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Chemokine (C-C Motif) Ligand 2 Engages CCR2+ Stromal Cells of Monocytic Origin to Promote Breast Cancer Metastasis to Lung and Bone

Chemokine (C-C Motif) Ligand 2 Engages CCR2+ Stromal Cells of Monocytic Origin to Promote Breast... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 42, pp. 29087–29096, October 16, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Chemokine (C-C Motif) Ligand 2 Engages CCR2 Stromal Cells of Monocytic Origin to Promote Breast Cancer Metastasis to Lung and Bone Received for publication, June 22, 2009, and in revised form, August 29, 2009 Published, JBC Papers in Press, August 31, 2009, DOI 10.1074/jbc.M109.035899 ‡1 ‡§2 Xin Lu and Yibin Kang ‡ § From the Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 and the Breast Cancer Program, Cancer Institute of New Jersey, New Brunswick, New Jersey 08903 Metastatic spread of cancer to distant vital organs, including bone pain, and hypercalcemia (3–5), whereas lung metastasis is lung and bone, is the overwhelming cause of breast cancer mor- accompanied by cough, bloody sputum, rib cage pain, and, tality and morbidity. Effective treatment of systemic metastasis eventually, failure of the respiratory functions (6). Colonization relies on the identification and functional characterization of of different secondary organs by breast cancer is believed to be metastasis mediators to multiple organs. Overexpression of the a complex, multigenic process that depends on productive chemokine (C-C motif) ligand 2 (CCL2) is frequently associated interactions between tumor cells and stromal microenviron- with advanced tumor stage and metastatic relapse in breast can- ments through concerted actions of organ-specific metastasis cer. However, the functional mechanism of CCL2 in promoting genes (7, 8). Functional genomic analysis of preclinical models organ-specific metastasis of breast cancer has not been rigor- of breast cancer to bone, lung, and brain have identified distinct ously investigated. Here, we used organ-specific metastatic sub- sets of organ-specific metastasis genes (9–11), providing novel lines of the MDA-MB-231 human breast cancer cell line to dem- mechanistic insights into key rate-limiting steps of metastasis onstrate that overexpression of CCL2 promotes breast cancer to different organs. However, as advanced breast cancer metastasis to both lung and bone. Conversely, blocking CCL2 patients often suffer from metastases at several secondary function with a neutralizing antibody reduced lung and bone organs, identifying genes that are capable of instigating metas- metastases. The enhancement of lung and bone metastases by tasis to multiple sites may provide the ideal targets for thera- CCL2 was associated with increased macrophage infiltration peutic intervention of systemic metastasis. and osteoclast differentiation, respectively. By performing func- Chemokines are small (8–14 kDa) proteins classified into tional assays with primary cells isolated from the wild type, four conserved groups (CXC, CC, C, and CX3C) based on the CCL2 and CCR2 knock-out mice, we showed that tumor cell- position of the first two cysteines that are adjacent to the amino derived CCL2 depends on its receptor CCR2 (chemokine, CC terminus (12). They are chemotactic cytokines that stimulate motif, receptor 2) expressed on stromal cells to exert its function directed migration of leukocytes in response to inflammatory in promoting macrophage recruitment and osteoclast differen- signals. Chemokines are also involved in the maintenance of tiation. Overall, these data demonstrated that CCL2-expressing hematopoietic homeostasis, regulation of cell proliferation, tis- breast tumor cells engage CCR2 stromal cells of monocytic sue morphogenesis, and angiogenesis (13). Chemokines bind to origin, including macrophages and preosteoclasts, to facilitate the seven-transmembrane domain receptors to elicit down- colonization in lung and bone. Therefore, CCL2 and CCR2 are stream molecular events that coordinate cell movement. Even promising therapeutic targets for simultaneously inhibiting though chemokines are unlikely to be a contributing factor for lung and bone metastasis of breast cancer. tumor initiation, they can have pleiotropic effects on tumor progression (13, 14). Among more than 50 human chemokines, CCL2 is of particular importance. CCL2, also called monocyte Breast cancer is the most common malignancy in women in chemoattractant protein-1 (MCP-1), is a potent chemoattrac- the United States, with an estimated 182,000 new cases and tant for monocytes, memory T lymphocytes, and natural killer 40,000 deaths in 2008 (1). Late stage breast cancer patients cells (15). It is involved in a number of inflammatory conditions develop metastases in bone, lung, liver, brain, and other organs, associated with monocyte recruitment, including delayed which are responsible for most breast cancer-related mortality hypersensitivity reactions, bacterial infection, arthritis, and and morbidity (2). Severe complications from bone metastasis renal disease (15). The importance of CCL2 in cancer was man- include debilitating bone fractures, nerve compression and ifested by its overexpression in a variety of tumor types, includ- ing glioma, ovarian, esophagus, lung, breast, and prostate can- * This work was supported, in whole or in part, by National Institutes of Health cers (15–17). In prostate cancer, CCL2 expression levels was Grant R01CA134519 (to Y. K.). This work was also supported by Depart- associated with advanced pathological stage (16). Importantly, ment of Defense Era of Hope Scholar Award BC051647 and American Can- CCL2-neutralizing antibodies inhibit bone resorption in vitro cer Society Research Scholar Award RSG MGO-110765. Recipient of a Harold W. Dodds Fellowship from Princeton University. and bone metastasis in vivo (18–20). In lung cancer, serum Investigator of the Champalimaud Metastasis Program at Princeton Univer- CCL2 levels were elevated in lung cancer patients with bone sity. To whom correspondence should be addressed: Dept. of Molecular metastasis compared with localized diseases. Neutralizing anti- Biology, WA Road, LTL 255, Princeton University, Princeton, NJ 08544. Tel.: 609-258-8834; Fax: 609-258-2340; E-mail: [email protected]. bodies against CCL2 also inhibited the tumor conditioned This is an Open Access article under the CC BY license. OCTOBER 16, 2009• VOLUME 284 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29087 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis media-induced osteoclast formation in vitro and bone metasta- EXPERIMENTAL PROCEDURES sis in vivo (17). Taken together, these findings suggested a role Cell Culture and Reagents—The SCP28, LM2, and 1833 cell of CCL2 in bone metastasis. lines were derived from the parental cell line MDA-MB-231 A potential role of CCL2 in breast cancer progression and (American Type Culture Collection) (9, 10). These sublines, metastasis has been indicated by the analysis of CCL2 expres- their genetically modified variants, the retroviral packing cell sion of tumor and serum samples from breast cancer patients. line H29, and the mouse monocyte/preosteoclast cell line Serum CCL2 levels were significantly higher in postmeno- RAW264.7 (American Type Culture Collection) were main- pausal breast cancer patients than in age-matched controls tained in Dulbecco’s modified Eagle’s medium supplemented (21). Over 50% of breast cancer tumor samples had intense with 10% fetal bovine serum and antibiotics. The human cell staining of CCL2 in tumor cells (22). Prognostic analysis further line hFOB1.19 (American Type Culture Collection) was main- revealed that high expression of CCL2 was correlated with tained as preosteoblasts in Dulbecco’s modified Eagle’s medi- advanced tumor stage, lymph node metastasis (23), and early um:F12 without phenol red supplemented with 0.3 mg/ml relapse (24). CCL2 up-regulation in breast tumors was also G418 and 10% fetal bovine serum at 34 °C. When switched to associated with the infiltration of tissue-associated macro- 39.5 °C for 2 days after confluence, the cells differentiated into phages (TAMs) and with increased microvessel density (22, mature osteoblasts. Recombinant human CCL2, murine recep- 24). TAMs have been known to contribute to primary tumor tor activator of NF- B ligand and murine macrophage colony progression and metastasis of breast cancer (25), which is sup- stimulating factor were purchased from PeproTech. ported by epidemiological evidence showing that TAM infiltra- Generation of CCL2-overexpressing Cells—The human CCL2 tion portended a poor clinical outcome (26, 27). However, coding sequence was inserted into the HpaI site of the retroviral whether the function of CCL2 in modulating activity of macro- vector pMSCVhyg (Clontech). pMSCVhyg-CCL2 or the vector phages and possibly other cell types is important for breast was transfected into the packaging cell line H29. After 48 h, tumor organotropic metastasis has not been rigorously investi- viruses were collected, filtered, and used to infect target cells in gated. CCL2 may engage organ-specific cell types derived from the presence of 5 g/ml polybrene. The infected cells were the same bone marrow myelomonocytic progenitors. These selected with 1.5 mg/ml hygromycin. To avoid clonal variations, a progenitors differentiate into osteoclast precursors in bone or pooled population of at least 500 independent colonies from each into blood monocytes that eventually become mature macro- transduction was used to generate each stable cell line. phages in different tissues, like alveolar macrophages in lung Western Blot Analysis—To detect CCL2 in conditioned (28). These stromal cell types of myelomonocytic origin may medium by Western blotting, cells were grown to confluence, contribute to different functions in different organ-specific changed to serum-free medium for 24 h before the medium was metastases. Another unresolved question regarding the func- collected, concentrated with Amicon centrifugal filter units tion of CCL2 in tumor-stroma interaction is the functional (Millipore), and applied to immunoblotting. The primary anti- involvement of CCL2 receptors. CCL2 can bind to both CCR2 body was mouse anti-human CCL2 (MAB279, R&D Systems). and CCR4 (29, 30). Loss of function studies in mice showed Anti-mouse IgG secondary antibody conjugated to horseradish CCL2 and CCR2 knock-out mice displayed similar impair- peroxidase was used for chemiluminescence detection (GE ments in monocyte migration (31, 32), suggesting that CCR2 is Healthcare). the major functional receptor for CCL2. Understanding ELISA—To detect CCL2 in the conditioned medium by whether CCR2 deficiency in stromal cells leads to compromised enzyme-linked immunosorbent assay (ELISA), cells were monocyte engagement by CCL2-expressing tumor cells may grown to confluence, changed to fresh medium and cultured have important implications in designing targeting therapeu- for 24 h before the medium was collected. Human CCL2 ELISA tics against the CCL2/CCR2 axis. kit (eBioscience) was used to measure CCL2 concentration fol- In this study, we used the recently developed organ-specific lowing the manufacturer’s instructions. metastatic sublines of the human breast