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The role of tumor-associated macrophages in gastric cancer development and their potential as a therapeutic target

  • Author Footnotes
    1 Both authors contributed equally as first authors.
    V. Gambardella
    Footnotes
    1 Both authors contributed equally as first authors.
    Affiliations
    Department of Medical Oncology, INCLIVA Biomedical Research Institute, University of Valencia, Valencia, Spain

    Instituto de Salud Carlos III, CIBERONC, Madrid, Spain
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  • Author Footnotes
    1 Both authors contributed equally as first authors.
    J. Castillo
    Footnotes
    1 Both authors contributed equally as first authors.
    Affiliations
    Department of Biochemistry and Molecular Biology, University of Valencia, Valencia, Spain
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  • N. Tarazona
    Affiliations
    Department of Medical Oncology, INCLIVA Biomedical Research Institute, University of Valencia, Valencia, Spain

    Instituto de Salud Carlos III, CIBERONC, Madrid, Spain
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  • F. Gimeno-Valiente
    Affiliations
    Department of Medical Oncology, INCLIVA Biomedical Research Institute, University of Valencia, Valencia, Spain
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  • C. Martínez-Ciarpaglini
    Affiliations
    Instituto de Salud Carlos III, CIBERONC, Madrid, Spain

    Department of Pathology, INCLIVA Biomedical Research Institute, Spain
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  • M. Cabeza-Segura
    Affiliations
    Department of Medical Oncology, INCLIVA Biomedical Research Institute, University of Valencia, Valencia, Spain
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  • S. Roselló
    Affiliations
    Department of Medical Oncology, INCLIVA Biomedical Research Institute, University of Valencia, Valencia, Spain

    Instituto de Salud Carlos III, CIBERONC, Madrid, Spain
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  • D. Roda
    Affiliations
    Department of Medical Oncology, INCLIVA Biomedical Research Institute, University of Valencia, Valencia, Spain

    Instituto de Salud Carlos III, CIBERONC, Madrid, Spain
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  • M. Huerta
    Affiliations
    Department of Medical Oncology, INCLIVA Biomedical Research Institute, University of Valencia, Valencia, Spain
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  • Author Footnotes
    2 Both authors contributed equally as senior authors.
    A. Cervantes
    Footnotes
    2 Both authors contributed equally as senior authors.
    Affiliations
    Department of Medical Oncology, INCLIVA Biomedical Research Institute, University of Valencia, Valencia, Spain

    Instituto de Salud Carlos III, CIBERONC, Madrid, Spain
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  • Author Footnotes
    2 Both authors contributed equally as senior authors.
    T. Fleitas
    Correspondence
    Corresponding authors at: Department of Medical Oncology, INCLIVA Biomedical Research Institute, University of Valencia, Blasco Ibañez, 17, 46010 Valencia, Spain.
    Footnotes
    2 Both authors contributed equally as senior authors.
    Affiliations
    Department of Medical Oncology, INCLIVA Biomedical Research Institute, University of Valencia, Valencia, Spain

    Instituto de Salud Carlos III, CIBERONC, Madrid, Spain
    Search for articles by this author
  • Author Footnotes
    1 Both authors contributed equally as first authors.
    2 Both authors contributed equally as senior authors.
Open AccessPublished:March 23, 2020DOI:https://doi.org/10.1016/j.ctrv.2020.102015

      Highlights

      • Tumor immune microenvironment signalling influences tumor progression.
      • Tumor associated macrophages are characterized by a dual pro- and anti-tumor activity.
      • Tumor associated macrophages emerge as a potential target for cancer treatment in gastric cancer.

      Abstract

      Gastric cancer (GC) represents the fifth cause of cancer-related death worldwide. Molecular biology has become a central area of research in GC and there are currently at least three major classifications available to elucidate the mechanisms that drive GC oncogenesis. Further, tumor microenvironment seems to play a crucial role, and tumor-associated macrophages (TAMs) are emerging as key players in GC development. TAMs are cells derived from circulating chemokine- receptor-type 2 (CCR2) inflammatory monocytes in blood and can be divided into two main types, M1 and M2 TAMs. M2 TAMs play an important role in tumor progression, promoting a pro-angiogenic and immunosuppressive signal in the tumor. The diffuse GC subtype, in particular, seems to be strongly characterized by an immuno-suppressive and pro-angiogenic phenotype. No molecular targets in this subgroup have yet been identified. There is an urgent need to understand the molecular pathways and tumor microenvironment features in the GC molecular subtypes. The role of anti-angiogenics and checkpoint inhibitors has recently been clinically validated in GC. Both ramucirumab, a fully humanized IgG1 monoclonal anti-vascular endothelial growth factor receptor 2 (VEGFR2) antibody, and checkpoint inhibitors in Epstein Bar Virus (EBV) and Microsatellite Instable (MSI) subtypes, have proved beneficial in advanced GC. Nevertheless, there is a need to identify predictive markers of response to anti-angiogenics and immunotherapy in clinical practice for a personalized treatment approach. The importance of M2 TAMs in development of solid tumors is currently gaining increasing interest. In this literature review we analyze immune microenvironment composition and signaling related to M1 and M2 TAMs in GC as well as its potential role as a therapeutic target.

      Graphical abstract

      Keywords

      The role of microenvironment in GC development and progression

      Gastric cancer (GC) is characterized by high incidence and mortality and it represents the sixth most common cancer type and fifth leading cause of cancer death worldwide [
      • Bray F.
      • Ferlay J.
      • Soerjomataram I.
      • et al.
      Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.
      ]. When diagnosed in an advanced stage, GC is characterized by very poor prognosis, with a five-year overall survival rate of about 5% [
      • Noone A.M.
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      Cancer incidence and survival trends by subtype using data from the surveillance epidemiology and end results program, 1992–2013.
      ]. Historically GC has been classified into two main subgroups, intestinal and diffuse, according to different microscopic features [

      Lauren P. The two histological main types of gastric carcinoma: diffuse and so-called intestinal-type carcinoma. An attempt at a histo-clinical classification. Acta Pathol Microbiol Scand 1965;64:31–49.

