Phagocyte extracellular matrix crosstalk empowers tumor development and dissemination

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1 REVIEW ARTICLE Phagocyte extracellular matrix crosstalk empowers tumor development and dissemination Chen Varol 1,2 and Irit Sagi 3 1 The Research Center for Digestive Tract and Liver Diseases, Tel-Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel-Aviv University, Israel 2 Department of Clinical Microbiology and Immunology, Sackler Faculty of Medicine, Tel Aviv University, Israel 3 Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel Keywords extracellular matrix; matrix metalloproteinases; tumor microenvironment; tumor-associated macrophages; tumor-associated neutrophils Correspondence C. Varol, Research Center for Digestive Tract and Liver Diseases, Tel-Aviv Sourasky Medical Center, Sackler Faculty of Medicine, Tel-Aviv University, Tel-Aviv 64239, Israel Tel/Fax: chenv@tlvmc.gov.il and I. Sagi, Department of Biological Regulation, Weizmann Institute of Science, Rehovot 76100, Israel Tel: irit.sagi@weizmann.ac.il Phagocytes, such as tumor-associated macrophages (TAMs) and tumorassociated neutrophils (TANs), are abundant in the stroma of experimental and human tumors and are locally educated to mediate important biological functions that profoundly affect tumor initiation, growth, and dissemination. Of considerable importance is the noncellular component of the tumor microenvironment, namely the extracellular matrix (ECM). This milieu is often overlooked due to its complexity and vast heterogeneity. Biophysical and biomechanical cues provided by the dynamically evolving tumorigenic ECM fundamentally modulate every behavioral facet of the cancer cells and of associated stromal cells. In this review, we discuss the intricate interplay between phagocytes and ECM that are lined up to support tumor progression. TAMs and TANs shape the tumorigenic ECM by providing key matrix-remodeling enzymes and structural proteins and in turn, the altered tumor ECM modulates their migration and function. A better mechanistic comprehension of this reciprocal dependence has exciting implications for the development of new therapeutic options for cancer. (Received 5 July 2017, revised 1 October 2017, accepted 31 October 2017) doi: /febs Abbreviations 3D, Three-dimensional; ADAM, a disintegrin and metalloproteinase; ADAMTS, a disintegrin and metalloproteinase with thrombospondin motifs; COX2, cyclooxygenase 2; CSF-1, colony-stimulating factor 1; DDR1, 2, discoidin domain receptor 1, 2; ECM, extracellular matrix; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; G-CSF, granulocyte colony-stimulating factor; GPNMB, glycoprotein nonmetastatic melanoma protein B; HA, hyaluronan; HBEGF, Heparin-binding EGF-like growth factor; HIF, hypoxia-inducible factor; LAIR-1, leukocyte-associated immunoglobulin-like receptor-1; LOXL1 or 2, LOX-like protein-1 or -2; LOX, Lysyl oxidase; MMPs, matrix metalloproteinases; MSF, migration stimulation factor; NETs, neutrophil extracellular traps; NK, natural killer; OPN, osteopontin; P4HA1, Prolyl 4-hydroxylase a-subunit isoform 1; PLOD-1, -3, procollagen-lysine 2-oxoglutarate 5-dioxygenase-1 and -3; SDF1, stromal-derived factor 1; SPARC, secreted protein acidic and rich in cysteine; TAMs, tumor-associated macrophages; TANs, tumor-associated neutrophils; TGFa, TGFb, transforming growth factor alpha and beta; TGM1, 2, transglutaminase-1 and -2; THBS1, thrombospondin-1; TILs, tumor-infiltrating T lymphocytes; TIMP-1, tissue inhibitor of metalloproteinases-1; TLRs, Toll-like receptors; Treg, regulatory T cells; TSG-6, tumor necrosis factor-stimulated gene 6; u-pa, Urokinase-type plasminogen activator; VEGF, vascular endothelial growth factor. 734 The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies

2 C. Varol and I. Sagi The tumorigenic phagocyte-ecm crosstalk Introduction The complex relationship between tumors and the immune system has been the subject of ongoing studies for many years. Genetic and epigenetic alterations are hallmarks of all cancers and provide a diverse set of tumor-specific antigens, which render the tumors more exposed to immune surveillance. Indeed, higher density of tumor-infiltrating T lymphocytes has been associated with a favorable clinical prognosis in various human cancers [1]. In marked contrast, epidemiological meta-analyses persuasively have indicated a correlation between the density of distinct phagocytic cells and poor prognosis in many human cancers. This has been established in particular for tumor-associated macrophages (TAMs) [2,3] and more recently, also for tumor-associated neutrophils (TANs) [4]. Once arriving at the tumor microenvironment, these cells usually adopt a protumoral phenotype and advance tumor initiation, progression, and metastasis [5 9]. TAM and TAN biochemical and signaling programs are significantly affected by cues generated from their microenvironment, among which are cues derived from the extracellular matrix (ECM) that surrounds each cellular compartment with distinct morphology typical to individual organs. The ECM is composed of a complex network of macromolecules that assemble into three-dimensional (3D) supramolecular structures, which provide the operational foundation for tissue function and mechanical integrity [10]. During organ development and homeostatic maintenance, multiple regulatory mechanisms act in concert to ensure normal ECM dynamics, collectively dictated by its production, degradation, and remodeling [11,12]. Deregulated and disorganized ECM has become a hallmark of diseases such as fibrosis and cancer [13]. The hallmarks of cancer, as originally established by Hanahan and Weinberg [14], encompass fundamental biological capabilities acquired during the development of human cancers including sustained proliferation, evasion of growth suppression, death resistance, replicative immortality, induced angiogenesis, initiation of invasion and metastasis as well as dysregulated cellular metabolism and evasion of immune destruction. A growing body of evidence indicates that abnormal biochemical and biomechanical properties acquired by the tumor ECM influence each of these cancer hallmarks and are critical for malignancy [15]. In this review, we first briefly