Cellular and Molecular Immunology (2018) 15, 335–345 & 2018 CSI and USTC All rights reserved 2042-0226/18 $32.00 www.nature.com/cmi

REVIEW

The MCP-1 (CCL2) in the host interaction with cancer: a foe or ally?

Teizo Yoshimura

Macrophages are one of the most abundant leukocyte populations infiltrating tumor tissues and can exhibit both tumoricidal and tumor-promoting activities. In 1989, we reported the purification of chemoattractant -1 (MCP-1) from culture supernatants of mitogen-activated peripheral mononuclear cells and tumor cells. MCP-1 is a potent monocyte-attracting chemokine, identical to the previously described -derived chemotactic factor or tumor-derived chemotactic factor, and greatly contributes to the recruitment of blood into sites of inflammatory responses and tumors. Because in vitro-cultured tumor cells often produce significant amounts of MCP-1, tumor cells are considered to be the main source of MCP-1. However, various non- tumor cells in the tumor stroma also produce MCP-1 in response to stimuli. Studies performed in vitro and in vivo have provided evidence that MCP-1 production in tumors is a consequence of complex interactions between tumor cells and non-tumor cells and that both tumor cells and non-tumor cells contribute to the production of MCP-1. Although MCP-1 production was once considered to be a part of host defense against tumors, it is now believed to regulate the vicious cycle between tumor cells and that promotes the progression of tumors. Cellular and Molecular Immunology (2018) 15, 335–345; doi:10.1038/cmi.2017.135; published online 29 January 2018

Keywords: ; ; inflammation; macrophages;

INTRODUCTION and in vitro. In a guinea pig transplantable tumor model, Macrophages play important roles in host defense by present- antigenically unrelated tumor cells were rejected at sites of ing antigens to or by participating in immune DTH reactions induced by another tumor line.4 This responses as effector cells. Macrophages that infiltrate sites of suggested that the immune response to antigenically unrelated inflammation are derived from blood monocytes, which are tumor cells was nonspecific and mediated by activated macro- attracted by locally produced chemotactic factors. A previous phages at the skin site. It was also shown that macrophages had focus on the cellular motility and accumulation of nonsensi- the capacity to kill tumor cells in vitro if they were properly tized inflammatory cells in delayed-type hypersensitivity activated.5,6 Activated macrophages were therefore assigned a (DTH) has led to the first description of a - critical role in the destruction of tumors by the host. On the migration inhibitory factor.1,2 This was followed by a series other hand, there was evidence suggesting that tumor- of studies in the early 1970s reporting that chemotactic associated macrophages (TAMs) might stimulate tumor growth activity for monocytes (lymphocyte-derived chemotactic factor; or connective tissue development.7–9 Again, neither the role of LDCF) was produced and released by antigen-stimulated TAMs nor molecules attracting TAMs into tumors had been sensitized lymphocytes or mitogen-stimulated non-sensitized clarified.10,11 lymphocytes.3 However, the molecules responsible for these In 1989, we and others reported the purification of mono- activities remained unidentified. cyte chemoattractant protein-1 (MCP-1; also termed by others Infiltration of leukocytes into cancer tissues could also be a as monocyte chemotactic and activating factor; MCAF) from result of a host immune reaction against a tumor-specific culture supernatants of tumor cell lines and activated periph- antigen. Many laboratories explored this possibility both in vivo eral blood mononuclear cells (PBMCs).12–14 Isolation of

Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Kita-ku, Okayama 700-8558, Japan Correspondence: Dr T Yoshimura, MD, PhD, Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata, Kita-ku, Okayama 700-8558, Japan. E-mail: [email protected] Received: 30 July 2017; Revised: 13 October 2017; Accepted: 18 October 2017 Chemokine MCP-1 in the host interaction with cancer TYoshimura