cancer cell MDA-MB- Reverse Transcription (RT)-PCR and Quantitative RT-PCR— 231 (9, 10, 33) and showed that overexpression of CCL2 pro- Total RNA was isolated from adherent cells using RNeasy kit motes both lung and bone metastases. This function was asso- (Qiagen) and reverse-transcribed with Superscript III kit ciated with increased TAM infiltration in lung metastasis and (Invitrogen) according to the manufacturer’s instructions. increased osteoclast differentiation in bone metastasis, respec- Primers used for amplifying cDNA fragments are: human CCR2 tively. Furthermore, by using macrophages and bone marrow (364 bp), 5-gacaagccacaagctgaaca-3 (forward) and 5-gagccc- cells isolated from wild type, CCL2-deficient, and CCR2-defi- acaatgggagagta-3 (reverse); human GAPDH (475 bp), 5-gtca- cient mice, we showed that CCR2 expression in stromal cells is tccatgacaactttggtatc-3 (forward) and 5-ctgtagccaaattcgttgtca- essential for tumor-derived CCL2 to recruit macrophages and tac-3 (reverse); mouse CCL2 (248 bp), 5-aggtccctgtcatgctt- promote osteoclastogenesis. Targeting tumor-derived CCL2 by ctg-3 (forward) and 5-tctggacccattccttcttg-3 (reverse); a neutralizing antibody significantly reduced metastasis forma- mouse CCR2 (252 bp), 5-ggtcatgatccctatgtgg-3 (forward) and tion in both bone and lung. 5-ctgggcacctgatttaaagg-3 (reverse); mouse GAPDH (977 bp), 5-ggtcggtgtgaacggatttgg-3 (forward) and 5-satgtaggccatgag- The abbreviations used are: TAM, tissue-associated macrophage; BLI, biolu- gtccacc-3 (reverse). Quantitative PCR was performed using minescence imaging; TRAP, tartrate-resistant acid phosphatase; ELISA, the SYBR Green PCR Master Mix (Applied Biosystems) with enzyme-linked immunosorbent assay; DAPI, 4,6-diamidino-2-phenylin- the ABI Prism 7900HT thermocycler (Applied Biosystems). dole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 29088 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 42 •OCTOBER 16, 2009 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis The following primers were used: human CCL2 (66 bp): 5- 1640 containing 100 ng/ml recombinant human CCL2 or RPMI cagccagatgcaatcaatgc-3 (forward) and 5-gcactgagatcttcctatt- 1640 conditioned by confluent tumor cells for 24 h. ggtgaa-3 (reverse); Human GAPDH (225 bp), 5-gaaggtgaagg- Bone Marrow Osteoclastogenesis Assay—Bone marrow cells tcggagtc-3 (forward) and 5-gaagatggtgatgggatttc-3 (reverse). were flushed out from femora and tibiae from 6-week-old mice CCL2 mRNA level was normalized with GAPDH level. and plated in basal culture medium (minimum essential In Vitro Growth Curve and Anchorage-independent Growth— medium  supplemented with 10% fetal bovine serum, 2 mM To establish growth curves, 2  10 cells were seeded in each L-glutamine, and antibiotics) overnight. The next day, the non- well of 6-well plates with triplicate for each time point. Medium adherent cells were plated at 1  10 /well in 24-well plates in was replaced every 2 days. Cell number was determined with basal culture medium supplemented with 30 ng/ml receptor dead cell exclusion using trypan blue every 2 days. For soft agar activator of NF-B ligand and 30 ng/ml macrophage colony growth, a bottom layer of 0.6% agar in growth medium was stimulating factor to induce osteoclast differentiation. When added to 6-well plates with triplicate for each cell line. 2  10 the effect of CCL2 was tested, 100 ng/ml CCL2 was added to the cells were seeded in 0.3% top agar containing growth medium medium or the medium was preconditioned with confluent to each well. Three weeks later, colonies were stained with crys- tumor cells for 24 h. Medium was changed at day 3. TRAP tal violet for visualization. staining was performed on day 6 using a leukocyte acid phos- Tumor Xenografts and Bioluminescence Imaging (BLI) phatase kit (Sigma). Nuclei were stained with 1 g/ml 4,6- Analyses—All procedures involving mice, such as housing and diamidino-2-phenylindole (DAPI). TRAP cells with 3–10 care, and all experimental protocols were approved by Institu- nuclei or 10–30 nuclei were separately quantified and reported tional Animal Care and Use Committee of Princeton Univer- as the number per microscopic field. 5 4 sity. For intracardiac injections, 10 cells were injected into the RAW264.7 Osteoclastogenesis Assay—2  10 RAW264.7 left cardiac ventricle of female nude mice (National Cancer cells were seeded to each well of 24-well plates. Receptor acti- Institute) as described (9). For intravenous injection, 2  10 vator of NF-B ligand (40 ng/ml) was added to the medium to cells were injected into the tail vein of nude mice as described induce differentiation. When the effect of CCL2 was tested, (10). Development of metastases in long bones and lungs was different concentrations of CCL2 was added to the medium. monitored by BLI with the IVIS Imaging System (Xenogen) as Medium was changed at day 2. TRAP staining, DAPI staining, described (10). Analysis was performed with Living Image soft- and quantification were performed on day 4 similar to the bone ware (Xenogen) by measuring photon flux of the region of marrow assay. interest. Data were normalized to the signal obtained immedi- Blocking CCL2 with Neutralizing Antibody—Neutralizing ately after injection (Day 0). X-ray radiography analysis of bone antibody to human CCL2 (MAB679, R&D Systems) or control lesions was performed as described previously (9). mouse IgG were injected intraperitoneally to mice every 4 days Histological Analysis—Hind limb bones and lungs were from the date of tumor cell inoculation to the end of the mouse excised, fixed in 10% neutral-buffered formalin, decalcified (for experiment at dose 10 g/mouse (36). bone only), and embedded in paraffin for hematoxylin and Statistical Analysis—Results were reported as average S.D. eosin staining (9), Goldner’s trichrome staining (34) or tartrate- or average  S.E., indicated in the figure legend. Two-sided resistant acid phosphatase (TRAP) staining (35) as described independent Student’s t test without equal variance assump- previously. The osteoclast number was assessed as multinucle- tion or nonparametric Mann-Whitney test was performed to ated TRAP cells along the tumor-bone interface and reported analyze the data with p  0.05 considered as statistically as number/mm of interface. Immunohistochemical analysis significant. was performed with heat-induced antigen retrieval. The pri- RESULTS mary antibody used was rabbit anti-human von Willebrand Factor (DAKO), rat anti-mouse F4/80 (Abcam), rat anti-mouse CCL2 Overexpression Promotes Lung Metastasis—Two organ- Ly6C (ER-MP20, Abcam), or ER-MP58 as myeloid precursor otropic metastatic sublines of MDA-MB-231 were used for this cells antibody (Abcam). Biotinylated secondary antibody was study: LM2 for lung metastasis (10) and SCP28 for bone metas- used with Vectastain ABC kit (Vector Laboratories) and DAB tasis (9). To evaluate the function of CCL2 in metastasis, human detection kit (Zymed Laboratories, Inc.) to reveal the positively CCL2 was stably overexpressed in LM2 and SCP28, as con- stained cells with nuclei counterstained with hematoxylin. firmed by quantitative RT-PCR (Fig. 1A) and Western blot / / Knock-out Mice—CCL2 and CCR2 mice were gener- analysis of conditioned media (Fig. 1B). CCL2 overexpression ated previously (31, 32) and obtained from The Jackson Labo- did not cause any significant change in the proliferation rate in ratory in C57BL/6 background. The colony was maintained by vitro (Fig. 1C) and anchorage-independent growth on soft agar crossing homozygous siblings. Wild type C57BL/6 mice were (Fig. 1D). These results suggest that CCL2 does not have as used as control. direct role in promoting tumor cell proliferation. Macrophage Chemotaxis Assay—Resident and thioglycol- Functional involvement of CCL2 in lung metastasis was late-elicited peritoneal monocytes/macrophages were har- tested by using LM2 cells with or without CCL2 overexpression vested by peritoneal lavage as described previously (32). Che- in lung metastasis assays. To facilitate noninvasive BLI of met- motaxis assay was performed with 12-well cell culture inserts astatic tumor growth, LM2 and SCP28 had been previously (BD Falcon) following previous methods (31). Migrated cells labeled with a firefly luciferase reporter (37). LM2-Vec (vector were stained with crystal violet for visualization. The agonist control) and LM2-CCL2 were intravenously injected into nude (lower chamber) was either RPMI 1640 medium alone or RPMI mice, and the development of lung metastases were monitored OCTOBER 16, 2009• VOLUME 284 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29089 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis weekly by BLI. As early as 1 week after injection, LM2-CCL2 cells already showed 5-fold stronger signals than LM2-Vec (p  0.027; Fig. 2, A and B), and this trend per- sisted for 5 weeks (p  0.05 for all time points), up to the point when the mice were sacrificed for histo- logical analyses. Lung metastasis nodule quantification and hematox- ylin and eosin staining confirmed the increased pulmonary metastasis burden in mice injected with LM2- CCL2 (Fig. 2, C and D). Lung contains several different types of macrophage, including alveolar, interstitial, intravascular, and airway macrophages (38). As an important function of CCL2 is to mobilize cells of monocyte lineage, we tested whether CCL2 overex- FIGURE 1. Characterization of CCL2 overexpression in MDA-MB-231 sublines. A, CCL2 mRNA level in vector pression caused an increase of infil- control (Vec) and CCL2-overexpressing (CCL2) derivatives of SCP28 and LM2, as measured by quantitative RT-PCR. B, Western blot of the conditioned medium showing the overexpressed CCL2 (doublet bands). Control trating macrophages in lung metas- denotes cells without transfection of plasmid. C and D, in vitro proliferation and anchorage-independent tases. We used an F4/80 antibody, growth of control and CCL2-overexpressing tumor cells. Scale bar, 200 m. In A, C, and D, data represent which recognizes a cell surface gly- average  S.D. coprotein specifically expressed in FIGURE 2. CCL2 overexpression promotes lung metastasis and macrophage infiltration. A, in vivo lung metastasis assay of LM2 cells with or without CCL2 overexpression. BLI data represent average S.E., *, p 0.05 by Mann-Whitney test on each time point. B, representative BLI images showing the development of lung metastasis in mice at different time points after LM2 injection. C, quantification of lung metastasis nodules with a diameter of1 mm during necropsy. p  0.0087 with Mann-Whitney test. D, hematoxylin and eosin staining of lung tissue from the mice injected with LM2 derivatives. T, tumor nodule; L, lung parenchyma. Scale bar, 400 m. E and F, immunohistochemical staining of F4/80, Ly6C, ER-MP58, and von Willebrand Factor (vWF) on the lung sections for quantification of mature macrophages, immature macrophages, and vessel density. Scale bar, 200 m. Data represent average  S.E.; p values by two-sided Student’s t test. Vec, vector control. 