Cancer immunotherapy relies on either restoring or activating the function of adaptive immune cells, mainly CD8+ T lymphocytes. Despite impressive clinical success, cancer immunotherapy remains ineffective in many patients due to the establishment of tumor resistance, largely dependent on the nature of tumor microenvironment. There are several cellular and molecular mechanisms at play, and the goal is to identify those that are clinically significant. Among the hematopoietic-derived cells, monocytes are endowed with high plasticity, responsible for their pro- and anti-tumoral function. Indeed, monocytes are involved in several cancer-associated processes such as immune-tolerance, metastatic spread, neoangiogenesis, and chemotherapy resistance; on the other hand, by presenting cancer-associated antigens, they can also promote and sustain anti-tumoral T cell response. Recently, by high throughput technologies, new findings have revealed previously underappreciated, profound transcriptional, epigenetic, and metabolic differences among monocyte subsets, which complement and expand our knowledge on the monocyte ontogeny, recruitment during steady state, and emergency hematopoiesis, as seen in cancer. The subdivision into discrete monocytes subsets, both in mice and humans, appears an oversimplification, whereas continuum subsets development is best for depicting the real condition. In this review, we examine the evidences sustaining the existence of a monocyte heterogeneity along with functional activities, at the primary tumor and at the metastatic niche. In particular, we describe how tumor-derived soluble factors and cell-cell contact reprogram monocyte function. Finally, we point out the role of monocytes in preparing and shaping the metastatic niche and describe relevant targetable molecules altering monocyte activities. We think that exploiting monocyte complexity can help identifying key pathways important for the treatment of cancer and several conditions where these cells are involved.

Keywords: monocytes heterogeneity, monocyte continuum, primary tumor, metastatic niche, targeting of monocytes

# INTRODUCTION

Monocyte diversity is well-recognized but the biologic and clinical significance of the different monocyte subtypes is far from being completely elucidated. The main hallmark of monocytes is their plastic nature, whereby they can exert multiple roles during the course of the immune response including cytokine production, pathogen clearance, antigen presentation, wound healing, and pro/anti-tumoral response (1–3). The original classification of monocytes into classical (in

#### Edited by:

Wei Zheng, Albert Einstein College of Medicine, United States

#### Reviewed by:

Robin Parihar, Baylor College of Medicine, United States Shentong Fang, Wihuri Research Institute, Finland

> \*Correspondence: Vincenzo Bronte<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology

> Received: 17 May 2019 Accepted: 16 July 2019 Published: 06 August 2019

#### Citation:

Canè S, Ugel S, Trovato R, Marigo I, De Sanctis F, Sartoris S and Bronte V (2019) The Endless Saga of Monocyte Diversity. Front. Immunol. 10:1786. doi: 10.3389/fimmu.2019.01786

**79**

humans: CD14high , CD16−; in mice: Ly6Chigh), intermediate (in humans: CD14high, CD16low), and non-classical (in humans CD14low, CD16high; in mice: Ly6Clow) is currently being replaced by evidences supporting the existence of a "monocyte continuum" rather than stepwise differences between the different subtypes (4). Indeed, in mice under steady state, circulating classical monocyte subsets have been shown to switch into non-classical monocytes over time (5–7). However, it remains to be shown what relationship exists among the human monocytic subsets, and whether and how pathological conditions, like inflammation and cancer, impact this process.

Circulating monocytes have been viewed for many years, as precursor cells that provide tissue macrophages and dendritic cell (DCs) populations (8, 9); however, mounting evidence suggests that monocytes have their own effector functions in the blood and at peripheral sites throughout the body (10). The emerging data that distinct monocyte subsets, carrying different genetic, epigenetic, transcriptional, and metabolic arrangements, are committed to become macrophages and DCs seems to contradict the general accepted view of monocytes responding to a particular environmental stimuli and then differentiating into multifaceted macrophages and DCs. The intriguing evidence, both in mice and humans (11–13), of trained-monocytes, both present as mature and precursor cells, seems to strongly support the former hypothesis and reinvigorate the idea that monocytes have specific functions beyond being precursor cells. In this review, by combining the most recent advances in the field of monocytes' genetic, epigenetic, transcriptomic, and metabolomic, we outline and evaluate the changes occurring in monocyte subsets that underlie the aforementioned plasticity and heterogeneity. Secondly, we discuss new concepts in the monocyte field, like trained immunity and reprogramming and highlight the targetable pathways controlling monocyte fate and function.

We think that combining the information of single-cell transcriptome profiling, metabolomics array and epigenetic studies will elucidate complex relationships between cell types, thus solving limitations in the existing classification that relies on a relatively small number of markers.

# MONOCYTE PHENOTYPICAL AND TRANSCRIPTIONAL PLASTICITY

# Inflammatory and Patrolling Monocytes

Studies over the past two decades have delineated two major subsets of monocytes in mice and humans. Inflammatory monocytes (iMo), characterized by the high expression of the chemokine receptor CCR2, are repeatedly released from the bone marrow into the circulation. These cells, alternatively known as classical monocytes, are Ly6Chi in mice and correspond to the CD14hiCD16lo monocyte subset in humans. The fate of these cells is strictly dependent on the state of the body. Under steady state conditions, extravasated iMo and their derived-cells enrich in nearly all tissues throughout the body, where they form a small yet significant group of the so called local tissue-resident macrophages (7). The gradual accumulation of monocyte-derived macrophages in tissues is generally associated with the slow but progressive replacement of embryonic macrophages, in both quantitative and qualitative fashion (14). Monocyte-derived macrophages present sustained gene modifications as compared to their circulating counterparts, as they shape to the tissue microenvironment. They acquire transcriptional signatures resembling resident macrophages of embryonic origin, even though a certain level of differences remains at the epigenetic, transcriptional and functional levels (15–17). Whether monocyte-derived macrophages that infiltrate tissues under steady-state condition gain a self-renewal ability, comparable to their embryonic counterparts, is still a matter of debate and seems to strictly depend on the type of infiltrated tissue. On the other hand, iMo can also conserve their monocyte-like state inside the tissues without differentiating into macrophages, thereby acting as a local monocyte reservoir (18) (**Table 1**).

Besides the aforementioned pathway of maturation, iMo can either remain in the blood, or transition into patrolling monocytes (pMo) by the setting up of de novo enhancers and activation of "frosted" enhancers (19, 20) (**Table 1**). The mechanisms driving the conversion of iMo into pMo are just beginning to be elucidated. It appears that Delta-like 1 (Dll1) signal from endothelial cells by interacting with NOTCH2 only iMo favors their switch into pMo cells (21). These data clearly indicate that iMo and pMo monocytes are biologically intertwined, corroborating observation obtained at the epigenetic level, which indicated that both monocyte subsets use the same promoter repertoire and minimally differ in their chromatin organization (19). Of course, this scenario raises several questions: are the iMo infiltrating the tissues able to reprogram into pMo? Is this switch tissue dependent? Can we interfere with this reprogramming to control the transition? Is there any factor maintaining iMo reservoir? Are pMo thus originated able to re-enter the blood stream? Are monocyte-derived macrophages transcriptionally similar to pMo? In mice, iMo can give rise to pMo, even though this does not rule out the presence of an alternative route for pMo development, independent from the iMo subset (8). Indeed, genetic evidence for this transition do exist. Two myeloid-determining transcription factors, like interferon regulatory factor 8 (IRF8), and the downstream Kruppel-like factor (KLF4), have been shown to regulate iMo generation without affecting the pMo numbers. Moreover, studies conducted on either global IRF8−/−mice, or fetal liver transplant of KLF4−/−cells into irradiated wild type mice, indicate a drastic reduced numbers of iMo in the bone marrow, while maintaining relatively normal pMo numbers (22). These findings suggest a pathway for pMo development untied from the iMo subset, probably originating directly from the common monocyte progenitor (cMoP). The identification of the transcription factors nerve growth factor IB (NR4A1) has helped to withstand the hypothesis that pMo can arise independently from iMo monocytes. On the other hand, recent single-cell RNA sequencing of murine and human monocytes indicate that circulating iMo and pMo represent, under physiological conditions, a nearly homogenous populations (19). Interestingly, data recently published (23) combining single TABLE 1 | Summary of monocyte subsets presented in this review, highlighting their markers and function in both humans (top part of the table) and mice (bottom part of the table).

profile and functional and phenotypic characterization, showed that monocytes subsets (defined as classical, intermediate, and non-classical) isolated from peripheral blood of both healthy mice and humans, can be further divided into two additional populations: one group expressing classical monocyte genes and also cytotoxic genes and the other one with undefined activity. Other studies conducted in human and mouse lung cancer samples (24) have showed that several tumor-infiltrating myeloid populations (TIM) and among those monocytes are uniquely associated with the disease and with clinical progress, highlighting the potential to use TIM as immunotherapeutic targets. We think that the multiple cell subsets identified in the aforementioned manuscripts, should be tested for their functional relevance in tumor progression, in disease progression and their abundance should be correlated with therapeutic response. Of course, the correlation between human and mouse TIM will help to achieve these goals with the ultimate purpose of gaining more insight into monocytes and monocytedependent therapies.

In contrast, patrolling monocytes (pMo) represent a more differentiated subset and are marked by the higher surface expression of CX3CR1. pMo express low levels of Ly6C in mice and are CD14loCD16hi in humans; they routinely check the vessels under physiological conditions through the engagement of an LFA/ICAM-dependent crawling mechanism with resting endothelial cells (25, 26). This patrolling behavior of pMo can be observed throughout the interdigitated system of capillaries, arterioles, and venules. Similarly, human CD14lo CD16hi pMo show patrolling behavior when adoptively transferred into immuno-compromised mice (27). The crawling features of pMo allows them to efficiently sense particles, on the one hand, and on the other hand to monitor of endothelial cell integrity. However, these cells are not restricted to the vessels as pMo also undergo diapedesis and can be identified within the parenchyma of multiple tissues (7). pMo display a longer lifespan at the steady state compared to iMo and they are, also for this reason, found in the blood, at any given time, more abundant than their counterpart (5, 28). Interestingly, pMo cells are strongly susceptible of the physiological status of the organism and therefore they might represent a potential diagnostic tool (29). Nevertheless, how the fine balance between iMo and pMo levels and differentiation capacity is maintained/regulated during severe inflammation, autoimmune diseases, and cancer is just began to be elucidated.

Under pathological conditions, such inflammation and cancer, the rapid recruitment of myeloid cells to sites of injury stimulates a constant development and mobilization of cells from the bone marrow, causing a state of "emergency" that might generate monocytes from different ways or precursors. These include monocytes that have circumvented the canonical MDP-cMoP-monocyte developmental axes and resemble neutrophil-like iMo derived from GMP precursors (30). An additional example of a recently described monocyte subset that appears under inflammatory conditions is the segregated nucleus-containing atypical pMo (SatM) (31). SatM and neutrophil-like monocytes represent a minor pool of monocyte subsets under steady-state (19, 30, 31) (**Table 1**), yet become conspicuous during inflammation (30, 31). At present, the lack of reliable surface markers, associated with a deep epigenetic and transcriptional profile unable to make a clear distinguish between neutrophil-like iMo identified using GFI1/IRF8 reporter mice (30) from classical iMo and SatM (identified as Ly6ClowCeacam1hiF4/80−Mac1hi), from non-classical pMo (31). The limited whole-cell proteomic data available so far showed, for example, that SatM cells contain granules expressing granulocyte-related protein, like myeloperoxidase (MPO) and neutrophil elastase (NE) (31) (**Figure 1**). These cells are related to fibrosic responses; in fact, adoptive transfer of SatM monocytes into bleomycin-treated mice exacerbates fibrosis. Furthermore, it was shown that chimeric mice, lacking the CCAAT/enhancerbinding protein beta (Cebpb) in the hematopoietic progenitors were resistant to fibrosis when exposed to bleomycin but they had unaltered inflammatory response, further supporting the role of SatM in sustaining the mechanism of fibrosis (31). Nevertheless, more studies will be required to uncover the origin of these subsets and their involvement in different pathologies. To add more complexity, a recent work by Hanna et al. (32), showed that a subset of circulating pMo, but not iMo, accumulate at the site of tumor where they display an anti-tumoral role, by directly engulfing cancer cells and by releasing factors which in turn activate cytotoxic natural killer cells (NK). Are these extravasating pMo similar to their blood counterpart? How the findings from Hanna et al. (32) correlate with observations that extravasating pMo can differentiate into macrophages during cancer? Are pMo "corrupted" by tumor cells once they have extravasated within tissues and switched to pro-tumoral cells? Are iMo and pMo competing for the access to the tumor site? Are iMo suppressing the anti-tumor function of pMo? Indeed, this mechanism is also consistent with the data from the pMo adoptive transfer experiments since pMo appear to act early during seeding but not after establishment of metastatic foci despite their continued accumulation. Therefore, under pathological conditions it still remains possible that pMo derive from either blood iMo, via the formation of an intermediate Ly6Cint monocyte, or from bone marrow Ly6C+ monocytes, from an independent bone marrow monocyte progenitor, or from a combination of all these pathways (**Figure 1**). In order to solve all these issues a detailed understanding of the factors and pathways regulating the development and survival of both iMo and pMo populations in specific inflammatory settings is necessary.

