One of the most important mechanisms by which cancer fosters its own development is the generation of an immune microenvironment that inhibits or impairs antitumor immune responses. A cancer permissive immune microenvironment is present in a large proportion of the patients with cancer who do not respond to immunotherapy approaches intended to trigger preexisting antitumor immune responses, for instance, immune checkpoint blockade. High circulating levels of IL8 in patients with cancer quite accurately predict those who will not benefit from checkpoint-based immunotherapy. IL8 has been reported to favor cancer progression and metastases via different mechanisms, including proangiogenesis and the maintenance of cancer stem cells, but its ability to attract and functionally modulate neutrophils and macrophages is arguably one of the most important factors. IL8 does not only recruit neutrophils to tumor lesions, but also triggers the extrusion of neutrophil extracellular traps (NET). The relevance and mechanisms underlying the contribution of both neutrophils and NETs to cancer development and progression are starting to be uncovered and include both direct effects on cancer cells and changes in the tumor microenvironment, such as facilitating metastasis, awakening micrometastases from dormancy, and facilitating escape from cytotoxic immune cells. Blockade of IL8 or its receptors (CXCR1 and CXCR2) is being pursued in drug development, and clinical trials alone or in combination with anti-PD-L1 checkpoint inhibitors are already ongoing.

Immune evasion in cancer is multifactorial. The issue has become more conspicuously important as resistance mechanisms are concerned with the advent of efficacious immunotherapy interventions by means of anti-PD-(L)1 and anti-CTLA-4 checkpoint inhibitors. Cumulative evidence in mouse and human models concludes that myeloid leukocytes, including polymorphonucelar leukocytes, mainly behave as immunosuppressive actors in the tumor microenvironment. The chemokines involved in attracting and modulating the function of such myeloid leukocytes constitute an important topic in clinical cancer research both as a source of biomarkers and potential immunotherapy targets. The attraction of neutrophils to tumor tissue and the ability of neutrophils to die extruding their genomic DNA to form neutrophil extracellular traps (NET) are currently the focus of much preclinical and clinical cancer research.

IL8 is a chemokine of the CXC glutamic acid-leucine-arginine motif bearing (ELR+) family that acts via CXCR1 and CXCR2 to chemoattract neutrophils and other myeloid leukocytes. A particular characteristic of IL8 (also called CXCL8) is that it can activate both CXCR1 and CXCR2, unlike most of the other ELR+ chemokines that mainly engage only CXCR2 (1).

Many chemokines of the CXC ELR+ family are overexpressed in cancer (CXCL1, 2, 5, 6, or 8). IL8 is a frequently upregulated chemokine in human malignant tissues and a fraction of patients shows increased circulating IL8 levels in advanced stages (2). IL8 in the tumor microenvironment is produced not only by cancer cells but also by myeloid cells and fibroblasts infiltrating tumors (3, 4).

To date, several protumor roles of IL8 have been described (Fig. 1). In some instances, cancer cells do express the IL8 receptors CXCR1 or CXCR2, which have been reported to be upregulated in cancer stem cells (5).

Figure 1.

IL8 in cancer. IL8 (red) is expressed in tumors by cancer cells (CK, green), and also by other stromal cells (DAPI only, blue) as MPO-positive cells (magenta). Representative image of a lung adenocarcinoma paraffin section stained by tissue multiplex immunofluorescence. Scale bar, 50 μm. IL8 activates both CXCR1 and CXCR2 chemokine receptors in different cellular compartments in the tumor microenvironment. IL8 can induce angiogenesis by activating endothelial cells in tumors and induces the recruitment of different myeloid immune cells to the malignant tissue microenvironment. In neutrophils and GrMDSCs, IL8 induces NETosis. In some cancer cells expressing IL8 receptors, IL8 promotes stemness and EMT.

Figure 1.

IL8 in cancer. IL8 (red) is expressed in tumors by cancer cells (CK, green), and also by other stromal cells (DAPI only, blue) as MPO-positive cells (magenta). Representative image of a lung adenocarcinoma paraffin section stained by tissue multiplex immunofluorescence. Scale bar, 50 μm. IL8 activates both CXCR1 and CXCR2 chemokine receptors in different cellular compartments in the tumor microenvironment. IL8 can induce angiogenesis by activating endothelial cells in tumors and induces the recruitment of different myeloid immune cells to the malignant tissue microenvironment. In neutrophils and GrMDSCs, IL8 induces NETosis. In some cancer cells expressing IL8 receptors, IL8 promotes stemness and EMT.