      ]. Several molecular classifications based on comprehensive analyses have been proposed to gain better understanding of GC biology and for a personalized medicine approach [
      • Cristescu R.
      • Lee J.
      • Nebozhyn M.
      • et al.
      Molecular analysis of gastric cancer identifies subtypes associated with distinct clinical outcomes.
      ,
      • Cancer Genome Atlas Research N
      Comprehensive molecular characterization of gastric adenocarcinoma.
      ,
      • Lei Z.
      • Tan I.B.
      • Das K.
      • et al.
      Identification of molecular subtypes of gastric cancer with different responses to PI3-kinase inhibitors and 5-fluorouracil.
      ]. Findings from these classifications identified at least two phenotypes that benefit from checkpoint inhibitors including the EBV positive and MSI-H profiles. PD-L1 expression, CPS score and the presence of TILs (Tumor infiltrate lymphocytes) along several solid tumors were associated with response to checkpoint inhibitors, nevertheless, their prognostic role need to be further explored [
      • Zheng X.
      • Song X.
      • Shao Y.
      • et al.
      Prognostic role of tumor-infiltrating lymphocytes in gastric cancer: a meta-analysis.
      ].
      There is increasing interest in the role of the immune microenvironment in GC [
      • Plummer M.
      • de Martel C.
      • Vignat J.
      • et al.
      Global burden of cancers attributable to infections in 2012: a synthetic analysis.
      ,
      • Bang Y.J.
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      Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, open-label, randomised controlled trial.
      ,
      • Kim S.T.
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      • Bass A.J.
      • et al.
      Comprehensive molecular characterization of clinical responses to PD-1 inhibition in metastatic gastric cancer.
      ]. Moreover, chronic inflammation and H. pylori infection are the classical determinants in GC development [
      • Plummer M.
      • de Martel C.
      • Vignat J.
      • et al.
      Global burden of cancers attributable to infections in 2012: a synthetic analysis.
      ].
      In this scenario, the role of macrophages is fascinating. Presence and density of tumor-associated macrophages (TAMs) has been correlated with prognosis and resistance to treatment. The immunosuppressive and pro-angiogenic phenotype that TAMs promote could be of primary interest especially in diffuse GC and in the genomically stable subgroup, where the presence of M2 Macrophages was found to be higher and could contribute to an immunosuppressive phenotype [
      • Ge S.
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      A proteomic landscape of diffuse-type gastric cancer.
      ,
      • Chung H.W.
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      ,
      • Joyce J.A.
      • Pollard J.W.
      Microenvironmental regulation of metastasis.
      ,
      • Balkwill F.R.
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      Cancer-related inflammation: common themes and therapeutic opportunities.
      ]. This review aims to analyze the role of tumor-associated macrophages (TAMs) in GC to pave the way for new molecular research and therapeutic approaches.

      Tumor-Associated Macrophage in solid tumors

      Macrophages are players in the innate immune response and a major component of the leukocyte infiltrate present in solid tumors. TAMs play a dominant role in cancer-related inflammation and constitute important regulators of tumorigenesis. Recent evidence supports the hypothesis that these cells are characterized by a dual pro- and anti-tumor activity. Macrophages were originally found to be involved in antitumor immunity, as they are able to identify non-self cells and finally phagocytize them. Nevertheless, increasing pre-clinical and clinical evidence shows that TAMs could paradoxically also enhance tumor development and metastatic capabilities. Paradoxically, a predominant role in supporting multiple aspects of tumor progression such as immune tolerance and tumor cell activation by paracrine signaling loops has been detected [
      • Wyckoff J.B.
      • Wang Y.
      • Lin E.Y.
      • et al.
      Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors.
      ,
      • Condeelis J.
      • Pollard J.W.
      Macrophages: obligate partners for tumor cell migration, invasion, and metastasis.
      ] and they have also been linked to cancer treatment resistance.

      The dual role of TAMs: M2 macrophages and cancer development and progression

      This dual role of macrophages in cancer has been justified by their functional plasticity, that may explain their differing behavior: “classically-activated” M1 macrophages produce type I proinflammatory cytokines such as IL-1β, IL-1α, IL-12, TNF-α, and GFAP [
      • Wynn T.A.
      Type 2 cytokines: mechanisms and therapeutic strategies.
      ,
      • Gordon S.
      Alternative activation of macrophages.
      ]. Conversely, “alternatively-activated” M2 macrophages produce type II cytokines, such as IL-4, IL-6, IL-10 promoting anti-inflammatory responses, and have pro-tumorigenic functions [
      • Gordon S.
      Alternative activation of macrophages.
      ,
      • Balkwill F.R.
      • Mantovani A.
      Cancer-related inflammation: common themes and therapeutic opportunities.
      ,
      • Mosser D.M.
      The many faces of macrophage activation.
      ,
      • Mosser D.M.
      • Edwards J.P.
      Exploring the full spectrum of macrophage activation.
      ,
      • Murray P.J.
      • Wynn T.A.
      Obstacles and opportunities for understanding macrophage polarization.
      ,
      • Qian B.-Z.
      • Pollard J.W.
      Macrophage diversity enhances tumor progression and metastasis.
      ,
      • Pollard J.W.
      Tumour-educated macrophages promote tumour progression and metastasis.
      ].
      During the transition from M1 to M2, the microenvironment appears to be dominated by cytokines and growth factors, such as IL-4 synthesized by CD4 + T cells and/or tumor cells [

      Coussens LM, Zitvogel L, Palucka AK. Neutralizing tumor-promoting chronic inflammation: a magic bullet? Science (New York, N.Y.) 2013;339:286–291.

      ,
      • Gocheva V.
      • Wang H.-W.
      • Gadea B.B.
      • et al.
      IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion.
      ], growth factors such as CSF-1 [
      • Lin E.Y.
      • Gouon-Evans V.
      • Nguyen A.V.
      • et al.
      The macrophage growth factor CSF-1 in mammary gland development and tumor progression.
      ] and Granulocyte-macrophage colony-stimulating factor (GM-CSF) [
      • Satoh T.
      • Xu R.H.
      • Chung H.C.
      • et al.
      Lapatinib plus paclitaxel versus paclitaxel alone in the second-line treatment of HER2-amplified advanced gastric cancer in Asian populations: TyTAN–a randomized, phase III study.
      ]. This environment (Th2) is enriched in transforming growth factor-β1 (TGF-β1) and Arginase 1, as well as an increased number of CD4 + T cells [
      • DeNardo D.G.
      • Barreto J.B.
      • Andreu P.
      • et al.
      CD4(+) T cells regulate pulmonary metastasis of mammary carcinomas by enhancing protumor properties of macrophages.
      ]. All these factors cause a switch in the polarization of macrophages from TAM-M1 to an alternatively immunoregulatory TAM-M2 (Fig. 1) [
      • Mantovani A.
      • Sica A.
      Macrophages, innate immunity and cancer: balance, tolerance, and diversity.
      ]. Certain extracellular matrix (ECM) molecules and their proteolytic fragments including elastin fragments, denatured and fragmented collagen I or soluble biglycan have been shown to act as inflammatory stimuli for recruitment of macrophages [
      • Chanmee T.
      • Ontong P.
      • Konno K.
      • et al.
      Tumor-associated macrophages as major players in the tumor microenvironment.
      ]. Tumor-derived hyaluronic acid (HA) fragments have also been shown to promote development of immunosuppressive M2 macrophages by triggering a transient early activation of monocytes [
      • Kuang D.-M.
      • Wu Y.
      • Chen N.
      • et al.
      Tumor-derived hyaluronan induces formation of immunosuppressive macrophages through transient early activation of monocytes.
      ]. Moreover, transcriptomic profiling of fluorescence-activated cell sorting (FACS)-isolated TAMs revealed a distinct ECM-catabolic signature, characterized by high expression of ECM-degrading enzymes and low expression of ECM proteins [
      • Madsen D.H.
      • Jurgensen H.J.
      • Siersbaek M.S.
      • et al.
      Tumor-associated macrophages derived from circulating inflammatory monocytes degrade collagen through cellular uptake.
      ,
      • Umakoshi M.
      • Takahashi S.
      • Itoh G.
      • et al.
      Macrophage-mediated transfer of cancer-derived components to stromal cells contributes to establishment of a pro-tumor microenvironment.
      ]. In general, metabolism differs depending on TAM phenotype: M1 TAMs are characterized by exhibit enhanced glycolysis, pentose phosphate pathway (PPP) and Fatty acid (FA) synthesis, with a truncated tricarboxylic acid cycle leading to accumulation of succinate and citrate. In contrast, M2 TAM-activated macrophage metabolism is characterized by oxidative phosphorylation, decreased glycolysis and PPP, and FA oxidation [
      • Mills E.L.
      • O'Neill L.A.
      Reprogramming mitochondrial metabolism in macrophages as an anti-inflammatory signal.
      ,
      • Palmieri E.M.
      • Menga A.
      • Martin-Perez R.
      • et al.
      Pharmacologic or Genetic targeting of glutamine synthetase skews macrophages toward an M1-like phenotype and inhibits tumor metastasis.
      ]. Emerging evidence reveals that macrophage metabolic features are closely associated with their immune functions.
      Figure thumbnail gr1
      Fig. 1A. Monocyte differentiation into M1 and M2 macrophages phenotypes. Proinflammatory M1 or “activated macrophages” promotes type I T helper (Th1) antitumoral immune response by producing IL-6, IL-12, IL-8 and TNFα. In contrast, alternatively activated M2 macrophages are involved in Th2 immune responses including humoral immunity and wound healing. In solid tumors, M2 macrophages, promotes tumor progression and invasion by inducing angiogenesis and suppressing the host immune response. B. Histopathological section of a diffuse-type gastric tumor with signet ring cells (HE, 200×). C. CD163 immunostaining highlights the presence of numerous M2 macrophages between tumor cells (CD163, 200×).

