-α induce the expression of MET in neutrophils. When activated by ligand hepatocyte growth factor (HGF), MET+ neutrophils release nitric oxide (NO) to limit tumor growth and metastasis. Neutrophils directly engage with tumor cells to exert their antitumor activity. For instance, neutrophils with enhanced expression of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and Fas ligand (FasL) induce apoptosis through direct contact with cancer cells. IL-17 has been shown to increase the expression of TRAIL as well as ROS, and IFN-γ to bolster the direct killing ability of neutrophils. Moreover, antibody-dependent cytotoxicity (ADCC), triggered by the binding of the Fc region of monoclonal antibodies (mAbs) to activated Fc receptors (FcRs) on neutrophils, can exhibit strong antitumor effects (Fig. 2).

7Neutrophils also play crucial roles in activating and regulating both innate and adaptive immunity. Recent findings suggest that T cells perform immune surveillance by recognizing specific antigens expressed by tumor cells. Simultaneously, activated T cells signal neutrophils to eliminate tumor escape variants with antigen heterogeneity. Therefore, neutrophils are indispensable for the complete eradication of tumors, and this effect is partially dependent on iNOS. Early research discovered that under the influence of IFN-γ and GM-CSF, immature neutrophils in the tumor microenvironment differentiate into hybrid neutrophils with antigen-presenting cell (APC) characteristics to stimulate anticancer T-cell responses. Subsequent studies have explained more details of this process. During the pretransfer stage, APC-like neutrophils capture tumor antigens, migrate to the tumor-draining lymph nodes (LNs), and form synapses with T cells, presenting antigens to T cells and triggering antitumor immune responses. Recent research shows that successful immunotherapy triggers a notable increase in tumor-infiltrating neutrophils, defining a distinct antitumor immune state termed the Sellhi state. The production of IL-12, a crucial cytokine, primarily relies on dendritic cells (DCs) and macrophages. Upon IL-12 activation, T cells release IFNγ, which triggers the expression of interferon response transcription factor (IRF1) in neutrophils, thereby exerting antitumor effects. In turn, antitumor neutrophils further drive macrophages to release IL-12, which promotes unconventional αβ T cell type I activation and IFN-γ production, leading to the inhibition of sarcoma progression in mice (Fig. 3).

8Neutrophils express a wide range of cytotoxic factors or apoptosis-related ligands that mediate direct killing effects on tumors. Since neutrophils have a short lifespan, this immune response could be transient. However, neutrophil interactions with other components of the immune system induce long-term adaptive immune responses (refer to Table 2 for details).

9Neutrophils, often drive tumor progression by fostering local tumor initiation and proliferation, promoting angiogenesis, facilitating tumor metastasis, and orchestrating networks of immune suppression within the tumor microenvironment (refer to Table 3 for details).

10Chronic inflammation and genetic instability caused by neutrophils promote cancer development. In infected mice, neutrophils were observed to gather and release high levels of NO in the colon, speeding up intestinal inflammation and cancer formation. Additionally, neutrophils release harmful substances that damage DNA and heighten cancer risk. In early intestinal tumor stages, ROS from bone marrow cells cause DNA harm, aiding carcinogenic changes. Independent of the ROS mechanism, neutrophil-derived particles with proinflammatory microRNAs (miR-23a and miR-155) also contribute to double-strand breaks in intestinal epithelial cells. Interestingly, depleting the entire neutrophil population using Ly6G antibodies weakens the carcinogenic effects in chemically induced and spontaneous cancer models, underscoring the significant potential of neutrophils in carcinogenesis.

11During tumor progression, mediators derived from neutrophils directly or indirectly facilitate tumor growth. NE derived from neutrophils can degrade insulin receptor substrate 1 (IRS1), a negative regulator, leading to the activation of phosphoinositide 3-kinase (PI3K) and promoting the proliferation of lung cancer cells both in vivo and in vitro. In a zebrafish model driven by RAS for tumor formation, neutrophils promoted cancer cell proliferation and growth by releasing PGE2. In a mouse model of prostate cancer, tumor cell-released apolipoprotein E (APOE) interacts with triggering receptor expressed on myeloid cells 2 (TREM2) on neutrophils, triggering the senescence process in neutrophils. Nevertheless, such senescent-like neutrophils generate a spectrum of bioactive molecules collectively referred to as the senescence-associated secretory phenotype (SASP), can create an immunosuppressive environment and inhibit the activation of NK cells and cytotoxic T lymphocytes, fostering chronic inflammation and driving tumor progression. Tumor-infiltrating neutrophils not only regulate their own senescence but also shield proliferating tumor cells from senescence, leading to sustained tumor cell growth. This antagonistic senescence response is attributed to the release of interleukin-1 receptor antagonist (IL-1RA) by neutrophils (see Fig. 4 for illustration).

