CCL2 in tumor microenvironments

Cytokine xxx (2017) xxx–xxx

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Review article

The production of monocyte chemoattractant protein-1 (MCP-1)/CCL2 in tumor microenvironments Teizo Yoshimura ⇑ Department of Pathology and Experimental Medicine, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Japan

a r t i c l e

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Article history: Received 6 December 2016 Accepted 1 February 2017 Available online xxxx Keywords: Chemokine MCP-1 CCL2 Tumor microenvironment

a b s t r a c t Infiltration of leukocytes is one of the hallmarks of the inflammatory response. Among the leukocyte populations, neutrophils are the first to infiltrate, followed by monocytes and lymphocytes, suggesting the presence of mediators that specifically recruit these cell types. Cytokine-like chemoattractants with monocyte chemotactic activity, such as lymphocyte-derived chemotactic factor (LDCF) or tumorderived chemotactic factor (TDCF), were reported as molecules that could play a critical role in the recruitment of monocytes into sites of immune responses or tumors; however, their identities remained unclear. In the 1980s, researchers began to test the hypothesis that leukocyte chemotactic activity is a part of the wider activities exhibited by cytokines, such as interleukin-1 (IL-1). In 1987, we demonstrated, for the first time, the presence of a cytokine like chemoattractant with cell type-specificity (now known as the chemokine interleukin-8 or CXC chemokine ligand 8) that was different from IL-1. This led us to the purification of the second such molecule with monocyte chemotactic activity. This monocyte chemoattractant was found identical to the previously described LDCF or TDCF, and termed monocyte chemoattractant protein-1 (MCP-1). Isolation of MCP-1 created a revolution in not only inflammation but also cancer research that continues today, and MCP-1 has become a molecular target to treat patients with many diseases. In this review, I will first describe a history associated with the discovery of MCP-1 and then discuss complex mechanisms regulating MCP-1 production in tumor microenvironments. Ó 2017 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Roads leading to the purification of MCP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . MCP-1 production in tumor microenvironments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Constitutive MCP-1 production by tumor cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Non-tumor stromal cells as the primary source of MCP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Activated tumor cells as the major source of MCP-1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction ⇑ Address: 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 address: [email protected]

One of the mechanisms that lead to the infiltration of leukocytes into sites of inflammatory responses or cancer is the production of chemotactic molecules that diffuse out from the site of

http://dx.doi.org/10.1016/j.cyto.2017.02.001 1043-4666/Ó 2017 Elsevier Ltd. All rights reserved.

Please cite this article in press as: T. Yoshimura, The production of monocyte chemoattractant protein-1 (MCP-1)/CCL2 in tumor microenvironments, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.02.001

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T. Yoshimura / Cytokine xxx (2017) xxx–xxx

release and form concentration gradient to which leukocytes respond and migrate. In 1987, we purified the first cytokine-like chemoattractant, monocyte-derived neutrophil chemotactic factor (MDNCF, also known as the chemokine interleukin-8 or CXCL8) [1]. In 1989, we and others reported the purification of the second chemokine monocyte chemoattractant protein-1 (MCP-1)/ macrophages chemotactic and activating factor (MCAF)/monocyte chemotactic protein (MCP) [2–5]. This chemoattractant is now widely known as MCP-1 or CC chemokine ligand 2 (CCL2) [6]. The identification of MCP-1 and its receptor CCR2 [7] greatly contributed to the studies to examine the mechanisms of monocyte trafficking and the role of monocytes/macrophages during inflammatory responses or cancer development. Several reviews concerning the role of MCP-1 in the pathogenesis of many inflammatory diseases and cancer are already available elsewhere [8–10]. In this review, I will first introduce earlier studies that led us to the purification of MCP-1 and then discuss the results of our recent studies analyzing the complex mechanisms by which MCP-1 production is up-regulated in tumor microenvironments.

