1. Introduction
Of all types of malignant tumors, lung cancer has the highest morbidity and mortality rates, and its occurrence and development are closely related to the lung microenvironment [
1], [
2], [
3], [
4], [
5], [
6]. Lung cancer can be classified into two groups, small cell lung cancer (17%) and non-small cell lung cancer (NSCLC; 83%); these groups comprise several subtypes, among which adenocarcinoma is the most common, followed by squamous cell carcinoma (SqCC) and large-cell lung carcinoma, while other types are rare [
7], [
8], [
9]. Neutrophils are the most abundant immune cells in the tumor microenvironment (TME) and the earliest immune cells to respond to lung injury and inflammation [
10]. Increasing attention has been paid to the relationship between neutrophils and lung cancer development, and many studies have found that neutrophil numbers influence the prognosis of patients with lung cancer [
11], [
12]. Moreover, neutrophil abundance affects patient responses to treatment regimens [
13], [
14]. The underlying mechanisms and factors influencing neutrophils in the context of lung cancer require further exploration.
Neutrophil origin, development, and maturation are complex processes. Previous studies have divided neutrophils into N1 and N2 subtypes, and some researchers have proposed that the proportion of N1 and N2 neutrophils in the TME determines the promotion or inhibition of cancer progression by neutrophils. Neutrophil classification has changed with the development of single-cell sequencing and mass cytometry [
15], [
16], leading to a more detailed understanding of neutrophil origin, development, classification, and function.
The development of lung cancer is a continuous and dynamic process that is influenced by neutrophils in various ways. During lung cancer formation, neutrophils can promote tumor cell colonization, survival, and growth [
17], dissolve the extracellular matrix (ECM), and increase tumor cell migration and invasion ability. Moreover, neutrophils can promote cancer growth by facilitating micro-vessel formation within tumors [
18]. Neutrophil elastase (NE) and neutrophil extracellular traps (NETs) also promote the distant metastasis of lung cancer [
19], [
20]. The cytotoxicity of neutrophils is the most critical mechanism underlying their antitumor effects, and some proteases and cytokines produced by neutrophils also contribute to this process. Similarly, as a type of immune cell, neutrophils can influence the roles of other tumor cells in tumor immunity through various mechanisms [
21], [
22], [
23]. The close association of neutrophils with the process of malignant lung tumor occurrence and development has led to a research focus on relevant therapeutic measures targeting neutrophils.
The lung is an organ composed of loose, porous connective tissue with abundant blood flow, and these characteristics make it a target organ for the metastasis of other malignant tumors [
24]. Lung metastases of breast cancer, melanoma, and colon cancer have been reported; however, the role of neutrophils in the metastasis process requires further investigation and summary.
In this review, we summarize the impact of neutrophils on patient prognosis and treatment outcomes, update the origin and classification of neutrophils, and discuss the impact of neutrophils on lung cancer development and the promotion of lung metastasis of other cancers by neutrophils. Neutrophils have potential as a new target for lung cancer management and treatment in the future, and the rapid development of nanotechnology has provided new strategies for cancer treatment targeting neutrophils [
25], [
26], [
27], [
28], [
29]. Furthermore, although many treatments targeting neutrophils are currently at a theoretical stage [
30], [
31], [
32], [
33], their development has been remarkable.
2. Neutrophils in patients with lung cancer
2.1. High pretreatment neutrophil count is associated with low patient survival rate
Over recent decades, numerous studies have focused on the tumor immune microenvironment; almost all immune cells play unique roles in tumor development and metastasis [
34], [
35], [
36], [
37], [
38], [
39]. Neutrophils, which are the most abundant immune cells in the body, are a research hotspot. A strong correlation between high neutrophil levels and low survival time in patients with lung cancer has been detected by many researchers [
40], [
41], [
42], [
43], [
44], [
45] (
Table 1 [
11], [
12], [
40], [
43], [
44], [
46], [
47], [
48], [
49], [
50], [
51], [
52], [
53]). To determine the effect of pretreatment peripheral blood neutrophil, lymphocyte, and monocyte counts, as well as the neutrophil-to-lymphocyte ratio (NLR), on patient survival, a randomized controlled trial enrolled 388 chemotherapy (CT)-naïve patients with stage IIIB or IV NSCLC. The study used proportional hazards regression models to estimate hazard ratio (HR) values after adjustment for covariates. The results showed that an elevated pretreatment neutrophil count was associated with shorter overall survival (OS) and shorter progression-free survival (PFS), suggesting that an elevated pretreatment neutrophil count is an independent prognostic factor in patients with advanced lung cancer [
54]. Another retrospective study analyzed the gene-expression profiles of tumor tissue samples from 19 NSCLC cohorts, including 18 microarray datasets and one RNA-Seq dataset from The Cancer Genome Atlas lung adenocarcinoma cohort. The percentage of neutrophil infiltration was significantly higher in patients with high recurrence risk than in those with low recurrence risk (5.6% vs 1.8%), demonstrating that neutrophil infiltration is an independent risk factor in patients with NSCLC [
55].
2.2. High neutrophil count is associated with low survival following traditional therapy
In comparison with immunotherapy, which has yet to become fully established, conventional treatments, including surgery, CT, radiotherapy, and targeted therapy (TT), have gained more acceptance. TT and CT are whole-body treatment modalities for lung cancer, similar to immunotherapy, and several clinical trials have shown that high neutrophil counts are strongly associated with poor prognosis of patients undergoing these treatment approaches [
46], [
47], [
48] (
Table 1). In a brief report, five immune checkpoint inhibitor (ICI) trials and six TT trials, comprising 1368 patients treated with ICIs and 1072 patients treated with CTs in ICIs trials (total
n = 2440), were analyzed. The researchers used the derived neutrophil-to-lymphocyte ratio (dNLR), calculated as absolute neutrophil count/(white blood cell count - absolute neutrophil count), as an evaluation index. High dNLR values were associated with shorter OS (HR = 1.64,
p < 0.001) and PFS (HR = 1.26,
p = 0.008) in patients treated with ICIs and were an independent prognostic factor. For patients treated with CT, dNLR ≤ 3 was associated with a longer survival time (HR = 0.49,
p < 0.001). Multivariate analysis showed that albumin level, lactate dehydrogenase (LDH) level, and dNLR could serve as independent prognostic factors. In an analysis of six TT trials, patients with low dNLR had a median survival of 46.5 months, compared with 16.6 months for those with high dNLR treated with TTs (HR = 0.28). Furthermore, among patients receiving CT, those with a low dNLR value had more prolonged survival than those with high dNLR (estimated median survival, 33.4 vs 17.1 months; HR = 0.41), while high dNLR and LDH levels were independently associated with poor OS (TTs,
p < 0.001; CTs,
p < 0.001) and PFS (TTs,
p < 0.001; CTs,
p < 0.001) in a multivariate analysis [
49]. A total of 182 patients with NSCLC treated with CT were enrolled in another study, where the NLR was defined as the absolute neutrophil count divided by the absolute lymphocyte count. The resulting data indicated that low pretreatment NLR (≤ 2.63) (odds ratio (OR) = 2.043,
p = 0.043) and decreased post-treatment NLR (OR = 2.368,
p = 0.013) were associated with the response to CT. Pretreatment NLR was also an independent prognostic factor for PFS (HR = 1.807,
p = 0.018) and OS (HR = 1.761,
p = 0.020) [
50]. Patients needing CT or TT typically have stage III or IV disease. Hence, these results may be due to either advanced cancer, in which the baseline neutrophil count is high, or neutrophil-mediated suppression of T lymphocytes. Therefore, data from large patient cohorts—particularly those with early-stage lung cancer—are required.
Surgery and radiotherapy are local treatment methods for malignant tumors and are the first choice for early- and mid-stage lung cancer therapy. Similar conclusions have been obtained for surgery and radiotherapy. A retrospective study on 485 surgically resected patients with solitary lung SqCC found that high levels of tumor-infiltrating CD10
+ neutrophils were associated with worse patient prognosis (
p = 0.021) (
Table 1). The researchers also constructed tissue microarrays and performed immunostaining for cluster of differentation 3 (CD3), protein tyrosine phosphatase receptor type C, CD8, CD4, forkhead box P3 (FoxP3), CD20, CD68, C-X-C motif chemokine ligand 12 (CXCL12), C-X-C motif chemokine receptor 4 (CXCR4), C-C motif chemokine receptor 7 (CCR7), interleukin-7 (IL-7) receptor, and IL-12. The results of an analysis of biologically relevant immune cell combinations showed that patients with CD10
high neutrophils and CD20
low lymphocytes had significantly worse OS (5-year OS, 42%) than those with other CD10 and CD20 combinations (5-year OS, 62%;
p < 0.001). Furthermore, multivariate analysis demonstrated that the CD10
high/CD20
low immune cell combination was an independent predictor of OS in both the training (HR = 1.61,
p = 0.006) and validation (HR = 1.75,
p = 0.043) cohorts [
43].
