Neutrophil phenotypes and functions in cancer: A consensus statement

1Neutrophils are polymorphonuclear granulocytes of the innate immune system that are the first line of defense to fight infection and maintain tissue homeostasis. They differentiate within the bone marrow (BM) to yield short-lived cytotoxic cells whose ebbs and flows in the vasculature and tissues are diurnally regulated. Neutrophils are highly abundant in circulation, accounting for up to ∼70% of all peripheral leukocytes in humans and ∼10–20% in mice. Rough estimates suggest humans produce ∼1 billion neutrophils daily per kilogram of body weight at steady state, and this may extend to 10 billion under inflammatory conditions. Neutrophils contain granules with an arsenal of cytotoxic factors, such as antimicrobial compounds, serine proteases, lysozyme, defensins, metalloproteases, and enzymes that mediate oxidative burst. Their principal innate functions include degranulation, phagocytosis, and release of neutrophil extracellular traps (NETs; expelled DNA webs decorated with microbicidal proteins such as myeloperoxidase, elastase, and defensins). In addition, emerging studies indicate that some neutrophils can present antigens, co-regulate T cell responses, and kill in an antibody-dependent manner. Usually, neutrophil cytotoxicity is beneficial and necessary to fight off infection; however, under chronic conditions, it can cause collateral tissue damage, particularly within highly vascularized tissues.

2Neutrophils are recruited to sites of sterile injury in large numbers, challenging the notion that these cells are exclusively antimicrobial and raising the possibility that they actively orchestrate tissue repair. This includes cancer, where various pro-tumorigenic functions of neutrophils have been described. Notably, neutrophils promote almost every aspect of cancer progression, such as primary tumor growth and metastasis, cancer stem cell maintenance, exit from dormancy and cell cycle progression, impaired immunosurveillance, and therapeutic resistance. Nevertheless, other studies have found that neutrophils can have an anti-tumorigenic role, including cytotoxicity against tumor cells and enhanced tumor cell clearance, particularly in early-stage disease. Given the discordant mechanisms by which neutrophils can influence cancer, it is apparent that we lack a fundamental understanding of how neutrophil biology shifts in the context of malignancy.

3This consensus statement follows a meeting at the Banbury Center at Cold Spring Harbor Laboratory focused on new and emerging concepts in the field of neutrophils in cancer. Here we summarize and expand on those discussions by reviewing current literature on neutrophil heterogeneity, discrepancies in the field, and open questions requiring further investigation.

4Classical views of neutrophils in cancer have adopted a binary classification system that compartmentalizes neutrophils as either pro- or anti-tumorigenic. For many years, this has served as a satisfactory working hypothesis; however, with emerging research, it is clear that this polarized paradigm is inadequate. Akin to our evolving understanding of macrophage diversity, recent research has identified diverse neutrophil states with widespread functionality. The effects of neutrophils on tumor biology have been covered by several excellent reviews. Therefore, we focus our discussion on the reciprocal effect, i.e., how the tumor and host environments regulate neutrophil heterogeneity (quantity and quality) to yield highly diverse cellular states with broad functionality.

5Tumor-induced neutrophilia requires signals to expand neutrophil progenitor pools and mediate chemotaxis. Two CSFs critical for granulopoiesis are granulocyte (G)-CSF (CSF-3) and GM-CSF (CSF-2; ). Early studies identified that highly metastatic tumors were capable of secreting G-CSF to stimulate the accumulation of neutrophils to promote metastasis. The effect of tumor- or stroma-derived G-CSF on Ly6G+ or Gr1+/hi granulocytes has since been confirmed by many additional groups, and it is now known that mechanistically, tumor-derived G-CSF skews hematopoiesis within BM toward the myeloid lineage resulting in elevated systemic frequencies of both immature and mature neutrophils with immunosuppressive properties in mice. G-CSF production by tumors can be regulated upstream by IL-23 and IL-17 supplied by phagocytes and T cells, respectively. Additional studies have suggested a similar role for tumor- or stroma-derived GM-CSF in promoting the expansion of neutrophils and their progenitors in association with cancer progression. The central role of these CSFs in tumor-associated inflammation mirrors that of emergency granulopoiesis, a survival mechanism to systemically control disseminated infections when the immune system becomes maximally challenged. As such, questions have emerged over the use of G/GM-CSF in cancer patients following chemotherapy-induced myelosuppression. This is now being explored in retrospective studies, in which, fortunately, G-CSF does not appear to exacerbate brain metastasis in patients with de novo stage IV breast cancer; however, these analyses need to be expanded to additional cancer contexts.

6Activation of the chemokine receptor CXCR2 is a key event for neutrophil mobilization from BM, whereas BM retention is regulated by CXCR4. The coordination of CXCR2 and CXCR4 is regulated diurnally and underlies circadian pattern of neutrophil flux in steady state. In human cancer, the CXCR2 ligand CXCL8 (IL-8) is abundantly secreted by various tumor types and is sufficient to regulate neutrophil recruitment and NETosis. Consistently, CXCR2 inhibitors can reduce tumor-associated NETs in models of melanoma, breast cancer, and colorectal cancer. Neutrophil CXCR2 signaling has been linked to adaptive immune responses by CD8+ T cells, and there is interest in combining CXCR2 inhibitors with immunotherapy. For example, in models of colitis-induced tumorigenesis, CXCR2 signaling within Ly6G+ myeloid cells suppresses tumoricidal functions of CD8+ T cells, as assessed by CD107a, IFNɣ, Prf1, and Gzmb. In murine oral, renal, and pancreatic tumor models, CXCR2 inhibition results in neutrophil depletion coinciding with increased survival, T cell infiltration, and immunotherapy response. In preclinical models of childhood cancers such as rhabdomyosarcoma, CXCR2+Ly6G+ cells mediate local immunosuppression, while CXCR2 inhibition improves immune checkpoint blockade efficacy. Similarly, retrospective studies have shown that serum IL-8 is an independent biomarker of reduced efficacy of immune checkpoint inhibitors in patients, and coincides with increased neutrophils within tumors. Preclinical studies have implicated other CXCR2 ligands in neutrophil recruitment to tumors, and it is possible that these may serve as additional biomarkers in patients. In the era of cancer immunotherapy, these findings highlight the importance of understanding neutrophil dynamics as they relate to anti-tumor T cell responses.