2. Roads leading to the purification of MCP-1 Macrophages play important roles in host defense by presenting Ag to lymphocytes or by participating in efferent limb immune responses as effector cells or secreting cytokines. Macrophages infiltrating sites of inflammation are derived from blood monocytes, which are attracted by chemotactic factors produced at inflammatory sites. We were interested in monocyte chemoattractants that accounts for the predominant infiltration by monocytes in most delayed hypersensitivity reactions [11–13]. It was of historical interest that a focus on the cellular motility and accumulation of nonsensitized inflammatory cells in DTH led to the first description of a lymphokine - migration inhibitory factor (MIF) [14,15]. This was followed by a series of reports in the early 1970s that chemotactic activity for macrophages or monocytes (lymphocyte-derived chemotactic factor; LDCF) was elaborated by antigen-stimulated sensitized lymphocytes or by mitogenstimulated non-sensitized lymphocytes [16]. However, neither MIF nor LDCF had been purified to homogeneity. The infiltration of leukocytes into cancer tissues could also be a result of a host immune reaction against tumor-specific antigen. Many laboratories previously explored in vivo and in vitro aspects of this hypothesis. It was shown by experiments with transplantable tumors in inbred guinea pigs that at dermal sites of delayed hypersensitivity reactions to one tumor cell line, antigenically unrelated tumor cells were also destroyed [17]. This suggested that the response to the antigenically unrelated tumor cells was immunologically nonspecific, and was mediated by activated macrophages infiltrating the site. It was shown that macrophages were capable of destroying tumor cells in vitro, provided that they were activated [18,19]. The activated macrophages were therefore assigned a critical role in host destruction of tumors. On the other hand, there was evidence suggesting that tumorassociated macrophages might stimulate tumor growth or connective tissue development [20–23]. Thus, neither the role of tumorassociated macrophages (TAMs) nor the mechanism of TAM infiltration was clarified [23,24]. As a possible mechanism of macrophage infiltration into tumors, Meltzer et al. reported the presence of a macrophage chemotactic factor in the culture supernatants of five murine sarcoma cell lines whose molecular weight was different from that derived from activated murine lymphocytes [25]. After several quiet years, Bottazzi et al. reported the production of a different degree of monocyte/macrophage chemotactic activity by human

and murine tumor cell lines. They also found a significant correlation between the amount of monocyte/macrophage chemotactic activity and tumor macrophage content. These findings strongly suggested that tumor-derived chemotactic factor (TDCF) plays a critical role in the recruitment of TAMs. Bottazzi et al. also detected monocyte chemotactic activity in the culture supernatants of human and murine embryo fibroblasts [22]; however, it was not clarified whether tumor cells and fibroblasts produced a same chemotactic molecule. In 1987, we purified the first chemokine IL-8, based on its neutrophil chemotactic activity, from the culture supernatant of lipopolysaccharide (LPS)-activated human peripheral blood cells (PBMC) [1,26]. During the process, we also noted the presence of monocyte chemoattractant in the same supernatant. Since a similar monocyte chemoattractant was also present in the culture supernatant of mitogen-activated human PBMC, we speculated that the monocyte attractant found in the culture supernatant of LPS-activated PBMC might be identical to the previously described LDCF. We attempted to purify the molecule to homogeneity from culture supernatants of activated PBMC by using column chromatography; however, we were not successful due to the limited availability of PBMC culture supernatants. Our problem was soon solved when we found that human malignant glioma cell lines produced a low to high level of monocyte chemotactic activity that could not be distinguished from that produced by activated PBMC. Among the cell lines, U-105MG cells released the highest amount of monocyte chemotactic activity [27]. In 1988, we purified the monocyte chemoattractant from the culture supernatants of both U-105MG cells and mitogenactivated PBMC. When the N-terminal amino acid sequence of the protein was analyzed by Edman degradation, we found that the N-terminus was blocked. By a combination of Edman gradation and mass spectrometry and cDNA cloning, it was established that MCP-1 comprises 76 amino acid residues, beginning at the Nterminus with pyroglutamic acid. In 1989, we reported all these results in three papers [2,3,28,29], and replaced the names LDCF and GDCF with molecule monocyte chemoattractant protein-1 (MCP-1), anticipating later discovery of MCP-2, 3, and so on. Matsushima et al. simultaneously reported the purification of the identical molecule (macrophage activating and chemotactic factor; MCAF) using the culture supernatant of activated THP-1 human monocytic leukemia cells [4]. Van Damme et al. also purified MCP-1 from the culture supernatants of double stranded RNAactivated MG-63 osteosarcoma cell line and LPS-activated human monocytes [5], and later structurally related chemoattractants, MCP-2 and 3 [30]. When the amino acid sequence of human MCP-1 was compared with those in a database, we noted that it had a significant amino acid sequence similarity to the protein coded by the mouse JE gene that was inducible in fibroblasts by platelet-derived growth factor [31]. Subsequently, MCP-1 was found identical to the product of the human orthologue of the mouse JE gene [32]. Finally, Bottazzi et al. demonstrated the production of MCP-1 by tumor cell lines which they used for the detection of TDCF, and concluded that MCP-1 was identical to TDCF [33]. Thus, MCP-1 is produced by either non-tumor cells or tumor cells, and it contributes to the development of not only inflammatory diseases but also cancer by promoting the recruitment of monocytes.