In the field of radiotherapy, a report on 59 patients with NSCLC treated with salvage stereotactic ablative radiotherapy (SABR) found that a high NLR after SABR predicted a low survival rate (HR = 1.25,
p = 0.012) [
51]. Due to the specific role of neutrophils in tumors, another retrospective study on patients undergoing SABR found that an appropriate NLR cutoff value for survival analysis was 2.98, based on receiver operating characteristic (ROC) curve analysis. Similarly, neutrophils are associated with genetic mutations in NSCLC [
52], [
56]. Although neutrophils have been shown to have antitumor effects in many experiments, in addition to promoting tumor progression, few clinical trials have provided evidence to conclusively support the antitumor influence of neutrophils.
2.3. High neutrophil count is associated with low survival following immunotherapy
Immunotherapy, which aims to inhibit tumor progression by improving the immune microenvironment in patients with cancer, has emerged as a new direction in the last decade [
38], [
39]. Numerous studies have shown the importance of neutrophil levels in determining the effectiveness of various treatment methods, including ICIs, an indispensable type of immunotherapy [
46], [
47], [
48], [
57] (
Table 1). Eleven randomized clinical trials were analyzed, including 4614 patients with metastatic NSCLC (mNSCLC). To strengthen the prognostic power of dNLR, a surrogate for NLR in NSCLC, lung immune prognostic index (LIPI) composite scores, calculated based on the dNLR (absolute neutrophil count/(white blood cell count - absolute neutrophil count)) and LDH level, were used as a key evaluation indicator in patients with mNSCLC who received ICIs—more specifically, programmed death-1 (PD-1)/programmed death-ligand 1 (PD-L1) inhibitors. The LIPI includes dNLR > 3 and LDH levels above the upper limit of normal (ULN) and divides patients into three risk groups: good, intermediate, and poor. A good LIPI score (LDH level < ULN and dNLR ≤ 3) was associated with longer OS compared with a poor LIPI score in the ICI-treated group, with estimated median survival times of 15.6 vs 4.5 months (HR = 0.34, 95% confidence interval (95% CI): 0.28-0.42). A similar prognostic association was observed in patients receiving CT, where those with good LIPI scores survived longer than those with poor scores (estimated median survival, 10.4 vs 5.3 months; HR = 0.49, 95% CI: 0.40-0.60). Furthermore, multivariate analysis showed that albumin levels, LDH levels < ULN, and dNLR ≤ 3 were all associated with longer OS and PFS [
49]. In another multicenter retrospective study, researchers found that pretreatment dNLR and LDH levels were correlated with ICI treatment outcomes in patients with NSCLC. The study was conducted in eight European centers and divided patients into three groups, according to therapy methods, as follows: test (
n = 161) and validation (
n = 305) groups treated with PD-1/PD-L1 inhibitors; and a control cohort (
n = 162), treated with CT only. Complete blood counts and LDH and albumin levels were measured prior to ICI treatment, and a LIPI was developed based on dNLR > 3 and LDH > ULN and was used to classify patients into three groups (good, intermediate, and poor). In the pooled ICI treatment cohort (
n = 466), dNLR > 3 and LDH > ULN were independently associated with OS (
p < 0.001, HR = 2.22, 95% CI: 1.23-4.01 and
p < 0.001, HR = 2.51, 95% CI: 1.32-4.76, respectively). Median OS times for patients with poor, intermediate, and good LIPI were 3 (95% CI: 1 to not reaching (NR)), 10 (95% CI: 8 to NR), and 34 (95% CI: 17 to NR) months, respectively, and median PFS times were 2.0 (95% CI: 1.7-4.0), 3.7 (95% CI: 3.0-4.8), and 6.3 (95% CI: 5.0-8.0) months (all
p < 0.001). However, no significant differences in survival were detected in the CT cohort [
12].
In another clinical study involving nivolumab (an anti-programmed death receptor-1 antibody), including 52 patients with mNSCLC [
11], a Cox regression analysis was used to investigate the prognostic value of NLR for OS and PFS. The study found that a high NLR was associated with poor OS (HR = 3.64, 95% CI: 1.78-7.46,
p < 0.001). The researchers concluded that NLR was only an independent prognostic factor for mNSCLC treated with nivolumab.
In a prospective clinical study, data—including complete blood count at baseline within one week prior to the first nivolumab infusion—from eligible patients with metastatic or non-resectable NSCLC who could not receive curative treatment were analyzed retrospectively. Patients with co-infections, including human immunodeficiency virus or hepatitis, receiving systemic steroids or combined radiotherapy, or with previous or ongoing autoimmune disease, were excluded. Median OS and PFS were 9.6 (95% CI: 6.0-14.0) and 2.1 (95% CI: 1.8-6.4) months, respectively, in the entire group of patients included in the analysis. Higher NLR was associated with trends toward shorter OS and PFS; however, significant differences in OS were detected among patients classified into three groups based on NLR quartiles. A higher than median NLR was also associated with worse OS (NR vs 5.1 months; HR = 3.3, 95% CI: 1.3-8.5,
p = 0.013 and 4.8 vs 13.2 months; HR = 4.1, 95% CI: 1.5-11.3,
p = 0.006, respectively). Correspondingly, the relationship between NLR and response to nivolumab treatment was significant in a model that included NLR and first-line response as predictors (
p = 0.028) [
53].
In summary, almost all published findings suggest that NLR or dNLR may be valuable tools for predicting the efficacy of immunotherapy or TT and informing decisions on whether more effective combination therapies should be used for patients with lung cancer. Hence, treatment effects may be improved by reducing patient neutrophil count before and during the ICI treatment. Therefore, investigation of whether the regulation of neutrophils can influence the effect of ICI treatment is warranted to develop new types of immunotherapy that combine ICI treatment with neutrophil-targeting agents, with the potential to provide better outcomes for patients with lung cancer.
3. Classification of neutrophils in lung cancer
3.1. Neutrophil origin and differentiation in lung cancer
Neutrophils differentiate in the bone marrow from common myeloid progenitor cells to typical granulocyte-monocyte progenitor cells; this process is followed by several further stages, including myeloblasts, promyelocytes, myelocytes, metamyelocytes, and mature neutrophils [
58], [
59], [
60]. Neutrophils are short-lived white blood cells of the innate immune system that require constant replenishment by newly differentiated cells from the bone marrow. During this process, various granules are formed, which store different components including primary azurophilic granules, secondary specific granules, tertiary gelatinase granules, and secretory vesicles [
59], [
61]. The differential release of various granules from neutrophils contributes to the heterogeneity, multifunctionality, and plasticity of the neutrophils observed in various pathophysiological conditions. Exposure to different combinations of cytokines and chemokines regulates the activation state mature, normal, and urgent neutrophils. Depending on the stimuli and stage of differentiation, neutrophil contents are discharged into the surrounding environment via cytosolic action, while their membrane components are expressed on the cell surface [
62], [
63], [
64], allowing neutrophils to respond rapidly to stimuli in their surrounding environment.
Numerous clinical studies have found that neutrophils can have pro-tumor-growth and metastasis effects and have demonstrated neutrophils’ pro-metastatic roles by eliminating neutrophils in animal cancer models, leading to decreased metastatic seeding [
65], [
66], [
67], [
68] (
Table 2 [
65], [
66], [
67], [
68], [
69], [
70], [
71], [
72], [
73], [
74], [
75], [
76], [
77], [
78], [
79], [
80]). Nevertheless, other studies have reported that the similar elimination of neutrophils in some animal models leads to increased metastasis, suggesting that neutrophils can also have anti-metastatic effects [
81], [
82], [
83] (
Table 3 [
81], [
82], [
83], [
84], [
85], [
86], [
87], [
88], [
89], [
90], [
91], [
92], [
93], [
94], [
95]). When neutrophil depletion is performed using the same antibody, it can result in tumor progression and/or tumor suppression outcomes (
Table 2,
Table 3). The effects of neutrophil depletion in opposing tumor growth and metastasis can be partly explained by the differential activation of neutrophils by tumor cells and other cells in the tumor environment, which results in different ratios of pro-tumor and antitumor neutrophils [
96], [
97]. Hence, neutrophils require more precise classification according to their specific structures and functions.