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IL8 promotes the epithelial-to-mesenchymal transition (EMT) of cancer cells (6) and favors the acquisition of a stem cell phenotype (5). Another major role of IL8 in cancer progression is the direct proangiogenic effect reported in numerous studies (2), which may play a key angiogenic role as a compensatory escape mechanism upon VEGF (7) or VEGFR blockade (8).

IL8 was first described as a potent chemotactic factor for neutrophils (9, 10). In tumors, IL8 and its receptors, CXCR1/CXCR2, have been largely implicated in the recruitment of myeloid-derived suppressor cells (MDSC; ref. 11), and neutrophils, with major implications for antitumor immunity (12). Therapeutic blockade of CXCR1 and CXCR2 in mouse tumor models can favor anticancer immune responses (12–14). The role of these chemokine receptors and their cognate ligands in the recruitment of such immunosuppressive myeloid populations is also related to other cancer-promoting functions of these leukocytes apart from their immunomodulatory functions, including extracellular matrix remodeling and angiogenesis (15). IL8 is absent from the genome in rodents, thus complicating incisive animal experimentation to evaluate in detail the biology of the IL8/CXCR pathway. Yet, human IL8 is able to act through mouse CXCR1 and CXCR2 allowing xenograft-based experiments (11) and the exploration of the biology of this chemokine system in vivo in myeloid leukocyte populations present in immunodeficient mice when using xenografted tumors. However, conclusively investigating the modulation of anticancer immune responses by IL8 in vivo is cumbersome. A transgenic mouse expressing IL8 is available. Such mice have a protumorogenic phenotype and can be used to assess in vivo the importance of IL8 in a fully immunocompetent mouse (16). It is important to acknowledge that the biology of CXCR1 and CXCR2 is complex and that IL8 may not induce the same signaling pathways in mouse neutrophils as in human neutrophils. It remains unclear what the functional orthologue of CXCL8 in mice is because the functions, tissue distribution, and signaling correspondences of CXC ELR+ chemokines across species are difficult to match (17). However, CXCR1 and CXCR2 blockade similarly inhibits neutrophil functions in humans and mice, making rodents a suitable preclinical research tool to investigate the consequences of more broadly blocking this axis.

The biological implications of high IL8 production in the tumor microenvironment have led to investigations into IL8 as a potential biomarker in oncology and cancer immunotherapy (2). High IL8 levels frequently correlate with adverse prognosis and, to some extent, with tumor burden (18). Reductions in circulating IL8 levels are associated with objective responses to PD-1 blockade in non–small cell lung cancer (NSCLC) and melanoma (4). IL8 has been investigated as a biomarker to predict sensitivity and resistance to checkpoint-based immunotherapy. Two large studies including data from phase III registrational clinical trials identified elevated baseline serum IL8 as a prominent predictor of poor survival benefit (3, 19). Importantly, baseline IL8 levels predict clinical outcome independently of tumor mutation burden or PD-L1 positivity in the tumor, thus increasing their value as a potential clinically useful biomarker. Mechanistically, IL8 levels strongly correlate with increased monocytic and neutrophil infiltration (3) and with phenotypic changes in circulating and tumor-infiltrating myeloid leukocyte populations (3, 19). In this complex scenario, two relevant questions emerge: first, the extent to which IL8 is just a biomarker or whether it also represents a targetable pathway; and second, which factors determine the variability in IL8 production that result in different circulating levels across patients with the same type of tumor. The latter issue is still a matter of investigation, but inflammatory cytokines, such as TNFα, IL1β, and IL17 (20, 21), as well as hypoxia are likely contributors (22). Of note, the expression of IL8 seems to be mostly transcriptionally controlled and mainly dependent on NF-κB and AP-1 (23). Interestingly, allelic polymorphisms in the promoter region of the IL8 gene could account for different expression levels, and there is also preliminary evidence for epigenetic regulation by DNA methylation (24, 25). Ascertaining the phenomenology leading to variable IL8 expression in solid tumors is thus paramount.

Both in animal models and in patients with cancer, the presence of neutrophils within the tumor microenvironment has been shown to exert protumorigenic roles and correlate with worse disease prognosis (Fig. 2). However, a few studies have identified antitumor functions promoted by neutrophils in preclinical cancer models of tumorigenesis and metastases. The mechanisms underlying the ability of tumor-associated neutrophils (TAN) to promote both antitumor and protumor responses are unknown, but it has been postulated that the great plasticity exhibited by neutrophils is behind this dual role. Some authors have proposed the existence of a tumor-associated N1 neutrophil population with anticancer properties and a tumor-promoting N2 neutrophil population. For a better understanding of neutrophil plasticity and its crucial impact on cancer development, we recommend a recent review on cancer-associated neutrophils (TANs) by Mantovani and colleagues (26).

Figure 2.