7In addition to specific tumor-supplied factors, genetic events within cancer cells underlie a broader shift in the tumor secretory profile with compounded effects on microenvironmental composition. One such event is Tp53 loss, which has pronounced effects on the tumor myeloid landscape. In breast cancer, loss of Tp53 upregulates the secretion of WNT ligands, which stimulate macrophages to produce IL-1β, mediate neutrophilic inflammation, and potentiate metastatic progression. In lung cancer, Tp53 loss and Kras activation lead to elevated levels of the receptor for advanced glycation end products (RAGE) within blood, which educates distant osteoblasts to stimulate the expansion of long-lived pro-tumorigenic SiglecFhi neutrophils (in contrast, SiglecFlo neutrophils are enriched in healthy lung;; ). In Kras-driven pancreatic cancer, loss of Tp53 stimulates the production of myeloid chemokines, including CXCR2/3 ligands, resulting in the recruitment of immunosuppressive CD11b+ myeloid cells. In prostate cancer, the combination of Pten and Tp53 loss enhances CXCL17 secretion and recruitment of CD11b+Gr1+ immunosuppressive neutrophils. Combined loss of Pten and Tp53 is also linked to CD11b+Gr1+ myeloid cell accumulation in breast cancer models. These studies demonstrate that loss of Tp53 in cancer cells, either alone or combined with additional genetic events, leads to an infiltrative myeloid microenvironment coinciding with tumor progression. Other genetic driver mutations might influence the neutrophilic immune landscape, such as oncogenic Kras mutations that enhance IL-8 production. However, in comparing each study, neutrophil phenotypes were highly diverse, underscoring the regulatory relationship between tumor genotypes and neutrophil states in cancer.

8One of the first preclinical studies to discriminate between neutrophil states in cancer described N1/anti-tumorigenic and N2/pro-tumorigenic neutrophils within tumors, whereby transition to N2 was regulated by TGF-β. TGF-β acts in part through its ability to regulate pro-inflammatory cytokines and neutrophil chemoattractants, such as CXCR2 ligands CXCL1/2/5, and may even synergize with CSFs in BM. Although TGF-β can be supplied by the tumor itself, it can also be derived from microenvironmental sources, including myeloid cells. Subsequent studies ascribed the terms high-density neutrophils (also known as “normal density”) or low-density neutrophils to anti/pro-tumoral subsets, based on buoyant density, granularity, and maturation state. These density fractions were first described in early work in autoimmunity and later refined using mass cytometry (cytometry time of flight [CyTOF]) to identify subpopulations within each fraction associated with cancer outcomes. Like N2 neutrophils, the low-density neutrophil subset is driven by TGF-β and exhibits immunosuppressive properties in cancer, and is associated with advanced disease. These early discoveries were among the first to challenge our understanding of neutrophil plasticity and heterogeneity in cancer.

9Since then, many studies have identified unique neutrophil phenotypes in cancer. Single-cell RNA sequencing (RNA-seq) of patient lung tumors identified five neutrophil clusters that only partially overlap with those in blood. Of these, three clusters are conserved in mice, including one that is uniquely found in tumors and not healthy tissue, with high levels of Ctsb and Ccl3 (termed N5; see Fig. 2 A;; ). Similar observations were made with CyTOF on blood samples from melanoma and lung cancer patients, where at least five neutrophil states were identified. Specific neutrophil states are associated with patient outcomes; for example, N5 neutrophils (detected by immunostaining for peptidase inhibitor 3) are associated with tumor growth and worse prognosis in lung cancer patients, and partially resemble pro-tumorigenic SiglecFhi neutrophils in mice. Moreover, distinct functional proficiencies can be ascribed to each state, including differential capacities for phagocytosis and ROS production. However, studies exhaustively testing additional functions, such as NETosis, have been limited, as most research is focused on the bulk analysis of neutrophils. As single-cell technologies have gained considerable momentum in recent years, expanding our mechanistic understanding of how distinct neutrophil states may yield unique effects in cancer immunology will be important for developing neutrophil-targeted immunotherapies. Moreover, it will be critical to understand the contribution of distinct neutrophil states to immunotherapy-related adverse events, which often necessitate discontinuation of these therapies.

10ROS production by neutrophils has several functional consequences, one of which is NETosis. In laboratory models, neutrophils from mice with leukemia, breast cancer, or lung cancer are prone to NETosis compared with those from healthy mice. Similar observations have been reported in humans; in esophageal, gastric, and lung cancer patients, NETs are elevated in blood compared with healthy individuals. Although these observations are associative, emerging work is now addressing the possibility that tumors play a causative role in NETosis. Moreover, it is becoming clear that NETs that form in response to malignancy have multifaceted effects. Early studies have shown that cancer-induced NETs act largely within the circulation, where they facilitate cancer-associated thrombosis and sequester circulating tumor cells to escort metastases. Subsequent studies revealed that NETs affect essentially every step of the metastatic cascade, including primary tumor progression, invasion and migration, survival in the circulation, chemotaxis to secondary niches, extravasation, metastatic colonization, and outgrowth of metastatic tumor cells. However, a remaining knowledge gap is how tumors trigger NETosis—Is this a direct consequence of tumor-derived factors? Is it a systemic response to cancer-associated inflammation? Or is it a broader shift in neutrophil developmental and aging programs? Co-culture experiments with tumor cells and NETosing neutrophils have hinted toward tumor-supplied factors, such as G-CSF, IL-8, or cathepsin C, but deeper mechanistic insights are needed to dissect contributions from developmental and diurnal neutrophil states. To date, there are no clinical trials testing NET inhibitors in cancer patients; however, clinical trials with recombinant human DNase1 are being conducted in patients with COVID-19 (NCT04409925, NCT04359654, NCT04445285, NCT04541979, NCT04432987, and NCT04402944), which will provide a foundation for translating NET-targeted therapies to immuno-oncology.

