Cyclooxygenase-2 Inhibition Blocks M2 Macrophage Differentiation and Suppresses Metastasis in Murine Breast Cancer Model

Tumor cells are often associated with abundant macrophages that resemble the alternatively activated M2 subset. Tumor-associated macrophages (TAMs) inhibit anti-tumor immune responses and promote metastasis. Cyclooxygenase-2 (COX-2) inhibition is known to prevent breast cancer metastasis. This study hypothesized that COX-2 inhibition affects TAM characteristics potentially relevant to tumor cell metastasis. We found that the specific COX-2 inhibitor, etodolac, inhibited human M2 macrophage differentiation, as determined by decreased CD14 and CD163 expressions and increased TNFα production. Several key metastasis-related mediators, such as vascular endothelial growth factor-A, vascular endothelial growth factor-C, and matrix metalloproteinase-9, were inhibited in the presence of etodolac as compared to untreated M2 macrophages. Murine bone marrow derived M2 macrophages also showed enhanced surface MHCII IA/IE and CD80, CD86 expressions together with enhanced TNFα expressions with etodolac treatment during differentiation. Using a BALB/c breast cancer model, we found that etodolac significantly reduced lung metastasis, possibly due to macrophages expressing increased IA/IE and TNFα, but decreased M2 macrophage-related genes expressions (Ym1, TGFβ). In conclusion, COX-2 inhibition caused loss of the M2 macrophage characteristics of TAMs and may assist prevention of breast cancer metastasis.


Introduction
Tumor associated macrophages (TAMs) and the factors they release amplify various aspects of cancer, including anti-tumor immune suppression, stimulation of tumor progression, and formation of metastases [1][2][3]. High levels of TAMs are often correlated with bad prognosis, and several recent studies have highlighted a link between their abundance and the metastatic process [4][5][6]. Macrophage population ablation by genetic and pharmacological approaches can counter subsequent cancer development [7][8][9][10].
Analogous to the Th1 and Th2 dichotomy of T cell polarization, macrophages can be polarized by the microenvironment to mount specific M1 (classically activated) or M2 (alternatively activated) functional programs [11][12][13]. TAMs exhibit a predominantly M2-like phenotype [13,14]. This preferential polarization is due to the abundance of M2 stimuli as well as the absence of M1-orienting signals in the tumor, such as IFN-c or bacterial components. Indeed, M2-macrophages differentiate from monocytes in response to specific growth factors released by both malignant and stromal tumor compartments, including CCL2, M-CSF, vascular endothelial growth factor (VEGF) and CXCL12 (also known as SDF1) [2,15,16]. Macrophage polarization is also regulated by post-stimulations such as the M2a stimulants IL-4 and IL-13, M2b stimulants including immune complexes/TLR ligands, and IL-10 and glucocorticoids polarize macrophages into the M2c subtype [13,17]. In general, the hallmarks of M2macrophages are production of IL-10 high , IL-12 low , IL-1RA high , IL-1decoyR high , CCL17 high , and CCL22 high . In addition, M2macrophages exhibit high expression of mannose, scavenger and galactose-type receptors, poor antigen-presenting capability, wound healing promotion, debris scavenging, angiogenesis, and tissue remodeling through high expression of VEGF, cyclooxygenase-2 (COX2), epidermal growth factor receptor (EGFR), and metalloproteinases (MMPs) [18][19][20]. In a tumor context, macrophages act as a guardian and induce anti-tumor immune responses in the early stages but in the later stages as the tumor progresses, macrophages enhance tumorigenesis and metastasis [19]. Macrophage polarization is the key step that accelerates tumor aggressiveness. However, data regarding the molecular mechanisms of macrophage polarization remain sparse.
Epidemiological studies have shown that regular intake of nonsteroidal anti-inflammatory drugs (NSAIDs) such as aspirin, the prototypic inhibitor of cyclooxygenase (COX), can reduce the risk of development of some cancers [21]. Thus far, several mechanisms by which COX-2 contributes to cancer progression have been reported, including stimulation of proliferation and inhibition of apoptosis of cancer cells, stimulation of cancer cell invasion and angiogenesis, and suppression of immune responses [22,23]. In addition to its effects on tumor cells, COX is the key enzyme induced when macrophages are activated for synthesis of inflammatory mediators. These mediators include prostaglandin E2, prostacyclin I2, and thromboxane A2. Other tumor-related molecules, such as VEGF-A and VEGF-C, can also be induced by COX-2, as demonstrated by data that COX-2 inhibition suppresses lymph node metastasis via reduction of macrophagemediated lymphangiogenesis [24]. These data provide evidence that COX-2 participates in macrophage polarization, but its exact role has not been elucidated.
Macrophages are related to tumor growth, metastasis, and relapse. Macrophage-mediated immune suppression is correlated with increased CD4 + CD25 + regulatory T cell infiltration and reduced CD8 + cytotoxic T cell function. The proteinase secretion capacities of macrophages directly lead tumor cells for moving through the extracellular matrix degradation [25]. Additionally, recruitment of CD11b + myeloid cells facilitates tumor regrowth after local irradiation therapy [26]. Therefore, various therapeutic applications to enhance tumor immunity have been attempted, including anti-TGFb antibodies, anti-CCR4 antibodies, anti-CTLA4 or Programmed Death-1 (PD-1) inhibitory molecules, TLR7 or Bacillus Calmette-Guerin (BCG) boosting therapy, and IL-2 or IFNc treatment.
To clarify the role of COX-2 inhibition in macrophage function in tumor context, we conducted human and mouse macrophage differentiation with COX-2 inhibitor, etodolac. Here we demonstrate that COX-2 inhibition blocks M-CSF-induced M2 macrophage differentiation and drives pro-inflammatory activities in human and murine macrophages. Regular etodolac intake inhibits breast cancer metastasis in relation to reducing M2 macrophage functions. Our finding suggest that COX-2 inhibition may inhibit M2 macrophage differentiation and polarization, thus suppressing tumor metastasis.