12Prolonged chronic inflammation and the resulting tissue damage create a favorable environment for tumor formation, leading to tumors being referred to as “nonhealing wounds.” However, the inflammatory response of neutrophils in the development of tumors still needs further investigation.

13Angiogenesis is critical for tumor growth and metastasis, as it supplies nutrients and eliminates metabolites. During tumor development, neutrophils release Bv8 and vascular endothelial growth factor (VEGF), effectively promoting tumor angiogenesis. Moreover, neutrophil-secreted MMP-9 degrades the extracellular matrix (ECM), releasing VEGF. When exposed to GM-CSF from breast cancer cells, neutrophils release large amounts of oncostatin M (OSM), which induces VEGF expression through activation of the Janus-activated kinase/signal transducer and activator of transcription (JAK-STAT) pathway in cancer cells. However, in VEGF-induced tumor models, liver fibroblast growth factor 2 (FGF2) can compensate for angiogenesis. FGF2, found in neutrophils and the ECM in liver metastases, can be released by neutrophil-secreted heparanase, promoting angiogenesis. Therefore, Neutrophils seem to produce FGF2 directly and trigger its release from the ECM (Fig. 4). Chemokines containing the ELR tripeptide motif (CXCL1–3, CXCL5–8) promote angiogenesis, and those lacking this motif (CXCL4, CXCL9–11, and CXCL14) inhibit angiogenesis (see review). The ELR+ subset may promote angiogenesis by directly binding to the receptor CXCR2 expressed on tumor blood vessels or indirectly recruiting leukocyte subsets. Thus, the balance between ELR+ and ELR− chemokines is crucial for regulating tumor angiogenesis.

14Overall, Neutrophils can boost proangiogenic factors, making patients less responsive to antiangiogenic treatments. In addition, Antiangiogenic therapy-induced hypoxia triggers mechanisms of drug resistance in tumors, such as promoting invasion and metastasis, coopting the normal vascular system for oxygen and nutrients (co-selection of blood vessels), and the transformation of tumor cells into vascular-like or endothelial-like cells involved in angiogenesis (vascular mimicry). Therefore, further research is necessary to explore the potential mechanisms underlying angiogenesis or its substitution.

15Neutrophils play crucial roles in promoting tumor migration, formation of the pre-metastatic niche and awakening dormant cancer cells in distant sites. Epithelial-mesenchymal transition (EMT) decreases cell adhesion, aiding tumor cells in migrating from the primary site to distant areas. Neutrophils secrete mediators such as IL-17, TGF-β, and NE, all of which bolster tumor EMT. Exosome transporter high-mobility group box 1 (HMGB1) derived from gastric cancer cells induces neutrophil autophagy via the HMGB1/Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling pathway, releasing pro-tumor cell migration factors, such as IL-1β and OSM. Even after successful removal of primary tumors, patients may face renewed cancer spread years later due to dormant cancer cells. In mice model of breast cancer, persistent lung inflammation was found to trigger the transformation of dormant cancer cells into invasive metastatic tumors. Mechanistically, inflammation induces NE and MMP-9 release during NET formation. These enzymes cleave adhesion proteins, generating integrin α3β1-activated epitopes that revive dormant cancer cells. Furthermore, amyloid protein β secreted by cancer-associated fibroblasts (CAFs) also drives the formation of NETs, supporting tumor development.

16Neutrophils support cancer metastasis by acting as adhesion substrates and energy suppliers. Tumor cells must withstand immune attacks and blood flow shear forces to survive when spreading to distant locations. For breast cancer patients, high levels of circulating tumor cell-neutrophil clusters indicate a greater risk of metastasis. Melanoma cells release IL-8, attracting neutrophils to bind with cancer cells. This process involves β2 integrins on neutrophils and intercellular adhesion molecule-1 on tumor cells, which stabilize the tumor cells and enhance their spread to the lungs. Moreover, NETs trap circulating tumor cells, helping them evade immune cell attacks. Neutrophils also provide energy for metastatic tumor cells. In a breast cancer study, lung mesenchymal cells (MCs) prompted neutrophils to store lipids that could be transferred to tumor cells, supporting their growth and survival, leading to lung metastasis (Fig. 4).

