Neutrophil Dynamics in Response to Cancer Therapies

Simple Summary

Neutrophils are a type of white blood cell best known for fighting infections, but recent studies show they also play complex roles in cancer. Within tumors, neutrophils can adopt opposing identities: the “N1” type combats cancer by killing tumor cells and stimulating immune activity, while the “N2” type does the opposite—supporting tumor growth, blocking immune responses, and aiding disease progression. In this review, we explore how neutrophils respond to different cancer treatments, including chemotherapy, radiotherapy, immune cell therapy, and therapies using viruses or bacteria. We highlight how different therapeutic environments can drive neutrophils toward either the beneficial N1 or the harmful N2 state. Understanding what determines this polarization is key to improving outcomes. By understanding when neutrophils act in favor of or against treatment, scientists can design better strategies to fight cancer. This knowledge could lead to new therapies that guide neutrophils to support the immune system, reduce tumor spread, and improve the efficacy of current cancer treatments.

Abstract

Neutrophils are increasingly recognized as key players in the tumor microenvironment (TME), displaying functional plasticity that enables them to either promote or inhibit cancer progression. Depending on environmental cues, tumor-associated neutrophils (TANs) may polarize toward antitumor “N1” or protumor “N2” phenotypes, exerting diverse effects on tumor growth, metastasis, immune modulation, and treatment response. While previous studies have focused on the pathological roles of TANs in cancer, less attention has been given to how cancer therapies themselves influence the behavior of TANs. This review provides a comprehensive synthesis of current knowledge regarding the dynamics of TANs in response to major cancer treatment modalities, including chemotherapy, radiotherapy, cell-based immunotherapies, and oncolytic viral and bacterial therapies. We discuss how these therapies influence TAN recruitment, polarization, and effector functions within the TME, and highlight key molecular regulators involved. By consolidating mechanistic and translational insights, this review emphasizes the potential to therapeutically reprogram TANs to enhance treatment efficacy. A deeper understanding of context-dependent TAN roles will be essential for developing more effective, neutrophil-informed cancer therapies.

1. Introduction

Constituting 50–70% of circulating leukocytes [1,2], neutrophils are traditionally recognized as innate immune granulocytes but have more recently been identified as significant components of the tumor microenvironment (TME) [3,4,5]. Clinical observations consistently associate substantial neutrophil infiltration within tumors with enhanced disease progression and unfavorable patient prognosis [6]. These pro-tumoral effects are mediated through multiple mechanisms, including angiogenesis stimulation via Vascular Endothelial Growth Factor (VEGF) secretion, facilitation of tumor cell migration, and immune suppression through the release of neutrophil elastase (NE), interleukin 8 (IL-8), and arginase-1 (ARG1) [7,8,9,10,11,12]. Such tumor-promoting neutrophils, often classified as “N2” tumor-associated neutrophils (TANs), further support cancer progression by forming neutrophil extracellular traps (NETs), secreting pro-inflammatory cytokines such as IL-17 and tumor necrosis factor alpha (TNF-α), remodeling ECM components, and inhibiting T-cell-mediated immunity [13,14,15].

Conversely, neutrophils can also exhibit potent antitumor properties, commonly referred to as “N1” neutrophils. Their tumoricidal capacity involves reactive oxygen species (ROS) generation, cytotoxic enzyme release, antibody-dependent cellular cytotoxicity (ADCC), and activation of lymphocyte populations including cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells [16,17,18]. These antitumoral “N1” neutrophils directly eliminate malignant cells through multiple effector mechanisms such as ROS and TNF-Related apoptosis-inducing ligand (TRAIL), while simultaneously enhancing adaptive immune responses [19,20,21].

The functional polarization of TANs exists along a continuum between these opposing phenotypes, with their behavior dynamically regulated by tumor microenvironmental cues [22]. Key regulatory factors include transforming growth factor-β (TGF-β), which promotes immunosuppressive N2 polarization, and type I interferons (IFN-β) that induce antitumoral N1 characteristics [23,24,25,26]. This phenotypic plasticity emphasizes the dual nature of neutrophils in cancer pathogenesis and illustrates the importance of understanding how therapeutic interventions influence neutrophil behavior, given their capacity to either compromise or potentiate treatment efficacy [27,28].

In summary, neutrophils in the TME are a double-edged sword, capable of promoting tumor growth and immune evasion, yet also potentially executing cytotoxic and immunostimulatory functions. While numerous reviews have summarized the tumor-promoting and tumor-suppressive functions of TANs, relatively few have examined how different cancer therapies modulate neutrophil phenotypes and functions, and what consequences these changes hold for therapeutic efficacy and resistance. The following sections review how neutrophil dynamics change in response to major cancer therapies—chemotherapy, radiotherapy (RT), cell-based immunotherapies, and oncolytic virotherapy (OVT)—integrating evidence from clinical studies on animal models. Where possible, we highlight emerging hypotheses and strategies to exploit or tame neutrophils for better therapeutic outcomes.

2. Polarization and Function Roles of TANs

TANs exhibit remarkable functional plasticity, polarizing into either an anti-tumorigenic (N1) or pro-tumorigenic (N2) phenotype in response to cues from the TME. N1 TANs display hypersegmented nuclei and mature morphology [29,30], and are typically induced by IFN-β [26], blockade of TGF-β signaling [23], or β-glucan stimulation [31]. They are associated with enhanced cytotoxic activity, immunostimulatory functions, and a shorter lifespan [32,33]. Moreover, N1 TANs upregulate inducible nitric oxide synthase (iNOS), TNF-α, intercellular adhesion molecule-1 (ICAM-1), and Fas, while expressing lower levels of ARG1 and VEGF [23,26,30]. They also exhibit increased production of ROS, nitric oxide (NO) [34], and NETs [26]. Importantly, N1 TANs enhance CD8+ T cell recruitment and activation through the secretion of chemokines such as C-C motif chemokine ligand 3 (CCL3), C-X-C motif chemokine ligands 9 and 10 (CXCL9, CXCL10) [35], as well as proinflammatory cytokines including IL-12, TNF-α, and granulocyte-macrophage colony-stimulating factor (GM-CSF), thereby promoting anti-tumor immunity (Figure 1) [23,34].

In contrast, N2 TANs possess regular or circular nuclei and are induced by a wide array of tumor- and stroma-derived factors, including TGF-β, IL-6, IL-17, CXCL2, prostaglandin E2 (PGE2), granulocyte colony-stimulating factor (G-CSF), GM-CSF, and hyaluronic acid (HA) fragments [23,29,36,37,38,39,40]. These cells are characterized by an immature morphology, extended lifespan, and an immunosuppressive phenotype [29,30]. N2 TANs exhibit high expression of ARG1, along with low levels of iNOS, TNF-α, ICAM-1, and reduced NET formation [23,26,30]. They also secrete chemokines such as C-X-C Motif Chemokine Receptor 4 (CXCR4), CCL2, and CCL5, as well as VEGF and S100 calcium-binding proteins A8/A9 (S100A8/A9), which facilitate tumor progression, angiogenesis, and the recruitment of other immunosuppressive cells [23,29,41,42]. As aforementioned, Type I interferons, particularly IFN-β, are critical regulators of N1 polarization [26]. Mice deficient in IFN-β develop predominantly N2-like TANs, whereas melanoma patients treated with type I IFN therapy exhibit a shift toward N1-like neutrophil profiles, underscoring the relevance of these mechanisms in both murine models and human cancer (Figure 1) [26].