Ethics statement
All human blood acquisitions were approved by the Institutional Review Board of Seoul National University, Korea (SNUIBC-R120713-1). Documented written informed consents provided by Ethics Committee were obtained from all participates in this study. All animal procedures were performed according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals prepared by the Institution of Animal Care and Use Committee of Seoul National University, Korea. The protocol was approved by Seoul National University Institute Animal Care and Use Committee (Approval Number SNU-10009-2). All surgical procedure was performed under zoletil (Virbac)/xylazine (Bayer) anesthesia, and all efforts were made to reduce unnecessary pain.

Human and murine macrophage differentiation and reagents
Human peripheral blood mononuclear cells (PBMC) were isolated from buffy coats of normal donors over a Ficoll-paque PLUS (GE Healthcare) gradient, according to standard procedures. Monocytes were purified from buffy coats by magnetic cell sorting using the human monocyte isolation kit II (Miltenyi Biotec). Monocytes (.90% CD14 + cells) were cultured for 7 days at a density of 10 6 /ml in RPMI media (Thermo) supplemented with 10% FBS (Gibco), 10 units/ml penicillin, and 10 mg/ml streptomycin and 2 mM L-glutamine (Gibco) (hereafter termed complete media) at 37uC in a humidified atmosphere with 5% CO 2 . For differentiating human M2 macrophages, 20 ng/ml human M-CSF (Prospec) were supplemented into complete media and for M1 macrophages, 25 ng/ml human GM-CSF (Prospec) were used. Murine bone marrow cells were obtained from 7,10 weeks of BALB/c female and differentiated into mature macrophages during 7 days in 10% L929 murine fibrosarcoma cell line culture supernatants supplemented complete media. Etodolac (Yuhan Corporation, Korea) was dissolved in DMSO and treated at 20 mM for monocyte differentiation. Where indicated, macrophages were activated for 12 hours (hrs) with LPS (100 ng/ml; Salmonella enterica, Sigma Aldrich) and IFNc (25 ng/ml, Prospec). 15-deoxy-D12,14-PGJ 2 in methyl acetate was obtained from Cayman Chemicals and added twice (at the indicated concentrations, 0.1-2 mM), at the start of the culture and on day 5 of macrophage differentiation.
4T1 syngeneic mouse breast cancer model. The 4T1 murine breast cancer cell line was obtained from ATCC and maintained in RPMI complete media. The 4T1_GFP cell line was generated through copGFP viral particle (Santa Cruz Biotechnology) infection followed by selection in 4 mg/ml puromycin for 2 weeks. Six-week-old female BALB/c mice were purchased from Orient Bio (Korea) and maintained in pathogen-free housing. For orthotopic implantation of tumor cells, mice were anesthesized with zoletil (Virbac)/xylazine (Bayer) and a total of 10 5 4T1 cells (suspended in 100 ml ice cold PBS) were injected into the right inguinal mammary fat pad of seven-week old mice. Etodolac was fed at 500 ppm throughout the experimental period. Animal weight and tumor size were measured twice per week. Tumor volumes were calculated as 0.56length6width 2 . Necropsy was performed 16 and 23 days after tumor cell implantation. After administration of anesthetic overdose, blood was collected by cardiac puncture and mice were sacrificed by cervical dislocation. Lungs were removed from each mouse, surface lung nodules and sizes which could be identified grossly were blindly measured. In the case of 4T1_GFP cells, lung images were captured using a fluorescence microscope (Leica M165FC) and GFP intensity was quantified using Image J software. Resected lung, spleen, and the primary tumor mass were processed for further examination.