Procancer roles of neutrophils and NETs. There are neutrophil interactions with the tumor at all the different stages. Tumors can induce enhanced neutrophil production and mobilization from the bone marrow and recruitment to the tumors. In primary tumors, neutrophils can inhibit antitumor T-cell responses and induce angiogenesis and matrix remodeling. Metastases are favored by neutrophils by promoting tumor cell migration, survival in the circulation, and survival and intravasation in the premetastatic niche. Within the metastatic niche, neutrophils continue supporting immunosuppression and can activate dormant cancer cells. Several molecules are involved in these neutrophil actions (depicted in the figure in black) and can be targeted by available immunotherapeutic drugs (in red). HGF, hepatocyte growth factor; PGE2, prostaglandin E2; Arg 1, arginase 1; inh, inhibitor; ITGs, integrins, ITG B1, integrin β1.

Figure 2.

Procancer roles of neutrophils and NETs. There are neutrophil interactions with the tumor at all the different stages. Tumors can induce enhanced neutrophil production and mobilization from the bone marrow and recruitment to the tumors. In primary tumors, neutrophils can inhibit antitumor T-cell responses and induce angiogenesis and matrix remodeling. Metastases are favored by neutrophils by promoting tumor cell migration, survival in the circulation, and survival and intravasation in the premetastatic niche. Within the metastatic niche, neutrophils continue supporting immunosuppression and can activate dormant cancer cells. Several molecules are involved in these neutrophil actions (depicted in the figure in black) and can be targeted by available immunotherapeutic drugs (in red). HGF, hepatocyte growth factor; PGE2, prostaglandin E2; Arg 1, arginase 1; inh, inhibitor; ITGs, integrins, ITG B1, integrin β1.

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The antitumor functions of TANs have been mostly described in tumor metastasis models (27–30), where tumor neutrophils are believed to directly kill tumor cells mainly by the production of reactive oxygen species (ROS; ref. 28), although additional mechanisms have also been proposed, including TRAIL, TNF secretion, and trogoptosis of opsonized cancer cells (27, 29, 31). Some experimental models have also identified antitumor effects of neutrophils in primary malignancies. In 3-methylcholanthrene–induced sarcomas, neutrophils promoted antitumor immune responses via activation of unconventional T cells (32). In a model of PTEN-deficient uterine cancer, the presence of neutrophils delayed cancer development by means of extracellular matrix degradation, promoting the anoikis of cancer cells (33). The antitumor effects of radiotherapy have been shown to be partially dependent on neutrophils and radiotherapy itself is able to modulate ROS production of TANs to favor cancer cell death (34). Epigenetic changes induced with β-glucan, as an example of training innate immunity, can promote granulocytic polarization toward an antitumor phenotype through a type I IFN mechanism (35). These observations point to the conclusion that, appropriate functional polarization of neutrophils can potentiate antitumor functions. Importantly, the presence of neutrophils in the tumor microenvironment, assessed by gene signatures or IHC, has been correlated with a better overall survival in some types of cancers, such as sarcomas (32) and colorectal cancer (32, 36).

However, there are abundant published reports that support the protumoral roles of neutrophils. Importantly, the expansion of neutrophils in patients with cancer, frequently measured by the circulating neutrophil-to-lymphocyte ratio (NLR), has been extensively associated with poor clinical outcome in terms of progression-free survival or overall survival in a wide range of cancer types, including advanced colorectal cancer (37), metastatic melanoma (38), breast cancer (39), and NSCLC (40, 41). In many of these studies, high NLRs correlated with poor responses to checkpoint-based immunotherapy as well (42). However, there are no human studies correlating the high numbers of circulating neutrophils with higher neutrophil infiltration, although in mouse models both parameters are actually correlated (43). Studies using the CIBERSORT algorithm, to computationally deconvolute mRNA transcripts to determine the relative abundance of tumor-infiltrating leukocyte populations, have identified neutrophils as the cell type associated with the worst clinical outcome across all cancer types (44). Numerous studies using different approaches have confirmed this observation (45, 46).

Many transplantable mouse tumor models, such as the LLC or 4T1 cancer cell lines, reflect this neutrophil expansion and their infiltration into tumors (43). In these models, aberrantly expanded neutrophils often have immunosuppressive capacities. Elegant studies in transgenic breast cancer models have dissected the mechanisms of how such neutrophil expansions favor metastatic potential. The mechanism involves the secretion of G-CSF in response to IL17 produced by gamma-delta T cells, which in turn are stimulated by IL1β (47, 48).

