Galectin-3 Accelerates M2 Macrophage Infiltration and Angiogenesis in Tumors
2013; Elsevier BV; Volume: 182; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2013.01.017
ISSN1525-2191
AutoresWeizhen Jia, Hiroyasu Kidoya, Daishi Yamakawa, Hisamichi Naito, Nobuyuki Takakura,
Tópico(s)Immune cells in cancer
ResumoIt is widely accepted that robust invasion of tumor-associated macrophages resembling M2 macrophage correlates with disease aggressiveness by affecting cancer cell invasion, metastasis, and angiogenesis. Many chemokines that induce migration of macrophages have been identified during inflammatory responses; however, further precise analysis of macrophage migration in the tumor microenvironment is required. Here, we analyzed the function of galectin-3 (Gal-3; gene LGALS3, alias Gal3) for macrophage chemotaxis using Gal3−/− mice as hosts, and a tumor allograft model. We engineered a concentration gradient of Gal-3 produced by the tumor. In this model, we found that macrophage infiltration was enhanced in tumors developing in these Gal3−/− mice relative to the Gal3+/+ animals. This was accompanied by enhanced tumor angiogenesis and tumor growth in Gal3−/− mice. We found that macrophages of the M2 phenotype were dominant in infiltrates in the Gal3−/− mice and that they expressed only low levels of Gal-3. Gal3 knockdown by siRNA in macrophages resulted in enhanced chemotaxis. These data suggest that M2-like macrophages migrate into the tumor along a Gal-3 gradient and that high-level Gal-3 expression in the tumor results in acceleration of angiogenesis and tumor growth. Therefore, Gal-3 could be a potential target for the development of new treatments to inhibit tumor growth. It is widely accepted that robust invasion of tumor-associated macrophages resembling M2 macrophage correlates with disease aggressiveness by affecting cancer cell invasion, metastasis, and angiogenesis. Many chemokines that induce migration of macrophages have been identified during inflammatory responses; however, further precise analysis of macrophage migration in the tumor microenvironment is required. Here, we analyzed the function of galectin-3 (Gal-3; gene LGALS3, alias Gal3) for macrophage chemotaxis using Gal3−/− mice as hosts, and a tumor allograft model. We engineered a concentration gradient of Gal-3 produced by the tumor. In this model, we found that macrophage infiltration was enhanced in tumors developing in these Gal3−/− mice relative to the Gal3+/+ animals. This was accompanied by enhanced tumor angiogenesis and tumor growth in Gal3−/− mice. We found that macrophages of the M2 phenotype were dominant in infiltrates in the Gal3−/− mice and that they expressed only low levels of Gal-3. Gal3 knockdown by siRNA in macrophages resulted in enhanced chemotaxis. These data suggest that M2-like macrophages migrate into the tumor along a Gal-3 gradient and that high-level Gal-3 expression in the tumor results in acceleration of angiogenesis and tumor growth. Therefore, Gal-3 could be a potential target for the development of new treatments to inhibit tumor growth. Neovascularization is an indispensable event for tissue/organ development. New blood vessel formation observed under pathologic and physiological conditions occurs mainly by sprouting angiogenesis (ie, the development of a new branch from pre-existing vessels).1Carmeliet P. Angiogenesis in health and disease.Nat Med. 2003; 9: 653-660Crossref PubMed Scopus (3554) Google Scholar In sprouting angiogenesis, proangiogenic factors released from hypoxic regions or sites of inflammation directly induce migration and proliferation of endothelial cells (ECs). Moreover, nonvascular cells infiltrating into the region produce angiogenic factors that induce angiogenesis indirectly.2Carmeliet P. Angiogenesis in life, disease and medicine.Nature. 2005; 438: 932-936Crossref PubMed Scopus (2871) Google Scholar, 3Coussens L.M. Werb Z. Inflammation and cancer.Nature. 2002; 420: 860-867Crossref PubMed Scopus (11719) Google Scholar We previously reported that hematopoietic stem/progenitor cells and CD11blow immature myeloid cells/monocytes induce angiogenesis and maturation of newly developed blood vessels by secretion of angiopoietin-1, a ligand for receptor tyrosine kinase Tie2 expressed on ECs.4Takakura N. Watanabe T. Suenobu S. Yamada Y. Noda T. Ito Y. Satake M. Suda T. A role for hematopoietic stem cells in promoting angiogenesis.Cell. 2000; 102: 199-209Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar, 5Yamada Y. Takakura N. Physiological pathway of differentiation of hematopoietic stem cell population into mural cells.J Exp Med. 2006; 203: 1055-1065Crossref PubMed Scopus (55) Google Scholar In addition, it is widely accepted that matrix metalloproteinase derived from mast cells, neutrophils, and macrophages promotes angiogenesis by matrix remodeling, and that vascular endothelial growth factor secreted by these cell populations also regulates angiogenesis.6Kessenbrock K. Plaks V. Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment.Cell. 2010; 141: 52-67Abstract Full Text Full Text PDF PubMed Scopus (3764) Google Scholar Among hematopoietic lineages, the relationships of macrophages to angiogenesis have been examined extensively and crucial roles, especially of tumor-associated macrophages and Tie2-expressing macrophages, for tumor angiogenesis have been reported.7Coffelt S.B. Tal A.O. Scholz A. De Palma M. Patel S. Urbich C. Biswas S.K. Murdoch C. Plate K.H. Reiss Y. Lewis C.E. Angiopoietin-2 regulates gene expression in TIE2-expressing monocytes and augments their inherent proangiogenic functions.Cancer Res. 2010; 70: 5270-5280Crossref PubMed Scopus (252) Google Scholar, 8Mazzieri R. Pucci F. Moi D. Zonari E. Ranghetti A. Berti A. Politi L.S. Gentner B. Brown J.L. Naldini L. De Palma M. Targeting the ANG2/TIE2 axis inhibits tumor growth and metastasis by impairing angiogenesis and disabling rebounds of proangiogenic myeloid cells.Cancer Cell. 2011; 19: 512-526Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar Monocyte chemoattractant protein-1 (also known as chemokine ligand 2) and colony-stimulating factor-1 (also known as macrophage-colony-stimulating factor) are well-known chemoattractants for monocytes/macrophages, but other factors such as placental growth factor, chemokine ligand 3 (macrophage inflammatory protein 1), chemokine ligand 4, chemokine ligand 5 (regulated on activation normal T-cell expressed and secreted), and vascular endothelial growth factor also have been reported to act this way.9Qian B.Z. Pollard J.W. Macrophage diversity enhances tumor progression and metastasis.Cell. 2010; 141: 39-51Abstract Full Text Full Text PDF PubMed Scopus (3661) Google Scholar, 10Grivennikov S.I. Greten F.R. Karin M. Immunity, inflammation, and cancer.Cell. 2010; 140: 883-899Abstract Full Text Full Text PDF PubMed Scopus (7884) Google Scholar Therefore, further precise investigation is required to identify the molecules inducing monocyte/macrophage migration. Here, we investigated relationships among galactose-binding lectin-3 (alias galectin-3; Gal-3), monocytes/macrophages, and angiogenesis. Galectins are a family of lectins containing conserved carbohydrate-recognition domains specific for β-galactoside.11Barondes S.H. Castronovo V. Cooper D.N. Cummings R.D. Drickamer K. Feizi T. Gitt M.A. Hirabayashi J. Hughes C. Kasai K. Leffler H. Liu F. Lotan R. Mercurio A.M. Monsigny M. Pillai S. Poirer F. Raz A. Rigby P.W. Rini J.M. Wang J.L. Galectins: a family of animal beta-galactoside-binding lectins.Cell. 1994; 76: 597-598Abstract Full Text PDF PubMed Scopus (1124) Google Scholar Gal-3 is a 32-kDa protein containing one carbohydrate-recognition domain and an N-terminal nonlectin domain. Different functions for Gal-3 in development, immune reactions, and tumorigenesis have been reported.12Dumic J. Dabelic S. Flögel M. Galectin-3: an open-ended story.Biochim Biophys Acta. 2006; 1760: 616-635Crossref PubMed Scopus (882) Google Scholar In macrophages, expression of Mac-2 (another name for Gal-3) has been reported. Published evidence suggests that Gal-3 may influence the migration of monocytes/macrophages.13Sano H. Hsu D.K. Yu L. Apgar J.R. Kuwabara I. Yamanaka T. Hirashima M. Liu F.T. Human galectin-3 is a novel chemoattractant for monocytes and macrophages.J Immunol. 2000; 165: 2156-2164PubMed Google Scholar However, the precise function of Gal-3 for monocyte/macrophage migration in the context of angiogenesis has not been clarified. Thus far, although it has been reported that the mobility of ECs is enhanced by scaffolding generated through the binding of Gal-3 to integrin, in vivo roles of Gal-3 in angiogenesis have been minimally investigated. Because it has been reported that tumor cells express abundant Gal-312Dumic J. Dabelic S. Flögel M. Galectin-3: an open-ended story.Biochim Biophys Acta. 2006; 1760: 616-635Crossref PubMed Scopus (882) Google Scholar, 14Radosavljevic G. Volarevic V. Jovanovic I. Milovanovic M. Pejnovic N. Arsenijevic N. Hsu D.K. Lukic M.L. The roles of Galectin-3 in autoimmunity and tumor progression.Immunol Res. 2012; 52: 100-110Crossref PubMed Scopus (96) Google Scholar and tumor angiogenesis is one available model for investigating Gal-3 function, we examined the role of Gal-3 in angiogenesis using Gal3-deficient mice. C57BL/6 mice (7 to 8 weeks of age) were purchased from Japan SLC (Shizuoka, Japan). Gal3 knockout (KO) mice (7 to 8 weeks of age) were generated as previously described.15Hsu D.K. Yang R.Y. Pan Z. Yu L. Salomon D.R. Fung-Leung W.P. Liu F.T. Targeted disruption of the galectin-3 gene results in attenuated peritoneal inflammatory responses.Am J Pathol. 