Neutrophils can be divided into different groups according to various classification methods. Neutrophils are commonly classified according to their cell surface markers. For example, neutrophils with the cell surface markers CD15
+, CD16
+, CD66b
+, and CD14
- are categorized as human neutrophils, while neutrophils with the markers CD11b
+, Ly6G
+, and Ly6C
- are mouse neutrophils [
98]. The names of neutrophils vary widely, depending on these different classification methods, particularly in malignant tumors. The most commonly reported neutrophil populations closely associated with lung cancer include N1/N2 neutrophils, tumor-associated neutrophils (TANs), low-density neutrophils (LDNs), and polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). During tumor progression, granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are secreted into the blood circulation by cancer tissues and stimulate bone marrow hematopoietic cells to secrete LDNs, high-density neutrophils (HDNs), PMN-MDSCs, monocytic myeloid-derived suppressor cells (M-MDSCs), and other types of neutrophil. All of these neutrophil types have remarkably similar morphological features and surface biological markers and can transform into other types in response to tumor-secreted cytokines, making the study of the evolution of neutrophils and their functional mechanisms in tumor progression challenging (
Table 4 [
99], [
100], [
101], [
102], [
16], [
103], [
104], [
105], [
106], [
107], [
108], [
109], [
110], [
111], [
112], [
113], [
114], [
115], [
116], [
117], [
118], [
119], [
120], [
121], [
122], [
123], [
124] and
Table 5 [
114], [
115], [
116], [
118], [
119], [
120]).
Regardless of the cell type, LDN, HDN, PMN-MDSC, M-MDSC, or N1 and N2 TANs, definitions of cell populations are based on cell origin and the specific acquisition method. There is no complete cytological definition of neutrophil surface and biological markers. Furthermore, cell types can intersect; for example, immunosuppressive LDNs identified in the peripheral blood of patients with cancer have a phenotype consistent with that of PMN-MDSCs [
98], [
125], [
126]. CD10 (also known as common acute lymphoblastic leukemia antigen, neutrophil endopeptidase, or enkephalinase) can be used as a marker to clearly distinguish between mature CD66b
+CD10
+ and immature CD66b
+CD10
- neutrophils in heterogeneous populations of circulating CD66b
+ neutrophils that emerge under inflammatory conditions [
127]. CD66b
+CD10
+ neutrophils inhibit T cell proliferation and interferon-γ (IFN-γ) production through the CD18-mediated release of contact arginase 1 (Arg1). In contrast, CD66b
+CD10
− neutrophils can manifest the opposite behavior, promoting T cell survival, proliferation, and IFN-γ production via CD18-mediated contact-dependent mechanisms. Given this complexity, we surveyed cell surface and biological markers for several species of neutrophils commonly found in tumors (
Table 4). Unfortunately, we did not identify a clear basis for neutrophil delineation, which may require more studies of subcellular populations.
Temporal physiological processes are major sources of neutrophil heterogeneity; numerous studies have found that tumor-associated aged neutrophils (Nageds; CXCR4
+CD62L
low) have a robust pro-metastatic capacity [
128], [
129], [
130], [
131]. Nageds (CXCR4
+CD62L
low neutrophils) can be obtained by downregulating CD62L and up-regulating CXCR4 in CXCR4
lowCD62L
high naïve neutrophils [
128]. They promote tumor migration and support tumor metastasis by increasing the release of several pro-metastatic factors, including NETs, reactive oxygen species (ROS), vascular endothelial growth factor (VEGF), and matrix metalloproteinase 9 (MMP9). Another study focused on the presence and biological function of Nageds and found that they can generate sirtuin 1 (SIRT1)-induced NETs dominated by mitochondrial DNA [
129]. Based on these results, various interventions centered on SIRT1-Nageds have been conducted; for example, vitamin B3 inhibits the SIRT1 activation of neutrophils by tumor cells, Mdivi-1 blocks mitosis and promotes Naged death, TRO19622 blocks DNA release from mitochondrial permeability transition pore (mPTP) channels, and deoxyribonuclease I (DNase I) clears NETs. These drugs can prevent tumor metastasis to varying degrees but do not have noticeable effects on primary tumors, indicating that methods to deliver drugs directly to tumors are urgently required.
3.2. Polymorphonuclear myeloid-derived suppressor cells (MDSCs)
MDSCs are heterogeneous cell populations derived from bone marrow progenitors and immature myeloid cells. To date, two significant subpopulations of MDSCs have been identified: M-MDSCs and PMN-MDSCs [
117], [
121], [
132]. PMN-MDSCs share phenotypic and morphological features with neutrophils, whereas M-MDSCs are similar to monocytes. Mouse MDSCs have a CD11b
+Gr1
+ phenotype and are divided into two subtypes: CD11b
+Ly6G
+Ly6C
- granulocytic MDSCs (also referred to as PMN-MDSCs) and CD11b
+Ly6G
-Ly6C
+ M-MDSCs [
133]. Human MDSCs are defined as M-MDSCs (human leukocyte antigen (HLA)-DR
-CD11b
+CD14
+CD15
-CD33
+CD66b
-) and PMN-MDSCs (HLA-DR
-CD11b
+CD14
-CD15
+CD33
+CD66b
+). MDSCs exert immunosuppressive functions, mainly through the production of Arg1, nitric oxide synthase 2 (NOS2), and ROS [
122]. PMN-MDSCs, which are an essential subpopulation of MDSCs mainly found in peripheral blood, were initially identified in mouse tumor models and have immunosuppressive effects that assist tumor progression (
Fig. 1). Relative to neutrophils, PMN-MDSCs show similar surface marker expression but lack specific surface antigens, and there is now a consensus that they should have at least the surface markers CD66b
+, CD16
+, CD15
+, CD11b
+, and CD33b
mild, and be HLA-DR-negative [
134], [
135], [
136]. Traditionally, PMN-MDSCs can be divided into subgroups: CD11b
+Ly6G
+ and Ly6C
- [
137], [
138]. Although PMN-MDSCs are primarily considered to foster cancer development, despite wielding an arsenal of cytotoxic agents, a surprising inhibitory role played by PMN-MDSCs in epithelial carcinogenesis was described based on experiments using a mouse model of phosphatase and tensin homolog (PTEN)-deficient uterine cancer [
139]. By inducing tumor cells to detach from the basement membrane, PMN-MDSCs impede early tumor growth and inhibit malignant tumor progression. Unexpectedly, PMN-MDSC recruitment and tumor growth control occur independently of lymphocyte and cellular senescence but are part of the innate inflammatory response of tumors to hypoxia. In another study, three populations of PMNs were detected in tumor-bearing mice: classical PMNs, PMN-MDSCs, and activated PMN-MDSCs (aPMN-MDSCs), where aPMN-MDSCs have potent immunosuppressive activity and are only found in tumors.
PMN populations in patients with cancer and in tumor-bearing mice have striking similarities. Two distinct PMN populations found in tumor-bearing mice, with the characteristics of classic PMNs and PMN-MDSCs, are also present in the peripheral blood of patients with cancer. In contrast, the genetic profiles of PMN-MDSCs in human tumors are similar to those of aPMN-MDSCs in mice and are associated with unfavorable clinical outcomes in patients with cancer. These findings demonstrate that aPMN-MDSCs may be a unique PMN population with tumor immunosuppressive functions. Furthermore, single-cell sequencing of CD45
+Ly6G
+Ly6C
low PMN cell populations from tumor-bearing mice showed that aPMN-MDSCs can be classified according to CD14 level—that is, as CD14
-, CD14
int, or CD14
high—and confirmed that CD14
high aPMN-MDSCs are associated with negative clinical outcomes [
123]. However, a prospective analysis of peripheral blood neutrophils and PMN-MDSCs (CD66b
+CD11b
+CD15
+CD14
-) contradicted these results [
124]. The researchers found that patients with NSCLC had a higher proportion of PMN-MDSCs and neutrophils than healthy individuals (
p < 0.0001). Moreover, patients with low PMN-MDSCs had better OS (22.1 (95% CI: 4.3-739.7) months) than those with high PMN-MDSCs (9.3 (95% CI: 0-18.8) months). Hence, based on these contrasting findings, it is unclear whether CD14 can serve as a marker for PMN-MDSCs. These discrepant findings may be attributable to multiple unknown types of PMN-MDSCs and their different levels in peripheral blood versus tumor tissue. The observation that patients with low PMN-MDSCs had better OS was also supported by another study comprising a single-cell RNA sequencing analysis of tissue biopsy samples from 42 patients with stage III/IV NSCLC. The study identified two distinct clusters of TANs [
99], both of which expressed various potential PMN-MDSC-related genes, including lipoxygenase 1 (
LOX-1); thus, it demonstrated that PMN-MDSCs are present in tumors and challenging to distinguish from TANs. Another study found that neutrophils were more abundant in human lung SqCC than in lung adenocarcinoma.