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MCP-1 greatly contributed not only to inflammation but also JE .18 Finally, Bottazzi et al.21 confirmed that the MCA to cancer research that continues today, and MCP-1 has they had previously detected in the culture supernatants of become a molecular target for treating many diseases. In this tumor cell lines was indeed due to the production of MCP-1 by review, the biological activity of MCP-1 is first described, and the cells. Identification of MCP-1, along with IL-8, was the then, the complex interactions between tumor cells and host beginning of the ‘chemokine era’. cells that result in MCP-1 production in tumor microenviron- ments are discussed. BIOLOGICAL ROLE OF MCP-1 The main activity of MCP-1 is to attract blood monocytes. In MCP-1 IS IDENTICAL TO LDCF, TUMOR-DERIVED an in vitro assay, MCP-1 attracts human blood − 9 CHEMOTACTIC FACTOR AND THE PRODUCT OF THE monocytes at its optimal concentration of 10 M,and GENE JE ~30–40% of input cells respond and migrate.12,13 The magni- As a possible mechanism of monocyte/ infiltration tude of the response of human monocytes to MCP-1 is similar into tumors, Meltzer et al. reported the production of MCF by to that of the bacterial peptide N-formyl-methionyl-leucyl- five murine sarcoma cell lines. The molecular weight of tumor phenylalanine (FMLF). There is no significant chemotactic cell-derived monocyte/macrophage chemotactic factor (MCF) response by human , although they responded well was in the range of 15 000 on a Sephadex G-100 column, to FMLF in the same assay. whereas murine LDCF was eluted in the molecular weight The presence of the MCP-1 on human monocytes region of 40 000, suggesting that tumor cell-derived MCF was was explored by a -binding assay using radioiodinated different from LDCF.15 Bottazzi et al. followed this report by MCP-1. Freshly isolated human monocytes possess 1700 ± 600 22 demonstrating the production of monocyte/macrophage che- binding sites per cell with a Kd of 1.9 ± 0.2 nM. Similarly, motactic activity (MCA) by human and murine tumor cell undifferentiated THP-1 cells possess 1175 ± 387 binding sites 23 lines (termed tumor-derived chemotactic factor). Peak MCA per cell with a Kd of 1.53 ± 0.35 nM. When monocytes were was eluted from a Sephadex G-75 column in the molecular induced to differentiate into macrophages, the number of weight range of 12 000. Importantly, there was a significant MCP-1-binding sites was significantly reduced and the cells correlation between the amount of MCA and macrophage poorly responded to MCP-1.22 The loss of MCP-1 binding was content in tumors. Similar to tumor cells, human and murine also detected after the differentiation of THP-1 cells induced by embryo fibroblasts also released MCA into the culture phorbol 12-myristate 13-acetate (PMA).23 The chemokine supernatants,9,16 suggesting that tumor cells and non-tumor receptor CCR2 was later cloned as the receptor for cells produce the same MCF. MCP-1,24,25 and downregulation of CCR2 expression during Our main interest was to identify LDCF, which is considered the differentiation of monocytes into macrophages was verified to be the mediator responsible for the infiltration of monocytes by reverse transcription-PCR and flow cytometry using mono- into sites of DTH reactions. After purification of the first cytes and THP-1 cells.26 Consistent with the results obtained chemokine, -8 (IL-8)/CXCL8, from the culture using in vitro-cultured cells, peritoneal exudate macrophages supernatants of (LPS)-activated human obtained from animals responded poorly to MCP-1 in a PBMCs,17 we immediately began to search for MCA in the chemotaxis assay.27 These findings support the notion that same culture supernatants and successfully purified a second MCP-1 is a potent chemoattractant for circulating blood chemokine, MCP-1, from the culture supernatants of not only monocytes via binding to its receptor CCR2, but is not a mitogen-activated human PBMC but also malignant human potent chemoattractant for differentiated monocyte-derived glioma cells.12,13 MCF produced by both PBMC and malignant macrophages. glioma cells had apparent molecular masses of 15 and 13 kDa, In addition to its monocyte chemotactic activity, MCP-1/ respectively, according to SDS-polyacrylamide gel electrophor- MCAF was shown to activate monocytes for tumor cell killing esis; this difference is likely due to different degrees of over a concentration range that is optimal for chemotaxis N-glycosylation.18 Thus, we demonstrated, for the first time, in vitro.14 Indeed, transplantation of Chinese hamster ovary that non-tumor host cells and tumor cells produce an identical (CHO) cells engineered to express MCP-1 into nude mice led molecule with MCA. to marked monocyte infiltration, and the transplanted cells Matsushima et al.14 simultaneously reported the purification failed to form a tumor.28 Furthermore, co-transplantation of of MCP-1/MCAF from the culture supernatants of activated MCP-1-producing CHO cells with MCP-1-non-producing THP-1 human monocytic leukemia cells. Van Damme et al.19 CHO cells or HeLa cells also suppressed tumor formation by also purified MCP-1 from the culture supernatants of double- these cells. When rat tumor cells that were transfected to stranded RNA-activated MG-63 osteosarcoma cells and LPS- produce MCP-1 were transplanted into syngeneic rats, the activated human monocytes. When the amino-acid sequence of number of macrophages expressing macrophage activation human MCP-1 was compared with those in a database, we markers, such as Ia antigens, was increased and the rate of noted that the sequence of MCP-1 had significant similarity to tumor growth was significantly reduced.29 the protein encoded by the mouse JE gene that was induced in There are two types of macrophages based on their fibroblasts by -derived .20 Subsequently, polarization states—macrophages activated by LPS ± inter- MCP-1 was found to be identical to the product of the human feron-γ (M1) or Th2-derived cytokines (M2), which are

