Controversy surrounds neutrophil function in cancer because neutrophils were shown to provide both pro- and antitumor functions. We identified a heterogeneous subset of low-density neutrophils (LDNs) that appear transiently in self-resolving inflammation but accumulate continuously with cancer progression. LDNs display impaired neutrophil function and immunosuppressive properties, characteristics that are in stark contrast to those of mature, high-density neutrophils (HDNs). LDNs consist of both immature myeloid-derived suppressor cells (MDSCs) and mature cells that are derived from HDNs in a TGF-β-dependent mechanism. Our findings identify three distinct populations of circulating neutrophils and challenge the concept that mature neutrophils have limited plasticity. Furthermore, our findings provide a mechanistic explanation to mitigate the controversy surrounding neutrophil function in cancer.

The role and characteristics of tumor-associated neutrophils (TAN) in cancer are poorly defined. We have recently shown that TAN can have anti-tumorigenic (N1) or pro-tumorigenic (N2) functions. An interesting unanswered question is how the phenotype of TAN is influenced by the ongoing evolvement of tumor microenvironment. We therefore studied the phenotype and effects of TAN at different time points during tumor progression. We used two models of murine tumor cancer cell lines-Lewis lung carcinoma (LLC) and AB12 (mesothelioma). Neutrophils were studied at early and late stages and compared to each other and to neutrophils from bone marrow/periphery of naïve mice. Although there was no difference in the number of neutrophils entering the tumor, we found that at early stages of tumor development, neutrophils were almost exclusively at the periphery of the tumor. Only at later stages, neutrophils were also found scattered among the tumor cells. We further found that TAN from early tumors are more cytotoxic toward tumor cells and produce higher levels of TNF-α, NO and H2O2. In established tumors, these functions are down-regulated and TAN acquire a more pro-tumorigenic phenotype. In line with this phenotype, only depletion of neutrophils at later stages of tumor development inhibited tumor growth, possibly due to their central location in the tumor. Our work adds another important layer to the understanding of neutrophils in cancer by further characterizing the changes in TAN during time. Additional research on the functional role of TAN and differences between subsets of TAN is currently underway.

The mechanisms by which tumor-associated neutrophils (TANs) affect tumor growth are to a large extent unknown. Regulatory T-cells (T-regs) are functionally immune-suppressive subsets of T-cells. Depletion or inhibition of T-regs can enhance antitumor immunity. We demonstrated both by RT-PCR and by ELISA that murine TANs secrete significant amounts of the T-regs chemoattractant, CCL17, much more than circulating or splenic neutrophils, and at a level progressively increasing during tumor development. Migration assays, both in vitro and in vivo, showed recruitment of T-regs by TANs, which was inhibited with anti-CCL17 monoclonal antibodies. Systemic neutrophil depletion in tumor-bearing mice using anti-Ly6G monoclonal antibodies reduced the migration of T-regs into the tumors. We further showed, using flow cytometry, that CCL17 secretion by TANs is not limited to mouse models of cancer but is also relevant to human TANs. Our results suggest a new indirect mechanism by which TANs may inhibit antitumor immune activity, thus promoting tumor growth. We further describe, for the first time, a clear link between TANs and T-regs acting together to impair antitumor immunity.Neutrophils make up a significant portion of the inflammatory cell infiltrate in many models of cancer. It is becoming increasingly clear that tumor-associated neutrophils (TANs) play a major role in cancer biology. 1,2 Despite some recent advancement in appreciating the role of neutrophils in cancer, the exact mechanisms by which TANs affect tumor growth are to a large extent unknown. In untreated tumors, neutrophils have been reported to suppress the antitumor immune response. 3 Depletion of these TANs inhibits tumor growth 4-6 and reduces the level of immunosuppression in the tumor microenvironment. 4 As mentioned above, we have recently demonstrated that in untreated tumors, neutrophils assume a protumorigenic state, which by analogy to tumor-associated macrophages is termed the "N2" phenotype. We, however, also noted that under certain conditions (e.g., after Transforming Growth Factor beta (TGF-b) blockade), TANs can take on an "N1" phenotype, which is proinflammatory and anti-immunogenic. 4 Gregory and Houghton raised the interesting question of whether the differences between N1 and N2 TANs were due to two unique transcriptional programs or instead represent two states of activation. 7 Although our work suggests a unique transcriptional program, we were, thus far, unable to identify clear markers for N1 or N2 TANs, somewhat limiting the separation between these neutrophil subsets, and their functions.The role of chemokines in the pathogenesis of lung cancer, as well as other cancers, is being increasingly appreciated. 8 One example is the involvement of CCL5 (Rantes) in tumor invasiveness. 9 Another example that we have investigated is the role of CCL2 (MCP-1), which was found to have protumorigenic function in lung cancer by reducing the recruitment and/ or polarization of M2 macrophages. 10 We have previously found that TANs overexpress many ...