29090 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 42 •OCTOBER 16, 2009 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis phage recruitment during lung metastasis. Is CCR2 essential for tumor cell-derived CCL2 to recruit macrophages? Can macrophages lacking CCL2 still be recruited? Answering these questions may have important clinical implications regarding whether targeting CCR2 and/or CCL2 in stroma may achieve the same therapeutic effects as tar- geting CCL2 in tumor cells. We first isolated resident perito- neal monocytes/macrophages from / / wild type, CCL2 , and CCR2 mice and assessed the expression of CCL2 and CCR2 using RT-PCR (Fig. 3A). Wild type macrophages express both genes, whereas those isolated from knock-out mice lack the expression of the corresponding gene. Next, we performed chemo- FIGURE 3. CCL2 in tumor-conditioned medium mobilizes macrophages in a CCR2-dependent manner. taxis assay of isolated resident mac- A, RT-PCR showing the expression pattern of CCL2 and CCR2 in the resident monocytes/macrophages from rophages with recombinant human wild type and knock-out (KO) mice. GAPDH was constitutively expressed in all cases. B, chemotaxis assay of CCL2 or tumor cell-conditioned resident macrophages with recombinant CCL2 or conditioned medium (CM) (by LM2 derivatives) as the ago- nist. C, chemotaxis assay of thioglycollate-elicited macrophages with recombinant CCL2 or conditioned medium as attractants (Fig. 3B). For medium as the agonist. Data represent average S.D., *, p 0.05, **, p 0.01, and ***, p 0.001 by two-sided macrophages isolated from wild Student’s t test (B and C). D, representative images of migrated monocytes/macrophages of the assay in C. Scale type mice, both recombinant CCL2 bar, 100 m. Vec, vector control. and conditioned medium from LM2-CCL2 significantly increased mature tissue macrophages (39). Indeed, CCL2 overexpression the migratory activity (2.7-fold and 2.8-fold, respectively) com- led to increased F4/80 macrophage infiltration by 3-fold pared with CCL2-free medium or medium conditioned by (Fig. 2, E and F). Immature macrophages were stained with two LM2-Vec. This indicates that CCL2 secreted by LM2-CCL2 different myeloid precursor markers Ly6C and ER-MP58 (40), cells elicits similar macrophage-mobilizing potency as recom- and no significant differences in their numbers were observed binant CCL2. Similar macrophage chemotaxis-promoting between two groups of samples (Fig. 2, E and F). CCL2 has also effect was observed when macrophages isolated from CCL2 been reported to affect angiogenesis (41). However, we did mice were used in the experiment (Fig. 3B, CCL2-KO), suggest- not observe significant change of microvessel density ing that endogenous CCL2 expression is not required for mac- between control and CCL2-overexpressing pulmonary rophage migration in response to CCL2. In contrast, CCR2 metastases, as revealed by von Willebrand Factor staining deficiency led to complete loss of CCL2-induced macrophage (Fig. 2, E and F), suggesting that elevated angiogenesis is not mobility (Fig. 3B, CCR2-KO). This result suggests that CCR2 is the cause of the increased lung metastasis burden in our a receptor essential for increased macrophage migration experimental model. Instead, the observation that CCL2 induced by CCL2-overexpressing tumor cells. overexpression already led to more lung metastasis at early Resident macrophage behavior may not necessarily reflect time point (week 1) suggests that the enhancement of tumor the behavior of TAMs as the latter is usually associated with invasion and initial lung colonization mediated by CCL2- inflammation. Therefore, we performed the same assays using recruited macrophages may be the main reason for the over- macrophages elicited by thioglycollate-induced nonspecific all increased lung metastasis burden. inflammation. Monocytes/macrophages, which constitute CCL2-overexpressing Tumor Cells Promote Macrophage most of the elicited leukocyte population 72 h after the intra- Chemotaxis in a CCR2-dependent Manner—Both of the CCL2 peritoneal thioglycollate instillation (32), were harvested from / / receptors, CCR2 (29) and CCR4 (30), are widely expressed in a wild type, CCL2 , and CCR2 mice. Consistent with pre- variety of cell types, including monocytes and macrophages (42, vious reports, the recruitment of macrophages was significantly 43). CCR2- and CCR4-deficient mice displayed similar defects reduced in the two mutant mice (data not shown). Neverthe- in macrophage recruitment in experimental inflammation con- less, equal numbers of cells were collected and applied to the ditions (31, 43). Similar impairments in monocyte migration chemotaxis assays. Similar to resident macrophages, thiogly- was also observed in mice lacking CCL2 (32, 44), suggesting collate-elicited macrophages were mobilized by recombinant that CCL2 may have a cell autonomous function in monocyte CCL2 or tumor cell-derived CCL2, and such an effect was again mobilization. These observations prompted us to investigate found to be dependent on the intact expression of CCR2, but two related questions about the function of CCL2 in macro- not CCL2, on macrophages (Fig. 3, C and D). Taken together, OCTOBER 16, 2009• VOLUME 284 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29091 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis bone-tumor interface in metastatic lesions formed by SCP28-CCL2 as compared with bone metastasis of SCP28-Vec (Fig. 4D). Because oste- oclast activity is critical for the establishment of osteolytic bone metastasis (3–5), we hypothesize that CCL2-overexpressing breast cancer cells become more efficient in colonizing the bone through increased activation of osteoclasts. CCL2-overexpressing Tumor Cells Promote Osteoclast Differentiation in a CCR2-dependent Manner—Previ- ous studies have shown that recom- binant CCL2 protein can promote osteoclast fusion and differentiation in vitro (45–48). Here, we wanted to test whether tumor cell-derived CCL2 could similarly elicit oste- oclast activation and whether this is dependent on CCR2 expressed on osteoclast progenitor cells. Primary bone marrow cells isolated from wild type mice expressed both CCL2 and CCR2, which were lost in CCL2 FIGURE 4. CCL2 overexpression enhances osteolytic bone metastasis and associated osteoclasts. A, in and CCR2 knock-out mice, respec- vivo bone metastasis assay of SCP28 cells with or without CCL2 overexpression. BLI data represent average S.E., *, p 0.05, **, p 0.01, and ***, p 0.001 by Mann-Whitney test on each time point. B, representative BLI tively, as confirmed with RT-PCR images showing the development of bone metastasis in mice at different time points after SCP28 injection. (Fig. 5A, left panel). Bone marrow Arrows, bone metastases in the hind limbs. C, representative x-ray radiographs of the hind limbs at week 6 after cells isolated from different geno- injection showing the osteolytic lesions (arrows) in proximal tibia. D, Goldner’s trichrome and TRAP staining (with quantification) of proximal tibia from the mice injected with SCP28 derivatives. B, bone matrix; T, tumor types were applied to an in vitro area. Scale bar, 200 m. Data represent average  S.E., p values by two-sided Student’s t test. Vec, vector osteoclastogenesis assay. Bone mar- control. row growth medium precondi- these results suggest that tumor cell-derived CCL2 induces tioned with SCP28-Vec or SCP28-CCL2 cells for 24 h were directional migration of monocyte/macrophages in a CCR2- added to the bone marrow culture to compare their ability to dependent manner, which may be responsible for the elevated induce the formation of multinucleated TRAP osteoclasts. infiltration of macrophage in the lung metastases formed by Recombinant CCL2 was used as the positive control. The num- LM2-CCL2 cells. ber of nuclei in the osteoclasts indicates the efficiency of cell CCL2 Overexpression Promotes Osteolytic Bone Metastasis— fusion during differentiation. Therefore, we classified oste- To test whether CCL2 overexpression could also increase bone oclasts into two types: cells with 3–10 nuclei (less efficient metastasis formation, vector control and CCL2-overexpressing fusion) and cells with 10–30 nuclei (more efficient fusion) by derivatives of SCP28 were injected into the left ventricle of nude counting DAPI-stained nuclei. In the wild type bone marrow, mice, and bone metastases development in the hind limbs were we found the number of TRAP osteoclasts with 10–30 nuclei quantitatively monitored by BLI (Fig. 4, A and B). Similar BLI was expanded significantly by recombinant CCL2, whereas the intensities were found in both groups 1 week after injection. number of osteoclasts with 3–10 nuclei was unaffected (Fig. 5, However, starting from week 2, bone metastasis burden in the A and B, wild type). When bone marrow cells from mutant mice CCL2 overexpression group became significantly higher than were used, both CCR2- and CCL2-deficient osteoclast progen- that in the vector group until the end of the experiment. Cor- itor cells could be induced to form TRAP osteoclasts with responding to the increased bone metastasis burden, mice inoc- 3–10 nuclei. However, bone marrow cells deficient in CCR2, ulated with SCP28-CCL2 cells also had a larger area of bone unlike wild type and CCL2-deficient cells, significantly lost the lesion as revealed by x-ray radiography (Fig. 4C). Consistently, responsiveness to recombinant CCL2 to form larger osteoclasts the tumor-bone interface in bone metastasis lesions formed by with 10–30 nuclei, indicating the critical role of CCR2 as the SCP28-CCL2 were typically more rugged than that those receptor for the function of CCL2 in stimulating osteoclast formed by SCP28-Vec, as shown by Goldner’s trichrome stain- fusion. Importantly, we observed that CCL2 derived from ing highlighting the collagen-rich bone matrix in green (Fig. 4D, SCP28-CCL2 medium was equally able to promote osteoclast Trichrome). Furthermore, when we used TRAP staining to differentiation in a CCR2-dependent manner, supporting our compare osteoclast density in the metastases, significantly more (6-fold) TRAP osteoclasts were observed along the hypothesis that CCL2-overexpressing tumor cells promote 29092 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 42 •OCTOBER 16, 2009 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis FIGURE 5. CCL2 in tumor-conditioned medium promotes osteoclast differentiation in a CCR2-dependent manner. A, RT-PCR analysis of CCL2 and CCR2 expression and osteoclastogenesis assay of nonadherent bone marrow cells from wild type and knock-out (KO) mice. The differentiation medium was either supplemented with recombinant CCL2 or preconditioned by SCP28 derivatives. At day 6, TRAP cells with 3–10 nuclei (middle panel) or 10 –30 nuclei (right panel) were quantified and reported as number per field (under a 5 objective lens). Data represent average S.D. ***, p 0.001 by two-sided Student’s t test. B, representative images of TRAP osteoclasts with nuclei stained by DAPI (under a 10 objective lens). Scale bar, 100 m. C, RT-PCR showing CCR2 expression of mature osteoblast hFOB1.19 (left) and preosteoclast RAW264.7 (right). Positive () control was universal reference RNA (Stratagene), and negative () control was water. GAPDH was constitutively expressed in all cases. D, histograms and representative images of differentiated RAW264.7 cells with cell fusion enhanced by increasing concentrations of recombinant CCL2. Scale bar, 400 m. CM, conditioned medium; Vec, vector control. osteoclast activation during