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The accumulation of circulating low-density neutrophils (LDN) has been described in cancer patients and associated with tumor-supportive properties, as opposed to the high-density neutrophils (HDN). Here we aimed to evaluate the clinical significance of circulating LDN in lung cancer patients, and further assessed its diagnostic vs prognostic value. Using mass cytometry (CyTOF), we identified major subpopulations within the circulating LDN/HDN subsets and determined phenotypic modulations of these subsets along tumor progression. LDN were highly enriched in the low-density (LD) fraction of advanced lung cancer patients (median 7.0%; range 0.2%-80%, n = 64), but not in early stage patients (0.7%; 0.05%-6%; n = 35), healthy individuals (0.8%; 0%-3.5%; n = 15), or stable chronic obstructive pulmonary disease (COPD) patients (1.2%; 0.3%-7.4%, n = 13). Elevated LDN (>10%) remarkably related with poorer prognosis in late stage patients. We identified three main neutrophil subsets which proportions are markedly modified in cancer patients, with CD66b+ /CD10low /CXCR4+ /PDL1inter subset almost exclusively found in advanced lung cancer patients. We found substantial variability in subsets between patients, and demonstrated that HDN and LDN retain a degree of inherent spontaneous plasticity. Deep phenotypic characterization of cancer-related circulating neutrophils and their modulation along tumor progression is an important advancement in understanding the role of myeloid cells in lung cancer.

In recent years, the role of neutrophils in cancer biology has been a matter of increasing interest. Many patients with advanced cancer show high levels of neutrophilia, tumor neutrophils are connected to dismal prognosis, and the neutrophil-to-lymphocyte ratio has been introduced as a significant prognostic factor for survival in many types of cancer. Neutrophils constitute an important portion of the infiltrating immune cells in the tumor microenvironment, but controversy has long surrounded the function of these cells in the context of cancer. Multiple evidences have shown that neutrophils recruited to the tumor can acquire either protumor or antitumor function. These findings have led to the identification of multiple and heterogeneous neutrophil subsets in the tumor and circulation. In addition, tumor-associated neutrophils (TANs) were shown to demonstrate functional plasticity, driven by multiple factors present in the tumor microenvironment. In this review, we examine the current knowledge on cancer-related circulating neutrophils, their source and the function of the different subtypes, both mature and immature. We then discuss the pro vs antitumor nature of TANs in cancer, their functional plasticity and the mechanisms that regulate neutrophil recruitment and polarization. Although the vast majority of the knowledge on neutrophils in cancer comes from murine studies, recent work has been done on human cancer-related neutrophils. In the final paragraphs, we expand on the current knowledge regarding the role of neutrophils in human cancer and examine the question whether cancer-related neutrophils (circulating or intratumoral) could be a new possible target for cancer immunotherapy.

Neutrophils play a major role in tumor biology. Among other functions, neutrophils can release extracellular traps (NETs), mesh-like structures of decondensed chromatin fibers, in a process termed NETosis. Originally characterized as an antimicrobial mechanism, NETosis has been described in cancer, but cancer-related predisposition is not clear. In the current study, we investigated the predisposition of circulating neutrophils to release NETs in lung cancer and the impact of G-CSF on this function, comparing circulating neutrophils isolated from cancer patients to the LLC and AB12 mouse models. We find that neutrophils from both healthy donors and cancer patients display high NETotic potential, with 30–60% of cells undergoing NETosis upon PMA stimulation. In contrast, neutrophils isolated from tumor-bearing mice displayed only 4–5% NETotic cells, though significantly higher than naive controls (1–2%). Despite differential mechanisms of activation described, Ionomycin and PMA mainly triggered suicidal rather than vital NETosis. G-CSF secreting tumors did not increase NETotic rates in murine neutrophils, and direct G-CSF stimulation did not promote their NET release. In contrast, human neutrophils strongly responded to G-CSF stimulation resulting also in a higher response to PMA + G-CSF stimulation. Our data show clear differences in NETotic potentials between human and murine neutrophils. We do not find a predisposition of neutrophils to release NETs in lung cancer patients compared to healthy controls, whereas cancer may modulate neutrophils' NETotic potential in mice. G-CSF secreted from tumors differentially affects murine and human NETosis in cancer. These important differences should be considered in future studies of NETosis in cancer.

The role and importance of neutrophils in cancer has become increasingly apparent over the past decade. Neutrophils accumulate in the peripheral blood of patients with cancer, especially in those with advanced-stage disease, and a high circulating neutrophil-to-lymphocyte ratio is a robust biomarker of poor clinical outcome in various cancers. To date, most studies investigating the role of neutrophils in cancer have involved animal models or investigated the function of circulating human neutrophils. Thus, only limited information is available on the roles of intratumoural neutrophils (also known as tumour-associated neutrophils (TANs)) in patients with cancer. In this Review, we initially describe the evidence correlating the neutrophil-to-lymphocyte ratio with prognosis, followed by a discussion on the predictive value of TANs, which remains debatable, with conflicting data from different cancer types, including variations based on neutrophil location within and/or around the tumour. We then explore available data on the implications of TAN phenotypes and functions for cancer development and progression, highlighting the reported effects of various treatments on TANs and how neutrophils might affect therapeutic efficacy. Finally, we examine the various compounds capable of modulating neutrophils and suggest future research directions that might ultimately enable the manipulation of TANs in patients with cancer.