2.1. Anti-Tumor Functions of TANs

N1 TANs mediate anti-tumor effects through direct cytotoxicity, ADCC, enhancement of adaptive immunity, improved responses to immunotherapy, and NET-mediated tumor cell killing (Figure 1; Table 1) [41].

For direct cytotoxicity, N1 TANs release ROS and matrix metalloproteinases 9 (MMP-9), which degrade the epithelial basement membrane, dismantling tumor support structures [43]. Particularly, hydrogen peroxide (H₂O₂) secreted by TANs induces tumor apoptosis by triggering Ca²⁺ influx via the TRPM2 channel (transient receptor potential cation channel, subfamily M, member 2) [44]. Moreover, β-glucan–trained neutrophils produce elevated ROS for enhanced cytotoxic activity [31], and NE cleaves the CD95 death domain to selectively kill tumor cells [45]. MET-activated neutrophils are stimulated by TNF-α and its ligand hepatocyte growth factor (HGF). These cells generate NO via iNOS, which suppresses tumor growth and metastasis [46]. Additionally, N1 TANs express death ligands TRAIL and FasL, whose expression can be enhanced by IL-17 to promote apoptosis in tumor cells [20,40,47].

In the context of ADCC, Fc receptors on neutrophils bind the Fc region of tumor-targeting monoclonal antibodies (mAb), initiating antibody-mediated cytotoxicity against tumor cells [48]. Neutrophils also facilitate adaptive immune responses by enhancing CD8⁺ T cell activity via NE [45], cooperating with T cells through iNOS-mediated pathways [49], and differentiating into antigen-presenting neutrophils under the influence of IFN-γ and GM-CSF. These antigen-presenting neutrophils can capture tumor antigens, migrate to tumor-draining lymph nodes, and present antigens to activate T cells, initiating immune responses [50,51].

N1 TANs also enhance responses to immunotherapy through cytokine feedback loops. IL-12 from dendritic cells and macrophages activates T cells to secrete IFN-γ, which induces interferon regulatory factor (IRF1) in neutrophils, boosting their anti-tumor activity and reinforcing IL-12 production by macrophages [52,53]. Additionally, NETs immobilize circulating tumor cells (CTCs) via β1-integrin interactions and deliver cytotoxic proteins such as myeloperoxidase (MPO), NE, and MMPs to disrupt tumor membranes and prevent metastasis [54].

Table 1. Functional Mechanisms of N1 and N2 Neutrophils in the TME.

Neutrophil Phenotype	Function/ Tumor State Categories	Effector	Mechanism	References
N1	Direct Cytotoxicity	ROS, MMP9	TANs release ROS and MMP-9, degrading the epithelial basement membrane and inducing apoptosis via H2O2-triggered Ca2+ influx via TRPM2 channels.	[31,43,44]
		NE	NE secreted by TANs cleaves CD95 death domain, selectively killing tumor cells.	[45]
		NO	HGF and TNF-α activate MET. MET-activated TANs produce NO to inhibit tumor proliferation and metastasis.	[46]
		Death Ligands (TRAIL, FasL)	TANs expressing TRAIL and FasL induce apoptosis in tumor cells via death receptor signaling, enhanced by IL-17.	[20,40,47]
	ADCC	FcRs	TANs bind the Fc region of mAbs via Fc receptors (FcRs), triggering ADCC-mediated killing	[48]
	Enhancing Adaptive Immunity	NE	NE enhances activation of CD8+ T cells at distant sites.	[45]
		T cell cooperation, iNOS	T cells detect tumor antigens and activate neutrophils to eliminate tumor escape via iNOS.	[49]
		Antigen presentation	Immature neutrophils differentiate into antigen-presenting TANs under IFN-γ and GM-CSF, capturing tumor antigen, migrating to lymph nodes, and activating T cells.	[50,51]
	Immunotherapy Response	Cytokine feedback loop	IL-12 released by Dendritic cells and macrophages stimulates T cells to produce IFN-γ, which activates IRF1 in neutrophils and thus amplifies antitumor activity and feedback to macrophages and T cells.	[52,53]
	NET-Mediated Tumor killing	NETs	NETs trap CTCs via β1-integrin interactions, limiting metastatic spread. NETs also carry cytotoxic proteins including NE and MPO that can damage tumor cells.	[55]
N2	Tumor Initiation	Genetic instability caused by NO, ROS, and Oncogenic miRNAs (miR-23a & miR-155)	Chronic NO/ROS cause DNA damage. Neutrophil-derived vesicles deliver miR-23a and miR-155, inducing DNA double-strand breaks and promoting carcinogenesis.	[56,57,58]
	Tumor Proliferation	NE	NE degrades IRS-1, increasing PI3K-PDGFR interaction thus promoting cell proliferation.	[59]
		PGE2	Neutrophil-secreted PGE2 promotes RAS-driven proliferation	[60]
		Neutrophil senescence, APOE-TREM2 interaction, SASP, IL-1RA	APOE produced by tumor cells binds to TREM2, activating the downstream DAP12/SYK pathway and promoting neutrophil senescence. These senescent neutrophils adopt the SASP phenotype, secreting pro-inflammatory cytokines as well as IL-1RA, thus promoting tumor proliferation.	[61,62,63]
		NET	NETosis is triggered by tumor-released IL-8, RAGE ligands, and Amyloid β and can promote tumor cell proliferation via the NF-κB signaling pathway.	[64,65,66,67]
	Tumor Angiogenesis	Proangiogenic factors (Bv8/Prok2, VEGF, MMP-9, OSM, FGF2)	Neutrophils release VEGF, Bv8/Prok2, and FGF2; MMP-9 liberates ECM-bound VEGF; OSM activates JAK–STAT to upregulate VEGF in tumors.	[68,69,70,71,72]
	Tumor Metastasis	EMT Inducers (IL-17, TGF-β, and NE)	Neutrophil-released IL-17, TGF-β, and NE induce EMT, reduce adhesion, and enhance tumor invasion.	[73,74,75]
		NETs	NETs remodel ECM via NE and MMP-9, awaken dormant tumor cells, and trap CTCs.	[65,76]
		Adhesion and Energy Transfer	Neutrophils bind to CTCs via β2 integrin–ICAM-1, protect from shear and immune attack, and transfer lipids to fuel metastasis.	[77,78,79]
	Immune Suppression	Nutrient Depletion, cytokines, PD-L1	Neutrophils consume glucose, produce lactic acid and PGE2, express PD-L1, and secrete IL-10/IL-1β, thereby suppressing T cells and promoting macrophages polarization.	[15,41,55,80,81,82,83]

Table 1. Functional Mechanisms of N1 and N2 Neutrophils in the TME.