the formation of osteolytic bone resultant multinucleated TRAP osteoclasts (Fig. 5D), we metastasis. observed a clear dose-dependent increase of nuclear number in CCL2 may exert its function in promoting osteoclast differ- the induced osteoclasts by CCL2, supporting its direct role in entiation directly through preosteoclasts or indirectly through promoting cell fusion during osteoclast differentiation. osteoblasts, which is often the cell type required for paracrine Inhibition of CCL2 Reduces Bone and Lung Metastases—We activation of osteoclast differentiation. To differentiate these wished to evaluate whether inhibiting CCL2 function could two possible mechanisms, we determined the levels of CCR2 reduce metastasis to bone and lung. To this end, we used a mRNA expression in osteoblast cell line hFOB1.19 and preos- neutralizing antibody for human CCL2 to block the function of teoclast cell line RAW264.7 with RT-PCR (Fig. 5C). Expression tumor-derived CCL2 in metastasis assays in vivo. Two strongly of CCR2 was not detected in hFOB1.19, consistent with the metastatic sublines derived from in vivo selection of MDA-MB- previous observation in another osteoblast line, UMR 106–01 231 (the lung-tropic LM2 and the bone-tropic 1833 (9)) were (46). In contrast, CCR2 is expressed in RAW264.7 cells. This used in the experiment. The 1833 subline has a much stronger result indicates that CCL2 should function directly through bone metastasis ability than SCP28 and is therefore more CCR2 on preosteoclasts to promote its cell fusion and differen- appropriate to be used for testing the possible effect of CCL2 tiation. Indeed, when we treated RAW264.7 cells under the inhibition on reducing bone metastasis. 1833 and LM2 sublines differentiation culture condition with an increasing dose of express considerable amount of CCL2 at the basal level, as recombinant CCL2 and profiled the number of nuclei in the measured by ELISA of the conditioned media (2.41  0.42 OCTOBER 16, 2009• VOLUME 284 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29093 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis FIGURE 6. Alteration of organotropic metastasis behavior by targeting CCL2 with neutralizing antibody in highly metastatic cells and by overexpressing CCL2 in weakly metastatic cells. A, in vivo bone metastasis assay of highly bone metastatic cell line 1833 with mice treated with control IgG or neutralizing antibody against human CCL2. B, in vivo lung metastasis assay of highly lung-seeking line LM2 with mice treated with control IgG or neutralizing antibody against human CCL2. C, in vivo lung metastasis assay of weakly lung-metastatic cell line SCP28 with or without the overexpression of CCL2. D, in vivo bone metastasis assay of weakly bone metastatic cell line LM2 with or without the overexpression of CCL2. In A–D, BLI data represent average  S.E. *, p  0.05, **, p  0.01, and ***, p  0.001; with no asterisk shown, p  0.05 by Mann-Whitney test on each time point. Vec, vector control. ng/ml for 1833 and 0.77  0.01 ng/ml for LM2), whereas fresh metastasis potential by CCL2 overexpression. However, medium does not contain any detectable level of human CCL2. CCL2 overexpression was not sufficient to enhance bone 1833 and LM2 were inoculated into nude mice by intracardiac metastasis for the otherwise weakly bone metastatic subline or intravenous injections, respectively, to generate bone and LM2 (Fig. 6D). Because metastasis ability is known to rely on lung metastases. Antibodies or isotype IgG controls were concerted function of multiple genes (9, 10), it is not surpris- injected into mice from the date of tumor inoculation until the ing to observe variable results in this test. The ability of end of the experiment. Both 1833-generated bone metastasis CCL2 to confer organotropic metastasis potential may and LM2-generated lung metastasis were reduced significantly depend on the genetic background of the cell lines (i.e. the by antibody treatment (Fig. 6, A and B). These results indicate existence of cooperating organotropic metastasis genes). that inhibition of CCL2 is effective in suppressing metastasis DISCUSSION formation in both bone and lung. Overexpression of CCL2 Alone in Weakly Metastatic Cells Is Suf- Chemokines and their receptors play diverse roles in malig- ficient to Promote Lung but Not Bone Metastasis Potential— nant tumor progression, particularly as key mediators of Although overexpression of CCL2 promotes metastasis to tumor-stroma interactions (12, 13, 49). The best characterized lung or bone in cell lines that already display significant basal example is the function of SDF-1/CXCR4 axis in mediating metastatic abilities to the respective organs (e.g. SCP28 to site-specific metastasis for various cancer types (50). Data pre- bone and LM2 and lung, Figs. 2A and 4A), we wished to test sented here demonstrate that CCL2 has dual activity to pro- whether CCL2 overexpression can render an otherwise mote both lung and bone metastases of breast cancer cells weakly metastatic or non-metastatic cell capable of metasta- through distinct functions. When CCL2 was overexpressed, sizing to bone or lung. We injected vector control or CCL2- lung metastasis development was accelerated with increased overexpressing variants of SCP28 (weakly lung metastatic) to infiltration of F4/80 macrophages. Similarly, CCL2-overex- the tail vein (Fig. 6C) and found a significant increase of lung pressing tumor cells developed more extensive osteolytic 29094 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 42 •OCTOBER 16, 2009 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis bone lesions with increased recruitment and activation of bone microenvironment (the “metastasis niche”) (2) or to form TRAP osteoclasts in the tumor-bone interface. Impor- small clusters of micrometastases. Neither of these events may tantly, by using primary cells isolated from wild type, be dependent on CCL2. Instead, CCL2 promotes bone metas- / / CCL2 , and CCR2 mice, we showed that tumor-derived tasis expansion through inducing osteoclast differentiation and CCL2 increases the directional migration of macrophages and establishment of the “vicious cycle” of bone metastasis (2), promotes osteoclast differentiation in a CCR2-dependent man- which occurs after the initial adaptation. Recognizing the ner. Furthermore, we demonstrated that targeting CCL2 in organ-specific modes of metastatic function of CCL2 has tumor cells with a neutralizing antibody inhibited metastasis potential clinical implications, as treatment of lung metastasis formation in lung and bone. Overall, these results suggest that may benefit from CCL2-targeting therapy only if the treatment CCL2 and CCR2 as targets for therapeutic intervention of bone is applied early, whereas macroscopic bone metastasis may still and lung metastasis. be sensitive to the inhibition of CCL2. CCL2 expression can be activated in advanced breast cancer It is generally believed that macrophages and bone marrow through genetic or epigenetic mechanisms. An infrequent poly- osteoclasts share a common monocytic hematopoietic progen- morphism in the CCL2 promoter region associated with itor. Several lines of evidence support this hypothesis. op/op increased CCL2 expression is overrepresented in metastatic mice (spontaneous colony-stimulating factor 1 mutation) have breast cancer patients (49). Several pathways involved in epi- severe deficiency of both osteoclasts and macrophages (55). thelial-mesenchymal transition, including dysadherin (51) and Knock-out of transcription factor PU.1 in mice caused absence -catenin/TCF-4 (52), promote CCL2 up-regulation. Here, we of both cell types, suggesting defects at an early common mye- used ectopic overexpression and blocking antibody to assess its loid differentiation stage (56). It is conceivable that common pro-metastatic function in the MDA-MB-231 breast cancer molecule(s) involved in regulating the homeostasis of both metastasis models. We chose this model because of the unique macrophages and osteoclasts can be hijacked by tumor cells to repertoire of organ-specific metastatic sublines that was simultaneously modulate the activity of both cell types in the recently developed (9–11). CCL2-mediated tumor- and metas- tumor microenvironment to promote tumor growth. Indeed, tasis-promoting functions have been investigated in other pre- our results showed that breast tumor cells are able to recruit clinical models. For example, CCL2-mediated macrophage macrophages in the lung parenchyma as well as enhancing oste- infiltration and increased tumor growth were reported in the oclast differentiation in the bone marrow by engaging the same gastric carcinoma nude mouse model (53). CCL2 inhibition by CCL2/CCR2 axis on these cells. Thus, pharmacological inhibi- short hairpin RNA-mediated knockdown or by systemic deliv- tors targeting CCL2 or CCR2 in breast cancer patients whose ery of neutralizing antibodies inhibit the development of bone tumor tissue expresses a high level of CCL2 may bring clinical metastasis by prostate cancer (19, 54). However, the functional benefits for controlling distant metastases in multiple organs. importance and cellular mechanism of CCL2 in promoting The CCL2/CCR2 axis may have prometastasis functions organ-specific metastasis of breast cancer metastasis has not beyond the direct engagement of stromal cells of monocytic been rigorously investigated. In addition to using the MDA- origin by tumor cells. CCL2/CCR2 may mediate a cascade of MB-231 in vivo models to study the functional importance of signaling events to enhance tumor malignancy. For example, CCL2 in promoting bone and lung metastasis, our current MDA-MB-231 cells can induce osteoblast hFOB1.19 and study also took advantage of the existing CCL2 and CCR2 MC3T3-E1 to up-regulate CCL2 (57), and CCL2 produced by knock-out mice and identified CCR2 as the essential receptor osteoblasts can promote osteoclast differentiation (47, 48). Fur- for mediating the function of CCL2 in macrophage recruitment thermore, tumor-derived CCL2 can exert function on other and osteoclast activation by breast cancer. CCR2 stroma cells, such as endothelial cells (41) and mesen- Careful comparison of the vector control and CCL2-overex- chymal stem cells (21). Our strategy of combining xenograft pressing metastasis signal curves in lung (Fig. 2A) and bone (Fig. models with genetically modified mice may help resolve the 4A) revealed an interesting difference in the modes of metasta- functional importance of these intercellular cross-talks during sis enhancement by overexpressing CCL2 in two organs. Lung tumor progression. metastasis burdens of mice injected with LM2-Vec and LM2- Acknowledgments—We thank Min Yuan and Euphemia Mu for tech- CCL2 already displayed a marked difference 1 week after tumor nical assistance. inoculation but maintained similar rates of increase afterward, suggesting that CCL2 may provide an initial advantage in met- astatic seeding in lung but did not confer further growth advan- REFERENCES tages. On the other hand, bone metastasis signals generated by 1. Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J., Murray, T., and Thun, M. J. 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Cell Res. 314, 173–183 29096 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 42 •OCTOBER 16, 2009 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Chemokine (C-C Motif) Ligand 2 Engages CCR2+ Stromal Cells of Monocytic Origin to Promote Breast Cancer Metastasis to Lung and Bone