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337 considered to be proinflammatory or anti-inflammatory, activation or tissue damage associated with the activation of respectively.30,31 Similar types of macrophages can be generated monocytes. Interestingly, additional phenotypes were observed in vitro by incubating monocytes in the presence of when mice were given exogenous stimuli. Contact hypersensi- granulocyte-macrophage colony-stimulating factor (GM-CSF) tivity reactions to 2,4-dinitro-1-fluorobenzene were enhanced or macrophage-colony-stimulating factor (M-CSF), in the skin (model 1), and inflammatory responses in the respectively.32 As described above, monocytes that infiltrated upon intravenous (i.v.) injection of LPS or yeast wall glucan tumors after injection of MCP-1-transfected tumor cells were also enhanced (model 4). When model 5 mice were showed antitumor activity; therefore, they appeared to be in crossed with model 2 mice, the resulting mice showed no the M1 polarization state. On the other hand, TAMs that monocyte infiltration, indicating that high systemic MCP-1 accumulated in response to MCP-1 produced in tumor tissues levels could prevent blood monocytes from responding to are generally in the M2 polarization state.33 MCP-1 was shown locally produced MCP-1. From these observations, it can be to promote M2 polarization of macrophages in vitro,31 whereas concluded that blood monocytes infiltrate tissues in response to M2 polarization of human monocytes induced by the culture local production, perhaps a low level of MCP-1, and then supernatants of human glioma cells was independent of differentiate into macrophages in loco.However,theirfate, MCP-1.34 Thus, it is debatable whether MCP-1 directly affects whether they become immune-activating (M1) or suppressing macrophage polarization. (M2) macrophages, is likely determined by other mediators To determine the activity of MCP-1 in vivo, we previously present at the sites. injected recombinant rat MCP-1 into rat skin.35 Intradermal In addition to MCP-1, other chemokines also induce injection of 1 μg of MCP-1 induced both intra- and extra- monocyte chemotaxis in vitro, including MCP-3/CCL7 MCP- vascular accumulation of mononuclear cells, characterized by -4/CCL13 (MCP-5/CCL12 in mice), and macrophage inflam- the expression of the blood monocyte marker ED1, 3 h after matory peptide (MIP)-1α/CCL3,41 suggesting that functionally MCP-1 injection. Accumulation of mononuclear cells, most of redundant chemokines are present and the loss of MCP-1 which were TRPM-3+ and ED3+, characteristic of exudate might be compensated by other chemokines. However, the macrophages, peaked at 12–24 h. These results indicated that infiltration of monocytes in MCP-1-deficient mice in response MCP-1 also attracts blood monocytes in vivo. However, this to a peritoneal injection of thioglycollate, LPS or zymosan was MCP-1-induced accumulation of mononuclear cells was tran- significantly reduced, indicating a non-redundant role of sient and did not result in tissue damage. There was also a MCP-1 in the infiltration of monocytes during inflammatory small increase in the number of OX8+ (CD8+) T cells. responses in vivo.42,43 The in vivo function of MCP-1 was further examined in five MCP-1 is also implicated in the recruitment of other cell MCP-1 transgenic mouse models (Table 1).36–40 In a model in types. Expression of the MCP-1 receptor CCR2 was detected in which MCP-1 was expressed in the epidermis (model 1) or various types of cells, such as T lymphocytes, B lymphocytes, several organs at high levels (model 2), no monocyte infiltrates natural killer cells, neutrophils,41 hematopoietic stem cells44 were observed in MCP-1-expressing organs. Instead, the and fetal microchimeric cells.45 In unchallenged MCP-1 numbers of dendritic cells and Langerhans cells were increased transgenic mice, minor infiltration of lymphocytes, including in model 1. By contrast, in models 3, 4 and 5, in which MCP-1 CD4+ T cells and CD8+ T cells (model 4 and 5), and B220+ B expression was targeted to a specific organ, such as the cells (model 5), was also noted; however, the role of MCP-1 in or central nervous system (model 3), type II alveolar epithelial the recruitment of these cell types in diseases remains unclear. cells in the lung (model 4) or pancreatic islets (model 5), MCP-1 was shown to possess other roles during tumor respectively, monocyte infiltration was detected at MCP-1- progression. Human endothelial cells from both the umbilical producing sites; however, there was no sign of monocyte cord vein and dermal microvasculature expressed CCR2 and

Table 1 Characteristics of MCP-1-transgenic mice

Models Authors Sites of expression (promoter) Cell infiltrates (naive) Other observations

1 Nakamura et al.31 Epidermis (Keratin 14) DCs, Langerhans cells Enhanced contact hypersensitivity reactions to DNFB 2Rutledgeet al.32 Systemic (MMTV-LTR) Negative Enhanced sensitivity to L. monocytogenes or M. tuberculosis 3 Fuentes et al.33 Thymus (Lck), CNS (MBP)MonocytesEnhanced monocyte infiltration in response to LPS Macrophages 4Gunnet al.34 Type II alveolar epithelial cell (surfac- Monocytes Enhanced inflammatory responses to LPS or yeast wall tant protein C) T-lymphocytes glycan 5 Grewal et al.35 Pancreatic islets of Langerhans (insulin)Monocytes No cell infiltrate after crossing to model 2 T- and B-lymphocytes

Abbreviations: CNS, central nervous system; DNFB, 2,4-dinitro-1-fluorobenzene; Lck, lymphocyte cell-specific protein tyrosine kinase; MBP, myelin basic protein; MMTV, mouse mammary tumor long terminal repeat.