Neutrophil Phenotype	Function/ Tumor State Categories	Effector	Mechanism	References
N1	Direct Cytotoxicity	ROS, MMP9	TANs release ROS and MMP-9, degrading the epithelial basement membrane and inducing apoptosis via H2O2-triggered Ca2+ influx via TRPM2 channels.	[31,43,44]
		NE	NE secreted by TANs cleaves CD95 death domain, selectively killing tumor cells.	[45]
		NO	HGF and TNF-α activate MET. MET-activated TANs produce NO to inhibit tumor proliferation and metastasis.	[46]
		Death Ligands (TRAIL, FasL)	TANs expressing TRAIL and FasL induce apoptosis in tumor cells via death receptor signaling, enhanced by IL-17.	[20,40,47]
	ADCC	FcRs	TANs bind the Fc region of mAbs via Fc receptors (FcRs), triggering ADCC-mediated killing	[48]
	Enhancing Adaptive Immunity	NE	NE enhances activation of CD8+ T cells at distant sites.	[45]
		T cell cooperation, iNOS	T cells detect tumor antigens and activate neutrophils to eliminate tumor escape via iNOS.	[49]
		Antigen presentation	Immature neutrophils differentiate into antigen-presenting TANs under IFN-γ and GM-CSF, capturing tumor antigen, migrating to lymph nodes, and activating T cells.	[50,51]
	Immunotherapy Response	Cytokine feedback loop	IL-12 released by Dendritic cells and macrophages stimulates T cells to produce IFN-γ, which activates IRF1 in neutrophils and thus amplifies antitumor activity and feedback to macrophages and T cells.	[52,53]
	NET-Mediated Tumor killing	NETs	NETs trap CTCs via β1-integrin interactions, limiting metastatic spread. NETs also carry cytotoxic proteins including NE and MPO that can damage tumor cells.	[55]
N2	Tumor Initiation	Genetic instability caused by NO, ROS, and Oncogenic miRNAs (miR-23a & miR-155)	Chronic NO/ROS cause DNA damage. Neutrophil-derived vesicles deliver miR-23a and miR-155, inducing DNA double-strand breaks and promoting carcinogenesis.	[56,57,58]
	Tumor Proliferation	NE	NE degrades IRS-1, increasing PI3K-PDGFR interaction thus promoting cell proliferation.	[59]
		PGE2	Neutrophil-secreted PGE2 promotes RAS-driven proliferation	[60]
		Neutrophil senescence, APOE-TREM2 interaction, SASP, IL-1RA	APOE produced by tumor cells binds to TREM2, activating the downstream DAP12/SYK pathway and promoting neutrophil senescence. These senescent neutrophils adopt the SASP phenotype, secreting pro-inflammatory cytokines as well as IL-1RA, thus promoting tumor proliferation.	[61,62,63]
		NET	NETosis is triggered by tumor-released IL-8, RAGE ligands, and Amyloid β and can promote tumor cell proliferation via the NF-κB signaling pathway.	[64,65,66,67]
	Tumor Angiogenesis	Proangiogenic factors (Bv8/Prok2, VEGF, MMP-9, OSM, FGF2)	Neutrophils release VEGF, Bv8/Prok2, and FGF2; MMP-9 liberates ECM-bound VEGF; OSM activates JAK–STAT to upregulate VEGF in tumors.	[68,69,70,71,72]
	Tumor Metastasis	EMT Inducers (IL-17, TGF-β, and NE)	Neutrophil-released IL-17, TGF-β, and NE induce EMT, reduce adhesion, and enhance tumor invasion.	[73,74,75]
		NETs	NETs remodel ECM via NE and MMP-9, awaken dormant tumor cells, and trap CTCs.	[65,76]
		Adhesion and Energy Transfer	Neutrophils bind to CTCs via β2 integrin–ICAM-1, protect from shear and immune attack, and transfer lipids to fuel metastasis.	[77,78,79]
	Immune Suppression	Nutrient Depletion, cytokines, PD-L1	Neutrophils consume glucose, produce lactic acid and PGE2, express PD-L1, and secrete IL-10/IL-1β, thereby suppressing T cells and promoting macrophages polarization.	[15,41,55,80,81,82,83]

2.2. Pro-Tumor Functions of TANs

N2-polarized TANs support tumor progression through multiple mechanisms, including promoting tumor initiation and proliferation, enhancing angiogenesis and metastasis, suppressing anti-tumor immunity, and facilitating therapy resistance (Figure 1; Table 1) [41].

TANs promote tumor initiation through the induction of genetic instability and a pro-inflammatory environment that favors tumor development. Particularly, TANs produce high levels of NO during chronic inflammation and induce ROS such as H₂O₂, which cause DNA damage and oxidative stress, resulting in genetic instability and promoting cancer formation in colorectal and intestinal tumor models [56,57]. Additionally, neutrophil-derived extracellular vesicles carrying oncogenic miRNAs such as miR-23a and miR-155 are delivered to epithelial cells, where they induce double-strand DNA breaks and contribute to tumor initiation [58].

TANs further contribute to tumor proliferation through the secretion of PGE2 in a zebrafish RAS-driven tumor model [60] and through NE-mediated degradation of insulin receptor substrate-1 (IRS-1), which enhances interactions between phosphatidylinositol 3-kinase (PI3K) and the potent mitogen platelet-derived growth factor receptor (PDGFR), promoting tumor cell proliferation [59]. In prostate cancer, tumor-derived apolipoprotein E (APOE) binds to triggering receptor expressed on myeloid cells 2 (TREM2) on neutrophils, activating DAP12/SYK signaling. This pathway promotes neutrophil senescence characterized by the senescence-associated secretory phenotype (SASP), which suppresses NK cells and CD8+ T cells and drives chronic inflammation and tumor proliferation [61,62,63]. In a murine and human breast cancer model, senescent TANs promote therapy resistance by secreting SASP-associated exosomes containing piRNA-17560, regulated through the STAT3 signaling pathway [84]. TANs also release interleukin-1 receptor antagonists (IL-1RA) to shield tumor cells from entering senescence, sustaining tumor proliferation [62]. Moreover, tumor-derived IL-8, RAGE ligands, and amyloid β induce NET formation via ROS and inflammatory signaling, promoting tumor proliferation through the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [64,65,85].

In addition to supporting tumor growth, TANs contribute significantly to tumor angiogenesis by releasing proangiogenic factors such as VEGF, prokineticin-2 (Bv8/Prok2), and fibroblast growth factor 2 (FGF2) [68,69,70]. TANs also secrete MMP-9, which liberates VEGF from the (extracellular matrix) ECM, boosting angiogenesis [71]. In breast cancer, TAN-derived oncostatin M (OSM) activates the JAK-STAT pathway in tumor cells, promoting VEGF expression and angiogenesis [72].

For tumor metastasis, TANs facilitate epithelial–mesenchymal transition (EMT) via IL-17, TGF-β, and NE [73,74,75], decreasing cell adhesion and enhancing tumor cell motility. In breast, pancreatic, and melanoma cancer, chronic inflammation and cancer-associated fibroblasts (CAFs) can trigger NET formation. The NET-associated enzymes NE and MMP-9 remodel laminin, reactivating dormant tumor cells and promoting metastasis [65,66,76]. TANs also act as adhesion partners and escort CTCs to distant sites by forming β2 integrin–ICAM-1 bonds that protect CTCs from shear stress and immune clearance [77,78]. Additionally, TANs accumulate lipids due to suppressed activity of adipose triglyceride lipase (ATGL) in neutrophils, which are later delivered to metastatic tumor cells, supporting energy metabolism and outgrowth in the lung niche [79].

Finally, TANs mediate immune evasion by depleting nutrients like glucose [80], releasing immunosuppressive metabolites such as lactic acid [81] and PGE2 [82], upregulating PD-L1 in response to tumor-derived signals such as TNF-α, GM-CSF, high mobility group box 1 (HMGB1), IL-6, and CCL20 [86,87,88,89,90,91,92], and inducing immunosuppressive cytokines including IL-10 and interleukin-1 beta (IL-1β) [15,63,83].