Lu, Xin; Kang, Yibin
Journal of Biological Chemistry – Oct 1, 2009
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10.1074/jbc.m109.035899
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 42, pp. 29087–29096, October 16, 2009 © 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Chemokine (C-C Motif) Ligand 2 Engages CCR2 Stromal Cells of Monocytic Origin to Promote Breast Cancer Metastasis to Lung and Bone Received for publication, June 22, 2009, and in revised form, August 29, 2009 Published, JBC Papers in Press, August 31, 2009, DOI 10.1074/jbc.M109.035899 ‡1 ‡§2 Xin Lu and Yibin Kang ‡ § From the Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544 and the Breast Cancer Program, Cancer Institute of New Jersey, New Brunswick, New Jersey 08903 Metastatic spread of cancer to distant vital organs, including bone pain, and hypercalcemia (3–5), whereas lung metastasis is lung and bone, is the overwhelming cause of breast cancer mor- accompanied by cough, bloody sputum, rib cage pain, and, tality and morbidity. Effective treatment of systemic metastasis eventually, failure of the respiratory functions (6). Colonization relies on the identification and functional characterization of of different secondary organs by breast cancer is believed to be metastasis mediators to multiple organs. Overexpression of the a complex, multigenic process that depends on productive chemokine (C-C motif) ligand 2 (CCL2) is frequently associated interactions between tumor cells and stromal microenviron- with advanced tumor stage and metastatic relapse in breast can- ments through concerted actions of organ-specific metastasis cer. However, the functional mechanism of CCL2 in promoting genes (7, 8). Functional genomic analysis of preclinical models organ-specific metastasis of breast cancer has not been rigor- of breast cancer to bone, lung, and brain have identified distinct ously investigated. Here, we used organ-specific metastatic sub- sets of organ-specific metastasis genes (9–11), providing novel lines of the MDA-MB-231 human breast cancer cell line to dem- mechanistic insights into key rate-limiting steps of metastasis onstrate that overexpression of CCL2 promotes breast cancer to different organs. However, as advanced breast cancer metastasis to both lung and bone. Conversely, blocking CCL2 patients often suffer from metastases at several secondary function with a neutralizing antibody reduced lung and bone organs, identifying genes that are capable of instigating metas- metastases. The enhancement of lung and bone metastases by tasis to multiple sites may provide the ideal targets for thera- CCL2 was associated with increased macrophage infiltration peutic intervention of systemic metastasis. and osteoclast differentiation, respectively. By performing func- Chemokines are small (8–14 kDa) proteins classified into tional assays with primary cells isolated from the wild type, four conserved groups (CXC, CC, C, and CX3C) based on the CCL2 and CCR2 knock-out mice, we showed that tumor cell- position of the first two cysteines that are adjacent to the amino derived CCL2 depends on its receptor CCR2 (chemokine, CC terminus (12). They are chemotactic cytokines that stimulate motif, receptor 2) expressed on stromal cells to exert its function directed migration of leukocytes in response to inflammatory in promoting macrophage recruitment and osteoclast differen- signals. Chemokines are also involved in the maintenance of tiation. Overall, these data demonstrated that CCL2-expressing hematopoietic homeostasis, regulation of cell proliferation, tis- breast tumor cells engage CCR2 stromal cells of monocytic sue morphogenesis, and angiogenesis (13). Chemokines bind to origin, including macrophages and preosteoclasts, to facilitate the seven-transmembrane domain receptors to elicit down- colonization in lung and bone. Therefore, CCL2 and CCR2 are stream molecular events that coordinate cell movement. Even promising therapeutic targets for simultaneously inhibiting though chemokines are unlikely to be a contributing factor for lung and bone metastasis of breast cancer. tumor initiation, they can have pleiotropic effects on tumor progression (13, 14). Among more than 50 human chemokines, CCL2 is of particular importance. CCL2, also called monocyte Breast cancer is the most common malignancy in women in chemoattractant protein-1 (MCP-1), is a potent chemoattrac- the United States, with an estimated 182,000 new cases and tant for monocytes, memory T lymphocytes, and natural killer 40,000 deaths in 2008 (1). Late stage breast cancer patients cells (15). It is involved in a number of inflammatory conditions develop metastases in bone, lung, liver, brain, and other organs, associated with monocyte recruitment, including delayed which are responsible for most breast cancer-related mortality hypersensitivity reactions, bacterial infection, arthritis, and and morbidity (2). Severe complications from bone metastasis renal disease (15). The importance of CCL2 in cancer was man- include debilitating bone fractures, nerve compression and ifested by its overexpression in a variety of tumor types, includ- ing glioma, ovarian, esophagus, lung, breast, and prostate can- * This work was supported, in whole or in part, by National Institutes of Health cers (15–17). In prostate cancer, CCL2 expression levels was Grant R01CA134519 (to Y. K.). This work was also supported by Depart- associated with advanced pathological stage (16). Importantly, ment of Defense Era of Hope Scholar Award BC051647 and American Can- CCL2-neutralizing antibodies inhibit bone resorption in vitro cer Society Research Scholar Award RSG MGO-110765. Recipient of a Harold W. Dodds Fellowship from Princeton University. and bone metastasis in vivo (18–20). In lung cancer, serum Investigator of the Champalimaud Metastasis Program at Princeton Univer- CCL2 levels were elevated in lung cancer patients with bone sity. To whom correspondence should be addressed: Dept. of Molecular metastasis compared with localized diseases. Neutralizing anti- Biology, WA Road, LTL 255, Princeton University, Princeton, NJ 08544. Tel.: 609-258-8834; Fax: 609-258-2340; E-mail: [email protected]. bodies against CCL2 also inhibited the tumor conditioned This is an Open Access article under the CC BY license. OCTOBER 16, 2009• VOLUME 284 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29087 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis media-induced osteoclast formation in vitro and bone metasta- EXPERIMENTAL PROCEDURES sis in vivo (17). Taken together, these findings suggested a role Cell Culture and Reagents—The SCP28, LM2, and 1833 cell of CCL2 in bone metastasis. lines were derived from the parental cell line MDA-MB-231 A potential role of CCL2 in breast cancer progression and (American Type Culture Collection) (9, 10). These sublines, metastasis has been indicated by the analysis of CCL2 expres- their genetically modified variants, the retroviral packing cell sion of tumor and serum samples from breast cancer patients. line H29, and the mouse monocyte/preosteoclast cell line Serum CCL2 levels were significantly higher in postmeno- RAW264.7 (American Type Culture Collection) were main- pausal breast cancer patients than in age-matched controls tained in Dulbecco’s modified Eagle’s medium supplemented (21). Over 50% of breast cancer tumor samples had intense with 10% fetal bovine serum and antibiotics. The human cell staining of CCL2 in tumor cells (22). Prognostic analysis further line hFOB1.19 (American Type Culture Collection) was main- revealed that high expression of CCL2 was correlated with tained as preosteoblasts in Dulbecco’s modified Eagle’s medi- advanced tumor stage, lymph node metastasis (23), and early um:F12 without phenol red supplemented with 0.3 mg/ml relapse (24). CCL2 up-regulation in breast tumors was also G418 and 10% fetal bovine serum at 34 °C. When switched to associated with the infiltration of tissue-associated macro- 39.5 °C for 2 days after confluence, the cells differentiated into phages (TAMs) and with increased microvessel density (22, mature osteoblasts. Recombinant human CCL2, murine recep- 24). TAMs have been known to contribute to primary tumor tor activator of NF- B ligand and murine macrophage colony progression and metastasis of breast cancer (25), which is sup- stimulating factor were purchased from PeproTech. ported by epidemiological evidence showing that TAM infiltra- Generation of CCL2-overexpressing Cells—The human CCL2 tion portended a poor clinical outcome (26, 27). However, coding sequence was inserted into the HpaI site of the retroviral whether the function of CCL2 in modulating activity of macro- vector pMSCVhyg (Clontech). pMSCVhyg-CCL2 or the vector phages and possibly other cell types is important for breast was transfected into the packaging cell line H29. After 48 h, tumor organotropic metastasis has not been rigorously investi- viruses were collected, filtered, and used to infect target cells in gated. CCL2 may engage organ-specific cell types derived from the presence of 5 g/ml polybrene. The infected cells were the same bone marrow myelomonocytic progenitors. These selected with 1.5 mg/ml hygromycin. To avoid clonal variations, a progenitors differentiate into osteoclast precursors in bone or pooled population of at least 500 independent colonies from each into blood monocytes that eventually become mature macro- transduction was used to generate each stable cell line. phages in different tissues, like alveolar macrophages in lung Western Blot Analysis—To detect CCL2 in conditioned (28). These stromal cell types of myelomonocytic origin may medium by Western blotting, cells were grown to confluence, contribute to different functions in different organ-specific changed to serum-free medium for 24 h before the medium was metastases. Another unresolved question regarding the func- collected, concentrated with Amicon centrifugal filter units tion of CCL2 in tumor-stroma interaction is the functional (Millipore), and applied to immunoblotting. The primary anti- involvement of CCL2 receptors. CCL2 can bind to both CCR2 body was mouse anti-human CCL2 (MAB279, R&D Systems). and CCR4 (29, 30). Loss of function studies in mice showed Anti-mouse IgG secondary antibody conjugated to horseradish CCL2 and CCR2 knock-out mice displayed similar impair- peroxidase was used for chemiluminescence detection (GE ments in monocyte migration (31, 32), suggesting that CCR2 is Healthcare). the major functional receptor for CCL2. Understanding ELISA—To detect CCL2 in the conditioned medium by whether CCR2 deficiency in stromal cells leads to compromised enzyme-linked immunosorbent assay (ELISA), cells were monocyte engagement by CCL2-expressing tumor cells may grown to confluence, changed to fresh medium and cultured have important implications in designing targeting therapeu- for 24 h before the medium was collected. Human CCL2 ELISA tics against the CCL2/CCR2 axis. kit (eBioscience) was used to measure CCL2 concentration fol- In this study, we used the recently developed