summarize decades of research on the contribution of TAMs and TANs to tumor growth and dissemination. We then discuss the role of ECM remodeling in cancer progression, highlighting key structural alterations as well as matrix proteolytic and cross-linking pathways associated with tumor growth and dissemination. We will mainly elaborate on the matrix-related signature acquired by TAMs and TANs in the tumor microenvironment and how they participate in the shaping of the tumorigenic ECM niche. Reciprocally, we will describe how specific alterations within the tumor ECM structure and composition regulate the protumoral activity of these phagocytic cells. TAMs and TANs promote tumor development and dissemination Tumor-associated macrophages represent a major component of the lymphoreticular infiltrates in solid tumors. They perform various protumoral functions that affect every hallmark of cancer progression [16]. TAMs predominantly rely on the active recruitment of classical Ly6C hi monocytes [17,18], which respond to tumor cell production of macrophage growth factors and chemoattractants, among which colony-stimulating factor 1 (CSF-1) (reviewed in Ref. [7]) and the chemokine CCL2 [17 19] were shown to be instrumental. In general, macrophages in tumors are biased away from the classically activated (M1) macrophage phenotype to the alternatively activated type named M2 [20]. Yet, TAMs are considered to be a heterogeneous cell population with multidimensional functional plasticity and often exhibit markers that are characteristic of both types. M1-like TAMs are more common in cases of chronic inflammation-induced cancers, and produce reactive nitrogen and oxygen intermediates, inflammatory cytokines and growth factors, which create a mutagenic tumor-initiating environment [5,8,21]. Once the tumors progress toward malignancy, TAMs acquire M2-like properties that qualify them to subvert adaptive immunity and foster the adequate immunological environment required for tumor progression [22]. This is typically manifested by down-regulation of the proinflammatory cytokine IL-12 and up-regulation of the anti-inflammatory cytokines IL-10 and TGFb [23] as well as by their release of chemokines that preferentially attract Th1, Th2 lymphocytes and regulatory T cells (Treg), devoid of cytotoxic functions [24]. Tumors require angiogenesis to grow beyond a certain size. It has been well established that TAMs orchestrate the so-called angiogenic switch required for malignant transition by producing neoangiogenic molecules that increase vascular density [25]. They can also induce lymphangiogenesis [26]. Monocytes expressing the angiopoietin receptor TIE-2 have also been demonstrated to possess potent proangiogenic The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies 735

3 The tumorigenic phagocyte-ecm crosstalk C. Varol and I. Sagi properties in a number of spontaneous or orthotopic mouse tumor models [27] and in human patients [28]. Hypoxia is a major driver of angiogenesis, and TAMs tend to cluster in hot spots in hypoxic avascular areas with a high level of angiogenesis [29]. Under hypoxic conditions, activation of the transcription factors hypoxia-inducible factor -1a (HIF-1a) and -2a (HIF- 2a) further promotes the proangiogenic activity of TAMs [30,31]. Tumor-associated neutrophils also make up a significant portion of the inflammatory cell infiltrate in many types of cancer, and are suggested to play an important role in all aspects of tumor development [9]. TANs are cells with high functional plasticity. Tumorderived TGFb skews neutrophils towards a tumor-promoting (N2) phenotype [32,33], while IFNb supports the development of an antitumor (N1) phenotype [34]. Depending on their N1 or N2 polarization, TANs can differentially affect T cell subsets. Accordingly, N1 TANs found in early stage tumors promote CD4 + and CD8 + T-cell recruitment and activation by provision of T cell-attracting chemokines and proinflammatory cytokines [32,35]. In contrast, N2 TANs preferentially recruit CD4 + Tregs [36]. Furthermore, in response to tumor-derived IL-8, TANs produce arginase 1, which directly suppresses cytotoxic CD8 + T cells [37]. Depletion of TANs in a mouse model of pancreatic cancer markedly reduces angiogenic switching in areas of dysplasia, highlighting their pivotal contribution to tumor angiogenesis [38]. The formation of neutrophil extracellular traps (NETs) is also considered to favor tumor growth by yet unclear mechanism [39,40]. Both TAMs and TANs play pivotal roles in the metastatic process. Intravital imaging has demonstrated remarkable interactions between tumor cells, TAMs, and blood vessels, supporting the notion that TAMs enhance tumor cell migration and invasion [41]. Specifically, a CSF1-epidermal growth factor (EGF) paracrine loop between TAMs and tumor cells has been implicated in the promotion of tumor cell invasion and intravasation in breast cancer [42,43]. A number of studies have demonstrated that myeloid cells recruited to metastatic sites create an environment optimized for rapid development of micrometastases, also known as the premetastatic niche. In this regard, myeloid cells expressing the receptor for vascular endothelial growth factor (VEGF)-A are recruited to premetastatic lung sites before metastatic spread and their removal prevents cluster formation and tumor metastasis [44]. Similarly, CD11b + Gr1 + myeloid cells support hepatic metastasis through down-regulation of the anti-angiogenic molecule angiopoietin-like 7 in cancer cells [45]. In a more cell-specific manner, the CCL2-CCR2 axis has shown to be instrumental in metastatic colorectal [46] and breast [47 49] cancer for the recruitment and retention of Ly6C hi monocytes in their respective seeding sites. In contrast, Ly6C lo patrolling non-classical monocytes reduce tumor metastasis via their tumor scavenging and natural killer (NK) cell recruitment and activation [50]. With respect to TANs, the concerted interplay between IL-17- producing gamma delta T cells and granulocyte colony-stimulating factor-dependent expansion and polarization of TANs suppresses CD8 + cytotoxic T-cell response against metastatic cancer cells [51]. TANs also facilitate metastatic tumor cell survival by inhibiting NK cell-mediated