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particularly important for induction of this gene in response to proinflammatory responses, such as LPS and (TNF)-α.54–56 We evaluated the role of NF-κBin constitutive MCP-1 production by tumor cells using the U-105MG and U-373MG human glioma cell lines.58 As shown in Figure 2a, U-105MG cells produced an ~ 40-fold higher level of MCP-1 compared with U-373MG cells. An NF-κBinhibitor, caffeic acid phenethyl ester, markedly inhibited MCP-1 pro- duction by both cell lines (Figure 2b); therefore, constitutive MCP-1 production by these cells is dependent on constitutively κ IL-8 Figure 1 Cis-elements involved in the transcription of the human active NF- B. Because transcription of the gene is also MCP-1 gene. Binding of transcription factors to multiple cis- dependent on NF-κB,59 U-105MG cells may produce a higher elements in the 5ʹ-untranslated region of the human MCP-1 gene level of IL-8 than U-373MG cells. In contrast to this hypoth- regulates transcription of this gene. Binding of Sp1 to the GC box esis, U-105MG cells produced an ~ 10-fold lower level of IL-8 in the proximal region regulates basal transcription. In response to than U-373MG cells. It is well established that transcription of PMA, LPS, TNF-α or IL-1, NF-κB dimers, such as RelA/c-Rel cis κ is regulated by multiple -elements and transcription heterodimers or RelA/RelA homodimers, bind to two NF- B-binding κ sites (A1 and A2) in the distal region,54–56 whereas signal factors; thus, constitutively active NF- B is only one mechan- transducer and activator of transcription 1 (STAT1) homodimers ism whereby U-105MG cells produce MCP-1 at a high level. bind to the IFN-γ activation site (GAS) after IFN-γ stimulation.57 Activation of the NF-κB pathway in human MDA-MB-231 Transcription of the MCP-1 gene in response to IFN-γ depends on a breast cancer cells also substantially contributed to expression balance between a positive enhancement mechanism mediated of MCP-1 mRNA via a cancer-associated membrane glycopro- through the GAS element and an inhibitory mechanism mediated tein, dysadherin.60 through the IFN response-inhibitory sequence (IRIS) site. Region 1 fi is similar to site A in the murine MCP-1 gene 55 and is occupied Speci city protein 1 (Sp1) regulates transcription of several by an unknown protein before stimulation, and its function remains genes involved in inflammation and tumorigenesis, including unclear. IFN-γ, -γ; IL-1, interleukin-1; LPS, vascular endothelial growth factor (VEGF), urokinase plasmi- lipopolysaccharide; NF-κB, nuclear factor-κB; PMA, phorbol 12- nogen activator (uPA), uPA receptor and epithelial growth α α myristate 13-acetate; TNF- , tumor necrosis factor- . factor receptor.61,62 Sp1 also regulates basal transcription of the MCP-1 gene by binding to a GC-box located in the proximal region of the 5′-untranslated region (Figure 1).54 Therefore, we migrated in response to MCP-1 in vitro. In vivo,MCP-1 evaluated the role of Sp1 in constitutive, high-level MCP-1 induced in the chick chorioallantoic membrane mRNA expression in U-105MG cells. As we speculated, assay and rat aortic ring sprouting assay in a manner that was fl 46 treatment with mithramycin A, an inhibitor of Sp1, markedly independent of in ammatory cells. Thus, MCP-1 can directly MCP-1 contribute to angiogenesis via CCR2, but neither MCP-1 − / − reduced the level of mRNA expression in U-105MG nor CCR2− / − mice have an abnormal angiogenic phenotype.47 cells (Figure 2c), suggesting that Sp1 overexpression may be Certain types of tumor cells, such as prostate cancer 48 and another mechanism of the constitutive high-level MCP-1 ovarian cancer cells,49 have been shown to express CCR2. In production by U-105MG cells. prostate cancer cells, MCP-1 binds to CCR2 and directly Epithelial to mesenchymal transition (EMT) is a biological stimulates their proliferation and migration, as well as protects process that leads to acquisition of invasiveness and subsequent them from autophagic death, greatly contributing to prostate of cancer cells. Several molecular processes, includ- fi tumorigenesis.48 ing activation of transcription factors, expression of speci ccell surface molecules, expression and reorganization of cytoskeletal REGULATION OF MCP-1 PRODUCTION IN TUMOR , production of that degrade ECM proteins MICROENVIRONMENTS and changes in expression of particular microRNAs, are Regulation of constitutive MCP-1 production by tumor cells engaged to initiate and complete EMT.63 It was recently Nuclear factor of κB(NF-κB) is a transcription factor that demonstrated that transcription factors, such as Twist 1 or regulates the transcription of many genes involved in inflam- Snail, which induce EMT, have the capacity to induce MCP-1 matory and immune responses.50,51 NF-κB is constitutively production in epithelial cells,64,65 suggesting that tumor cells activated in most cancer cells,52 resulting in the expression of that have undergone EMT may acquire the ability to consti- anti-apoptotic genes and prolonged cancer cell survival. It also tutively produce MCP-1. The expression status of Twist 1 or regulates the genes involved in the proliferation, invasion and Snail in U-105MG cells is currently unknown. metastasis of cancer cells.52,53 Thus, activation of NF-κBis Another signaling molecule involved in the upregulation of closely associated with all phases of cancer progression. MCP-1 expression is Kras.66 Kras mutations occur in one-third The promoter region of the human MCP-1 gene contains of human cancers, including malignant glioma.67,68 Arecent multiple cis-elements for different transcription factors study demonstrated that MCP-1 production was upregulated (Figure 1). Two NF-κB sites located in the distal region are in tumor cells harboring activating Kras mutations.66 This may