3. MCP-1 production in tumor microenvironments Tumor tissues contain a variety of non-tumor stromal cells, including fibroblasts, endothelial cells, myocytes and inflammatory cells, such as myeloid-derived suppressor cells, regulatory T cells, macrophages and dendritic cells. The interaction of tumor cells

Please cite this article in press as: T. Yoshimura, The production of monocyte chemoattractant protein-1 (MCP-1)/CCL2 in tumor microenvironments, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.02.001

T. Yoshimura / Cytokine xxx (2017) xxx–xxx

with stromal cells leads to the production of an array of mediators that provide the soil in which tumor cells grow, invade and metastasize. These mediators include matrix metalloproteinases, growth factors, cytokines and chemokines, such as MCP-1 [34–36]. Since a number of tumor cell lines constitutively produce MCP1 in vitro, tumor cells were originally thought to be the primary source of MCP-1 in established tumors. However, recent studies strongly suggested that stromal cells were the primary cell source of MCP-1 in some tumors [37,38]. There are three potential mechanisms by which MCP-1 production is increased in tumors; (1) tumor cells constitutively produce MCP-1, (2) tumor cells produce MCP-1 in response to stimuli, and (3) stromal cells produce MCP-1 in response to stimuli, such as a tumor cell product(s). 3.1. Constitutive MCP-1 production by tumor cells Nuclear factor of jB (NF-jB) is a transcription factor regulating the transcription of many genes involved in the inflammatory and immune responses [39,40]. NF-jB is constitutively activated in most cancer cells [41], resulting in the expression of antiapoptotic genes and prolonged cancer cells survival. It also regulates the genes involved in the proliferation, invasion and metastasis of cancer cells [41,42]. Thus, activation of NF-jB is closely associated with all phases of cancer progression. The transcription of the MCP-1 gene is shown to increase in response to stimuli, such as TNF or LPS, by the binding of NF-jB dimers to two distal NF-jB binding sites [43–45]. This led to a hypothesis that constitutive activation of NF-jB in tumor cells may play a role in their high level MCP-1 production. We previously demonstrated constitutive production of MCP-1 by tumor cells, such as malignant glioma [27] or malignant fibrous histiocytoma [46] in vitro. Among them, as described above, the human malignant glioma cell line U-105MG produced the highest level of MCP-1 and U-373MG cells produced a low level [27]. We examined the mechanisms of MCP-1 production by the two cell lines. Both cell line cells released MCP-1 without any additional stimuli; however, U-105MG released approximately 30-fold higher levels than U-373MG by ELISA. The constitutive MCP-1 release was not due to an autocrine effect by TNF because anti-TNF neutralizing antibody had no effect. Activation with recombinant TNF resulted in a further increase in MCP-1 release by both cell line cells, indicating that they also have the capacity to respond to proinflammatory stimuli and produce higher levels of MCP-1. To evaluate the role of NF-jB in the constitutive MCP-1 production by U-105MG cells, we pharmacologically inhibited NF-jB. A NF-jB inhibitor, caffeic acid phenethyl ester, markedly inhibited MCP-1 production. We, therefore, compared NF-kB activities in these cell lines by a luciferase reporter assay using the constructs containing the NF-jB binding sequence of the human IgG j-chain (pNF-jB) or the 3.8 kb human MCP-1 gene promoter sequence containing two distal NF-jB sites known to regulate the MCP-1 gene transcription (pGL-3.8k) [44]. High levels of luciferase activities were detected in both cell lines transfected with pNF-jB; however, high levels of luciferase activities were detected only in U-105MG cells when they were transfected with pGL-3.8k. The important role of the NF-jB sites in pGL-3.8k was confirmed using an additional construct in which both NF-jB sequences in the MCP-1 promoter were mutated. These results indicate that the constitutive, high level MCP-1 production by U-105MG cells is regulated by constitutively active NF-jB but this is just one of the mechanisms whereby they produce MCP-1 at a high level (Yoshimura and Yang, unpublished). Specificity protein 1 (Sp1) regulates the transcription of several genes involved in inflammation and tumorigenesis, including vascular endothelial growth factor (VEGF), urokinase plasminogen activator (uPA), uPA receptor and epithelial growth factor receptor

