ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH
Vol. 38, No. 10 October 2014
Ethanol Supports Macrophage Recruitment and Reinforces Invasion and Migration of Lewis Lung Carcinoma Keke Yu, Jinlian Yang, Fei Wang, Li Chen, Yanmin Lu, Jia Luo, and Siying Wang
Background: Inflammation plays a critical role in cancer progression, and our data suggested that ethanol (EtOH) could promote the progression of breast cancer via increased monocyte chemo-attractant protein-1 (MCP-1). Thus, we investigated the effects of EtOH on lung cancer growth and metastasis to explore whether immunosuppression had a role. Methods: C57BL/6 mice (n = 10) implanted with Lewis lung cancer (LLC) cells were used to model physiologically relevant EtOH intake on tumor growth and inflammation after macrophage polarization. Tumors were isolated and measured, and MCP-1 expression was measured via immunohistochemistry and Western blot. Recruitment of macrophages using CD11b and F4/80 antibodies was detected with immunohistochemistry and flow cytometry. Changes in arginase I and inducible nitric oxide synthase (iNOS) expression were measured with immunofluorescent microscopy. EtOH’s effect on in vitro tumor angiogenesis was evaluated in culture, and the tumor microvessel density was assessed with CD31 immunohistochemistry. Matrix metalloproteinase 9 and interleukin 10 expressions were measured by Western blot, ELISA, and immunohistochemistry. Finally, we treated a macrophage cell line RAW264.7 with EtOH and measured changes in arginase I and iNOS expression. Results: With EtOH exposure, macrophage density was positively correlated with MCP-1 expression. Macrophages infiltrated the tumor site, becoming tumor-associated macrophages that polarized to M2 phenotypes (ArgIhigh/iNOSlow) after EtOH treatment. Cancerous cells interacted with immune cells, especially M2 macrophages, and promoted tumor angiogenesis, progression, and invasiveness. RAW264.7 cells stimulated with EtOH expressed more arginase I and less iNOS than controls. These results agreed with the features of M2 phenotype macrophages (ArgIhigh/iNOSlow). Conclusions: Data show that EtOH induced M2 phenotype macrophages, suggesting that progression and metastasis of LLC may be mediated by recruitment of M2 macrophages, especially under the influence of EtOH. Key Words: Ethanol, Lewis Lung Carcinoma, Tumor-Associated Macrophages, Monocyte Chemo-Attractant Protein-1, M2 Macrophages.
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THANOL (ETOH) CONSUMPTION is a key risk factor for development and progression of diverse cancers such as that of the liver, breast, and colon (Boffetta et al., 2006). Mechanisms underlying these cancers include alcohol metabolism, changes in retinoic acid concentration, estrogen metabolism, and oxidative stress (Seitz and Stickel, 2007). Meanwhile, long-term EtOH consumption inhibits immune function. Lung cancer is the most common cause of cancer deaths in recent years (Gadqeel, 2013). Most studies have focused From the Department of Immunology (KY, FW, SW), Anhui Medical University, Hefei, Anhui, China; Department of Biobank (KY), Shanghai Chest Hospital, Shanghai JiaoTong University, Shanghai, China; Department of Pathophysiology (KY, JY, LC, YL, SW), Anhui Medical University, Hefei, Anhui, China; and Department of Internal Medicine (JL), University of Kentucky College of Medicine, Lexington, Kentucky. Received for publication February 23, 2014; accepted July 8, 2014. Reprint requests: Siying Wang, Department of Immunology, Anhui Medical University, Hefei, Anhui, China; Tel.: +86-0551-6513-7833; Fax: +86-0551-6513-7833; E-mail:[email protected] Copyright © 2014 by the Research Society on Alcoholism. DOI: 10.1111/acer.12512 Alcohol Clin Exp Res, Vol 38, No 10, 2014: pp 2597–2606
on primary tumor events, often with metastasis as an end point. In Lewis lung cancer (LLC), macrophages are activated and interleukin-6 (IL-6) and tumor-necrosis factor-a are secreted via activation of the Toll-like receptor family (Zajac et al., 2013). Tumors secrete chemokines that direct bone marrow-derived monocytes toward the tumor site. These myeloid cells are CD11b- and VEGFR1-positive, and they secrete matrix metalloproteinase 9 (MMP-9) that releases matrix-bound vascular endothelial growth factor (VEGF), whose function is required for increased metastatic efficiency (Kaplan et al., 2005). Resident and newly infiltrating macrophages are key leukocyte populations that regulate production of inflammatory mediators to warn of injury and initiate tissue repair during cancer-associated inflammation. Inflammatory cells such as macrophages can interact with cancer cells and express angiogenic factors. In the metastatic position, macrophages prepare for the arrival of tumor cells, and different subsets of macrophages promote tumor cell extravasation, survival, and growth. Clinical studies suggest a strong association between poor survival and increased macrophage density in thyroid, lung, and hepatocellular cancers (Duluc et al., 2007). Tumor-associated macrophages 2597
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(TAMs) the most abundant immunosuppressive cells in the tumor microenvironment, originate from blood monocytes and exhibit IL-10high IL-12low. They can release diverse cytokines and inflammatory mediators and are correlated with cancer cell proliferation. In some cases, the tumor microenvironment suppresses many immune responses and alters the phenotype to promote tumor growth. Studies suggest that macrophages can produce IL-10, which in turn, induces monocytes to express the costimulatory molecule programed death ligand (PD)-L1 (Kuang et al., 2009). Macrophages in human ovarian cancers can regulate the influx of regulatory T cells (Tregs) that suppress cytotoxic T cell responses (Green et al., 2009). The implications of these associations for EtOH have not been fully elucidated, but our previous studies of breast cancer (Wang et al., 2012) confirmed that vascular generation and increased monocyte chemo-attractant protein-1 (MCP-1) expression correlated with tumor progression. Thus, we sought to evaluate the importance of macrophages in innate and adaptive immunity and to investigate whether EtOH can stimulate cancer via supporting the recruitment of TAMs and M2 polarization.