11Neutrophils can adopt immunosuppressive functions both systemically and within the tumor microenvironment. This is partially due to metabolic reprogramming of neutrophils in the context of cancer, where nutrient sharing between cells and tissues becomes subverted to accommodate the high energy demands of a rapidly growing tumor. The prevailing dogma is that neutrophils are almost exclusively glycolytic, and while this is true in some cancer settings, it is not always the case. In the 4T1 transplantable mammary tumor model, the glucose-restricted microenvironment causes neutrophils to utilize mitochondrial fatty acid oxidation, leading to enhanced ROS production, T cell suppression, NETosis, and liver metastasis. Moreover, in the spontaneous MMTV-PyMT breast cancer model, neutrophils secrete leukotrienes, which are lipid products of Alox5-mediated oxidation of arachidonic acid, to promote tumor survival and colonization within the pre-metastatic niche. Changes in lipid metabolism in neutrophils have also been observed in mouse models of lung cancer, colon cancer, and rhabdomyosarcoma, and in patients in association with immunosuppression. Lipid uptake by neutrophils has been reported in several cancer models via fatty acid transport protein 2 (FATP2) or adipose triglyceride lipase, which not only causes immunosuppression of antigen-specific CD8+ T cells, but also creates an energy reservoir for metastasizing cells. Lipid uptake can also be regulated by tumor-derived G/GM-CSF. Elegant models using a cell-penetrant fluorescent labeling system to study cellular neighbors of metastatic breast cancer cells in the lung microenvironment have shown that neutrophils within the immediate niche of the tumor exhibit elevated oxidative phosphorylation and ROS production, although immunosuppressive functions in this context have not been evaluated. Of note, the immunosuppressive consequences of low glucose levels in the tumor microenvironment are not restricted to granulocytes and may extend to regulatory T cells, dendritic cells, and M2-like macrophages.

12Neutrophil biology is strongly influenced by variables in host physiology, such as sex, age, circadian rhythms, and anatomical location (Fig. 1). For example, peripheral neutrophils with an immature phenotype are elevated in young men compared with young women and exhibit increased mitochondrial metabolism. In contrast, women have more mature neutrophils with a heightened capacity for activation, including hyperresponsiveness to type I IFNs and enhanced capacity for NETosis. Of note, ex vivo NETosis assays with mouse neutrophils have reported the opposite effect. Additionally, neutrophil phenotypes are not static; sex dimorphism is lost with aging, and in females, the immature neutrophil phenotype is enhanced during pregnancy as estrogen levels rise. Interestingly, estrogen signaling can promote intratumoral immunosuppressive activity of estrogen receptor (ER)–positive Ly6CloLy6G+ myeloid cells and enhance cancer progression in mice, even in ER-negative tumors. At the molecular level, multi-omics analyses of male versus female BM neutrophils in mice have confirmed sex dimorphic differences: transcriptomics revealed a female bias for extracellular matrix and cell surface–related pathways and a male bias for chromatin and cell cycle–related pathways; metabolomics showed differences in nucleotide and amino acid metabolism; and lipidomics showed a male bias for increased lipid storage. These fundamental differences are likely to mechanistically underlie sex dimorphic functional variations observed in steady-state, autoimmunity, and infection; however, the impact on tumor biology remains unexplored.

13Although neutrophils are highly sex dimorphic throughout life, the effects of aging on neutrophils appear to be similar, regardless of sex. Aging is associated with chronic, low-grade inflammation leading to a gradual decline in immune function over time, which likely underlies the higher incidence of infection, autoimmunity, and cancer in the elderly. In mouse models, hematopoietic stem cells (HSCs) in BM are skewed toward myeloid lineage specification both in steady-state and following transplantation of aged HSCs into young mice, suggesting that the age-related myeloid bias is cell intrinsic. Similar observations have been made in humans by comparing elderly (65–85 yr) and young (20–31 yr) individuals. In mice, analysis of male and female BM neutrophils from young (4–5 mo) and old (20–21 mo) mice has shown that aging is associated with significant changes to neutrophil gene expression, with relatively minimal changes to metabolomic or lipidomic profiles. Aging downregulates pathways related to chromatin organization, despite no functional differences in cell-cycle phase distribution, suggesting possible relevance to NETosis. Pathways related to autophagy, which are critical for normal neutrophil differentiation and function, are also upregulated with aging. Within some lymphoid organs, including BM, LNs, and spleen, neutrophil frequencies increase in aged mice (22–24 mo; equivalent to 60–70 human yr) compared to young mice (2–3 mo; equivalent to 18 human yr), yet become functionally dysregulated (e.g., display reduced phagocytosis, increased senescence, and dysregulated NETosis;;;; ). It remains unclear how phenotypes reported in young mice (which are typically used in cancer research) compare with those in aged mice where neutrophil function may be intrinsically different. It is possible that this variable contributes to the differences observed between neutrophils in mouse models and humans (discussed in Neutrophils with an anti-tumorigenic…; ).

14In both males and females, neutrophils are replenished diurnally, and the circadian regulation of neutrophil aging translates to distinct phenotypes at different times of the day. In mice, neutrophils newly released into the circulation (“fresh” neutrophils) exhibit enhanced migratory programs including an enhanced capacity for vascular rolling, adhesion, and extravasation into inflamed tissues, whereas neutrophils toward the end of their lifecycle (“aged” neutrophils) show enhanced antimicrobial activity and can be retained within healthy tissues during periods of organismal activity (note that for mice, the active period is at night;; ). In steady-state, circulating neutrophils “disarm” their cytotoxic functions as they age by progressively degranulating; this phenomenon likely makes them less harmful once they reach tissues and may help offset potential collateral damage to highly vascularized niches, including lung. These changes are dependent on CXCR2, which, in addition to its functions above, is a master regulator of neutrophil diurnal activation. A functional consequence of these time-dependent properties is that metastatic events may be diurnally regulated. Elegant circadian experiments have shown that B16F1 melanoma cells injected intravenously into syngeneic mice in the morning form overt lung metastases while injections in the evening yield minimal metastatic disease, and this difference is mitigated when neutrophils are depleted. These findings raise the possibility that the efficacy of neutrophil-targeted therapies may be different depending on the time of administration, and may even extend to other therapies that are influenced by neutrophil function. This concept was recently explored in the context of immune checkpoint blockade in melanoma patients, where it was suggested that daytime infusions may be more effective than evening infusions.