Reverse Transcription Polymerase Chain Reaction (RT-PCR)
For real-time RT-PCR, mRNA from maturated macrophages or CD11b-positive magnetic cell sorted (Miltenyi Biotec) murine spleen macrophages and tumor-associated macrophages [27] were analyzed in duplicate. cDNA was synthesized from 1 mg total RNA using Maxime RT Premix (Intronbio, Korea) [28]. The primers and probes used for PCR analyses were: human TGFbeta

Histology
Lung tissues were resected, formalin-fixed, and paraffinembedded using standard methods. Tissue sections were evaluated microscopically for tumor progression indices using H&E staining.

ELISA
Differentiated macrophages were stimulated with appropriate concentrations of LPS/IFNc for 12 hrs. Supernatants were tested for the presence of cytokines using a commercially available multiplex ELISA for IL-1b. ELISA was performed for IL-6 (R&D Systems).

Confocal microscopy and image analysis
A total of 105 human monocytes per chamber were seeded onto Lab-Tek four-chamber slides (Nunc) and allowed to differentiate for 7 days. Macrophages were washed once with PBS and fixed with 4% paraformaldehyde. Chambers were washed 3 times (5 min each) with PBS followed by 300 nM DAPI (Invitrogen) staining for 1 min at room temperature in the dark. Chambers were washed 3 times (5 min each), plastic chamber inserts were removed, and slides were coverslipped with ProLong Gold antifade reagent (Invitrogen). Slides were evaluated and captured with an OLYMPUS FluoView 1000 Confocal Microscope (OLYMPUS). Merged images were created with OLYMPUS FLUOVIEW Viewer Software, version 2.0. Attached macrophages were quantified using Image J software and plotted using GraphPad software, version 5.0.

Bacterial infection
Human mononocytes (0.5610 6 ) in 500 ml of complete media were differentiated for 7 days on a 24-well plate with or without etodolac treatment. Staphylococcus aureus (serotype V) was grown in Luria broth with agitation at 37uC to an OD 600 of 0.4, which is equivalent to 1610 8 cfu/ml. A total 0.2610 6 cfu of unwashed S. aureus was used to infect each well. Plates were centrifuged for 5 min at 4006g and incubated for 1 hr. The resulting supernatants were subjected to serial 10-fold dilution in water prior to dispensing 100 ml of each dilution onto agar plates. The plates were incubated upside down at 37uC, and bacterial colonies were counted 24 hrs later.

Serum High Performance Liquid Chromatography (HPLC)
HPLC was performed on a Hewlett-Packard 1100 series (Korea Basic Science Institute, Seoul). The etodolac standard and samples were separated on OP-C18 (25064.6 mm, 5 mm, RStech Corporation) and detected by absorbance at 274 nm. The mobile phase was 0.05% H 3 PO 4 : acetonitrile (1:1) and the flow rate was 1.0 ml/ min. Injection volume was 100 ml. For quantification of etodolac in samples, a linear calibration plot was obtained in the concentration range 0.5-5.0 mM.