MATERIALS AND METHODS Cell Culture and EtOH Exposure LLC and RAW264.7 cell lines were grown with DMEM (GIBCO, Gaithersburg, MD) medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT), penicillin (100 U/ml)/streptomycin (100 U/ml). All cell lines were incubated at 37°C with 5% CO2. EtOH solutions were made daily, and the appropriate amount of EtOH (0.1%) was added to the culture medium (Luo and Miller, 1997). Tumor Model C57BL/6 mice (male and female, 6 to 8 weeks of age) were purchased from the experimental animal center of Anhui Province, housed on hardwood bedding with 12-hour light/dark cycles. Mice were given 1% EtOH in drinking water for 12 hours overnight, and this was replaced with regular water drinking during the remaining 12 hours each day for 4 weeks. Control mice received regular drinking water only. In the second week, all mice were injected subcutaneously in the right flank with ~5 9 105 LLC cells in 0.1 ml phosphate-buffered saline (PBS). Both groups were comprised of 10 mice. Finally, mice were sacrificed, all tumors were excised, and tumor diameters were measured with calipers. Tumor volume (TV) was calculated as follows: TV = length 9 width 9 depth 9 0.52 (mm3). Immunohistochemical Staining for Microvessels and Immune Cells Perfused tumors were fixed in formalin and embedded in paraffin, cut into 4-lm sections, then stained with hematoxylin and eosin (H&E), and photographed under a microscope. Five sections were examined per mouse with each of the following antibodies: rat polyclonal CD11b antibody (1:200 dilution, bs-1014R; Bioss, Beijing, China) to stain macrophages, rat anti-mouse MCP-1 (Sc-7269; 1:100 dilution) and IL-10 (ab33471; 1:300 dilution), goat polyclonal MMP-9 antibody (sc-6840; 1:300 dilution), and rabbit polyclonal CD31 (platelet endothelia cell adhesion molecule 1) antibody
(BA2211; Boster, Wuhan, HB, China) to stain microvessels. Microvessel densities (MVDs) were counted by scanning sections stained with anti-mouse CD31 antibody. Any CD31+-stained endothelial cell or cluster that was clearly separated from adjacent microvessels, tumor cells, and connective elements was counted as 1 microvessel. The mean microvessel count of the 5 most vascular areas was taken as the MVD, which was expressed as the absolute number of microvessels per 1.485 mm2 (200 9 field; Tan et al., 2007). Western Blots Cells were collected, washed, and resuspended in lysis buffer. Samples were boiled and separated by SDS-PAGE then transferred to a 0.22-lm PVDF membrane (Millipore Co., Billerica, MA) by electro-blotting. Primary antibodies included rat antimouse MCP-1 (Sc-7269), IL-10 (ab33471), and rabbit anti-mouse inducible nitric oxide synthase (iNOS) (ProteinTech-14142, Chicago, IL), and Arginase I (ProteinTech-16001). b-actin (Sc-47778) was used for control protein loading. Secondary antibodies were peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG. Proteins were detected with enhanced chemi-luminescent. Lymphocyte Isolation from Tumors and Bone Marrow Mice were sacrificed, and tumors and femurs were removed and weighed. Tumors were isolated and washed, passed through a wire mesh screen into ice-cold PBS, and centrifuged at 4°C, 2,000 9 g for 10 minutes. Cells were resuspended and layered onto 5 ml lympholyte-M in a tube. The tube was centrifuged at room temperature at 2,000 9 g for 20 minutes. Tumor-infiltrated lymphocytes at the interface were collected and counted. One femur from each C57BL/6 mouse was removed, and the 2 ends of the bone were cut to expose the bone cavity. The marrow was flushed with 5 to 10 ml PBS. Bone marrow cells were resuspended and layered onto 10 ml lympholyte-M to remove red blood cells. Flow Cytometry Analysis Previous immunohistochemistry data were qualitative, so we measured the proportion of positive cells by flow cytometry. Cell phenotypes were analyzed using PE-labeled-CD11b and FITC-labeled-F4/80 (all from eBioscience, San Diego, CA). Cells were preincubated for 30 minutes at 4°C with antibody and then analyzed on a FACScan (BD Inc., San Diego, CA). Isotype control monoclonal antibodies were from BD Inc. Cell counts were expressed as % gated (percentage of CD11b- and F4/80positive cells in the tumor). Immunofluorescent Analysis of Macrophage Polarization States Cell numbers were measured, and 20,000 cells were cytospun onto a slide for 20 minutes. Three slides per mouse were examined for both control and EtOH-treated samples. Primary antibody concentrations were as follows: iNOS (1:100), arginase I (1:100), and CD-11b-PE (1:200). Then, cells were washed and treated with secondary antibody (1:1,000) and rinsed 3 times. Immunofluorescent images were examined with a LEICA-inverted confocal microscope (Wetzlar, Germany), and fluorescent signals were measured with the same pinhole, detector gain, and amplifier offset. Three-Dimensional Endothelial Cell and Tumor Coculture System To investigate the effect of EtOH on tumor angiogenesis, we utilized a 3-dimensional (3D) model of endothelial cell and
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Fig. 1. Ethanol (EtOH) consumption significantly increased Lewis lung cancer (LLC) tumor volume and attenuated invasive tumor growth patterns. (A, B) Comparison of in situ tumor growth and tumor volume with/without 1% EtOH treatment in a LLC C57BL/6 mice model 22 days after tumor implantation. Tumor sizes were measured every 2 days. (C) Comparisons of tumor weight between control and EtOH treatments. (D) Lung metastasis in LLC tumor model was increased by EtOH treatment. Percentages of mice with metastatic lung carcinoma nodes are presented. N = 10 per group. *p < 0.05, compared with control group by Student’s t-test. (E) Histological features of lungs from mice with/without 1% EtOH treatment. Metastatic carcinoma nodules were present in lungs of the 1% EtOH-exposed group.
tumor cell cocultures adapted from a previous study with some modifications (Chen et al., 2009). In brief, SVEC cells were trypsinized and mixed with Cytodex beads (3 9 103; Sigma, St. Louis, MO) in DMEM medium. The mixtures were incubated for 8 hours. Mixtures were washed, resuspended, and added to 24-well cell culture plates precoated with 0.625 U thrombin (Sigma-Aldrich, St. Louis, MO). The fibrinogen/bead solution was allowed to coagulate for 5 minutes, and then, it was incubated. For coculture, endothelial cells/macrophages were layered atop the fibrin gels (Xu et al., 2010). Serum IL-10 Quantitation by ELISA Because IL-10 regulates immune and inflammatory responses and because it is an inhibitory cytokine that mediates suppression by regulatory or suppressor T cells (Tsuji-Takayama et al., 2008), we assayed serum IL-10 by ELISA, according to the manufacturer’s instructions. MTT Assay With an MTT assay, cytotoxicity of CD11b+ TAMs was measured. Cells were plated into 96-well plates and exposed to 0.1% EtOH or PBS. After treatment, 10 ll of MTT reagent was added into each well, and the plates were incubated. The culture was solubilized, and spectrophotometric absorbance was measured at 595 nm using a microtiter plate reader.