organ-specific lowing the manufacturer’s instructions. metastatic sublines of the human breast cancer cell MDA-MB- Reverse Transcription (RT)-PCR and Quantitative RT-PCR— 231 (9, 10, 33) and showed that overexpression of CCL2 pro- Total RNA was isolated from adherent cells using RNeasy kit motes both lung and bone metastases. This function was asso- (Qiagen) and reverse-transcribed with Superscript III kit ciated with increased TAM infiltration in lung metastasis and (Invitrogen) according to the manufacturer’s instructions. increased osteoclast differentiation in bone metastasis, respec- Primers used for amplifying cDNA fragments are: human CCR2 tively. Furthermore, by using macrophages and bone marrow (364 bp), 5-gacaagccacaagctgaaca-3 (forward) and 5-gagccc- cells isolated from wild type, CCL2-deficient, and CCR2-defi- acaatgggagagta-3 (reverse); human GAPDH (475 bp), 5-gtca- cient mice, we showed that CCR2 expression in stromal cells is tccatgacaactttggtatc-3 (forward) and 5-ctgtagccaaattcgttgtca- essential for tumor-derived CCL2 to recruit macrophages and tac-3 (reverse); mouse CCL2 (248 bp), 5-aggtccctgtcatgctt- promote osteoclastogenesis. Targeting tumor-derived CCL2 by ctg-3 (forward) and 5-tctggacccattccttcttg-3 (reverse); a neutralizing antibody significantly reduced metastasis forma- mouse CCR2 (252 bp), 5-ggtcatgatccctatgtgg-3 (forward) and tion in both bone and lung. 5-ctgggcacctgatttaaagg-3 (reverse); mouse GAPDH (977 bp), 5-ggtcggtgtgaacggatttgg-3 (forward) and 5-satgtaggccatgag- The abbreviations used are: TAM, tissue-associated macrophage; BLI, biolu- gtccacc-3 (reverse). Quantitative PCR was performed using minescence imaging; TRAP, tartrate-resistant acid phosphatase; ELISA, the SYBR Green PCR Master Mix (Applied Biosystems) with enzyme-linked immunosorbent assay; DAPI, 4,6-diamidino-2-phenylin- the ABI Prism 7900HT thermocycler (Applied Biosystems). dole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. 29088 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 42 •OCTOBER 16, 2009 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis The following primers were used: human CCL2 (66 bp): 5- 1640 containing 100 ng/ml recombinant human CCL2 or RPMI cagccagatgcaatcaatgc-3 (forward) and 5-gcactgagatcttcctatt- 1640 conditioned by confluent tumor cells for 24 h. ggtgaa-3 (reverse); Human GAPDH (225 bp), 5-gaaggtgaagg- Bone Marrow Osteoclastogenesis Assay—Bone marrow cells tcggagtc-3 (forward) and 5-gaagatggtgatgggatttc-3 (reverse). were flushed out from femora and tibiae from 6-week-old mice CCL2 mRNA level was normalized with GAPDH level. and plated in basal culture medium (minimum essential In Vitro Growth Curve and Anchorage-independent Growth— medium  supplemented with 10% fetal bovine serum, 2 mM To establish growth curves, 2  10 cells were seeded in each L-glutamine, and antibiotics) overnight. The next day, the non- well of 6-well plates with triplicate for each time point. Medium adherent cells were plated at 1  10 /well in 24-well plates in was replaced every 2 days. Cell number was determined with basal culture medium supplemented with 30 ng/ml receptor dead cell exclusion using trypan blue every 2 days. For soft agar activator of NF-B ligand and 30 ng/ml macrophage colony growth, a bottom layer of 0.6% agar in growth medium was stimulating factor to induce osteoclast differentiation. When added to 6-well plates with triplicate for each cell line. 2  10 the effect of CCL2 was tested, 100 ng/ml CCL2 was added to the cells were seeded in 0.3% top agar containing growth medium medium or the medium was preconditioned with confluent to each well. Three weeks later, colonies were stained with crys- tumor cells for 24 h. Medium was changed at day 3. TRAP tal violet for visualization. staining was performed on day 6 using a leukocyte acid phos- Tumor Xenografts and Bioluminescence Imaging (BLI) phatase kit (Sigma). Nuclei were stained with 1 g/ml 4,6- Analyses—All procedures involving mice, such as housing and diamidino-2-phenylindole (DAPI). TRAP cells with 3–10 care, and all experimental protocols were approved by Institu- nuclei or 10–30 nuclei were separately quantified and reported tional Animal Care and Use Committee of Princeton Univer- as the number per microscopic field. 5 4 sity. For intracardiac injections, 10 cells were injected into the RAW264.7 Osteoclastogenesis Assay—2  10 RAW264.7 left cardiac ventricle of female nude mice (National Cancer cells were seeded to each well of 24-well plates. Receptor acti- Institute) as described (9). For intravenous injection, 2  10 vator of NF-B ligand (40 ng/ml) was added to the medium to cells were injected into the tail vein of nude mice as described induce differentiation. When the effect of CCL2 was tested, (10). Development of metastases in long bones and lungs was different concentrations of CCL2 was added to the medium. monitored by BLI with the IVIS Imaging System (Xenogen) as Medium was changed at day 2. TRAP staining, DAPI staining, described (10). Analysis was performed with Living Image soft- and quantification were performed on day 4 similar to the bone ware (Xenogen) by measuring photon flux of the region of marrow assay. interest. Data were normalized to the signal obtained immedi- Blocking CCL2 with Neutralizing Antibody—Neutralizing ately after injection (Day 0). X-ray radiography analysis of bone antibody to human CCL2 (MAB679, R&D Systems) or control lesions was performed as described previously (9). mouse IgG were injected intraperitoneally to mice every 4 days Histological Analysis—Hind limb bones and lungs were from the date of tumor cell inoculation to the end of the mouse excised, fixed in 10% neutral-buffered formalin, decalcified (for experiment at dose 10 g/mouse (36). bone only), and embedded in paraffin for hematoxylin and Statistical Analysis—Results were reported as average S.D. eosin staining (9), Goldner’s trichrome staining (34) or tartrate- or average  S.E., indicated in the figure legend. Two-sided resistant acid phosphatase (TRAP) staining (35) as described independent Student’s t test without equal variance assump- previously. The osteoclast number was assessed as multinucle- tion or nonparametric Mann-Whitney test was performed to ated TRAP cells along the tumor-bone interface and reported analyze the data with p  0.05 considered as statistically as number/mm of interface. Immunohistochemical analysis significant. was performed with heat-induced antigen retrieval. The pri- RESULTS mary antibody used was rabbit anti-human von Willebrand Factor (DAKO), rat anti-mouse F4/80 (Abcam), rat anti-mouse CCL2 Overexpression Promotes Lung Metastasis—Two organ- Ly6C (ER-MP20, Abcam), or ER-MP58 as myeloid precursor otropic metastatic sublines of MDA-MB-231 were used for this cells antibody (Abcam). Biotinylated secondary antibody was study: LM2 for lung metastasis (10) and SCP28 for bone metas- used with Vectastain ABC kit (Vector Laboratories) and DAB tasis (9). To evaluate the function of CCL2 in metastasis, human detection kit (Zymed Laboratories, Inc.) to reveal the positively CCL2 was stably overexpressed in LM2 and SCP28, as con- stained cells with nuclei counterstained with hematoxylin. firmed by quantitative RT-PCR (Fig. 1A) and Western blot / / Knock-out Mice—CCL2 and CCR2 mice were gener- analysis of conditioned media (Fig. 1B). CCL2 overexpression ated previously (31, 32) and obtained from The Jackson Labo- did not cause any significant change in the proliferation rate in ratory in C57BL/6 background. The colony was maintained by vitro (Fig. 1C) and anchorage-independent growth on soft agar crossing homozygous siblings. Wild type C57BL/6 mice were (Fig. 1D). These results suggest that CCL2 does not have as used as control. direct role in promoting tumor cell proliferation. Macrophage Chemotaxis Assay—Resident and thioglycol- Functional involvement of CCL2 in lung metastasis was late-elicited peritoneal monocytes/macrophages were har- tested by using LM2 cells with or without CCL2 overexpression vested by peritoneal lavage as described previously (32). Che- in lung metastasis assays. To facilitate noninvasive BLI of met- motaxis assay was performed with 12-well cell culture inserts astatic tumor growth, LM2 and SCP28 had been previously (BD Falcon) following previous methods (31). Migrated cells labeled with a firefly luciferase reporter (37). LM2-Vec (vector were stained with crystal violet for visualization. The agonist control) and LM2-CCL2 were intravenously injected into nude (lower chamber) was either RPMI 1640 medium alone or RPMI mice, and the development of lung metastases were monitored OCTOBER 16, 2009• VOLUME 284 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29089 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis weekly by BLI. As early as 1 week after injection, LM2-CCL2 cells already showed 5-fold stronger signals than LM2-Vec (p  0.027; Fig. 2, A and B), and this trend per- sisted for 5 weeks (p  0.05 for all time points), up to the point when the mice were sacrificed for histo- logical analyses. Lung metastasis nodule quantification and hematox- ylin and eosin staining confirmed the increased pulmonary metastasis burden in mice injected with LM2- CCL2 (Fig. 2, C and D). Lung contains several different types of macrophage, including alveolar, interstitial, intravascular, and airway macrophages (38). As an important function of CCL2 is to mobilize cells of monocyte lineage, we tested whether CCL2 overex- FIGURE 1. Characterization of CCL2 overexpression in MDA-MB-231 sublines. A, CCL2 mRNA level in vector pression caused an increase of infil- control (Vec) and CCL2-overexpressing (CCL2) derivatives of SCP28 and LM2, as measured by quantitative RT-PCR. B, Western blot of the conditioned medium showing the overexpressed CCL2 (doublet bands). Control trating macrophages in lung metas- denotes cells without transfection of plasmid. C and D, in vitro proliferation and anchorage-independent tases. We used an F4/80 antibody, growth of control and CCL2-overexpressing tumor cells. Scale bar, 200 m. In A, C, and D, data represent which recognizes a cell surface gly- average  S.D. coprotein specifically expressed in FIGURE 2. CCL2 overexpression promotes lung metastasis and macrophage infiltration. A, in vivo lung metastasis assay of LM2 cells with or without CCL2 overexpression. BLI data represent average S.E., *, p 0.05 by Mann-Whitney test on each time point. B, representative BLI images showing the development of lung metastasis in mice at different time points after LM2 injection. C, quantification of lung metastasis nodules with a diameter of1 mm during necropsy. p  0.0087 with Mann-Whitney test. D, hematoxylin and eosin staining of lung tissue from the mice injected with LM2 derivatives. T, tumor nodule; L, lung parenchyma. Scale bar, 400 m. E and F, immunohistochemical staining of F4/80, Ly6C, ER-MP58, and von Willebrand Factor (vWF) on the lung sections for quantification of mature macrophages, immature macrophages, and vessel density. Scale bar, 200 m. Data represent average  S.E.; p values by two-sided Student’s t test. Vec, vector control. 