15Neutrophil aging is regulated in part by intrinsic factors, such as the molecular clock transcription factor Bmal1, and also extrinsic factors, such as the microbiome, which neutrophils can sense through signaling from their pattern recognition receptors. The microbiome regulates neutrophil differentiation and function during infection. Germ-free and antibiotic-treated mice have reduced frequencies of circulating aged neutrophils, whereas this phenomenon is reversed by fecal transplantation or LPS administration. These studies suggest that dysbiosis, obesity, antibiotic use, or other factors that modify the microbiome may alter neutrophil activity both in steady-state and in response to inflammatory stimuli, including cancer. A role for the gut microbiome in regulating cancer progression and response to immunotherapy is also emerging in humans. For instance, two-phase I/II clinical trials found that fecal microbiota transplantation can sensitize previously refractory cancer patients to immune checkpoint inhibitors. These responses were associated with improved T cell infiltration and activation within tumors, and it is likely that innate immune components, including neutrophils, are also involved, given their ability to directly sense bacteria.

16Neutrophils patrol healthy tissues in steady-state, and there is tissue specificity to the dynamics of neutrophil infiltration and phenotype. At homeostatic baseline, neutrophils are abundant in BM, spleen, and lung, and with lower frequencies in liver, intestine, muscle, skin, and white adipose tissue. Studies have also identified neutrophils that recirculate through LNs where they protect against infection within the lymphatics. Parabiosis experiments with CD45.1 and CD45.2 mice showed that blood-derived neutrophils accumulate and are retained in most tissues in the evening, whereas the intestine and liver show minimal rhythmicity. Neutrophil spatial patterning is also tissue specific, where distribution within tissues appears to be somewhat random in most organs, whereas the intestine and spleen exhibit a more purposeful localization pattern. Functionally, neutrophil half-life is dramatically distinct between tissues; the total lifespan ranges from 28.9 h in liver (8.7 h half-life) to 67.1 h in BM (20.2 h half-life), with blood, spleen, lung, intestine, and skin ranging in between. Similarly in humans, a lifespan of up to 5.4 d has been reported, though this remains controversial. Moreover, tissue-specific analyses of RNA, protein, and chromatin have revealed striking heterogeneity between mouse neutrophils from distinct anatomical sites, including BM, lung, intestine, skin, spleen, and blood. Together these findings strongly challenge the prevailing dogma that neutrophils are functionally uniform cells with rapid turnover. Despite neutrophils being the most abundant myeloid cell type in the body, a comparison of how tissue-specific properties of neutrophils might differentially impact cancers arising in different organs has not been explored.

17Pathologic conditions add further complexity to the influence of host physiology on immune function (Fig. 1). One condition with high relevance to oncology is infection, given the relationship between postoperative infection and poor outcomes. Surgical stress, itself even in the absence of infection, is sufficient to trigger NETosis in liver ischemia-reperfusion models, leading to accelerated metastatic progression. In a mouse model of intra-abdominal sepsis by cecal ligation and puncture, it has been shown that neutrophils exhibit enhanced NETosis, which facilitates trapping of tumor cells in blood and aids in the establishment of hepatic metastases. Interactions between NETs and circulating tumor cells were achieved through the expression of β1-integrins on tumor cells, which are upregulated in response to infection. Infection-enhanced metastasis can be mitigated by treatment with DNase1 in vivo, which degrades extracellular DNA to prevent NETs from escorting tumor cells into the metastatic niche. Additional studies have shown that inflammation following treatment with bacterial LPS promotes neutrophil-mediated pulmonary metastasis. This occurs in part by triggering NETosis, which enables dormant metastatic cells to re-engage their proliferative capacity or by enhanced degranulation, resulting in secretion of proteases, such as elastase and cathepsin-G, that cleave anti-tumorigenic thrombospondin-1 (Tsp-1; ). Given the diversity of neutrophil states, an open question is whether neutrophils uniformly respond to infection. Single-cell RNA-seq has shown that although bacterial infection primes neutrophils for activation at the transcriptional level, it does not affect core gene signatures that distinguish subpopulation identities. However, it is unclear which subpopulation of neutrophils is primarily responsible for cancer-associated NETosis and how this may change during infection. Outside the context of cancer, it is established that bacterial infection and sepsis promote NETosis; for example, in mouse models of hemorrhagic and septic shock, extracellular cold-inducible RNA-binding protein, an endogenous damage-associated molecular pattern, stimulates a unique subset of ICAM1+ neutrophils to exhibit enhanced NETosis and reverse-transmigration. Whether ICAM1+ neutrophils play a specialized role during cancer metastasis by virtue of their inflammatory properties remains unknown.

18Cigarette smoke is another external stimulus that has systemic effects on host immune responses and is the leading preventable risk factor for cancer mortality, accounting for ∼30% of all cancer deaths. In addition to direct genotoxic effects of cigarettes on the lung epithelium, there are dramatic effects on the lung immune landscape. Nicotine promotes the recruitment of N2-like neutrophils to the pre-metastatic lung, where they support mesenchymal-to-epithelial transition of incoming tumor cells, thereby facilitating colonization. Exposing mice to nicotine or tobacco also causes neutrophils to undergo NETosis. Proteases within NETs, including neutrophil elastase and MMP9, subsequently cleave laminin within the extracellular matrix to facilitate integrin signaling and proliferation of dormant cancer cells within the lung. Genotoxic properties of additional components of cigarette smoke, such as urethane, may also be amplified by neutrophils. Urethane directly induces neutrophil ROS, which exacerbates DNA damage and proliferation of the lung epithelium. As a consequence, urethane-induced lung tumorigenesis is blunted in neutropenic Gcsf−/− mice, and remarkably, when neutrophils are transiently replenished with recombinant G-CSF treatment during urethane exposure (∼1 wk), this is sufficient to rescue lung tumorigenesis up to 4 mo later. These findings implicate neutrophils in tumor initiation in smokers’ lungs and corroborate previous work showing a role for neutrophils during neoplastic transformation in other tissues.