Statistical Analysis Where indicated, data are presented as means SEM of duplicate experiments (n = 10/group). Statistically significant differences in means between groups were examined by an unpaired Student’s t-test. A p < 0.05 was considered statistically significant. All statistical calculations were performed using SPSS software (SPSS Inc., Chicago, IL).
RESULTS EtOH Consumption Significantly Increased LLC Tumor Volume and Attenuated Invasive Tumor Growth Patterns One week before LLC cell implantation, C57BL/6 mice were given EtOH as described in the Materials and Methods section. EtOH exposure significantly increased tumor volumes and weight after LLC cell implantation (Fig. 1A). Lung metastasis in a LLC model was increased by EtOH treatment. More metastatic nodules appeared in the EtOHtreated group and the rate increased from 35% in the control to 75% in the EtOH-treated group (Fig. 1B). Under light microscopy and H&E staining, pulmonary congestion was present, as were metastatic carcinoma nodules after EtOH treatment (Fig. 1C).
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EtOH Supported Recruitment of Immune Cells Especially TAMs into Tumors Immunohistochemical staining revealed that CD11b+ TAMs accumulated at tumor sites (Fig. 3A). Surface expression of CD11b and F4/80 in cell populations obtained from tumors by fluorescence-activated cell sorting (Fig. 3B) indicated that the number and percentage of CD11b+/F4/80+ TAMs increased significantly in the tumor from the EtOHtreated group.
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Fluorescent intensity was scanned, and the ratio of ArgI/ iNOS was calculated and statistically analyzed. EtOH supports macrophages displayed M2 (ArgIhigh/iNOSlow) polarized during LLC development. This mirrored the M2 activation state of macrophages after EtOH treatment. M2 macrophages, another immune-suppressing leukocyte population (Marttila-Ichihara et al., 2009), had poor antigen-presenting capacity, prevented T cell activation, and promoted the recruitment of Tregs (Fig. 4).
Temporal Changes in TAM Activation States
Overexpression of IL-10 in Serum and Tumor
Because the stage of tumor progression affected the activation state of macrophages, we hypothesized that EtOH consumption changed the tumor microenvironment to promote macrophage activation. Macrophages were stained with anti-CD11b to readily distinguish them from other lymphocytes. For all samples, monocytes displaying no detectable arginase I or iNOS were designated as ArgIlow/iNOSlow. M2 macrophages were stained as ArgIhigh/iNOSlow, and M1 macrophages were stained as ArgIlow/iNOShigh. Colocalization of the binding of different antibodies to the same cell was detected with multiple fluorescent stains. These activation state changes in macrophages were shown in control and EtOH-treated tumors. Immunoblots were not different with regard to iNOS expression between control and EtOH-treated tumors, but arginase I expression was significantly increased in the EtOH-treated group.
Overexpression of IL-10 in serum has been reported in several other malignancies: melanoma and ovarian carcinoma (Wang et al., 2011). The product of tumor cells, including extracellular matrix components, IL-10, and chemokines such as MCP-1 can activate macrophages and cause M2 polarization, which is a cancer-promoting mode. We assayed IL-10 contents in serum by ELISA and expression of IL-10 in the tumor by Western blot and immunohistochemistry. Data show that IL-10 in the EtOH-treated group increased significantly (Fig. 5A–D), suggesting that EtOH promoted MCP-1 and IL-10 expression in the tumor. Effect of EtOH on In Vitro Tumor Angiogenesis To test the hypothesis that TAMs may mediate EtOHinduced tumor angiogenesis, we used a 3D angiogenic
Fig. 4. Temporal changes in tumor-associated macrophage (TAM) activation states. (A) Two-color immunohistochemical detection of CD11b+ (red) and arginase I+ (green) or inducible nitric oxide synthase (iNOS)+ (green) cells in tumors with/without 1% ethanol (EtOH) treatment in a Lewis lung cancer (LLC) C57BL/6 mice model 22 days after tumor implantation. (B) Fluorescent intensity was scanned, and the ratio of ArgI/iNOS was calculated and analyzed statistically. The results showed that EtOH-treated macrophages displayed polarized M2 (ArgIhigh/iNOSlow) TAMs during LLC development.
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EtOH Supported Recruitment of Immune Cells Especially TAMs into Tumors Immunohistochemical staining revealed that CD11b+ TAMs accumulated at tumor sites (Fig. 3A). Surface expression of CD11b and F4/80 in cell populations obtained from tumors by fluorescence-activated cell sorting (Fig. 3B) indicated that the number and percentage of CD11b+/F4/80+ TAMs increased significantly in the tumor from the EtOHtreated group.
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Fluorescent intensity was scanned, and the ratio of ArgI/ iNOS was calculated and statistically analyzed. EtOH supports macrophages displayed M2 (ArgIhigh/iNOSlow) polarized during LLC development. This mirrored the M2 activation state of macrophages after EtOH treatment. M2 macrophages, another immune-suppressing leukocyte population (Marttila-Ichihara et al., 2009), had poor antigen-presenting capacity, prevented T cell activation, and promoted the recruitment of Tregs (Fig. 4).
Temporal Changes in TAM Activation States
Overexpression of IL-10 in Serum and Tumor
Because the stage of tumor progression affected the activation state of macrophages, we hypothesized that EtOH consumption changed the tumor microenvironment to promote macrophage activation. Macrophages were stained with anti-CD11b to readily distinguish them from other lymphocytes. For all samples, monocytes displaying no detectable arginase I or iNOS were designated as ArgIlow/iNOSlow. M2 macrophages were stained as ArgIhigh/iNOSlow, and M1 macrophages were stained as ArgIlow/iNOShigh. Colocalization of the binding of different antibodies to the same cell was detected with multiple fluorescent stains. These activation state changes in macrophages were shown in control and EtOH-treated tumors. Immunoblots were not different with regard to iNOS expression between control and EtOH-treated tumors, but arginase I expression was significantly increased in the EtOH-treated group.
Overexpression of IL-10 in serum has been reported in several other malignancies: melanoma and ovarian carcinoma (Wang et al., 2011). The product of tumor cells, including extracellular matrix components, IL-10, and chemokines such as MCP-1 can activate macrophages and cause M2 polarization, which is a cancer-promoting mode. We assayed IL-10 contents in serum by ELISA and expression of IL-10 in the tumor by Western blot and immunohistochemistry. Data show that IL-10 in the EtOH-treated group increased significantly (Fig. 5A–D), suggesting that EtOH promoted MCP-1 and IL-10 expression in the tumor. Effect of EtOH on In Vitro Tumor Angiogenesis To test the hypothesis that TAMs may mediate EtOHinduced tumor angiogenesis, we used a 3D angiogenic
Fig. 4. Temporal changes in tumor-associated macrophage (TAM) activation states. (A) Two-color immunohistochemical detection of CD11b+ (red) and arginase I+ (green) or inducible nitric oxide synthase (iNOS)+ (green) cells in tumors with/without 1% ethanol (EtOH) treatment in a Lewis lung cancer (LLC) C57BL/6 mice model 22 days after tumor implantation. (B) Fluorescent intensity was scanned, and the ratio of ArgI/iNOS was calculated and analyzed statistically. The results showed that EtOH-treated macrophages displayed polarized M2 (ArgIhigh/iNOSlow) TAMs during LLC development.