MTT assay
4T1 cells (10 4 /500 ml complete media) were seeded on a 24-well plate and allowed to attach overnight. Etodolac was added at 0, 10, 20, 50, 100, and 500 mM. An MTT assay was performed at each time point (24,48, or 72 hrs). Fifty microliters MTT (Sigma Aldrich) was added to each well and further incubated for 4 hrs at 37uC with 5% CO 2 . After washing off supernatants, 500 ml of 100% DMSO was added and the plate was shaken for 5 min. The absorbance at 560 nm was measured using a Beckman ELISA reader.
Statistical analyses. Student's t-tests were performed to determine statistically significant differences between groups using GraphPad Prism (GraphPad Software, CA, USA). A P-value ,0.05 was considered significant.

Etodolac inhibits human alternatively activated macrophage phenotype
To mimic the tumor microenvironment in vitro culture conditions, we used M-CSF as a tumor-associated, macrophageassisted growth factor. Normal human peripheral blood monocytes were isolated using magnetic bead negative selection. After differentiation for 7 days with 20 ng/ml M-CSF in the presence or absence of etodolac, macrophages were examined for surface marker expressions (Fig. 1). Scatter plots showed that etodolactreated macrophages have distinct subpopulations compared to those treated with M-CSF alone (Fig. 1A). M-CSF-induced macrophages had CD14/CD163 double positive population which represents M2 polarization (Fig. 1B) but etodolac abolished CD163 induction almost completely, and partially abolished CD14 induction. Etodolac also increased CD11b expression (Fig. 1C). As shown in Figure S1A and S1B, GM-CSF-induced classically activated macrophages from human monocytes do not have CD14/CD163 double positive population but had higher CD11b expression compared to M-CSF-induced M2 macrophages [29]. We supposed from these results that COX-2 activity might be required for M2 macrophage differentiation. Other macrophage characteristics were also examined. Macrophage morphology often represents their activation state, and we confirmed that etolodac induced different macrophage morphologies (Fig. S1C). M-CSF-treated macrophages at 20 ng/ml concentration exhibited predominantly a smaller and crumpled morphology whereas etodolac-treated macrophages were larger and elongated. Etodolac increased macrophage attaching ability but reduced macrophage phagocytic ability upon Staphylococcus aureus infection (Fig.  S1D), indicating reduced scavenger receptor expression, which is associated with the classically activated macrophages [28]. Collectively, these in vitro data suggest that etodolac altered human M2 macrophage differentiation process and that COX-2 might be required to normal alternatively activated macrophage development.

Etodolac induces pro-inflammatory cytokines but inhibits pro-metastatic molecules in human macrophages
Classically activated macrophages induce rapid inflammation, mediated mainly by various early cytokines, such as TNFa, IL-1b, and IL-6 upon stimuli. In contrast, alternatively activated macrophages secrete IL-10 or TGFb and contribute to tissue homeostasis. To determine whether etodolac-treated macrophages exhibit a different cytokine panel, we first examined intracellular TNFa accumulation during 5 hrs upon LPS stimulation ( Fig. 2A) using FACS. As expected, macrophages produced increased TNFa in the presence of etodolac. Basal IL-10 synthesis levels were lower in the etodolac treated macrophages. IL-1b and IL-6 in the culture supernatant of M-CSF/etodolac-treated macrophages after LPS/INFc stimulation also increased than control (Fig. S1E). In addition to cytokines, several tumor metastasisrelated genes showed the same tendency. Etodolac reduced the gene expression levels of TGFb, VEGFA, VEGFC, MMP-9, and MMP-1 in macrophages (Fig. 3). Taken together, these data further indicate that COX-2 is required for normal M2 or tumorassociated macrophage differentiation.

Etodolac induced more immune activated murine macrophages
To determine if these effects of COX-2 inhibition are also applied in murine macrophages, BALB/c bone marrow derived macrophages were tested for their markers and cytokine expressions. M-CSF secreting cell line L929 culture supernatants were supplemented in 10% as tumor microenvironmental monocyte differentiating factors, and these condition gives about 50% of CD11b F4/80 double positive mature macrophage population (Fig. 4A). Etodolac induced more MHCII IA/IE and co-receptors CD80 and CD86 on macrophage surfaces both in 20 and 100 mM (Fig. 4B). In the case of cytokines, TNFa expression levels were higher but IL-10 was lower in etodolac treated macrophages. These results showed that COX-2 inhibition also reduced murine M2 macrophage function.