3.3. Tumor-associated neutrophils
TANs were originally identified in mouse tumors and, similar to macrophages, are classified into two potential phenotypes—namely, antitumor N1 and pro-tumor N2—which have distinct functional cytokine and gene expression profiles [
100], [
101], [
102], [
140]. The TME can affect the balance of N1 and N2 subpopulations through the secretion of cytokines, including TGF-β, IL-6, G-CSF, and IL-35, which induce pro-tumor polarization, while IFN-β and IL-12 induce antitumor polarization [
69], [
84], [
141], [
142] (
Fig. 2). In the TME, tumor cells attract TANs using chemokines such as CXCL8, which promotes CXCR1 and CXCR2 expression, contributing to angiogenesis and tumor progression [
84], [
140]. TANs can also regulate other lymphocytes. N2 TANs can inhibit natural killer (NK) cell tumor cell killing function, thus promoting tumor metastasis [
143], [
144] and promoting tumor growth by recruiting regulatory T cells through the secretion of C-C motif chemokine ligand 17 (CCL17) [
145], [
146]. In contrast, N1 TANs recruit and activate CD8
+ T cells by producing chemokines, thus providing antitumor effects [
147], [
148], [
149], [
150]. In a recent study, neutrophils were divided into five subpopulations in humans (hN1-5) and six subpopulations in mice (mN1-6) by spectral clustering [
16]. Ratios of these subpopulations differed significantly between tumors and normal lung tissue; mN1s and mN2s were enriched in healthy lungs, while mN4s and mN5s were more abundant than mN1s and mN2s in tumor tissue, and mN3s and mN6s were only present in tumor tissue. The hN1 and mN1 subtypes expressed many typical neutrophil markers (i.e., MMP8, MMP9, S100A8, S100A9, and ADAM8), while the hN2 and mN2 cells expressed type I interferon-responsive genes, including interferon induced protein with tetratricopeptide repeats 1 gene (
IFIT1), interferon regulatory factor 7 gene (
IRF7), and radical S-adenosyl methionine domain containing 2 gene (
RSAD2). The hN5 and mN5 subtypes expressed the cytokines cystatin B (CSTB, a stefin that functions as an intracellular thiol protease inhibitor), cathepsin B (CTSB, a lysosomal cysteine protease with both endopeptidase and exopeptidase activity, which may play a role in protein turnover), CCL3 (which contributes to inflammatory responses by binding to the receptors CCR1, CCR4, and CCR5), and colony-stimulating factor 1 (CSF1, which encodes a cytokine that controls macrophage production, differentiation, and function). mN6 neutrophils are very similar to mN4 neutrophils in that both express hexosaminidase subunit beta (HEXB, the beta subunit of the lysosomal enzyme beta-hexosaminidase, which catalyzes degradation of the ganglioside GM2 and of other molecules containing terminal
N-acetyl hexosamines) and prothymosin alpha (PTMA, which is involved in negative regulation of apoptosis). Furthermore, mN6 neutrophils uniquely express ficolin B (Fcnb, which is involved in complement activation and the lectin pathway) and neutrophilic granule protein (Ngp, which is involved in negative regulation of angiogenesis and lymphangiogenesis).
As different neutrophil types have contrasting functions, artificial alteration of the abundances of neutrophil populations may have unexpected results; for example, inhaled cowpea mosaic virus nanoparticles (CPMV-NPs) comprise 60 copies of self-assembling virus-like NPs that generate a 30 nm icosahedral structure composed of differently sized capsid protein units. It can inhibit the lung metastasis of certain cancers by increasing the frequency of CD11b
+CD86
high tumor-infiltrating neutrophils (
Table 6). CD11b
+CD86
high neutrophils can kill tumor cells by releasing reactive oxygen intermediates, priming CD4
+ T cells and inducing them to a helper T (Th) 1 phenotype, and modulating NK cell function. Moreover, CD11b
+CD86
high neutrophils can produce CXCR3, CXCL9, and CXCL10, which recruit CD4
+ and CD8
+ T cells and generate an antitumor immune microenvironment. This CPMV-NPs represent a new pathway that could be targeted for treating malignant tumors [
151].
3.4. Low-density neutrophils
Numerous clinical trials have demonstrated that the numbers of circulating neutrophils are increased and that high NLR values are associated with aggressive outcomes in patients with lung cancer. The density gradient centrifugation of peripheral blood separates HDNs and LDNs, which are respectively found in the high- and low-density fractions [
103], [
104], [
109], [
110], [
111], [
112], [
152], [
153]. HDNs account for approximately 95% of peripheral blood neutrophils and express CD66b, CD11b, CD15, CD16, and CD10 in humans and CD11b, Gr1, and Ly6G in mice; however, in tumor-bearing mice, the number of LDNs increases with tumor progression [
154]. HDNs are reported to have antitumor effects, while LDNs have pro-tumor effects [
152], [
153], [
154].
In vitro experiments demonstrated that HDNs accumulate in the premetastatic lung and inhibit tumor cell implantation by releasing ROS; in tumor-bearing mice, tumors can be suppressed by HDN injection [
154]. LDNs are a heterogeneous group associated with various diseases, including asthma, sepsis, rheumatoid arthritis, systemic lupus erythematosus, and cancers [
104], [
105]. Furthermore, LDNs can promote tumor development by downregulating the expression of inflammatory factors and limiting CD8
+ T cell proliferation. LDNs lack specific biological markers and share the same surface markers as PMN-MDSCs and TANs. Moreover, like TANs, LDNs display pro-tumor properties in malignant tumors, making it difficult to distinguish these subgroups [
106] (
Fig. 2).
To clarify the specific role of LDNs in lung cancer, a clinical trial analyzed the composition of peripheral blood from patients with lung cancer and healthy subjects and found that LDNs were elevated in patients with advanced lung cancer and associated with poor clinical prognosis. Phenotypes of HDNs and LDNs were also characterized in patients with advanced lung cancer (stages IIIB-IV) by applying a unique, newly developed mass cytometry method; two HDN subpopulations (CD66b
highCD10
intCXCR4
int/lowPDL1
low and CD66b
highCD10
highCXCR4
intPDL1
low) were detected in both healthy individuals and patients, while one unique LDN subpopulation (CD66b
highCD10
lowCXCR4
highPDL1
int) was exclusively found in patients with cancer [
107]. In another study, a high-dimensional human cell surface marker screen identified 12 down-regulated and 41 up-regulated surface markers on LDNs compared with HDNs, and finally selected CD36, CD41, CD61, and CD226 as surface markers of LDNs [
113]. Although this study identified new markers of LDNs, indicating that LDNs are a distinct neutrophil population, much remains unknown about this cell type, and further future exploration is warranted.
4. The role of neutrophils in lung cancer development and their corresponding nanotechnology therapeutic targets
4.1. The dual role of neutrophils in the development and progression of lung cancer and tumor-targeting nanotechnologies
There are diverse immune cells in the NSCLC TME, and flow cytometry has demonstrated that neutrophils are the dominant immune cell type in this context, accounting for almost 20% of all CD45
+ cells. Myeloid lineage cells represent > 50% of tumor-infiltrating CD45
+ cells, while the percentage of macrophages is significantly lower. B cells comprise only 5% of CD45
+ cells in NSCLC. Combined T cell receptor (TCR)-β sequencing and experimental data indicate that the proportion of T cells in lung cancer varies greatly [
155]. Neutrophils have essential roles in lung cancer occurrence and development. Cancer is a specific type of inflammatory condition [
156] in which endothelial cells are exposed to products of inflammation, such as ROS and reactive nitrogen species produced by neutrophils, leading to the increased oxidation of proteins, lipids, and nucleic acids. If the proteasome cannot degrade those oxidized proteins, advanced glycation end-products accumulate and activate receptors for advanced glycation end-products (RAGE), resulting in tumor cell proliferation and carcinogenesis [
97]. In non-small cell carcinomas and adenocarcinomas, basal RAGE expression is remarkably high, but it is rapidly down-regulated on malignant transformation, and this is associated with the highly aggressive phenotypes of these types of lung cancer [
157].
Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, can degrade basement membrane, the ECM, and non-matrix substrates, thereby acting as an angiogenic switch and contributing to tumorigenesis and tumor progression [
158], [
159]. Neutrophils also contribute to the regulation of tumor angiogenesis, which is pivotal to the progression of early-stage tumors. The STAT3 signaling pathway activates neutrophil pro-angiogenic functions by controlling the transcription of vascular endothelial growth factor A, fibroblast growth factor 2, and MMP9 (
Fig. 3). An inhibitor of STAT3 was encapsulated in NPs and used to treat a murine
HER-2+ breast cancer model, resulting in STAT3 inhibition and consequent immune activation via an increase of IFN-γ, GM-CSF, p-signal transducer and activator of transcription (pSTAT)-1, IL-15, IL-2, and IL-12b in the TME, which activated CD8
+ T cells and decreased tumor angiogenesis, tumor growth, and metastasis [
160] (
Table 6). MMP9 controls late tumor angiogenesis [
161], and bone-marrow-derived neutrophils are the primary source of MMP9 in tumor-bearing lung tissues following tumor cell inoculation. An orthotopic lung cancer model showed that MMP9 promotes tumor cell survival at a very early stage of tumor establishment; however, its inability to influence tumor size suggests that its effect on subsequent tumor growth is limited [
162]. When neutrophils are activated or destroyed, proteinases stored in neutrophil granules are released. NE, which is encoded by
Elane, is among the most potent neutrophil proteinases; it can degrade the ECM and may contribute to lung cancer development. Molecular genetics analysis has shown that two single nucleotide polymorphisms (-903 T and -741 G) in the
Elane promoter might accelerate lung cancer development due to an imbalance of the α1-antitrypsin (AT)/NE system [
163]. NE can also promote lung cancer cell growth and proliferation by degrading insulin receptor substrate 1 (IRS1), which is stored in the endosomal compartment in tumor cells. Hence, NE degradation of IRS1 must occur within cells. During NE degradation of IRS1, the interaction between phosphatidylinositide 3-kinases (PI3K) and platelet-derived growth factor receptor (PDGFR) is increased, modulating the P13K axis toward promoting cancer cell proliferation; however, NE cannot induce lung cancer cell proliferation directly [
164]. The MiR-30 family is an important regulator of signaling networks and suppresses the expression of molecules involved in cell proliferation, such as epidermal growth factor receptor (EGFR), type 1 insulin-like growth factor receptor (IGF1R), IRS1, and
E2F transcription factor 7 (E2F7); it may play a pivotal role in microRNA (miRNA) replacement nanomedicine therapy [
165].
Neutrophils induce tumor cell detachment by altering cell-to-cell contact, which involves several membrane-bound molecules, including tumor necrosis factor-α (TNF-α)/TNF-α receptor inhibitor, intercellular adhesion molecule-1 (ICAM-1)/lymphocyte function-associated antigen-1 (LFA-1), interleukin-1α (IL-1α)/IL-1α receptor, and NE [
166] (
Fig. 4). In patients with adenocarcinoma bronchioloalveolar carcinoma (BAC) features, neutrophil tumor shedding is crucial to aerogenous cancer cell spread [
167]. Hepatocyte growth factor (HGF), a pleiotropic cytokine produced by neutrophils, also contributes to the aerogenous spread of BAC by activating cellular-mesenchymal epithelial transition factor (c-met). HGF can be detected in bronchoalveolar lavage fluid from most patients with BAC but not in healthy controls, which indicates that HGF is produced locally. The TME of BAC can attract neutrophils from peripheral blood, and neutrophil-derived HGFs induce BAC tumor cell migration and promote cancer cell spread [
168].
Genomic and transcriptomic technologies have enabled a better understanding of the molecular basis of lung cancer and have revealed that inflammation is a prominent characteristic of lung cancer. As the dominant immune cells in lung cancer, neutrophils may be able to suppress lymphocytes in SqCC [
169]. A
Kras mutant lung cancer animal model showed that IL-17A recruits neutrophils to tumor sites by inducing expression of the proinflammatory cytokines IL-6, G-CSF, and MFGE8, as well as the chemokine CXCL1, leading to reduced recruitment of CD4 T cells, CD8 T cells, and B cells. PEGylated liposome doxorubicin NPs (PLD@NEs)—a model chemotherapeutic nano-drug using neutrophils as carriers—significantly increase the concentration of inflammatory factors (CXCL1/ keratinocyte chemoattractant (KC)) in tumor sites, which is essential to PLD@NEs infiltration [
170] (
Table 6). The neutrophil count is negatively correlated with T cell count in patients with NSCLC [
171]; furthermore, neutrophil and CD8
+ cell content are negatively correlated in cancer but not in adjacent normal tissues, suggesting that this phenomenon is tumor-specific.
Moreover, a negative correlation between neutrophils and CD4
+ lymphocyte content—particularly Th1 and Th17 subsets—has been detected [
155]. Lung adenocarcinoma with a mutation of serine/threonine kinase 11 (
STK11), a tumor suppressor gene, exhibits a marked reduction of dendritic cells, NK T cells, and macrophages. Neutrophil degranulation is a potential immunosuppressive mechanism associated with
STK11 mutation; however, the evidence supporting this hypothesis is limited to proteomics data [
172].
Tumor recurrence seriously affects the survival of patients with lung cancer, and neutrophils can reactivate dormant tumor cells by releasing proinflammatory S100A8/S100A9 proteins under the control of stress hormones. S100A8/S100A9 release activates myeloperoxidase (MPO), leading to the accumulation of oxidized lipids in neutrophils. Releasing these lipids from neutrophils activates the FGF pathway, allowing dormant tumor cells to form new tumors; hence, high S100A8/S100A9 expression results in early lung cancer recurrence [
173] (
Fig. 3).
AuPB@mPDA, a nanoplatform for efficient loading and the photoregulated release of DNase I, can disrupt NET formation, thereby enhancing the therapeutic efficacy of tumor immunotherapy and moderating metastatic spread [
174] (
Fig. 3). In response to inflammatory cues to trap and kill pathogens, DNA webs derived from neutrophils can awaken dormant cancer cells. Two NET-associated proteases, NE and MMP9, sequentially cleave and remodel laminin, resulting in the proliferation of dormant cancer cells by activating α3β1 integrin signaling in response to sustained inflammation [
175]. NE is involved in NETs formation, which facilitates lung cancer progression. Sivelestat, a small-molecule NE inhibitor, can function
in vitro but does not work
in vivo [
176]. A new kind of lipid-based NP-mediated delivery system was found to improve sivelestat efficiency. In a mouse model, interbilayer-crosslinked multilamellar vesicles delivered sivelestat, which was taken up by neutrophils and inhibited NETs formation [
177] (
Table 6).
As mentioned above, neutrophils comprise heterogeneous populations with pro-tumor and antitumor functions. Neutrophils play their antitumor roles mainly via neutrophil cytotoxicity. H
2O
2, a type of ROS secreted by neutrophils, is an essential mediator of tumor cell killing that induces Ca
2+ channel activation, resulting in Ca
2+ influx into tumor cells [
65]. Transient receptor potential cation channel, subfamily M, member 2 (TRPM2), an H
2O
2-dependent Ca
2+-permeable channel, is relatively likely to be found on tumor cells that have undergone epithelial-to-mesenchymal transition (EMT) but is reduced on cells that have undergone mesenchymal-to-epithelial transition (MET). Cells expressing high levels of TRPM2 are more susceptible to neutrophil cytotoxicity, indicating that EMT promotes tumor cell sensitivity to neutrophil cytotoxicity [
178]. Neutrophils also exert antitumor effects via tumor necrosis factor-related apoptosis-inducing ligands (TRAILs). In response to IFN-γ stimulation, neutrophils exert their cytotoxic function by elevating TRAIL expression and release, which promotes cancer cell apoptosis [
179].
The loss of testosterone can impair neutrophil antitumor function. In castrated mice,
Cxcr2 messenger RNA (mRNA) levels are reduced, while
Cxcr4 and
Vla-4 are increased, which is opposite to the situation during neutrophil maturation and leads to an increased likelihood of tumor formation in the lung [
180]. RAGE plays a pro-tumor role under inflammatory conditions; however, it also has antitumor functions. When RAGE on tumor cells interacts with and is recognized by cathepsin G on neutrophils, the neutrophil-mediated killing of tumor cells is activated [
181]. These phenomena illustrate the complex dual role of neutrophils in cancer.