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model in which endothelial cells and macrophages from mouse bone marrow or mouse TAMs (CD11b+) were cultured together. Endothelial cells attached to Cytodex beads were formed a 3D capillary tube-like network indicative of angiogenesis. As shown in Fig. 6, SVEC attached, sprouted, and formed narrow cordlike structures. The inclusion of macrophages in this system modestly increased the number and the length of spouts from SVEC, but EtOH significantly increased the spouts (spouts were 4.8% in SVEC cultures alone; 11.3% in cocultured with macrophages; 68.6% in cocultured with macrophages treated with EtOH). Spout percentages were 22.1% in SVECs cocultured with CD11b+ TAMs and 70.8% with EtOH treatment.
and chemokines, such as VEGF and MMP-9, which directly promote angiogenesis and tumor metastasis (Mantovani and Sica, 2010). In EtOH-treated groups, after tumor implantation, expression of intracellular MMP-9 protein increased (Fig. 8). MMP-9 expression significantly increased after EtOH exposure, especially in hypoxic areas of tumor necrosis. EtOH Suppressed Cytotoxicity of TAMs Macrophages in the tumor can produce cytokines which regulate the influx of Tregs to suppress responses of cytotoxic T cells and TAMs. Our data revealed cytotoxicity of TAMs in the EtOH-treated group decreased more than controls as measured by MTT (Fig. 9).
EtOH Promoted Tumor Angiogenesis in a LLC Model Vascular endothelial cells were stained with PECAM-1 (CD31). Tumor MVDs were calculated by new vessel formation using CD31 staining, and microvessels appeared as brown linear fragments. With EtOH treatment, tumor MVDs increased markedly (Fig. 7). Macrophages in the tumor microenvironment facilitate tumor invasion and angiogenesis by secreting several inflammatory cytokines
EtOH Induced RAW264.7 Cells to M2 Polarized To confirm that EtOH induced M2 macrophage polarization, we treated RAW264.7 macrophage cells with 0.1% EtOH and unstimulated cells were used as controls. Slight arginase I staining was consistently seen in unstimulated RAW264.7 cells. After cell stimulation for 24, 48, and 72 hours, cells were probed for membrane protein CD11b
Fig. 5. Overexpression of interleukin 10 (IL-10) in serum and tumor. IL-10 in serum and in tumors was assayed by ELISA, Western blot, and immunohistochemistry. (A) Serum IL-10 concentration histogram and statistical analysis. (B) IL-10 expression in tumor detected by Western blot. (C) Comparison of IL-10 in tumors from mice with/without ethanol (EtOH) treatment was detected by immunohistochemistry of IL-10 antibody. (D) Quantitation was calculated as the IL-10 positive area/total area. Columns represent means of 10 samples in each group n = 10 per group, *p < 0.05. IL-10 in serum and tumor increased significantly after 1% EtOH treatment.
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and changes in arginase I and iNOS expression. Colocalization experiments indicated that the same cells stained for both proteins. Arginase I expression was significantly higher in RAW264.7 cells after EtOH exposure compared with control cells. Polarization was more apparent with a longer induction time (Fig. 10). DISCUSSION Alcohol is a known dietary component and is usually only of concern when it is abused, at which point it becomes immunosuppressive. Nonetheless, immune response regulation by EtOH is complex and dose-dependent as well as dependent on the exposure duration and the cell type targeted (Crews et al., 2006). Little is known about how EtOH exposure induces or alters immune cells in a lung cancer microenvironment during tumor progression and metastasis.
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Tumors consist of both tumor and stromal cells and create a unique microenvironment essential for tumor progression and invasion. The tumor microenvironment can actively program immune dysfunction allowing the tumor cell to escape immunologic attack (Rabinovich et al., 2007). TAMs, CD4+CD25+ Tregs, and myeloid-derived suppressor cells are known to inhibit host antitumor immune activity (Finn, 2008; Zhang and Meadows, 2008). TAMs have 2 activation phenotypes that are regulated by varied cytokines. M1 is classically polarized by lipopolysaccharide, interferon-c and releases IL-6 and IL-12. M1 up-regulate iNOS. M2, an alternative polarized phenotype by IL-4, IL-13, releases anti-inflammatory IL-10. M2 up-regulate arginase I and mannose receptors. During wound healing, M2 can facilitate angiogenesis and tissue remodeling. Arginase I catalyzes the formation of ornithine, which leads to synthesis of polyamines necessary for DNA synthesis and the stimulation of cell
Fig. 6. Effect of ethanol (EtOH) on in vitro tumor angiogenesis. (A) In vitro tumor angiogenesis in mouse bone marrow macrophages after EtOH treatment. (B) In vitro tumor angiogenesis in mouse tumor-associated macrophages (CD11b+) after EtOH treatment.
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Fig. 7. Ethanol (EtOH) promoted tumor angiogenesis in a Lewis lung cancer model. (A) Tumor microvessel densities (MVDs) were calculated by new vessel formation using CD31 staining. Microvessels appeared as brown linear fragments. Comparison of MVDs by CD31+ tumor endothelial cells by hematoxylin and eosin staining (1,2) and immunostaining (3,4) between mice with/without EtOH treatment. (B) Comparison of MVDs between control and EtOH treatment groups, *p < 0.05.
Fig. 9. Ethanol (EtOH) suppressed cytotoxicity of tumor-associated macrophages (TAMs). TAM cytotoxicity was assayed by MTT and EtOH increased cytotoxicity.