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Figure 2 Role of NF-κB and Sp1 in the constitutive production of MCP-1 by human glioma cell lines. U-105MG or U-373MG human malignant glioma cells were incubated in six-well culture plates for 2 days in the presence or absence of an indicated inhibitor, and then, the concentration of MCP-1 in the culture supernatant or expression of MCP-1 mRNA by glioma cells was examined by -linked immunosorbent assay or Northern blotting, respectively. (a) Constitutive production of MCP-1 by U-105MG or U-373MG cells. (b)Effectof − 8 − 7 the NF-κB inhibitor CAPE (10 μM). ***Po0.001. (c) Effect of the Sp1 inhibitor mythramycin (10 or 10 M). MCP-1, monocyte chemoattractant protein-1; NF-κB, nuclear factor-κB; Sp1, specificity protein 1. provide, at least partly, a direct mechanism by which TAMs establishment of prostate cancer in the bone infiltrate tumors with Kras mutations. microenvironment.73 Prostate tumor cells release parathyroid hormone-related protein, which stimulates MCP-1 expression Regulation of MCP-1 production by the host–tumor cell by osteoblasts, and this osteoblast-derived MCP-1 causes interaction increased osteoclastic bone resorption and binds to its receptor In addition to constitutive MCP-1 production by tumor cells, on prostate tumor cells, stimulating proangiogenic factor MCP-1 production can be further upregulated in tumor cells VEGF-A release from tumor cells.74 Thus, MCP-1 derived by inflammatory mediators, such as IL-1, IL-6, TNF-α or from stromal cells and osteoblasts, in this case, plays a critical 69 transforming growth factor-β. In human ovarian cancer cells, role in cancer development. autocrine production of TNF-α by cancer cells was shown to The potential roles of MCP-1 in the development of breast stimulate a constitutive network of cytokines, angiogenic cancer have been extensively studied, and significant associa- factors and chemokines, including MCP-1.70 In a murine tions between MCP-1 production and the progression of breast chronic colitis-associated carcinogenesis model induced by cancer have been found.75–77 In a MDA-MB-231 human breast azoxymethane and dextran sulfate sodium, MCP-1 protein cancer transplantation model, MCP-1 was detected in macro- was detected by immunohistochemistry in mononuclear cells, phages, fibroblasts and endothelial cells, and MCP-1 staining particularly macrophages, infiltrating the lamina propria and was most evident in macrophages. In 128 human breast cancer submucosal regions and also in endothelial cells at the early tissues examined, MCP-1 was detected in both tumor cells and phase; and in carcinoma cells at a later phase.71 Production of stromal cells, mainly CD68-positive monocytic cells (likely MCP-1 in this model was almost completely inhibited when macrophages). Furthermore, macrophage infiltration was sig- hematopoietic cells could not produce TNF-α,72 strongly nificantly correlated with MCP-1 staining in stromal cells, but suggesting that tumors cells activated by macrophage-derived not in tumor cells.78 These results suggest that MCP-1 TNF-α are the primary source of MCP-1 in this chemically produced by stromal cells, but not by tumor cells, plays a induced colon cancer model. Thus, TNF-α, among the several critical role in the recruitment of macrophages into breast cytokines noted above, whether it is produced by stromal cells cancer tissues. or tumor cells, is a common molecule that upregulates production of MCP-1 by tumor cells in the tumor REGULATION OF MCP-1 PRODUCTION IN MURINE microenvironment. TRANSPLANTABLE TUMOR MODELS The host–tumor cell interaction also leads to MCP-1 Lewis lung carcinoma model production by stromal cells. Osteoclastogenesis and bone Lewis lung carcinoma (LLC or LLC1) is a non-small-cell lung resorption are two independent steps that lead to the develop- cancer (NSCLC) cell line adapted to culture from a lung ment of skeletal metastases and are mutually essential for the carcinoma that arose in a C57BL mouse.79 LLC cells are known