3. Neutrophils in Chemotherapy

Chemotherapy remains a foundational strategy in cancer treatment, primarily functioning by disrupting DNA replication, mitosis, and metabolic pathways essential for tumor cell survival and proliferation [93,94]. However, accumulating evidence suggests that its impact extends beyond direct tumor cytotoxicity. Many chemotherapeutic agents inflict collateral damage on surrounding stromal and immune cells within the TME, resulting in tissue injury and initiating a form of non-infectious, therapy-induced inflammation known as sterile inflammation [95]. This inflammatory response is characterized by the release of damage-associated molecular patterns (DAMPs) from dying or stressed cells, including molecules like HMGB1, adenosine triphosphate (ATP), and calreticulin, alongside the production of pro-inflammatory cytokines such as IL-1β and TNF-α, and chemokines including CXCL1, CXCL2, and CXCL8/IL-8 [95,96,97,98,99]. These inflammatory mediators serve as chemoattractants that recruit innate immune cells, particularly neutrophils. Although traditionally regarded as passive bystanders in cancer therapy, neutrophils are now recognized as dynamic responders to chemotherapy-induced inflammation [100]. Within TME, TANs undergo context-dependent phenotypic programming shaped by cytokine gradients, tumor-derived signals, or the specific chemotherapeutic agents administered. This plasticity enables neutrophils to adopt a functional spectrum ranging from anti-tumorigenic N1-like to pro-tumorigenic N2-like phenotypes (Figure 2; Table 2) [23,32,101].

Table 2. Chemotherapy-induced mechanisms of neutrophil polarization in the TME.

Chemo Agent/ Effector	Tumor Model (Species)	Polarization Mechanism	Phenotype	References
Oxaliplatin + Lipid A (OM-174)	Colorectal tumor; PROb (Rat), CT26 (mouse)	Oxaliplatin induces SASP and chemokines (CXCL1/2/8). Lipid A, a TLR4 agonist, promotes iNOS expression and N1 polarization, leading to over 95% tumor regression.	N1	[102,103]
Cisplatin	NSCLC (A549, human)	Cisplatin-induced ferroptosis in tumor cells triggers the release of CXCL1/2 and DAMPs, recruiting neutrophils and polarizing TANs to N1 characterized by the upregulation of TNF-α, granzyme B, and NE. N1 TANs enhance T cell infiltration, CD4+ T cell differentiation, and CD8+ T cell activation and migration.	N1	[45,104,105,106]
CB-839 + 5-FU/Capecitabine	PIK3CA-mutant CRC (mouse, human clinical phase II trial)	CB-839 and 5-FU/Capecitabine (oral form of 5-FU) combination treatment upregulate IL-8/CXCL5 (human/mice), leading to neutrophil recruitment. This increases ROS and induces NET formation, releasing CTSG that promotes tumor apoptosis via BAX activation.	N1	[107]
DNA damage (cGAS-STING)	Murine tumors, melanoma patients (human)	Chemotherapy-induced DNA damage activates the cGAS-STING pathway and IFN-β signaling, promoting N1 polarization and enhancing cytotoxicity in tumors.	N1	[26,41,108,109]
5-FU	4T1 (mouse, TNBC lung metastasis)	5-FU induces ROS and activates NF-κB, upregulating CXCL1/2 thereby recruiting TANs that express Prok2. Promotes angiogenesis and metastasis.	N2	[110]
Docetaxel + Carboplatin (TCb)	Human and mouse breast cancer	Docetaxel and TCb combination therapy upregulates the Slc11a1 gene in neutrophils, releases Fe2+ and ROS, promoting NET formation, which damages endothelium and supports metastasis.	N2	[111]
Doxorubucin	Breast cancer MCF-7, MDA-MB-231 (human); xenograft (mouse)	Doxorubicin induces neutrophil senescence and exosome release via STAT3. Exosomal piR-17560 stabilized FTO and upregulated ZEB1 in tumor cells, promoting EMT and chemoresistance.	N2	[84]
G-CSF (post-chemo)	4T1 (mouse), human lung metastasis	G-CSF restores neutrophils but primes them for NET release and N2 polarization. This promotes metastasis in both human and mouse models.	N2	[112,113,114,115,116]

Table 2. Chemotherapy-induced mechanisms of neutrophil polarization in the TME.

Chemo Agent/ Effector	Tumor Model (Species)	Polarization Mechanism	Phenotype	References
Oxaliplatin + Lipid A (OM-174)	Colorectal tumor; PROb (Rat), CT26 (mouse)	Oxaliplatin induces SASP and chemokines (CXCL1/2/8). Lipid A, a TLR4 agonist, promotes iNOS expression and N1 polarization, leading to over 95% tumor regression.	N1	[102,103]
Cisplatin	NSCLC (A549, human)	Cisplatin-induced ferroptosis in tumor cells triggers the release of CXCL1/2 and DAMPs, recruiting neutrophils and polarizing TANs to N1 characterized by the upregulation of TNF-α, granzyme B, and NE. N1 TANs enhance T cell infiltration, CD4+ T cell differentiation, and CD8+ T cell activation and migration.	N1	[45,104,105,106]
CB-839 + 5-FU/Capecitabine	PIK3CA-mutant CRC (mouse, human clinical phase II trial)	CB-839 and 5-FU/Capecitabine (oral form of 5-FU) combination treatment upregulate IL-8/CXCL5 (human/mice), leading to neutrophil recruitment. This increases ROS and induces NET formation, releasing CTSG that promotes tumor apoptosis via BAX activation.	N1	[107]
DNA damage (cGAS-STING)	Murine tumors, melanoma patients (human)	Chemotherapy-induced DNA damage activates the cGAS-STING pathway and IFN-β signaling, promoting N1 polarization and enhancing cytotoxicity in tumors.	N1	[26,41,108,109]
5-FU	4T1 (mouse, TNBC lung metastasis)	5-FU induces ROS and activates NF-κB, upregulating CXCL1/2 thereby recruiting TANs that express Prok2. Promotes angiogenesis and metastasis.	N2	[110]
Docetaxel + Carboplatin (TCb)	Human and mouse breast cancer	Docetaxel and TCb combination therapy upregulates the Slc11a1 gene in neutrophils, releases Fe2+ and ROS, promoting NET formation, which damages endothelium and supports metastasis.	N2	[111]
Doxorubucin	Breast cancer MCF-7, MDA-MB-231 (human); xenograft (mouse)	Doxorubicin induces neutrophil senescence and exosome release via STAT3. Exosomal piR-17560 stabilized FTO and upregulated ZEB1 in tumor cells, promoting EMT and chemoresistance.	N2	[84]
G-CSF (post-chemo)	4T1 (mouse), human lung metastasis	G-CSF restores neutrophils but primes them for NET release and N2 polarization. This promotes metastasis in both human and mouse models.	N2	[112,113,114,115,116]

3.1. Chemotherapy-Induced Polarization Toward Antitumor N1 Neutrophils

Several chemotherapeutic agents have been reported to promote N1-like polarization. For instance, in murine colorectal tumor models (rat PROb and mouse CT26), oxaliplatin induces tumor cell senescence characterized by the SASP. This pro-inflammatory state involves elevated mRNA expression of IL-6, IL-8, and MMP-3; increased protein levels of IFN-γ, IL-1β, and TNF-α; and upregulation of neutrophil-attracting chemokines such as CXCL1, CXCL2, and IL-8/CXCL8. These chemokines facilitate neutrophil recruitment into the TME. Upon administration of lipid A (OM-174), a Toll-like receptor 4 (TLR4) agonist, TANs are reprogrammed toward an N1-like phenotype, marked by iNOS activation [102]. These N1 neutrophils enhance local cytotoxic activity, and the combination therapy leads to complete tumor regression in over 95% of treated animals [103].