TANs have been shown to participate both in the development of the primary tumors and metastases, although they seem to be more prominent in metastases. It is clear that whether termed TANs or granulocytic myeloid suppressor cells (GrMDSCs), these polymorphonuclear leukocytes mediate direct immunomodulatory functions on T cells involved in antitumor adaptive immune responses. These activities include arginine depletion by Arginase 1 (49–51), inhibition of T-cell receptor signaling mediated by oxidative mechanisms (52), and production of prostaglandin E2 (53). Some of the proteins released from neutrophil granules may also play immunomodulatory roles in the cancer microenvironment. Matrix metalloproteinase (MMP9) secreted by neutrophils, for example, impairs T-cell infiltration in tumors (54). To support the successful formation of metastases, neutrophils act on different steps of the metastatic cascade. First, favoring tumor cell survival within the circulation (55), then facilitating adhesion and migration within the vascular niche, and finally through inhibiting several immune functions against the arriving or recently arrived tumor cells to successful metastatic foci (56). The different mechanisms used by neutrophils to support tumor growth and metastases may be a consequence of great neutrophil plasticity, as it has been shown that they can acquire different phenotypes when leaving circulation (57) and within tumors (58).

Neutrophils can undergo a unique form of cell death termed NETosis that consists of the extrusion of their nuclear DNA content and lytic proteins to the extracellular space in the form of so-called NETs. This mechanism of death was first described by Brinkmann and colleagues (59) and plays an important role in controlling infection by certain pathogens, such as bacteria and fungi (60). In essence, the adhesive double-stranded DNA (dsDNA) entraps and immobilizes microorganisms, and the antibacterial polypeptides from the neutrophil granules adsorbed onto the extracellular DNA exert a microbiocidal effect.

A number of extracellular substances have been shown to induce NETosis, and some of these are overexpressed in cancer. NETosis-inducing stimuli include Toll-like receptor 4 (TLR4) agonists, such as HMGB1, C5a, and formyl peptides, among many others (61). In spite of the intensive study of NETosis phenomena over recent years, the molecular signaling events that elicit and control NETosis are not well understood and probably differ across different stimuli (62). How and when these stimuli, which are known to induce other functions in neutrophils, such as chemotaxis and degranulation, trigger NETosis instead of activating other neutrophil functions remain unclear. NETosis is commonly induced following ROS production and ERK signaling. These two signals lead to the cytosolic release of neutrophil granule proteases that degrade the cytoskeleton and the nuclear membrane, letting dsDNA expand into the cytoplasm by partially degrading histones and nucleosomes. In parallel, histone 3 citrullination of its arginines catalyzed by peptyl diamine deaminase-4 (PAD4) determines chromatin decondensation (63, 64). These DNA expansion processes generate interstitial pressures able to disrupt the plasma membrane and to project the NETs (63). Very importantly, DNA adsorbs many of the proteins present in the cytosol or granules of the neutrophil while undergoing NETosis, retaining many of their enzymatic functions, as in the case of myeloperoxidase (MPO) or proteases, such as MMP9 and neutrophil elastase (NE; ref. 65). Both NE- and Pad4-knockout mice are defective in NETosis, both in vivo and ex vivo (65, 66).

Apart from their implication in immune responses against pathogens, NETosis and NETs are being identified as playing important pathogenic roles in a number of different diseases, including thrombosis (67, 68), biliary lithiasis (69), chronic inflammation, and autoimmunity (70). In cancer, NETs are emerging in experimental studies as a major mechanism by which TANs promote cancer progression.

The presence of NETs in patients with cancer has only been studied recently because of the lack of robust techniques to detect them in tissue or circulation. Most studies have focused on detecting circulating MPO-dsDNA nucleosomes, or citrullinated H3 (CitH3), detected either as a single parameter or as coupled to dsDNA (71). Although some studies quantitate NETs in cancer tissue by the immunostaining of CitH3 alone (72, 73), multi-parametric techniques to codetect neutrophil lytic enzymes and dsDNA are recommended to document the origin of the NETs from neutrophils because H3 citrullination can also occur in other cellular processes, such as mitosis.

Although more and more studies are being published evaluating circulating NETs in cancer and other diseases, it is important to clarify that the methods to quantify circulating NETs are mostly semiquantitative and can be blighted by prominent issues, such as the size of the circulating nucleosomes. Importantly, the dynamics of NET formation/biodistribution/degradation, and whether circulating NETs directly reflect the amount of NETosis occurring in tissues are unclear. Therefore, new technology and validated techniques are required to measure and understand NETosis in patients with cancer. A summary of the methods to assess the presence of NETs is depicted in Fig. 3. So far, NETosis has been described in several diseases, such as lung adenocarcinoma, large B-cell lymphoma, breast cancer, and ovarian cancer (summarized in Table 1; refs. 74, 75). Importantly, in many of these diseases, NETosis preferentially appears in advanced stages and, at least, in breast and colon cancer, seems more prominent in the liver metastases of these patients. According to most of these studies, the abundance of NETs correlates with worse prognosis (76) and disseminated disease (72). Importantly, two studies have shown circulating IL8 to correlate with circulating NETs (71, 76).