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to produce a significant amount of MCP-1 in vitro.80,81 As MCP-1 mRNA in LLC tumors that grew in TNF-α−/− mice was described above, it was recently demonstrated that LLC cells significantly reduced, and the serum MCP-1 concentrations −/− harbor the activating KrasG12C mutation, which upregulates were lower in tumor-bearing TNF-α mice. Furthermore, the −/− MCP-1 production, potentially linking Kras mutations and size and volume of tumors in TNF-α mice were significantly cancer-associated inflammation.66 MCP-1 production by these smaller than those in WT mice. These results indicate that cells can be further increased in response to activation by the TNF-α is a critical macrophage-produced mediator that TLR4 ligand LPS or TNF-α.80,82 Intrapleural injection of LLC elevates MCP-1 production by tumor cells in vivo.Cordero 85 cells expressing MCP-1-specific short hairpin (sh)RNA resulted et al. reported that eiger, the sole member of the TNF Drosophila in a reduced level of pleural fluid accumulation,82 and superfamily in expressed by tumor-associated Drosophila neutralization of MCP-1 by an antibody (Ab) reduced the hemocytes (leukocytes in ), was necessary and fi α growth of subcutaneously injected LLC cells.83 These in vivo suf cient to trigger TNF- signaling and express dMMP1 in studies demonstrated the critical role of MCP-1 in the tumor cells. The chemokine/ system does fl 86 progression of LLC and suggests its role in human NSCLC. not appear to be present in the y. We examined the mechanisms of MCP-1 production in LLC It was previously demonstrated that the extracellular matrix fl protein versican released by LLC cells induces TNF-α produc- tumors by injecting LLC cells into the ank of WT or 87 MCP-1−/− mice.84 The absence of MCP-1 in host stromal cells tion in myeloid cells through activation of TLR2. MyD88 is a −/− critical signaling molecule located downstream of TLR2, and (in MCP-1 mice) did not affect the level of MCP-1 detected we found that macrophages from MyD88−/− mice failed to in the tumors or sera of tumor-bearing mice, indicating that elevate MCP-1 mRNA expression by LLC cells in co-culture, tumor cells are the main MCP-1-producing cells. The high- leading to the hypothesis that macrophage production of TNF- level MCP-1 production detected in LLC tumors was not due α may be due to activation of TLR2. However, macrophages to the constitutive MCP-1 mRNA expression in tumor cells but −/− from TLR2 mice were as efficient as those from WT mice to the activation of tumor cells in the microenvironment and elevated MCP-1 expression by LLC cells. Macrophages because LLC cells isolated from tumors grown in either wild- from TLR4−/− or IL-1R1−/− mice or TLR9−/− mice also elevated MCP-1−/− type (WT) or mice produced a level of MCP-1 MCP-1 mRNA expression by LLC cells. Thus, LLC cells appear in vitro in vitro similar to that found in -cultured cells. to elevate MCP-1 production in macrophages by a mechanism We next examined whether the interaction between LLC independent of TLR2, TLR4, TLR9 or IL-1R1, involving cells and host cells, particularly macrophages, upregulates MyD88. Alternatively, LLC cells may activate macrophages MCP-1 production by LLC cells. Co-culture with WT mouse via more than one TLR. Taken together, our results indicate MCP-1 macrophages markedly increased the level of mRNA that the interaction of LLC cells with macrophages results in expression by LLC cells, whereas co-culture with macrophages the production of TNF-α by macrophages, and this TNF-α −/− −/− from TNF-α mice or MyD88 mice did not. Neutralization subsequently upregulates MCP-1 production by tumor cells in of TNF-α in the co-culture almost completely blocked the LLC tumors (Figure 3).84 Additional studies are required to effect of WT macrophages to elevate MCP-1 production by determine the exact mechanism of macrophage activation by LLC cells. Consistent with the in vitro results, expression of LLC cells.

4T1 mouse breast cancer model As noted above, a study of human breast cancer tissues suggestedanimportantroleforhostcell-derivedMCP-1in the infiltration of macrophages into tumors.78 4T1 breast cancer cells were established from a spontaneous mammary tumor of Balb/cC3H mice. After orthotopical injection into the mammary pads of Balb/c mice, 4T1 cells form tumors at the injected site and spontaneously metastasize to distant tissues, such as the lung, liver and bone, thus providing a clinically relevant model to characterize the mechanisms that are 88 Figure 3 Mechanisms involved in the production of MCP-1 in LLC important for tumor growth and metastasis. Furthermore, tumors. LLC cells harbor the activating KrasG12C mutation, which unlike LLC cells, 4T1 cells are polyclonal and contain mixed causes constitutive MCP-1 production.66 An unidentified product of populations, providing an experimental model of tumor LLC cells activates tumor-infiltrating macrophages to produce and heterogeneity.89 release TNF-α.ThisTNF-α activates LLC cells to further upregulate We therefore analyzed the biological role of MCP-1 in the MCP-1 production. The level of MCP-1 produced by LLC tumors is 4T1 breast cancer model (Figures 4).80 4T1 cells constitutively much higher than that in other tumors, including 4T1 and B16 in vitro fi tumors. We did not examine the biological significance of MCP-1 produced MCP-1 , and its production was signi cantly α production in this model. LLC, Lewis lung carcinoma; MCP-1, increased in response to activation with TNF- or LPS; monocyte chemoattractant protein-1; TNF-α, tumor necrosis however, these cells produced a markedly lower level of factor-α. MCP-1 than LLC cells. After intra-mammary injection of