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[47,48]. Sp1 also regulates the basal level transcription of the MCP1 gene by binding to a GC-box located in the proximal region of the 50 -UTR [43]. To evaluate the role of Sp1 in MCP-1 expression in U105MG and U-373MG cells, we treated them with mithramycin A that inhibits Sp1 activity by binding to GC-rich sequences. Interestingly, mithramycin A markedly reduced the level of MCP-1 mRNA expression by both cell lines. In contrast, the expression of IL-8 mRNA of which expression is independent of Sp1 was not affected. Furthermore, the expression of Sp1 is highly elevated in U-105MG. These results suggest that overexpression of Sp1 may be another mechanism of the high level, constitutive MCP-1 expression by U-105MG cells (Yoshimura and Yang, unpublished). Epithelial mesenchymal transition (EMT) is a biological process leading to the acquisition of invasiveness and subsequent metastasis of cancer cells. A number of molecular processes, including activation of transcription factors, expression of specific cell-surface proteins, reorganization and expression of cytoskeletal proteins, production of ECM-degrading enzymes and changes in the expression of specific microRNAs, are engaged in order to initiate EMT and enable it to reach completion [49]. It was recently demonstrated that transcription factors, such as Twist 1 or Snail that induce EMT, have the capacity to induce MCP-1 in epithelial cells [50,51], suggesting that tumor cells that have undergone EMT may acquire the ability to constitutively produce MCP-1. The expression of Twist 1 or Snail in U-105MG cells is unknown. Additional studies are required to determine the exact mechanisms of constitutive MCP-1 production by tumor cells. 3.2. Non-tumor stromal cells as the primary source of MCP-1 Osteoclastogenesis and bone resorption are independent steps leading to the development of skeletal metastases and are mutually essential for prostate cancer establishment in the bone microenvironment [52]. In addition to its chemotactic activity, MCP-1 is reported to directly support prostate cancer cell growth [53]. Li et al. investigated the mechanistic role of MCP-1 in prostate cancer growth in the bone and found that prostate tumor cells release parathyroid hormone-related protein, which stimulates MCP-1 expression by osteoblasts. This osteoblast-derived MCP-1 causes increased osteoclastic bone resorption and also binds to its receptor on prostate tumor cells and stimulates the proangiogenic factor VEGF-A release from tumor cells [37]. Thus, MCP-1 derived from stromal cells, osteoblasts in this case, plays a critical role in cancer development. Fujimoto et al. transplanted the human MDA-MB-231 breast cancer cells into SCID mice and examined the source of MCP-1 in tumors by immunohistochemistry. MCP-1 was detected in macrophages, fibroblasts and endothelial cells, among which MCP-1 staining was more evident in macrophages. They also examined 128 human breast cancer tissues and detected MCP-1 in both tumor cells and stromal cells, mainly CD68-positive monocytic cells (likely macrophages). However, macrophage infiltration significantly correlated with MCP-1 staining only in stromal cells, but not in tumor cells [38]. These results suggest that the production of MCP-1 by stromal cells, but not tumors cells, plays a significant role for the recruitment of macrophages in breast cancer tissues. The 4T1 breast cancer cells were isolated from a spontaneous mammary tumor of a Balb/cC3H mouse. When the cells are orthotopically injected into mammary pads of Balb/c mice, they form tumors and metastasize spontaneously to tissues, such as lung, liver and bone, providing an excellent model to elucidate the mechanisms involved in tumor growth and metastasis [54]. Using this model, we examined the relative contribution of stromal cells to the production of MCP-1 and subsequent tumor progression [55].