17Therefore, neutrophils are pivotal in all phases of tumor spread. Interestingly, organs where metastases occur are not just passive targets; they are actively chosen and modified by the primary tumor before spreading. Common sites for metastasis include the lungs, liver, brain, bones, and lymph nodes, with specific integrins in tumor-derived exosomes guiding organ selection. For example, α6β4 and α6β1 integrins in exosomes are linked to lung metastasis, while αvβ5 integrin is associated with liver metastasis. The precise link between specific exosomes and various metastatic organs is still under active investigation.

18Metabolic abnormalities within the TME, such as abnormal lactate metabolism, lipid metabolism, and amino acid metabolism, significantly impact the antitumor activity of immune cells. Tumors compete with T cells for glucose uptake, leading to T cell depletion and immune evasion. Tumor cells relying on glycolysis produce lactic acid, shaping neutrophils and macrophages towards a pro-tumor state, creating immune-suppressing surroundings. During the development of tumors, lipids are not only energy sources of tumor cells and structural components of membranes but also alter the metabolic crosstalk between different immune cells in the TME. Neutrophil derived myeloperoxidase (MPO) induces lipid peroxidation, hindering DCs’ antigen presentation to CD8+ T cells. Neutrophils expressing FATP2 absorb arachidonic acid, boosting PGE2 production that hampers T cell function. Additionally, in the TME, neutrophils undergoing ferroptosis release lipid mediators, such as PGE2, that limit T cell activity. Tumor-activated neutrophils release arginase 1 (ARG1), reducing L-arginine levels and impacting T cell function. Recent research has shown that neutrophil lineage cells, rather than non-monocytes or macrophages, are the primary sources of ARG1 expression in human non-small cell lung cancer.

19Neutrophils in the tumor microenvironment induce autoimmune checkpoint ligand expression, exerting immunosuppressive effects. Tumor-driven factors like TNF-α, GM-CSF, HMGB1, IL-6, CCL20, and hypoxia boost PD-L1 levels in neutrophils, dampening immune responses, particularly T cells. For instance, GM-CSF from gastric cancer, HMGB1 via extracellular vesicles, and IL-6 from HCC-associated CAFs enhance PD-L1 expression in neutrophils through JAK-STAT3 signaling. Moreover, both tumors and healthy tissues express CD47, a “don’t eat me” signal, binding to SIRP receptors on macrophages and neutrophils, aiding tumors in evading immune eradication.

20Neutrophils influence other immune cells through cytokine secretion, hindering anti-tumor responses. In mice, the complement factor C5a drives neutrophil migration to tumors, releasing ROS and reactive nitrogen species (RNS) that hamper T cell function. Altering TCR recognition and reducing chemokine receptor expression, along with nitrosylation of tyrosine, obstruct T cell movement towards tumors, creating an RNS-based barrier. The RNS-induced immune “cold” environment orchestrates the preferential entry of marrow cells with immunosuppressive characteristics, compromising cancer immunotherapy for various solid tumors, including pancreatic ductal adenocarcinoma. Clearing RNS could enhance current treatment effectiveness. Immunosuppressive neutrophils producing IL-10 lower IL-12 from macrophages, switching them from M1 to M2. Microbiota TLR signals prompt neutrophils to release IL-1β and IL-23, activating lung-resident γδ T cells. Nevertheless, IL-17 from γδ T cells recruits neutrophils, suppressing CD8+ T cell action in breast tumors. This reciprocal interaction between γδ T cells and neutrophils forms amplification loops within the tumor microenvironment, effectively promoting tumor progression (Fig. 5).

21Thus, neutrophils shape the immune system to aid tumor development, particularly impacting T cells. Essentially, neutrophils assist tumors in evading immune monitoring by changing metabolism, adjusting immune checkpoints, and modulating immune cell subsets via cytokines.