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4T1 cells in WT or MCP-1−/− mice, the tumor size at the mice; (2) the serum MCP-1 levels were increased in tumor- injected sites was increased at a similar rate in both strains, and bearing WT mice, but not in MCP-1−/− mice; (3) the MCP-1 −/− there was no difference in their size or weight at 4 weeks. mRNA level in tumors of MCP-1 mice was comparable to However, the number of metastatic tumors in the of that expressed by 4T1 cells cultured in vitro; and (4) the MCP-1 MCP-1−/− mice was significant smaller than that in the lungs of mRNA level in tumors of WT mice was clearly higher than that WT mice, and MCP-1−/− mice survived significantly longer expressed by 4T1 cells activated in vitro with LPS or TNF-α. than WT mice. Host cells in the tumor stroma were the Although MCP-1 from either hematopoietic or non- primary source of MCP-1 in this model for the following hematopoietic cells in the tumor stroma promoted lung reasons: (1) MCP-1 mRNA was easily detected in tumors of metastasis of 4T1 cells, MCP-1 from hematopoietic cells, such WT mice, but was hardly detectable in tumors of MCP-1−/− as macrophages, was sufficient. MCP-1 promotes lung metas- tasis of breast cancer cells by recruiting pro-angiogenic monocytes.90 We recently found that the cell-free culture supernatants of 4T1 cells (4T1-sup) directly activate mouse inflammatory macrophages for MCP-1 production (Figure 4).91 4T1-sup also elevates the expression of other MCP genes, such as the MCP-3/CCL7 and MCP-5/CCL12 genes, in macrophages. However, unlike LPS, which highly upregulates the expression of -attracting chemokine genes, such as the KC/CXCL1 or MIP-2/CXCL2, 4T1-sup only modestly upregu- lates the expression of these chemokine genes. A 4T1 cell product with a size of ~ 20–30 kDa was responsible for the upregulation of MCP-1 mRNA expression by macrophages. Figure 4 Role and mechanism of MCP-1 production in 4T1 breast α cancer. Various cell types, including hematopoietic cells, fibroblasts Although neutralization of TNF- in the culture did not affect and endothelial cells, are present in the primary tumor. Compared MCP-1 production by macrophages, neutralization of GM-CSF with LLC cells, the capacity of 4T1 cells to produce MCP-1 is almost completely abolished the MCP-1 mRNA expression and significantly lower. However, 4T1 cells can activate stromal cells to MCP-1 production induced by 4T1-sup. Furthermore, activa- produce a sufficient amount of MCP-1 for lung metastasis. For tion with recombinant GM-CSF markedly increased MCP-1 example, they produce and release GM-CSF, which upregulates the production by macrophages at a concentration as low as 1 ng/ production of MCP-1 and other cytokines/chemokines by inflammatory macrophages. Activated CAFs and endothelial cells ml. By contrast, neutralization of M-CSF had no effect, and could be additional sources of MCP-1. MCP-1 production by recombinant M-CSF exhibited only modest activity to upre- stromal cells results in further recruitment of macrophages and gulate MCP-1 expression at 100 ng/ml. Expression of GM-CSF angiogenesis and promotes lung metastasis. Tumor cell production mRNA was detected in 4T1 cells in vitro and 4T1 tumors of MCP-1 appears to promote the survival and seeding of tumor in vivo. These results indicate that 4T1 cells can directly cells, potentially by recruiting metastasis-associated increase MCP-1 production by macrophages by producing 80,90 fi macrophages. CAF, cancer-associated broblast; DC, dendritic and releasing GM-CSF (Figure 2). An important role of GM- cells; EC, endothelial cells; GM-CSF, granulocyte-macrophage colony-stimulating factor; LLC, Lewis lung carcinoma; MCP-1, CSF in the progression of human breast cancer was also 92 monocyte chemoattractant protein-1; MDSC, myeloid-derived reported by others. However, in our study, neutralization of suppressor cell; Treg, regulatory T cells; TAM, tumor-associated GM-CSF did not reduce the level of MCP-1 production or lung macrophages. metastasis in tumor-bearing WT mice, indicating that GM-

Figure 5 Expression of MCP-1 mRNA in mouse breast cancer models. (a)ExpressionofMCP-1 mRNA in mammary and prostate tumors that arose in transgenic mice carrying a rat C3(1)-simian virus 40 large tumor antigen fusion gene was examined by Northern blotting. Ten micrograms of total RNA isolated from Normal mammary grand tissues, mammary tumor tissues, mammary tumor cell lines (M6, 6c, 8 and 8c), normal prostate tissue and prostate tumor cell lines (Pr-12 and 14) was used. (b) Expression of MCP-1 mRNA by 4T1 cells and 4T1-derived clones was examined by quantitative reverse transcription-PCR.