The interaction between C-type lectin receptors in neutrophils and tumor Nidogen-1 can also enhance neutrophil cytotoxicity toward mouse tumor cells [
182]. In addition to MMP9, neutrophils produce MMP8, which has an antitumor function. MMP8 has been shown to increase the adhesion between tumor cells and ECM protein levels, thereby reducing the invasive ability of tumor cells through Matrigel and inhibiting tumor metastasis, as well as regulating the inflammatory responses induced by carcinogens [
183].
Neutrophils interact with other immune cells, as well. TANs isolated from lung tumors can activate T cell proliferation, increasing costimulatory molecule expression on the surface of neutrophils. This positive-feedback loop demonstrates that TANs exert their antitumor effects by activating T cell responses [
184]. The H
2O
2 produced by neutrophils is vital in killing cancer cells and enables neutrophils to inhibit cancer cell seeding in the lung [
82]. A core-shell supramolecular hybrid nano-gel has been designed to mimic the function of neutrophil lysosomes; it can be used to treat tumors through a cascade reaction involving superoxide dismutase and chloroperoxidase [
185].
4.2. Nanotherapeutics targeting the mechanisms underlying neutrophil promotion of lung cancer metastasis
Lung cancer metastasis is an important factor affecting patient prognosis. Neutrophils accelerate lung cancer metastasis through two main pathways. The first pathway is a contact-dependent mechanism. Selectins expressed on endothelial cells (E-selectin), platelets (P-selectin), and leukocytes (L-selectin) bind to selectin ligands, such as sialyl Lewis-a (sLe
a) and sialyl Lewis-x (sLe
x), expressed on circulating tumor cells (CTCs), increasing the adhesion between cancer cells and distant organs [
186] (
Fig. 4).
Salmonella exhibits antitumor behavior through various mechanisms; however, neutrophils recruited into tumor sites by
Salmonella directly eliminate the bacteria, limiting the antitumor efficacy of
Salmonella; hence, a neutrophil-depleting strategy is required to improve its efficacy. Silver NPs (AgNPs) decorated with PEGylated sialic acid were designed to recognize the L-selectin expressed on neutrophils, enabling the AgNPs to accumulate at neutrophil-rich tumor sites and deplete neutrophils locally. These AgNPs were then applied in combination with
Salmonella in a neutrophil-depleting model to treat tumors. Moreover, AgNPs have intrinsic antitumor activity; hence, these NPs can exert their antitumor effects both by improving the efficacy and safety of
Salmonella and through neutrophil depletion [
187] (
Table 6).
Lipopolysaccharide-induced systemic inflammation may activate neutrophils and influence cell adhesion, leading to cancer cell spread. Photodynamic therapy (PDT)-induced acute inflammation in deep tumor tissues can be used as tumor therapy. PDT-induced inflammation accelerates neutrophil infiltration in tumor tissues, enabling nanotherapeutics to incorporate neutrophils during the design of drug delivery systems [
188]. In more than 30% of patients with lung cancer metastasis, the metastasis is to the liver. Activated neutrophils within inflamed liver sinusoids can facilitate CTC binding with liver sinusoids and mediate tumor cell arrest. Interactions among selectin-sLe
x-neutrophils increase the liver metastasis of lung cancer [
189]. The molecular mechanism underlying neutrophil interaction with cancer cells has also been explored. Under inflammatory conditions, neutrophils are activated by inflammatory stimuli, potentially leading to endothelial cell dysfunction, with changes in cell adhesion molecule (CAM) expression on the neutrophils surface. Mac-1, a type of CAM, changes its expression on the surface of neutrophils, affecting their adherence and migratory abilities within potential metastatic tissues.
Moreover, the expression of the binding ligand of Mac-1, ICAM-1, is increased on CTCs, and interaction between the Mac-1 expressed on neutrophils and the ICAM-1 expressed on CTCs promotes cancer cell arrest in distant organs, leading to distant metastasis of lung cancer [
70]. Based on this mechanism, neutrophil-mimicking (NM)-NPs were designed by coating neutrophil membranes onto a poly (lactic-
co-glycolic acid) (PLGA) surface to preserve the bio-binding ability of the neutrophils. These NM-NPs have high CTC-capture ability. Furthermore, combining NM-NPs with carfilzomib, a second-generation proteasome inhibitor, selectively neutralized CTCs and prevented the formation of a premetastatic niche in a breast cancer murine model [
190] (
Fig. 4 and
Table 6). Neutrophils can also accelerate liver metastasis through the formation of NETs. Extracellular RNAs released from Lewis lung carcinoma cells enhance the formation of NETs by activating epithelial cells and increasing their IL-1β release and the CAM-1 expression on their surface [
191]. A mathematical model also showed that NETs play important roles in regulating lung cancer invasion and metastasis [
192]. Under conditions of severe postoperative infection, NETs can trap CTCs, increasing the adhesion between tumor cells and distant organ sites and facilitating lung cancer metastasis [
193]. Neutrophils have also been used as carriers of liposomes containing paclitaxel (PTX). Under inflammatory-factor stimulation, NETs and PTX liposomes are released from neutrophils and then enter glioma tumor cells, where PTX has cytotoxic effects and inhibits tumor development [
194].
The second pathway is contact independent. IL-8, a member of the α-chemokine family and a highly potent neutrophil chemoattractant released by tumor cells, is associated with cancer progression and metastasis. In response to IL-8, neutrophils release enzymes that can hydrolyze ECM components and remodel ECM, accelerating tumor angiogenesis and metastasis [
195]. Neutrophils can also induce IL-8 release. An experiment using human NSCLC A549 cells showed that NE could dose-dependently increase IL-8 release, and that the underlying mechanism mainly involves the mitogen-activated protein kinase (MAPK) pathway. p38 MAPK, an activator of many transcription factors, can be activated by NE and upregulates nuclear factor-κB (NF-κB) and activator protein 1 (AP-1); the latter binds with the IL-8 promoter and increases its transcription [
196]. Nuclear translocation of NF-κB is involved in carcinogenesis via activating inflammatory signaling pathways. Astaxanthin (AX) can reduce the oxidative imbalance in inflammation. To reduce NF-κB expression, solid lipid NPs containing AX (AX-SLN) were prepared and resulted in antiproliferative, antioxidant, chemotherapeutic, and anti-inflammatory effects in breast-cancer-bearing rats [
197] (
Table 6).
5. Neutrophils enhance both the lung metastasis of other cancers and their counterpart nanotherapeutics
5.1. Neutrophils promote breast cancer metastasis to the lungs
The lung is the preferred metastatic organ for breast cancer, melanoma, and colon cancer, and neutrophils play critical roles in the metastasis of these cancers to the lung. Primary tumors may change the paracrine characteristics of distant organs to facilitate the metastatic colonization of resident cancer cells in structures referred to as premetastatic niches. Premetastatic niche formation is essential to colonize cancer cells in distant organs, and lung mesenchymal stromal cells (LMSCs) contribute to establishing such niches. Complement 3 (C3) is upregulated in LMSCs and can recruit neutrophils and promote the formation of NETs. Th2 cytokines can induce C3 under STAT6 regulation; hence, the STAT6-Th2-C3-NETs cascade promotes breast cancer metastasis to the lungs [
198] (
Fig. 4). A well-designed nanocarrier named mP-NPs-DNase/PTX, which contains a PTX core and an MMP9 and DNase I shell, can release DNase I to degrade NETs in murine lung cancer and breast cancer tumor tissues, while the PTX core exerts a cytotoxic effect on tumor cells; together, these two aspects function to inhibit tumor growth and distant metastasis [
199] (
Table 6). Eukaryotic translation initiation factor 4E (eIF4E) is a well-known translation control factor whose phosphorylation promotes EMT and the translation of pro-survival and pro-invasion mRNAs, such as B-cell lymphoma-2 (BCL2), myeloid cell leukemia-1 (MCL1), SNAIL, and MMP3 [
200], [
201], thereby enhancing cancer invasion and metastasis; BCL2 and MCL1 are also anti-apoptotic proteins [
202]. In mouse breast cancer models, eukaryotic initiation factor 4E (eIF4E) mutation leads to the loss of eIF4E phosphorylation, resulting in decreased expression of BCL2 and MCL1, influencing neutrophil survival in the premetastatic niche, and preventing metastatic progression
in vivo. These results demonstrate that neutrophils in the lung favor metastasis to the lung [
203] and support the initiation of lung metastasis of cancers.