Similarly, in non-small cell lung cancer (NSCLC) models, cisplatin triggers ferroptosis in tumor cells (A549 human lung cancer cells), as evidenced by lipid peroxidation. This process induces the upregulation of chemokines CXCL1 and CXCL2, which recruit TANs into the TME. Cisplatin-triggered ferroptotic tumor cells reprogram TANs toward an N1-like phenotype, characterized by the upregulation of cytotoxic effectors such as TNF-α, granzyme B, and NE, as well as chemokines and cytokines including CCL2, CCL3, CXCL9, CXCL10, CXCL11, IL-12A, and IL-12B. These N1 TANs exert antitumor effects not only through direct tumor cytotoxicity but also by enhancing immune responses. Specifically, N1 TANs promote CD8⁺ T cell activation and migration through upregulation of T cell–recruiting chemokines (CXCL9/10/11) and facilitate Th1 differentiation of CD4⁺ T cells via IL-12A and IL-12B signaling. Co-culture experiments reveal that CD4⁺ T cells exposed to these TANs exhibit elevated mRNA expression of IFNA2, IFNB, IFNG, TNF-β, IL-2, and CCR5, along with increased IFN-γ secretion, collectively supporting a robust Th1-skewed immune response [45,104,105,106].

Furthermore, the combination of CB-839, a glutaminase inhibitor, and 5-fluorouracil (5-FU)—or its oral prodrug capecitabine used in a Phase II clinical trial—has been shown to suppress the growth of PIK3CA-mutant colorectal cancers (CRCs) in both murine models and metastatic CRC patients. This chemotherapy regimen induces an N1-like neutrophil response by upregulating IL-8 (human) or CXCL5 (mouse), thereby promoting neutrophil recruitment into the TME. The drug combination increases ROS in neutrophils, triggering NET formation (NETosis) and the release of cathepsin G (CTSG), which enters tumor cells via RAGE, cleaves 14-3-3ε, and activates BAX-dependent mitochondrial apoptosis [117].

Moreover, chemotherapy-induced DNA damage can activate the cyclic GMP-AMP synthase–stimulator of interferon genes (cGAS–STING) pathway by generating cytosolic double-stranded DNA fragments, which trigger a signaling cascade through TANK-binding kinase 1 (TBK1) and IRF3 that results in the transcription and secretion of IFN-β [108]. Type I IFNs, particularly IFN-β, promote neutrophil polarization toward an N1 phenotype, enhancing their antitumor activity in both murine models and human patients [41,109]. In mice, IFN-β deficiency results in N2-skewed TANs and accelerated tumor growth. In melanoma patients treated with type I IFN therapy, neutrophils exhibit increased N1 markers, reduced chemokine receptor expression, and enhanced cytotoxic features, supporting the conserved role of IFN-β in driving N1 polarization across species [26].

3.2. Chemotherapy-Induced Polarization Toward Antitumor N2 Neutrophils

On the other hand, some chemotherapeutic agents can paradoxically promote N2-like polarization. In a mouse triple-negative breast cancer (4T1) lung metastasis model, 5-FU elevated ROS in tumor cells, activating NF-κB signaling and upregulating CXCL1 and CXCL2 to recruit neutrophils. These N2-like TANs expressed Prok2, which binds Prokr1 on 4T1 tumor cells, supporting metastatic outgrowth and angiogenesis [110].

Additionally, in a neoadjuvant breast cancer model involving both human and mouse systems, combination therapy with docetaxel and carboplatin (TCb) was shown to induce NET formation through upregulation of the Slc11a1 gene in neutrophils. Slc11a1 encodes NRAMP1, a transporter responsible for increasing ferrous iron (Fe²⁺) influx into neutrophils, which in turn enhances intracellular ROS production and promotes NET release. These chemotherapy-induced NETs contribute to vascular endothelial injury, as evidenced by elevated levels of endothelial damage markers such as Syndecan-4 and von Willebrand Factor (vWF), decreased expression of VE-cadherin and CD31, and increased expression of the pro-apoptotic marker Bax in human umbilical vein endothelial cells (HUVECs). Together, these findings suggest a polarization toward an N2-like neutrophil phenotype, characterized by pro-metastatic and tissue-damaging activities [111].

Similarly, oxaliplatin was shown to robustly induce NET formation by activating neutrophils through elevated ROS, MPO, and Peptidyl Arginine Deiminase 4 (PAD4) signaling pathways. This process is initiated by oxaliplatin-induced damage to the gut lining, which allows bacterial lipopolysaccharide (LPS) to leak into the bloodstream, further stimulating neutrophil activation. The resulting NETosis contributes to microcirculatory disruption and tissue hypoxia [118].

Recent studies have identified senescent neutrophils as key contributors to chemoresistance in breast cancer. In response to doxorubicin, neutrophils undergo senescence—evidenced by the upregulation of p16 ^INK4A and elevated expression of SASP factors such as colony stimulating factors 3 (CSF3), CCL3, CXCL8, and IL-1α. Senescent neutrophils exhibit enhanced exosome secretion. Activated STAT3 signaling in these neutrophils drives the packaging of PIWI-interacting RNA piR-17560 into exosomes. Once transferred to breast cancer cells, piR-17560 stabilizes FTO mRNA, increasing FTO protein levels and enabling m6A demethylation of ZEB1 mRNA, which prevents its degradation by the m6A reader YTHDF2. This results in increased ZEB1 expression, which promotes EMT, characterized by downregulation of E-cadherin and upregulation of Vimentin, ultimately enhancing tumor cell invasiveness and resistance to docetaxel [84].

Beyond its direct cytotoxic effects, chemotherapy can also influence neutrophil behavior through downstream consequences such as treatment-induced neutropenia, which is defined as a significant reduction in circulating neutrophils due to bone marrow suppression [119]. This condition increases susceptibility to opportunistic infections and often necessitates dose delays or reductions [120]. To counteract this, G-CSF is commonly administered to stimulate neutrophil production and restore immune competence [112,113,114]. However, emerging evidence suggests that G-CSF not only increases neutrophil counts but also promotes their polarization toward an N2-like, pro-tumorigenic phenotype characterized by enhanced NET formation. In the 4T1 murine breast cancer model, tumor-derived G-CSF was shown to prime neutrophils for NET release, which could be reversed by anti-G-CSF treatment. Moreover, neutrophils from healthy mice also became NET-prone upon treatment with recombinant G-CSF, indicating a direct role for G-CSF in driving this phenotype [115]. A subsequent study extended these findings to humans, reporting elevated NET levels in lung metastases compared to primary breast tumors and confirming that recombinant G-CSF could induce NET formation in human neutrophils ex vivo, further supporting its role in promoting pro-metastatic neutrophil behavior [116].

3.3. Therapeutic Strategies Targeting N2 Neutrophils to Enhance Chemotherapy Response

The recognition of neutrophils’ pro-tumorigenic roles in chemotherapy has spurred the development of several therapeutic strategies aimed at reprogramming or inhibiting N2-like phenotypes. One effective approach targets NET formation. PAD4 inhibitors, such as Cl-amidine and GSK484, inhibit chromatin citrullination, thereby reducing NET production, metastatic potential, and enhancing chemotherapy responsiveness in preclinical models [116,121]. Likewise, Deoxyribonuclease 1 (DNase1) has been shown to safely degrade NETs across various murine models, including breast cancer, pulmonary inflammation, and autoimmune diseases, with no significant toxicity reported in non-cancer clinical use [122]. Another strategy is the use of combination chemo–immunotherapy regimens, which have shown potential to reduce N2-associated TANs while enhancing antitumor immune responses. For example, in colorectal carcinoma models, combining oxaliplatin with ATR inhibition and anti-PD-1 therapy significantly decreased neutrophil infiltration and facilitated the expansion of stem-like, IFN-γ–producing CD8⁺ T cells, resulting in complete tumor regression [123]. Together, these findings reinforce the therapeutic promise of targeting neutrophil plasticity to enhance chemotherapy efficacy and limit tumor progression.