22Neutrophils exhibit a dual role in the tumor microenvironment, with both antitumor and protumor effects. On the one hand, neutrophils serve as immune system defenders and exert inhibitory effects on tumors. However, under the influence of the tumor microenvironment, the release of immature neutrophils or phenotypic changes in neutrophils promotes tumor growth. The intricate relationship between neutrophils and cancer progression depends on multiple factors, such as tumor characteristics, endogenous influences, exogenous therapeutic interventions, and the overall health status of patients (Fig. 6).

23The characteristics of tumors within the host, including cancer type, developmental stage, tissue specificity, and metabolic reprogramming, closely influence the phenotypic state of neutrophils. The role of neutrophils in cancer is highly intricate, and there may be variations across different cancer types. In most cancer patients, high levels of TAN are generally associated with poorer treatment outcomes. However, in colorectal cancer patients, TAN infiltration is linked to a favorable prognosis. We hypothesize that different cancers may exhibit varying levels of inflammation, leading to differences in the recruitment and infiltration of neutrophils into tumor tissue. The developmental stage of cancer, whether early or late, significantly impacts the phenotype and characteristics of neutrophils. For instance, LNs are crucial sites for initiating adaptive immune responses and serve as important locations for tumor cell metastasis. In the early N0 stage, neutrophil infiltration is associated with improved patient survival. Conversely, in the late N1–3 stage, a high presence of neutrophils indicates a decrease in the patient survival rate. The phenotypic transformation of neutrophils may be attributed to changes in the immune microenvironment at different developmental stages. Additionally, neutrophils exhibit distinct functional characteristics in different target tissues within the context of cancer. Neutrophils in the lungs and intestines demonstrate significant angiogenic properties, while they are nearly absent in the intestines and skin under sterile conditions. Metabolic reprogramming plays a beneficial role in regulating energy metabolism and facilitating rapid cell growth and proliferation and is considered an emerging marker of cancer. However, the competitive consumption of abundant oxygen and nutrients by cancer cells typically impairs the metabolic adaptability of immune cells infiltrating tumors. Concurrently, abnormal metabolites or intermediates in tumors ultimately influence the effects and functions of immune cells, thereby promoting immune evasion within the tumor microenvironment. When tumors consume glucose, they generate a substantial amount of lactic acid, leading to the formation of an acidic extracellular tumor microenvironment that hampers the antitumor effects of various immune cells. Furthermore, the establishment of a hypoxic environment promotes the upregulation of PD-L1 in neutrophils. Thus, the presence of high levels of lactic acid, low pH, and hypoxia in the tumor microenvironment exacerbates the negative impact on immune cell functionality.

24Endogenous factors such as mediator secretion, genetic aberrations, and microorganisms play crucial regulatory roles in neutrophils. Mediators derived from cancer cells and their surrounding stromal cells, such as IL-6, PGE2, TGF-β, hyaluronic acid fragments (HA), and G-CSF/GM-CSF, contribute to the reprogramming of neutrophils into a tumor-promoting phenotype. CircPACRGL, derived from colorectal cancer (CRC), is a circRNA that increases TGF-β expression upon uptake of miR-142-3P or miR-506-3P, leading to the polarization of neutrophils to the N2 phenotype and eventual tumor progression. Conversely, IFN-γ (type II IFN) induces IRF1 expression, which is essential for the tumor-suppressive effect of neutrophils in the context of successful immunotherapy. Moreover, genetic aberrations in the tumor itself alter the cytokine secretion profile, thereby inducing the accumulation of immunosuppressive neutrophils. In a mouse model with Tp53-deficient tumors, WNT ligands secreted by cancer cells induced IL-1β secretion by tumor-associated macrophages, ultimately leading to the aggregation of immunosuppressive neutrophils. Unexpectedly, microorganisms play essential roles in regulating the functional heterogeneity of neutrophils. Local microorganisms not only prolong the lifespan of neutrophils under inflammatory conditions but also promote neutrophil-mediated inflammation and tumor growth. For example, CRC-derived Escherichia coli breaks the intestinal vascular barrier and translocates to the liver, further promoting the accumulation of neutrophils and leading to the formation of an ecological niche before tumor metastasis. The regulatory effects of various endogenous factors on neutrophils are complex and diverse, playing an important role in regulating neutrophil function and inflammatory immunity.