      TAMs and immune response

      One of the most critical roles TAMs play in tumor initiation and progression is their ability to induce immune tolerance. This is a highly complex process in which T lymphocytes, macrophages and other cells may have a primary role [

      Coussens LM, Pollard JW. Leukocytes in mammary development and cancer. Cold Spring Harbor perspectives in biology;3:a003285.

      ,
      • Gajewski T.F.
      • Schreiber H.
      • Fu Y.-X.
      Innate and adaptive immune cells in the tumor microenvironment.
      ,
      • Movahedi K.
      • Laoui D.
      • Gysemans C.
      • et al.
      Different tumor microenvironments contain functionally distinct subsets of macrophages derived from Ly6C(high) monocytes.
      ,
      • Eissmann M.F.
      • Dijkstra C.
      • Jarnicki A.
      • et al.
      IL-33-mediated mast cell activation promotes gastric cancer through macrophage mobilization.
      ]. Ligand expression of programmed cell death protein 1 (PD-1) and cytotoxic T-lymphocyte antigen 4 (CTLA-4) inhibitory receptors could also contribute to this. These inhibitory ligands are normally upregulated in activated immune effector cells such as T cells, B cells, and NK T cells, as part of a safety mechanism that controls the intensity of the immune response the Blockade of PD-1 and CTLA-4 by their ligands (PD-L1, PD-L2, and B7-1 [D80], B7-1 [CD86], respectively) directly inhibits T-Cell Receptor and B-Cell Receptor signaling. This activation also inhibits cytotoxic T cell function, regulating their cell cycles. PD-L1 and PD-L2 are differentially expressed, with PD-L1 constitutively expressed by immune cells including T cells, B cells, macrophages, DCs, non-hematopoietic cells and cancer cells. In contrast, PD-L2 expression is limited to antigen-presenting cells (APCs) and its expression induced in monocytes and macrophages by CSF-1, IL-4, and interferon- α (INF-α) [
      • Loke P.
      • Allison J.P.
      PD-L1 and PD-L2 are differentially regulated by Th1 and Th2 cells.
      ]. Both PD-L1 and L2 are regulated in TAMs and myeloid-derived suppressor cells [
      • Loke P.
      • Allison J.P.
      PD-L1 and PD-L2 are differentially regulated by Th1 and Th2 cells.
      ,
      • Belai E.B.
      • de Oliveira C.E.
      • Gasparoto T.H.
      • et al.
      PD-1 blockage delays murine squamous cell carcinoma development.
      ,
      • Duraiswamy J.
      • Freeman G.J.
      • Coukos G.
      Therapeutic PD-1 pathway blockade augments with other modalities of immunotherapy T-cell function to prevent immune decline in ovarian cancer.
      ], PD-L1 and PD-L2 are up-regulated in macrophages as dynamic response to cytokines. PD-L1 is highly expressed on inflammatory Macrophages. In contrast, PD-L2 is not expressed on inflammatory macrophages but can be induced by alternative activation via IL-4 suggesting that PD-L1 and PD-L2 might have different functions in regulating type 1 and type 2 responses [
      • Loke P.
      • Allison J.P.
      PD-L1 and PD-L2 are differentially regulated by Th1 and Th2 cells.
      ,
      • Belai E.B.
      • de Oliveira C.E.
      • Gasparoto T.H.
      • et al.
      PD-1 blockage delays murine squamous cell carcinoma development.
      ,
      • Duraiswamy J.
      • Freeman G.J.
      • Coukos G.
      Therapeutic PD-1 pathway blockade augments with other modalities of immunotherapy T-cell function to prevent immune decline in ovarian cancer.
      ].
      TME alteration also enables development of immunoediting, a dynamic process during which cancer cells able to immune surveillance [
      • Shankaran V.
      • Ikeda H.
      • Bruce A.T.
      • et al.
      IFNγ and lymphocytes prevent primary tumour development and shape tumour immunogenicity.
      ,
      • Schreiber R.D.
      • Old L.J.
      • Smyth M.J.
      Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion.
      ]. Immunoediting can be promoted by tumor cells secreting cytokines and chemokines to recruit MDSCs, regulatory T cells (Tregs), and TAMs.
      Another important factor in tumor initiation is neo-angiogenesis [
      • Hanahan D.
      • Weinberg R.A.
      Hallmarks of cancer: the next generation.
      ]. It has been suggested that environmental conditions such as tumor hypoxia may mediate this phenomenon. Indeed, TAMs accumulate in regions of hypoxia in growing tumors [
      • Shen Z.
      • Kauttu T.
      • Seppänen H.
      • et al.
      Both macrophages and hypoxia play critical role in regulating invasion of gastric cancer in vitro.
      ] mediated by an upregulation of macrophage chemo-attractants including endothelin-2 and vascular endothelial growth factor (VEGF). Of note, TAM accumulation in these regions correlates with the subsequent acquisition of an invasive phenotype [
      • Shen Z.
      • Kauttu T.
      • Seppänen H.
      • et al.
      Both macrophages and hypoxia play critical role in regulating invasion of gastric cancer in vitro.
      ], causing a switch in macrophage polarization [
      • Quail D.F.
      • Joyce J.A.
      Microenvironmental regulation of tumor progression and metastasis.
      ]. Several anti VEGF and VGFR inhibitors have been tested in GC; nevertheless, only ramucirumab, a selective VRGFR2 monoclonal antibody, was able to improve clinical outcomes in advanced disease. Ramucirumab was also observed to act against TAMs and its inhibition of VEGFR2 could cause the decrease in TAM immune infiltration, cytokine and chemokine release, that reduces tumor growth and proliferation [

      Javle M, Smyth EC, Chau I. Ramucirumab: Successfully Targeting Angiogenesis in Gastric Cancer. Clinical Cancer Research 2014:clincanres.1071.2014.