In mouse breast cancer models, CD11b
+Ly6G
+ neutrophils are the most frequent immune cells in the premetastatic lung. Moreover, neutrophils accumulate earlier than cancer cells [
71], and the depletion of neutrophils within premetastatic niches decreases spontaneous metastasis, indicating that neutrophils promote breast cancer metastatic initiation. CD11b
+Ly6G
+ neutrophils are essential for the internalization of anti-CD11b-coated NPs and thus provide a platform for gold nanorods-anti-CD11b therapy [
204]. Neutrophil-derived factors—including the lipids leukotriene B4 and the cysteinyl leukotrienes C4, D4, and E4 (LTC/D/E4), which are produced by the arachidonate 5-lipoxygenase (Alox5)—mediate metastatic initiation by providing a selective proliferative advantage to cancer cells and transforming them into highly metastatic cells, by enhancing their metastatic competence [
71]. Chronic exposure to nicotine also contributes to premetastatic niche formation by recruiting pro-tumor N2 neutrophils. Within the lungs, N2 neutrophils secrete the glycoprotein STAT3-activated lipocalin 2 (LCN2), which induces breast cancer cell EMT, thereby promoting colonization and metastatic outgrowth [
72]. CD140a
+ mesenchymal cells in the lung produce prostaglandin E2 (PGE2), which binds with the PGE2 receptors expressed on neutrophils in lung tissue, leading to lipid accumulation in the lung and the reduction of adipose triglyceride lipase expression. PGE2 expression by mesenchymal cells can, in turn, be induced by the cytokine IL-1β, which is produced by neutrophils. Lipids from neutrophils promote the metastasis of breast tumors to the lungs [
205]. Hypoxia-inducible factor-1α (HIF-1α) overexpression in the TME contributes to tumor cell growth. HIF-1α siRNA-loaded superparamagnetic iron oxide-trimethyl chitosan (TMC)-hyaluronate (HA) NPs were designed to treat breast cancer, colorectal cancer, and melanoma cell lines, and were found to significantly inhibit the HIF-1α/COX2/PGE2/EP4 signaling pathways, thereby inhibiting tumor cell proliferation, migration, and invasion [
206] (
Table 6).
Triple-negative breast cancer (TNBC), which is one of the most common types of breast cancer, is characterized by an inflammatory TME that is mainly composed of cytokines and chemokines. This inflammation stimulates the growth and distant metastasis of cancer cells. IL-26 is a cytokine produced by TNBC cells that belongs to the IL-10 cytokine family; it has an amphipathic nature that allows it to bind to extracellular DNA—particularly NETs. The interaction between NETs and IL-26 induces the expression of other cytokines, such as IL-6/IL-8 and CXCL1, which elicits inflammation in the TME, activating Janus-family tyrosine kinase (JAK)-STAT3 signaling and promoting cancer progression and TNBC metastasis to the lung [
207]. Active-targeted HA-recoated
N,N,N-TMC NPs were used to deliver siRNAs targeting IL-6 and STAT3. The NPs showed high IL-6/STAT3 inhibition capacity, which decreased proliferation, migration, colony formation, and angiogenesis in breast, colorectal, and melanoma cancer cell lines [
208]. Moreover, tumor-secreted protease cathepsin C (CTSC) promotes neutrophil recruitment and the formation of NETs, resulting in enhanced breast-to-lung metastasis. The underlying mechanism involves the CTSC activation of neutrophil membrane-bound proteinase 3 (PR3) through enzymolysis, and the subsequent PR3-mediated activation of IL-1β release and NF-κB expression, as well as the upregulation of IL-6 and CCL3, resulting in neutrophil recruitment. The CTSC-PR3-IL-1β axis also activates p38 and subsequently induces neutrophil ROS production and NETs formation, leading to thrombospondin-1 (Tsp-1) degradation and the metastatic growth of breast cancer cells in the lungs [
73]. Sphingomyelin nanosystems (SNs) were designed to associate with the cell-binding domain adhesive peptide (4N1K) peptide, a ten-amino-acid peptide derived from the Tsp-1 protein. SNs-4N1Ks inhibit cancer cell colony formation [
209]. Furthermore, breast cancer cells can induce NETs formation without infection and promote breast cancer cell invasion and migration [
210]. In late-stage breast cancer, the formation of NETs is associated with venous thrombi in the lung, which provide an environment conducive to lung colonization by cancer cells [
211].
5.2. Neutrophils promote melanoma and colorectal cancer metastasis to the lungs
The extracellular nucleotide uridine diphosphate (UDP) is a signaling molecule that binds with and activates its receptor, purinergic receptor P2Y, G-protein-coupled, 6 (P2y6), to promote breast cancer and melanoma invasion. Moreover,
P2y6 knockout was found to significantly reduce neutrophil infiltration and slightly alter NK cell, macrophage, T cell, and dendritic cell infiltration in melanoma premetastatic niches, altering the immune cell composition in the TME (
Fig. 4). This finding underlines the pivotal role of neutrophils in promoting tumor metastasis [
212]. Under inflamed conditions, neutrophils recruited to the lungs degranulate azurophilic granules to release the serine proteases, cathepsin G, and elastase, causing the hydrolysis of the antitumorigenic factor Tsp-1. This finding provides a new perspective on how neutrophil-mediated lung inflammation contributes to melanoma metastasis to the lung [
74]. Anti-CD47 antibodies can improve cancer cell phagocytosis by inhibiting CD47 from binding with its ligands, such as Tsp-1 and signal regulatory protein α [
213]. IL-8 also plays an important role in melanoma metastasis. Cancer cells injected in the lungs secrete IL-8, which increases β2 integrin expression and activates neutrophils. The ICAM-1 expressed on melanoma cells interacts with the β2 integrin expressed on neutrophils, enhancing cell adhesion, which results in an increased accumulation of cancer cells in the endothelium and retention in the lungs, thereby increasing lung metastasis development [
214]. Anti-ICAM NPs have been exploited to deliver inflammation inhibitors and therapeutic enzymes to epithelial cells, resulting in decreased neutrophil infiltration [
215].
Approximately 5%-15% of colorectal cancers metastasize to the lungs; hence, the molecular mechanisms involved have attracted the attention of researchers. Patients with colorectal cancer and deficiency of contraction of Sma and Mad 4 (SMAD4) (a cancer suppressor) have high CCL15 expression and CCR1
+ TAN accumulation in lung metastases, demonstrating that the loss of SMAD4 promotes the lung metastasis of colorectal cancer through the accumulation of CCR1
+ TANs via the CCL15-CCR1 axis [
216]. In patients with differentiated thyroid carcinoma and distant metastasis, an NLR > 3 is strongly associated with poor cause-specific survival [
217]. In human anaplastic thyroid carcinoma (ATC) cells, neutrophil gelatinase-associated lipocalin (NGAL, which belongs to a large family of lipocalins and is also known as LCN2) [
218] mediates NF-κB oncogenic activity, as well as influencing ATC cell invasion ability by increasing MMP9 enzymatic activity. Cells with NGAL knocked down exhibit a reduced ability to form lung metastasis in nude mice [
219].
NETs can also trap the CTCs generated by lung cancer, carrying them to distant organs, increasing their contact with metastatic tissues, and facilitating their avoidance of destruction by the immune system, thereby promoting lung cancer metastasis (
Fig. 4). For the lung metastasis of breast cancer, gastric cancer, and colorectal cancer to occur, the formation of a pre-metastasis niche and the survival and growth of metastatic tumor cells are important [
210], [
220], [
221].