Figure 3.

Methods to assess NETosis. Several methods are in place to assess the relevance of NETosis in experimental mouse models and in samples from patients with cancer. Sandwich ELISAs can detect circulating nucleosomes of DNA formed during NETosis, different immunodetection strategies identify NETs by citrullination in H3 histone in neutrophils or neutrophil-rich areas. Ex vivo ability to form NETs of neutrophils isolated from cancer-bearing hosts can be tested with different stimuli, host sera, or tumor cells culture supernatants. Circulating neutrophils undergoing NETosis can also be detected by flow cytometry. In experimental models, live imaging of neutrophils and extracellular DNA or NE activity enables observations of NET formation, localization, and functions. Several animal models have defects in NETosis, including selective knockout of PAD4 in granulocytes only.

Figure 3.

Methods to assess NETosis. Several methods are in place to assess the relevance of NETosis in experimental mouse models and in samples from patients with cancer. Sandwich ELISAs can detect circulating nucleosomes of DNA formed during NETosis, different immunodetection strategies identify NETs by citrullination in H3 histone in neutrophils or neutrophil-rich areas. Ex vivo ability to form NETs of neutrophils isolated from cancer-bearing hosts can be tested with different stimuli, host sera, or tumor cells culture supernatants. Circulating neutrophils undergoing NETosis can also be detected by flow cytometry. In experimental models, live imaging of neutrophils and extracellular DNA or NE activity enables observations of NET formation, localization, and functions. Several animal models have defects in NETosis, including selective knockout of PAD4 in granulocytes only.

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Table 1.

Evidence of NETs in cancer mouse models and patients with cancer.

MalignancyModelTechniqueObservationsReferences
Mouse models 
Ovarian cancer ID8 cell line H3-Cit IF Invasion of the omentum (92) 
Large B-cell lymphoma A20 cell line H3-Cit IF Cancer cell proliferation, migration (76) 
BC 4T1 cell line H3-Cit IF, IVM in mets Impairment of immune–tumor cell contact, immune suppression (85) 
  H3-Cit IF tumor Metastatic cell invasion (91) 
Lung cancer LLC cell line H3-Cit IF Tumor growth (81) 
  H3-Cit IF of tumors, IVM of liver Impairment of immune–cancer cell contact/killing (85) 
  MPO-DNA ELISA Metastatic cell capture (87) 
Pancreatic cancer KPC mice H3-Cit IF tumors, mets  (90) 
  Ex vivo NET induction Immune suppression (21) 
 PancO2 H3-Cit IF tumors  (77) 
 AsPC-1 cell line MPO-DNA ELISA Increased metastases (79) 
Liver cancer NASH/STAM mouse model H3-Cit WB Development of liver cancer (94) 
Multiple myeloma DP42 cell line Ex vivo NET induction Tumor progression (80) 
Colorectal cancer APCMin/+ mice Ex vivo NET induction  (78) 
Patients with cancer 
Ovarian cancer  H3-Cit IF N/A (92) 
Large B-cell lymphoma  H3-Cit IF, MPO-DNA ELISA Increased NETs in advanced stages, worse PFS, OS (76) 
Lung adenocarcinoma  MPO-DNA ELISA Increased NETs in advanced stages, correlation with liver metastases (87) 
Esophagogastric adenocarcinoma  MPO-DNA ELISA Increased NETs in advanced stages (87) 
BC  MPO-DNA ELISA Increased liver metastases, increased NETs in triple-negative BC (91) 
Colorectal cancer  MPO-DNA ELISA Correlation with procoagulation parameters (75) 
Gastric adenocarcinoma  NE-DNA ELISA Correlation with worse PFS in HER2-negative patients only (74) 
Oral squamous cell carcinoma  MPO-DNA ELISA Increased NETs in advanced stages (84) 
Pancreatic cancer  H3-Cit IF Association with bad clinical prognosis after surgery (73) 
Liver metastases of colon and BC  H3-Cit IF, MPO-DNA ELISA Correlation with metastasis-free survival (72) 
Pan-cancer  H3-Cit and MPO-DNA ELISAs Increased in patients with cancer, correlates with bad poor prognosis (71) 
MalignancyModelTechniqueObservationsReferences
Mouse models 
Ovarian cancer ID8 cell line H3-Cit IF Invasion of the omentum (92) 
Large B-cell lymphoma A20 cell line H3-Cit IF Cancer cell proliferation, migration (76) 
BC 4T1 cell line H3-Cit IF, IVM in mets Impairment of immune–tumor cell contact, immune suppression (85) 
  H3-Cit IF tumor Metastatic cell invasion (91) 
Lung cancer LLC cell line H3-Cit IF Tumor growth (81) 
  H3-Cit IF of tumors, IVM of liver Impairment of immune–cancer cell contact/killing (85) 
  MPO-DNA ELISA Metastatic cell capture (87) 
Pancreatic cancer KPC mice H3-Cit IF tumors, mets  (90) 
  Ex vivo NET induction Immune suppression (21) 
 PancO2 H3-Cit IF tumors  (77) 
 AsPC-1 cell line MPO-DNA ELISA Increased metastases (79) 
Liver cancer NASH/STAM mouse model H3-Cit WB Development of liver cancer (94) 
Multiple myeloma DP42 cell line Ex vivo NET induction Tumor progression (80) 
Colorectal cancer APCMin/+ mice Ex vivo NET induction  (78) 
Patients with cancer 
Ovarian cancer  H3-Cit IF N/A (92) 
Large B-cell lymphoma  H3-Cit IF, MPO-DNA ELISA Increased NETs in advanced stages, worse PFS, OS (76) 
Lung adenocarcinoma  MPO-DNA ELISA Increased NETs in advanced stages, correlation with liver metastases (87) 
Esophagogastric adenocarcinoma  MPO-DNA ELISA Increased NETs in advanced stages (87) 
BC  MPO-DNA ELISA Increased liver metastases, increased NETs in triple-negative BC (91) 
Colorectal cancer  MPO-DNA ELISA Correlation with procoagulation parameters (75) 
Gastric adenocarcinoma  NE-DNA ELISA Correlation with worse PFS in HER2-negative patients only (74) 
Oral squamous cell carcinoma  MPO-DNA ELISA Increased NETs in advanced stages (84) 
Pancreatic cancer  H3-Cit IF Association with bad clinical prognosis after surgery (73) 
Liver metastases of colon and BC  H3-Cit IF, MPO-DNA ELISA Correlation with metastasis-free survival (72) 
Pan-cancer  H3-Cit and MPO-DNA ELISAs Increased in patients with cancer, correlates with bad poor prognosis (71) 