25Exogenous therapeutic interventions, including surgery, chemotherapy/radiotherapy, immune agonists (such as β-glucan and BCG), and immunosuppressants, also modulate the functional properties of neutrophils. The surgical process may lead to the shedding and dissemination of tumor cells, ultimately resulting in cancer recurrence in postoperative patients. However, surgical trauma-induced inflammatory reactions activate neutrophils and create a tumor microenvironment conducive to tumor growth and spread. Moreover, surgical stress often triggers the formation of NETs, which act as protective barriers, facilitating the metastasis of tumor cells to distant sites. Therefore, precise resection or postoperative adjuvant therapy helps eliminate tumor recurrence. Radiation exposure induces acute lung injury and promotes neutrophilic pro-metastatic responses, enhancing the colonization of tumors at distant sites. Disseminated tumors after chemotherapy cessation induce neutrophil recruitment into the metastatic site of the liver and promote increased metastatic growth. Mechanistically, growth arrest-specific 6 (Gas6) expressed by neutrophils activates AXL receptors on tumor cells, promoting their regeneration. Hence, combining chemotherapy with the inhibition of the Gas6/AXL signaling axis may be effective in reducing cancer recurrence. In fact, β-glucan and BCG vaccines act as agonists of cellular immunity, inducing neutrophils to form antitumor innate immunological memory at the bone marrow level. β-glucan-trained granulocytes possess the ability to limit tumor growth, and this immunological trait is retained at the bone marrow level and can be transferred to untrained recipient mice. BCG vaccination not only promotes the generation of bone marrow lineages biased toward granulocytes but also induces the long-term functional transformation of circulating neutrophils, resulting in enhanced antibacterial function of neutrophils under the co- stimulation of multiple pathogens in vitro. Currently, the BCG vaccine is used as a first-line adjuvant treatment for bladder cancer. Furthermore, targeting key immune checkpoints and immunosuppressive molecules enhances the antitumor activity of neutrophils. Activation of the STAT3 signaling pathway typically leads to the upregulation of PD-L1 in neutrophils, enabling tumors to evade neutrophil surveillance and clearance. Therefore, the development of targeted inhibitors of PD-L1 and STAT3 would be beneficial in restoring the antitumor function of neutrophils.

26The state of patients can vary with age, sex, physiological rhythm, and obesity, which have diverse effects on immune cell function. Generally, immune function tends to gradually decline with age, which may explain why elderly individuals are more susceptible to infections, cancer, and immune-related diseases. During the aging process, dysfunction of neutrophils occurs due to weakened signal transduction of specific receptors. Additionally, young women exhibit higher levels of mature neutrophils and stronger activation responses than do young men. In pregnant women, the frequency of immature neutrophils in the blood is higher than that in healthy women, and these immature neutrophils may play beneficial roles in maintaining maternal-fetal tolerance. In the process of melanoma-induced metastasis, a clear diurnal pattern has been observed, where many metastatic tumors appear in the lungs during cell injection in the morning, with an almost nonexistence of metastatic lesions during injection at night. This pattern aligns with the circadian rhythm of neutrophil aging. Notably, the depletion of neutrophils leads to the alleviation of tumor metastasis, providing new evidence for targeted neutrophil immunotherapy and time-controlled drug delivery. Obesity and alcoholism exacerbate increases in CXCL1, resulting in the increased infiltration of neutrophils into the liver and synergistically promoting the occurrence of steatohepatitis. In an obese mouse model, adipose tissue was shown to induce an increase in lung neutrophils, thereby facilitating cancer cell metastasis. Smoking exposure is recognized as a risk factor for various cancers. The inflammatory response triggered by smoking induces changes in the number and function of neutrophils, implicating them as potential participants in cancer progression. Nicotine in cigarette smoke stimulates N2-neutrophil polarization through the STAT3 pathway. Subsequently, these neutrophils release exosomal miR-4466, promoting the metabolic transformation of brain cancer cells and ultimately accelerating the metastasis of lung cancer to the brain. Furthermore, persistent lung inflammation caused by tobacco smoke exposure promotes the formation of neutrophil extracellular traps (NETs), which can aid in the recovery of dormant cancer cells. Therefore, the inflammatory response induced by cigarette smoke can enhance the tumor-promoting effect of neutrophils.