As mentioned before, adoptive transfer and fate-mapping studies support the hypothesis that monocytes develop along a differentiation continuum in which inflammatory monocytes give rise to the patrolling subset in the circulation (3, 5). Development of monocytes from bone marrow progenitors combines the regulated expression of numerous transcription factors, with the contribution of growth factors and cytokines (33). Monocytes and neutrophils are both derived from hematopoietic stem cells (HSCs) via common myeloid progenitors (CMPs), which can originate from granulocytemonocyte progenitor (GMP) and monocytes/macrophages, DC precursors (MDPs) (34, 35) (**Figure 1**). The commitment toward monocytes is characterized by three major lineage-determining factors (LDTF): PU.1, CCAAT/enhancer-binding protein beta (Cebpb) and Cebpa. PU.1 is a master regulator of myeloid and lymphoid cell development (36). The genetic ablation in mice of PU.1 promotes a lethal embryonic phenotype and the transfer of PU.1 mutated stem cells favors an altered myelopoiesis characterized by a robust contraction in both monocytes and DCs (37, 38). By binding closed chromatin through its Cterminal DNA-binding domain (39), PU.1 acts as a coordinator for the activation of selected genomic regions in collaboration with monocyte-associated transcriptional factors such as the IRF8 and KLF4 (40). Moreover, PU.1 synergistically cooperates with C/EBP-δ to activate the promoters of interleukin-6 (IL-6) and CCL5 (41) and transactivates the human macrophage colony-stimulating factor receptor (M-CSFR) promoter via the c-Jun pathway (42). Proteins of the C/EBP family radically impact the myeloid cells development. Since in Cebpa-deficient mice the transition from CMPs to GMPs is completely abrogated, these mice lack the granulocytic compartment (43, 44) indicating that C/EBPα is the master regulator of steady-state granulopoiesis.

Additionally, during myelopoiesis, C/EBPα controls and activates the myeloid-associated gene expression program by binding to either promoters or enhancers of myeloid-related genes, such as colony stimulating factor 3 receptor (CSF3R), growth factor independent-1 (GFI-1), interleukin 6 receptor (IL-6R), or C/EBPε, both in mice and in human stem cells (45). C/EBPα inhibits specific transcriptional factors attenuating the expression of non-myeloid lineage genes (46, 47). Moreover, genetically enforced inducible C/EBPα expression in GMPs by tamoxifen administration favors monocyte development demonstrating the critical role of this transcriptional factor during monopoiesis (48). By contrast, C/EBPβ is not necessary for steady-state granulopoiesis. However, C/EBPβ was recently identified as the key factor during the epigenetic default differentiation of iMo monocytes into pMo cells under steady state condition, highlighting the multifaceted role of this transcriptional factor during monocyte development (19). In this regard, a key contribution of C/EBPβ to mylopoiesis is highlighted by data showing that mice knockout for Cebpb have a dramatic reduction in circulating monocytes (19). C/EBPβ is also the master regulator of emergency myelopoiesis; in fact, inflammatory signals (i.e., cytokine stimulation) strongly induce the downregulation of all members of C/EBP family except for C/EBPβ (43). Under pathological condition, like fibrosis, a C/EBPβ-associated gene program in FcεR1+ GMPs progenitors promote the development of alternative monocytes, like SatM, previously described. As we will discuss below, C/EBPβ-driven programs are also activated in cancer-educated myeloid cells.

 Continuous lines indicate events occurring during normal myelopoiesis while shaded lines indicate events in emergency myelopiesis (e.g., cancer and inflammation). Briefly, under steady state cMop precursors originate both inflammatory (1) and patrolling (2) monocytes, both in humans and mice. However, it has been reported that during emergency myelopoiesis cMop precursors can also differentiate into M-MDSC (3) and into not yet defined immature cells (4). Particularly, during infection, inflammatory monocytes acquire a trained phenotype (5) and also switch into Ly6Cint cells (6) only identified in mice and with not fully defined function, transcriptional regulators, (Continued) FIGURE 1 | and markers. During fibrosis a novel subset of monocytes, so called SatM (8), have been characterized, in mice, defined as Ly6C+ and expressing proteins typical of the neutrophil granules (MPO and NE). These cells, together with neutrophil-like monocytes (7), found in peripheral blood of mice during microbial infection and in the bone morrow in steady-state condition, originate from GMP/FcεRI+ precursors cells in the bone marrow. In pathological conditions, like cancer, inflammatory monocytes infiltrating the tissue give rise to TAM (9) which in turn represent a fultifaced population of macrophages. Additionally, inflammatory monocytes can also differentiate into classical DC (10) expressing the costimulatory molecules CD80 and CD86 and TipDC (11) expressing high level of NOS2 and TNFα.

# Myeloid-Derived Suppressor Cells

In cancer, tumor-derived soluble factors, such as growth factors, cytokines, chemokines, and tumor-derived exosomes, not only support an increased recruitment of monocytes from bone marrow to tumor-microenvironment bypassing the canonical monocyte development but, also, favor the acquisition of immunosuppressive features in myeloid cells. To highlight these acquired functional properties, myeloid cells comprising monocytes, neutrophils, and immature cells were named myeloid-derived suppressor cells (MDSCs) (49, 50). Although this terminology generated some controversies, it represents a useful ground for scientific researches on altered hematopoiesis. It relies on the concept that myelopoiesis in pathology might give rise to cellular subsets that can share some markers with the cells present under steady state but are functionally and molecularly distinct, sharing the property of negatively regulating effectors of adaptive and innate immunity. The monocytic-MDSC (M-MDSC) subset is broadly defined in mice as Ly6C+CD11b+ cells and in human as CD14+HLA-DR− or CD14+CD124+ cells and is endowed with a stronger ability to arrest T cell response, when compared to the granulocytic-MDSC (G-MDSC) counterpart (51, 52), in part dependent on the activation of two enzymes, arginase 1 (ARG1) and inducible nitric oxide synthases (NOS2/iNOS), which are directly regulated by C/EBPβ expression (53) (**Figure 1**; **Table 1**). We demonstrated that, in tumor-bearing mice, both the expansion and the immunosuppressive function of MDSCs are abrogated in the absence of C/EBPβ, resulting in restricted tumor spread (53). These data confirm the central role of C/EBPβ in tumor-associated inflammation, underscoring it as promising therapeutic target to develop new approach to limit cancer progression. Both human and mouse M-MDSCs secrete immunoregulatory cytokines, like IL-10, TGFβ, and IL-6 and present and array of molecules, such as ARG1, FADD-like IL-1β-converting enzyme-inhibitory protein (c-FLIP), indoleamine 2,3-dioxygenase 1 (IDO1), and nitric oxide synthase 2 (NOS2), which can contribute to the suppressive activity of these cells. Mechanistically, IL-6, for example, activates PI3Kγ, which stimulates mTOR, S6Kα, and C/EBPβ-mediated antiinflammatory gene expression and inhibits NFkB-mediated proinflammatory gene expression, thereby promoting the immune suppressive function of these cells mediated by, but not limited to, IL-10, TGFβ, and ARG1 (54, 55). In particular, Arg1 gene in MDSCs is strictly controlled by several inducible transcriptional factors able to recognize sequences characterized by high content in GC that impacts the nucleosomal stability (56), such as signal transducer and activator of transcription 3 (STAT3), IRF8, as well as CHOP, PU.1, KLF4, and activator-protein 1 (AP-1) (57). Moreover, STAT3 promotes both expansion and survival of M-MDSCs through Bcl-XL, c-Myc, and Cyclin D1 expression (58) as well as the induction of several immune regulatory mediators like bFGF, HGF, VEGF, IL-1β, MMP9, CCL2, and CXCL2 (50). Interestingly, phosphorylated STAT3 binds to multiple sites in the Arg1 promoter, suggesting that STAT3 inhibitors, like Stattic, could reduce ARG1 dependent immunosuppression by dampening the expression of Arg1 mRNA (59). Within the tumor environment, ARG1 can cooperate with NOS2 to produce high levels of superoxide anion (O− 2 ) that can react with either nitric oxide (NO) or H2O generating reactive–nitrogen species (RNS), such as peroxinitrites (ONOO−), which damage both the function and migration of T cells to tumor site (60), and reactive-oxygen species (ROS), such as H2O2 which decreases T cellular CD3ζ expression limiting the activation of T cells, respectively (61). However, ARG1 has a hierarchically dominant negative role compared to NOS2 in developing an immunosuppressive tumor microenvironment by limiting the activity of monocyte-derived NOS2-expressing and TNF-producing dendritic cells (defined as Tip-DCs) that can sustain and favor the anti-tumor effect of transferred T lymphocytes (62). An alternative way to reprogram MDSC differentiation and function is through the expression of p53. It was recently demonstrated that M-MDSC can be driven to differentiate into potent antigen-presenting, defined as Ly6C+CD103+ DCs by inflammation-induced activation of p53. In fact, mice with a targeted deletion of p53 in myeloid cells specifically loose the Ly6C+CD103+ population and became unresponsive to different forms of immunotherapy and immunogenic chemotherapy (63).

Recently, we demonstrated the ability of c-FLIP, which controls the extrinsic apoptotic pathway and caspase 8 activation (64), to re-program monocytes into MDSC-like cells (65). In fact, FLIP-expressing monocytes displayed impressive regulatory features both in vitro, constraining the activated T cell proliferation, and in vivo, controlling the development of graft vs. host disease in a xenogeneic mouse model. Indeed, enforced expression of c-FLIP in monocytes up-regulates MDSCassociated immunosuppressive genes, such as CD273, CD124, IL-6, IL-10, CFS3, PTGS2, and IDO1, as a result of a "steered" NF-kB activation induced by the nuclear co-localization of c-FLIP with NF-kB p50 (65). During the course of a disease, like cancer, MDSCs infiltrate the tumor, differentiating into tumorassociated macrophages (TAMs) which can sustain primary tumor growth and contribute to the pre-metastatic niche formation (**Figure 1**).

# Trained Immunity and Metabolic Landscape of Monocyte Subsets

Recent studies have shown that during infection with some pathogens iMo can undergo extensive epigenetic, transcriptional, and metabolic reprogramming, with the functional consequence of an enhanced immune reactivity upon a second encounter, in other words they acquire an immunological memory. The existence of this innate immune memory was initially suggested by studies in mice deficient for functional T and B cells and exposed to mild C. albicans infection, which show protection against C. albicans reinfection by increased responsiveness of monocytes (66). Even though the requirements for monocyte training has been primarily investigated either in vitro or under in vivo steady state, trained monocytes seem to originate from iMo during emergency hematopoiesis by a profound epigenetic and metabolic rewiring (**Table 1**). Exposure of iMo to either C. albicans or β-glucan in vitro, induce profound genome-wide changes in epigenetic marks, including, but not restricted, histone H3 lysine 4 monomethylation (H3K4me1), trimethylation (H3K4me3), and H3 lysine 27 acetylation (H3K27ac) (67) as a consequence of Dectin-1/AKT/mTOR/HIF-1α signaling pathway activation and secretion of IL-6, IL-1β, and TNFα. Other studies instead, identified Bacille Calmette-Guérin (BCG) and peptidoglycan as potent inducers of the aforementioned trained-related epigenetic modifications, though a different mechanism dependent on nucleotide-binding oligomerization domain-containing protein 2 (NOD2) pathway and activation of NF-kB (68). Concomitantly to these epigenetic changes, a metabolic switch also occurs. Trained monocytes are mainly glycolytic (aerobic glycolysis) with impairment of the oxidative phosphorylation, production of lactate and disruption of the Krebs cycle at the level of both citrate, which is withdrawn for fatty acid biosynthesis, and succinate, which activates HIF-1α and consequently up regulates the expression of several proinflammatory cytokines, mainly IL-1β and TNF-α (69).

Beside microbial particles, products of the lipid metabolism were found to be activators of the trained immunity. Oxidizedlow density lipoprotein (oxLDL), a damage-associated molecular pattern (DAMP), interacts with CD36 on myeloid cells leading to the activation of NLRP3 inflammasome and consequent production of IL-1β (70–72).

Although the role of epigenetic programing as a mechanism required to insure innate immune memory is becoming more clear, one crucial aspect still remains unanswered: what is the cellular process that induces and maintains such epigenetic changes? Initial evidences seem to suggest that metabolites might play a role since they can act as cofactors for the enzymes (mainly methylases, methyltransferases, histone deacetylases, and histone acetylases) involved in epigenetic modulation of gene transcription (67, 73, 74). Of course, more studies are required to fill the gap and also to deeply examine the role that different chromatin modifications have on the stability of the chromatin. It is expected that stable histone modifications (e.g., histone methylation) would be more suitable to maintain a functional modification than those with short half-life (e.g., histone acetylation). Thus, the long-lasting persistence of some histone modifications could reflect both the stability of such modifications or the persistent activation status of the signaling pathways and transcription factors upstream (75, 76). Understanding these regulations is a sine qua non for designing therapeutic intervention aimed at modulating trained immunity, to dampen it when in excess (e.g., organ rejection, autoimmunity, allergy, atherosclerosis) or enhance it when defective (e.g., cancer, infection).

A growing body of evidence suggests that the development of immune cells and their different effector functions are the results of a dynamic changes occurring at the metabolic level (77, 78). In mouse models of cancer, myeloid cells are metabolically influenced by tumor-derived factors to become MDSCs, helping to protect tumor from the effects of chemotherapy (79). Specifically, mouse MDSCs undergo a major metabolic reprogramming by switching off glycolysis and enhancing fatty acid β oxidation (FAO) pathway. This metabolic reprogramming is generally characterized by an up-regulation of lipid uptake receptors CD36 and Mrs1, an increase in FAO enzymes, mainly carnitine palmitoyltransferase 1 (CPT1) and 3-hydroxyacyl-Coa dehydrogenase (HADHA), and an increase in oxygen consumption. These events are associated with the activation of immunosuppressive pathways, namely upregulation of ARG1 and NOS2 synthesis and production of ONOO−, contributing to dampen T cell proliferation and IFNγ secretion (80–82). Blockade of FAO, both in vitro and in vivo in different tumor models, decreased the incorporation of fatty acid and ATP production, holding up the development of suppressive MDSCs (82) and leading to increased efficacy of chemotherapy and adoptive T cell therapy. Interestingly, fatty acid oxidation also plays an important role in regulating the inflammatory properties of iMo. Increased intracellular level of unsaturated fatty acid (arachidonic acid) was shown to stimulate the secretion of pro-inflammatory IL-1α by uncoupling the mitochondrial respiration (83, 84) thus exacerbating the pathogenesis of atherosclerosis (85). The relevance of changes in the lipid metabolism occurring during myeloid cells differentiation, was also recently demonstrated by Mitroulis et al., in an vivo model of trained immunity (13). Treatment with β-glucan determines an increase in gene expression of several enzymes involved in cholesterol biosynthesis and decrease in expression of Abca1, a transporter regulating cholesterol efflux (86). Consistently, β-glucan administration in mice not only upregulates CD131, a subunit of the receptor for IL-3, IL-5, and GM-CSF, expression in myeloid precursors, but also activates downstream signaling, as demonstrated by STAT5 phosphorylation. The capacity of β-glucan to enhance the biosynthesis of cholesteryl esters and significantly decrease glycerophospholipid-containing arachidonic fatty acid chains highlight the capacity of cells to alter their lipidome and, thus, the physicochemical features of their membranes. This adaptive response has direct consequences in the composition of cellular membranes (87) and consequently in cell signaling (88). In this regard, alterations in the quantitative and qualitative cholesterol composition of the membrane can impact the localization of CD131, its signaling (86, 89) and consequently the differentiation of specific myeloid subsets.