Please cite this article in press as: T. Yoshimura, The production of monocyte chemoattractant protein-1 (MCP-1)/CCL2 in tumor microenvironments, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.02.001

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4T1 cells constitutively expressed a low level of MCP-1 mRNA and protein in vitro. The production of MCP-1 was significantly increased in response to TNF or LPS. However, the level of MCP-1 produced by 4T1 cells was markedly lower than that by Lewis lung carcinoma (LLC) cells previously shown to produce a high level of MCP-1 [56]. Thus, 4T1 cells had the capacity to produce MCP-1, but at a low level. After intra-mammary injection of 4T1 cells, tumor size increased at a similar rate in both WT and MCP-1/ mice at the injected sites and there was no difference in their size or weight at 4 weeks. Interestingly, however, the number of metastatic tumor nodules in the lung of MCP-1/ mice was significant lower compared to that of WT mice, and MCP-1/ mice survived significantly longer than WT mice. MCP-1 mRNA was readily detectable in tumors of WT mice at 2, 3 and 4 weeks after tumor cell injection, but it was hardly detectable in tumors of MCP-1/ mice. Serum MCP-1 levels were increased in tumor-bearing WT, but not MCP1/ mice. The level of MCP-1 mRNA detected in tumors of WT mice was clearly higher than that of 4T1 cells stimulated in vitro with TNF or LPS. The level of MCP-1 mRNA expressed in tumors of MCP-1/ mice was comparable to that of in vitro stimulated 4T1 cells. These results indicated that stromal cells were the primary source of MCP-1 in this tumor model and MCP-1 regulates not only carcinogenesis, as previously reported [57], but also metastatic spread. Furthermore, MCP-1 produced by both hematopoietic and non-hematopoietic cells promoted the lung metastasis of 4T1 cells, and MCP-1 produced by hematopoietic cells, likely macrophages, was sufficient. Qian et al. examined the cellular source of MCP-1 in mammary tumors by using PyMT mice in which PyMT oncogene is expressed under the control of MMTV-LTR promoter [58]. By immunohistochemistry, primary tumors were heterogeneously stained for MCP-1, whereas metastatic tumors in the lung were homogeneously stained. In a tumor transplantation model in which the human MDA-MB-231 breast cancer cells were transplanted in SCID mice, MCP-1 produced by both tumor cells and stromal cells was important for the lung metastasis of tumor cells because either anti-human MCP-1 or anti-mouse MCP-1 antibody inhibited the lung metastasis of MDA-MB-231 cells. We previously examined

the expression of MCP-1 mRNA in three cell lines established from mammary tumors that arose in C3(1)/SV40 T-antigen transgenic mice [59]. MCP-1 expression was undetectable in one cell line, whereas a low to moderate level of MCP-1 was detected in other two lines by Northern blotting (Yoshimura and Green, unpublished), strongly suggesting heterogeneous MCP-1 expression also in mammary tumors of C3(1)/SV40 T-antigen transgenic mice. As for the survival and metastatic seeding of circulating tumor cells, tumor cell production of MCP-1 appears to play a critical role (Fig. 1). Qian et al. demonstrated that treatment of Met-1 cells (a cell line generated from a PyMT-induced mammary tumor) with anti MCP-1 antibody shortly before i.v. injection reduced the number of lung metastases, via the inhibition of metastasis-associated macrophage recruitment [58]. In our study, 4T1 cells isolated from lung metastases of MCP-1/ mice expressed higher levels of MCP1 than originally transplanted cells [55]. It remains unclarified whether injected 4T1 cells contained a population of cells with the ability to express a higher level of MCP-1 or 4T1 cells acquired the ability to express higher level of MCP-1 after transplantation. It is interesting to note that lung or bone metastasis of MDA-MB-231 cells was inhibited by the blockade of MCP-1, but liver metastasis of Met-1 cells was not [58], indicating that MCP-1-independent mechanisms are involved in the metastasis of mammary tumor cells to the liver. We recently analyzed the potential mechanisms by which MCP1 production is upregulated in macrophages infiltrating 4T1 tumors [60]. Cell-free culture supernatants of 4T1 cells (4T1-sup) markedly upregulated MCP-1 production by mouse peritoneal inflammatory macrophages. 4T1-sup also upregulated other MCPs, such as MCP-3/CCL7 and MCP-5/CCL12, but modestly neutrophil chemotactic chemokines, such as KC/CXCL1 or MIP-2/CXCL2. A 4T1 cell product with an approximately 2–3 kDa molecular mass was responsible for the upregulated MCP-1 expression by macrophages. A neutralizing antibody against granulocyte-macrophagecolony stimulating factor (GM-CSF), almost completely abrogated MCP-1-inducing activity of 4T1-sup, and recombinant GM-CSF markedly up-regulated MCP-1 production by macrophages at as low as 1 ng/ml. In contrast, a neutralizing antibody against macrophage-colony stimulating factor (M-CSF) had no effect and