2000; 156: 1073-1083Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar Animals were housed in environmentally controlled rooms of the animal experimentation facility at Osaka University. All experiments were performed in accordance with the guidelines of Osaka University Committee for Animal and Recombinant DNA Experiments. Mice were handled and maintained according to Osaka University guidelines for animal experimentation. Cell lines, including B16 (mouse melanoma), mouse Lewis lung carcinoma (LLC), and J774 (murine macrophage), were purchased from the Riken cell bank (Tsukuba, Japan). The B16 and LLC cell lines were cultured in Dulbecco's modified Eagle's medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin. The J774 cell line was cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, and 2 mol/L l-glutamine (Gibco, Life Technologies, St. Paul, Brazil). Single-cell suspensions from tumor tissue were produced as previously described.16Naito H. Kidoya H. Sakimoto S. Wakabayashi T. Takakura N. Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels.EMBO J. 2011; 31: 842-855Crossref PubMed Scopus (136) Google Scholar Murine bone marrow–derived macrophages (CD45high CD11bhigh F4/80high) were isolated and prepared as previously described16Naito H. Kidoya H. Sakimoto S. Wakabayashi T. Takakura N. Identification and characterization of a resident vascular stem/progenitor cell population in preexisting blood vessels.EMBO J. 2011; 31: 842-855Crossref PubMed Scopus (136) Google Scholar and were cultured in RPMI-1640 (Sigma) supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin, 2 mmol/L l-glutamine, and 50-ng/mL murine M-colony-stimulating factor (PeproTech, Rocky Hill, NJ). These bone marrow–derived macrophages were maintained for 7 days in a CO2 incubator. Total RNA was extracted from cells and tumor tissues using RNeasy-plus mini kits (Qiagen, Hilden, Germany) and was reverse-transcribed using the PrimeScript RT reagent Kit (Takara, Kyoto, Japan) according to the manufacturer's protocol. Real-time PCR analysis was performed using Platinum SYBR Green qPCR SuperMix-UDC (Invitrogen, Carlsbad, CA) and an Mx3000p QPCR System (Stratagene, La Jolla, CA). The baseline and threshold were adjusted according to the manufacturer's instructions. The level of the target gene expression was normalized to that of glyceraldehyde-3-phosphate dehydrogenase in each sample. We used the following primer sets for mouse genes: 5′-CCACGTCGTAGCAAACCACCA-3′ (forward) and 5′-AGGAGCACGTAGTCGGGGCA-3′ (reverse) for tumor necrosis factor-α, 5′-TCCTCTCTGCAAGAGACTTCCATCC-3′ (forward) and 5′-GGGAAGGCCGTGGTTGTCACC-3′ (reverse) for IL6, 5′-AGGCTCATCCAGAGCCCGGAG-3′ (forward) and 5′-AGGGTGGTGCGGCTGGACTT-3′ (reverse) for inducible nitric oxide synthase, 5′-TCGGTGGACTGTGGACGAGCA-3′ (forward) and 5′-TCCCGCCTTTCGTCCTGGCA-3′ (reverse) for macrophage mannose receptor 1 (MRC1), 5′-CCCCAGGCAGAGAAGCATGGC-3′ (forward) and 5′-GGGGAGAAATCGATGACAGCGCC-3′ (reverse) for IL10, 5′-TCAGCCAGATGCAGTTAACGCCC-3′ (forward) and 5′-GCTTCTTTGGGACACCTGCTGCT-3′ (reverse) for monocyte chemoattractant protein-1, 5′-TGCCCTATGACCTGCCCTT-3′ (forward) and 5′-TCCTGCTTCGTGTTACACACAA-3′ (reverse) for Gal3 and, finally, 5′-TGGCAAAGTGGAGATTGTTGCC-3′ (forward) and 5′-AAGATGGTGATGGGCTTCCCG-3′ (reverse) for glyceraldehyde-3-phosphate dehydrogenase (Gapdh). Methods for Western blotting were as previously described.17Kidoya H. Naito H. Takakura N. Apelin induces enlarged and nonleaky blood vessels for functional recovery from ischemia.Blood. 2010; 115: 3166-3174Crossref PubMed Scopus (101) Google Scholar Briefly, lysates from whole cells were resolved in SDS-PAGE. Proteins electrophoretically separated using 12.5% SDS-PAGE gels were transferred to nylon membranes (Amersham, Buckinghamshire, UK) by a wet blotting procedure and incubated with the following antibodies: rat anti-mouse Gal-3/MAC-2 (Cedarlane, Ontario, Canada); and anti-mouse glyceraldehyde-3-phosphate dehydrogenase (Millipore, Temecula, CA). Proteins were detected with horseradish-peroxidase–conjugated goat anti-rat IgG, goat anti-mouse IgG (Jackson Laboratories, Bar Harbor, ME) secondary antibodies and ECL reagents (Amersham). The blots were scanned with an imaging densitometer LAS-3000 mini (Fujifilm, Tokyo, Japan). siRNA specific to mouse Gal-3 and negative control siRNA were purchased from Sigma and transfected into J774 cells using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. The effect of siRNA on Gal-3 expression was observed using Western blotting with an anti–Gal-3/MAC-2 antibody (Cedarlane) and real-time PCR. Migration analysis was performed using 5-μm pore-size cell culture inserts in 24-well plates (Corning, Chelmsford, UK). J774 cells or bone marrow–derived macrophages (1 × 104) were seeded into the top of the Transwell chambers precoated with fibronectin (Corning, Tokyo, Japan), and 8 μg/mL recombinant Gal-3 (R&D Systems, Minneapolis, MN) was added into the lower well. After 8 hours of incubation, cells on the upper membrane surface were removed with a cotton swab. Cells on the lower membrane surface were fixed with 4% formaldehyde and stained with Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA). B16 or LLC tumor cells (1 × 106 per mouse in 0.1 mL PBS) were inoculated subcutaneously into wild-type C57BL/6 or Gal3 KO mice (7 to 8 weeks of age). Tumors were dissected 18 days after implantation (in allografts using bone marrow–transplanted chimeric mice, discussed later (see Bone Marrow Transplantation)). Tumor volumes were measured with calipers every 3 days and were calculated as follows: width × width × length × 0.52.18Huang X. Yamada Y. Kidoya H. Naito H. Nagahama Y. Kong L. Katoh S.Y. Li W.L. Ueno M. Takakura N. EphB4 overexpression in B16 melanoma cells affects arterial-venous patterning in tumor angiogenesis.Cancer Res. 2007; 67: 9800-9808Crossref PubMed Scopus (34) Google Scholar Immunostaining analysis was performed on 10-μm cryostat sections of mouse tumor tissue. Procedures for tissue fixation and staining of sections with antibodies were as described previously.4Takakura N. Watanabe T. Suenobu S. Yamada Y. Noda T. Ito Y. Satake M. Suda T. A role for hematopoietic stem cells in promoting angiogenesis.Cell. 2000; 102: 199-209Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar For immunohistochemistry, rat anti-mouse F4/80 antibody (AbD Serotec, Raleigh, NC), rat anti-mouse CD206 (MRC1) antibody (AbD Serotec), rat anti-mouse CD31 antibody (BD Pharmingen, San Jose, CA), and hamster anti-mouse CD31 antibody (Millipore) were used for staining and anti-rat IgG Alexa Fluor 488 (Invitrogen), anti-rat IgG Alexa Fluor 546 (Invitrogen), and anti-Armenian hamster FITC (eBioscience, San Diego, CA) as the secondary antibodies. Cell nuclei were visualized with TO-PRO-3 (Invitrogen). To measure hypoxia in tumor tissues, 60 mg/kg HypoxyProbe-1 (Hypoxyprobe, Inc, Burlington, MA) was injected intraperitoneally 1 hour before sacrificing the mice. Tumor sections were stained using an anti-HypoxyProbe antibody. Sections were observed by conventional microscopy (brightfield) (DM5500 B; Leica, Wetzlar, Germany) or confocal microscopy (TCS/SP5; Leica), and images were acquired with a digital camera (DFC500; Leica). In all analyses, an isotype-matched control Ig was used as a negative control and it was confirmed that the positive signals were not derived from a nonspecific background. Images were processed using Photoshop CS2 software version 9.0.2 (Adobe Systems, San Jose, CA). All images shown are representative of six or more independent experiments. Flow cytometric analysis was performed as described previously.5Yamada Y. Takakura N. Physiological pathway of differentiation of hematopoietic stem cell population into mural cells.J Exp Med. 2006; 203: 1055-1065Crossref PubMed Scopus (55) Google Scholar Fluorescence-labeled anti-mouse antibodies specific for F4/80, CD206 (MRC1) (AbD Serotec), CD11b, and CD45 (BD Pharmingen) were used. Stained cells were analyzed with a FACSCalibur or FACSAria (BD Biosciences) using FlowJo software version 7.6.1 (TreeStar, Ashland, OR) and sorted by a FACSAria. Dead cells were excluded by propidium iodide staining or analyses using the two-dimensional profile of the forward versus side scatter. Bone marrow cells from wild-type mice were transplanted into wild-type C57BL/6 or Gal3 KO mice (7 to 8 weeks of age). Bone marrow cells were obtained by flushing the tibias and femurs of age-matched donor wild-type C57BL/6 mice. Bone marrow transplantation (BM-T) was performed using lethally irradiated (10.0 Gy) wild-type C57BL/6 or Gal3 KO mice by intravenous infusion of 1 × 106 donor whole bone marrow cells. Four weeks after transplantation, BM chimeric mice received an inoculation of B16 or LLC tumor cells (1 × 106 per mouse in 0.1 mL PBS) subcutaneously, and tumor tissues were dissected 18 days after implantation. Tumor volumes were measured with calipers every 3 days and calculated as follows: width × width × length × 0.52.18Huang X. Yamada Y. Kidoya H. Naito H. Nagahama Y. Kong L. Katoh S.Y. Li W.L. Ueno M. Takakura N. EphB4 overexpression in B16 melanoma cells affects arterial-venous patterning in tumor angiogenesis.Cancer Res. 2007; 67: 9800-9808Crossref PubMed Scopus (34) Google Scholar All data are presented as means ± SD. Statistical analysis was performed by the Tukey-Kramer multiple comparison test using the statcel version 2 software package (OMS, Saitama, Japan). When only two groups were compared, a two-sided Student's t-test was used. A P value less than 0.05 was considered statistically significant. It has been suggested that Gal-3 induces angiogenesis by directly promoting chemotaxis of ECs.19Nangia-Makker P. Honjo Y. Sarvis R. Akahani S. Hogan V. Pienta K.J. Raz A. Galectin-3 induces endothelial cell morphogenesis and angiogenesis.Am J Pathol. 