Abbreviations: BC, breast cancer; IF, immunofluorescence; IVM, intravital microscopy; Mets, metastases; OS, overall survival; PFS, progression-free survival; WB, Western blotting.

The presence of NETs in mouse cancer models has also been reported previously (Table 1; refs. 77–80). In mouse tumor models producing high amounts of G-CSF, neutrophils are poised to undergo NET extrusion (81, 82). NETs are found in murine models, not only in primary tumors, but also in the premetastatic niche before any malignant cells can be detectable (72). NETosis in cancer-bearing animals has been associated with several cancer-associated disorders, including vascular damage (83) and alterations in coagulation (84). The main inducers of NETosis in cancer may differ between the different models, but both CXCR1 and CXCR2 agonists (11, 76, 85) and HMGB1 (86) have been demonstrated to stimulate cancer-associated NETosis.

To date, experimental approaches studying NETosis in cancer models have identified a role for NETs in metastasis promotion (Fig. 2). NETs in the liver premetastatic niche are induced by experimental sepsis in animals with established tumor lesions elsewhere. NETs in the liver sinusoids in these conditions increase the number of metastatic cancer cells entrapped in this organ (87, 88). Capture of arriving tumor cells by the NETs (87, 88), induction of chemotaxis and adhesion mediated by a DNA receptor that directly interacts with NETs (CCDC25; ref. 72), and integrin functions are invoked as the underlying mechanisms (89). NETs also favor the development of liver metastases in pancreatic ductal adenocarcinoma models by modulating proliferation of cancer-associated fibroblasts (90). Furthermore, we have recently shown that in the liver sinusoids, NETs surrounding cancer cells impede contact with cytotoxic T and natural killer cells. In this way, the NETs would protect trafficking tumor cells from immune cytotoxicity when they are vulnerable just when they are adhering and transmigrating to the tissue (85).

Proteases adhered to the NETs are also likely to play a role in promoting metastatic cell intravasation because NETs have been shown to mediate extracellular matrix degradation and promote 4T1 tumor cell migration, resulting in increased spontaneous metastases in vivo (91).

In an orthotopic model, ovarian cancer colonization of the omentum by peritoneal dissemination has been shown to be supported by neutrophils and NETs, which reportedly promote the adhesion of cancer cells (92). NETosis not only helps tumor metastases to form the premetastatic niche, but also has been shown to awaken preexisting metastatic dormant cells when induced by lung inflammation following lung irritation with tobacco smoke (93).