Amino acids, besides being the building block of several molecules, serve as essential precursors of different metabolites. Different studies have shown that glutamine metabolism into glutamate, α-ketoglutarate and succinate semialdehyde can fuel the synthesis of fumarate and succinate for the tricarboxylic acid cycle (TCA) (90). Inhibition of glutaminolysis, in mice, down regulates the production of pro-inflammatory cytokines in monocytes exposed to C. albicans, dampening the development of efficient trained monocytes triggered by β-glucan. In line with these observations, the biochemical catheterization of βglucan trained monocytes has revealed that upon induction of the signaling cascade Dectin-1-Akt-mTOR-HIF-1α, a metabolic shift occurs leading to an increase aerobic glycolysis, glucose usage, lactate production, and TNF-α secretion (67).

Thus, it appears that modulation of metabolic landscape represents a fundamental step to unravel the functional consequences of different monocytes subsets helping to identify new strategies of intervention for the treatment of several pathophysiological conditions. It remains instead undefined whether cancer-derived factors can also generate trained monocytes and if these cells contribute to dampen the anti-tumor response or favor metastatic spread.

# MONOCYTE FUNCTIONS

# Monocytes at the Primary Tumor

Tumor derived factors (TDFs) are key mediators in the crosstalk between monocytes and tumor cells. They are involved in monocyte recruitment from the hematopoietic organs in adult life, i.e., bone marrow and in part the spleen, survival, and differentiation within the tumor site. Tumor-released monocyte chemoattractant protein-1 (MCP-1, also known as CCL2) was identified as the major TDF involved in iMo recruitment, through the CCL2-CCR2 axis, into several mouse and human tumors (91). Indeed, inhibition of CCL2-CCR2 signaling in a mouse model of breast cancer significantly impair iMo infiltration and reduce tumor growth and metastases (92). Several studies have described the presence of other chemokines within the tumor microenvironment (TME), including CCL3, CCL4, CCL5, CXCL12, and growth factors such as colony stimulating factor-1 (CSF1), which may also contribute to monocyte recruitment to tumors (93). Indeed, in both mouse (94) and human (91) tumors, cells secrete high level of CSF1 that is involved in recruitment, survival, and differentiation of monocytes. The inhibition of CSF1 signaling in an experimental model of lung carcinoma significantly reduced the number of mature TAMs due to impaired recruitment, proliferation and maturation of iMo cells (95). Moreover, CSF1R signaling blockade can also reprogram immunosuppressive TAMs prompting the differentiation of iMo in anti-tumoral M1-like TAMs (96). However, abrogation of CSF1R signaling, by either small molecules (97) or monoclonal antibodies (98), even though appealing, have so far demonstrated a limited antitumor effects. Only a recent work done by Kumar et al. (99) highlighted that the CSF1-dependent cross-talk between tumor cells and cancer-associated fibroblasts (CAF) might explain the inefficacy of such a treatment. Thus, combinatorial therapy targeting both CSF1R and CXCR2 seems to have more chances to generate an effective anti-tumor T cell response. Thus, the therapeutic focus has shifted to combinations of CSF1R inhibitors with other agents. Indeed, treatment with CSF1 inhibitor in combination with either paclitaxel or radiotherapy, is showing to improve the survival of mouse model of breast or prostate cancer, respectively (79, 100) and improve the efficacy of ACT when combined with anti-PD-1 and anti-CTLA4 in a pancreatic mouse model (101). It is becoming clear that cytotoxic therapies, for example, induce mammary epithelial cells to produce monocyte/macrophage recruitment factors, including CSF1 and interleukin-34 (IL-34), which together enhance CSF1R-dependent monocytes/macrophage infiltration, making its inhibition more effective.

One of the strongest stimuli inducing the secretion of TDFs is hypoxia. Indeed, during tumor growth the level of available O2 is significantly reduced, especially in the inner part of the neoplastic mass. This hypoxic microenvironment triggers HIF-1α stabilization in tumor cells and the consequent release of proangiogenic factors, such as growth factors (VEGF, PDGF, PIGF, ANG-2), chemokines (CXCL8, CXCL12), cytokines (TNFα, IL1β, TGFβ), and metalloproteases (MMPs), which results in the sprouting of new vessels supporting cancer cells growth (102, 103). In addition, hypoxia is a powerful monocyte and macrophage attractant. Through the release of VEGFB and PIGF, tumor cells can enhance haematopoiesis and monocyte recruitment (104). In addition, angiopoietin-2 (ANG-2) is able to recruit circulating Tie2-expressing monocytes (TEMs) that inhibit apoptosis in both tumor and endothelial cells, by mechanisms depending on TNFα release (105), and exhibits an essential pro-angiogenetic role with a not completely clarified mechanism. Interestingly, it was recently discovered that a small subgroup of recruited iMo can be educated by VEGF, and exert their proangiogenic function, supporting the formation of capillaries and larger vessels, as short-lived monocytes without becoming macrophages (106). Secreted VEGF promotes the acquisition of immunosuppressive features in monocytes generating M-MDSCs by upregulating both ARG1 and iNOS through hypoxia response elements and NF-kB (107). In addition, MDSCs can fuel this circuit by releasing MMP-9, which induces VEGF release from ECM (108). Therefore, targeting VEGF/VEGFR has received attention as a strategy to interfere with monocyte-driven angiogenesis. Moreover, blocking this axis will affect monocytes recruitment to the tumor site (109), and favor the conversion from predominant suppressive to antitumoral monocytes (110). An additional way to interfere with MDSC differentiation is by interfering with p53 expression, as mentioned before (63).

In the last few years, extracellular vesicles (EVs) emerged among the TDFs as additional determinants in the formation of TME, both at primary and metastatic sites (111). EVs are a heterogeneous group of membrane vesicles mainly composed of exosomes and microvesicles. Interestingly, EVs released by tumor cells (tEVs) and tumor-derived exosomes (TEX) interact with immune cells inducing their switch toward a pro-tumoral phenotype. Particularly, exosomes target monocytes altering their normal function by several mechanisms. In melanoma and colon cancer, TEX block peripheral iMo differentiation into DCs, and favor the acquisition of a peculiar phenotype reminiscent of M-MDSCs and characterized by decreased expression of HLA-DR and costimulatory molecules (112). A similar modulation of monocytes has been described in many other malignancies, including pancreatic cancer, bladder carcinoma, glioblastoma, and multiple myeloma (113–115) and it is often associated with increased cytokine secretion, i.e., CCL2, CCL4, and IL-6, as well as programmed death-ligand 1 (PD-L1) expression (116). Moreover, glioblastoma-derived EVs may skew the differentiation of peripheral blood monocytes to alternatively activated M2 macrophages inducing the expression of elevated levels of VEGF, IL6, Cox2, ARG1, and PD-L1 through STAT3 activation (117). Interestingly, in gastric cancer, TEX effectively educated monocytes to differentiate into a peculiar type of M2 TAM expressing PD-1, which induce T cell dysfunction through IL-10 secretion by interacting directly with PD-L1+ cells and thereby promote tumor progression (118). TEX have been described to contain miRNA which can be transferred to target cells and modulate cellular function (119). These data strongly highlight the role of TEX as additional mediators of monocyte dysfunction in TME. In the last few years, significant advances in understanding the mechanisms associated with exosome biogenesis/release have been obtained identifying some possible targets to interfere with this cell-cell communication. Recent studies in mouse models demonstrated that RAB27A or RAB35 inhibition significantly impair TEX secretion in HeLa cervical carcinoma and Oli-Neu oligodendroglial precursor cell lines (120), respectively. Moreover, RAB27A deficient tumor cell lines displayed reduced growth due to impaired recruitment of bone-marrow derived, pro-tumoral immune cells (121).

In addition to be modulated by soluble factors released by the tumor cells, monocytes can shape their effector function also in a contact-dependent manner. For instance, breast cancer stem cells express CD90 and Ephrin A4 receptor (EphA4R) that interact with CD11b and EphA4 present on tumor-associated monocytes and macrophages, respectively, leading to the secretion of inflammatory cytokine (IL-6, IL-8, GM-CSF), which in turn sustain tumor stem cell fate (122).

Among factors that could shape monocyte plasticity, TGFβ, a key multifunctional cytokine, involved in both cancer and inflammation, appears to play a key role. Besides being targeted in a number of human diseases (123), TGFβ has a very well-recognized ability to regulate T cell responses (124), supporting Th9 (125, 126) and Th17 (127) differentiation, and promoting regulatory T cell function (128–130). However, how TGFβ regulates innate immune responses just began to be appreciated. Many TME-associated cells, and among those monocytes and macrophages, express high amount of latent LTGFβ. The recent work by Kelly et al. (131), demonstrates that iMo, beside tumor cells, express high levels of αvβ8 integrin responsible for the activation TGFβ from LTGFβ form (131, 132). Additionally, monocyte-derived macrophages, integrin expression and TGFβ signaling are generally maintained in anti-inflammatory macrophages but down-modulated in pro-inflammatory macrophages. To sustain the immunosuppressive microenvironment, tumor cells exploit this regulatory mechanism upregulating the expression of integrin αvβ8 and activating TGFβ from LTGFβ-expressing monocytes and macrophages (133).

# Monocytes at the Metastatic Niche

Cancer metastasis is a multi-step process of the neoplastic progression termed "invasion-metastasis cascade" (134, 135). Monocytes are, among other myeloid cells (e.g., neutrophils), corrupted to foster tumor progression and metastasis. Accumulating evidence indicates that monocytes (primarily iMo) are essential pre-metastatic promoters being rapidly recruited from the bone marrow to the pre-metastatic niche, mainly by CCL2/CCR2 axis (136, 137), where they promote tumor colonization by secreting angiogenic factors, like VEGFA (92, 138). Indeed, in the MMTV-PyMT mouse model of spontaneous breast cancer, CCL2, released by either tumor cells or stromal cells at the metastatic lung niche, induces the recruitment of CCR2-expressing iMo, which in turn favor the extravasation of tumor cells, through the release of VEGFA (92). Consequently, the inhibition of CCL2-CCR2 signaling axis abrogated the recruitment of monocytes thus reducing metastasis formation. Similarly, in a mouse model of metastatic melanoma (B16F10 model), accumulation of CXCR3+monocytes/macrophages in the lung was a prerequisite to mediate melanoma engraftment and metastatic disease (139). However, how this process takes place remains undefined. In a different set of experiments employing B16F10 melanoma model, it was shown that M-MDSCs were recruited to the pre-metastatic niche, mainly by CCL12 expression. By releasing IL-1β, these cells promoted the expression of E-selectin on endothelial cells thus promoting the adhesion of tumor cells to the vascular endothelium (140).

At the metastatic targeted-organs, monocytes can offer survival stimuli for cancer cells. Metastatic cells in the lung, from either mouse or human breast cancer, overexpress vascular cell adhesion molecule-1 (VCAM-1) and shRNA-mediated depletion of VCAM-1 inhibited metastasis formation. Moreover, monocytic cells expressing α4-integrin can bind VCAM-1 present on the surface of tumor cells. Thus, upon α4-integrin engagement on monocytes, VCAM-1 delivers anti-apoptotic signals into breast cancer cells through the PI3K/Akt pathway favoring tumor cell survival (141).

EVs can cross the basal lamina of alveolar capillaries in the lung. In the lungs, alveolar and interstitial macrophages upon taking up these EVs start to secret CCL2 favoring the recruitment of iMo, which in turn differentiate into macrophages, mostly M2-like cells, promoting tumor growth by the secretion of IL-6 and deposition of fibrin (142). Similarly, in colorectal cancer (CRC) patients the high expression of serum exosomal-derived miR-203 was associated with increased probability to develop distant metastases. It was shown that TEX-derived miR-203 uptaken by monocytes promoted their differentiation into M2 like macrophages, in vitro. Furthermore, mice injected with CRC cells transfected with miR-203 developed significantly more liver metastases than the control group (143).

We have previously underlined the plasticity of monocytes and how iMo and pMo play different roles in cancer progression and surveillance. In line with these observations, it has been demonstrated that TEX from poorly-metastatic melanoma cells are taken up by bone-marrow monocytes, promoting their differentiation into pMo, which in turn migrate at the metastatic niche clearing tumor cells by direct engulfment or by activating cytotoxic NK cells (144). Interestingly, TEX from poorly-metastatic tumors caused macrophage alteration toward

M1-like cells expressing TRAIL, which competed with NK cells for tumor killing. These findings suggest that prior to the acquisition of the metastatic capacity, tumors continuously alert host immune system by producing vesicles that affect innate immune responses and support the concept of developing new cancer immunotherapeutic approach based on TEX to deliver specifically immune triggers.