Fig. 1. The role of MCP-1 derived from stromal cells or tumor cells in the lung metastasis of breast cancer cells. A variety of cell types, including hematopoietic cells, fibroblasts and endothelial cells, are present in primary tumors. The interaction of tumor cells with stromal cells results in the production of MCP-1 by stromal cells. For example, tumors cells produce and release GM-CSF, which up-regulates MCP-1 production by tumor-infiltrating macrophages. Cancer-associated fibroblasts are also likely source of MCP-1. This MCP-1 production leads to the recruitment of additional macrophages and angiogenesis. Tumor cells invade blood vessel and reach the lung. Tumor production of MCP-1 appears to promote the survival and seeding of tumor cells potentially by recruiting metastasis-associated macrophages.

Please cite this article in press as: T. Yoshimura, The production of monocyte chemoattractant protein-1 (MCP-1)/CCL2 in tumor microenvironments, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.02.001

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Fig. 2. Different mechanisms are involved in the production of MCP-1 in a 4T1 and LLC tumor microenvironment. A. In a 4T1 tumor model, 4T1 cells produce and release GMCSF that activates macrophages to produce MCP-1 (shown by green dots). However, this mechanism accounts for a small part of MCP-1 production in this tumor. Cancerassociated fibroblasts could be an important source of MCP-1. B. In a LLC tumor model, a product of LLC cells (undetermined) activates tumor-infiltrating macrophages to produce and release TNF. This TNF then activates LLC cells to produce MCP-1. The level of MCP-1 produced in LLC tumors is much higher than that in 4T1 tumors. However, the low level MCP-1 production in 4T1 tumors is sufficient to promote their metastasis to the lung.

recombinant M-CSF only modestly upregulated MCP-1 expression at 100 ng/ml. The expression of GM-CSF was detected in 4T1 tumors in vivo. These results indicate that 4T1 cells can directly up-regulate MCP-1 production by macrophages by releasing GMCSF (Fig. 2A). An important role of GM-CSF in the progression of human breast cancer was also reported by others [61]. However, treatment of mice with anti-GM-CSF antibody did not reduce MCP-1 production or lung metastasis in tumor-bearing mice in our study, indicating that other mechanisms are also involved in increased MCP-1 levels in the 4T1 tumor microenvironment. As described above, mouse orthologue of human MCP-1 was cloned from PDGF-activated fibroblasts [31] and human fibroblasts produce MCP-1 [32,62]. Therefore, fibroblasts are another likely source of MCP-1 in the 4T1 tumor microenvironment. Tsuyada et al. reported that products of breast cancer cells increased the expression of MCP-1 by human primary cancer-associated fibroblasts (CAF) in a STAT3-dependent manner [63]. The nature of the cancer cell product that activates CAF remains unidentified. In addition to 4T1 cells, we also examined the source of MCP-1 in B16 melanoma. Similar to 4T1 tumors, MCP-1 mRNA expression was detectable in B16 tumors of WT mice but it was almost undetectable in tumors of MCP-1/ mice, indicating that stromal cells are the major MCP-1 producing cells also in B16 tumors [64]. Redon et al. also injected B16 melanoma cells into MCP-1/ mice to examine whether tumors cause changes in distant tissues [65]. Consistent with our result, serum MCP-1 level was elevated in tumor-bearing WT mice, but no measurable amount of MCP-1 was detected in sera of tumor-bearing MCP-1/ mice. Interestingly, two serious types of DNA damage, double-strand breaks (DSBs) measured by c-H2AX focus formation and oxidatively induced non-DSB clustered DNA lesions (OCDLs), were elevated in tissues distant from the tumor site in tumor-bearing WT mice, but strikingly, MCP-1/ mice lacked increased levels of DSBs and OCDLs in tissues distant from implanted tumors. Thus, MCP-1 produced by non-tumor cells plays a critical role in B16 tumorinduced changes in distant tissues. The mechanisms involved in increased MCP-1 production in B16 tumors are uncharacterized. 3.3. Activated tumor cells as the major source of MCP-1 The production of MCP-1 can be up-regulated in a wide variety of tumor cell line cells in response to stimuli, such as IL-1, IL-6, TNF or TGF-b [66]. In human ovarian cancer cells, autocrine production of TNF by cancer cells stimulated a constitutive network of cytokines, angiogenic factors and chemokines, including MCP-1 [67]. To