29090 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 42 •OCTOBER 16, 2009 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis phage recruitment during lung metastasis. Is CCR2 essential for tumor cell-derived CCL2 to recruit macrophages? Can macrophages lacking CCL2 still be recruited? Answering these questions may have important clinical implications regarding whether targeting CCR2 and/or CCL2 in stroma may achieve the same therapeutic effects as tar- geting CCL2 in tumor cells. We first isolated resident perito- neal monocytes/macrophages from / / wild type, CCL2 , and CCR2 mice and assessed the expression of CCL2 and CCR2 using RT-PCR (Fig. 3A). Wild type macrophages express both genes, whereas those isolated from knock-out mice lack the expression of the corresponding gene. Next, we performed chemo- FIGURE 3. CCL2 in tumor-conditioned medium mobilizes macrophages in a CCR2-dependent manner. taxis assay of isolated resident mac- A, RT-PCR showing the expression pattern of CCL2 and CCR2 in the resident monocytes/macrophages from rophages with recombinant human wild type and knock-out (KO) mice. GAPDH was constitutively expressed in all cases. B, chemotaxis assay of CCL2 or tumor cell-conditioned resident macrophages with recombinant CCL2 or conditioned medium (CM) (by LM2 derivatives) as the ago- nist. C, chemotaxis assay of thioglycollate-elicited macrophages with recombinant CCL2 or conditioned medium as attractants (Fig. 3B). For medium as the agonist. Data represent average S.D., *, p 0.05, **, p 0.01, and ***, p 0.001 by two-sided macrophages isolated from wild Student’s t test (B and C). D, representative images of migrated monocytes/macrophages of the assay in C. Scale type mice, both recombinant CCL2 bar, 100 m. Vec, vector control. and conditioned medium from LM2-CCL2 significantly increased mature tissue macrophages (39). Indeed, CCL2 overexpression the migratory activity (2.7-fold and 2.8-fold, respectively) com- led to increased F4/80 macrophage infiltration by 3-fold pared with CCL2-free medium or medium conditioned by (Fig. 2, E and F). Immature macrophages were stained with two LM2-Vec. This indicates that CCL2 secreted by LM2-CCL2 different myeloid precursor markers Ly6C and ER-MP58 (40), cells elicits similar macrophage-mobilizing potency as recom- and no significant differences in their numbers were observed binant CCL2. Similar macrophage chemotaxis-promoting between two groups of samples (Fig. 2, E and F). CCL2 has also effect was observed when macrophages isolated from CCL2 been reported to affect angiogenesis (41). However, we did mice were used in the experiment (Fig. 3B, CCL2-KO), suggest- not observe significant change of microvessel density ing that endogenous CCL2 expression is not required for mac- between control and CCL2-overexpressing pulmonary rophage migration in response to CCL2. In contrast, CCR2 metastases, as revealed by von Willebrand Factor staining deficiency led to complete loss of CCL2-induced macrophage (Fig. 2, E and F), suggesting that elevated angiogenesis is not mobility (Fig. 3B, CCR2-KO). This result suggests that CCR2 is the cause of the increased lung metastasis burden in our a receptor essential for increased macrophage migration experimental model. Instead, the observation that CCL2 induced by CCL2-overexpressing tumor cells. overexpression already led to more lung metastasis at early Resident macrophage behavior may not necessarily reflect time point (week 1) suggests that the enhancement of tumor the behavior of TAMs as the latter is usually associated with invasion and initial lung colonization mediated by CCL2- inflammation. Therefore, we performed the same assays using recruited macrophages may be the main reason for the over- macrophages elicited by thioglycollate-induced nonspecific all increased lung metastasis burden. inflammation. Monocytes/macrophages, which constitute CCL2-overexpressing Tumor Cells Promote Macrophage most of the elicited leukocyte population 72 h after the intra- Chemotaxis in a CCR2-dependent Manner—Both of the CCL2 peritoneal thioglycollate instillation (32), were harvested from / / receptors, CCR2 (29) and CCR4 (30), are widely expressed in a wild type, CCL2 , and CCR2 mice. Consistent with pre- variety of cell types, including monocytes and macrophages (42, vious reports, the recruitment of macrophages was significantly 43). CCR2- and CCR4-deficient mice displayed similar defects reduced in the two mutant mice (data not shown). Neverthe- in macrophage recruitment in experimental inflammation con- less, equal numbers of cells were collected and applied to the ditions (31, 43). Similar impairments in monocyte migration chemotaxis assays. Similar to resident macrophages, thiogly- was also observed in mice lacking CCL2 (32, 44), suggesting collate-elicited macrophages were mobilized by recombinant that CCL2 may have a cell autonomous function in monocyte CCL2 or tumor cell-derived CCL2, and such an effect was again mobilization. These observations prompted us to investigate found to be dependent on the intact expression of CCR2, but two related questions about the function of CCL2 in macro- not CCL2, on macrophages (Fig. 3, C and D). Taken together, OCTOBER 16, 2009• VOLUME 284 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29091 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis bone-tumor interface in metastatic lesions formed by SCP28-CCL2 as compared with bone metastasis of SCP28-Vec (Fig. 4D). Because oste- oclast activity is critical for the establishment of osteolytic bone metastasis (3–5), we hypothesize that CCL2-overexpressing breast cancer cells become more efficient in colonizing the bone through increased activation of osteoclasts. CCL2-overexpressing Tumor Cells Promote Osteoclast Differentiation in a CCR2-dependent Manner—Previ- ous studies have shown that recom- binant CCL2 protein can promote osteoclast fusion and differentiation in vitro (45–48). Here, we wanted to test whether tumor cell-derived CCL2 could similarly elicit oste- oclast activation and whether this is dependent on CCR2 expressed on osteoclast progenitor cells. Primary bone marrow cells isolated from wild type mice expressed both CCL2 and CCR2, which were lost in CCL2 FIGURE 4. CCL2 overexpression enhances osteolytic bone metastasis and associated osteoclasts. A, in and CCR2 knock-out mice, respec- vivo bone metastasis assay of SCP28 cells with or without CCL2 overexpression. BLI data represent average S.E., *, p 0.05, **, p 0.01, and ***, p 0.001 by Mann-Whitney test on each time point. B, representative BLI tively, as confirmed with RT-PCR images showing the development of bone metastasis in mice at different time points after SCP28 injection. (Fig. 5A, left panel). Bone marrow Arrows, bone metastases in the hind limbs. C, representative x-ray radiographs of the hind limbs at week 6 after cells isolated from different geno- injection showing the osteolytic lesions (arrows) in proximal tibia. D, Goldner’s trichrome and TRAP staining (with quantification) of proximal tibia from the mice injected with SCP28 derivatives. B, bone matrix; T, tumor types were applied to an in vitro area. Scale bar, 200 m. Data represent average  S.E., p values by two-sided Student’s t test. Vec, vector osteoclastogenesis assay. Bone mar- control. row growth medium precondi- these results suggest that tumor cell-derived CCL2 induces tioned with SCP28-Vec or SCP28-CCL2 cells for 24 h were directional migration of monocyte/macrophages in a CCR2- added to the bone marrow culture to compare their ability to dependent manner, which may be responsible for the elevated induce the formation of multinucleated TRAP osteoclasts. infiltration of macrophage in the lung metastases formed by Recombinant CCL2 was used as the positive control. The num- LM2-CCL2 cells. ber of nuclei in the osteoclasts indicates the efficiency of cell CCL2 Overexpression Promotes Osteolytic Bone Metastasis— fusion during differentiation. Therefore, we classified oste- To test whether CCL2 overexpression could also increase bone oclasts into two types: cells with 3–10 nuclei (less efficient metastasis formation, vector control and CCL2-overexpressing fusion) and cells with 10–30 nuclei (more efficient fusion) by derivatives of SCP28 were injected into the left ventricle of nude counting DAPI-stained nuclei. In the wild type bone marrow, mice, and bone metastases development in the hind limbs were we found the number of TRAP osteoclasts with 10–30 nuclei quantitatively monitored by BLI (Fig. 4, A and B). Similar BLI was expanded significantly by recombinant CCL2, whereas the intensities were found in both groups 1 week after injection. number of osteoclasts with 3–10 nuclei was unaffected (Fig. 5, However, starting from week 2, bone metastasis burden in the A and B, wild type). When bone marrow cells from mutant mice CCL2 overexpression group became significantly higher than were used, both CCR2- and CCL2-deficient osteoclast progen- that in the vector group until the end of the experiment. Cor- itor cells could be induced to form TRAP osteoclasts with responding to the increased bone metastasis burden, mice inoc- 3–10 nuclei. However, bone marrow cells deficient in CCR2, ulated with SCP28-CCL2 cells also had a larger area of bone unlike wild type and CCL2-deficient cells, significantly lost the lesion as revealed by x-ray radiography (Fig. 4C). Consistently, responsiveness to recombinant CCL2 to form larger osteoclasts the tumor-bone interface in bone metastasis lesions formed by with 10–30 nuclei, indicating the critical role of CCR2 as the SCP28-CCL2 were typically more rugged than that those receptor for the function of CCL2 in stimulating osteoclast formed by SCP28-Vec, as shown by Goldner’s trichrome stain- fusion. Importantly, we observed that CCL2 derived from ing highlighting the collagen-rich bone matrix in green (Fig. 4D, SCP28-CCL2 medium was equally able to promote osteoclast Trichrome). Furthermore, when we used TRAP staining to differentiation in a CCR2-dependent manner, supporting our compare osteoclast density in the metastases, significantly more (6-fold) TRAP osteoclasts were observed along the hypothesis that CCL2-overexpressing tumor cells promote 29092 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 42 •OCTOBER 16, 2009 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis FIGURE 5. CCL2 in tumor-conditioned medium promotes osteoclast differentiation in a CCR2-dependent manner. A, RT-PCR analysis of CCL2 and CCR2 expression and osteoclastogenesis assay of nonadherent bone marrow cells from wild type and knock-out (KO) mice. The differentiation medium was either supplemented with recombinant CCL2 or preconditioned by SCP28 derivatives. At day 6, TRAP cells with 3–10 nuclei (middle panel) or 10 –30 nuclei (right panel) were quantified and reported as number per field (under a 5 objective lens). Data represent average S.D. ***, p 0.001 by two-sided Student’s t test. B, representative images of TRAP osteoclasts with nuclei stained by DAPI (under a 10 objective lens). Scale bar, 100 m. C, RT-PCR showing CCR2 expression of mature osteoblast hFOB1.19 (left) and preosteoclast RAW264.7 (right). Positive () control was universal reference RNA (Stratagene), and negative () control was water. GAPDH was constitutively expressed in all cases. D, histograms and representative images of differentiated RAW264.7 cells with cell fusion enhanced by increasing concentrations of recombinant CCL2. Scale