clearance of tumor cells from initial sites of dissemination [52]. In another study, it has been demonstrated that metastatic breast cancer cells can induce the formation of neutrophil NETs that further stimulate the invasion and migration of breast cancer cells in vitro [53]. However, there is also evidence that supports inhibitory activity for neutrophils at the premetastatic sites. For example, metastasis-incompetent tumors generate metastasissuppressive microenvironments in distant organs by inducing thrombospondin-1 (THBS1) expression in bone marrow-derived Gr1 + myeloid cells [54]. In another study using a breast cancer model, it has been shown that neutrophils inhibit metastatic seeding in the lungs by generating H 2 O 2, and that tumor-secreted CCL2 is a critical mediator of their antimetastatic activity [55]. Therefore, these studies highlight the importance of tumor cell-derived cues in dictating the pro- or antimetastatic activity of neutrophils. Collectively, TAMs and TANs actively participate in various aspects of tumor growth and dissemination. Below we discuss those related to their ECM-remodeling properties. ECM dysregulation contributes to neoplastic progression and metastasis The tumor cellular eco-system is nourished by its ECM, comprising a 3D supramolecular network of proteins, glycoproteins, proteoglycans, and polysaccharides. Alterations within tumor ECM actively promote cancer by providing critical biomechanical and biochemical cues that drive tumor cell growth, survival, invasion, and metastasis, and by regulating angiogenesis and immune function. The tumor ECM differs significantly from its normal tissue counterpart ECM [56], an outcome of aberrantly expressed or modified structural proteins and remodeling events orchestrated by specific proteolytic and protein cross-linking enzymes [57]. The importance of the ECM in tumor 736 The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies

4 C. Varol and I. Sagi The tumorigenic phagocyte-ecm crosstalk biology has been reviewed extensively elsewhere [13,15,58,59] and therefore will be briefly discussed here. Tumors often display enhanced ECM stiffness, ensuing from altered deposition, cross-linking, and geometrical organization (e.g., linearization) of fibrous proteins, especially of collagen fibers that are often positioned perpendicular to the tumor boundary [60,61]. The resultant rigidity has been implicated in many of the hallmarks of cancer [15]. It fosters angiogenesis by providing a conduit for endothelial cell migration, and serves as a reservoir for pro- and antiangiogenic factors. For example, collagen type IV can directly promote angiogenesis and neovessel survival in a dose-dependent manner [62]. Moreover, ECM mechano-sensing pathways in endothelial cells instruct their branching morphogenesis [63] and proliferation [64]. It has also been suggested that stiffened ECM can impede antitumorigenic T cell function [65], enhance tumor cell invasion and metastasis by inducing focal adhesion assemblies, and promote epithelialto-mesenchymal transition [60,66,67]. The establishment of a metastatic niche within distant tissue supports the survival and growth of circulating tumor cells, and is considered a key feature of metastasis and tumor progression [68]. The tissuespecific pattern of metastasis has been intriguing ever since Paget first introduced the seed and soil hypothesis over a century ago [69]. It is becoming clear that ECM morphology and composition in the metastatic site is crucial for the seeding and growing ability of metastatic cells [58]. For example, increased ECM stiffening facilitates colonization of cancer cells and infiltration of myeloid cells at the metastatic site [70,71]. Remodeling of the ECM prior to receipt of disseminated tumor cells can be induced by factors secreted systemically from primary tumors and immune cells or locally by resident and infiltrating cells, supporting together the formation of premetastatic niches. In this respect, instigator tumors can secrete endocrine factors such as osteopontin (OPN) that mediate the mobilization and recruitment of BMderived granulin-expressing hematopoietic progenitor cells to sites of metastasis. There, granulin increases the expression of a variety of ECM components and their modifying enzymes by tissue-resident fibroblasts contributing to a prometastatic desmoplastic response [72]. Another example is tumor-released exosomes that play an important role in the premetastatic niche via their modulation of local ECM [73 75]. Emerging findings also highlight a clear link between hypoxia and ECM remodeling events in the primary tumors resulting in the release of factors that modulate the metastatic niche [76]. The ECM is also suggested as a gatekeeper in the transition from dormancy to metastatic growth [77,78]. Collectively, the primary tumor and metastatic site ECM are typically characterized by compositional and organizational alterations that converge to support tumor growth and dissemination. ECM proteolysis is a master switch in cancer progression and dissemination An additional hallmark of tumoral ECM is high levels of proteolytic degradation of physical barriers between cells, which facilitates the invasion of malignant and endothelial cells and promotes the activation and release of cryptic proteins, which directly stimulate tumor cell survival, proliferation, motility, and the neoangiogenic switch [59,79]. In addition, the high proteolytic activity results in degradation of ECM proteins thus creating biologically active ECM fragments termed matrikines [80]. Matrikines are reported to play a role in cancer progression by modulating cell proliferation, migration, protease production, or apoptosis. Thus, the tumoral ECM provides a complex, dynamic signaling moiety that utilizes combinatorial signaling programs, presumably mediated by profound cellular and mechanical feedback events [12]. There is mounting evidence for a critical involvement of matrix metalloproteinases (MMPs) in mediating tumorigenesis and especially the susceptibility of distant sites to metastatic seeding. These are a family of zinc-dependent endopeptidases that play a key role in the molecular communication between tumor and stroma. Selected examples of their effects on