4. Neutrophils in Radiotherapy (RT)

RT is another fundamental treatment modality in oncology that employs ionizing radiation, such as high-energy X-rays or subatomic particles, to destroy cancer cells. This is primarily achieved by inducing double-strand DNA breaks, thereby disrupting the cell cycle and leading to mitotic catastrophe, a form of cell death caused by irreparable division failure [124,125,126]. Although normal tissues are also exposed, their greater capacity for DNA repair allows selective survival when treatment is fractionated over time [127]. Depending on the clinical scenario, RT may be applied externally (external beam radiation) or internally (brachytherapy), either as a standalone therapy or in combination with surgery, chemotherapy, or immunotherapy [128,129]. RT triggers a robust cytokine and chemokine cascade within the TME that plays a central role in neutrophil recruitment. Across multiple preclinical tumor models, RT has been shown to increase the expression of cytokines such as IL-1β, GM-CSF (CSF2), and G-CSF (CSF3), along with chemokines including CXCL1, CXCL2, CXCL5, CCL2, and CCL5 [130]. IL-1β can further amplify this response by upregulating CXCL chemokines, thereby enhancing neutrophil-attracting signals. In parallel, RT elevates the expression of chemokine receptors CXCR2 and CXCR4 on neutrophils, which bind to CXCL1 and CXCL2, promoting their directed migration into the irradiated TME [130,131,132,133,134,135]. Beyond the local tumor site, RT can also alter systemic cytokine levels, supporting neutrophil mobilization and expansion in the circulation. Clinical observations align with these findings; in patients with pancreatic cancer, elevated levels of CCL2, CCL4, TGF-β, and VEGF were detected in the blood after RT, suggesting that radiation not only reshapes the TME but also exerts systemic immunomodulatory effects that may influence neutrophil behavior [136] (Figure 2; Table 3).

Table 3. Radiotherapy-induced mechanisms of neutrophil polarization in the TME.

Tumor Model (Species)	Polarization Mechanism	Phenotype	References
LLC model (Mouse)	RT-induced DNA damage increases CXCL1, CXCL2, and CCL5 expression, recruiting ROS-producing neutrophils. G-CSF also enhances neutrophil recruitment. ROS generation in combination with RT suppresses PI3K/Akt/Snail signaling, inhibiting EMT and promoting MET.	N1	[137]
RM-9 prostate, EG7 thymoma, 4T1 breast (Mouse)	RT rapidly recruits CD11b+Gr-1 high+ neutrophils, which produce ROS that triggers tumor apoptosis and initiate sterile inflammation, enhancing CTL activation.	N1	[75]
In vitro (Human or Mouse); thymoma, breast, prostate, pancreatic (Mouse)	Higher RT doses enhance neutrophil ROS production, contributing to tumor regression; ROS inhibition reduces the antitumor effect.	N1	[138,139,140]
MC38 colorectal and RM-9 prostate (Mouse)	RT activates cGAS and AIM2 pathways, increasing IL-1β expression, which in turn elevates CXCL chemokines and drives neutrophil infiltration.	N1	[131]
Lung tissue pre-metastatic niche with breast cancer cells (Mouse)	RT recruits activated neutrophils to irradiated lung tissue, which promotes Notch–Sox9 signaling in infiltrating cancer cells, inducing stem-like, pro-metastatic traits.	N2	[141]
Cervical cancer (Human)	High peripheral neutrophil counts during treatment correlate with poor local control and survival, suggesting a protumorigenic role for TANs in clinical settings.	N2	[142]
Bladder cancer (Mouse and Human)	RT induces robust NET formation that physically blocks CD8+ T cells from accessing tumor cells and impairs cytotoxicity, contributing to immune evasion and treatment resistance.	N2	[143]
In vitro colon carcinoma spheroids (Human)	Low RT dose (e.g., 0.25 Gy) stimulates NET formation that restricts immune cell-mediated tumor killing.	N2	[144]
Prostate and pancreatic cancer (Mouse); Rectal cancer (Human)	RT increases expression of IDO1 and ARG1 in TANs, which deplete tryptophan and L-arginine, suppressing CD8+ T cells and NK cell functions and weakening antitumor immunity.	N2	[136,145,146]

Table 3. Radiotherapy-induced mechanisms of neutrophil polarization in the TME.

Tumor Model (Species)	Polarization Mechanism	Phenotype	References
LLC model (Mouse)	RT-induced DNA damage increases CXCL1, CXCL2, and CCL5 expression, recruiting ROS-producing neutrophils. G-CSF also enhances neutrophil recruitment. ROS generation in combination with RT suppresses PI3K/Akt/Snail signaling, inhibiting EMT and promoting MET.	N1	[137]
RM-9 prostate, EG7 thymoma, 4T1 breast (Mouse)	RT rapidly recruits CD11b+Gr-1 high+ neutrophils, which produce ROS that triggers tumor apoptosis and initiate sterile inflammation, enhancing CTL activation.	N1	[75]
In vitro (Human or Mouse); thymoma, breast, prostate, pancreatic (Mouse)	Higher RT doses enhance neutrophil ROS production, contributing to tumor regression; ROS inhibition reduces the antitumor effect.	N1	[138,139,140]
MC38 colorectal and RM-9 prostate (Mouse)	RT activates cGAS and AIM2 pathways, increasing IL-1β expression, which in turn elevates CXCL chemokines and drives neutrophil infiltration.	N1	[131]
Lung tissue pre-metastatic niche with breast cancer cells (Mouse)	RT recruits activated neutrophils to irradiated lung tissue, which promotes Notch–Sox9 signaling in infiltrating cancer cells, inducing stem-like, pro-metastatic traits.	N2	[141]
Cervical cancer (Human)	High peripheral neutrophil counts during treatment correlate with poor local control and survival, suggesting a protumorigenic role for TANs in clinical settings.	N2	[142]
Bladder cancer (Mouse and Human)	RT induces robust NET formation that physically blocks CD8+ T cells from accessing tumor cells and impairs cytotoxicity, contributing to immune evasion and treatment resistance.	N2	[143]
In vitro colon carcinoma spheroids (Human)	Low RT dose (e.g., 0.25 Gy) stimulates NET formation that restricts immune cell-mediated tumor killing.	N2	[144]
Prostate and pancreatic cancer (Mouse); Rectal cancer (Human)	RT increases expression of IDO1 and ARG1 in TANs, which deplete tryptophan and L-arginine, suppressing CD8+ T cells and NK cell functions and weakening antitumor immunity.	N2	[136,145,146]