      ]. This potential role against TAM could be one of the cornerstones of ramucirumab activity across the different GC subtypes.
      In addition, it was recently shown that TAMs in hypoxic tumor regions upregulate PD-L1 expression as a consequence of hypoxia-induced factor-1 α (HIF-1α) signaling [
      • Noman M.Z.
      • Desantis G.
      • Janji B.
      • et al.
      PD-L1 is a novel direct target of HIF-1α, and its blockade under hypoxia enhanced MDSC-mediated T cell activation.
      ]. CSF-1-regulating macrophages have been recognized as playing a pivotal role in tumor neo-angiogenesis through VEGF production [
      • Lin E.Y.
      • Pollard J.W.
      Tumor-Associated Macrophages Press the Angiogenic Switch in Breast Cancer.
      ]. Several experiments in solid tumor models have confirmed that macrophage-synthesized WNT7B targets vascular endothelial cells, stimulating their VEGF production, resulting in neo-angiogenic development. These angiogenic TAMs express Tie2, a tyrosine-protein kinase that acts as a cell-surface receptor for angiopoietins, which regulate angiogenesis, endothelial cell survival, proliferation, migration, adhesion and cell spreading. Inhibition of this population ultimately blocks the neo-angiogenesis process in several cancer models [
      • Yeo E.-J.
      • Cassetta L.
      • Qian B.-Z.
      • et al.
      Myeloid WNT7b mediates the angiogenic switch and metastasis in breast cancer.
      ,
      • Du R.
      • Lu K.V.
      • Petritsch C.
      • et al.
      HIF1alpha induces the recruitment of bone marrow-derived vascular modulatory cells to regulate tumor angiogenesis and invasion.
      ,
      • De Palma M.
      • Venneri M.A.
      • Galli R.
      • et al.
      Tie2 identifies a hematopoietic lineage of proangiogenic monocytes required for tumor vessel formation and a mesenchymal population of pericyte progenitors.
      ,
      • Mazzieri R.
      • Pucci F.
      • Moi D.
      • et al.
      Targeting the ANG2/TIE2 axis inhibits tumor growth and metastasis by impairing angiogenesis and disabling rebounds of proangiogenic myeloid cells.
      ,
      • Forget M.A.
      • Voorhees J.L.
      • Cole S.L.
      • et al.
      Macrophage colony-stimulating factor augments Tie2-expressing monocyte differentiation, angiogenic function, and recruitment in a mouse model of breast cancer.
      ], Interestingly, CSF-1 upregulates Tie2 in TAMs [
      • Forget M.A.
      • Voorhees J.L.
      • Cole S.L.
      • et al.
      Macrophage colony-stimulating factor augments Tie2-expressing monocyte differentiation, angiogenic function, and recruitment in a mouse model of breast cancer.
      ] indicating a link between CSF-1, Tie2 + macrophages and neo-angiogenesis induction. Moreover, Tie2 + macrophages aligned along the vessels also could promote tumor cell intravasation into the circulation [
      • Wyckoff J.B.
      • Wang Y.
      • Lin E.Y.
      • et al.
      Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors.
      ], leading to metastasis. In fact, macrophage infiltration correlates significantly with tumor vascularity in human GC and promotes directional tumor cell migration and invasion via a paracrine loop consisting of tumor-cell-synthesized CSF-1, which induces epidermal growth factor (EGF) expression by macrophages, creating a positive feedback loop [
      • Wyckoff J.B.
      • Wang Y.
      • Lin E.Y.
      • et al.
      Direct visualization of macrophage-assisted tumor cell intravasation in mammary tumors.
      ,
      • Condeelis J.
      • Pollard J.W.
      Macrophages: obligate partners for tumor cell migration, invasion, and metastasis.
      ,
      • Ohta M.
      • Kitadai Y.
      • Tanaka S.
      • et al.
      Monocyte chemoattractant protein-1 expression correlates with macrophage infiltration and tumor vascularity in human gastric carcinomas.
      ,
      • Guiet R.
      • Van Goethem E.
      • Cougoule C.
      • et al.
      The process of macrophage migration promotes matrix metalloproteinase-independent invasion by tumor cells.
      ].
      Macrophages recruitment is also an important step for pre-metastatic niche formation. LOX, a copper-dependent amine oxidase secreted from tumor cells, forms the cross-links of collagen IV in the basement membranes at the pre-metastatic sites where TAMs then adhere to the cross-linked collagen IV and produce MMP-2. This positive feed-forward loop eventually increases extracellular matrix remodelling and contributes to forming the pre-metastatic niche [
      • Erler J.T.
      • Bennewith K.L.
      • Cox T.R.
      • et al.
      Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premetastatic niche.
      ]. The potential impact of TAMs on cancer cell metastases has also been linked to epithelial mesenchymal transition (EMT) promotion. This phenomenon is mediated by TAM production of osteonectin (SPARC), cathepsin proteases and TGF-β, fundamental for cancer cell migration and invasion [
      • Quail D.F.
      • Joyce J.A.
      Microenvironmental regulation of tumor progression and metastasis.
      ,
      • Sangaletti S.
      • Di Carlo E.
      • Gariboldi S.
      • et al.
      Macrophage-Derived SPARC Bridges Tumor Cell-Extracellular Matrix Interactions toward Metastasis.
      ,
      • Laoui D.
      • Movahedi K.
      • Van Overmeire E.
      • et al.
      Tumor-associated macrophages in breast cancer: distinct subsets, distinct functions.
      ,
      • Bonde A.-K.
      • Tischler V.
      • Kumar S.
      • et al.
      Intratumoral macrophages contribute to epithelial-mesenchymal transition in solid tumors.
      ]. All these factors support tumor-associated angiogenesis, promotion of tumor cell invasion, migration, and intravasation and suppression of antitumor immune responses [
      • Condeelis J.
      • Pollard J.W.
      Macrophages: obligate partners for tumor cell migration, invasion, and metastasis.
      ,
      • Pollard J.W.
      Tumour-educated macrophages promote tumour progression and metastasis.
      ,
      • Pathria P.
      • Louis T.L.
      • Varner J.A.
      Targeting Tumor-Associated Macrophages in Cancer.
      ].