2000; 156: 899-909Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar Functional chemotaxis also may involve other cell types, such as monocytes/macrophages, because of the known function of Gal-3 in macrophage migration.13Sano H. Hsu D.K. Yu L. Apgar J.R. Kuwabara I. Yamanaka T. Hirashima M. Liu F.T. Human galectin-3 is a novel chemoattractant for monocytes and macrophages.J Immunol. 2000; 165: 2156-2164PubMed Google Scholar, 20Hsu D.K. Chernyavsky A.I. Chen H.Y. Yu L. Grando S.A. Liu F.T. Endogenous galectin-3 is localized in membrane lipid rafts and regulates migration of dendritic cells.J Invest Dermatol. 2009; 129: 573-583Crossref PubMed Scopus (85) Google Scholar Macrophages act as proangiogenic accessory cell components in tumors by secreting several angiogenic factors such as vascular endothelial growth factor, matrix metalloproteinases, and others.21Leek R.D. Harris A.L. Tumor-associated macrophages in breast cancer.J Mammary Gland Biol Neoplasia. 2002; 7: 177-189Crossref PubMed Scopus (314) Google Scholar, 22Lin E.Y. Li J.F. Bricard G. Wang W. Deng Y. Sellers R. Porcelli S.A. Pollard J.W. Vascular endothelial growth factor restores delayed tumor progression in tumors depleted of macrophages.Mol Oncol. 2007; 1: 288-302Abstract Full Text Full Text PDF PubMed Scopus (139) Google Scholar, 23Giraudo E. Inoue M. Hanahan D. An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis.J Clin Invest. 2004; 114: 623-633Crossref PubMed Scopus (566) Google Scholar Therefore, it was hypothesized that Gal-3 in the tumor environment accelerates tumor angiogenesis via macrophage chemotaxis, resulting in tumor growth. To test this, Gal3 mutant (Gal3−/−) mice were used as tumor-bearing hosts because macrophages themselves produce Gal-3 and chemotaxis caused by tumor-derived Gal-3 would not be assessable. Although Gal-3 production from tumor stromal cells is absent in Gal3−/− mice, tumor cell–derived Gal-3 should generate a concentration gradient from the tumor, enabling visualization of Gal3-deficient macrophage infiltration into tumor tissue. We used two cancer cell lines, B16 mouse melanoma and LLC mouse lung cancer cells for the allograft model. We inoculated these cells into Gal3+/+ (wild-type) and Gal3−/− mice and evaluated tumor growth. These results showed enhancement of tumor growth in Gal3−/− mice compared with Gal3+/+ mice in both B16 (Figure 1, A–C) and LLC tumors (Figure 1, D–F). Both B16 and LLC tumor tissues were found to express Gal-3 more strongly than normal cells such as those from skin, spleen (B cells), liver, and lung, both at the mRNA (Figure 1G) and protein levels (Figure 1H). This suggests that cancer cell–derived Gal-3 does form a concentration gradient in Gal3−/− mice and may induce migration of monocytes/macrophages into tumors. The number of infiltrated F4/80-positive macrophage lineage cells was significantly higher in tumors that developed in Gal3−/− mice than in Gal3+/+ mice (Figure 2, A and B ). Next, we investigated if exogenous Gal-3 induces chemotaxis of macrophages and if endogenous Gal-3 in macrophages would obscure this chemotaxis. We used J774, a macrophage cell line, and silenced Gal-3 expression by siRNA (Figure 2C). As expected, knockdown of endogenous Gal-3 enhanced transmigration of J774 macrophages (Figure 2, D and E). We next isolated CD45high CD11bhigh F4/80high macrophages from the bone marrow of Gal3+/+ or Gal3−/− mice and confirmed a lack of Gal-3 in those from Gal3 KO animals (Figure 2F). By using these primary macrophages, we observed chemotaxis stimulated by Gal-3. As was seen in the J774 cell line, a lack of endogenous Gal-3 resulted in enhanced macrophage chemotaxis along a Gal-3 gradient (Figure 2, G and H). Because we found abundant F4/80-positive macrophage lineage cells migrating into tumors developing in Gal3−/− mice, we identified their subtype. We confirmed that a population of CD11bhigh F4/80high macrophages was more highly abundant in both B16 and LLC tumors developing in Gal3−/− mice than in Gal3+/+ mice (Figure 3A). Macrophages in tumors from Gal3−/− mice expressed MRC1 (CD206), an M2 macrophage marker, more strongly than in Gal3+/+ mice (Figure 3B). Moreover, they expressed IL10 and monocyte chemoattractant protein-1 strongly as well as MRC1 at the mRNA level, a characteristic M2 macrophage gene signature (Figure 3C).24Satoh T. Takeuchi O. Vandenbon A. Yasuda K. Tanaka Y. Kumagai Y. Miyake T. Matsushita K. Okazaki T. Saitoh T. Honma K. Matsuyama T. Yui K. Tsujimura T. Standley D.M. Nakanishi K. Nakai K. Akira S. The Jmjd3-Irf4 axis regulates M2 macrophage polarization and host responses against helminth infection.Nat Immunol. 