the tumor microenvironment have been comprehensively reviewed [79]. One of the most studied MMPs in cancer is MMP9 (gelatinase B), which plays an important role in tumor development and metastasis [79]. In particular, MMP9 plays a critical role in tumor angiogenesis by turning on the angiogenic switch in avascular tumors and mediating the development and maintenance of neovascular networks sustaining tumor cell intravasation [81]. For example, MMP9 regulates the bioavailability of VEGF [82], the most potent inducer of tumor angiogenesis. Noteworthy however, in some tumors MMP9 can also generate ECM fragments like tumstatin, a fragment of the collagen IV a3 chain and a potent suppressor of tumor vasculature formation [83]. These opposing roles for MMP9 in angiogenesis illustrate the biocomplexity of MMP enzymes, a matter that should be carefully considered in developing MMP inhibitor-based therapeutics. While the cellular source of MMP9 has not been The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies 737

5 The tumorigenic phagocyte-ecm crosstalk C. Varol and I. Sagi addressed in these studies, various cells in the primary tumor and metastatic sites express it. As is discussed below, both TAMs and TANs are prominent sources of this enzyme. Closely related to the MMPs are the so-called ADAM (a disintegrin and metalloproteinase) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) families of metzincin proteinases. Most of these enzymes are anchored to the membrane and function in the pericellular space, thus influencing many aspects of tumourigenesis [84]. ADAM proteinases are considered as key regulators of epidermal growth factor receptor (EGFR) pathway [85]. Accordingly, ADAM-10 triggers the release of soluble EGFR ligands such as b-cellulin and EGF, whereas ADAM-17 is a major sheddase of the EGFR and other Erbb-receptor ligands [86]. Moreover, through their regulation of cellular adhesion, cytokine, and chemokine activation, ADAM proteinases can contribute to the immunosuppressive environment of the tumor [84]. For example, proteolytic shedding of tumor-associated major histocompatibility complex class I-related proteins MICA and MICB by ADAM- 17 can potently suppress NK cell-mediated cytotoxicity toward the cancer cells [87]. Another important family of proteolytic enzymes is the cathepsins, a group of lysosomal proteinases or endopeptidases, which play different roles in distinct tumorigenic processes such as proliferation, angiogenesis, metastasis, and invasion [88 90]. Phagocyte-ECM interplay is instrumental for solid tumor development and dissemination Tumor-associated macrophages and TANs are actively involved in the biodynamic alterations of ECM composition and structure during cancer growth and spread. Reciprocally, changes within the ECM can affect the protumoral polarization of these cells [91]. In the following sections we discuss a growing body of evidence supporting an interdependent cross-talk between TAMs and TANs and the tumor ECM in supporting cancer progression. TAMs and TANs mediate tumorigenic ECM proteolysis In cancer, altered matrix proteolysis leads to unregulated tumor growth, tissue remodeling, inflammation, tissue invasion, and metastasis [79,81,89]. TAMs and TANs are a major source of matrix proteolytic enzymes in the tumor microenvironment [91,92]. CSF- 1-mediates the metalloprotease-dependent release of EGF from the TAM cell surface [93]. It also induces the formation of ventral membrane protrusions and invaginations called podosomes, which have ECMdegrading activity and may be essential for tumor invasiveness [94]. MMPs represent the most studied and prominent family of proteinases associated with tumorigenesis [79]. As mentioned above, MMP9 plays a critical role in tumor angiogenesis and metastasis [81]. Coussens et al. [95] have initially reported that MMP9 expression by tumor-infiltrating, BM-derived hematopoietic cells promotes metastatic growth. Moreover, MMP9 originating from BM-derived immune cells triggers BM hyperplastic hematopoiesis through VEGF availability and induces myeloid-derived suppressor cell expansion in a mammary tumor model [96]. Further studies have attributed MMP9-governed angiogenic activity to TAMs. Accordingly, adoptively transferred WT splenocytes giving rise to MMP9 + TAMs are sufficient to reconstitute the impaired angiogenesis and tumorigenicity of human ovarian cancer cells implanted in Mmp9 / mice [97]. Moreover, MMP9 expression in premetastatic lung endothelial cells and TAMs is elicited by distant primary tumors via VEGFR-1 Flt-1 tyrosine kinase interactions [98]. In models of cervical cancer [99] and glioblastoma [100], the release of sequestered VEGF has been credited to TAM-derived MMP9. TAMs can also regulate the expression of MMP9 by tumor cells via their secretion of immune modulators such as transforming growth factor beta-1 (TGFb1) [101]. In contrast, other studies have highlighted TANs as a major source of MMP9 in the tumor microenvironment. Accordingly, MMP9-expressing TANs are predominantly found inside angiogenic islets during early stages of pancreatic carcinogenesis and are pivotal for VEGF activation [38]. Moreover, direct comparison between TANs and TAMs revealed that the former are more effective in the immediate supply of angiogenic MMP9 zymogen, which is uniquely unencumbered by tissue inhibitor of metalloproteinases-1 (TIMP-1) [102]. Thus, neutrophil MMP9 could serve as a therapeutic target in human cancers in which neutrophil infiltration is associated with enhanced tumor angiogenesis and poor prognosis. In contrast to MMP9, other MMPs may have antitumor functions. MMP8 contributed by TANs has a tumor-suppressing role in a mouse model of carcinogen-induced skin cancer [103] and in the Lewis lung carcinoma model of lung metastasis [104]. Moreover, MMP12 delivered by macrophages suppresses lung metastasis growth by inhibiting angiogenesis [105]. These examples illustrate a possible conflict between the activities of distinct 738 The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies

6 C. Varol and I. Sagi The tumorigenic phagocyte-ecm crosstalk MMPs brought by phagocytes to the tumor. Importantly, tumor fate decisions may be dictated by various cues in the microenvironment affecting MMP activity. Proteolytic activity of MMPs can be regulated at different levels: gene expression, compartmentalization, conversion from zymogen to active enzyme, and, finally, the presence of specific inhibitors, such as TIMPs [79]. Moreover, regulation of the availability of the physiological substrates for each MMP could serve as a driving force for tumor growth or arrest. These parameters may vary according to the developmental phase of the tumor, its changing cellular composition and the resulted dynamic repertoire of matrix proteases, their inhibitors and substrate availability. ADAM17 from tumor-associated leukocytes, mainly TAMs, plays a role in the initiation of mammary tumors via its positive regulation of cyclooxygenase 2 (COX2) expression [106]. Interestingly, the release of CSF-1 from breast tumor cells is mediated by ectodomain shedding through ADAM17 and this further stimulates proangiogenic macrophages [107]. We have recently shown in an orthotopic colorectal cancer model that TAMs express ADAM17 at the gene and protein levels [17], but its direct contribution to tumor development remains elusive. These colorectal TAMs also express ADAM8, 9, 10, and 15 [17]. Among these, ADAM8 is expressed in positive correlation with tumor invasiveness, metastasis, and poor prognosis in various experimental and human cancers, including pancreatic [108] and invasive breast cancer [109], where it has been shown to stimulate both angiogenesis via the release of VEGF-A and transendothelial cell migration via b1-integrin activation. Similarly to MMPs, ADAMs can be regulated at multiple levels. Therefore, when judging the pathophysiological relevance of increased expression of these matrix proteases in TAMs and TANs, their activity has to be verified. Another family of matrix proteases is the cysteine cathepsins, shown to function in proteolytic pathways that increase neoplastic progression [88,89]. Tumorsecreted IL-4 induces cathepsins B and S activity in TAMs to promote pancreatic tumor growth, angiogenesis, and invasion [110]. Inhibition of cathepsins B, L, and S triggers TAM death owing to increased oxidative stress [111]. TAM-derived cathepsin B has also been implicated in the development of lung metastasis in a mouse model of breast cancer [112] as well as in chemoprotective effects [113]. We have shown that Ly6C hi monocytes up-regulate cathepsin B, D, and L gene and protein expression upon their differentiation into colorectal TAMs [17]. Moreover, cathepsin H is expressed in TAMs localizing in proximity to blood vessels, and its deletion leads to a significant reduction in vessel area and pancreatic islet cell tumor growth [114]. Urokinase-type plasminogen activator (u-pa)- mediated ECM breakdown also plays important role in promoting the metastatic potential of cancer cells through increased tumor angiogenesis and cancer cell intravasation [115]. u-pa and its receptor were found to be predominantly synthesized by TAMs in a number of different cancers and their levels correlate with high microvessel density and poor prognosis [ ]. Imbalance of neutrophil elastase serine protease and its inhibitor alpha 1-antitrypsin also plays a significant role in cancer development and progression of malignant tumors, such as liver, lung, or colorectal cancer [119]. Mechanistically, it has been shown that neutrophil elastase can directly induce tumor cell proliferation in both human and mouse lung adenocarcinomas [120]. TAMs as builders of the tumorigenic ECM niche Extracellular matrix deposition (desmoplasia) is a hallmark of various solid tumors, and it ensues from altered deposition and geometrical organization of matrix proteins [13]. TAMs produce matrix proteins, some of which are typical of neoplastic tissues. Using molecular profiling approaches, two recent studies on human ovarian carcinoma TAMs [92] and orthotopic implanted colorectal TAMs [17] have greatly contributed to our comprehension of TAM-mediated contribution to the composition of tumor ECM core and affiliated proteins. Specifically, in both studies, TAMs exhibited up-regulated expression of the matricellular glycoproteins OPN, osteoactivin (also called glycoprotein nonmetastatic melanoma protein B (GPNMB), fibronectin, and secreted protein acidic and rich in cysteine (SPARC) [92]. OPN is one of the most highly expressed genes in various types of cancer [121] and has been linked with malignancy by being involved in protease activation and ECM remodeling, cell adhesion and migration, angiogenesis, and regulation of inflammation and immunity [122]. GPNMB is also overexpressed in various malignancies, is involved in the promotion of angiogenesis and tumor invasiveness and represents an attractive target in cancer immunotherapy [123,124]. Interestingly, the expression of OPN and GPNMB is also highly up-regulated in both murine and human glioma-associated microglia/ macrophages and has been associated with poor prognosis [125]. In glioblastoma, TANs also express OPN, which mediates their recruitment via its Arg-Gly-Asp (RGD) integrin-binding domain [121]. The structure function relationship of fibronectin in advancing The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies 739

7 The tumorigenic phagocyte-ecm crosstalk C. Varol and I. Sagi tumorigenesis has also been the subject of many studies [126]. Specifically, an oncofetal-truncated isoform of fibronectin known as migration stimulation factor is expressed in M2-polarized TAMs and stimulates the in vitro migration of tumor cells [127]. We have demonstrated the expression of THBS1 glycoprotein in colorectal TAMs [17], although its role in the tumor microenvironment is controversial with reports on both pro- and antitumorigenic affects [128]. Collagen is the major component of the ECM and the tumor microenvironment actively promotes degradation and redeposition of collagen to promote tumor progression [129]. Intravital imaging studies revealed an abundance of TAMs at the collagen-rich border of the tumor [130], suggesting their involvement in collagenous matrix remodeling. Indeed, we have recently found that colorectal Ly6C hi monocyte-derived TAMs play a pivotal role in the buildup of the tumorigenic collagenous niche by directly contributing to the deposition, crosslinking, and