      Inflammation, microenvironment and TAMs in GC development

      Inflammation is one of the hallmarks of cancer [
      • Colotta F.
      • Allavena P.
      • Sica A.
      • et al.
      Cancer-related inflammation, the seventh hallmark of cancer: links to genetic instability.
      ] and many types of solid tumors are preceded by a chronic inflammatory process, mostly initiated by infections or exposure to environmental factors. One characteristic of the tumor microenvironment (TME) in GC is chronic inflammation derived from infection, such as H. pylori, which cause the upregulation of pathways that promote cell survival, activate stem cells and epithelial proliferation [
      • Polk D.B.
      • Peek Jr., R.M.
      Helicobacter pylori: gastric cancer and beyond.
      ,
      • Cristescu R.
      • Lee J.
      • Nebozhyn M.
      • et al.
      Molecular analysis of gastric cancer identifies subtypes associated with distinct clinical outcomes.
      ]. H. pylori and other pathogens impair M1 macrophage responses inducing an M2-like state, and increase the rate of ROS-induced macrophage apoptosis, enhancing the risk of disease progression [
      • Hardbower D.M.
      • Asim M.
      • Murray-Stewart T.
      • et al.
      Arginase 2 deletion leads to enhanced M1 macrophage activation and upregulated polyamine metabolism in response to Helicobacter pylori infection.
      ].
      TME alteration also enables development of immunoediting, the process by which the immune system can either block or promote cancer development, supporting the increase of tumor cells with reduced immunogenicity [
      • Shankaran V.
      • Ikeda H.
      • Bruce A.T.
      • et al.
      IFNγ and lymphocytes prevent primary tumour development and shape tumour immunogenicity.
      ,
      • Schreiber R.D.
      • Old L.J.
      • Smyth M.J.
      Cancer immunoediting: integrating immunity's roles in cancer suppression and promotion.
      ]. Immunoediting can be promoted by tumor cells secreting cytokines and chemokines to recruit MDSCs, regulatory T cells (Tregs), and TAMs. High density of M2 TAM macrophages has been associated with worse overall survival in several malignancies, including GC [
      • Zhang Q-w
      • Liu L.
      • Gong C-y
      • et al.
      Prognostic significance of tumor-associated macrophages in solid tumor: a meta-analysis of the literature.
      ,
      • Liu J.-Y.
      • Peng C.-W.
      • Yang G.-F.
      • et al.
      Distribution pattern of tumor associated macrophages predicts the prognosis of gastric cancer.
      ,
      • Mantovani A.
      Macrophages, neutrophils, and cancer: a double edged sword.
      ]. TAM infiltration differs essentially when tumor tissue and normal tissue are compared, with worse survival in cases with high abundance of M2 TAMs [
      • Yin S.
      • Huang J.
      • Li Z.
      • et al.
      The prognostic and clinicopathological significance of tumor-associated macrophages in patients with gastric cancer: a meta-analysis.
      ]. When the microenvironment was better characterized, it was possible to observe that the co-existence of TAMs and TGF-β was associated with aggressive cancer features, leading to poor prognosis, and could therefore be used as an independent prognostic factor in GC [
      • Yan Y.
      • Zhang J.
      • Li J.-H.
      • et al.
      High tumor-associated macrophages infiltration is associated with poor prognosis and may contribute to the phenomenon of epithelial-mesenchymal transition in gastric cancer.
      ,
      • Okita Y.
      • Tanaka H.
      • Ohira M.
      • et al.
      Role of tumor-infiltrating CD11b<sup>+</sup> antigen-presenting cells in the progression of gastric cancer.
      ,
      • Wang X.L.
      • Jiang J.T.
      • Wu C.P.
      Prognostic significance of tumor-associated macrophage infiltration in gastric cancer: a meta-analysis.
      ]. M2 TAMs were also correlated to worse OS in resected GC patients with lymph node metastases. Moreover, research suggests that a higher number of CD204-positive macrophages (a M2-polarized macrophage receptor) in stroma could be related to GC carcinogenesis. Another interesting observation warranting further investigation is the relationship between TAMs and tumor-infiltrating lymphocytes (TILs) as a potential prognostic biomarker [
      • Zhang Q-w
      • Liu L.
      • Gong C-y
      • et al.
      Prognostic significance of tumor-associated macrophages in solid tumor: a meta-analysis of the literature.
      ,
      • Taniyama D.
      • Taniyama K.
      • Kuraoka K.
      • et al.
      Long-term follow-up study of gastric adenoma; tumor-associated macrophages are associated to carcinoma development in gastric adenoma.
      ]. PD-1 overexpression in TAMs has also been demonstrated as a mechanism associated with reduction in the phagocytic capacity of macrophages and tumor progression and impaired NK response through TGF-β activation [
      • Gordon S.R.
      • Maute R.L.
      • Dulken B.W.
      • et al.
      PD-1 expression by tumour-associated macrophages inhibits phagocytosis and tumour immunity.
      ,
      • Peng L-s
      • Zhang J-y
      • Teng Y-s
      • et al.
      Tumor-associated monocytes/macrophages impair NK-cell function via TGFβ1 in human gastric cancer.
      ].
      M2 macrophages level was descripted to be lower in signet ring cell carcinoma and mucinous adenocarcinoma but ample in poor-differentiated adenocarcinoma [
      • Liu X.
      • Xu D.
      • Huang C.
      • et al.
      Regulatory T cells and M2 macrophages present diverse prognostic value in gastric cancer patients with different clinicopathologic characteristics and chemotherapy strategies.
      ]. A meta-analysis showed that the number of infiltrating M2 macrophages and total TAMs could be negative prognostic factors for GC patients, while M1 macrophage infiltration may be associated with a favorable survival rate [
      • Wang X.L.
      • Jiang J.T.
      • Wu C.P.
      Prognostic significance of tumor-associated macrophage infiltration in gastric cancer: a meta-analysis.
      ]. In a recent investigation, the PDL1 expression and TAMs were analyzed according the different molecular subtypes of GC. Among the PDL1-high patients analyzed in this cohort, the CD68 + macrophages were predominant in the GS and diffuse subtypes, whereas the CD68 + CD206++ (M1) macrophages were enriched in the MSI and intestinal subtype. In addition, TAMs in the GS and diffuse cancer subtypes had significantly lower median PDL1 expression. Nevertheless, no prognostic differences were identified [
      • Huang Y.K.
      • Wang M.
      • Sun Y.
      • et al.
      Macrophage spatial heterogeneity in gastric cancer defined by multiplex immunohistochemistry.
      ].
      To better characterize [

      Zeng D, Li M, Zhou R, et al. Tumor microenvironment characterization in gastric cancer identifies prognostic and immunotherapeutically relevant gene signatures. Cancer Immunol Res 2019:canimm.0436.2018.