Simple Summary Myeloid-derived suppressor cells (MDSC) represent a heterogeneous myeloid cell population that is expanded in tumor bearing hosts and substantially contributes to immunosuppression, representing thereby a valuable therapeutic target. Our study analyzes polymorphonuclear (PMN) and monocytic (M) MDSC subsets regarding their immunosuppressive capacity and recruitment mechanisms in murine melanoma. The immunosuppressive activity of both subsets was comparable. We identified the C-X-C Motif Chemokine Receptor (CXCR) 2/chemokine C-X-C motif ligand (CXCL) 1 axis as an important mediator of PMN-MDSC recruitment. Inhibition of CXCR2 resulted in a decreased infiltration of tumors with PMN-MDSC and increased survival of melanoma bearing mice. Furthermore, adjuvant treatment of mice with resected tumors reduced the infiltration of pre-metastatic sites with PMN-MDSC and the occurrence of distant metastasis. The decrease in PMN-MDSC infiltration was accompanied by an increase in natural killer (NK) cell frequency, suggesting an important role of PMN-MDSC in suppressing the NK cell-mediated anti-tumor response. Abstract Background: Despite recent improvement in the treatment of malignant melanoma by immune-checkpoint inhibitors, the disease can progress due to an immunosuppressive tumor microenvironment (TME) mainly represented by myeloid-derived suppressor cells (MDSC). However, the relative contribution of the polymorphonuclear (PMN) and monocytic (M) MDSC subsets to melanoma progression is not clear. Here, we compared both subsets regarding their immunosuppressive capacity and recruitment mechanisms. Furthermore, we inhibited PMN-MDSC migration in vivo to determine its effect on tumor progression. Methods: Using the RET transgenic melanoma mouse model, we investigated the immunosuppressive function of MDSC subsets and chemokine receptor expression on these cells. The effect of CXCR2 inhibition on PMN-MDSC migration and tumor progression was studied in RET transgenic mice and in C57BL/6 mice after surgical resection of primary melanomas. Results: Immunosuppressive capacity of intratumoral M- and PMN-MDSC was comparable in melanoma bearing mice. Anti-CXCR2 therapy prolonged survival of these mice and decreased the occurrence of distant metastasis. Furthermore, this therapy reduced the infiltration of melanoma lesions and pre-metastatic sites with PMN-MDSC that was associated with the accumulation of natural killer (NK) cells. Conclusions: We provide evidence for the tumor−promoting properties of PMN-MDSC as well as for the anti-tumor effects upon their targeting in melanoma bearing mice.

We have recently shown that neutrophil antitumor cytotoxicity is Ca2+ dependent and is mediated by TRPM2, an H2O2-dependent Ca2+ channel. However, neutrophil antitumor activity is dependent on context and is manifested in the premetastatic niche, but not at the primary site. We therefore hypothesized that expression of TRPM2 and the consequent susceptibility to neutrophil cytotoxicity may be associated with the epithelial/mesenchymal cellular state. We found that TRPM2 expression was upregulated during epithelial-to-mesenchymal transition (EMT), and mesenchymal cells were more susceptible to neutrophil cytotoxicity. Conversely, cells undergoing mesenchymal-to-epithelial transition (MET) expressed reduced levels of TRPM2, rendering them resistant to neutrophil cytotoxicity. Cells expressing reduced levels of TRPM2 were protected from neutrophil cytotoxicity and seeded more efficiently in the premetastatic lung. These data identify TRPM2 as the link between environmental cues at the primary tumor site, tumor cell susceptibility to neutrophil cytotoxicity, and disease progression. Furthermore, these data identify EMT as a process enhancing tumor-cell immune susceptibility and, by contrast, MET as a novel mode of immune evasion. Significance: EMT is required for metastatic spread and concomitantly enhances tumor cell susceptibility to neutrophil cytotoxicity. Cancer Res; 78(17); 5050–9. ©2018 AACR.

Neutrophils play a critical role in cancer, with both protumor and antitumor neutrophil subpopulations reported. The antitumor neutrophil subpopulation has the capacity to kill tumor cells and limit metastatic spread, yet not all tumor cells are equally susceptible to neutrophil cytotoxicity. Because cells that evade neutrophils have greater chances of forming metastases, we explored the mechanism neutrophils use to kill tumor cells. Neutrophil cytotoxicity was previously shown to be mediated by secretion of H2O2. We report here that neutrophil cytotoxicity is Ca2+ dependent and is mediated by TRPM2, a ubiquitously expressed H2O2-dependent Ca2+ channel. Perturbing TRPM2 expression limited tumor cell proliferation, leading to attenuated tumor growth. Concomitantly, cells expressing reduced levels of TRPM2 were protected from neutrophil cytotoxicity and seeded more efficiently in the premetastatic lung. Significance: These findings identify the mechanism utilized by neutrophils to kill disseminated tumor cells and to limit metastatic spread. Cancer Res; 78(10); 2680–90. ©2018 AACR.