extend our knowledge about the interaction between tumor cells and non-tumor stromal cells in tumor microenvironments, we subcutaneously injected LLC cells into the flank of WT or MCP-1/ mice, and examined the effects of MCP-1-deficiency. It was previously demonstrated that the blockade of MCP-1 significantly slowed the growth of primary tumors in mouse non-small cell lung carcinoma models, including a LLC model [68]; however, the lack of MCP-1 in stromal cells did not interfere with local tumor growth in our study, indicating that stromal cell-derived MCP-1 does not play a critical role in tumor growth in this tumor model. We next compared the level of MCP-1 expressed by LLC tumors with that expressed by in vitro cultured LLC cells. Much higher levels of MCP-1 mRNA were detected in all tumors that grew in both WT and MCP-1/ mice. Serum MCP-1 levels were also elevated in tumor-bearing WT and MCP-1/ mice. Similar to subcutaneous tumors, lung tumors formed in both WT and MCP-1/ mice by intravenous injection of LLC cells expressed high levels of MCP-1 mRNA. These results indicated that tumor cells were the primary source of MCP-1 in LLC tumors. LLC tumors expressed markedly higher levels of MCP-1 compared to 4T1 or B16 tumors as described above, with approximately 10-fold higher MCP-1 mRNA in LLC tumors than in 4T1 tumors. There are two possibilities which could explain increased MCP1 expression in LLC cells in tumors; one is that tumor cells constitutively express high levels of MCP-1, and the other is that tumor cells express high levels of MCP-1 in response to stimuli present in a tumor microenvironment. We, therefore, isolated LLC cells from tumors grown in either WT or MCP-1/ mice, cultured in vitro, and examined the level of MCP-1 mRNA. The levels of MCP-1 mRNA expressed by LLC cells isolated from tumors either from WT or MCP-1/ mice were low and comparable to that expressed by in vitro cultured original LLC cells, supporting the hypothesis that MCP-1 expression was not constitutively elevated in tumor cells, but rather, activation of tumor cells in a tumor microenvironment caused elevated MCP-1 expression by tumor cells in vivo. In fact, LLC cells, in response to LPS or TNF, expressed high levels of MCP-1 mRNA in vitro. These results indicate that LLC cells expressed higher levels of MCP-1, once they formed tumors in vivo and were exposed to factors in a tumor microenvironment. To analyze the potential mechanisms by which MCP-1 expression is elevated in LLC cells in a tumor microenvironment, we cocultured LLC cells with macrophages in vitro. Interestingly, coculture with macrophages markedly increased the level of MCP-1 mRNA expression by LLC cells. TLR ligands released from tumor cells can activate macrophages to produce proinflammatory

Please cite this article in press as: T. Yoshimura, The production of monocyte chemoattractant protein-1 (MCP-1)/CCL2 in tumor microenvironments, Cytokine (2017), http://dx.doi.org/10.1016/j.cyto.2017.02.001