19Finally, obesity is another major contender for the top modifiable risk factor for cancer incidence and mortality, estimated to be responsible for up to ∼20% of all cancer deaths. The adipose tissue microenvironment undergoes widespread immunological remodeling during weight gain, which regulates systemic inflammatory changes that contribute to metabolic syndrome. In the non-tumor bearing setting, obesity stimulates myelopoiesis to yield elevated neutrophils and Gr1+ cells in multiple organs. Lung neutrophils from obese and lean mice have a highly divergent transcriptome, with a significant enrichment in pathways related to oxidative stress in obese hosts coinciding with enhanced NETosis. In cancer, both genetic- and diet-induced obesity models have elevated peripheral and pulmonary neutrophils that promote breast cancer metastasis to the lung in a GM-CSF–dependent manner. Similar findings have been reported in murine models of high fat diet–induced hypercholesterolemia, where the cholesterol metabolite 27-hydroxycholesterol supports neutrophil recruitment to distal sites to promote metastasis. Consistently, it has been shown that a high-fat diet promotes the accumulation of Gr1+ myeloid cells within multiple tissues concomitant with suppressed CD8+ T cells and enhanced cancer progression. In a liver cancer model driven by non-alcoholic fatty liver disease/non-alcoholic steatohepatitis, live imaging studies have shown that a high-fat diet promotes neutrophil infiltration into the liver, and that treatment with the anti-diabetic drug metformin is sufficient to reverse this effect and reduce early cancer progression. Human studies have similarly found that obesity is associated with peripheral neutrophilia concomitant with elevated markers of neutrophil activation such as myeloperoxidase and neutrophil elastase. Importantly, obesity interventions, such as bariatric surgery, exercise, or diet, have been linked to reduced cancer incidence and/or mortality in association with decreased circulating inflammatory markers.

20Taken together, many host conditions influence neutrophil heterogeneity and function—this includes infection, smoking, and obesity as discussed, but also extends to other chronic inflammatory conditions, seasonal viral infections, the gut microbiome, severe COVID-19, and stress, among other factors. The combination of pathogens and inflammatory stimuli we are exposed to throughout life is unique between individuals and contributes to trained immunity. Moreover, the effects of cancer genotypes compounded with diverse host conditions and environmental exposures underlie highly complex neutrophil heterogeneity, as has been observed by many groups. The role that each of these different factors plays in tumor progression is a growing area of investigation.

21It has been paradoxically observed that tumor-associated neutrophils can be protective against cancer. In some cases, tumor cells succumb to neutrophil cytotoxicity and are thus effectively cleared. Neutrophil tumoricidal functions can be blunted by catalase, suggesting a role for H2O2. Consistently, elevated expression of the H2O2-dependent calcium channel, transient receptor potential cation channel-M2, sensitizes metastatic tumor cells to neutrophil cytotoxicity, while sparing cells at the primary tumor site. The discrepancy in susceptibility to neutrophil cytotoxicity in primary versus secondary tumors can be explained in part by cellular functions required for dissemination; metastatic tumor cells become susceptible to neutrophil cytotoxicity following epithelial-to-mesenchymal transition, a process regulated by TGF-β signaling, echoing earlier studies implicating this pathway in neutrophil functional diversity. Beyond oxidative stress, neutrophils utilize additional ammunition to kill tumor cells, including granule enzymes such as elastase or cathepsin-G. In addition, neutrophils can kill antibody-opsonized tumor cells via trogocytosis, which could translate to tumor-targeting antibody therapeutics. These studies suggest that harnessing neutrophil tumoricidal functions, including innate immune checkpoints, may help combat cancer. However, they also exemplify that some neutrophil-derived mediators, such as elastase or ROS, can elicit both pro- and anti-tumoral effects depending on context—a concept that is still a puzzle in the field.

22Complementing cytotoxicity, neutrophils can conspire with the adaptive immune system to facilitate tumor cell recognition and clearance. Co-culture experiments using cells from early-stage human lung cancer have shown that tumor-associated neutrophils enhance T cell activation compared to their blood-borne counterparts. In turn, activated T cells prolong the lifespan of neutrophils in vitro and upregulate co-stimulatory molecules on the neutrophil plasma membrane, leading to a positive-feedback loop that perpetuates T cell stimulation. In subsequent work, a specialized subset of tumor-associated HLA-DR+ neutrophils was identified in early-stage tumors, which is capable of cross-presenting exogenous tumor antigens to CD8+ T cells to stimulate tumor-specific effector T cell responses. Others have similarly reported that FcɣR engagement converts neutrophils into antigen presenting cells that cross-present to CD8+ T cells to induce anti-tumor immunity in melanoma models. However, over time, the ability of tumor-associated neutrophils to engage adaptive immune pathways declines as tumors progress. These data support a model where neutrophils undergo an immunogenic “switch” from anti-tumorigenic to pro-tumorigenic as cancer advances; however, this temporal response may be context specific. For example, in mouse models of melanoma, neutrophils maintain some anti-tumorigenic properties in the advanced disease under specific therapeutic settings. Combining a triple therapy of chemotherapy, infusion of CD4+ T cells specific to the melanoma antigen Trp1, and co-stimulation or immune checkpoint blockade is sufficient to eliminate tumors, due to a significant infiltration of neutrophils that exhibit anti-tumor behavior. The increase in these anti-tumorigenic neutrophils was associated with an increase in cutaneous immune adverse events. In human melanoma samples, treatment with immune checkpoint inhibitors is associated with enhanced NETosis, which has been proposed as a potential mechanism to eradicate tumor antigen escape variants that arise in response to treatment selection pressure (often in concordance with immune-related adverse events; ). Although many studies have characterized the role of neutrophils in cancer progression, how neutrophils are altered in response to specific therapies at different stages of disease remains unanswered and needs further exploration.

23Many anti-tumorigenic neutrophil functions have been described in humans, raising questions about species-specific roles in cancer. Single-cell RNA-seq of human and mouse neutrophils from lung tumors showed considerable, but not complete, overlap between species. At the functional level, several differences have been reported. First, the NETosis potential of circulating neutrophils from mice versus patients with cancer is distinct, including their ability to release NETs in response to G-CSF. Second, the tumoricidal functions of neutrophils may be species specific, based on differences in their granule/secretory profile or utilization of distinct ROS pathways. Third, mouse and human neutrophils differ in their expression of Arg-1 and ability to metabolize arginine. This could affect T cell responses, as decreased arginine availability in the tumor microenvironment is associated with T cell immunosuppression. Another explanation for the protective role of neutrophils in cancer is the possibility of stage-dependent enrichment for specific neutrophil states. For example, when comparing the immune infiltrate of highly and poorly metastatic murine tumors, both increase myeloid infiltration in the pre-metastatic lung; however, poorly metastatic tumors also secrete prosaposin, which induces Tsp-1 expression in lung-infiltrating Gr1+ cells to impair metastasis. Similar mechanisms between early and advanced tumors in patients have yet to be compared.