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CSF-independent mechanisms are also involved in increased CONCLUDING REMARKS MCP-1 production in the 4T1 tumor microenvironment.91 Tumors are wounds that do not heal;97 thus, there is no secret As described above, the mouse ortholog of the human to the production of the chemokine MCP-1 in the tumor MCP-1 gene was cloned from platelet-derived growth factor- microenvironment. Because MCP-1 production in tumors activated fibroblasts as the gene JE,20 and human fibroblasts facilitates the accumulation of immune-suppressive, tumor- produce MCP-1.18,93 Therefore, fibroblasts are another likely promoting TAMs, it has become a molecular target for cancer source of MCP-1 in the 4T1 tumor microenvironment. It was treatment.98 The effects of the blockade of the MCP-1/CCR2 previously shown that the products of breast cancer cells pathway on the progression of breast cancer or prostate cancer increased the expression of MCP-1 by human primary have been tested in clinical trials using anti-MCP-1 antibodies cancer-associated fibroblasts (CAF) in a STAT3-dependent or CCR2 antagonists alone or in combination with other manner.94 The nature of the cancer cell product that activated agents, and positive responses have been reported.99,100 On the CAF remains unidentified. other hand, it was recently reported that interruption of anti- In mammary tumors arising in PyMT mice in which the MCP-1 Ab treatment markedly increased lung metastasis of PyMT oncogene is expressed under the control of the MMTV- breast cancer cells and subsequent death in mouse syngeneic LTR promoter, primary tumors were heterogeneously stained cancer models. Further analysis of this model led to the finding for MCP-1, whereas metastatic tumors in the lung were that interruption of Ab treatment resulted in monocyte release homogeneously stained by immunohistochemistry. In a tumor from the , an increase in cancer cell mobilization transplantation model in which human MDA-MB-231 breast from the primary tumor, formation and prolifera- cancer cells were orthotopically transplanted into SCID mice, tion of metastatic cells in the lung in a manner dependent on total blockade of MCP-1 using anti-human and anti-mouse IL-6 and VEGF. Inhibition of MCP-1 along with IL-6 MCP-1 antibodies inhibited spontaneous lung metastasis. prevented the promotion of lung metastases after interruption When MDA-MB-231-derived, metastatic 4173 cells were of anti-MCP-1 Ab therapy.101 Thus, blockade of the MCP-1/ intravenously injected, either an anti-human or anti-mouse CCR2 pathway needs to be carried out with caution, and MCP-1 Ab inhibited lung metastasis.90 These results suggest additional information is clearly needed for an effective cancer that the primary mammary tumors consist of cells with therapy using MCP-1 or CCR2 antagonists. different degrees of MCP-1-producing capacity and that Over the past several years, we attempted to determine the MCP-1-producing tumor cells may preferentially metastasize mechanisms regulating the production of MCP-1in the tumor to the lung and MCP-1 produced by both tumor cells and microenvironment using transplantable mouse tumor models. stromal cells is important to promote lung metastasis. In fact Our results, along with those by others, indicate that the we found that tumor cell lines established from mammary production of MCP-1 is a consequence of the interaction 95 tumors that arose in C3(1)/SV40 T-antigen transgenic mice between host cells and tumor cells and that both tumor cells MCP-1 expressed different levels of mRNA (Figure 5a). and host stromal cells can contribute to the production of this Similarly, clones isolated from the original 4T1 cells showed chemokine. TNF-α, a well-known tumor promoter, is a MCP-1 heterogeneous mRNA expression (Figure 5b). frequently found mediator that upregulates production of Regarding the role of tumor cell-derived MCP-1, treatment MCP-1 by tumor cells, whereas tumor cell-derived mediators, of Met-1 cells (a cell line generated from a PyMT-induced such as GM-CSF, act on host cells, such as macrophages, to mammary tumor) with an anti-MCP-1 Ab shortly before i.v. induce production of MCP-1 and other cytokines/chemokines. injection reduced the number of lung metastases via inhibition The serum MCP-1 level is increased in some cancer patients 90 of metastasis-associated macrophage recruitment. In our and may correlate with the progression of the disease, but its study, 4T1 cells isolated from lung metastases of WT mice meaning remains unclear. It would be helpful if we could expressed various levels of MCP-1, whereas cells isolated from determine from the serum MCP-1 level the nature of the host– lung metastases of MCP-1−/− mice tended to express high levels 80 tumor cell interaction that occurs inside a tumor. The of MCP-1, supporting the hypothesis that a population of information that we have obtained to date is still limited, and tumor cells expressing higher levels of MCP-1 has a better additional studies are obviously needed. Our goal is to identify chance to metastasize to the lung. It is important to note that the mechanisms of the tumor cell–stromal cell interaction to lung or bone metastasis of MDA-MB-231 cells was inhibited by better understand the tools tumor cells use for their progres- the blockade of MCP-1, but liver metastasis of Met-1 cells was sion and to provide a new means to save the lives of patients not,90 indicating that MCP-1-independent mechanisms are with cancer. involved in the metastasis of mammary tumor cells to the liver. Similar to 4T1 tumors, MCP-1 mRNA expression was CONFLICT OF INTEREST detectable in mouse B16 melanoma growing in WT mice, Theauthordeclaresnoconflict of interest. but was almost undetectable in tumors in MCP-1−/− mice, indicating that stromal cells are the major MCP-1-producing ACKNOWLEDGEMENTS 84 cells in B16 tumors. A similar observation was made by I am grateful to Drs Hideo Hayashi, Edward J Leonard, Kouji others.96 However, the host-tumor cell interaction regulating Matsushima and Joost J Oppenheim for their invaluable input during MCP-1 production in B16 melanoma remains uncharacterized. my studies on inflammation. I am also grateful to Drs Naoya Yuhki,

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