Few studies in mouse models have described the role of NETs in primary tumor growth. LLC lung adenocarcinomas that show abundant NETosis in the tumor microenvironment grow slower in PAD4-deficient mice (81) and mouse models of lymphoma have shown that NETs promote tumor growth and dissemination because of their TLR9 agonistic activity exerted on the lymphoma cells (76).

Links between NETosis and tumorigenesis have also been substantiated because NETosis plays a direct role in the progression of nonalcoholic steatohepatitis (NASH) to hepatocellular carcinogenesis (94) and in the etiopathology/pathogenesis of pancreatitis (95). In this regard, it will be important to ascertain whether NETosis could play a role in escape from immune surveillance.

In further support of a dominant role of NETs in adaptive antitumor immune evasion, we recently reported that pharmacologic inhibition of PAD4 sensitized the breast cancer cell line, 4T1, to combined immunotherapy with anti-PD-1 and anti-CTLA-4 checkpoint inhibitors (85). Our interpretation from a mechanistic standpoint is that tumor-associated NETs constitute at least a mechanical/physical barrier that prevents interactions between tumor and effector immune cells (85). This protection disappears upon NETosis impairment by PAD4 inhibitors, suggesting a feasible immunotherapy combination to test clinically. Intravital microscopy time-lapse imaging studies strongly supported this interpretation of contact restriction. However, the presence of NETs in tumors and metastases can mediate other negative immunomodulatory functions based, for instance, on their protease activity and on the capacity of NETs to activate TLR9. Recently, CCDC25 has been identified as a receptor for NET DNA (72). CCDC25 is expressed by immune cells, which indicates that immune cell suppression may be directly regulated by NETs via this receptor or other yet unidentified receptors.

The implications of the ability of NETs to impair immunotherapy have been recently been made clear (Fig. 2). DNAse I produced in the liver by AAV vectors reduced the presence of NETs in colon cancer metastases and enhanced local CD8+ T-cell infiltration (96). Furthermore, orthotopic tumors using Kras-induced pancreatic cancer cells (KPC cells) engrafted in Pad4-deficient mice showed increased infiltration of activated CD8+ T cells and were more sensitive to PD-1 blocking mAbs (21). This report also stressed the importance of IL17 in production of NETs via induction of CXCR1/2 agonist chemokines, which attract neutrophils and elicit NETosis (21).

The protumor effects of neutrophils and NETs have opened up new avenues for diagnostics and immunotherapeutic intervention in cancer. Indeed, several immunotherapies have been developed to interfere with neutrophil recruitment into tumors. Because of the CXCR1/2–IL8 axis hierarchical importance in neutrophil chemoattraction and the several protumor functions that this axis mediates, CXCR1/2–IL8 becomes an especially attractive therapeutic target. Some of the approaches being clinically tested, such as Navarixin, SX-682, and reparixin, can simultaneously target both CXCR1 and CXCR2. An ongoing phase II study in advanced solid tumors is evaluating Navarixin in combination with pembrolizumab (NCT03473925). SX-682 is currently being tested in phase I studies in patients with advanced melanoma in combination with pembrolizumab, as well as in myelodysplastic syndromes (NCT03161431 and NCT04245397). Reparixin showed tolerable safety in combination with paclitaxel in a phase Ib study in patients with HER2-negative metastatic breast cancer (97, 98) and has entered a phase II clinical study in patients with metastatic triple-negative breast cancer (NCT02370238).

CXCR2 alone has also been selectively targeted using different inhibitors in preclinical models. Targeting CXCR2 alone only induces modest delays in tumor growth, but sensitizes tumors to checkpoint blockade immunotherapy in various tumor models (12–14). The AstraZeneca inhibitor, AZD5069, is currently being evaluated in combination with enzalutamide in patients with castration-resistant prostate cancer (NCT03177187).

Another strategy to target the IL8 pathway is to directly block the ligand/receptor signaling. In this regard, a humanized anti-IL8 antibody, termed HuMax-IL8, has shown an adequate safety profile as a single agent in a phase I trial (99) and is being tested in a phase II clinical trial in combination with the anti-PD-1 mAb, nivolumab (NCT02536469).

The blockade of other TAN functions is also being pursued in clinical trials and preclinical research (26, 100, 101). To target NETosis, two preclinical strategies have been tested with potential for translation. The first of these is the inhibition of the enzymes required for NET formation. Specific inhibitors targeting both NE and PAD4 have been developed, and inhibition of both enzymes has shown NETosis blockade in cancer mouse models (85, 87, 91). The second strategy involves the injection of clinical-grade DNAse I that is able to degrade already formed NETs. DNAse perfusion has been shown to degrade cancer-associated NETosis and has been functionalized in microparticles to extend its half-life and the duration of the effect in animal models (91).