      ] TME infiltration patterns, in a recent study analyzing 1,524 gastric cancer patients, three TME phenotypes were identified using principal component analysis algorithms. The high TME score subtype exhibited immune activation and response to virus and IFN-α, while activation of TGF-β, EMT angiogenesis, TGFb signaling and hypoxia pathways were observed in the low TME score subtype, which are considered T-cell suppressive and may be responsible for significantly worse prognosis in GC.
      In another study mRNA expression of GC was evaluated, and in the diffuse subtype there was notable high expression of ECM, angiogenesis, intracellular and cytoskeleton and collagen related genes. Moreover, ECM-related modules showed inverse correlation with immune response gene expression and this was identified as the key factor associated with the aggressive features of diffuse gastric tumours, which indicates tumour progression involving mechanisms to evade immune surveillance in diffuse tumours [
      • Kalamohan K.
      • Periasamy J.
      • Bhaskar Rao D.
      • et al.
      Transcriptional coexpression network reveals the involvement of varying stem cell features with different dysregulations in different gastric cancer subtypes.
      ]. Furthermore, at single-cell gene expression level, a recent study had shown impressive cellular changes in GC tumor samples compared to matched normal mucosa, with increased stromal cells and cytotoxic T cells in tumor samples with heterogenous profiles of exhaustion and expression of multiple immune checkpoint and costimulatory molecules as well as an heterogenous population of macrophages not confined to M1/M2 stages [
      • Sathe A.
      • Grimes S.M.
      • Lau B.T.
      • et al.
      Single cell genomic characterization reveals the cellular reprogramming of the gastric tumor microenvironment.
      ].
      More translational studies are necessary to elucidate the complex relationship of the immune microenvironment with GC in order to apply a more rational and personalized therapeutic approach that could improve outcomes across different phenotypes.

      Limitations of the current evidence on TAMs

      Despite the attractive role of TAMs and their implication in the immune response, their role and therapeutic implication need to be further validated. Moreover, TAMs evaluation in different subgroups of GC, should be further clarified to provide theoretical for GC immunotherapy.

      TAMs as a potential target for cancer treatment

      In the era of immunotherapy, there is an increasing need to identify new treatment strategies for a personalized treatment approach and to overcome resistance to check-point inhibition. Moreover, the potential role of macrophages in tumor development has driven the development of new anti-cancer treatments. Indeed, several strategies have been proposed to deplete TAM or to reconvert TAM M2 into TAM M1. In some preclinical models, the reversion of TAMs back to a M1 phenotype was associated with tumor regression by disrupting NFkB signaling or interacting with TNF-α [
      • Hagemann T.
      • Lawrence T.
      • McNeish I.
      • et al.
      “Re-educating” tumor-associated macrophages by targeting NF-kappaB.
      ,
      • Shime H.
      • Matsumoto M.
      • Oshiumi H.
      • et al.
      Toll-like receptor 3 signaling converts tumor-supporting myeloid cells to tumoricidal effectors.
      ]. The nuclear factor κB (NF-κB) signaling pathway is important in cancer-related inflammation and malignant progression. Recent preclinical studies in mouse models of colon and liver cancer have defined an important role for NF-κB activation in driving cancer-associated inflammation. Cytokines of the TNF family trigger a variety of NF-κB-dependent responses that can be specific to both cell type and signaling pathway. The cytokine tumor necrosis factor (TNF) initiates tissue inflammation, a process mediated by the NF-κB transcription factor. In response to TNF, latent cytoplasmic NF-κB is activated, enters the nucleus, and induces expression of inflammatory and anti-apoptotic gene expression programs. Recently it has been shown that NF-κB displays two distinct activation modes, monophasic and oscillatory, depending on stimulus duration [
      • Tian B.
      • Nowak D.E.
      • Brasier A.R.
      A TNF-induced gene expression program under oscillatory NF-kappaB control.
      ].
      Another interesting strategy was reported, demonstrating the inhibition of intratumoral macrophage M2 polarization through endostatin (an antiangiogenic) gene therapy in renal cell carcinoma [
      • Foguer K.
      • Braga Md.S.
      • Peron J.P.S.
      • et al.
      Endostatin gene therapy inhibits intratumoral macrophage M2 polarization.
      ].

      Direct inhibition of TAM

      The most important pathway associated with TAM recruitment and proliferation is (CSF-1)/CSF-1 receptor (CSF-1R) signaling, essential for macrophage survival and for the transition from TAM M1 into TAM M2type. The CSF-1R belongs to the platelet-derived growth factor family. It is characterized by a highly glycosylated extracellular region comprised of five immunoglobulin domains, a transmembrane domain, and an intracellular domain comprised of a juxtamembrane domain and an intracellular tyrosine kinase domain. The known ligands for the CSF-1R are CSF-1 and IL-34. CSF-1R signaling promotes myeloid differentiation, monocytic commitment, and the survival, proliferation, and chemotaxis of macrophages [
      • Cannarile M.A.
      • Weisser M.
      • Jacob W.
      • et al.
      Colony-stimulating factor 1 receptor (CSF1R) inhibitors in cancer therapy.
      ].
      Several monoclonal antibodies and tyrosine kinase inhibitors targeting CSF-1/CSF-1R are under development in clinical trials and being tested as single agents or in combination (Table 1).
      Table 1Clinical trials targeting tumor-associated macrophages in solid tumors.
      Class of drugDrugTargetCompound characteristicsClinical trialTumor TypeTrial designTreatment
      Monoclonal antibodiesEmactuzumab (RG7155)CSF1RMactuzumab binds to CSF1R expressed on macrophages and inhibits binding of colony-stimulating factor-1 (CSF-1) to CSF1R.NCT02323191 NCT02760797 NCT02923739 NCT03369964 NCT03821246 NCT01494688 NCT03193190Locally advanced or metastatic solid tumorsPhase I Phase I Phase II Phase I Pahse II Phase I Phase ISingle agent Single agent Paclitaxel Bevacizumab Anti CD20. Enzalutatmide + Atezolizumab. Paclitaxel. Multiple immunotherapy
      Cabiralizumab (FPA008)Binds to CSF1R expressed on monocytes, macrophages, and osteoclasts and inhibits CSF1R ligands colony-stimulating factor-1 (CSF-1) and interleukin-34 (IL-34), from binding to CSF1RNCT02526017 NCT03336216 NCT03768531 NCT03335540Advanced Solid Tumors, Including But Not Limited to Lung Cancer Head and Neck Cancer Pancreatic Cancer Ovarian Cancer Renal Cell Carcinoma Malignant Glioma Biliary tractPhase I Phase I Phase I Phase INivolumab. Nivolumab ±CT. Single agent. Multiple immunotherapy
      IMC-CS4Bactuzumab binds to CSF1R expressed on macrophages and inhibits binding of colony-stimulating factor-1 (CSF-1) to CSF1R.NCT01346358 NCT03153410 NCT02265536Advanced solid tumors refractory to standard therapy, Pancreatic cancer, Breast Cancer, Prostate CancerPhase I Phase I Phase ISingle agent CY, Pembrolizumab, GVAX Single agent
      Tyrosine Kinase inhibitorsPexidartinib (PLX3397)CSF1R, cKIT, FLT3, PDGFRPexidartinib binds to and inhibits phosphorylation of stem cell factor receptor (KIT), colony-stimulating factor-1 receptor (CSF1R) and FMS-like tyrosine kinase 3 (FLT3NCT02584647 NCT02071940 NCT02452424 NCT01499043 NCT01349036 NCT01596751 NCT01525602Advanced solid tumors refractory to standard therapy, Sarcoma, Melanoma, Melanoma