6. Nanotechnology targeting neutrophils in cancer therapy
6.1. Nanotechnology targeting neutrophils in conventional cancer treatment and detection
Aside from the use of neutrophils as a prognostic or predictive biomarker for patients with cancer, the multiple functions of neutrophils in cancer biology make neutrophils useful as therapeutic targets. Nanotechnology is used to engineer, measure, assemble, and manufacture materials at a nanometer scale (1-100 nm) [
222]; hence, nanotechnology deals with and takes advantage of small things, and its precision and controllability in both drug delivery and biosensing are unique [
223], [
224], [
225], [
226], [
227]. Due to the natural propensity of neutrophils to reside at sites of inflammation, neutrophil-based assays and nanomedicine development have gained tremendous momentum in recent years [
1], [
228], [
229], [
230]. 5-Hydroxytryptamine (5-HT) assembled onto nanoparticles containing photosensitizers and Zileuton (HZ-5 NPs), a novel therapeutic nanomedicine targeting tumor neutrophils’ chemical and biological functions, can improve cancer therapy by providing sustained drug release. Leukotrienes released by neutrophils can promote the survival and translocation of circulating cancer cells in the lung and eventually cause lung metastasis formation [
71]. 5-HT was assembled onto NPs containing photosensitizers and Zileuton (a leukotriene inhibitor) to obtain MPO and neutrophil-targeting HZ-5 NPs. Synthesized
bis-5-HT has an MPO targeting function, where MPO is a neutrophil-specific enzyme [
231]. HZ-5 NPs inhibit neutrophil-mediated lung metastasis through the sustained release of Zileuton, thereby providing a novel strategy for improved lung cancer therapy and the exploration of a new nanomedicine drug design that exploits the TME [
232] (
Table 6).
Similarly, novel NPs for neutrophil detection have been extensively investigated. For example, protease-sensing NPs were used to monitor the dynamics of NE during lung infection and assess the efficacy of protease inhibitor therapy targeting NE in the treatment of α1 antitrypsin deficiency [
233]. Near-infrared NPs can detect infiltrating neutrophil MPO activity [
234]. Moreover, by combining human NE (HNE)-specific peptide substrates, quantum dots, and organic dyes, a fluorescence resonance energy transfer system capable of the
in vitro detection and
in vivo imaging of HNE was generated [
235].
6.2. Nanotechnology targeting neutrophils in immunotherapy
Research into the use of NPs as cancer therapeutics has focused on their use as delivery platforms for conventional chemotherapeutic agents [
26], [
236], [
237]. NP immunotherapy is among the least explored topics in this area and holds significant promise in oncology [
25], [
238]. NPs can interact with and be taken up by innate immune cells to become immune-stimulatory agents that modulate the characteristics of natural immune cell populations [
34], [
239]. A recent study found that inhaled empty self-assembled cowpea mosaic virus (eCPMV) reduced lung metastasis from melanoma while producing potent systemic antitumor immunity against the primary tumor. The CPMV self-assembled into a 30 nm icosahedral structure consisting of 60 copies, each containing a different size of capsid protein unit. Inhaled eCPMV NPs were rapidly taken up by neutrophils in the TME, increasing the frequency of CD11b
+Ly6G
+-activated neutrophils and CD11b
+CD86
high tumor-infiltrating neutrophils, which have potential antigen presentation and T cell priming ability. In this way, both the percentage (from 3% to 21%) and the total number of CD45
+ cells were significantly increased, inhibiting the tumor lung metastasis of melanoma by promoting regulatory T cell activation through modulating T cell receptor activation in response to antigen presentation. CD11b
+CD86
high neutrophils can kill tumor cells by releasing reactive oxygen intermediates, priming CD4
+ T cells and inducing them toward a Th1 phenotype, and modulating NK cell function. CD11b
+CD86
high neutrophils can produce CXCR3, CXCL9, and CXCL10, which recruit CD4
+ and CD8
+ T cells and generate an antitumor immune microenvironment [
151]. In addition, NP-mediated NE inhibition may improve the efficacy of drugs that inhibit NET formation and thus reduce tumor metastasis [
177].
Due to their inherent properties and drug-loading characteristics, NPs have become important tools in neutrophil detection and disease treatment. As research on NPs continues to intensify, we expect many new applications of NPs to be elucidated.
7. Conclusions and perspectives
Although multiple distinct populations of neutrophils have different functional characteristics, their lack of specific surface markers makes them challenging to distinguish. Neutrophils are highly plastic cell types that can respond rapidly to various stimuli in order to adapt to environmental changes, particularly in disease. Therefore, some researchers believe that neutrophil subpopulations may represent manifestations of neutrophil plasticity in response to disease stimulation rather than being naturally distinct populations. As research has progressed, discoveries have emerged that disprove traditional perceptions regarding neutrophils; for example, neutrophils have been classified into five (N1-5 or hN1-5) and six (mN1-6) subsets in humans and mice, respectively, rather than only being classified as N1 and N2 TANs [
16]. Furthermore, CD36, CD41, CD61, and CD226 may be new markers that can differentiate LDNs from HDNs [
113]; however, more evidence is needed to construct a new theoretical framework.
Neutrophils can promote tumor development through multiple pathways. After recruitment by the TME, neutrophils are stimulated by cytokines and chemokines, which alter the neutrophil properties. These altered neutrophils influence the recruitment and activation of inflammatory cells in the TME by releasing cytokines and enzymes. Subsequently, they create a beneficial immunosuppressive microenvironment for tumor development, metastasis, and angiogenesis, while playing an important role in the prognostic assessment of patients with tumors. Different types of neutrophils have varying functional mechanisms. PMN-MDSCs promote tumor progression primarily through immunosuppressive effects. In addition, they can induce T and NK cell incompetence, EMT, and MET, and can promote angiogenesis. TANs can promote tumor progression and inhibit antitumor immune responses through tumor suppressors, ROS, MMPs, NE, angiogenic elements, and numerous other factors [
240], [
241], [
242], [
243]. In some cases, TANs can also be converted to an antitumor phenotype [
244]. The antitumor activity of neutrophils is mainly attributable to the release of proinflammatory and immune-stimulatory molecules, such as IL-12 and TNF-α, or to TGF-β inhibition; these induce T cell recruitment and support tumor suppression. In addition, circulating CD66b
+ neutrophil populations can have immune-suppressive or proinflammatory functions in some acute and chronic inflammatory conditions and in the peripheral blood of patients with cancer, infections, or autoimmune diseases [
98], [
103], [
245].
Nano-medicines, which represent a new treatment approach in the field of cancer, have been extensively studied due to their potential advantages over traditional treatments [
230], [
246], [
247], [
248], [
249]. Nano-drug delivery systems can carry drugs to target sites and release them precisely to achieve better therapeutic effects and reduce drug side effects. Simultaneously, NP drug delivery systems can maintain stable blood levels in organisms by controlling the drug release rate. In recent decades, the rapid development of nanotechnology has led to the design and construction of nanoplatforms for various biomedical applications, including disease diagnosis and treatment [
250], [
251], [
252], [
253], [
254], [
255]. Meanwhile, extensive research has demonstrated that NP-mediated delivery improves drug efficacy
in vivo, decreases the clinical signs of lung injury, and reduces proinflammatory cytokines in the serum [
256], [
257], [
258], [
259].
Tumor cells release chemokines, which attract neutrophils from the peripheral blood to the local TME, generating a local inflammatory environment that promotes tumor progression. Therefore, detecting neutrophils in tumors using nanotechnology and directly targeting neutrophils through nano-drug delivery systems to inhibit tumor progression have become hot research topics [
232]. Many NPs have been used to detect neutrophil content and activity according to the NPs’ properties [
233], [
234], [
235], [
260]. For example, NE-based delivery vehicles can be primed with chemoattractants and migrate along a chemotactic gradient toward tumor cells infiltrating in the inflamed brain, then deliver drugs efficiently into tumor cells and induce cytotoxicity to inhibit tumor recurrence [
194]. MPO has been widely used to develop molecular-based imaging probes that are retained at sites of inflammation by MPO-induced dimerization and protein attachment [
261]. With continuous updating, we believe that nanotechnology will become an indispensable tool for treating lung malignancies.
Acknowledgments
This work was financially supported by the National Natural Science Foundation of China (31971318, 21876205, 22027810, and 32101091), China Postdoctoral Science Foundation (2021M690043), the Key-Area Research and Development Program of Guangdong Province (2020B0101020001), the Chinese Academy of Sciences (CAS) Key Research Program for Frontier Sciences (QYZDJSSW-SLH022), the CAS Interdisciplinary Innovation Team, and Big Data Program of PLA General Hospital (2017MBD-016).
Authors’ contribution
Jian Zhang, Shasha Jiang, Shilin Li, Jipeng Jiang, Jie Mei, Yandong Chen, Yongfu Ma, Yang Liu, and Ying Liu contributed to writing this paper. All authors read and approved the final manuscript.
Compliance with ethics guidelines
Jian Zhang, Shasha Jiang, Shilin Li, Jipeng Jiang, Jie Mei, Yandong Chen, Yongfu Ma, Yang Liu, and Ying Liu declare that they have no conflict of interest or financial conflicts to disclose.
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