27In brief, neutrophils play a dual role as both allies and adversaries in cancer development. However, comprehending the complex nature of neutrophils in cancer necessitates considering a myriad of factors. These factors encompass the intrinsic characteristics of the tumor, such as cancer type, stage of progression, tissue specificity, and metabolic alterations. Additionally, endogenous factors such as mediator secretion, genetic abnormalities, and microorganism presence, as well as exogenous therapeutic interventions such as surgery, chemotherapy, radiotherapy, immune agonists, and immunosuppressants, contribute to this intricate dynamic. Furthermore, the individual state of patients, including age, sex, physiological rhythms, obesity, alcoholism, and smoking, also impacts neutrophil behavior. There might be unidentified factors influencing the plasticity of neutrophils in terms of their transcriptional profile and functional attributes.

28The dual nature of neutrophils within the tumor microenvironment presents challenges for targeted therapy. To enhance treatment efficacy, we propose three strategies: 1) blocking the infiltration of neutrophils into local tumors, 2) targeting the immunosuppressive function of neutrophils, 3) improving the adjuvant anticancer efficacy of neutrophils (refer to Table 4 for details).

29Targeting pathways that attract neutrophils to tumors shows potential for halting cancer advancement. CXCR1/CXCR2 receptors play a crucial role in neutrophil recruitment throughout the body, responding strongly to IL-8. Thus, Blocking the IL-8 pathway and its receptors CXCR1/2 emerges as a promising cancer therapy strategy. CXCR1/CXCR2 inhibitors include Navarixin (NCT03473925), SX-682 (NCT05604560), Reparixin (NCT02370238), and AZD5069 (NCT03177187). The anti-IL-8 monoclonal antibody BMS-986253 demonstrated good safety and tolerability in a phase I study as a monotherapy for cancer patients. Currently, BMS-986253 has entered phase II clinical trial (NCT04050462). The C5a receptor (C5aR) on neutrophils is a key therapeutic target. Cancer-produced C5a is a potent neutrophil attractant and contributes to an immunosuppressive tumor environment by upregulating molecules like ARG1, CTLA4, and PD-L1. Blocking C5a/C5aR signaling holds promise as an effective anti-cancer approach. Clinical trials are underway to assess C5aR antagonists’ efficacy in cancer treatment, including IPH5401 (NCT03665129) and TJ210001 (NCT04947033).

30Adjusting the immune-dampening signals of neutrophils in the tumor setting to revive their natural anticancer role holds significant promise. One approach is to target critical immune checkpoints and suppressive molecules to bolster neutrophils’ anticancer functions. Blocking the CD47-SIRPα interaction could amplify neutrophils’ anticancer impact. Promising CD47 antibodies like Magrolimab and Lemzoparimab have shown early clinical benefits and good tolerability. The novel anti-SIRPα antibody CC-95251 (NCT03783403) and the SIRPα inhibitor BI 765063 (NCT03990233) have undergone Phase I study evaluations. Direct STAT3 activation fosters oncogene expression, spurring tumor advancement and immune suppression. Thus, targeting the key regulatory protein STAT3 holds significant promise as an anticancer therapy. Currently effective STAT3 inhibitors include TTI-101, Napabucasin, AZD9150. TGF-β is a key cytokine in the regulation of immune function and is essential for both neutrophil recruitment and reprogramming. In a phase II trial, the oral TGF-β receptor type I kinase inhibitor LY2157299, when used with neoadjuvant radiochemotherapy, improved complete remission to 32% with good tolerance. M7824 (bintrafusp alfa), a dual-action fusion protein inhibiting TGF-β and PD-L1, displayed promising anticancer effects in a Phase II study. M7824 (NCT03631706) and NIS793 (NCT04935359) have entered Phase III evaluation.

31Cancer chemotherapy often causes bone marrow suppression and low neutrophil count, raising infection risks. G-CSF, crucial for boosting neutrophil levels, is frequently used to prevent chemotherapy-induced neutropenia. In a Phase III study with breast cancer patients, F-627, a third-generation long-acting G-CSF drug, lowered infection and febrile neutropenia rates. Although G-CSF doesn’t directly fight cancer, it can improve treatment effectiveness when used alongside chemotherapy. Currently, F-627 has received approval in China for the treatment of chemotherapy-induced neutropenia. IL-6, linked to immune-related adverse events (irAEs) from immune checkpoint blockade (ICB) therapy, is key for neutrophil actions. Anti-CTLA-4 therapy triggers IL-6 release and gut neutrophil buildup, causing an inflammatory response disrupting the gut microbiome balance and promoting irAEs. Combining IL-6 inhibition with antibiotics has shown to reduce irAEs and enhance immune responses. Thus, blocking IL-6 during ICB therapy enhances tumor immunity and relieves irAE symptoms. Tocilizumab and siltuximab are two effective clinical IL-6 inhibitors.