# FUTURE PROSPECTIVE: TARGETING OF MONOCYTES AS THE NEW FRONTIER FOR CANCER IMMUNOTHERAPY

Myeloid cells are extremely plastic and can develop specialized functions in response to micro-environmental pathologic conditions such as infections, autoimmunity or cancer (145). Myeloid cell polarization into either tumor-suppressive or tumor-promoting phenotypes is fundamental for shaping TME. Once at the tumor site, these myeloid cells generally acquire a pro-tumor phenotype (146). Thus, one of the major goals of contemporary tumor immunotherapy is targeting tumorassociated myeloid cells by depletion, recruitment inhibition or reprogramming their polarization/activation status.

As mentioned above, inhibition of the CCL2-CCR2 axis, used to prevent the egression of monocytes from bone marrow, improved the efficacy of chemo-, radio- and immunetherapy in several preclinical models. Nevertheless, the use of either CCL2 or CCR2 inhibitors, in clinical trials, gave disappointing results, indicating the need of supplementary studies considering the presence of potential TME-dependent compensatory mechanisms acting on tumor-resident myeloid cells (146, 147). Moreover, although the continuous blockade of macrophages constrains tumor progression, cessation of the CCL2 blocking therapy stimulates them to a rapid rebound, leading to accelerated metastatic disease via a mechanism dependent on VEGF-A and IL-6 production monocyte-derived by macrophages (102).

# Targeting Trained Immunity

Trained immunity inducing factors were tested for their anti-tumor activity, both in vitro and in vivo, and some of them reached the clinical application. A β-glucan PAMP, Imprime PGG (Imprime), is currently in clinical development in combination with immune checkpoint inhibitors, tumortargeting antibodies, and anti-angiogenic antibodies. The results from a randomized phase 2 clinical trial of Imprime in combination with bevacizumab and carboplatin/paclitaxel vs. bevacizumab and chemotherapy alone in the 1st-line treatment of stage IV non-small cell lung cancer showed promising efficacy in terms of both objective tumor response and survival (ClinicalTrials.gov NCT 00874107, EudraCT 2008-006780-37). Earlier results have shown that both the M2 macrophages and DCs derived from Imprime-trained monocytes have higher expression of PD-L1 and CD86, rendering these cells suitable for treatment with anti-PD-1 antibody. Ex vivo treatment of T cells with Nivolumab, an anti-PD-1 antibody, enhanced proliferation in response to αCD3/αCD28 stimulation and co-culture with Imprime-trained monocytes-derived M2 macrophages or DCs further improved T cell expansion and increased production of several cytokines, including IFNγ, IL-2, TNF-α, and GM-CSF. Results were further validated in syngeneic mouse model, like the CT26 colon carcinoma. Bacillus Calmette-Guerin (BCG), another trained immunity inducer, is currently the only agent approved by the US Food and Drug Administration for first line treatment of carcinoma in situ of the bladder. BCG therapy reduces the risk of recurrence and maintenance therapy with BCG decreases the risk of progression in patients with highgrade, non–muscle invasive bladder cancer (148, 149). It has been speculated that the mechanism of action involves the autophagy. In fact, pharmacologic or genetic inhibition of autophagy blocks the epigenetic reprogramming of monocytes at the level of H3K4 trimethylation, arresting the mechanism of trained immunity induced in vitro by BCG. Single nucleotide polymorphisms associated with bladder cancer progression and recurrence, in the autophagy genes ATG2B (rs3759601) and ATG5 (rs2245214), affected both the in vitro and in vivo training effect of BCG (150).

Muramyl dipeptide (MDP), a synthetic peptide of N-acetyl muramic acid attached to a short amino acid chain of L-Ala-D-isoGln, is a bacterial cell wall peptidoglycan active as NOD2 agonist and contributing to the generation of trained monocytes (151). Interestingly, Paclitaxel conjugated to MDP showed not only antitumor activity, but also immune enhancement capacity. In fact, compared with either paclitaxel or MDP alone, the combination significantly increased the expression and secretion of TNFα and IL-12 from mouse peritoneal monocytes (152). Moreover, it was shown that MDP can upregulate PD-L1 in healthy monocytes, but in patients with Crohn's disease, carrying the Leu1007 frameshift mutation of the NOD2 gene, such effect was completely lost (153) (**Table 2**).

# Targeting Signaling Pathways

An alternative approach to target trained immunity is to inhibit the pathway Dectin-1-Akt-mTOR-HIF-1α. To this end, the beneficial effects of Metformin and Everolimus, an mTOR activator rapamycin analog, administration to patients with type 2 diabetes and cancer, respectively, were linked with the modulation of trained monocytes (67, 90, 154). Interestingly, inhibitors of other kinases, such as Raf-1, PI3K, and ERK are of particular interest in modulating trained monocytes because they represent downstream effectors of Dectin-1 and NOD2 activation (**Table 2**). In particular the knockout of PI3Kγ was reported to break tumor tolerance by MDSC reduction (54) as well as, the combination targeting of PI3Kδ in association with PD-L1- based immunotherapy better limited tumor progression (155, 156). Moreover, the pharmacological treatment using multi-kinase inhibitors carbozantinib and BEZ235, which limit MDSC accumulation, in combination with immune checkpoint therapy controlled more efficiently tumor growth in a castrationresistant prostate tumor model than the single agents (157) underlying the possibility to overcame de novo resistance to antibody blockade based therapy by limiting MDSCs.

The development of epigenetic modulators is acquiring increased interest due to the relevance of epigenetic changed in several diseases. The broad jumonji histone demethylase TABLE 2 | Inhibitors and their corresponding targets found to impact pathways regulating different aspects of monocyte biology.

inhibitor JIB-04 decreased trained immunity response by modulating of the methylation status of H3K9 (158). Interestingly, a clinically relevant small molecule of the BET family of bromodomains, I-BET151, was shown to prevent monocyte tolerance when administered concomitantly with LPS, but it was ineffective when administered after LPS stimulation. These results suggest that I-BET151 is not an effective treatment in monocytes that have already experienced an inflammatory response (159) (**Table 2**).

# Targeting Metabolic Pathways

Metabolic modulation also represents another interesting approach to target both trained monocytes and M-MDSCs. Some metabolites and metabolic enzymes function as either substrates or cofactors for chromatin modifying enzymes, thereby influencing the epigenetic landscape of target cells. Accordingly, fumarate can increase trained immunity by increasing H3K4me3 and H3K27ac and inhibiting the degradation of HIF-1α (90). Moreover, the decreased expression of lysine demethylase 5 family of HDAC (KDM5), responsible for H3K4 demethylation, is also inhibited by fumarate, maintaining then the accessibility of the chromatin. On the other hand, it was shown that tolerant monocytes lack the activity of KDM5, whose function can be restored by its cofactor, α-ketoglutarate (90). Mevalonate, intermediate of the cholesterol pathway has been shown to induce trained immunity (74), consequently, statins can be used to prevent this process under conditions in which accumulation of trained monocytes is detrimental, like patient with hyper-IgD syndrome or inflammatory conditions. Additionally, given the enhanced glutamynolysis associated to trained immunity (90), administration of 6-diazo-5-oxo-L-norleucine (DON), which inhibits glutamine uptake and metabolism, was shown to have favorable effects after organ transplant preventing rejection (160) (**Table 2**). Even though targeting metabolic pathways, to modulate trained monocyte function, is feasible, toxicity, and side effects represent the main drawbacks of metabolic drugs. Overcoming this limitation, for example, by delivering drugs trough nanoparticles/nanocarriers, could open the access to a variety of molecules that have already demonstrated their efficacy in vitro.

# Targeting Immunosuppressive Monocytes (M-MDSC)

Together with trained monocytes, MDSCs represent the other group of targetable cells. Proliferating T cells need a large supply of amino acid like L-arginine and L-tryptophan. MDSCs have developed a strategy to modulate local concentrations of these amino acids via the up regulation of enzymes involved in their degradation like ARG1, NOS2, and IDO. Developing inhibitors of these enzymes represent a field of intense research. To this end, nitroaspirin, consisting of a nitric oxide group covalently linked to aspirin, was shown to restore L-arginine levels in T cells, by suppressing the production of ROS and iNOS (161). Moreover, our laboratory showed that treatment with AT38 [3-(aminocarbonyl) furoxan-4-yl]methyl salicylate], decreased MDSC-induced nitration within the tumor environment, increasing CCL2 binding and T cell tumor infiltration in mice (60). N-hydroxy-L-arginine (NOHA) is an intermediate in the conversion of arginine to citrulline and NO by iNOS (162) (**Table 2**). It is a potent physiologic inhibitor of ARG1. Mice exposed to NOHA demonstrated inhibition of MDSC function, and mice with B cell lymphoma treated with NOHA had decreased numbers of circulating Treg cells and improved immune responses to the cancer (163). We also demonstrated that the production of polyamine by ARG1 activity promotes IDO1 activation through Src kinase signaling (164). Therefore, a combined targeting of ARG1 and IDO1 using pharmacological compounds (165) could be an effective treatment to constrain tumor-associated immunosuppression improving cancer immunotherapy. MDSCs generate an immunosuppressive environment also by producing prostaglandin E2 (PGE2), which levels are regulated through the enzyme Cox2 (166). PGE2 activates PGE2-R on MDSCs altering the differentiation of MDSCs. In the bone marrow, activation of PGE2-R hampers the differentiation of monocytes into antigen presenting cells, while increased PGE2 switch monocytes into MDSCs via increased expression of IDO, IL-4Rα, iNOS, and IL-10 (167, 168). In line with these findings, blocking the production of PGE2 in mice bearing lung carcinoma, with Cox2 inhibitors, decreased the expression of ARG1 in MDSC and tumor growth (169). Cox2 inhibitors may also provide other antitumor effects (**Table 2**). Celecoxib, a Cox2 inhibitors, was shown to decrease MDSC recruitment and increased CD8+ T cell tumor infiltration in gliomas and colon carcinoma by decreasing CCL2 production (170).

Methionine, an essential amino acid for normal T cells function, is generally supplied by antigen presenting cells. DCs and macrophages import cysteine to create methionine, which is then secreted; they additionally release thioredoxin converting cysteine to methionine. However, in the TME, MDSCs transport cysteine intracellularly thus depleting T cells of methionine (171, 172). Consequently, blocking thioredoxin could prevent T cells proliferation arrest. In this situation small molecules or neutralizing antibodies targeting extracellularly released enzymes could be beneficial to restore T cells function and used in combination with ACT of tumor specific T cells or with check point inhibitors.

Constitutive activation of the JAK-STAT pathway has been implicated in the proliferation of MDSCs via anti-apoptotic and pro-proliferative genes (173). Moreover, ARG1 and iNOS, immunosuppressive enzymes in MDSCs, are controlled via STAT1 and STAT3 (174). Consequently, inhibition of the JAK-STAT pathway has been of great interest. Many inhibitors of STAT1/STAT3 have been discovered and several of them already enter clinical trials. Among those one, Stattic, an inhibitor of pSTAT3, reduces the suppressive activity of MDSC in vitro both in mice and in humans (175, 176) (**Table 2**).

In the last 10 years, several approaches to target immunosuppressive monocyte referred as M-MDSC were developed using a large spectrum of pharmacological compounds and immunotherapeutic approaches with the aim to limit MDSC proliferation, function, and recruitment to tumor site. For instance, non-therapeutic low doses of conventional chemotherapeutic drugs such as gemcitabine and 5-fluorouracil [which does not induce immunogenic cancer-cell death of tumor cells (177)] were able to limit Ly6C+MDSC number and activity by inducing c-FLIP down-regulation (65). The M-MDSC elimination is essential to restore the immune response in tumor-bearing mice by rescuing the frequency of circulating anti-tumor T cells (51). These data obtained in preclinical cancer models were also confirmed in tumor patients. In fact, renal cell cancer (RCC) patients with low frequency of M-MDSCs showed a better disease control after anti-tumor peptide-based vaccination in combination with chemotherapy (178); moreover, the HPV16 long-peptide-based vaccination during CarboTaxol treatment was able to reduce circulating MDSCs and generated a strong immune response, confirming the relevance of M-MDSC elimination as a valid approach to enforce anti-tumor immune response (179). Based on these data, several chemotherapeutic compounds with different mechanisms of action were listed as M-MDSC-targeting drugs (173). M-MDSC elimination can be also achieved using immune compounds such cytokines and antibodies. In this line, the combination of IL-2 with anti-CD40 antibody was effective on cancer growth control in two different mouse tumor models by inducing M-MDSC elimination trough Fas-mediated apoptosis (180). Similarly, to improve the efficacy of CAR T cells immunotherapy in leukemia setting, by limiting transferred T cells-associated life-threatening cytokine-release syndrome (CRS) and neurotoxicity, either monocyte-depletion or infusion of IL-1 receptor antagonist anakinra were reported as effective strategies (181). Other therapeutic approaches aim at favoring MDSC differentiation into anti-tumoral cell subsets such as macrophages and DCs. For example, all-transretinoic acid (ATRA) treatment was reported to promote the differentiation of MDSC into mature anti-tumoral myeloid cells via the activation of the ERK1/2 signaling pathway (182) as well as the treatment with 25-hydroxyvitamin D3 was reported to reduce the frequency of immature immune suppressive cells in peripheral blood of HNSCC patients (183). Moreover, M-MDSC differentiation toward mature anti-tumoral monocytes-derived DCs can be achieved using pharmacological inhibitor of ATP-converting ectoenzyme ENTPD2, thus mitigating cancer growth and enhancing the efficiency of immune checkpoint inhibitors (184). Finally, some targeting approaches aim at blocking M-MDSC migration from bone marrow to tumor site. For example, amino-biphosphonates were able to prevent the activation of matrix metalloproteases (MMPs) limiting cancer aggressiveness and distal spread (185). Along the same line, pharmacological antagonist of chemokine receptors (i.e.,

# REFERENCES

S-265610, CXCR2-specific antagonist) were able to drastically reduce M-MDSC in tumor-bearing mice (186).