4.1. RT-Induced Polarization Toward Antitumor N1 Neutrophils

RT has been shown to actively reprogram TANs toward an antitumor, N1-like phenotype through multiple converging mechanisms involving DNA damage response, inflammatory signaling, and ROS production [130,147]. In mice bearing Lewis lung carcinoma (LLC), hypofractionated RT (8 Gy × 3) induced persistent DNA double-strand breaks, upregulated chemokines such as CXCL1, CXCL2, and CCL5, and recruited newly infiltrating neutrophils into the TME [137]. These RT-recruited TANs generated elevated levels of ROS, which suppressed the PI3K/Akt/Snail signaling cascade, thereby inhibiting EMT and promoting mesenchymal–epithelial transition (MET). This effect sensitized tumors to radiation and constrained progression. Co-administration of G-CSF further amplified this N1 phenotype by enhancing neutrophil recruitment, ROS generation, and MET induction [137]. Similarly, in syngeneic mouse models including RM-9 prostate cancer, EG7 thymoma, and 4T1 breast cancer, a single high dose of RT (15 Gy) caused rapid infiltration of CD11b⁺Gr-1 ^high+ neutrophils within 24 h. These TANs generated high levels of ROS, induced tumor cell apoptosis, and initiated sterile inflammation that facilitated activation of tumor-specific CTLs. G-CSF synergized with RT to further strengthen these effects [138]. In syngeneic murine colorectal (MC38) and prostate (RM-9) tumor models, RT activated the cGAS and AIM2 DNA-sensing pathways, leading to IL-1β upregulation and CXCL chemokine-mediated neutrophil recruitment [131]. Additionally, murine models of thymoma, breast, prostate, and pancreatic cancers demonstrated that RT significantly increased neutrophil-derived ROS within tumors, contributing to tumor regression in a dose-dependent manner [138,139,140]. Notably, in vitro studies revealed that single RT doses of 6, 12, or 18 Gy enhanced neutrophil ROS release, while fractionated RT reduced ROS production compared to unirradiated controls [148].

4.2. RT-Induced Polarization Toward Antitumor N2 Neutrophils

In contrast to its capacity for promoting N1 phenotypes, RT can also skew neutrophils toward a tumor-promoting, N2-like state, facilitating metastasis and radioresistance through diverse mechanisms [149]. In a thoracic RT model, Nolan et al. demonstrated that irradiation of healthy lung tissue led to the recruitment of activated neutrophils, which induced Notch–Sox9 signaling in infiltrating breast cancer cells, promoting stem-like and metastatic traits. Inhibiting neutrophil degranulation reversed this effect, implicating TAN-derived factors in the formation of pro-metastatic niches [141]. Clinically, elevated neutrophil levels have been linked to poor outcomes following RT. In cervical cancer patients treated with chemoradiotherapy, high peripheral neutrophil counts during treatment were associated with worse local control and survival. Using a genetically engineered mouse model of autochthonous sarcoma, Wisdom et al. showed that both genetic and antibody-mediated neutrophil depletion improved tumor sensitivity to image-guided focal irradiation, with corresponding downregulation of Mitogen-Activated Protein Kinase (MAPK) pathway activity. These findings support a functional role for neutrophils in promoting tumor resistance to radiation [142]. At the mechanistic level, NETs have emerged as key mediators of N2-like functions in irradiated tumors. Shinde-Jadhav et al. reported that RT induces NET formation in murine bladder tumors, particularly at high or fractionated doses. These fibrous traps impede CD8⁺ T cell infiltration and cytotoxic activity. Disruption of NETs via PAD4 knockout, DNase I, or NE inhibition significantly improved therapeutic efficacy and survival [143]. NETosis was also observed in vitro at RT doses as low as 0.25 Gy and increased with dose escalation [144]. In vivo, colon and bladder tumors exhibited NET-mediated immune exclusion, while bladder cancer patients with residual disease post-RT showed elevated NET levels and worse outcomes [143,144]. In parallel, RT upregulates immunosuppressive enzymes such as indoleamine 2,3-dioxygenase 1 (IDO1) and ARG1 in TANs, which deplete tryptophan and L-arginine—amino acids essential for CD8⁺ T and NK cell function. This phenomenon was demonstrated in murine prostate and pancreatic tumor models [136,145] and in rectal cancer patients where circulating neutrophils exhibited high ARG1 expression [146]. IDO1 also expands regulatory T cells, suppresses NK cell activity, and promotes angiogenesis [150,151,152,153]. Combining ARG1 inhibition with RT reduced TAN infiltration, restored CD8⁺ T cell presence, and delayed tumor growth [145,153].

4.3. Dose-Dependent Effects of RT on Neutrophil Function

The immunological effects of RT on neutrophils are highly dose- and context-dependent. In radiosensitive tumor models such as EG7-bearing C57BL/6 mice, a low dose of 1.3 Gy was sufficient to elicit antitumor responses via infiltration of CD11b⁺Gr-1^high⁺ neutrophils and suppression of tumor viability [138,154]. In contrast, more resistant models like 4T1 breast cancer required a higher dose (15 Gy) to achieve comparable ROS-mediated antitumor effects [138]. However, high-dose regimens (≥10 Gy) can also trigger NETosis, which may promote tumor progression. In a bladder cancer model, 10 Gy or 2 × 5 Gy radiation significantly increased NET formation, whereas 2 Gy failed to do so. These NETs formed extracellular barriers that physically excluded CD8⁺ T cells, thereby limiting T cell infiltration and cytotoxic activity and contributing to radioresistance [143]. Meanwhile, in the Lewis lung carcinoma (LLC) model, fractionated 8 Gy × 3 RT induced CXCL1/2/5 chemokines and robust ROS production, promoting mesenchymal-to-epithelial transition (MET) and enhancing tumor radiosensitivity [137]. Collectively, these findings highlight how both RT dose intensity and tumor type influence neutrophil polarization and function, with critical implications for therapeutic efficacy [154].

5. Neutrophils in Cell-Based Therapies

Cell-based cancer immunotherapies have introduced a new method in oncology by employing immune cells to selectively target and eliminate tumor cells [28]. These strategies include well-established modalities such as adoptive T cell transfer, chimeric antigen receptor (CAR) T-cell therapy, and newer approaches involving NK cells and macrophage-based therapies [155,156,157,158]. While these treatments primarily rely on direct immune-mediated tumor cell killing, their therapeutic outcomes are significantly influenced by the surrounding TME [159,160]. Among its many components, neutrophils, particularly TANs, have emerged as important regulators that can either support or impair the effectiveness of these immunotherapies (Figure 2) [52,161].

In CAR-T cell therapy, neutrophils have been implicated in both therapeutic response and treatment-related toxicities [162,163,164,165]. Several studies have reported that activated neutrophils, and particularly NETosis, are involved in the pathogenesis of cytokine release syndrome (CRS), an acute systemic inflammatory response triggered by activated CAR-T cells [162,163,164]. For instance, longitudinal plasma proteomics from patients treated with CAR-T cells revealed a temporal association between markers of neutrophil activation, NET formation, and the onset of CRS [165]. Clinical data further show that absolute neutrophil count (ANC) at baseline and during therapy correlates with CAR-T cell expansion kinetics and the likelihood of CRS, suggesting that neutrophils may serve as early biomarkers of treatment dynamics [162].

Moreover, neutrophil phenotypes appear to shape clinical outcomes. One study found that elevated frequencies of CD10⁻ immature neutrophils were associated with poor responses and inferior survival in patients receiving CD19-directed CAR-T therapy for B-cell acute lymphoblastic leukemia [166]. These immature neutrophils may contribute to an immunosuppressive environment that impairs CAR-T function. Notably, a recent single-cell study of ciltacabtagene autoleucel–treated myeloma patients demonstrated that CRS is accompanied by a wave of neutrophil activation that precedes clonal CAR-T cell re-expansion, suggesting a functional link between innate and adaptive cellular dynamics during therapy [165].