24A major source of neutrophil heterogeneity comes from the dynamics of their maturation and release from BM. Neutrophils arise from HSCs in BM, which give rise to multipotent progenitors, then common myeloid progenitors, granulocyte–monocyte progenitors (GMPs), and neutrophil-committed precursors (Table 1). This linear model may in part underlie our confounded understanding of neutrophil heterogeneity, as neutrophil ontogeny and phenotypic plasticity are likely to be more complex. Learning from other myeloid lineages, monocyte heterogeneity and functionality reflect both ontogeny and response to microenvironmental stimuli. Inflammatory monocytes can arise independently from both GMPs and monocyte–dendritic cell progenitors (MDPs), where GMPs give rise to neutrophil-like monocytes, while MDPs give rise to monocytes that can further differentiate into dendritic cells. Moreover, factors within the microenvironment confer a preference for monocyte maturation along one of these developmental trajectories; LPS favors neutrophil-like monocyte maturation from GMPs, while exposure to CpG favors the MDP lineage. These developmental mechanisms may in part explain the functional breadth of monocyte and macrophage identities. Indeed, the lessons that we have learned from monocytes may be relevant to a modified understanding of granulopoiesis and neutrophil plasticity.

25In recent years, single-cell technologies have made substantial advancements toward defining neutrophil maturation from GMPs. Across studies, several new terms have been coined for overlapping neutrophil developmental states, which are in need of consolidation (Table 1 and Fig. 2 A). In mouse BM, GMPs mature into pro-neutrophils (proNeu, corresponding to clusters G0/G1 in similar work) and then into highly proliferative, poorly motile precursors (preNeu) that drive expansion within spleen and BM. Cells defined as preNeu are transcriptionally similar to unipotent neutrophil progenitors (NeP; ), neutrophil precursors (NeuP; ), and to a G2 cluster that resembles myelocytes/metamyelocytes (; also see ). Subsequently, preNeu differentiate into immature (Ly6GintCXCR2−; immNeu) or mature (Ly6GhiCXCR2+; mNeu) neutrophils that are non-proliferative and mediate trafficking and effector functions. ImmNeu overlaps transcriptionally with a band cell-like G3 cluster that expresses low Cxcr2 and high secondary granule genes, and with late-stage progenitors (cluster C2; high Ly6G expression), as confirmed by adoptive transfer experiments with NeP. Mature neutrophils correspond to cluster G4 from, representing the most mature state in BM with high expression of Mmp8 and Cxcl2, while the G5 cluster was the most mature overall and predominant in blood. A summary of overlapping states can be found in Table 1.

26Neutrophil development coincides with shifts in chromatin accessibility and transcription factor activity. Developmental trajectory analysis of single-cell RNA-seq data unveiled a developmental continuum known as “neutrotime,” which spans the maturation spectrum from neutrophil precursors in BM to mature neutrophils in blood and spleen. The sharpest changes in neutrotime occur during granulopoiesis (transition from immature to mature) and mobilization (transition from BM to blood; ). Consistently, in steady-state and inflammation, neutrophil chromatin landscapes and transcription factor networks shift in waves. In steady-state, the initiation of granulopoiesis in BM involves RUNX1 and C/EBPα transcription factors within GMPs, followed by a shift to GFI1 and C/EBPε in preNeu during early neutrophil differentiation, and then C/EBPδ and PU.1 as mNeu enter the circulation. The temporal expression of C/EBP-family members mirrors the pattern of granule enzyme expression; primary granule enzymes (e.g., Mpo) are expressed at the GMP stage, secondary granules (e.g., Ltf) at the preNeu/immNeu stage, and tertiary granules (e.g., Mmp8) at the mNeu stage, corresponding to Cebpa, Cebpe, and Cebpd, respectively. In comparison, chromatin profiling of neutrophils combined with genetic validation approaches has identified transcription factors involved in neutrophil maturation during acute inflammation. For example, RUNX1 and KLF6 drive neutrophil maturation in BM, and after cells are mobilized, chromatin is remodeled to enable access by RFX2 and RELB to promote survival. Within tissues, another chromatin remodeling event enables the transcriptional activity of RELB, IRF5, and JUNB, which license innate effector functions that are pre-programmed in early developmental stages. These findings echo those from earlier work describing two major waves of chromatin remodeling between developmental transitions from (pro)myelocyte to metamyelocytes and from segmented neutrophils to polymorphonuclear neutrophils, and extend them by charting a transcriptional blueprint for each transition. The remaining knowledge gaps include understanding how environmental signals integrate with transcription factor landscapes to yield functional outputs, and how the release of neutrophils from BM at different maturation states is regulated.

27Functionally, there are still many unknowns surrounding the kinetics of neutrophil maturation, trafficking, and effector function. For example, some neutrophils are capable of reverse transmigration from inflamed tissues back into the circulation. Moreover, in cancer, multiple neutrophil developmental states co-exist: in orthotopic mouse models of pancreatic cancer, CD101− immNeu accumulate intratumorally in association with disease progression, corroborating studies that have reported a pro-tumorigenic role for neutrophils with a banded (immature) nuclear morphology. Consistently, in orthotopic melanoma models, NeP are elevated in BM, blood, and tumor, and they promote tumor growth in association with elevated expression of PD-L1. In melanoma patients, human NeP are found at higher frequencies in blood compared to healthy donors, where they are extremely rare. Even earlier developmental stages can be detected in blood and tumors of lung cancer patients, including precursors to NeP/preNeu states. Moreover, extramedullary granulopoiesis has been described in the context of cancer where neutrophil maturation in spleen yields an immunosuppressive phenotype. Therefore, shifts in the developmental states of granulocytes, their premature egress from BM at different stages (which may influence granule cargo), and their distinct functional contributions to tumor growth shed light on the high degree of neutrophil heterogeneity observed in cancer. This echoes findings in other inflammatory conditions, like systemic lupus erythematosus, where distinct neutrophil states identified through single-cell RNA-seq uniquely regulate disease pathogenesis.