32In summary, though advancements have been achieved in neutrophil-targeting immunotherapies, challenges remain. Precisely targeting distinct neutrophil groups while maintaining a balance between immune regulation and protective functions is vital for optimal treatment results.

33Neutrophils in cancer treatment often lack specificity and have a short duration of actionTargeting neutrophils inadvertently affects other cells or antitumor neutrophils, disrupting the normal immune system and making patients more susceptible to adverse reactions such as infections. For instance, the use of an anti-Gr1 antibody that recognizes Ly6G and Ly6C leads to the depletion of Ly6C-expressing cells such as monocytes and macrophages during treatment. Neutrophil targeting is short-lived due to compensatory mechanisms in the complex immune system that counteract neutrophil blockade within the tumor, resulting in rapid recovery. Anti-Ly6G specifically targets neutrophils, but its depletion triggers compensatory bone marrow release and even induces extramedullary spleen production of neutrophils, leading to a “rebound” effect. Interestingly, newly generated circulating neutrophils express low levels of Ly6G on their membrane, rendering anti-Ly6G treatment inadequate and short-lived. Thus, specific targeting of neutrophils is essential to enhance treatment efficacy and reduce toxic side effects.

34Most studies on neutrophil mechanisms have been conducted in mouse models since neutrophils have a short lifespan. Currently, mouse models for inducing tumor formation include genetic engineering models, chemical toxic drug models, implantation models, and humanized mouse models. However, preclinical models often fail to fully replicate the complex immune system environment and disease progression in humans, making the translation of research results from mouse models to clinical treatment a challenging and time-consuming task. Although humanized mouse models can bridge the gap between the human immune system and xenograft mice, they require advanced technical expertise. In the future, it is essential to establish more effective and innovative preclinical models to predict and assess the applicability of various antitumor drugs in humans.

35Responses to targeted neutrophil therapy may vary among individuals, highlighting the need for personalized treatment protocols. The effectiveness of treatment can be influenced by various factors, including tumor characteristics, endogenous and exogenous factors, and interpatient differences such as disease type and dietary habits. Additionally, variations in drug metabolism abilities and immune system status can contribute to differences in treatment responses. Therefore, the development of personalized treatment plans is crucial to ease the burden of targeted therapy and improve treatment efficacy.

36To optimize the efficacy of targeted neutrophil therapy, it is essential to develop precise methods that specifically target neutrophils while minimizing the impact on other cell types and reducing side effects. Therefore, the identification of highly specific biomarkers for immunosuppressive neutrophils becomes crucial in promoting treatment effectiveness and accuracy. NE, an essential physiological factor in neutrophil-mediated diseases, is exclusively present on the surface of active neutrophils and not in other leukocyte subpopulations. Researchers have discovered that an α1-antitrypsin-derived peptide specifically binds to NE on activated neutrophils. Coating nanoparticles with this peptide enables selective anchoring to activated neutrophils for localized drug delivery.

37Neutrophils have a dual role in tumor progression and participate in immune evasion and drug resistance in tumors. Thus, combining targeted neutrophil therapy with other treatment methods, such as chemotherapy and immunotherapy, can synergistically breakdown immune suppression and enhance antitumor immune effects. Chimeric antigen receptor (CAR)-T cell therapy based on neutrophils effectively improves therapeutic outcomes in solid tumors with heterogeneous antigen expression without additional immunotoxicity. The multifunctional pro-inflammatory neutrophil activating protein (NAP), derived from Helicobacter pylori, possesses potent immune regulatory properties. Using CAR-T cells carrying NAP can promote the recruitment and activation of neutrophils, leading to the eradication of antigenically heterogeneous tumors. However, primary neutrophils have a short lifespan and are resistant to genome editing, limiting their use in CAR-modified immunotherapy. CRISPR–Cas9 technology can be employed to knock in genes into human pluripotent stem cells, resulting in the generation of CLTX-T-CAR neutrophils that exhibit optimal antitumor activity against glioblastoma.