Fig. 8. Ethanol (EtOH) promoted tumor angiogenesis in a Lewis lung cancer model. Matrix metalloproteinase 9 (MMP-9) was detected by immunostaining. (A) MMP-9 expression-promoted tumor metastasis markedly increased in EtOH-treated mice, especially in hypoxic areas of tumor necrosis. (B) Quantitation was calculated as the MMP-9 positive area/total area. Columns represent means of 10 samples in each group, *p < 0.05 compared with the control group.
division while depleting the arginine substrate needed to produce NO. It has been hypothesized that immune cells produce a mutagenic environment by generating both reactive nitrogen and oxygen species (Cho et al., 2013; Redente et al., 2007). In our study, 4 weeks of EtOH exposure significantly increased tumor size and weight. EtOH exposure promoted tumor metastasis to the lungs; the metastatic rate increased
from 35% in the control to 75% in the EtOH-exposed group. MCP-1 is a chemoattractant for macrophages to the inflammatory areas, is related to a variety of inflammatory diseases, (Melgarejo et al., 2009) and is correlated with tumor angiogenesis (Wang et al., 2012). We confirmed that in a LLC mouse model, MCP-1 expression increased after EtOH treatment and CD11b+/F4/80+ TAMs significantly increased and infiltrated around the tumor stroma. Also, TAMs accumulate at tumor sites to maintain immune tolerance which circumvents tumor immunity (Zou, 2005). We report that after EtOH exposure, tumors progressed to malignancy, and macrophages increased and were polarized to M2 displaying ArgIhigh/iNOSlow. Classical M1 may be phenotypically characterized with expression of iNOS and alternative M2 with expression of arginase I. Macrophages stained with arginase I or iNOS had a temporal dependence on the stage of tumor development. RAW264.7 cells treated with EtOH stained more intensely for arginase I than controls, but iNOS
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expression diminished. These results were in accordance with the features of M2 macrophages and indicated that EtOH could polarize macrophages to the M2 phenotype. Using an in vitro 3D angiogenic model, we saw that macrophages and CD11b+ TAMs could promote tumor angiogenesis, and this ability was significantly enhanced with EtOH. Growth and expansion of tumor masses are strictly dependent on angiogenesis. Thus, we calculated tumor MVDs by new vessel formation using CD31 staining and found that tumor MVDs increased after EtOH treatment. In contrast, TAMs had high pro-angiogenic activity and can secrete MMP-9 actively, facilitating endothelial cell migration. Consistent with the phenotype of tumor growth and distant metastasis, MMP-9 expression also increased more markedly after EtOH exposure compared to control. The evidence suggested the effects of EtOH intake on MMP-9 expression may be linked to the mechanisms of EtOHinduced LLC tumor progression. After EtOH treatment, obvious tumor necrosis was observed. Macrophage accumulation and increased MMP-9 expression in hypoxic areas of the tumor were particularly associated with necrotic tissue. Tumor cells may be activated by TAMs and secrete angiogenic factors. The interaction between them was, in part, mediated through soluble factors involving the IL-10 pathway.
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An inflammatory microenvironment may increase mutation rates: activated inflammatory cells can serve as reactive nitrogen intermediate sources which can induce DNA damage and genomic instability (Grivennikov et al., 2010). TAM phenotype analysis suggests that transcriptional factors NFjB and HIF-1 are master regulators of their transcriptional programs and that these factors centrally regulate tumor progression and metastasis (Sica and Bronte, 2007). In this study, tumors in control samples invaded to fat and fascia, but in the EtOH-treated group, tumors invaded into deep tissue and skeletal muscle. In addition to TAMs, IL-10 increased in serum and tumors. Production of IL-10 may provide a mechanism for tumor evasion of T cell–mediated immune responses. Our results revealed cytotoxicity of TAMs as evidenced by decreased MTT in the EtOH-treated group. All of these changes could promote immunosuppression, tumor progression, and metastasis. In summary, our work suggests that chronic alcohol consumption promotes TAMs recruitment into microdomains, leading to immunosuppression. TAMs dysfunction was consistent with increased immunosuppression in the LLC EtOH-treated mice. TAMs especially M2 phenotypes constituted the major components of leukocyte infiltration. Progression and metastasis of LLC may be mediated by recruitment TAMs, especially under the influence of EtOH.
Fig. 10. Ethanol (EtOH) induced RAW264.7 cells to M2 polarized. (A) Two-color immunofluorescent detection of CD11b+ (red) and arginase I+ (green) or inducible nitric oxide synthase (iNOS)+ (green) in RAW264.7 cells with/without 1% EtOH treatment. RAW264.7 cells lost their M1 (iNOS-expressing) phenotype and immunostained exclusively for arginase I. RAW264.7 cells’ M2 polarization was more apparent after a longer period of induction. (B) Fluorescent intensity was scanned, and the ratio of ArgI/iNOS was calculated and statistically analyzed. The results showed that EtOH-treated macrophages displayed a polarized M2 phenotype (ArgIhigh/iNOSlow). (C) Immunoblotting of iNOS and arginase I in control and EtOH-treated RAW264.7 cells. iNOS decreased but arginase I increased after EtOH treatment. b-actin used as a loading control for the gel.
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These studies provide a theoretical basis and experimental proof for treatment of tumors with TAMs as therapeutic targets, and these data are meaningful to the study of tumor immunity. This work should offer a significant contribution for clarifying the carcinogenic mechanism of EtOH and establishing EtOH consumption a significant factor in cancer development. ACKNOWLEDGMENTS This work was supported by the Shanghai JiaoTong University, School of Medicine Biobank Fund (YBKL2013009) and the AnHui Young Talents in University Fund (2011SQRL059). REFERENCES Boffetta P, Hashibe M, La Vecchia C, Zatonski W, Rehm J (2006) The burden of cancer attributable to alcohol drinking. Int J Cancer 4:884–887. Chen Z, Htay A, Dos Santos W, Gillies GT, Fillmore HL, Sholley MM, Broaddus WC (2009) In vitro angiogenesis by human umbilical vein endothelial cells (HUVEC) induced by three-dimensional co-culture with glioblastoma cells. J Neurooncol 92:121–128. Cho E, Kim M, Ko YS, Lee HY, Song M, Kim MG, Kim HK, Cho WY, Jo SK (2013) Role of inflammation in the pathogenesis of cardiorenal syndrome in a rat myocardial infarction model. Nephrol Dial Transplant 11:2766–2778. Crews FT, Bechara R, Brown LA, Guidot DM, Mandreakar P, Oak S, Qin L, Szabo G, Wheeler M, Zou J (2006) Cytokines and alcohol. Alcohol Clin Exp Res 30:720–730. Duluc D, Delneste Y, Tan F, Moles MP, Grimaud L, Lenoir J, Preisser L, Anegon I, Catala L, Ifrah N, Descamps P, Gamelin E, Gascan H, Hebbar M, Jeannin P (2007) Tumor-associated leukemia inhibitory factor and IL6 skew monocyte differentiation into tumor-associated macrophages-like cells. Blood 110:4319–4330. Finn OJ (2008) Cancer immunology. N Engl J Med 358:2704–2715. Gadqeel SM (2013) New targets in non-small cell lung cancer. Curr Oncol Rep 4:411–423. Green CE, Liu T, Montel V, Hsiao G, Lester RD, Subramaniam S, Gonias SL, Klemke RL (2009) Chemoattractant signaling between tumor cells and macrophages regulates cancer cell migration, metastasis and neovascularization. PLoS One 4:e6713. Grivennikov SI, Greten FR, Katin M (2010) Immunity, inflammation, and cancer. Cell 140:883–899. Kaplan RN, Riba RD, Zacharoulis S, Branley AH, Vincent L, Costa C, MacDonald DD, Jin DK, Shido K, Kerns SA, Zhu Z, Hicklin D, Wu Y, Port JL, Altorki N, Port ER, Ruqqero D, Shmelkov SV, Jensen KK, Rafii S, Lyden D (2005) VEGFR1-positive haematopoietic bone marrow progenitors initiate the pre-metatatic niche. Nature 438:820–827. Kuang DM, Zhao Q, Peng C, Xu J, Zhang JP, Wu C, Zheng L (2009) Activated monocytes in peritumoral stroma of hepatocellular carcinoma foster
immune privilege and disease progression through PD-L1. J Exp Med 206:1327–1337. Luo J, Miller MW (1997) Differential sensitivity of human neuroblastoma cell lines to ethanol: correlations with their proliferative responses to mitogenic growth factors and expression of growth factor receptors. Alcohol Clin Exp Res 21:1186–1194. Mantovani A, Sica A (2010) Macrophages, innate immunity and cancer: balance, tolerance, and diversity. Curr Opin Immunol 22:231–237. Marttila-Ichihara F, Auvinen K, Elima K, Jalkanen S, Salmi M (2009) Vascular adhesion protein-1 enhances tumor growth by supporting recruitment of Gr-1+ CD11b+ myeloid cells into tumors. Cancer Res 69:7875– 7883. Melgarejo E, Medina MA, Sanchez-Jimenez F, Urdiales JL (2009) Monocyte chemoattractant protein-1: a key mediator in inflammatory processes. Int J Biochem Cell Biol 41:998–1001. Rabinovich GA, Gabrilocich D, Sotomayor EM (2007) Immunosuppressive strategies that are mediated by tumor cells. Annu Rev Immunol 25:267– 296. Redente EF, Orlicky DJ, Bouchard RJ, Malkinson AM (2007) Tumor signaling to the bone marrow changes the phenotypes of monocytes and pulmonary macrophages during urethane-induced primary lung tumorigenesis in A/J mice. Am J Pathol 170:693–708. Seitz HK, Stickel F (2007) Molecular mechanisms of alcohol-mediated carcinogenesis. Nat Rev Cancer 7:599–612. Sica A, Bronte V (2007) Altered macrophage differentiation and immune dysfunction in tumor development. J Clin Invest 117:1155–1166. Tan W, Bailey AP, Shparago M, Brsby B, Covinqton J, Johnson JW, Young E, Gu JW (2007) Chronic alcohol consumption stimulates VEGF expression, tumor angiogenesis and progression of melanoma in mice. Cancer Biol Ther 6:e1–e7. Tsuji-Takayama K, Suzuki M, Yamamo M, Harashima A, Okochi A, Otani T, Inoue T, Suqimoto A, Motoda R, Yamasaki F, Nakamura S, Kibata M (2008) IL-2 activation of STAT5 enhances production of IL-10 from human cytoxic regulatory T cells, HOZOT. Exp Hematol 36:181–192. Wang R, Lu M, Zhang J, Chen S, Luo X, Qin Y, Chen H (2011) Increased IL-10 mRNA expression in tumor-associated macrophage correlated with late stage of lung cancer. J Exp Clin Cancer Res 30:62. Wang S, Xu M, Li F, Wang X, Bower KA, Frank JA, Lu Y, Chen G, Zhang Z, Ke Z, Shi X, Luo J (2012) Ethanol promotes mammary tumor growth and angiogenesis: the involvement of chemoattractant factor MCP-1. Breast Cancer Res Treat 3:1037–1048. Xu M, Bower KA, Wang S, Frank JA, Chen G, Ding M, Wang S, Shi X, Ke Z, Luo J (2010) Cyanidin-3-glucoside inhibits ethanol-induced invasion of breast cancer cells overexpressing ErbB2. Mol Cancer 9:285. Zajac E, Schweiqhofer B, Kupriyanova TA, Juncker-Jensen A, Minder P, Quiqley JP, Deryuqina EI (2013) Angiogenic capacity of M1- and M2polarized macrophages is determined by the levels of TIMP-1 complexed with their secreted proMMP-9. Blood 25:4054–4067. Zhang H, Meadows GG (2008) Chronic alcohol consumption perturbs the balance between thymus-derived and myeloid-derived natural killer cells in the spleen. J Leukoc Biol 83:41–47. Zou W (2005) Immunosuppressive networks in the tumor environment and their therapeutic relevance. Nat Rev Cancer 5:263–274.
Vol. 38, No. 10 October 2014
Ethanol Supports Macrophage Recruitment and Reinforces Invasion and Migration of Lewis Lung Carcinoma Keke Yu, Jinlian Yang, Fei Wang, Li Chen, Yanmin Lu, Jia Luo, and Siying Wang
Background: Inflammation plays a critical role in cancer progression, and our data suggested that ethanol (EtOH) could promote the progression of breast cancer via increased monocyte chemo-attractant protein-1 (MCP-1). Thus, we investigated the effects of EtOH on lung cancer growth and metastasis to explore whether immunosuppression had a role. Methods: C57BL/6 mice (n = 10) implanted with Lewis lung cancer (LLC) cells were used to model physiologically relevant EtOH intake on tumor growth and inflammation after macrophage polarization. Tumors were isolated and measured, and MCP-1 expression was measured via immunohistochemistry and Western blot. Recruitment of macrophages using CD11b and F4/80 antibodies was detected with immunohistochemistry and flow cytometry. Changes in arginase I and inducible nitric oxide synthase (iNOS) expression were measured with immunofluorescent microscopy. EtOH’s effect on in vitro tumor angiogenesis was evaluated in culture, and the tumor microvessel density was assessed with CD31 immunohistochemistry. Matrix metalloproteinase 9 and interleukin 10 expressions were measured by Western blot, ELISA, and immunohistochemistry. Finally, we treated a macrophage cell line RAW264.7 with EtOH and measured changes in arginase I and iNOS expression. Results: With EtOH exposure, macrophage density was positively correlated with MCP-1 expression. Macrophages infiltrated the tumor site, becoming tumor-associated macrophages that polarized to M2 phenotypes (ArgIhigh/iNOSlow) after EtOH treatment. Cancerous cells interacted with immune cells, especially M2 macrophages, and promoted tumor angiogenesis, progression, and invasiveness. RAW264.7 cells stimulated with EtOH expressed more arginase I and less iNOS than controls. These results agreed with the features of M2 phenotype macrophages (ArgIhigh/iNOSlow). Conclusions: Data show that EtOH induced M2 phenotype macrophages, suggesting that progression and metastasis of LLC may be mediated by recruitment of M2 macrophages, especially under the influence of EtOH. Key Words: Ethanol, Lewis Lung Carcinoma, Tumor-Associated Macrophages, Monocyte Chemo-Attractant Protein-1, M2 Macrophages.