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cytokines, such as TNF, in a tumor microenvironment [69]. Therefore, we examined whether TNF produced by macrophages was involved in elevated MCP-1 expression by LLC cells. In contrast to WT macrophages, macrophages from TNF/ mice or MyD88/ mice did not increase MCP-1 expression or production by LLC cells. Neutralization of TNF almost completely inhibited the effect of WT macrophages to promote MCP-1 mRNA expression or production by LLC. The expression of TNF mRNA was detected in LLC tumors. Thus, it is highly likely that TNF produced by LLC cell-activated macrophages upregulates MCP-1 production by LLC cells in the LLC tumor microenvironment (Fig. 2B). We examined the role for host cell-derived TNF and MyD88 in MCP-1 production in vivo by transplanting LLC cells into the flank of WT, MyD88/ or TNF/ mice. Consistent with in vitro results, the expression of MCP-1 was markedly lower in LLC tumors that grew in TNF/ mice. Serum MCP-1 levels were also lower in tumor-bearing TNF/ mice. Importantly, the size and volume of tumors in TNF/ mice were significantly smaller than those in WT mice. These results indicate that TNF is a critical macrophagederived mediator that increases the production of MCP-1 by LLC cells in tumors. Cordero et al. also demonstrated by using a fly model that TNF/Egr expressed by tumor-associated hemocytes (leukocytes in fly) was necessary and sufficient to trigger TNF signaling in tumor cells for dMMP1 expression [70]. Although the expression of MCP-1 was also reduced in LLC tumors grown in MyD88/ mice, there was no significant difference in either serum MCP-1 level or tumor volume between MyD88/ and WT mice. Finally, we investigated the mechanistic basis for LLC cells to induce TNFa production by macrophages. It was previously demonstrated that LLC cells released an extracellular matrix protein versican that could activate myeloid cells to produce TNF via TLR2 [69]. MyD88 is a signaling molecule downstream of TLR2 and the loss of MyD88/ in macrophages reduced MCP-1 mRNA expression by LLC cells in vitro; therefore, TLR2 on macrophages may play a role in LLC cell-induced macrophage TNF production. Contrary to our hypothesis, however, macrophages from TLR2/ mice were as efficient as those from WT mice to increase MCP-1 expression by LLC cells. Macrophages from TLR4/ or IL-1R1/ mice or TLR9/ mice also increased MCP-1 mRNA expression by LLC cells. Thus, it appears that LLC cells activate macrophages by a mechanism independent of TLR2, TLR4, TLR9 or IL1R1 involving MyD88. It is also possible that LLC cells activate macrophages via more than one TLR. Popivanova et al. investigated the production of MCP-1 in the colon during colon cancer development using a mouse chronic colitis-associated carcinogenesis model induced by azoxymethane and dextran sulfate sodium. By immunohistochemistry, MCP-1 protein was detected at an early phase in mononuclear cells, particularly macrophages, infiltrating the lamina propria and submucosal regions and also in endothelial cells, and at a later phase in carcinoma cells [57]. The production of MCP-1 in this model was almost completely inhibited when TNF was deficient in hematopoietic cells [71]. These findings strongly suggest that tumors cells activated by macrophage-derived TNF are the primary source of MCP-1 also in this colon cancer model. As described above, TNF could also be produced by tumor cells, such as human ovarian cancer cells, for their production of MCP-1 [67]. Thus, TNF, whether it is produced by stromal cells or tumor cells, is a critical, common molecule up-regulating the production of MCP-1 by tumor cells in tumor microenvironments.

4. Concluding remarks Since the purification of MCP-1 almost 30 years ago, significant progress has been made to understand the mechanisms of

monocyte recruitment to sites of inflammatory responses and cancer, and the role of TAMs in tumor progression. In the past several years, we focused to determine the mechanisms regulating MCP-1 production in tumor microenvironments using transplantable mouse tumor models. Either non-tumor stromal cells or tumor cells are the primary source of MCP-1 depending on each tumor model. The level of MCP-1 production also varies among tumor models. The nature of tumor cells and crosstalk between tumor cells and stromal cells dictate both the primary cell source and the expression level of MCP-1. Tumor cell-derived GM-CSF is a unique activator of macrophages and TNF is a common mediator upregulating the production of MCP-1. Considering that naturally arising tumors are heterogeneous, multiple mechanisms may be simultaneously serving within a tumor. Our goal is to identify additional mechanisms of tumor cell-stromal cell interaction to better understand the tools tumor cells use for their progression and to provide a new means to target tumor microenvironments.

Acknowledgments I am grateful to Drs. Edward J. Leonard, Kouji Matsushima and Joost J. Oppenheim for their invaluable inputs during my studies at the National Cancer Institute. I am also grateful to Drs. Naoya Yuhki, Shuji Tanaka and Ettore Appella, and Ms. Elizabeth A. Robinson for their critical collaborations for the identification and cloning of MCP-1, and to Dr. Ji Ming Wang and members of the Laboratory of Molecular Immunoregulation, NCI, for their discussion and support.

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