38Neutrophils hold potential as carriers for delivering tumor drugs due to their high migratory ability during inflammatory responses and their ability to cross the blood–brain barrier, enhancing drug penetration and anticancer efficacy. Currently, there are two main delivery strategies based on neutrophils: (1) using neutrophils as carriers and (2) using nanovesicles derived from neutrophil membranes as carriers. Neutrophils loaded with paclitaxel liposomes can penetrate the brain and serve as postoperative adjuvant therapy for brain tumors. Neutrophil membrane-derived nanovesicles enhance drug delivery. A nanoscale neutrophil-mimicking drug delivery system (NM-NP) was developed by coating neutrophil membranes onto the surface of polylactic acid glycolic acid nanoparticles (NPs). This process preserves the biological binding activity of neutrophils. After loading the second-generation proteasome inhibitor carfilzomib, NM-NP-based nanoparticles selectively deplete CTCs, prevent early metastasis, and inhibit the formation of metastatic niches. However, there are limitations to drug delivery using neutrophils as carriers, such as the short in vitro lifespan of neutrophils, potential damage to neutrophil activity caused by drug loading, and challenges in controlling the storage and release profiles of nanoparticles within neutrophils.

39Overall, targeting neutrophils for cancer therapy presents multiple opportunities and challenges. Current challenges include limitations in drug therapy, preclinical models and individual patient variability. Future studies aim to enhance the effectiveness of targeted therapy by optimizing targeting strategies, combining therapies, and exploring the potential of neutrophils as drug delivery carriers.

40Neutrophils exhibit remarkable plasticity and complexity within the tumor microenvironment. As a subset of lymphocytes with phagocytic and cytotoxic functions, neutrophils can directly eliminate tumor cells by releasing various toxic substances or modulating the expression of apoptosis-related ligands. They also indirectly inhibit tumor progression by regulating immune system activation. However, when exposed to tumor-associated factors or exosomes, neutrophils undergo “functional remodeling,” acquiring biological properties that promote tumor immune escape and angiogenesis, thereby facilitating tumor growth and metastasis. Remodeled neutrophils contribute to tumor growth through various mechanisms and are closely associated with immune-related toxicity caused by immune checkpoint blockade. Therefore, inhibiting neutrophil migration into the tumor region not only enhances antitumor efficacy but also effectively mitigates the strong toxic response induced by immunotherapy.

41Determining whether neutrophils act as allies or adversaries in cancer immunotherapy requires considering the combined influence of various factors. Tumor characteristics, including cancer type, stage, tissue specificity, and metabolic reprogramming, influence the dual nature of neutrophils. Endogenous factors such as altered immune status, genetic aberrations, and the microbiota favor the formation of tumor-promoting neutrophils within the tumor microenvironment. Therapeutic interventions, such as surgically induced inflammatory responses, activate immunosuppressive neutrophils, leading to tumor recurrence. However, immune agonists such as β-glucan and BCG can induce antitumor properties in neutrophils. Additionally, individual patient characteristics such as age, sex, and lifestyle further influence the immune landscape. Thus, comprehensively evaluating the complex role of neutrophils in cancer requires considering these multiple factors and exploring other unknown factors to develop personalized and comprehensive treatment strategies.

42Neutrophils play a complex role in cancer immunotherapy, acting as a “double-edged sword” by participating in immune defense and immune tolerance maintenance. Targeting neutrophils in cancer treatment poses challenges due to potential immune dysfunction and adverse reactions, such as infections associated with new drugs or formulations. Therefore, a key focus of current clinical research is optimizing chemotherapy regimens to enhance neutrophil vitality while reducing toxicity. In future cancer immunotherapy, comprehensive approaches such as specific immunotherapy, combination therapy, and personalized therapy can be employed to maximize the therapeutic benefits of targeting neutrophils. Furthermore, leveraging cutting-edge technologies and platforms such as gene editing and nanotechnology enhances neutrophil activity and specificity, leading to superior efficacy. Bridging the gap between preclinical mouse models and human pathological environments, as well as developing personalized treatment plans for patients, will facilitate the advancement of the application of neutrophils in cancer treatment. Additionally, researchers should continue exploring the potential of neutrophils as drug delivery carriers in cancer treatment.