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THANOL (ETOH) CONSUMPTION is a key risk factor for development and progression of diverse cancers such as that of the liver, breast, and colon (Boffetta et al., 2006). Mechanisms underlying these cancers include alcohol metabolism, changes in retinoic acid concentration, estrogen metabolism, and oxidative stress (Seitz and Stickel, 2007). Meanwhile, long-term EtOH consumption inhibits immune function. Lung cancer is the most common cause of cancer deaths in recent years (Gadqeel, 2013). Most studies have focused From the Department of Immunology (KY, FW, SW), Anhui Medical University, Hefei, Anhui, China; Department of Biobank (KY), Shanghai Chest Hospital, Shanghai JiaoTong University, Shanghai, China; Department of Pathophysiology (KY, JY, LC, YL, SW), Anhui Medical University, Hefei, Anhui, China; and Department of Internal Medicine (JL), University of Kentucky College of Medicine, Lexington, Kentucky. Received for publication February 23, 2014; accepted July 8, 2014. Reprint requests: Siying Wang, Department of Immunology, Anhui Medical University, Hefei, Anhui, China; Tel.: +86-0551-6513-7833; Fax: +86-0551-6513-7833; E-mail:[email protected] Copyright © 2014 by the Research Society on Alcoholism. DOI: 10.1111/acer.12512 Alcohol Clin Exp Res, Vol 38, No 10, 2014: pp 2597–2606
on primary tumor events, often with metastasis as an end point. In Lewis lung cancer (LLC), macrophages are activated and interleukin-6 (IL-6) and tumor-necrosis factor-a are secreted via activation of the Toll-like receptor family (Zajac et al., 2013). Tumors secrete chemokines that direct bone marrow-derived monocytes toward the tumor site. These myeloid cells are CD11b- and VEGFR1-positive, and they secrete matrix metalloproteinase 9 (MMP-9) that releases matrix-bound vascular endothelial growth factor (VEGF), whose function is required for increased metastatic efficiency (Kaplan et al., 2005). Resident and newly infiltrating macrophages are key leukocyte populations that regulate production of inflammatory mediators to warn of injury and initiate tissue repair during cancer-associated inflammation. Inflammatory cells such as macrophages can interact with cancer cells and express angiogenic factors. In the metastatic position, macrophages prepare for the arrival of tumor cells, and different subsets of macrophages promote tumor cell extravasation, survival, and growth. Clinical studies suggest a strong association between poor survival and increased macrophage density in thyroid, lung, and hepatocellular cancers (Duluc et al., 2007). Tumor-associated macrophages 2597
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(TAMs) the most abundant immunosuppressive cells in the tumor microenvironment, originate from blood monocytes and exhibit IL-10high IL-12low. They can release diverse cytokines and inflammatory mediators and are correlated with cancer cell proliferation. In some cases, the tumor microenvironment suppresses many immune responses and alters the phenotype to promote tumor growth. Studies suggest that macrophages can produce IL-10, which in turn, induces monocytes to express the costimulatory molecule programed death ligand (PD)-L1 (Kuang et al., 2009). Macrophages in human ovarian cancers can regulate the influx of regulatory T cells (Tregs) that suppress cytotoxic T cell responses (Green et al., 2009). The implications of these associations for EtOH have not been fully elucidated, but our previous studies of breast cancer (Wang et al., 2012) confirmed that vascular generation and increased monocyte chemo-attractant protein-1 (MCP-1) expression correlated with tumor progression. Thus, we sought to evaluate the importance of macrophages in innate and adaptive immunity and to investigate whether EtOH can stimulate cancer via supporting the recruitment of TAMs and M2 polarization.
MATERIALS AND METHODS Cell Culture and EtOH Exposure LLC and RAW264.7 cell lines were grown with DMEM (GIBCO, Gaithersburg, MD) medium supplemented with 10% fetal bovine serum (HyClone, Logan, UT), penicillin (100 U/ml)/streptomycin (100 U/ml). All cell lines were incubated at 37°C with 5% CO2. EtOH solutions were made daily, and the appropriate amount of EtOH (0.1%) was added to the culture medium (Luo and Miller, 1997). Tumor Model C57BL/6 mice (male and female, 6 to 8 weeks of age) were purchased from the experimental animal center of Anhui Province, housed on hardwood bedding with 12-hour light/dark cycles. Mice were given 1% EtOH in drinking water for 12 hours overnight, and this was replaced with regular water drinking during the remaining 12 hours each day for 4 weeks. Control mice received regular drinking water only. In the second week, all mice were injected subcutaneously in the right flank with ~5 9 105 LLC cells in 0.1 ml phosphate-buffered saline (PBS). Both groups were comprised of 10 mice. Finally, mice were sacrificed, all tumors were excised, and tumor diameters were measured with calipers. Tumor volume (TV) was calculated as follows: TV = length 9 width 9 depth 9 0.52 (mm3). Immunohistochemical Staining for Microvessels and Immune Cells Perfused tumors were fixed in formalin and embedded in paraffin, cut into 4-lm sections, then stained with hematoxylin and eosin (H&E), and photographed under a microscope. Five sections were examined per mouse with each of the following antibodies: rat polyclonal CD11b antibody (1:200 dilution, bs-1014R; Bioss, Beijing, China) to stain macrophages, rat anti-mouse MCP-1 (Sc-7269; 1:100 dilution) and IL-10 (ab33471; 1:300 dilution), goat polyclonal MMP-9 antibody (sc-6840; 1:300 dilution), and rabbit polyclonal CD31 (platelet endothelia cell adhesion molecule 1) antibody