Etodolac inhibits lung metastasis in a murine syngeneic breast cancer model
Next, to examine the effects of etodolac on tumor progression, we used a murine syngeneic breast cancer model. Female BALB/c mice (age 6 weeks) were fed food containing 500 ppm etodolac, starting one week before injecting with 105 4T1 murine breast tumor cells in the right mammary fat pad until the final sacrifice date. We did not observe any side effects of etodolac treatments during the experimental periods, as represented by mouse feeding and weight data (Fig. S2A). There have been several reports regarding direct COX-2 mediated tumorigenesis [22,30], but we did not observe any COX-2-mediated cellular growth advantage in the 4T1 cell line (Fig. S2B). Consistent with these data, primary tumor volumes were not different between control and etodolactreated mice (Fig. 5A). However, metastatic lung nodules were significantly lower both in the number and size in etodolac treated group (Fig. 5B). 4T1_GFP cell implantation further confirmed that lung metastatic nodules of etodolac treated mice are fewer in number and smaller in size (Fig. 5C). H&E staining showed that the etodolac-treated group had fewer and smaller lung nodules than the non-treated group (Fig. 5D). We infered from these results that tumor cell metastasis may be inhibited due to COX-2 inhibition during macrophage differentiation.

Etodolac induced more immune activated macrophages in breast tumor
During tumor progression, tumor cells build up an immunosuppressive microenvironment in which TAMs [27], mostly M2 macrophages, promote angiogenesis and metastasis. To examine if etodolac change immune status of TAMs, we investigated TAMs and spleen macrophages for receptor expression and cytokine contents. Primary tumors had large CD11b F4/80 double positive macrophage population, up to 25,30% (Fig. 6A). In accordance with in vitro results, TAMs showed higher levels of MHCII IA/IE molecules in etodolac-treated mice (Fig. 6B), which correlated with increased macrophage immune activities [31]. TAMs contained less intracellular IL-10 (Fig. 6C), and splenic macrophages had more TNFa but less IL-10 due to etodolac (Fig. 6D). Next we isolated F4/80 positive macrophages and examined the expression levels of genes related to tumor metastasis. Etodolactreated spleen macrophages exhibited reduced Ym I gene expression, a murine M2 macrophage marker (Fig. 7A). Etodo-lac-treated TAMs had decreased levels of TGFb, VEGFA, and VEGFC mRNA (Fig. 7B). TGFb is well-known as a major immunosuppressive cytokine produced by alternatively activated macrophages and enhances tumor cell epithelial-to-mesenchymal transition, followed by metastasis [32]. VEGFA recruits angiogenic endothelial precursors, and VEGFC guides lymphangiogenesis [33]. Taken together, these data imply that etodolac may reduce M2 macrophage polarization of TAMs and thus inhibit breast tumors to lung metastasis.