In recent years, the role of immune cells in tumor progression has been a matter of increasing interest. Neutrophils constitute an important portion of the immune cells infiltrating the tumor microenvironment. Traditionally viewed as the first line of defense against infections, it is now well accepted that neutrophils also have an important role in multiple aspects of cancer biology. Multiple and heterogeneous neutrophil subsets have been identified in tumors and in circulation. Evidence from many studies now supports the notion that tumor-associated neutrophils (TANs) show functional plasticity driven by multiple factors present in the tumor microenvironment. In this review, we first concisely discuss the pro-tumor vs. anti-tumor nature of neutrophils in cancer, their functional plasticity, and the mechanisms that regulate neutrophil polarization. We then expand on the various crosstalks and mutual effects between TANs and other tumor-infiltrating immune cell types, emphasizing the active role of neutrophils as regulators of the immune system, promoting or inhibiting the establishment of a permissive tumor microenvironment. Finally, the possible modulation of cancer-related neutrophils by therapies directed toward immune checkpoints is discussed briefly.

Sickle cell anemia (SCA) is a chronic inflammatory disease, in which activated neutrophils play a role in initiating vaso-occlusion. Two populations of circulating neutrophils have been described, denominated as low-(LDN) and high-(HDN) density neutrophils. Circulating numbers of LDN (a less inflammatory subset) are normally minimal, but this population augments under inflammatory stress, such as that seen in cancer. Transforming growth factor beta-1 (TGF-β1) is a cytokine with anti-inflammatory properties that is elevated in SCA. In conditions such as Chron's disease, TGF-β1 has protective effects, mediated by its immuno-suppressive functions. In macrophages, it is thought to trigger the polarization from pro- (M1) to anti-inflammatory phenotypes (M2), which hypothetically occur in neutrophils too (N1 and N2). Moreover, dimethyl sulfoxide (DMSO) reportedly increases TGF-β receptors expression on epithelial cells. We aimed to characterize the subsets of circulating neutrophils in SCA patients and investigate the effects of TGF-β1 and DMSO on these cells. Neutrophils from healthy (CON) and SCA individuals, in steady state and without blood transfusion for 90 days, were isolated from peripheral blood by Ficoll-Paque density gradient centrifugation. HDN and LDN were obtained from the granulocyte and mononuclear layers, respectively, and stained with CD66b for neutrophil identification by flow cytometry. As no significant effect of hydroxyurea (HU) therapy on the data obtained was observed, patients' data were grouped together irrespective of HU use. Percentages of LDN were calculated based on the total of gated CD66b+ cells. SCA patients had higher levels of LDN than CON (3.2±0.9%, N=7 vs 1.3±0.3%, N=13; p=0.02). We next investigated the presence of CD66b+/CD206- and CD66b+/CD206+ cells, to infer the presence of N1 and N2 phenotypes, respectively. N2 were more frequent in the LDN than in the HDN subset (CON: 68.1±3.3% vs 52.0±4.4%, N=9, p=0.01; SCA: 77.6±8.9% vs 44.1±5.0%, N=3, p=0.03). To determine whether TGF-β1 and DMSO could shift HDN to a LDN profile, cells were treated (2h) with TGF-β1 (50pM) and/or DMSO (1.5%). Treatments with DMSO alone or combined with TGF-β1 increased the percentage of CD206+ cells in CON (45.7±2.1% vs 61.9±7.6 and 53.6±2.6% respectively, N=6, p=0.04), as well as CD206+ expression on each cell (mean fluorescence intensity, MFI) (137.5±16.9 MFI vs 293.6±71.2 MFI and 210.1±23.9 MFI, respectively, p=0.03). In SCA, only the combined TGF-β1/DMSO treatment increased the MFI of CD206 in HDN (115.7±10.2 vs 255.8±29.7 MFI, N=4, p=0.03). We next investigated whether TGF-β1/DMSO could reduce the adhesion of HDN to fibronectin ligand (FN, 20μg/mL) using static adhesion assays (30 min, 37ºC). HDN from CON and SCA were treated with TGF-β1 and/or DMSO (90min) and stimulated with TNF-α (200ng/mL, 30min). Although TGF-β1 alone did not reduce the adhesion of HDN to FN (p>0.05), the addition of DMSO decreased TNF-α-induced adhesion in CON (16.5±1.8% to 11.3±1.5%, p=0.03, N=10) and SCA HDN (38.9±23.9% to 13.9±1.5%, p=0.04, N=3). Subsequently, HDN were stimulated (4h) with LPS (100ng/mL) and INF-γ (20ng/mL), to induce N2 polarization, in the presence/absence of TGF-β1 and DMSO. The combined treatment again reduced adhesion in both groups (CON: 11.1±1.5% to 4.2±1.2%, p Disclosures No relevant conflicts of interest to declare.