28Finally, it remains unknown how trained immunity at the individual (host) level may influence this complex system. Studies in animal and plant models that lack adaptive immunity have shown that innate immune cells or their developmental precursors can become “trained” following exposure to an inflammatory stimulus, resulting in an altered response to secondary challenges even after cells have returned to a resting state. Trained immunity is thus different between individuals, depending on history of pathogen exposures, vaccination, cancer, or other inflammatory stimuli. Indeed, studies have identified β-glucan, Bacillus Calmette-Guérin, and other vaccines as prototypical agonists of trained immunity, mediated by education of myeloid cells within the BM and myeloid-bias progenitor expansion; however, trained immunity can also be achieved through sterile triggers, such as diet-induced changes to myeloid progenitor reprogramming. Recent studies have explored the role of trained immunity in cancer in the context of granulopoiesis. β-glucan induces myeloid expansion concomitant with elevated innate immune signaling mediators, such as IL-1β and GM-CSF, which mediate neutrophil expansion in tumors. Treatment of mice with β-glucan 1 wk prior to subcutaneous inoculation of B16F10 melanoma or Lewis lung carcinoma lung cancer cells into syngeneic hosts blunts tumor growth, even in Rag1−/− mice that lack T and B cells. Mechanistically, β-glucan causes epigenetic and transcriptomic reprogramming of granulopoiesis, yielding progenitor cells with enhanced IFN signaling and tumoricidal mature neutrophils with elevated ROS. Moreover, the anti-tumor effect of β-glucan is maintained and transferrable following BM transplantation or neutrophil adoptive transfer from β-glucan–treated donor mice into naive recipients. Outside the context of cancer, activated neutrophils can prime macrophages to exhibit long-term responses against parasitic infection, raising the possibility that neutrophils may participate in training of other myeloid cells in tumors. These findings support the notion that myeloid-targeted immunotherapies that aim to reprogram, rather than deplete, target cells are an attractive therapeutic approach to harnessing anti-tumor immunity.

29Owing to the complexity of neutrophil diversity, it is not surprising that we have encountered several experimental discrepancies as a field. First, amongst many possible functional states, neutrophils can be immunosuppressive, thus challenging the designation of granulocytic myeloid-derived suppressor cells (G-MDSCs; also known as PMN-MDSC) as a distinct population. G-MDSC and neutrophils are indistinguishable by archetypal cell surface markers and have identical granular and nuclear morphologies. Moreover, there are no genetic models to trace or target G-MDSCs due to lack of genetic differences from neutrophils. Instead, antibody depletion via anti-Gr1 or anti-Ly6G is often used to test their functional role in disease; however, these antibodies also deplete neutrophils, making causative functional assessment of MDSCs uninterpretable. For associative functional studies, it has been suggested that identification of MDSCs requires confirmation of immunosuppression ex vivo, but often cell surface markers are used alone. Single-cell RNA-seq studies have attempted to identify MDSC in tumors; however, putative G-MDSCs share transcriptional similarities with canonical neutrophils as defined by Immgen, as well as signatures of mature neutrophils across multiple tissues (; Fig. 2 B). Given the diversity of neutrophil identities, which can include immunoregulatory functions, it remains controversial whether G-MDSCs should simply be called “immunosuppressive neutrophils” and thus terminology is used inconsistently between groups. Although putative surface markers have been identified to distinguish G-MDSC and neutrophils such as CD84, CD14, FATP2, or LOX-1, these have not been used by the broader research community and they may be expressed by bona fide neutrophils under certain conditions. Given the lack of existing genetic and phenotypic markers to distinguish bona fide neutrophils from putative G-MDSC, it has been suggested that the monolithic MDSC terminology be dropped in favor of a more colorful view of myeloid biology that embraces the high degree of cellular and functional heterogeneity. We support this view and urge scientists to consider the literature on both neutrophils and G-MDSCs to inform ongoing research within these evolving fields.

30Second, antibody-mediated neutrophil depletion has context-dependent efficacy. The most common antibodies used are anti-Ly6G (clone 1A8, rat IgG2a) and anti-Gr1 (clone RB6-8C5, rat IgG2b); anti-Ly6G is often preferred since mature neutrophils are Ly6G+/hi, while Gr1 also targets monocytes, some immature myeloid cells, and even a subset of CD8+ memory T cells by virtue of Ly6C expression. However, in C57BL/6 mice, anti-Gr1 effectively ablates neutrophils, whereas anti-Ly6G is less effective because depletion is slower than neutrophil repopulation from BM (; Preprint). Despite this, numerous studies have used anti-Ly6G to deplete neutrophils successfully, suggesting that experimental design is key: first, dose and duration are variables that affect any mAb-based depletion approach. Short-term experiments (1–2 d) with anti-Ly6G result in highly effective neutrophil depletion; however, in longer trials (>3–7 d), neutrophil numbers may begin to rebound. Second, there are differences in neutrophil depletion efficacy depending on experimental context. Comparative analyses with anti-Gr1 and anti-Ly6G mAbs showed enhanced depletion in Balb/c and FVB/n backgrounds compared to C57BL/6, especially when C57BL/6 mice were aged (; Preprint). Moreover, neutrophils are more difficult to eliminate in certain tissues, such as BM. Third, for experimental contexts in which anti-Ly6G is ineffective, a more durable protocol has been developed via co-administration of rat anti-mouse Ly6G and anti-rat mAbs, which enhance the killing efficacy of anti-Ly6G to deplete neutrophils for at least 18 d in C57BL/6 models (; Preprint). Finally, neutrophil depletion efficacy cannot be validated using anti-Ly6G (1A8), as antigen masking produces false-negative staining. Alternative strategies include intracellular Ly6G staining, histology for myeloperoxidase (MPO) or neutrophil elastase (NE), or reporter mice, such as LysM-cre or Ly6G-cre, combined with Ly6Clo/int, and side scatter assessment. Genetic neutrophil depletion approaches, as discussed below, may also provide functional confirmation of antibody effects.