linearization of fibrillar collagens (Scheme 1). Accordingly, TAMs constitute a prominent source of collagen types I, VI, and XIV, and TAMdeficient colorectal tumors exhibit reduced deposition of these collagen subtypes and impaired collagen crosslinking and linearization [17]. A similar association between TAM presence and protumorigenic and metastatic collagen remodeling has been described in a model of breast cancer [131]. Genes encoding for collagen types I, V, and VI are also evident in TAMs isolated from human ovarian carcinoma [92]. Collagen VI synthesis has also been demonstrated in monocytes associated with triple-negative breast cancer xenografts [132]. More recently, it has been demonstrated that pancreatic ductal adenocarcinoma embryonically derived TAMs, but not Ly6C hi monocyte-derived TAMs, exhibit a profibrotic function manifested by their expression of genes encoding for various types of collagen and their direct ex vivo production of collagen type I [133]. The ability of macrophages to acquire a fibroblast-like phenotype expressed by their direct synthesis of specific collagen subtypes is further supported by studies in renal fibrosis [134] and atherosclerotic plaques [135]. In another study, the Pollard group has demonstrated that macrophage-deficiency results in impaired collagen fibrillogenesis and altered organization of terminal-end buds during mammary gland development [136]. In culture, TGFb1 induces abundant secretion of type VI collagen in human macrophages [137]. Therefore, these findings strongly support that TAMs can directly contribute to the deposition of specific collagen subtypes in the tumor microenvironment. The ability of TAMs to acquire a fibroblast phenotype may be tissue specific and depends on their ontogeny. Notably, TAMs also produce proteins associated with collagen assembly and organization (Scheme 1). A specific example is their expression of SPARC glycoprotein; a collagen chaperone and a master stromal regulator [17,92,133]. Immune cell-derived SPARC has shown to be important for the assembly and organization of collagenous matrix [138,139]. Colorectal TAMs also express enzymes involved with post-translational modifications of intracellular collagen and its assembly including prolyl 4-hydroxylase a-subunit isoform 1 (P4HA1) and procollagen-lysine 2-oxoglutarate 5-dioxygenase-1 and -3 (PLOD-1, -3) [17]. They also express the matrix cross-linker enzymes TGM1, TGM2, and coagulation factor XIII (F13A1) [17]. Interestingly, the expression of TGM1, TGM2, PLOD3, and F13A1 was higher in tumor-associated Ly6C hi monocytes in comparison with their more mature TAM descendants [17], suggesting that these enzymes may be involved in early phase tumor development. TGM2 expression in various cancer cell types has been linked to increased drug resistance, cell survival, invasiveness, metastasis and poor patient survival [140]. Moreover, pancreatic tumor embryonic-derived TAMs express genes encoding for the collagen cross-linkers lysyl oxidase (LOX) and LOX-like protein 1 (LOXL1) [133]. The TAMs can also contribute indirectly to the production and organization of collagenous matrix through their regulation of cancer-associated fibroblast (CAF) activity (Scheme 1). In alignment with this, CAFs from TAM-deficient colorectal tumors exhibit reduced expression of collagen type I [17]. Moreover, triple-negative breast cancer xenografts cotransplanted with monocytes exhibit higher collagen deposition and activation of stromal CAFs [132]. Macrophage-elicited profibrotic activity of fibroblasts has also been demonstrated in a model of skin injury, where IL-4 polarized macrophages induced the activity of the collagen crosslinker enzyme lysyl hydroxylase 2 in adjacent fibroblasts [141]. Additional examples are the profibrotic fibroblast cell activation by Ly6C hi monocyte-derived TGFb during fibrosis of the liver [142] and lung [143]. The tumor matrix is also a valuable repository for specific TAM-derived growth factors and cytokines that may activate the fibrotic activity of CAFs, including EGF, FGF, PDGF, and above all TGFb [92,144]. Reciprocally, activated CAFs can affect TAMs recruitment, retention and protumoral polarization through their collagenous matrix remodeling activity [145] and the secretion of stromal-derived factor 1 (SDF1) [146] or CXCL16 [132]. TAMs and CAFs also collaborate in the recruitment and activation of endothelial cells to drive de novo angiogenesis [146]. Collectively, the secretion, construction, and remodeling of the ECM are 740 The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies

8 C. Varol and I. Sagi The tumorigenic phagocyte-ecm crosstalk Blood 1 Recruited Ly6C hi monocytes acquire a fibrotic phenotype CCR2, M-CSF 9 ECM rigidity increases tumour cell EMT, survival, proliferation and metastasis 2 Embryonic TAMs may also acquire a fibrotic phenotype 3 TAMs produce collagen and molecules promoting its assembly 5 Pro-fibrotic TAMs -CAFs crosstalk 8 Collagen sensing promotes TAN adhesion and inhibits their NETosis 4 TAMs drive collagen crosslinking and linearization Interstitial Matrix 6 Collagen degradation by TAM and TAN-derived MMPs 7 Collagen sensing promotes TAM-M2 polarization and mesenchymal migration Tumour cell Ly6C hi monocyte Monocytederived TAM Embryonic TAM TAN CAF Collagen fibrillogenesis Scheme 1. Collagenous matrix remodeling as a model for the tumorigenic cross-talk between TAMs, TANs and ECM. Schematic illustration of the reciprocal interplay between phagocytes and collagenous ECM during tumor development and invasion. 1. Ly6C hi monocytes are recruited to the tumor in a CCR2 and M-CSF-dependent manner, where they acquire a fibroblast-like phenotype, while differentiating into TAMs. TAMs accumulate at the tumor margins. 2. In certain tumors, embryonic-derived TAMs differentiated from tissue-resident macrophages are more profibrotic than the Ly6C hi monocyte-derived TAMs. 3. In the tumor microenvironment, Ly6C hi monocytes and mature TAMs are involved with the deposition of specific types of collagen as well as enzymes and factors that facilitate collagen maturation, stability, and packaging. 4. TAMs promote collagen cross-linking and linearization. Collagen fibers are positioned perpendicular to the tumor boundary, supporting tumor invasion. 