# CONCLUDING REMARKS

Monocytes are relevant immunological cells connecting the innate and adaptive immune compartments. Here, we have attempted to integrate recent advances in the molecular, metabolic, and functional aspects of monocyte biology with the current state of understanding about the role of these cells in cancer growth and metastatic spread. Although a large body of evidence supports the notion that circulating monocytes serve only as precursor cells that replenish tissue macrophages and DC populations, the overwhelming complexity underlined by high throughput technologies, is supporting a direct contribution of monocyte to cancer development. Targeting monocytes at the immunological, metabolic, epigenetic, and transcriptional level is a promising strategy to treat both disease with impaired immune function, like cancer, or with over-reactive immune response, like autoimmune diseases. The advances in our knowledge on monocyte development, response, and reprogramming, particularly during cancer evolution and metastatic spread, will pave the way for the development of new therapeutic strategy with specificity and limited toxicity.

# AUTHOR CONTRIBUTIONS

SC, SU, RT, IM, FD, and SS wrote parts of the manuscript. SC and VB edited and finalized the manuscript.

# FUNDING

This work was supported by the Italian Association for Cancer Research (18603 and 12182 that refers to Special Program Molecular Clinical Oncology 5 per mille), by the Cariverona Fondation (Project call, 2017), by the Cancer Research Institute (Clinic and Laboratory Integration Program, CLIP-2017), and by Euronanomed III (Joint Translational Call 2017, Project resolve) to VB.

for experimental influenza A/Anhui/1/2013 (H7N9) infection in mice. Front Immunol. (2018) 9:869. doi: 10.3389/fimmu.2018.02471

diagnostic signature of chronic myelomonocytic leukemia. Blood. (2015) 125:3618–26. doi: 10.1182/blood-2015-01-620781

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Canè, Ugel, Trovato, Marigo, De Sanctis, Sartoris and Bronte. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Neutrophil Maturity in Cancer

John B. G. Mackey 1,2, Seth B. Coffelt 1,2 \* and Leo M. Carlin1,2 \*

*1 Institute of Cancer Sciences, University of Glasgow, Glasgow, United Kingdom, 2 Cancer Research UK Beatson Institute, Glasgow, United Kingdom*

Neutrophils are implicated in almost every stage of oncogenesis and paradoxically display anti- and pro-tumor properties. Accumulating evidence indicates that neutrophils display diversity in their phenotype resulting from functional plasticity and/or changes to granulopoiesis. In cancer, neutrophils at a range of maturation stages can be identified in the blood and tissues (i.e., outside of their developmental niche). The functional capacity of neutrophils at different states of maturation is poorly understood resulting from challenges in their isolation, identification, and investigation. Thus, the impact of neutrophil maturity on cancer progression and therapy remains enigmatic. In this review, we discuss the identification, prevalence, and function of immature and mature neutrophils in cancer and the potential impact of this on tumor progression and cancer therapy.

Keywords: neutrophil, cancer, myeloid, cancer inflammation, granulopoiesis

#### Edited by:

*Wei Zheng, Albert Einstein College of Medicine, United States*

#### Reviewed by:

*Siegfried Weiss, Helmholtz Center for Infection Research, Germany Viktor Umansky, German Cancer Research Center (DKFZ), Germany*

#### \*Correspondence:

*Seth B. Coffelt<EMAIL_ADDRESS>Leo M. Carlin<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Cancer Immunity and Immunotherapy, a section of the journal Frontiers in Immunology*

> Received: *09 May 2019* Accepted: *29 July 2019* Published: *14 August 2019*

#### Citation:

*Mackey JBG, Coffelt SB and Carlin LM (2019) Neutrophil Maturity in Cancer. Front. Immunol. 10:1912. doi: 10.3389/fimmu.2019.01912*

# INTRODUCTION

Neutrophils in cancer have received very little attention until recently, despite contributing 50–70% and 10–25% of circulating leukocytes in humans and laboratory mice, respectively (1). However, recent progress has renewed interest in these cells. In experimental cancer models, neutrophils have been implicated in nearly every stage of the oncogenic process and their role has been reviewed in detail (2–4). Neutrophils are able to mediate a broad range of anti- and pro-tumor activities from direct cancer cell killing to tumor cell proliferation, angiogenesis, metastasis, and orchestrating other immune responses. These recent studies have highlighted the complexity of neutrophils in cancer progression, with novel information on their previously unappreciated plasticity and heterogeneity. While neutrophil plasticity can be directly affected by the local microenvironment, neutrophil heterogeneity is also influenced by their maturation (5), age (6), suppressive properties (7), function [e.g., phagocytosis (8)], and reverse transendothelial migration (rTEM) (9). An underexplored aspect of this is the appearance of immature neutrophils in cancer.  However, the influence of neutrophil maturity on their anti- or pro-tumor properties remains understudied. In this review, we focus on the functional properties and relevance of immature neutrophils in cancer. We discuss methods used to identify neutrophils of different maturation states and explore their limitations. Finally, we postulate the impact that neutrophil maturity may have on the efficacy of cancer therapies.

# GRANULOPOIESIS

After birth, neutrophil production occurs primarily in the bone marrow (BM) where they are derived from hematopoietic stem cells (HSCs). During neutrophil differentiation in mice and humans the nucleus progresses from a banded to segmented morphology, allowing the

**97**

identification of neutrophils at distinct stages of maturity (11). Stages of neutrophil differentiation are also characterized by their unique expression of the transcription factors PU.1 and CCAAT enhancer binding protein (C/EBP)-α (12), C/EBPβ (13), and C/EBPε(14). Mature neutrophils are mitotically inactive with cell cycle arrest occurring during the myelocyte to metamyelocyte transition (15). The post-mitotic BM transit of neutrophils and release into the circulation takes between 5 and 8 days in humans (16) and 1–2 days in mice during homeostasis (17). Neutrophil granules, termed azurophillic (primary), specific (secondary), and gelatinase (tertiary), in addition to secretory vesicles, are formed at specific stages of neutrophil differentiation. Each granule type is composed of distinct proteins synthesized at the time of formation (18) and granules are released in reverse sequential order following neutrophil activation (19). As such, the proteome composition of immature and mature neutrophils is greatly different. It is important to also acknowledge that in disease, including cancer, granulopoiesis can occur outside of the medullary spaces of the BM, termed extramedullary hematopoiesis (EMH); however, little is known about the mechanisms regulating EMH and its influence on neutrophil development (20).

# ISOLATION AND IDENTIFICATION OF IMMATURE NEUTROPHILS

Despite the extensive data on neutrophils and their functions in homeostasis and disease, they remain a challenging cell population to study largely due to their short half-life [∼18.5 h in the circulation of humans during homeostasis (16)] and propensity for priming and activation. While neutrophil life span can be increased following their activation and extravasation, a small window of opportunity for in vitro experimentation remains in comparison to other cell types. Neutrophil properties derived from ex vivo experimentation can be difficult to accurately interpret and apply to their behavior in vivo. Developments of in vivo imaging techniques and identification of neutrophils (e.g., via in vivo injection of fluorescently conjugated anti-Ly6G antibody, clone 1A8 (9, 21, 22) and fluorescent reporter mice (23) have allowed their investigation without possible ex vivo manipulation-induced artifacts; however, these approaches still have their own caveats for example the undetermined function for Ly6G (23–25). Importantly, experimental analysis of immature neutrophil populations is an even greater challenge.

# Density Properties

Neutrophil density changes during development as a result of their increased granularity and changes in cell size (26). Therefore, density gradient purification is useful for enriching neutrophil populations at certain stages of maturation and allows for down-stream analysis. Immature neutrophils are typically found in low density (LD) fractions, whereas mature neutrophils are found in the normal/high density (N/HD) fractions (5) (**Tables 1**, **2**). Nevertheless, the neutrophil populations obtained by density gradient purification are not pure as N/HDNs can TABLE 1 | Methods for the identification of immature neutrophils in humans.

become LDNs following activation (55), making interpretation of the functional properties of neutrophil maturity challenging by this technique. For instance, LDNs isolated from the peripheral blood of 4T1 tumor-bearing mice make up ∼40% of morphologically mature neutrophils (5), LDNs obtained from the peripheral blood of mice bearing breast cancer liver metastasis were composed of 80% neutrophils with an immature nuclear morphology (56), and the nuclear morphology of LDNs from lung cancer patients represent both mature and immature neutrophils (5). Overall, this technique can be useful for enriching neutrophil populations; although, more specific methods of identification of neutrophil maturity are required for accurate interpretation of downstream functional analysis.

# Morphology and Cell Surface Markers

Nuclear segmentation is considered accurate for immature neutrophil identification in the peripheral blood of cancer patients (57) and mouse models of cancer (58) (**Tables 1**, **2**). However, cells cannot be isolated by this method for downstream experimentation. A major hindrance in neutrophil biology is the lack of a specific and robust marker of neutrophil maturity. Changes in cell surface receptor expression during maturation, such as the CXCR4:CXCR2 axis (59, 60), can be used to separate

immature and mature neutrophils (48) (**Table 2**). However, these surface receptors are prone to alteration following neutrophil activation [e.g., CD11b:CD18 (61)], tissue migration [e.g., CD62L (6, 62, 63)], and aging [e.g., CXCR4 (6)], resulting in a major challenge in the identification of efficient markers of maturity. In mice, immature and mature neutrophils can accurately be identified as Ly6GInt/LowCD11bPos and Ly6GHighCD11bPos respectively (13, 21, 51) (**Table 2**). However, the limitations of using Ly6G as a maturity marker include relatively small differences in expression of this molecule between immature and mature neutrophils, compounding the technical issues associated with fluorescence intensity comparisons in some readouts. Despite this, recently identified markers of neutrophil maturity with larger differences in expression, for example CD101 (48), could be useful candidates for development of fluorescent reporter models and in vivo identification. Here, CD101 expression can be used to identify CD101Neg (immature) and CD101Pos (mature) neutrophils (48); however, this marker requires further validation to ensure its accuracy in a wide range of pathologies. Another example is c-KIT/CD117, the expression of which has been shown to associate with neutrophil maturity in naïve mice, mice undergoing candida-induced emergency granulopoiesis (13), and a mouse model of breast cancer (64) (**Table 2**). However, although in the K14-Cre;Cdh1F/F ;Trp53F/F , and 4T1 mouse mammary tumor models, neutrophil c-KIT expression is enriched on immature neutrophils, it fails to completely correlate with maturation status (58, 65). In humans, immature and mature neutrophils are commonly identified as CD16LowCD10Neg and CD16HighCD10Pos, respectively (66) (**Table 1**). Expression of CD16 (FcγRIII) is initiated between the metamyelocyte and band stages of neutrophil maturation (67, 68). However, its expression can be reduced during apoptosis (69) and can be up-regulated on the cell surface following secretory granule cell membrane fusion (67). Distinct differences in the hematopoietic environment, local and systemic cytokine levels and the functional requirements for neutrophils will exist between naïve, emergency granulopoiesis and the more chronic "inflammation" present in cancer. Therefore, as neutrophils can exhibit plasticity in response to their environment, certain markers are likely to only be suitable in particular models and require efficient validation in each. Overall, the challenges associated with identifying and isolating populations of neutrophil maturity have hindered their study and our current understanding of their functional properties.

# FUNCTIONAL PROPERTIES OF MATURE AND IMMATURE NEUTROPHILS

## Immature Neutrophils in Cancer

The existence of immature neutrophils in the circulation and tissues is a consequence of cancer development in human patients and mouse models. For example, immature neutrophils are detectable in the circulation (and in some cases the primary tumors) of both injectable and transgenic mouse models of colon (70), skin (70), mammary (5, 58, 71, 72), lung cancer (5, 73), and mesothelioma (AB12) (5, 73).

# Drivers of Immature Neutrophil Appearance Outside of the Hematopoietic Niche

Premature release from the BM as observed in states of emergency granulopoiesis, as reviewed by others (75), is considered the main reason for the presence of immature neutrophils in the circulation. Emergency granulopoiesis commonly results from increased levels of granulocyte colonystimulating factor (G-CSF; also known as CSF-3) (76) that promotes the differentiation of hematopoietic precursors down the neutrophil lineage and release of neutrophils into the circulation (60, 71, 72, 77) (**Figure 1A**). Production of G-CSF is controlled by interleukin (IL)-23 and IL-17 (58, 78, 79) and can be increased in many cancer models and patients (56, 58, 71, 72, 80). Enhanced levels of G-CSF drive excessive production and release of neutrophils and their precursors into the circulation, leading to neutrophilia (58, 71, 72, 81). G-CSF is dispensable for emergency granulopoiesis and other cytokines, including granulocyte/macrophage (GM)-CSF (also known as CSF-2) (43, 75), can drive neutrophil production and release. Furthermore, neutrophil precursors can seed distant tissues and produce neutrophils in situ, as has been observed in cancer patients (82) (**Figure 1B**). TGFβ is another cytokine that favors the presence of immature neutrophils, since its inhibition converts neutrophils to a mature phenotype in transplantable models of mesothelioma (73). The N1/N2 nomenclature—which mirrors the Th1/Th2 nomenclature of T helper cells—was coined in this study based on the influence of TGFβ to modulate neutrophil phenotype and function. Neutrophils were named pro-tumor N2 cells or anti-tumor N1 cells after Th1/Th2 CD4 T cells and M1/M2 macrophages. However, evidence that neutrophils mediate type 1 or type 2 immunity is lacking, and additionally, how these phenotypes relate to the neutrophils found in patients is still under investigation [recently reviewed in Shaul and Fridlender (83)]. Therefore, this nomenclature may be confusing in the context of cancer at this time and future work will determine its appropriateness. In contrast to TGFβ, expression of Type 1 interferons (IFNα and IFNβ) in tumors favors mature neutrophils over immature neutrophils (57). Most likely, there are many other tumor-derived factors that influence neutrophil maturity and their discovery could lead to opportunities for therapeutic intervention.