31Common genetic approaches to target or deplete neutrophils in vivo include the Mrp8-cre model, either combined with a specific floxed allele or ROSA-DTA mice (cre-dependent expression of diphtheria toxin), respectively. The Mrp8-cre model targets ∼80% of mature neutrophils in vivo; however, Mrp8 is also expressed in 10–20% of GMPs. Alternatively, the Ela2-cre model targets mature neutrophils expressing elastase, a serine protease within neutrophil primary granules. Newer models, such as the Ly6G-cre mouse, offer a more specific alternative; however, the efficacy of recombination is purportedly allele-specific depending on the particular floxed strain being used. Further, activation of recombinase under control of this gene occurs late in neutrophil development, such that proteins already produced may persist for the short lifetime of the cell. For combinations with fluorescent reporter strains for intravital microscopy or other imaging modalities (such as the Ai9 cre reporter mouse), the Ly6G-cre mouse is an excellent option for neutrophil-specific tracing. However, for gene deletion studies, this model should be used in parallel with Mrp8-cre or Ela2-cre, and recombination should be carefully validated.

32Third, methods to target NETosis have raised debate within the field. Genetic knockout studies have shown that protein arginine deiminase 4 (PAD4) is required for nuclear decondensation and nuclear rupture preceding NETosis. Yet, some studies have found that targeting PAD4 is effective against NETosis, while others have not. There are several plausible explanations for this discrepancy: First, some defining features of NETosis can be mimicked in other contexts. Examples include leukotoxic hypercitrullination (involving non-bactericidal hyperactivation of PAD leading to DNA extrusion), constitutively defective mitophagy (resulting in mitochondrial DNA expulsion;;; ), or epigenetic regulation of pluripotency (whereby PAD4-mediated citrullination promotes an open chromatin structure; ). Thus, a standardized readout for NETosis is needed. Although citrullinated histone H3 (H3cit) is a reasonably specific marker for NETs in disease models since PAD4 deficiency/inhibition prevents citrullination of histones, histological assessment of NE or MPO associated with extracellular DNA is required to measure NETs in the context of PAD4 blockade. Second, NETosis may not always rely on PAD4, as observed in models of pneumosepsis. NETosis can be NOX2 dependent (induced by phorbol esters, LPS, etc.) or NOX2 independent (induced by calcium ionophores, UV light, etc.;;;;; ). Hypercitrullination of histones only occurs during calcium ionophore-activated NETosis, suggesting PAD4 may contribute specifically to NOX2-independent NETosis. Third, mouse background strain may influence PAD4 dependency. C57BL/6 mice have a Th1-type bias, whereas other strains tend to favor Th2 responses, therefore, we recommend that background strains be reported in publications. Finally, PAD4 reliance may be context dependent. For example, “aged” neutrophils exhibit lower NETosis than “fresh” neutrophils newly released from BM; neutrophils responding to bacterial infection exhibit pronounced NETosis compared with those responding to sterile injury; and PAD4 inhibitor efficacy may have species-specific variation. Of note, it is uncertain how targeting NETs might impact innate immune responses in humans, given their role in autoimmunity, aging, emergency granulopoiesis, vascular inflammation, and other inflammatory conditions. This question is being addressed for the first time in patients with severe COVID-19 who exhibit elevated NETosis (;;; Preprint), which will provide insight for cancer patients.

33Going forward, it will be critical to reconsider how neutrophils are classified and studied in the laboratory setting. First, there is a knowledge gap in reconciling the root cause of neutrophil heterogeneity. Adopting principles from the mononuclear phagocyte system, in which cells are classified developmentally and phenotypically, may help clarify tissue-specific neutrophil biology in steady-state and inflammation. Although neutrophils originate from definitive hematopoiesis, efforts in this direction are now emerging, and there is a growing appreciation that functionally distinct developmental identities can co-exist in cancer. We propose to use the terms “states” to refer to phenotypically distinct neutrophil populations, including those that are immunosuppressive since it is the environment that appears to drive neutrophil heterogeneity via shifts in maturation and/or activation.

34Second, there is a need to standardize techniques. This includes methods to deplete neutrophils with antibodies, discriminate between neutrophil states, and validate NET targeting approaches. Of note, currently, the preferred methods for NET detection are immunohistochemistry (association of MPO, NE, or Ly6G with extracellular DNA, or H3cit in the presence of MPO/DNA complexes) or dual-target ELISA (e.g., anti-elastase and anti-DNA-peroxidase), and new detection tools are emerging, including antibodies against histone H3 cleavage events specific to NETs in humans ( Preprint). However, alternative approaches are readily used, including H3cit ELISA/immunohistochemistry or flow cytometry for different combinations of MPO, H3cit, and/or SYTOX viability dyes, while these provide reasonable estimates, they are not conclusive. Although we see value in these techniques under certain circumstances, for example, to analyze limited patient material, we recommend that orthogonal approaches to validate findings are employed.

35Third, most studies are focused on bulk analysis of neutrophils. However, single-cell technologies can resolve shifts in the relative proportions of neutrophil states that co-exist outside the BM. Using these datasets to map differentiation trajectories and epigenetic regulation of neutrophil development using single-cell assay for transposase-accessible chromatin sequencing may also reveal relationships not evident from the transcriptome. These efforts will be particularly informative to understand molecular mechanisms and transcriptional regulators that govern neutrophil maturation and function. Moreover, they will allow us to resolve nomenclature for various neutrophil developmental stages (see Table 1 and ). In addition, in vivo techniques to trace or target individual cell subsets would be of value. For example, three neutrophil states are conserved within tumors in both humans and mice. Whether these neutrophil states are functionally plastic and/or whether they can be individually targeted has yet to be determined.

36Finally, our knowledge of neutrophils in cancer largely depends on data from murine models. However, it is clear that there are some species-specific differences in neutrophil regulation of tumor biology. In human studies, most experimental approaches are limited to ex vivo analyses of blood neutrophils, transcriptomic profiling of tumor-associated neutrophils, and basic correlations with clinical outcomes. Although these approaches are informative, they shed light on disease association and not causation. Therefore, a comprehensive understanding of the functional impact of human neutrophils on cancer is lacking and needs to be further elucidated to develop relevant therapeutic strategies.

37Taken together, our fundamental understanding of neutrophil maturation, heterogeneity, and function in the context of cancer has recently seen remarkable advances with emerging fate-tracing, high-parameter, and single-cell technologies that are now allowing us to study fundamental neutrophil biology at unprecedented depth. The next challenge is to reconcile existing data and unify nomenclature so that our collective discoveries can be integrated and directly compared. Ultimately, we aim to harness our emerging understanding of neutrophil phenotypic heterogeneity to characterize neutrophil states at the functional level, and effectively exploit them for therapeutic purposes.