2010; 11: 936-944Crossref PubMed Scopus (902) Google Scholar LLC but not B16 tumors in Gal3+/+ mice expressed tumor necrosis factor-α more abundantly than in Gal3−/− mice. M2 macrophages were more abundant in LLC than B16 tumors. Therefore, M1 macrophage-mediated effects may be more apparent in LLC tumors of Gal3+/+ mice. The earlier-described data suggest that cells similar to M2 macrophages may migrate selectively into the tumor parenchyma when Gal-3 production is higher in the tumor than in nontumor tissue, such that a concentration gradient occurs. Moreover, it was possible that Gal-3 expression in M2 macrophages was constitutively lower and they more easily migrated into Gal-3-producing areas. We therefore divided bone marrow CD11bhigh F4/80high macrophages into two fractions, that is, MRC1low and MRC1high (Figure 3D), and quantified their Gal-3 expression (Figure 3E). MRC1 mRNA expression correlated with the protein level in macrophages. As expected, Gal-3 mRNA expression was lower in MRC1high than in MRC1low macrophages. It is widely accepted that M2 macrophages promote angiogenesis.25Sica A. Allavena P. Mantovani A. Cancer related inflammation: the macrophage connection.Cancer Lett. 2008; 267: 204-215Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 26Solinas G. Germano G. Mantovani A. Allavena P. Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation.J Leukoc Biol. 2009; 86: 1065-1073Crossref PubMed Scopus (1138) Google Scholar Therefore, enhanced tumor growth in Gal3−/− is suggested to be induced by increased angiogenesis as a result of infiltration by M2-like macrophages. Hence, we investigated the localization of MRC1-positive macrophages in the tumor microenvironment. As shown in Figure 4, A–C, higher numbers of MRC-positive macrophages were observed in both B16 and LLC tumors developing in Gal3−/− than in Gal3+/+ mice. Moreover, co-localization of MRC1-positive cells with CD31-positive ECs indicated that more of these cells were interacting with blood vessels in both B16 and LLC tumors in Gal3−/− mice. The number of blood vessels identified as CD31+ was significantly higher in both B16 and LLC tumors developing in Gal3−/− than in Gal3+/+ mice (Figure 4, D and E). It is possible that a mere increase in the number of blood vessels does not necessarily correlate with effective blood circulation in tumors and that nonfunctional blood vessel formation increases severe hypoxia.27Noguera-Troise I. Daly C. Papadopoulos N.J. Coetzee S. Boland P. Gale N.W. Lin H.C. Yancopoulos G.D. Thurston G. Blockade of Dll4 inhibits tumour growth by promoting non-productive angiogenesis.Nature. 2006; 444: 1032-1037Crossref PubMed Scopus (895) Google Scholar We therefore evaluated hypoxic conditions and found that hypoxia was not more severe in tumors developing in Gal3−/− mice (Figure 4, F and G), suggesting that functional blood vessels are induced in tumors in Gal3−/− mice, enhancing tumor growth. Lack of Gal-3 in bone marrow macrophage lineage cells may be responsible for the enhancement of M2-like macrophage infiltration into tumors, resulting in induction of tumor angiogenesis and enhanced tumor growth. To test this hypothesis, we replaced the bone marrow of Gal3−/− mice with cells from Gal3+/+ mice by BM-T and analyzed tumor growth and angiogenesis using these chimeric mice [Gal3−/− (BM-T) mice] (Figure 5A). We generated control mice in which bone marrow of Gal3+/+ animals was replaced with that of Gal3+/+ mice [Gal3+/+ (BM-T) mice]. After BM-T, we confirmed that bone marrow cells did express Gal-3 in the Gal3−/− (BM-T) mice (Figure 5B). Enhancement of both B16 and LLC tumor growth was abrogated completely in Gal3−/− (BM-T) mice (Figure 5, C–H). In terms of macrophage infiltration, the number of F4/80-positive cells in the tumor microenvironment was not significantly different between Gal3−/− (BM-T) mice and Gal3+/+ (BM-T) mice (Figure 6, A and B ). This was also the case when the number of MRC1-positive cells and the number of these cells closely associating with CD31-positive blood vessels in the tumor microenvironment was quantified (Figure 6, C–E). Attenuation of enhancement of macrophage infiltration into tumors developing in Gal3−/− (BM-T) mice also was accompanied by restoration of angiogenesis to a level observed in Gal3+/+ (BM-T) host tumors (Figure 6, C–E). Therefore, we conclude that lack of Gal-3 in bone marrow cells is responsible for the enhanced migration of macrophages into the tumor microenvironment, and that this facilitates tumor growth. We analyzed the functional role of tumor environmental Gal-3 in tumor growth. Although several functions of Gal-3 in a variety of cells, such as immune cells, neuronal cells, epithelial cells, cancer cells, and others, have been suggested,12Dumic J. Dabelic S. Flögel M. Galectin-3: an open-ended story.Biochim Biophys Acta. 2006; 1760: 616-635Crossref PubMed Scopus (882) Google Scholar, 28Nangia-Makker P. Balan V. Raz A. Regulation of tumor progression by extracellular galectin-3.Cancer Microenviron. 