Discussion
Macrophages are a prominent component of the tumor stroma and infiltrate into human and experimental mouse tumors [7,15,[34][35][36][37][38]. In the relationship between macrophages and tumor cells, several molecules, including cytokines, growth factors, and proteolytic enzymes, are known to be essential for successful tumor cell invasion [3,34,37,39]. Accumulating data regarding macrophage polarization indicate the existence of opposite-characterized macrophage subsets known as anti-tumoral classically activated M1 macrophages and pro-tumoral alternatively activated M2 macrophages. TAMs have much in common with M2 macrophages because they promote angiogenesis through VEGF, enhance macrophage recruitment by M-CSF or TGFb secretion, and promote local invasion through cathepsin and metalloproteinases [34]. Moreover, tumor cell dispersion during early-stage macrophage migration may be linked to recurrence post-surgery [25]. The exact mechanisms of macrophage polarization need to be elucidated to determine their therapeutic potential and ability to pharmacologically control macrophage infiltration in tumors.
Macrophage polarization can be represented experimentally using M-CSF and GM-CSF. M-CSF-induced monocyte differentiation produces mature M2 type macrophages both in human and mice. Human M2 macrophages express high levels of the surface scavenger receptor CD163 and GPI-coupled receptor CD14 but lower levels of CD11b compared with M1 macrophages. Murine M2 macrophage markers includes YmI, FizzI and ArginaseI as well as several surface markers including CD206 and CD209. Both human and murine M1 macrophages express iNOS and IL-12 gene expressions. In our in vitro experimental systems, COX-2 inhibition did not reduce all of above M2 markers but partially changed several markers including CD163 (human), CD11b (human), and YmI (murine). Actually, IL-12 gene expression is the hallmark of M1 polarization, and COX-2 pathway does not seemed to regulate IL-12 under M2 differentiation signals (data not shown). And transient COX-2 blocking in already matured macrophages did not convert their phenotype into M1 type and only COX-2 inhibition during whole differentiation period showed the above polarizing effects. Thus we conclude that COX-2 signaling pathway during differentiation is one of required prerequisites for alternatively activated macrophage development and COX-2 blocking during differentiation could make some leakage in fully charged M2 macrophages. Human M2 macrophages differentiated with M-CSF appear crumpled and relatively slim, slightly attached to the flask, or float individually. These morphologies specifically extremes when human monocytes were co-cultured with several cancer cell lines including MDA-MB-231, HT29, MeWo (data not shown).
Functionally, they secrete lower levels of various inflammatory cytokines such as TNFa, IL-1b, IL-12, and IL-6 in response to LPS stimulation, but secrete more TGFb and IL-10 than do M1 macrophages. In contrast, GM-CSF induced M1 macrophages firmly attach to the flask, broaden their cytoplasm, and express low  CD14 and CD163 but high CD11b (Fig. S1A and S1B). They capture and phagocytose less bacteria than do M-CSF-oriented M2 macrophages (Fig. S1D) but secrete increased amounts of pro-inflammatory cytokines and chemokines. Based on these immune enhancing effects of classically activated macrophages, the GVAX cancer vaccine platform has been successful in several clinical trials involving administration of GM-CSF-secreting autologous tumor cells in that those results indicated enhanced anti-tumor immunity [40]. In this study, we found that COX-2 inhibition during M-CSF induced macrophage differentiation resulted in effects somewhat similar to those of GM-CSF. This indicates that during monocyte differentiation, COX-2 is the key enzyme for M2 polarization and that blocking this enzyme may be used as a therapy for induction of enhanced anti-tumor immunity.
The exact signaling pathways involved in M2 polarization remain largely unknown. The classic pathway involves the M2priming cytokines IL-4 and IL-13 inducing STAT6 phosphorylation [41]. Recently, the PI3K/Akt pathway was identified as an M2-inducing signaling pathway conferred by serum amyloid P [42]. IL-4 induces the production of PPAR ligands in macrophages by induction of 12/15 lipoxygenase followed by lipoxin A4 (LXA4) [43]. We confirmed STAT3 phosphorylation and COX-2 induction in M-CSF-treated macrophages (data not shown). No COX-2 band was detected, but increased levels of activated STAT3 were detected in etodolac-treated macrophages. Further study of COX-2 inhibition and STAT3 activation during macrophage polarization is necessary. Meanwhile, COX-2 induces PGE 2 at the early inflammatory stage, which has been suggested to induce angiogenesis. We attempted to examine the relationship between PGE 2 and macrophage polarization using protein kinase C and cAMP activator, but did not observe PGE 2 -mediated intracellular signaling effects on macrophage phenotype. However, 15d-PGJ 2 production during monocyte differentiation was interesting because it increased and peaked on day 5 of differentiation under M-CSF and was completely inhibited in etodolac-treated macrophages (Fig. S3). 15d-PGJ 2 has been shown to possess anti-inflammatory properties, which are conferred via different mechanisms including activation of the prostaglandin D2 receptor 2 (DP2), peroxisome proliferator activated receptor-c (PPAR-c), and covalent modification of cysteine thiols in target proteins, such as IkB kinase b (IKKb) and p65 of NF-kB [44,45]. Derek et al. also reported that inducible cyclooxygenase may have anti-inflammatory properties partially via 15d-PGJ 2 [46]. It will be interesting to determine whether 15d-PGJ 2 has the potential to differentiate monocytes into alternatively activated macrophages or just primes mature macrophages into the M2 type.