Beyond acting as modulators of other immune cell therapies, neutrophils themselves are now being explored as direct therapeutic agents [27]. Recent advances in stem cell engineering have enabled the generation of CAR-neutrophils derived from human pluripotent stem cells (hPSCs) [167,168,169]. These engineered neutrophils exhibit potent antitumor activity against solid tumors in vivo, including glioblastoma and prostate cancer models [167,169,170]. Notably, CAR-neutrophils retain their natural tumor-homing capacity while being redirected to specifically recognize and kill tumor cells through CAR-mediated signaling. In glioblastoma models, CAR-neutrophils have also been used as delivery vehicles for TME–responsive nanodrugs, providing a combinatorial platform for chemoimmunotherapy [170].

6. Neutrophils in Oncolytic Viral (OVT) and Bacterial Therapies

OVT represents another promising class of cancer treatment that utilizes replication-competent viruses to selectively infect, lyse tumor cells, and activate systemic antitumor immunity [171]. Several oncolytic viruses, including modified herpes simplex virus (HSV), adenovirus, vaccinia virus, have been developed, with some already approved for clinical use [172,173,174]. In addition to direct tumor lysis, OVT stimulates immunogenic cell death, releases tumor antigens, and recruits immune effector cells, thereby facilitating crosstalk between innate and adaptive immune responses [175,176]. Neutrophils have recently found a critical yet underappreciated role in shaping OVT outcomes (Figure 2) [177,178].

On the one hand, neutrophils can restrict OVT effectiveness by clearing therapeutic viruses prematurely. These effects are mediated through phagocytosis, inflammatory cytokine release, and NET formation. For example, in glioma models treated with oncolytic HSV, tumor-derived G-CSF triggered neutrophil-driven NETosis, which limited viral propagation and impaired tumor control. Inhibiting this pathway improved therapeutic outcomes. [179]. Similarly, transient blockade of neutrophil activity improved systemic delivery of oncolytic vaccinia virus by reducing early viral clearance [180]. Conversely, when properly activated, neutrophils can enhance OVT by mediating direct cytotoxicity and shaping downstream immunity. In a pulmonary melanoma model, oncolytic Orf virus (ORFV)–induced neutrophils contributed to tumor regression and supported viral amplification [181]. Neutrophils have also been shown to secrete TNF-α which facilitates antigen presentation and T cell priming during OVT, thereby linking innate and adaptive immunity [181].

In addition to oncolytic viruses, the use of Clostridium novyi-NT (C. novyi-NT) has emerged as a promising modality to treat hypoxic and treatment-resistant tumors (Figure 2). C. novyi-NT is an obligate anaerobe capable of selectively germinating within hypoxic tumor cores and inducing localized tumor necrosis [182,183]. Over the past decade, our group and others have demonstrated the potent antitumor effects of C. novyi-NT across multiple models, including orthotopic glioblastomas [184,185,186,187,188]. We also highlighted C. novyi-NT as a paradigm for hypoxia-targeting bacterial cancer therapy [182]. A critical insight from our work is that host neutrophil responses significantly influence the therapeutic outcomes of oncolytic bacterial therapy. This concept was directly demonstrated in our studies. Immunofluorescence analysis revealed that Ly6G⁺ neutrophils form a localized barrier around germinating C. novyi-NT spores following intratumoral injection, physically separating the bacteria from surrounding tumor tissue (Figure 3). Notably, neutrophil depletion markedly enhanced bacterial spread and improved tumor clearance in vivo, confirming that neutrophils can act as early barriers to effective bacterial-mediated oncolysis [189].

These findings mirror the challenges faced in OVT, emphasizing a broader principle across microbial therapies: while neutrophils are essential for containing infection and regulating inflammation, they may also hinder microbial propagation within tumors. Thus, modulating neutrophil timing and function represents a promising strategy to optimize the balance between microbial replication and anti-tumor immune activation.

7. Conclusions

The past decade has revealed neutrophils to be more than supporting players of innate immunity; they are dynamic, context-dependent regulators of cancer therapy outcomes. Across multiple modalities such as chemotherapy, radiotherapy, cell-based immunotherapy, and oncolytic virotherapy, neutrophils exhibit remarkable functional plasticity. They may adopt either anti-tumor or pro-tumor phenotypes depending on tumor microenvironmental cues. N1-like neutrophils can exert cytotoxicity and promote immune activation, whereas N2-polarized subsets often contribute to tumor progression through mechanisms such as immunosuppression, angiogenesis promotion, and remodeling of the ECM [23,190].

The complexity of neutrophil behavior becomes especially apparent in the context of therapy-induced reprogramming. For instance, type I interferons released following radiotherapy have been shown to promote a tumoricidal N1 phenotype [149,191], whereas cytokines such as IL-6 and G-CSF, which are commonly upregulated in treatment-induced inflammation, tend to reinforce immunosuppressive behaviors and NET formation [85]. These observations emphasize the paradoxical nature of neutrophils: they can either enhance or hinder therapeutic efficacy depending on the timing, location, and context of the treatment. Notably, neutrophil-related effectors, such as ROS, NO, NE, and NETs, along with broader tumor-associated mechanisms such as SASP, act as double-edged swords in cancer. On one hand, ROS can directly kill tumor cells through oxidative bursts and induce apoptosis via TRPM2-mediated calcium influx; on the other, excessive ROS in the TME suppress T cell proliferation and enhance T cell apoptosis. NE similarly promotes tumor cell apoptosis via death receptor cleavage, yet also enhances tumor growth through EGFR signaling and angiogenesis. While NETs can trap and kill CTCs, they more often aid metastasis by facilitating tumor adhesion and extravasation. SASP, which may trigger immune clearance of damaged cells, paradoxically promotes tumor relapse and EMT when persistently secreted after therapy. These examples underscore the challenge of therapeutic targeting in the TME, where context determines whether these neutrophil-driven factors act as allies or adversaries.

A central conceptual challenge moving forward is reconciling the dual roles of neutrophils and the context-dependent effects of their effector functions. While the N1/N2 framework provides a useful starting point, it is increasingly evident that neutrophils span a continuum of activation states, shaped by a dynamic interplay of cytokines, cellular interactions, and therapeutic cues. The traditional binary classification into N1 and N2 is insufficient to capture this complexity. Integrating high-resolution approaches, such as single-cell transcriptomics and spatial proteomics, will be crucial for identifying discrete neutrophil subsets with distinct therapeutic relevance [192,193]. Moreover, longitudinal profiling during therapy, rather than static snapshots, may reveal how neutrophils transition between states and thereby inform the timing of interventions aimed at modulating their activity [194]. In addition to mechanistic insights, peripheral biomarkers such as the neutrophil-to-lymphocyte ratio (NLR) offer a valuable window into neutrophil dynamics during therapy. Elevated NLR has been linked to poor prognosis across multiple tumor types, including hepatocellular carcinoma, NSCLC, and lymphoma [195,196,197,198]. Although NLR does not directly reflect neutrophil phenotype (e.g., N1 vs. N2), it is increasingly recognized as a practical surrogate for systemic inflammation and immune imbalance. This underscores the clinical relevance of tracking neutrophil-related changes during therapy, and further supports the need for therapeutic strategies that consider neutrophil modulation alongside treatment selection.

Ultimately, neutrophils exemplify a broader theme in cancer immunology: innate immune cells are not static tools but responsive, context-sensitive effectors. Deploying their therapeutic potential will require embracing their complexity, accounting for their microenvironment, and intervening with both precision and restraint. The future of neutrophil-targeted cancer therapy will depend not only on better tools, but also on better questions—ones that recognize their duality, adapt to their variability, and seek to direct their power rather than modulate it. This review adopts a pan-cancer perspective, drawing from studies in both solid and hematologic malignancies to illustrate how diverse cancer therapies influence neutrophil phenotypes and functions.