# Functional Properties of Immature Neutrophils

The degree of functional difference between immature and mature neutrophils remains an open question in the field. Due to the importance of neutrophil differentiation in their effector mechanisms, (e.g., production of granule proteins), there is a strong argument for functional differences. Immature neutrophils may in many cases fall under the myeloidderived suppressor cell (MDSC) umbrella, as these cells have been reported to inhibit T cells. MDSCs encompass a wide range of granulocytic and monocytic cell types at different stages of differentiation.  Recently, the classification and identification of MDSC subsets based on their phenotype and morphology has been improved, but these are still identified as CD11bPosLy6CLowLy6GPos (84). Nevertheless, we believe that the suppressive functions of immature and mature neutrophils is a pathological response to tumorigenesis rather than a completely separate granulocytic population, as discussed by others (86–88). Therefore, we refer to PMN-MDSCs as neutrophils in this article. Ex vivo suppression assays are the most common technique for identifying and analyzing suppressive neutrophils. Findings that have used this technique are challenging to interpret as they can be influenced by neutrophil survival, cytotoxicity, neutrophil:T cell ratio, and protocols used [e.g., CD3/CD28 microbeads or antibodies (89)]. Immature (90) and mature (91) neutrophils can be suppressive; however, differences in the suppressive capacities of these populations (66, 92) are likely influenced by disease, model, and neutrophil isolation and identification protocols used (**Figure 1D**). It should also be noted that not all tumorinfiltrating immature neutrophils possess T cell-suppressive abilities (93, 94).

Immunosuppression by neutrophils is not only important for primary tumor progression, but this mechanism can also promote metastasis formation. Neutrophils can be recruited by CXCR2 ligands to dampen anti-tumor immunity in premetastatic organs so that disseminated cancer cells can evade immune destruction (58, 95, 96). In these cases, it is the immature neutrophils that are thought to mediate immunosuppression and subsequent metastasis; although, this has not been formally shown. In addition, immature neutrophils and other myeloid progenitors can aid in the formation of the pre-metastatic niche via mechanisms other than T cell suppression (97–99).  Together, these data indicate that neutrophil maturity may be irrelevant to their pro-metastatic functions.

ROS production is important in several neutrophil effector mechanisms including their microbicidal (101), phagocytic (102) and suppressive capacity and contributes to neutrophil antiand pro-tumor functions [reviewed in Ohl and Tenbrock (103)]. One such pro-tumor function of neutrophil ROS in cancer is their promotion of tumor initiation at states of inflammation by damaging proliferating epithelial cells (104). In relation to neutrophil maturity the production of ROS can be variable between immature and mature cells. For example, immature neutrophils (Ly6GPosCD101Neg) display reduced ROS production compared to mature (Ly6GPosCD101Pos) in a mouse orthotopic pancreatic cancer model (48). Similarly, in a range of other transplantable mouse cancer models, LDNs which are enriched in morphologically immature neutrophils have reduced ROS production (5). However, the amount of ROS production may be context dependent and reliant on metabolism. In tumor-free mice (Ly6GIntc-KitPos) and ovarian cancer patients (CD10Int), immature neutrophils are dependent on oxidative mitochondrial metabolism rather than glycolysis, for ROS production (65). Recently, LDNs from mice bearing 4T1 mammary tumorliver metastasis have also been shown to have an increased oxidative metabolism (56). This dependency may have implications in the glucose-limited tumor microenvironment

FIGURE 1 | Immature neutrophils are present in cancer and have an altered functional capacity compared to mature that may influence tumor progression. Immature neutrophils can be present and significantly increased in the peripheral blood and tissues of cancer patients. This increase may result from: (A) promotion of their early release from their bone marrow (BM) haematopoietic niche by increased systemic chemokines, such as granulocyte colony-stimulating (G-CSF), e.g., tumor produced or as therapy. (B) release of neutrophil precursors from the BM and their extramedullary proliferation in the circulation or tissues. Immature neutrophils may have both anti- and pro-tumor properties. These include (C) altered localization resulting from their differential cell surface marker expression influencing their chemotactic capacity and/or less segmented nuclear morphology compared to mature neutrophils reducing their deformability and (D) different functional capacity compared to mature neutrophils including their reduced phagocytic capacity, altered suppressive properties, reduced NETosis, and reduced granularity. (E) Together, these differences in the properties and functions of immature neutrophils could lead to their negative influence when targeting neutrophils in cancer therapy. G-CSF, colony stimulating factor-3; GMP, granulocyte monocyte progenitor; ROS, reactive oxygen species; NET, neutrophil extracellular trap; NK, natural killer cell.

and affect neutrophil function (65). Therefore, while immature neutrophils can have reduced ROS production compared to mature neutrophils, this appears to be dependent on stimulus, their localization and the tissue microenvironment.

Neutrophil extracellular traps (NETs) are extracellular fibers composed of nuclear, mitochondrial, cytoplasmic and granule contents that can be released by neutrophils following their activation (105). NETs can capture circulating cancer cells in the mouse lung promoting their extravasation and metastasis formation (106, 107). Neutrophils can also aid in formation of the omental pre-metastatic niche and capture of circulating ovarian cancer cells, promoting their metastasis at this site (108). The ability of immature human neutrophil populations to release NETs is reduced following interferon priming (35) (**Figure 1D**). In addition, when isolated from the peripheral blood of acute myeloid leukemia (AML) patients, morphologically immature neutrophils show decrease capacity for NET formation following phorbol 12-myristate 13-acetate (PMA) stimulation (109). As NETs have been proposed to arise from the inability of terminally differentiated neutrophils to re-enter mitosis (110), it could be inferred that the increased mitotic capacity of immature populations contributes to these differences. ROS contribute to NETosis by promoting granule release and rupture of the nuclear envelope, as highlighted by the inability of neutrophils from chronic granulomatous disease patients to undergo NETosis (111, 112). Differences in ROS production with neutrophil maturity may also influence NETosis (65). Differences in granule composition of neutrophils at different maturity may also influence the functional capacity of their NETs. The tertiary granule component MMP-9 (113) has been implicated in NETinduced dormant cancer cell reactivation (114) and its possible reduced abundance in banded neutrophils and earlier neutrophil precursors present in cancer could reduce their ability to promote this reactivation.

Multiple studies have indicated a reduced migratory capacity of immature compared to mature neutrophils (5, 48) (**Figure 1C**). This may result from lower expression of chemokine receptors, such as CXCR1 and CXCR2 (30), and other genes involved in chemotaxis (48). In mice, proliferating neutrophil precursors, identified as Ly6GLowCXCR2Negc-KitPosCXCR4Pos, have reduced migration to laser-induced damage (48). High CXCR2 expression by neutrophils has been associated with poor outcome in human pancreatic ductal adenocarcinoma (PDAC) patients (95). Inhibition of CXCR2 in a mouse model of PDAC reduces neutrophil migration and delays tumor progression (95). Banded nuclear morphology, and thus reduced deformability, may promote immature neutrophil sequestration in capillaries and reduce their migratory capacity (115); although, banded nuclear morphology in immature human neutrophils does not affect transendothelial migration (TEM) when compared to segmented neutrophils ex vivo (62) (**Figure 1C**). It is therefore conceivable that their increased sequestration in off target tissues and ability to undergo TEM may result in unwanted immature neutrophil accumulation and the promotion of inflammation and/or metastasis. Additionally, neutrophil spontaneous migration is increased in the early compared to late stages of cancer in a mouse orthotopic lung cancer model (116). These changes in neutrophil function with tumor progression are present in BM cells, suggesting altered granulopoiesis over time (116). Therefore, while further investigation is required, differential trafficking of immature neutrophils could have the capacity to both antagonize and promote tumor development dependent on their localization.

The phagocytic capacity of immature, compared to mature, neutrophils is also reduced (5, 48) and could result from their altered cell surface receptor expression and decreased ROS production (**Figure 1D**). Fc receptors (FcRs) are important in mediating phagocytosis (117) with decreased expression of CD16 likely influencing their phagocytic capacity. Furthermore, immature neutrophils (CD16Int) are unable to kill tumor cells via FcγRI, but exhibit cytotoxicity via FcαRI (118). Activation of FcRs, integrins and G-protein coupled receptors (GPCRs) can trigger neutrophil ROS production and its extracellular or intracellular release into the phagolysosome, as reviewed in more detail by others (102, 119). Unsurprisingly, immature neutrophils have also been shown to have an increased life span and can mature ex vivo (120). It will be interesting to determine if neutrophil maturation after their release from the BM contributes to heterogeneity within the mature neutrophil population. However, despite differences in the functional capacity of immature and mature neutrophils, they are still capable of mediating innate immune functions (120). Overall, the effect of neutrophil maturity in cancer remains enigmatic and further investigation, coupled with accurate identification, is required.

# NEUTROPHIL MATURITY IN ANTI-CANCER THERAPY

Immunotherapy has shown great promise in cancer; however, only a minority of patients respond to certain therapies (121) and combinatorial therapies targeting a broad range of immune populations may be more beneficial. Therapies targeting neutrophils have received relatively little attention (**Figure 1E**). While the direct effect of therapies on neutrophils at different stages of maturation has not been investigated, we can consider ways in which the properties of immature neutrophils are relevant.

Neutrophils recruited to the tumor via CXCR2 can aid tumor progression (122) and inhibition of CXCR1 and CXCR2 has shown promise in mouse models (95) and human cancers (123). As CXCR2 expression increases with neutrophil maturation (48) inhibitors of CXCR2 may differentially influence immature and mature neutrophils affecting their efficacy. Therapies targeting immunosuppressive neutrophils enhance responses to checkpoint blockade by promoting tumor infiltration by T cells in mouse models (124–127). A greater understanding of the maturity composition of these cells could better aid targeting of this population. Furthermore, tyrosine kinase inhibitors that target the hepatocyte growth factor (HGF) receptor, cMET (e.g., Cabozantinib and Capmatinib) can extend survival by influencing neutrophil behavior in mouse melanoma and PTEN/p53-deficient prostate cancer models (128, 129). As tyrosine kinases (e.g., Bruton's tyrosine kinase; BTK) are important in regulating neutrophil development (49) and in neutrophil integrin signaling (130) it is possible that their inhibitors have altered effects on neutrophils of different maturity. Similarly, monoclonal antibody (mAb) based therapies, for example anti-gp75 (TA99) (131), anti-HER2 (Trastuzumab) (131), and anti-SIRPα (KWAR23) (132, 133), promote neutrophil-mediated destruction of cancer cells. The reduced phagocytic capacity (48) of immature neutrophils and differences in their FcR expression (118) may reduce the efficacy of these therapies. Furthermore, these properties may hamper their ability to deliver therapeutics, such as in nanoparticles (134), to the tumor (**Figure 1E**). Finally, adoptively transferred neutrophils can aid in the killing of cancer cells (135) and can be isolated from G-CSF-treated donors (136) (**Figure 1E**). Here, the activation (137) and potential retention of transferred immature neutrophils in off-target organs [e.g., the lung (138)] needs to be considered. In addition, G-CSF-driven immature neutrophil release, neutrophil accumulation, and alterations to neutrophil function in cancer (66, 100, 139) need to be further deliberated when treating neutropenic cancer patients with G-CSF (140, 141).

# CONCLUSIONS

To gain an accurate understanding of maturity on neutrophil functional capacity, consensus protocols for identification of neutrophil maturity are urgently required. However, as protocols and markers may not be transferable between models, detailed confirmation of maturity in each is essential (e.g., associated nuclear morphology, transcriptomics, proteomics, and surface protein expression data) allowing for proper comparison. Functional investigation needs to be further driven by in

# REFERENCES

vivo investigation to remove concerns associated with ex vivo manipulation. Of particular importance, investigating the localization and suppressive capacity of immature neutrophils in situ will aid in determining their influence on immunotherapy. Furthermore, more research on immature neutrophils in cancer patients should be carried out to determine where these cells appear. Correlations between immature neutrophils and mutational drivers need investigation to understand how these cells occur outside the bone marrow and to identify additional biomarkers of disease. Changes in neutrophil maturation status before, during and after anti-cancer therapy may provide insight into how these cells are regulated. Taken together, the available evidence suggests immature neutrophils in cancer inevitably influence tumor development and we emphasize the importance of improving methodologies for their study.

# AUTHOR CONTRIBUTIONS

JM, SC, and LC wrote and edited the manuscript.

# FUNDING

This work was supported by Core funding from Cancer Research UK (A23983 and A17196 to LC), Cancer Research UK Glasgow Center (A25142 to SC), Breast Cancer Now (2018JulPR1101 to SC), Wellcome Trust (208990/Z/17/Z to SC), Tenovus Scotland (Project S17-17 to SC).

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**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Mackey, Coffelt and Carlin. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Context Drives Diversification of Monocytes and Neutrophils in Orchestrating the Tumor Microenvironment

Juhee Jeong1 , Yoorock Suh1 and Keehoon Jung1,2,3 \*

*1 Lab of Cancer Immunology and In Vivo Imaging, Department of Biomedical Sciences, BK21 Plus Biomedical Science Project, Seoul National University College of Medicine, Seoul, South Korea, 2 Department of Anatomy and Cell Biology, Seoul National University College of Medicine, Seoul, South Korea, 3 Institute of Allergy and Clinical Immunology, Seoul National University Medical Research Center, Seoul, South Korea*

#### Edited by:

*Wei Zheng, Albert Einstein College of Medicine, United States*

#### Reviewed by:

*Markus Munder, Johannes Gutenberg University Mainz, Germany Raymond B. Birge, Rutgers University, The State University of New Jersey, United States*

\*Correspondence:

*Keehoon Jung<EMAIL_ADDRESS>
#### Specialty section:

*This article was submitted to Molecular Innate Immunity, a section of the journal Frontiers in Immunology*

Received: *03 May 2019* Accepted: *18 July 2019* Published: *16 August 2019*

#### Citation:

*Jeong J, Suh Y and Jung K (2019) Context Drives Diversification of Monocytes and Neutrophils in Orchestrating the Tumor Microenvironment. Front. Immunol. 10:1817. doi: 10.3389/fimmu.2019.01817* Recent preclinical/clinical studies have underscored the significant impact of tumor microenvironment (TME) on tumor progression in diverse scenarios. Highly heterogeneous and complex, the tumor microenvironment is composed of malignant cancer cells and non-malignant cells including endothelial cells, fibroblasts, and diverse immune cells. Since immune compartments play pivotal roles in regulating tumor progression via various mechanisms, understanding of their multifaceted functions is crucial to developing effective cancer therapies. While roles of lymphoid cells in tumors have been systematically studied for a long time, the complex functions of myeloid cells have been relatively underexplored. However, constant findings on tumor-associated myeloid cells are drawing attention, highlighting the primary effects of innate immune cells such as monocytes and neutrophils in disease progression. This review focuses on hitherto identified contextual developments and functions of monocytes and neutrophils with a special interest in solid tumors. Moreover, ongoing clinical applications are discussed at the end of the review.