2008; 1: 43-51Crossref Scopus (102) Google Scholar we focused on macrophages. It has been reported that recombinant Gal-3 induces chemotaxis of monocytes/macrophages; however, the in vivo relevance of this for tumor growth had not been analyzed. We report that Gal-3 produced by the tumor induces macrophage migration, resulting in the promotion of angiogenesis and tumor growth. There are two modes of action of Gal-3 (ie, mediated by intracellular Gal-3), especially in the nucleus, or secreted Gal-3. In the case of intracellular action, Gal-3 as a component of the heterogeneous nuclear ribonuclear protein is a factor in pre-mRNA splicing to control cell cycling and prevent apoptosis possibly mediated through interaction with Bcl-2 family members.29Dagher S.F. Wang J.L. Patterson R.J. Identification of galectin-3 as a factor in pre-mRNA splicing.Proc Natl Acad Sci U S A. 1995; 92: 1213-1217Crossref PubMed Scopus (365) Google Scholar, 30Haudek K.C. Spronk K.J. Voss P.G. Patterson R.J. Wang J.L. Arnoys E.J. Dynamics of galectin-3 in the nucleus and cytoplasm.Biochim Biophys Acta. 2010; 1800: 181-189Crossref PubMed Scopus (121) Google Scholar On the other hand, the secreted form of Gal-3 regulates cell adhesion and migration through cell-cell and cell-extracellular matrix interactions.31Califice S. Castronovo V. Van Den Brûle F. Galectin-3 and cancer (review).Int J Oncol. 2004; 25: 983-992PubMed Google Scholar, 32Perillo N.L. Marcus M.E. Baum L.G. Galectins: versatile modulators of cell adhesion, cell proliferation, and cell death.J Mol Med. 1998; 76: 402-412Crossref PubMed Scopus (588) Google Scholar In terms of myeloid cell lineages, it has been reported that exogenous Gal-3 affects apoptosis of neutrophils in a context-dependent manner33Karlsson A. Christenson K. Matlak M. Björstad A. Brown K.L. Telemo E. Salomonsson E. Leffler H. Bylund J. Galectin-3 functions as an opsonin and enhances the macrophage clearance of apoptotic neutrophils.Glycobiology. 2009; 19: 16-20Crossref PubMed Scopus (113) Google Scholar and induces mediator release from both IgE-sensitized and IgE-nonsensitized mast cells.34Frigeri L.G. Liu F.T. Surface expression of functional IgE binding protein, an endogenous lectin, on mast cells and macrophages.J Immunol. 1992; 148: 861-867PubMed Google Scholar, 35Liu F.T. Frigeri L.G. Gritzmacher C.A. Hsu D.K. Robertson M.W. Zuberi R.I. Expression and function of an IgE-binding animal lectin (epsilon BP) in mast cells.Immunopharmacology. 1993; 26: 187-195Crossref PubMed Scopus (30) Google Scholar Here, we analyzed Gal-3 secreted from tumors for its action on macrophages. It has been reported that Gal-3 production is observed in immune cells, epithelial cells, and neuronal cells.12Dumic J. Dabelic S. Flögel M. Galectin-3: an open-ended story.Biochim Biophys Acta. 2006; 1760: 616-635Crossref PubMed Scopus (882) Google Scholar, 31Califice S. Castronovo V. Van Den Brûle F. Galectin-3 and cancer (review).Int J Oncol. 2004; 25: 983-992PubMed Google Scholar To analyze the migration of macrophages into the tumor microenvironment more precisely, attenuation of Gal-3 expression by all cells except tumor cells is required. In our system, we used Gal3−/− mice as an allograft host and two Gal-3-producing cancer cell lines that established tumors generating a concentration gradient of Gal-3, enabling us to visualize macrophage migration. Macrophages in the tumor microenvironment are termed tumor-associated macrophages and their number correlates with the malignancy of the tumor.36Allavena P. Mantovani A. Immunology in the clinic review series; focus on cancer: tumour-associated macrophages: undisputed stars of the inflammatory tumour microenvironment.Clin Exp Immunol. 2012; 167: 195-205Crossref PubMed Scopus (314) Google Scholar It is widely accepted that tumor-associated macrophages promote angiogenesis, which stimulates tumor growth.25Sica A. Allavena P. Mantovani A. Cancer related inflammation: the macrophage connection.Cancer Lett. 2008; 267: 204-215Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 26Solinas G. Germano G. Mantovani A. Allavena P. Tumor-associated macrophages (TAM) as major players of the cancer-related inflammation.J Leukoc Biol. 2009; 86: 1065-1073Crossref PubMed Scopus (1138) Google Scholar In our present work, we found that the Gal-3 concentration gradient effectively induced migration of M2-like macrophages. It is well known that there are two types of macrophages. One is termed M1, which are macrophages differentiated and activated by lipopolysaccharide and the proinflammatory cytokine interferon-γ. M1 macrophages produce high levels of oxidative metabolites and proinflammatory cytokines for host defense and tumor cell death.37Mosser D.M. Edwards J.P. Exploring the full spectrum of macrophage activation.Nat Rev Immunol. 