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 HO We report here that neutrophil cytotoxicity is Ca dependent and is mediated by TRPM2, a ubiquitously expressed HO-dependent Ca 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. These findings identify the mechanism utilized by neutrophils to kill disseminated tumor cells and to limit metastatic spread. .

The role of neutrophils in tumor progression has become in recent years a subject of growing interest. Tumor-associated neutrophils (TANs), which constitute an important portion of the tumor microenvironment, promote immunosuppression in advanced tumors by modulating the proliferation, activation and recruitment of a variety of immune cell types. Studies which investigated the consequences of manipulating TAN polarization suggest that the impact of these neutrophils on tumor progression is considerably mediated by and dependent on the presence of CD8 T-cells. It has been previously shown that granulocytic myeloid regulatory cells, i.e. TANs and granulocytic myeloid-derived suppressor cells (G-MDSCs) are capable of suppressing CD8 T-cell proliferation and affect their activation. In the current study, we find that in addition, TANs isolated from different models of murine cancer promote immunosuppression by strongly inducing CD8 T-cell apoptosis. We demonstrate that the TNFα pathway in TANs is critical for the induction of apoptosis, and that the mechanism through which apoptosis is induced involves the production of NO, but not ROS. In the absence of pre-activation, TANs are capable of activating CD8 T-cells, but specifically induce the apoptosis of non-activated CD8CD69 cells. Despite this contradictive effect on T-cell function, we show that TANs suppress the anti-tumor effect of CD8 T-cells and abolish their ability to delay tumor growth. Our results add another important layer on the understanding of the possible mechanisms by which TANs regulate the anti-tumor immune response mediated by CD8 T-cells, therefore promoting a tumor-supportive environment.

It has been shown that inhibitors of the immune system reside in the spleen and inhibit the endogenous antitumor effects of the immune system. We hypothesized that splenectomy would inhibit the growth of relatively large non-small lung cancer (NSCLC) tumors by modulating the systemic inhibition of the immune system, and in particular Myeloid Derived Suppressor Cells (MDSC). The effect of splenectomy was evaluated in several murine lung cancer models. We found that splenectomy reduces tumor growth and the development of lung metastases, but only in advanced tumors. In immune-deficient NOD-SCID mice the effect of splenectomy on tumor growth and metastatic spread disappeared. Splenectomy significantly reduced the presence of MDSC, and especially monocytic-MDSC in the circulation and inside the tumor. Specific reduction of the CCR2+ subset of monocytic MDSC was demonstrated, and the importance of the CCL2-CCR2 axis was further shown by a marked reduction in CCL2 following splenectomy. These changes were followed by changes in the macrophages contents of the tumors to become more antitumorigenic, and by increased activation of CD8 Cytotoxic T-cells (CTL). By MDSC depletion, and adoptive transfer of MDSCs, we demonstrated that the effect of splenectomy on tumor growth was substantially mediated by MDSC cells. We conclude that the spleen is an important contributor to tumor growth and metastases, and that splenectomy can blunt this effect by depletion of MDSC, changing the amount and characteristics of myeloid cells and enhancing activation of CTL.

<p>Supplemental Figure S1. BAPTA protects tumor cells from H2O2 induced cell death. A. Extent of H2O2 (25 μM) cytotoxicity time course in 4T1 B. Annexin-V/PI analysis of vehicle and H2O2 treated (25 μM) 4T1 cells following overnight incubation. C. Effect of BAPTA-AM (5μM) on viability of 4T07, MC38, LLC, FARN and AT3 cells. D. Effect of BAPTA-AM (5μM) on neutrophil cytotoxicity in 4T07, MC38, LLC, FARN and AT3 cells. **p<0.01.</p>

<div>Abstract<p>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.</p><p><b>Significance:</b> These findings identify the mechanism utilized by neutrophils to kill disseminated tumor cells and to limit metastatic spread. <i>Cancer Res; 78(10); 2680–90. ©2018 AACR</i>.</p></div>