Continuous NSAID intake did not influence 4T1 cell proliferation in a BALB/c breast cancer model, but inhibited lung metastasis. TAMs and splenic macrophages showed higher MHCII and pro-inflammatory TNFa expression, indicating that systemic innate immunity was enhanced by etodolac intake. Actually, we observed macrophage enhancing activity even after one week of etodolac feeding before tumor cell injection (Fig. S4) in that these mice had more activated peritoneal macrophages and adipose tissue macrophages and consequently produced more serum TNFa and IL-12 against LPS challenge. Our results are in accordance with recently published article [47] in part, which shows COX-2 inhibition alters the phenotype of tumor-associated macrophages from M2 to M1 in Apc Min/+ mouse polyps. The author clarified the effect of celecoxib intake mainly on primary polyp size together with whole tumor cytokine mileu. Our study further extent these phenomenon more precisely showing individual TAM's characteristics in mouse breast cancer model, as well as in human monocyte derived macrophages. COX-2 inhibition potentiates macrophage's inflammatory cytokine responses but reduced IL-10 secretion thus might skew overall tumor microenvironment to favor Th1 immune responses which was confirmed by reduced regulatory T cells as well as myeloid derived suppressor cells in etodolac fed mice tumor mass (data not shown). Up to our knowledge, this is the first describes about NSAIDs mediated human macrophage polarization. Reducing M2 macrophage characteristics systemically may provide advantages to cancer patients because they express much lower levels of TGFb, VEGFA, VEGFC, MMP-9, and MMP-1 (Fig. 3). All of these molecules enhance tumor cell metastasis directly or indirectly [25]. This is significant because most cancer patients who undergo resection surgery have recurrences. Alternatively activated macrophages, which actively secrete the above molecules, could generate single circulating tumor cells before surgery, preventing tumor relapse. Targeting COX-2 and macrophage polarization may help reduce the rate of relapse.
We report that COX-2 inhibition reduces lung metastasis in an experimentally induced breast cancer model. Because COX-2 is an enzyme for differentiation of monocytes into alternatively activated macrophages, COX-2 inhibition may inhibit acquisition of tumor-promoting characteristics of TAMs, including immunesuppressive cytokine secretion, growth factor synthesis, as well as abundant metalloproteinase secretion into the tumor microenvironment. Successive adjuvant therapies targeting VEGF, CD47, CD40, or immune-inhibitory molecules such as PD-1 and CTLA-1, in combination with immune enhancing cytokines, are now considered clinically promising. The majority of these approaches are directly or indirectly associated with macrophage functions because macrophages are the harmful population in the tumor microenvironment. In conclusion, our study provides valuable insights regarding TAM regulation that will facilitate prevention of tumor metastasis. Attached cells were quantified (right graph) using Image J software from DAPI images at 406magnification. Data from three independent experiments, ***, p,0.001 by unpaired t-test. Panel D: Colonies indicating remnant Staphylococcus aureus in the culture supernatant after macrophage phagocytosis during (left image). Monocytes (0.5610 6 ) were differentiated in a 24-well plate. Infection was performed with 0.2610 6 cfu Staphylococcus aureus for 1 hr after 4 and 7 days of differentiation. A 1:100 dilution of 100 ml culture supernatants was cultured on LB agar plates and colonies were counted manually (right graph). A total of two independent experiments were performed. *, p,0.05 by Student's t-test. Panel E: Human IL-1b and IL-6 levels in macrophage culture supernatants were measured using ELISA. Human monocytes from five independent healthy donors were differentiated in vitro for 7 days. On the sixth day of differentiation, LPS/ IFNc (100 ng/ml, 25 ng/ml) was added to each group and incubated for another 24 hrs. **, p,0.01 by unpaired Student's t- expressions of peritoneal macrophages using FACS. Upper dot plots represents peritoneal macrophage gating strategies (CD45 + CD11b + F4/80 + ) and lower histogram and bar graph shows enhanced IA/IE expressions. *, p,0.05 by unpaired Student's t-test. n = 5. Panel B: Adipose tissues were examined for their M2 markers Arginase I, Fizz I, and Ym I gene expressions by real-time PCR. Adipose tissues from etodolac fed mice had reduced M2 marker expressions. **, p,0.01 by unpaired student's t-test. n = 3. Panel C: Mice were injected with 50 mg of LPS intraperitoneally and blood was collected one hour after injection. Serum TNFa and IL-12 were detected by ELISA. **, p,0.01 by unpaired Student's t-test. n = 6. (TIF)