Keywords: monocytes, neutrophils, tumor microenvironment, myeloid cell heterogeneity, innate immunity, cancer immunology

# MONOCYTES: FROM DEVELOPMENT TO DEPLOYMENT

# Monocyte Development

Monocytes originally stem from the bone marrow and constitute 10% of leukocytes in human blood and 4% of leukocytes in mouse blood, respectively (1). The development of blood monocytes is dependent on colony-stimulating factor 1 receptor, CSF-1R (also known as M-CSFR; macrophage colony-stimulating factor receptor) (1, 2). CSF-1R is a hematopoietic growth factor receptor expressed on monocytes, macrophages, dendritic cells and their progenitors (1, 2). CSF-1R interacts with its ligands CSF-1 (M-CSF) and IL-34 to regulate the development of monocytes in the bone marrow (1, 2). In mice deficient in CSF-1R and CSF-1, monocyte development is inhibited, and therefore the number of monocytes in blood is remarkably reduced (1, 2).

With knowledge of CSF-1R, it is possible to navigate the development process of monocytes. From the bone marrow, hematopoietic stem cells (HSCs) give rise to heterogeneous multipotent progenitors (MPPs) generating common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs) in a CSF-1 dependent manner (3). While lymphoid cells such as T lymphocytes, B lymphocytes, and natural killer cells are derived from CLPs, CMPs generate megakaryocyte and erythrocyte progenitors (MEPs) or granulocyte and macrophage progenitors (GMPs). Generated GMPs further go through a series of differentiation, firstly into macrophage, and DC progenitors (MDPs), then into common monocyte progenitors (cMoPs), and finally into monocytes (3). Differentiated monocytes can be divided into two main subpopulations defined as Ly6ChiCX3CR1low and Ly6ClowCX3CR1hi cells in mice and as CD14hiCD16+/− and CD14lowCD16hi cells in humans (4–8).

Ly6ChiCX3CR1low populations (hereinafter referred to as Ly6Chi monocytes) are named "classical" or "inflammatory" monocytes, whereas Ly6ClowCX3CR1hi populations (hereinafter referred to as Ly6Clo monocytes) are named "non-classical" or "patrolling" monocytes for their preferential patrolling behavior while circulating the blood stream (9, 10). Development of Ly6Chi monocytes occurs during the cMoP stage, dependent on GM-CSF, c-FLIP, IRF8, and KLF4 (10). The widely accepted hypothesis on Ly6Clo monocyte differentiation is that after generation of Ly6Chi monocytes from the bone marrow, a proportion of them differentiate into Ly6Clo monocytes as downregulation of Ly6C and upregulation of Nr4a1, C/EBPβ, CSF-1R, and CX3CR1 (4, 10, 11). However, this was questioned for some time in that deletion of transcription factors KLF4 and IRF8 hinders the development of Ly6Chi monocytes but not Ly6Clo monocytes (12–14). This finding led to controversy on whether Ly6Clo monocytes originate from Ly6Chi monocytes or not. The latter argues that Ly6Clo monocytes might have a distinct differentiation lineage in a Ly6Chi monocyte-independent way, namely direct differentiation from cMoPs.

Single-cell RNA sequencing provided an additional clue, reasserting that Ly6Chi monocyte population is the source of Ly6Clo monocytes (15). Application of such advanced technology revealed that steady-state Ly6Chi and Ly6Clo monocytes are homogenous populations, and C/EBPβ regulates the differentiation of Ly6Chi monocytes into Ly6Clo monocytes (15). This is also in line with remarkable expression/function of Nr4a1 on Ly6Clo monocyte development (16), as it was found that regulation of Nr4a1 is mediated by the expression of C/EBPβ and also KLF2 assisting conversion of Ly6Chi monocytes to Ly6Clo monocytes (12, 15–17). Besides Nr4a1, Ten-Eleven-Translation-3 (TET3), a target of hsa-miR-150, regulates differentiation of classical monocytes into non-classical monocytes in K562 human chronic myeloid leukemia and U937 human lymphoma (18). Upregulation of TET3 expression in classical monocytes following downregulation of hsa-miR150 rarely generates non-classical monocytes, but does not affect the survival of non-classical monocytes (18). Recently, singlecell RNA-seq has also led to the identification of two additional monocyte populations and their distinct relationships with other immune cells in human blood, highlighting the heterogeneity of myeloid cells (19). High-dimensional mass cytometry has further revealed heterogeneity within human non-classical monocytes, and has allowed distinguishing between two different nonclassical monocyte subsets, Slan+ and Slan−, with functional differences based on Slan expression (20).

Some developed monocytes can enter non-lymphoid organs such as skin and lung without differentiation and orchestrate the physiological condition, while some portion of developed monocytes undergoes differentiation into macrophages or dendritic cells (21–24). Of note, differentiated macrophages are conventionally classified into pro-inflammatory M1 type and anti-inflammatory (pro-tumoral) M2 type, and these macrophages differentially regulate tumor progressions and metastases (25). However, this binary classification of macrophages is insufficient to represent their multifaceted and plastic functions (25). On the other hand, monocyte-derived dendritic cells have been mainly regarded as immune activators in the tumor microenvironment, recruiting and stimulating immune effector cells (26). Nevertheless, dendritic cells are also highly heterogeneous, and cancer cells can recruit the immunosuppressive subset of dendritic cells and/or suppress their anti-tumoral functions (26). All this flexibility appears in a context-dependent manner. Likewise, differing individual functions of monocytes might result from different contexts of development. While it is well-accepted that the bone marrow is the primary source of production and supply of monocytes in physiological condition (1), there is substantial controversy whether the bone marrow serves the same role in cancer-derived pathological conditions. Splenic progenitor cells are reinforced to generate monocytes during KP lung carcinoma progression, which suggests that the spleen could be a critical organ to produce and amplify monocytes (27). The pivotal role of spleen as a source of monocytes has also been highlighted in a different inflammatory condition (28). Angiotensin II plays a central role in amplifying Ly6Chi monocytes and their precursors in the spleen red pulp of KP lung carcinoma-bearing mice as well as releasing monocytes from their splenic reservoir (28, 29). However, a conflicting view has been suggested in a different lung tumor model. During the development of Lewis lung carcinoma (LLC), the bone marrow primarily promotes monocyte production while the spleen plays a minor role in monocyte production (30). Monocytes produced from the bone marrow are more favored to migrate into and to be accumulated in the tumor region than those from the spleen (30). Although an increased accumulation of monocytes in the spleen is also detected in the LLC model, it is because the bone marrow primarily accelerates monocyte production and transfers the newly formed monocytes to the spleen; the spleen is not the primary source (30). As such, different context might have yielded the controversy on tumor monocyte development. Therefore, further studies need to be conducted in as many types of tumors as possible (31).

# Monocytes: Pro-tumoral vs. Anti-tumoral Functions in Solid Tumors

Other than the well-known feature as precursors of macrophage and dendritic cell populations, monocytes play a significant role per se in orchestrating the immune system not only in homeostatic condition (21), but also in tumor progression (7, 8, 32–35). Generally, high rate of monocyte infiltration into the tumor milieu indicates poor clinical prognosis of cancers (36, 37). Since each subset of monocytes has different functions in tumor progression depending on the context, it is momentous to decide which subset of monocytes should be targeted in each tumor. Distinct functions of Ly6Chi monocytes and Ly6Clo monocytes in solid tumors have been explored (**Table 1**; **Figure 1**). These monocytes play pro-tumoral or anti-tumoral roles, regulating diverse mechanisms ranging from angiogenesis to immune modulation in a context-dependent manner (**Table 1**; **Figure 1**).

### Recruitment of Classical Monocytes and Their Functions in Solid Tumors

Ly6Chi classical monocytes have been mostly reported to play pro-tumoral functions once recruited to the tumor microenvironment (**Table 1**; **Figure 1**). Ly6Chi monocytes express high levels of CCR2 on their surface (32). CCR2 mediates the migration of Ly6Chi monocytes from the bone marrow to CCL2-secreting tumor milieu in PyMT spontaneous breast carcinoma, KCKO pancreatic carcinoma, and MC38 colorectal carcinoma (29, 45, 46). These recruited classical monocytes release VEGFA (a major stimulator of angiogenesis) to facilitate tumor cell extravasation and lung metastasis (32, 47). In human pancreatic tumor as well as murine pancreatic lesion model, the tumor microenvironment releases CCL2 and thereby actively recruits CCR2-expressing CD14+CD16− classical monocytes from bone marrow to blood stream, which is a prognostic factor of worse outcome (45). In contrast, CCR2 inhibition attenuates the mobilization and thus leads to forming an anti-tumoral immune environment in KCKO pancreatic carcinoma and MC38 colorectal carcinoma (45, 46). In human RCC patients and xenograft models, the IL-1β/IL-1R interaction activates the MyD88-NF-kB signaling pathway, and thereby enables classical monocytes with pro-tumoral phenotypes to upregulate pro-tumoral genes such as VEGF, MMP-10, IL-8, TNF-α, and PTGS2 (38). Ly6Chi monocytes/CD14+CD16− monocytes also facilitate cancer cell invasion and metastases via expressing F13a1 to promote fibrin cross-linking not only in murine KLN205 lung squamous cell carcinoma but also in human lung cancer, implicating poor survivals (39). As such, in hepatocellular carcinoma (HCC), Gr-1+ myeloid cells which contain Ly6Chi monocyte population play pro-tumoral function supporting tumor fibrosis by secreting platelet-derived growth factor-beta (PDGF-β), a pro-fibrotic growth factor (40).

Moreover, classical monocytes play a major role in establishing a cancer therapy-resistant microenvironment (34, 48, 49). Doxorubicin treatment on MMTV-PyMT breast carcinoma, for induction of necrotic cell death, triggers the enhanced infiltration of CCR2-expressing monocytes. At later stages of cancer, this backfires; these monocytes have been revealed responsible for resistance against doxorubicin, promoting tumor relapse after treatment (48). In 4T1 and MMTV-PyMT breast carcinoma, paclitaxel treatment induces the secretion of tumorderived extracellular vesicles (EVs), and these EVs upregulate pulmonary CCL2 expression to elicit classical monocyte expansion establishing a lung pre-metastatic niche (34). Applying radiotherapy on KPC pancreatic carcinoma also leads to a significant increase in CCL2 production by tumor cells. Subsequent recruitment of classical monocytes thereby endows the tumor with resistance against the cancer treatment (49). Use of anti-CCL2 antibodies selectively restrains radiotherapydependent recruitment of classical monocytes, impeding tumor progression when combined with radiotherapy (49).

Based on these findings, treatment with anti-CCL2 antibody might sound attractive for tumor regression. However, the following study has proposed a caution for anti-CCL2 monotreatment. During anti-CCL2 treatment in 4T1, J110, and Met-1 mammary carcinoma, a large population of the classical monocytes is retained within the bloodstream, and their homing to the primary tumor or to the metastatic site is attenuated (50). However, after anti-CCL2 treatment cessation, monocytes initiate their migration to the lungs, and the level of IL-6 rises within the lungs. The increased level of IL-6 augments pro-angiogenic VEGF-A expression in classical monocytes, and thereby accelerates tumor metastasis (50). IL-6RA is largely expressed in Ly6Chi monocytes, and anti-IL-6R antibodies effectively target Ly6Chi monocytes (51, 52). Notably, IL-6- IL-6R interaction not only promotes VEGF-A secretion from classical monocytes but also activates the STAT3 signaling pathway in cancer cells, which enhances tumor cell proliferation in pancreatic ductal adenocarcinoma (PDAC) (50–53). IL-6 is also strongly induced in adipocytes and tumor-infiltrated myeloid cells after anti-VEGF treatment on overweight breast cancer patients. The upregulated IL-6 mediates resistance to anti-VEGF therapy, leading to the proliferation of cancer cells and dysfunctional angiogenesis (54). IL-6 inhibition increases the tumor microenvironment's sensitivity to chemotherapy and anti-angiogenic therapy and promotes tumor cell death (52, 54).

Contrary to the pro-tumoral properties of Ly6Chi/CD14+CD16− monocytes explicated above, it has also been reported that these classical monocytes play anti-tumoral functions in certain treatments (**Table 1**; **Figure 1**). Tumor fibrosis promotes tumor progression by increasing collagen deposition, reducing T cell infiltration, and inducing protumoral macrophage polarization (33, 55–58). A distinct class of Mac1+F4/80−Msr1+ceacam1+Ly6Clo monocytes has been recently discovered to promote fibrosis in C/EBPβ dependent manner (59). Meanwhile, Ly6Chi monocyte infiltration into KPC pancreatic adenocarcinoma via IFN-γ and CCL2 following anti-CD40 treatment has been reported to facilitate degradation of tumor fibrosis, increasing the efficacy of the chemotherapy on PDAC while Ly6Chi monocyte-containing Gr-1+ myeloid cells in HCC play pro-fibrotic roles (33, 40, 55).