2008; 8: 958-969Crossref PubMed Scopus (6554) Google Scholar, 38Benoit M. Desnues B. Mege J.L. Macrophage polarization in bacterial infections.J Immunol. 2008; 181: 3733-3739PubMed Google Scholar On the other hand, M2 macrophages activated by IL4 or IL13 promote angiogenesis and also matrix remodeling.39Bronte V. Zanovello P. Regulation of immune responses by L-arginine metabolism.Nat Rev Immunol. 2005; 5: 641-654Crossref PubMed Scopus (1370) Google Scholar, 40Mantovani A. Sica A. Sozzani S. Allavena P. Vecchi A. Locati M. The chemokine system in diverse forms of macrophage activation and polarization.Trends Immunol. 2004; 25: 677-686Abstract Full Text Full Text PDF PubMed Scopus (4732) Google Scholar, 41Stein M. Keshav S. Harris N. Gordon S. Interleukin 4 potently enhances murine macrophage mannose receptor activity: a marker of alternative immunologic macrophage activation.J Exp Med. 1992; 176: 287-292Crossref PubMed Scopus (1431) Google Scholar Therefore, a high frequency of M2 macrophages as observed in tumors developing in Gal3−/− mice correlates with robust angiogenesis in this model. Interestingly, we found that MRC1-positive M2-like macrophages express lower levels of Gal-3 than MRC-negative macrophages. When cells abundantly express Gal-3, they may not use remotely produced Gal-3 for their chemoattractive migration toward the Gal-3-producing area. Therefore, Gal-3 expression needs to be attenuated for testing cell migration toward Gal-3. In our analysis, J774 macrophages in which Gal-3 had been knocked down, or macrophages from Gal3−/− mice, more effectively migrate toward Gal-3 than the parental J774 cells or macrophages derived from Gal3+/+ mice, respectively. These results suggest that M2 macrophages have the ability to migrate along Gal-3 concentration gradients. In our present model, we injected LLC lung cancer cells subcutaneously, so this was a nonorthotopic model. Because melanoma occurs in the skin, subcutaneous B16 injection may be viewed as an orthotopic model. In tumor progression, interactions between host cells and tumor cells influence both angiogenesis and metastasis.42Fidler I.J. Critical determinants of metastasis.Semin Cancer Biol. 2002; 12: 89-96Crossref PubMed Scopus (356) Google Scholar Therefore, in addition to the nonorthotopic model using LLC cells, we inoculated these cells into the lung and observed the effects of Gal-3. The results suggest that Gal-3 from tumor cells induces migration of M2 macrophages and angiogenesis in an orthotopic model (Supplemental Figure S143Takizawa H. Kondo K. Toba H. Kenzaki K. Sakiyama S. Tangoku A. Fluorescence diagnosis of lymph node metastasis of lung cancer in a mouse model.Oncol Rep. 2009; 22: 17-21PubMed Google Scholar). This further suggests that Gal-3 from metastatic as well as primary tumor induces macrophage migration into the tumor. Our present model using Gal3−/− mice as a tumor host recapitulates the tumor condition in which expression of Gal-3 in the tumor increases. It has been reported that Gal-3 directly induces endothelial tube formation.19Nangia-Makker P. Honjo Y. Sarvis R. Akahani S. Hogan V. Pienta K.J. Raz A. Galectin-3 induces endothelial cell morphogenesis and angiogenesis.Am J Pathol. 2000; 156: 899-909Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar, 44Markowska A.I. Liu F.T. Panjwani N. Galectin-3 is an important mediator of VEGF- and bFGF-mediated angiogenic response.J Exp Med. 2010; 207: 1981-1993Crossref PubMed Scopus (250) Google Scholar Therefore, higher levels of Gal-3 induce angiogenesis directly affecting ECs within the tumor; however, macrophage recruitment by Gal-3 also may be involved in the acceleration of tumor angiogenesis. In summary, knowledge of Gal-3 expression may help in the assessment of cancer patient status. Indeed, one line of evidence suggests that Gal-3 expression levels are related to the degree of biological aggressiveness in human colorectal tumors.45Legendre H. Decaestecker C. Nagy N. Hendlisz A. Schüring M.P. Salmon I. Gabius H.J. Pector J.C. Kiss R. Prognostic values of galectin-3 and the macrophage migration inhibitory factor (MIF) in human colorectal cancers.Mod Pathol. 2003; 16: 491-504Crossref PubMed Scopus (89) Google Scholar Therefore, suppression of the function or expression of Gal-3 may be a promising approach for cancer therapy. We thank Dr. Daniel K. Hsu, Dr. Fu-Tong Liu (University of California), and Dr. Koichi Hiraga (University of Toyama, Japan) for providing Gal3−/− mice; Noriko Fujimoto for preparation of plasmid DNA; and Keisho Fukuhara for administrative assistance. Download .pdf (.11 MB) Help with pdf files Supplemental Figure S1
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