5. TAMs can also indirectly contribute to the production and organization of collagenous matrix through their regulation of CAF activity. Reciprocally, activated CAFs promote TAM recruitment, retention and their protumoural polarization. 6. TAMs and TANs can shape tumor collagenous matrix through their provision of specific collagen-degrading MMPs. 7. The increased tumor matrix rigidity, as a result of collagen fibrillogenesis, provides biomechanical and biochemical cues that promote M2-like TAM polarization and mesenchymal mode of macrophage migration. 8. Collagen sensing by TANs promotes their recruitment, adhesion and protumoral activation. It also inhibits NETosis via LAIR The increased tumor matrix rigidity support tumor cell EMT, survival, proliferation, and invasiveness. each regulated by a complex interplay between tumor cells, fibroblasts, and TAMs. Further studies are required to define the cooperative networks in this cellular triad, and how they are linked-up for the buildup of the tumorigenic ECM niche. Phagocytes can also contribute to ECM remodeling at the premetastatic niche. Ly6C hi monocytes recruited to premetastatic lung niches in the PyMT spontaneous breast cancer model produce the ECM proteoglycan versican, which stimulates mesenchymal-to-epithelial transition of metastatic tumor cells [147]. Tumor ECM regulates TAM and TAN recruitment and polarization Aberrant expression, release of proteolytic products or exposure of cryptic domains of ECM components at tumor sites can regulate the protumoral behavior and The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies 741

9 The tumorigenic phagocyte-ecm crosstalk C. Varol and I. Sagi function of TAMs and TANs [148]. For example, a cryptic peptide of laminin-10, a prominent component of basement membranes, is chemotactic for neutrophils and macrophages and induces the up-regulation of MMP9 and TNFa [149]. Particular attention has been given to proteolytic ECM fragments and the activation of Toll-like receptors (TLRs). Versican enhances the metastatic potential of lung carcinoma cells through its direct activation of TLR-2 and its coreceptors TLR6 and CD14 on macrophages [150], thus providing a link between TLR-driven inflammation and cancer metastasis. An additional well-studied example is the glycosaminoglycan hyaluronan (HA), which is avidly synthesized and degraded in the tumor microenvironment and is considered a key player in many cancerassociated processes [148]. The molecular mass of HA determines its activity in the tumor microenvironment. High-molecular weight HA assembles the ECM in association with binding molecules, maintains tissue structure and serves as a scaffold for cell adhesion and migration. In contrast, catabolized low-molecular weight HA fragments diffuse through tissues and initiate immune responses by binding HA receptors on adjacent immune cells [148]. Accordingly, synthetized HA fragments, as well as HA fragments of similar molecular mass extracted from serum of patients with acute lung injury, induce the production of several inflammatory chemokines in peritoneal and BMderived macrophages through activation of TLR4 and TLR2 [151]. In this study, HA also promoted the recovery from lung injury. Therefore, under the physiological settings of tissue injury HA may actually polarize macrophages toward a prorestorative M2-like phenotype. In other studies it has been demonstrated that tumor-derived HA stimulates M2-like TAM formation via interaction with CD44 [152,153]. Moreover, deactivation of human monocytes in the presence of tumor cells involves HA-mediated engagement of both CD44 and TLR4, leading to the up-regulation of the negative immunoregulator kinase IRAK-M [154]. While these studies seem to describe opposing roles for HA in determining macrophage polarization, a broader molecular and functional characterization is required to determine whether there are receptor-specific immune-regulatory properties for HA fragments in TAMs. HA-binding partners may also cooperate in its regulation of macrophage activity. In this respect, HA and its associated molecules form cable-like ECM structures that are involved in the adhesion and recruitment of monocytes through association with CD44 [155]. Such partners may include SHAP, tumor necrosis factor stimulated gene 6 (TSG-6) and versican [156]. With respect to the latter, HA-versican aggregates derived from stromal fibroblasts promote TAM mobilization in a cooperative fashion [157]. Changes in ECM composition are also important for the recruitment and activity of phagocytes at the metastatic niche. In lung metastases, increased fibronectin expression is essential for the recruitment and adherence of VEGFR1 + myeloid cells, which express the fibronectin receptor integrin a4b1 [44]. Physical and mechanical cues regulate macrophage phenotype and function [158]. Therefore, the altered deposition and geometrical organization of collagenous matrix, as evident in various solid tumors, may also affect the migration behavior and function of TAMs and TANs (Scheme 1). Macrophages must be able to probe and discern changes in the mechanical properties of their environment and transduce these changes into biochemical signals. Specifically, macrophages have been known to deploy alternative modes of adhesion to collagen. Generally, b 1 integrin is thought to be the main receptor for macrophage adhesion to collagen [159]. Moreover, collagen-driven activation of the tyrosine kinase receptor discoidin domain receptor 1 (DDR1) is necessary for macrophage infiltration into atherosclerotic plaques [160], and type I collagen fragments are chemotactic for monocytes [161] and neutrophils [162]. Recently it has been demonstrated that increased collagen deposition and SPARC expression in breast tumors promotes a CXCR4-mediated recruitment of myeloid-derived suppressor cells at the tumor site [163]. The tyrosine kinase collagen receptor DDR2 plays an important role in directing neutrophil migration within 3D collagen matrices by increasing secretion of metalloproteinases and local generation of collagen-derived chemotactic peptide gradients [164]. Macrophages can apply two main migration modes while moving through 3D matrices: amoeboid or mesenchymal [165]. Biophysical parameters of the matrix can control the switch between these migration modes; macrophages use either the amoeboid migration mode in fibrillar collagen I, or the mesenchymal migration mode in denser collagen matrices [166]. Tissue ECM may also dictate macrophage functional polarization. In this regard, collagen-rich ECM promotes human monocyte proliferation and activation and favors a protumorigenic M2 polarization phenotype [167,168], whereas a fibronectin-rich ECM promotes the M1 or antitumorigenic profile of macrophages [169]. Cell elasticity is a critical determinant of macrophage innate function. In view of this, the rigidity of the matrix on which macrophages are cultured determines macrophage cell elasticity and function. Accordingly, macrophages cultured on high rigidity polyacrylamide gels (150 kpa) exhibits increased 742 The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies

10 C. Varol and I. Sagi The tumorigenic phagocyte-ecm crosstalk phagocytosis and decreased inflammatory response to LPS in comparison with low rigidity gels (1.2 kpa) [170]. Effects of matrix rigidity on phagocyte cell behavior have also been reported in the premetastatic niche. Accordingly, LOX-governed ECM modification induces the recruitment, invasion and retention of CD11b + myeloid cells to premetastatic sites [70]. Tumor-associated neutrophils can also adhere and become activated in type I collagen matrices [171]. Collagens are high affinity ligands for the inhibitory leukocyte-associated immunoglobulin-like receptor-1 (LAIR-1) [172], broadly expressed on various immune cells. A recent study demonstrated a direct link between SPARC-governed collagenous matrix remodeling, LAIR-1 neutrophil engagement and the restraining of NET extrusion [173]. Notably, an immune-suppressive phenotype has also been attributed to LAIR-1 and PD- L1 highly expressed on tumor-associated dendritic cell population in late stage ovarian cancer [174]. Furthermore, cross-linking of LAIR-1 has been linked with suppression of cytotoxic T-cell activity [175]. Therefore, these findings highlight collagen LAIR-1 interactions as a detrimental in generating an immunosuppressive environment. In another study it has been shown that breast tumors induced in transgenic mouse models of collagen-dense mammary tumors exhibit a cytokine milieu, which supports neutrophil recruitment and activation [176]. Interestingly, neutrophil-specific ablation in these mice significantly diminished the formation of new tumors and reduced tumor burden and lung metastasis only in tumors arising in the collagen-dense tumor microenvironment [176], suggesting that tumor progression in collagen-dense microenvironments occurs at least partially through TAN-mediated protumoral activity. Concluding remarks and future applications It is increasingly acknowledged that extensive remodeling of ECM macromolecule networks has fundamental importance in the development of cancer and its progression. Similarly, among the tumor-infiltrating immune cells, TAMs and TANs are often polarized to perform various protumoral functions. Nevertheless, our comprehension of the interplay between these phagocytic cells, tumor cells and their noncellular milieu is still at its beginning. Emerging findings clearly indicate that TAMs and TANs can shape the protumorigenic ECM through their unique provision of matrix proteases, instructed collagen cross-linking and deposition of protumoral ECM components. In turn, the abnormal ECM can regulate their migration, polarization and function. This kind of networking appears to be a necessary factor in tumor development and metastatic dissemination. In particular, there is a great need for studies attempting to resolve how tumorigenic changes within the ECM structure and composition impact the recruitment, differentiation and activity of TAMs and TANs. A potentially interesting question is the identity of the egg and of the chicken in the interplay between phagocytes and ECM remodeling. As discussed above, TAMs and TANs bring to the tumor microenvironment a specific repertoire of ECM structural proteins and remodeling enzymes. Hence, these cells may be important for initiating specific ECM remodeling events at the tumor and the premetastatic niche. However, one may argue that earlier ECM remodeling events may be executed by nonphagocyte tissue-resident cells such as fibroblasts and tumor cells, and are required for the recruitment of monocytes and neutrophils. Later stages of tumor development are influenced by the dynamic nature of the cross-talk between ECM and these immune cells. Therefore, TAMs and TANs are pivotal conductors that not only orchestrate but also respond to ECM remodeling events in the dynamically evolving tumor microenvironment. Formidable challenges remain in identifying the diverse and novel roles of ECM remodeling reactions, especially with regard to their distinct biophysical, biochemical, and structural properties. Considering the heterogeneous, dynamic, and hierarchical nature of the ECM, in order to decipher such molecular details, a multidisciplinary integrated research scheme is required to provide a 3D functional molecular view of ECM remodeling with respect to morphological changes, and biochemical, signal transduction, and biomechanical cues. Such integrated experimental schemes should encompass various methods including optical imaging and electron microscopy spanning wide ranges of spatial and temporal resolutions that would allow in-depth characterization of the structural alterations within the native tumor ECM. Subsequently, image-guided site directed proteomics might be utilized to reveal molecular compositions in defined niches of the tumor. TAM- and TAN-inducible ablation tools can be integrated to study their impact on the evolving tumor ECM structure and composition at defined niches and during distinct phases of tumor development. Ex vivo reconstituted 3D-ECM tumor microenvironments supplemented with specific TAM or TAN subsets will enable bridging between remodeled ECM macromolecule architecture and composition, and functional cell macromolecular interactions. Mechanistic information derived from such analyses The FEBS Journal 285 (2018) ª 2017 Federation of European Biochemical Societies 743