      Non-small Cell Lung Cancer

      Squamous Cell Carcinoma of the Head and Neck

      Gastrointestinal Stromal Tumor (GIST)

      Ovarian Cancer
      Phase I Phase I Phase I Phase I Phase II Phase Ib/II Phase ISingle agent Single Agent Combined with Pembrolizumab Single Agent Single Agent Combined with Eribulin Single Agent
      ARRY-382,CSF-1RA small molecule and orally available inhibitor of CSF-1NCT01316822Advanced solid tumors refractory to standard therapyPhase ISingle agent
      PLX7486CSF-1R, TRKA-B-CThe tosylate salt form of PLX7486, a selective inhibitor of the receptor tyrosine kinases colony-stimulating factor-1 receptor (CSF1R; fms) and neurotrophic tyrosine kinase receptor types 1, 2 and 3 (TrkA, TrkB, and TrkC, respectively)NCT01804530Advanced solid tumors refractory to standard therapyPhase ISingle agent
      BLZ945CSF-1RBLZ945 selectively binds to CSF1R expressed on tumor-associated macrophages (TAMs), blocks CSF1R activity, and inhibits CSF1R-mediated signal transduction pathways.NCT02829723Advanced solid tumors refractory to standard therapyPhase I/IICombined with PDR001
      JNJ- 40,346,527CSF-1RA small molecule, orally available inhibitor of colony-stimulating factor-1 receptor (CSF1R; FMS) with potential antineoplastic activity. FMS tyrosine kinase inhibitor JNJ-40346527 blocks the receptor-ligand interaction between FMS and its ligand CSF1NCT03177460 NCT01054014Advanced solid tumors refractory to standard therapy, ProstatePhase I Phase Iversus Daratumumab. Single agent
      Emactuzumab is a humanized monoclonal antibody directed againstCSF-1R. It was studied as a single agent in a phase I trial enrolling patients with tenosynovial giant cell tumors, in which it showed no significant toxicity at the optimal immunomodulatory dose of 1000 mg every two weeks. In the dose-expansion phase, 24 (86%) of the 28 patients tested showed an objective response, and 2 (7%) achieved a complete response [
      • Cassier P.A.
      • Italiano A.
      • Gomez-Roca C.A.
      • et al.
      CSF1R inhibition with emactuzumab in locally advanced diffuse-type tenosynovial giant cell tumours of the soft tissue: a dose-escalation and dose-expansion phase 1 study.
      ].
      The same pathway was also inhibited by using Tyrosine kinase inhibitors (TKis) (Table 1). Results from phase I and phase II trials indicated that pexidartinib (PLX3397) was well tolerated; however, when it was tested in a phase II trial enrolling patients with recurrent glioblastoma, pexidartinib did not improve six-month progression-free survival rates compared with standard radiotherapy and temozolomide [
      • Butowski N.
      • Colman H.
      • De Groot J.F.
      • et al.
      Orally administered colony stimulating factor 1 receptor inhibitor PLX3397 in recurrent glioblastoma: an Ivy Foundation Early Phase Clinical Trials Consortium phase II study.
      ]. Nevertheless, another TKi, BLZ945 [
      • Pyonteck S.M.
      • Akkari L.
      • Schuhmacher A.J.
      • et al.
      CSF-1R inhibition alters macrophage polarization and blocks glioma progression.
      ], showed promising results in glioma when used in combination with inhibitors of insulin-like growth factor 1 receptor (IGF1R) and phosphoinositide 3 kinase (PI3K) [
      • Quail D.F.
      • Bowman R.L.
      • Akkari L.
      • Quick M.L.
      • Schuhmacher A.J.
      • Huse J.T.
      • et al.
      The tumor microenvironment underlies acquired resistance to CSF-1R inhibition in gliomas.
      ], and is currently under assessment in a trial in advanced-stage solid tumors as a single agent and in combination with the anti-PD1 antibody PDR001 (NCT02829723).
      Both monoclonal antibodies and TKis seem to be well tolerated with a favorable safety profile. Frequently reported adverse events (AEs) include fatigue, elevated liver enzymes, facial and peripheral edema, asthenia, pruritus, rash, nausea/vomiting, headache and dry skin [
      • Murray P.J.
      • Allen J.E.
      • Biswas S.K.
      • et al.
      Macrophage activation and polarization: nomenclature and experimental guidelines.
      ,
      • Liao X.
      • Sharma N.
      • Kapadia F.
      • et al.
      Krüppel-like factor 4 regulates macrophage polarization.
      ,
      • Covarrubias A.J.
      • Aksoylar H.I.
      • Horng T.
      Control of macrophage metabolism and activation by mTOR and Akt signaling.
      ,
      • Festuccia W.T.
      • Pouliot P.
      • Bakan I.
      • et al.
      Myeloid-specific Rictor deletion induces M1 macrophage polarization and potentiates in vivo pro-inflammatory response to lipopolysaccharide.
      ,
      • Bhattacharjee A.
      • Pal S.
      • Feldman G.M.
      • et al.
      Hck is a key regulator of gene expression in alternatively activated human monocytes.
      ,
      • Poh A.R.
      • Love C.G.
      • Masson F.
      • et al.
      Inhibition of hematopoietic cell kinase activity suppresses myeloid cell-mediated colon cancer progression.
      ,
      • Rutschman R.
      • Lang R.
      • Hesse M.
      • et al.
      Cutting Edge: Stat6-dependent substrate depletion regulates nitric oxide production.
      ,
      • Satoh T.
      • Takeuchi O.
      • Vandenbon A.
      • et al.
      The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection.
      ,
      • Chawla A.
      Control of macrophage activation and function by PPARs.
      ].
      Asymptomatic increases in liver enzymes seen in CSF-1R-targeting treatment are most likely caused by a decrease in physiologic clearance through partial depletion of sessile liver macrophages (CSF-1R + Kupffer cells) [
      • Radi Z.A.
      • Koza-Taylor P.H.
      • Bell R.R.
      • et al.
      Increased serum enzyme levels associated with kupffer cell reduction with no signs of hepatic or skeletal muscle injury.
      ].
      Potentially immune-related AEs have been described for monoclonal antibodies (mAbs), but serious liver injuries have not been reported. In contrast, pexidartinib caused serious non-fatal liver toxicity [
      • Poh A.R.
      • Ernst M.
      Targeting Macrophages in Cancer: From Bench to Bedside. Frontiers.
      ]. In line with the generally favorable safety profile of CSF-1R inhibitors, combination treatment studies have been initiated for both chemotherapies and targeted therapies or immunotherapies.
      Another way to inhibit macrophages is through the CCL2–CCR2 axis. CCL2 is a potent chemoattractant for monocytes, T cells and NK cells [
      • Deshmane S.L.
      • Kremlev S.
      • Amini S.
      • et al.
      Monocyte chemoattractant protein-1 (MCP-1): an overview.
      ,
      • Chun E.
      • Lavoie S.
      • Michaud M.
      • et al.
      CCL2 promotes colorectal carcinogenesis by enhancing polymorphonuclear myeloid-derived suppressor cell population and function.
      ,
      • Fang W.B.
      • Jokar I.
      • Zou A.
      • et al.
      CCL2/CCR2 chemokine signaling coordinates survival and motility of breast cancer cells through Smad3 protein- and p42/44 mitogen-activated protein kinase (MAPK)-dependent mechanisms.
      ,
      • Fang W.B.
      • Yao M.
      • Brummer G.
      • et al.
      Targeted gene silencing of CCL2 inhibits triple negative breast cancer progression by blocking cancer stem cell renewal and M2 macrophage recruitment.
      ,
      • Peña C.G.
      • Nakada Y.
      • Saatcioglu H.D.
      • et al.
      LKB1 loss promotes endometrial cancer progression via CCL2-dependent macrophage recruitment.
      ]. CCL2 released by tumor cells recruits classical monocytes that express the receptor CCR2 to the tumor sites, and its inhibition reduced tumor burden and metastasis in experimental models. High CCL2 levels in serum and in the tumor were associated with poor prognosis in different types of tumors [
      • Ueno T.
      • Toi M.
      • Saji H.
      • et al.
      Significance of Macrophage chemoattractant protein-1 in macrophage recruitment, angiogenesis, and survival in human breast cancer.
      ,
      • Lebrecht A.
      • Grimm C.
      • Lantzsch T.
      • et al.
      Monocyte chemoattractant protein-1 serum levels in patients with breast cancer.
      ]. For these reasons, several CCL2-neutralizing antibodies are now being tested in clinical trials (NCT00992186).