bar, 400 m. CM, conditioned medium; Vec, vector control. osteoclast activation during the formation of osteolytic bone resultant multinucleated TRAP osteoclasts (Fig. 5D), we metastasis. observed a clear dose-dependent increase of nuclear number in CCL2 may exert its function in promoting osteoclast differ- the induced osteoclasts by CCL2, supporting its direct role in entiation directly through preosteoclasts or indirectly through promoting cell fusion during osteoclast differentiation. osteoblasts, which is often the cell type required for paracrine Inhibition of CCL2 Reduces Bone and Lung Metastases—We activation of osteoclast differentiation. To differentiate these wished to evaluate whether inhibiting CCL2 function could two possible mechanisms, we determined the levels of CCR2 reduce metastasis to bone and lung. To this end, we used a mRNA expression in osteoblast cell line hFOB1.19 and preos- neutralizing antibody for human CCL2 to block the function of teoclast cell line RAW264.7 with RT-PCR (Fig. 5C). Expression tumor-derived CCL2 in metastasis assays in vivo. Two strongly of CCR2 was not detected in hFOB1.19, consistent with the metastatic sublines derived from in vivo selection of MDA-MB- previous observation in another osteoblast line, UMR 106–01 231 (the lung-tropic LM2 and the bone-tropic 1833 (9)) were (46). In contrast, CCR2 is expressed in RAW264.7 cells. This used in the experiment. The 1833 subline has a much stronger result indicates that CCL2 should function directly through bone metastasis ability than SCP28 and is therefore more CCR2 on preosteoclasts to promote its cell fusion and differen- appropriate to be used for testing the possible effect of CCL2 tiation. Indeed, when we treated RAW264.7 cells under the inhibition on reducing bone metastasis. 1833 and LM2 sublines differentiation culture condition with an increasing dose of express considerable amount of CCL2 at the basal level, as recombinant CCL2 and profiled the number of nuclei in the measured by ELISA of the conditioned media (2.41  0.42 OCTOBER 16, 2009• VOLUME 284 • NUMBER 42 JOURNAL OF BIOLOGICAL CHEMISTRY 29093 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis FIGURE 6. Alteration of organotropic metastasis behavior by targeting CCL2 with neutralizing antibody in highly metastatic cells and by overexpressing CCL2 in weakly metastatic cells. A, in vivo bone metastasis assay of highly bone metastatic cell line 1833 with mice treated with control IgG or neutralizing antibody against human CCL2. B, in vivo lung metastasis assay of highly lung-seeking line LM2 with mice treated with control IgG or neutralizing antibody against human CCL2. C, in vivo lung metastasis assay of weakly lung-metastatic cell line SCP28 with or without the overexpression of CCL2. D, in vivo bone metastasis assay of weakly bone metastatic cell line LM2 with or without the overexpression of CCL2. In A–D, BLI data represent average  S.E. *, p  0.05, **, p  0.01, and ***, p  0.001; with no asterisk shown, p  0.05 by Mann-Whitney test on each time point. Vec, vector control. ng/ml for 1833 and 0.77  0.01 ng/ml for LM2), whereas fresh metastasis potential by CCL2 overexpression. However, medium does not contain any detectable level of human CCL2. CCL2 overexpression was not sufficient to enhance bone 1833 and LM2 were inoculated into nude mice by intracardiac metastasis for the otherwise weakly bone metastatic subline or intravenous injections, respectively, to generate bone and LM2 (Fig. 6D). Because metastasis ability is known to rely on lung metastases. Antibodies or isotype IgG controls were concerted function of multiple genes (9, 10), it is not surpris- injected into mice from the date of tumor inoculation until the ing to observe variable results in this test. The ability of end of the experiment. Both 1833-generated bone metastasis CCL2 to confer organotropic metastasis potential may and LM2-generated lung metastasis were reduced significantly depend on the genetic background of the cell lines (i.e. the by antibody treatment (Fig. 6, A and B). These results indicate existence of cooperating organotropic metastasis genes). that inhibition of CCL2 is effective in suppressing metastasis DISCUSSION formation in both bone and lung. Overexpression of CCL2 Alone in Weakly Metastatic Cells Is Suf- Chemokines and their receptors play diverse roles in malig- ficient to Promote Lung but Not Bone Metastasis Potential— nant tumor progression, particularly as key mediators of Although overexpression of CCL2 promotes metastasis to tumor-stroma interactions (12, 13, 49). The best characterized lung or bone in cell lines that already display significant basal example is the function of SDF-1/CXCR4 axis in mediating metastatic abilities to the respective organs (e.g. SCP28 to site-specific metastasis for various cancer types (50). Data pre- bone and LM2 and lung, Figs. 2A and 4A), we wished to test sented here demonstrate that CCL2 has dual activity to pro- whether CCL2 overexpression can render an otherwise mote both lung and bone metastases of breast cancer cells weakly metastatic or non-metastatic cell capable of metasta- through distinct functions. When CCL2 was overexpressed, sizing to bone or lung. We injected vector control or CCL2- lung metastasis development was accelerated with increased overexpressing variants of SCP28 (weakly lung metastatic) to infiltration of F4/80 macrophages. Similarly, CCL2-overex- the tail vein (Fig. 6C) and found a significant increase of lung pressing tumor cells developed more extensive osteolytic 29094 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 42 •OCTOBER 16, 2009 CCL2/CCR2 Axis Promotes Breast Cancer Metastasis bone lesions with increased recruitment and activation of bone microenvironment (the “metastasis niche”) (2) or to form TRAP osteoclasts in the tumor-bone interface. Impor- small clusters of micrometastases. Neither of these events may tantly, by using primary cells isolated from wild type, be dependent on CCL2. Instead, CCL2 promotes bone metas- / / CCL2 , and CCR2 mice, we showed that tumor-derived tasis expansion through inducing osteoclast differentiation and CCL2 increases the directional migration of macrophages and establishment of the “vicious cycle” of bone metastasis (2), promotes osteoclast differentiation in a CCR2-dependent man- which occurs after the initial adaptation. Recognizing the ner. Furthermore, we demonstrated that targeting CCL2 in organ-specific modes of metastatic function of CCL2 has tumor cells with a neutralizing antibody inhibited metastasis potential clinical implications, as treatment of lung metastasis formation in lung and bone. Overall, these results suggest that may benefit from CCL2-targeting therapy only if the treatment CCL2 and CCR2 as targets for therapeutic intervention of bone is applied early, whereas macroscopic bone metastasis may still and lung metastasis. be sensitive to the inhibition of CCL2. CCL2 expression can be activated in advanced breast cancer It is generally believed that macrophages and bone marrow through genetic or epigenetic mechanisms. An infrequent poly- osteoclasts share a common monocytic hematopoietic progen- morphism in the CCL2 promoter region associated with itor. Several lines of evidence support this hypothesis. op/op increased CCL2 expression is overrepresented in metastatic mice (spontaneous colony-stimulating factor 1 mutation) have breast cancer patients (49). Several pathways involved in epi- severe deficiency of both osteoclasts and macrophages (55). thelial-mesenchymal transition, including dysadherin (51) and Knock-out of transcription factor PU.1 in mice caused absence -catenin/TCF-4 (52), promote CCL2 up-regulation. Here, we of both cell types, suggesting defects at an early common mye- used ectopic overexpression and blocking antibody to assess its loid differentiation stage (56). It is conceivable that common pro-metastatic function in the MDA-MB-231 breast cancer molecule(s) involved in regulating the homeostasis of both metastasis models. We chose this model because of the unique macrophages and osteoclasts can be hijacked by tumor cells to repertoire of organ-specific metastatic sublines that was simultaneously modulate the activity of both cell types in the recently developed (9–11). CCL2-mediated tumor- and metas- tumor microenvironment to promote tumor growth. Indeed, tasis-promoting functions have been investigated in other pre- our results showed that breast tumor cells are able to recruit clinical models. For example, CCL2-mediated macrophage macrophages in the lung parenchyma as well as enhancing oste- infiltration and increased tumor growth were reported in the oclast differentiation in the bone marrow by engaging the same gastric carcinoma nude mouse model (53). CCL2 inhibition by CCL2/CCR2 axis on these cells. Thus, pharmacological inhibi- short hairpin RNA-mediated knockdown or by systemic deliv- tors targeting CCL2 or CCR2 in breast cancer patients whose ery of neutralizing antibodies inhibit the development of bone tumor tissue expresses a high level of CCL2 may bring clinical metastasis by prostate cancer (19, 54). However, the functional benefits for controlling distant metastases in multiple organs. importance and cellular mechanism of CCL2 in promoting The CCL2/CCR2 axis may have prometastasis functions organ-specific metastasis of breast cancer metastasis has not beyond the direct engagement of stromal cells of monocytic been rigorously investigated. In addition to using the MDA- origin by tumor cells. CCL2/CCR2 may mediate a cascade of MB-231 in vivo models to study the functional importance of signaling events to enhance tumor malignancy. For example, CCL2 in promoting bone and lung metastasis, our current MDA-MB-231 cells can induce osteoblast hFOB1.19 and study also took advantage of the existing CCL2 and CCR2 MC3T3-E1 to up-regulate CCL2 (57), and CCL2 produced by knock-out mice and identified CCR2 as the essential receptor osteoblasts can promote osteoclast differentiation (47, 48). Fur- for mediating the function of CCL2 in macrophage recruitment thermore, tumor-derived CCL2 can exert function on other and osteoclast activation by breast cancer. CCR2 stroma cells, such as endothelial cells (41) and mesen- Careful comparison of the vector control and CCL2-overex- chymal stem cells (21). Our strategy of combining xenograft pressing metastasis signal curves in lung (Fig. 2A) and bone (Fig. models with genetically modified mice may help resolve the 4A) revealed an interesting difference in the modes of metasta- functional importance of these intercellular cross-talks during sis enhancement by overexpressing CCL2 in two organs. Lung tumor progression. metastasis burdens of mice injected with LM2-Vec and LM2- Acknowledgments—We thank Min Yuan and Euphemia Mu for tech- CCL2 already displayed a marked difference 1 week after tumor nical assistance. inoculation but maintained similar rates of increase afterward, suggesting that CCL2 may provide an initial advantage in met- astatic seeding in lung but did not confer further growth advan- REFERENCES tages. On the other hand, bone metastasis signals generated by 1. Jemal, A., Siegel, R., Ward, E., Hao, Y., Xu, J., Murray, T., and Thun, M. J. 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