### Recruitment of Non-classical Monocytes and Their Functions in Solid Tumors

On the other hand, Ly6Clo/CD14−CD16+ non-classical monocytes have independent mechanisms for infiltration to tumors, and their functions are context-dependent. In models of colorectal cancer, Jung et al. have firstly revealed immunosuppressive functions of non-classical monocytes in any context, including cancers (7, 8). Anti-angiogenic therapy leads to non-classical monocyte influx to CX3CL1-secreting tumor milieu. Then these recruited non-classical monocytes secrete CXCL5, and mediate a massive infiltration of CXCR2-expressing neutrophils through the highly specific chemokine axis (7). This

*Summarizes diverse protumoral and antitumoral functions of monocyte subsets (classical and non-classical monocytes) and their related factors in each model.*

finding echoes a previous finding also showing that non-classical monocytes recruit neutrophils, albeit mediated by CXCL1—not CXCL5—in a different disease condition outside oncology (60). These tumor-infiltrating non-classical monocytes and neutrophils release immunosuppressive cytokines including IL-10 which inhibits infiltration and activity of cytotoxic T lymphocytes in tumors (7, 8) (**Table 1**; **Figure 1**). Jung et al. also successfully developed several therapeutic strategies targeting these non-classical monocyte-mediated cascades by blocking their infiltration and activity (7, 8). Through a series of in silico and in vitro screening, novel siRNA sequences against CX3CL1 with potent knock-down efficacy were identified. The siRNA was formulated with nanoparticles particularly designed for endothelial cell-specific delivery, which resulted in inhibiting Ly6Clo monocyte infiltration and subsequently reduced tumor growth (7). Notably, CXCR4 was discovered to be a critical chemokine receptor expressed on non-classical monocytes and neutrophils (8). CXCL12/CXCR4 axis in these cells mediates restrained cytotoxic T cell infiltration and builds up immunosuppressive tumor microenvironment in CT26, SL4 colorectal carcinoma, and E0771, MCa-M3C mammary carcinoma (8, 61). Supporting this finding, AMD3100 which is a potent CXCR4 inhibitor, also known as plerixafor, efficiently hinders the recruitment of non-classical monocytes, improving the treatment efficacy of anti-VEGFR2 therapy. This suggests the potential of rapid clinical translation, since AMD3100 is already an FDA-approved CXCR4 blocker being used in the clinic for other uses (8, 61).

Despite the several pro-tumoral features of Ly6Clo/CD14−CD16+ non-classical monocytes, these monocytes also display anti-tumoral properties in different tumor/treatment conditions (**Table 1**; **Figure 1**). In B16F10 melanoma and MMTV-PyMT spontaneous mammary carcinoma, non-classical monocytes play a pivotal role in engulfing tumor material in the lung and attenuating tumor metastasis and activating NK cells (17, 35). In B16F10 and B16F0 melanoma, non-classical monocytes also activate NK cells by releasing IL-15, which is a determinant cytokine for NK cells' homeostasis, activation and effector function, preventing lung metastases in primary tumor-bearing mice (44). In B16F10 melanoma and A375 human melanoma xenograft models, exosomes secreted from non-metastatic cancer cells promoted the expansion of non-classical monocytes in the bone marrow (42). The expanded population of the non-classical monocytes leads to recruiting NK cells which function in cancer cell clearance at the pre-metastatic niche (42). This NK cell-recruiting function of non-classical monocytes have been reconfirmed in early stage lung cancer patients (43). Based on these findings, reduced CD16+ non-classical monocytes might be correlated with NK cell paucity in this lung tumor lesions (43). According to ex vivo study of patients with stage IV cutaneous melanoma, CD14−CD16+ non-classical monocytes kill regulatory T lymphocytes (Tregs) by assisting ipilimumab, anti-cytotoxic T lymphocyte associated antigen 4 (CTLA4) monoclonal antibody, -mediated ADCC (antibody-dependent cell-mediated cytotoxicity) (62).

Importantly, it had been widely believed that non-classical monocytes are not able to extravasate out of blood vessels. Instead, they were known to stay inside vasculature and patrol the endothelium, which gave these monocytes the nickname "patrolling monocytes" (9). However, recent studies strongly suggest that they do have the capability of transmigration and actively infiltrate into tissues, proven by state-of-the-art in vivo imaging techniques (7, 8). Supporting this, in DLD1

and HCT116 human colorectal carcinoma, recruited human patrolling monocytes in tumors secrete matrix metalloproteinase 9 (MMP9), a proteolytic enzyme fostering angiogenesis, triggering a release of matrix-bound VEGFA. This accelerates the extravasation and accumulation of these pro-angiogenic patrolling monocytes, promoting tumor progression (41). This also validates the first finding of non-classical monocyte extravasation directly visualized by intravital microscopic imaging (7, 8).

## Tie2-Expressing Monocytes

acquire chemosensitivity upon treatments.

Other than the traditional classification of monocytes by Ly6C expression level, another classification method by Tie2 (angiopoietin receptor) expression exists. Tie2-expressing monocytes (TEMs) are a monocyte population present in both human and mouse peripheral blood and tumor, and are localized in perivascular spaces but not incorporated with vascular endothelial cells (63, 64). Angiopoietin-1 (Ang-1), a Tie2 ligand, is likely to promote the recruitment of TEMs to tumor vasculature before the turn-on of the angiogenic switch in early stages of N202 breast carcinoma, Rip1-Tag2 pancreatic insulinoma and U87 human glioma (63, 65, 66). In a following study, it was also elucidated that Angiopoietin-2 (Ang-2), another Tie2 ligand upregulated in tumor hypoxia, can also recruit TEMs. The TEMs are then reprogrammed to show proangiogenic phenotypes (67, 68). Meanwhile, Collagen triple-helix repeat-containing 1 (CTHRC1) secreted by several malignant tumors has been reported to recruit TEMs to the tumor microenvironment through upregulation of Ang-2 in endothelial cells and promote metastasis in human MiaPaCa-2, CFPAC-1, and Panc-1 pancreatic cancers (69). Recruited TEMs promote angiogenesis via secretion of a proangiogenic molecule, basic fibroblast growth factor (bFGF) (63–66). Also, Ang-2 and hypoxia cause TEM influx into the tumor microenvironment, and the TEMs mediate downregulation of TNF-α supporting cancer cell survival and causing metastasis of the primary tumor (63, 67, 70). Blockade of Ang-2 impedes tumor angiogenesis in MMTV-PyMT breast carcinoma and Rip1-Tag2 pancreatic insulinoma through downregulation of Tie2 in TEMs (71).

# NEUTROPHILS: FROM DEVELOPMENT TO DEPLOYMENT

# Neutrophil Development

Neutrophils are another myeloid compartment which plays critical roles both in homeostatic condition and tumor context. There is a train of precursors to be passed through to generate mature neutrophils in the bone marrow (72). Hematopoietic stem cells (HSCs) give rise to multipotent progenitors (MPPs), lymphoid primed multipotent progenitors (LMPPs), and granulocyte/macrophage progenitors (GMPs) in this very order (72). There are several more stages to go to be differentiated to neutrophils, namely a series of myeloblasts, promyelocytes, myelocytes, metamyelocytes, band cells, and finally neutrophils (72). These steps for neutrophil generation occur under major regulation by the granulocyte-colony stimulating factor (G-CSF), granulocyte– macrophage-colony stimulating factor (GM-CSF) and also minor regulation by other molecules such as IL-6 and KIT ligand (KITL) (73). Differentiating neutrophils express the G-CSF receptor (G-CSFR) throughout the myeloid lineage (73). During development in the bone marrow, neutrophils acquire three types of granules sequentially; azurophil (primary) granules which retain myeloperoxidase regulated by transcription factors C/EBPα and Gfi-1, specific (secondary) granules which contain lactoferrin mostly regulated by C/EBPε, and gelatinase (tertiary) granules which contain MMP9 regulated by C/EBPβ, C/EBPδ, C/EBPγ, and PU.1 (74, 75). Of note, mass cytometry has recently found new proliferative precursors of neutrophils after GMP stage which further differentiate to immature neutrophils and mature neutrophils with regulation of C/EBPε (76). Although the bone marrow is primarily responsible for the neutrophil formation, the spleen can be an alternative source of neutrophils during emergency granulopoiesis derived from cancer progression (73). In KP lung adenocarcinoma, splenic hematopoietic stem cells, and progenitor cells produce neutrophils during tumor progression (27). Presence of cancer cells upregulates the expression of several factors accelerating neutrophil development. The expression of CXCL1, CXCL2, CXCL5, and CXCL8, which are CXCR2 ligands, and the expression of KITL and GM-CSF are strongly enhanced by KRAS signaling in cancer cells and tumor-derived hypoxia (73). Moreover, IL-1β-producing macrophages and IL-17-producing γδ T cells secrete G-CSF to promote neutrophil development in the tumor (73). Cancer cells accelerate secretion of these cytokines and chemokines to instigate overactive granulopoiesis and neutrophilia (73). The accelerated secretion of these factors promotes the release of immature neutrophils to the blood stream, resulting in an increased number of circulating neutrophils (73). In 4T07, 4T1 mammary carcinoma, LLC, and Kras-driven pancreatic carcinoma, G-CSF production is also facilitated via RAS/MEK/ERK pathway in cancer cells, promoting recruitment of neutrophils (77). Meanwhile, type I IFNs from tumor trigger differentiation of neutrophils to achieve an anti-tumoral phenotype, reducing not only CXCR4 expression in neutrophils which mediates tumor-homing, but also VEGF and MMP9 expression (78, 79). Moreover, type I IFNs suppress G-CSF signaling pathways in neutrophils, thereby reducing expression of Bv8, S100A8, S100A9, and MMP9 so that they can attenuate the formation of the pre-metastatic niche (78, 79). Inhibition of type I IFNs impairs cytotoxicity of neutrophils and promotes metastasis of B16F10 melanoma, MCA205 fibrosarcoma, 4T1 mammary carcinoma and LLC mediated by neutrophils (78, 79).

# Neutrophils: Pro-tumoral vs. Anti-tumoral Functions in Solid Tumors

Functions of neutrophils in the tumor microenvironment vary by context including types of tumor, stages of tumor progression, and different therapies (**Table 2**; **Figure 2**).

## Recruitment of Neutrophils to Tumor Milieu

As mentioned above, tumors promote the early release of neutrophils yet with immature phenotypes from the bone marrow. There are several ligand-receptor axes studied for neutrophil recruitment into the tumor. Upon research on diverse tumor models, it has been revealed that CXCR2 is a pressing chemokine receptor which recruits neutrophils to the tumor (7, 8, 119, 120). In KPC pancreatic carcinoma and inflammationdriven and spontaneous intestinal adenocarcinoma, the migration of myeloid cells, especially neutrophils, to the tumor microenvironment is impaired when CXCR2 signaling is suppressed (119, 120). This enhances tumor cell apoptosis and restrains tumorigenesis, resulting in a failure to set up a metastatic niche (119, 120). Moreover, activated neutrophils also express CCR7 on their membrane, which pushes those cells to tumor sites in response to CCL19, CCL21, and GM-CSF secretion (121). IL-17 also triggers neutrophil recruitment to tumor sites in 4T1 breast carcinoma (122), KRAS mutated lung carcinoma (123), and ovarian carcinoma (124). The recruited neutrophils present high expression of tumor-promoting genes such as TNF-α, CXCL1, MMP9, and VEGF (122). In zebrafish larvae model of glioblastoma initiation, neutrophils are actively recruited to KRAS-transformed cells very early in oncogenesis via the CXCL8-CXCR1 signaling axis, and this recruitment contributes to the proliferation of tumor-initiating cells (125). Gastrin-releasing peptide (GRP)-GRP receptor (GRPR) axis can also induce neutrophil migration in the tumor (126, 127). In A375 and M24met human melanoma, CXCL5 overexpression by tumor cells enhances neutrophil recruitment and infiltration into primary tumors and tumor lymphatic vessels (128). It triggers the proximal interaction between neutrophils and cancer cells near the lymphatic endothelial cells in order to help trans-endothelial migration of the cancer cells (128). In SL4 and CT26 colorectal cancer, tumor-infiltrated Ly6Clo monocytes induced by anti-VEGF therapy can also recruit CXCR2-expressing neutrophils to the tumor site via the CXCL5-CXCR2 and CXCL12-CXCR4 axes (7, 8). Albeit in a different disease setting, it has been

#### TABLE 2 | Context-dependent multifaceted functions of neutrophils.

#### TABLE 2 | Continued

*Summarizes multiple protumoral and antitumoral functions of neutrophils and their responsible factors in each different context.*

also reported that monocytes recruit neutrophils in a TLR7 dependent manner through CXCL1 (60), different from the case of cancer context where non-classical monocytes-derived CXCL5 was newly discovered as the key chemokine attracting neutrophils (7, 8).

## Pro-tumoral Functions of Neutrophils in Solid Tumors

Recruited neutrophils from the blood stream have potent influences on various components of tumor progression and metastasis, including tumor initiation, cancer cell survival/proliferation, immune modulation, angiogenesis, and intra/extravasation of cancer cells (**Table 2**; **Figure 2**).

### **Neutrophils and tumor initiation**

In Kras mutant mice, airway inflammation induces secretion of IL-8 by lung keratinocytes, recruiting CXCR2-expressing neutrophils to the inflammation site (80). Neutrophil elastase (NE), a powerful serine protease exclusively found in primary granules of neutrophils, facilitates inflammation-mediated tumor initiation in the lung (80). H. hepaicus-induced colitis triggers tissue infiltration of MPO+ neutrophils and macrophages into the infected sites (81). These neutrophils and macrophages generate reactive oxygen species (ROS) and reactive nitrogen species (RNS) which subsequently cause molecular damage, promoting tumorigenesis (81). Transcriptional analysis reveals that genes involved in repairing DNA damage were downregulated, but genes associated with reactive chemical species generation were upregulated in infected colons (with no impact on cell proliferation) (81).

### **Neutrophils and cancer cell survival/proliferation**