      Indirect inhibition of TAM

      Use of bisphosphonates has been related to inhibited proliferation, migration and invasion of macrophages, which share the same lineage with osteoclasts, causing apoptosis [
      • Rogers T.L.
      • Holen I.
      Tumour macrophages as potential targets of bisphosphonates.
      ,
      • Moreau M.F.
      • Guillet C.
      • Massin P.
      • et al.
      Comparative effects of five bisphosphonates on apoptosis of macrophage cells in vitro.
      ,
      • Zeisberger S.M.
      • Odermatt B.
      • Marty C.
      • et al.
      Clodronate-liposome-mediated depletion of tumour-associated macrophages: a new and highly effective antiangiogenic therapy approach.
      ,
      • Zhang W.
      • Zhu X.-D.
      • Sun H.-C.
      • et al.
      Depletion of tumor-associated macrophages enhances the effect of sorafenib in metastatic liver cancer models by Antimetastatic and Antiangiogenic Effects.
      ]. Zoledronic acid reduced tumor progression in mouse models of bone metastases from breast cancer [
      • Daubiné F.
      • Le Gall C.
      • Gasser J.
      • et al.
      Antitumor effects of clinical dosing regimens of bisphosphonates in experimental breast cancer bone metastasis.
      ] and modulated the TME by reducing the number of TAMs and their polarization status [
      • Coscia M.
      • Quaglino E.
      • Iezzi M.
      • et al.
      Zoledronic acid repolarizes tumour-associated macrophages and inhibits mammary carcinogenesis by targeting the mevalonate pathway.
      ]. It has also been demonstrated that treatment with zoledronic acid impairs macrophage polarization, reduces macrophage-induced angiogenesis and decreases tumor invasion in prostate cancer [
      • Comito G.
      • Pons Segura C.
      • Taddei M.L.
      • et al.
      Zoledronic acid impairs stromal reactivity by inhibiting M2-macrophages polarization and prostate cancer-associated fibroblasts.
      ].
      An important observation is that the efficacy of some chemotherapeutic agents such as trabectedin may be also related to their ability to kill TAMs. In preclinical experiments, it was shown that trabectedin maintained anticancer activity when resistant cells were transplanted in immunocompetent mice[
      • Germano G.
      • Frapolli R.
      • Belgiovine C.
      • et al.
      Role of macrophage targeting in the antitumor activity of trabectedin.
      ]. The drug activity was probably associated with the capability in decreasing of TAM density.
      Radiotherapy was also found to influence TAM: Low-dose irradiation of mouse pancreatic and colorectal cancers functionally reprogrammed TAMs to an antitumor phenotype, characterized by a NOS2-dependent increase in their T cell stimulatory properties [
      • Shree T.
      • Olson O.C.
      • Elie B.T.
      • et al.
      Macrophages and cathepsin proteases blunt chemotherapeutic response in breast cancer.
      ]. The changes in microenvironment observed with both chemotherapy and radiotherapy and their therapeutic impact on cancer confirm the importance of inhibiting TAM in solid malignancy.
      A summary of the different clinical trials targeting TAMs are described in Table 1.

      Conclusion

      This review summarizes the role of macrophages in solid tumors and in GC, in particular in the diffuse subtype where no targeted molecular alterations have been detected so far. In several translational studies the role of TAM2 was found to be predominant in this subgroup according to FACs evaluations and transcriptomic analyses. The prognostic value of TAMs seems to suggest a more aggressive phenotype, nevertheless perspective studies and further evaluations are needed to better clarify their role in resistance to chemotherapy and in the development of an immunosuppressive phenotype. As immunotherapy represents a relevant part of the treatment of many solid tumors where predictive markers such as PD-L1 expression, TILs and Tumor mutation border could help in identifying those patients who will benefit from this approach, the role of TME needs to be further understood. In particular when TAMs are significantly represented it would be necessary to investigate about their role and possibly acting by the use of specific inhibitors actually under development. Nevertheless, the impact of this treatment in GC should be further evaluated.

      Funding

      This study was supported by grants from the Carlos III Health Institute ( PI15/02180 and PI18/01909 to AC; PI18/01508 to TF). VG was supported by the ESMO 2014 fellowship program, and by Rio Hortega contract CM18/00241 from the Carlos III Health Institute ; NT was supported by Rio Hortega contract CM15/00246 from the Carlos III Health Institute ; DR was supported by Joan Rodes contract 16/00040 from the Carlos III Health Institute ; MC is supported by a pre-doctoral grant from the Spanish Cancer Association (AECC), Spain . TF is supported by Joan Rodes contract 17/00026 from the Carlos III Health Institute.

      Declaration of Competing Interest

      AC declares institutional research funding from Genentech, Merck Serono, BMS, MSD, Roche, Beigene, Bayer, Servier, Lilly, Novartis, Takeda, Astelas and Fibrogen and advisory board or speaker fees from Merck Serono, Roche, Servier, Takeda and Astelas in the last five years. The other authors declare no potential conflicts of interest.

      Acknowledgements

      The authors gratefully acknowledge the support of the Carlos III Institute and INCLIVA Research Health Institute – Hospital Clínico Universitario de Valencia.

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