Unfortunately, and despite progress in the detection of IL8 pathway biomarkers and NETs in cancer, most ongoing clinical studies are enrolling unselected patients with advanced PD-L1 refractory disease. The NCT02536469 trial testing anti-IL8 mAb with nivolumab is an exception because patients with more than 10 pg/mL of serum IL8 are being recruited. However, it is anticipated that analysis of samples from these studies using molecular pathway metrics will allow the interpretation of results and will eventually support the development of patient selection strategies.

IL8 is a central regulator of the presence and function of myeloid cells in human cancer. The protumor roles of neutrophils in advanced tumors are being confirmed and expanded in multiple experimental models (26). IL8 and its receptors, CXCR1 and CXCR2, play a critical role in the biology of TANs and this chemokine/chemokine receptor axis is clinically targetable with different drugs. Despite the limited success of many of these drugs in inflammatory disorders, IL8/CXCR1/2 axis blockade is being pursued in several cancer clinical trials, which include combinations with checkpoint inhibitors. Results from these clinical trials and biomarker work could shed light on opportunities for patient selection (3, 19). The potential compensatory resistance mechanisms that cancer may deploy upon IL8/CXCR1/2 blockade remain to be seen.

IL8 and its receptors are clearly involved in the induction of NETosis. NETs seem to be a major neutrophil mechanism to promote cancer metastasis and progression, by shaping the tissue microenvironment. Following conclusive evidence in mouse models, including xenografted mice (11), studies on the role of IL8 in NETosis and the impact of NETs on the anticancer immune responses in cancer are warranted. It is also important to consider in our investigations that NETs may also be detrimental for tumors as shown by neutrophils under certain polarizing conditions.

Few studies on patient samples have initially shown correlations between NETosis and IL8 in cancer (71, 76) and viral infection (102), but more extensive studies should be performed to further confirm this connection across cancer types and in larger clinical series. Other mediators produced in the tumor microenvironment can induce NET extrusion and IL8 might not be the main driver of NETosis in all patients. Importantly, the main role of IL8 in the sequence of events that gives rise to NETs in the tumor microenvironment is actually myeloid leukocyte recruitment. Once in the tumor, neutrophils may exert a number of immunomodulatory functions even if NETs are not formed.

IL8 has emerged as a potent biomarker to predict checkpoint blockade responses in patients and its upstream role in neutrophil modulation and its pleiotropic protumor effects make IL8 and its receptors suitable therapeutic targets. The value of NETs as biomarkers is yet to be fully characterized and technical issues need to be solved to be both quantitatively and reliably reproducible (De Andrea and colleagues, in preparation), but they may serve to predict different clinical aspects, such as, perhaps, the presence of liver metastases (72).

All things considered, these concepts offer the opportunity for biomarker identification and development both in tissue and in plasma samples. More importantly, translation to the clinic is feasible because safe therapeutic tools are already at hand.

K.A. Schalper reports personal fees from Celgene, Moderna Therapeutics, Shattuck Labs, Pierre-Fabre, AstraZeneca, EMD Serono, Ono Pharmaceuticals, Clinica Alemana de Santiago, Dynamo Therapeutics, PeerView, AbbVie, Fluidigm, Takeda, Merck, Bristol-Myers Squibb, Agenus, and Torque Therapeutics and grants from Navigate Biopharma, Genentech/Roche, Tesaro Inc., Moderna Therapeutics, Takeda, Surface Oncology, Pierre-Fabre Research Institute, Merck, Bristol-Myers Squibb, AstraZeneca, Ribon Therapeutics, and Eli Lilly outside the submitted work. M.F. Sanmamed reports grants from Roche outside the submitted work. I. Melero reports grants and personal fees from Bristol-Myers Squibb during the conduct of the study and Roche, Alligator, AstraZeneca, Genmab, and PharmaMar and personal fees from F-Star, Numab, and Gossamer outside the submitted work. No disclosures were reported by the other authors.

This project was supported by MINECO SAF2017-83267-C2-1-R (AEI/FEDER, UE, to I. Melero). This project has received funding from the European Union’s Horizon 2020 Research and Innovation Program (grant agreement no. 635122 - PROCROP), Fundación de la Asociación Española Contra el Cáncer, and Cancer Research UK (accelerator project: local radioimmunotherapy). A. Teijeira has received financial support through “la Caixa” Banking Foundation (LCF/BQ/LR18/11640014).

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