(BA2211; Boster, Wuhan, HB, China) to stain microvessels. Microvessel densities (MVDs) were counted by scanning sections stained with anti-mouse CD31 antibody. Any CD31+-stained endothelial cell or cluster that was clearly separated from adjacent microvessels, tumor cells, and connective elements was counted as 1 microvessel. The mean microvessel count of the 5 most vascular areas was taken as the MVD, which was expressed as the absolute number of microvessels per 1.485 mm2 (200 9 field; Tan et al., 2007). Western Blots Cells were collected, washed, and resuspended in lysis buffer. Samples were boiled and separated by SDS-PAGE then transferred to a 0.22-lm PVDF membrane (Millipore Co., Billerica, MA) by electro-blotting. Primary antibodies included rat antimouse MCP-1 (Sc-7269), IL-10 (ab33471), and rabbit anti-mouse inducible nitric oxide synthase (iNOS) (ProteinTech-14142, Chicago, IL), and Arginase I (ProteinTech-16001). b-actin (Sc-47778) was used for control protein loading. Secondary antibodies were peroxidase-conjugated goat anti-rabbit IgG or goat anti-mouse IgG. Proteins were detected with enhanced chemi-luminescent. Lymphocyte Isolation from Tumors and Bone Marrow Mice were sacrificed, and tumors and femurs were removed and weighed. Tumors were isolated and washed, passed through a wire mesh screen into ice-cold PBS, and centrifuged at 4°C, 2,000 9 g for 10 minutes. Cells were resuspended and layered onto 5 ml lympholyte-M in a tube. The tube was centrifuged at room temperature at 2,000 9 g for 20 minutes. Tumor-infiltrated lymphocytes at the interface were collected and counted. One femur from each C57BL/6 mouse was removed, and the 2 ends of the bone were cut to expose the bone cavity. The marrow was flushed with 5 to 10 ml PBS. Bone marrow cells were resuspended and layered onto 10 ml lympholyte-M to remove red blood cells. Flow Cytometry Analysis Previous immunohistochemistry data were qualitative, so we measured the proportion of positive cells by flow cytometry. Cell phenotypes were analyzed using PE-labeled-CD11b and FITC-labeled-F4/80 (all from eBioscience, San Diego, CA). Cells were preincubated for 30 minutes at 4°C with antibody and then analyzed on a FACScan (BD Inc., San Diego, CA). Isotype control monoclonal antibodies were from BD Inc. Cell counts were expressed as % gated (percentage of CD11b- and F4/80positive cells in the tumor). Immunofluorescent Analysis of Macrophage Polarization States Cell numbers were measured, and 20,000 cells were cytospun onto a slide for 20 minutes. Three slides per mouse were examined for both control and EtOH-treated samples. Primary antibody concentrations were as follows: iNOS (1:100), arginase I (1:100), and CD-11b-PE (1:200). Then, cells were washed and treated with secondary antibody (1:1,000) and rinsed 3 times. Immunofluorescent images were examined with a LEICA-inverted confocal microscope (Wetzlar, Germany), and fluorescent signals were measured with the same pinhole, detector gain, and amplifier offset. Three-Dimensional Endothelial Cell and Tumor Coculture System To investigate the effect of EtOH on tumor angiogenesis, we utilized a 3-dimensional (3D) model of endothelial cell and
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Fig. 1. Ethanol (EtOH) consumption significantly increased Lewis lung cancer (LLC) tumor volume and attenuated invasive tumor growth patterns. (A, B) Comparison of in situ tumor growth and tumor volume with/without 1% EtOH treatment in a LLC C57BL/6 mice model 22 days after tumor implantation. Tumor sizes were measured every 2 days. (C) Comparisons of tumor weight between control and EtOH treatments. (D) Lung metastasis in LLC tumor model was increased by EtOH treatment. Percentages of mice with metastatic lung carcinoma nodes are presented. N = 10 per group. *p < 0.05, compared with control group by Student’s t-test. (E) Histological features of lungs from mice with/without 1% EtOH treatment. Metastatic carcinoma nodules were present in lungs of the 1% EtOH-exposed group.
tumor cell cocultures adapted from a previous study with some modifications (Chen et al., 2009). In brief, SVEC cells were trypsinized and mixed with Cytodex beads (3 9 103; Sigma, St. Louis, MO) in DMEM medium. The mixtures were incubated for 8 hours. Mixtures were washed, resuspended, and added to 24-well cell culture plates precoated with 0.625 U thrombin (Sigma-Aldrich, St. Louis, MO). The fibrinogen/bead solution was allowed to coagulate for 5 minutes, and then, it was incubated. For coculture, endothelial cells/macrophages were layered atop the fibrin gels (Xu et al., 2010). Serum IL-10 Quantitation by ELISA Because IL-10 regulates immune and inflammatory responses and because it is an inhibitory cytokine that mediates suppression by regulatory or suppressor T cells (Tsuji-Takayama et al., 2008), we assayed serum IL-10 by ELISA, according to the manufacturer’s instructions. MTT Assay With an MTT assay, cytotoxicity of CD11b+ TAMs was measured. Cells were plated into 96-well plates and exposed to 0.1% EtOH or PBS. After treatment, 10 ll of MTT reagent was added into each well, and the plates were incubated. The culture was solubilized, and spectrophotometric absorbance was measured at 595 nm using a microtiter plate reader.
Statistical Analysis Where indicated, data are presented as means SEM of duplicate experiments (n = 10/group). Statistically significant differences in means between groups were examined by an unpaired Student’s t-test. A p < 0.05 was considered statistically significant. All statistical calculations were performed using SPSS software (SPSS Inc., Chicago, IL).
RESULTS EtOH Consumption Significantly Increased LLC Tumor Volume and Attenuated Invasive Tumor Growth Patterns One week before LLC cell implantation, C57BL/6 mice were given EtOH as described in the Materials and Methods section. EtOH exposure significantly increased tumor volumes and weight after LLC cell implantation (Fig. 1A). Lung metastasis in a LLC model was increased by EtOH treatment. More metastatic nodules appeared in the EtOHtreated group and the rate increased from 35% in the control to 75% in the EtOH-treated group (Fig. 1B). Under light microscopy and H&E staining, pulmonary congestion was present, as were metastatic carcinoma nodules after EtOH treatment (Fig. 1C).
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EtOH Supported Recruitment of Immune Cells Especially TAMs into Tumors Immunohistochemical staining revealed that CD11b+ TAMs accumulated at tumor sites (Fig. 3A). Surface expression of CD11b and F4/80 in cell populations obtained from tumors by fluorescence-activated cell sorting (Fig. 3B) indicated that the number and percentage of CD11b+/F4/80+ TAMs increased significantly in the tumor from the EtOHtreated group.
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Fluorescent intensity was scanned, and the ratio of ArgI/ iNOS was calculated and statistically analyzed. EtOH supports macrophages displayed M2 (ArgIhigh/iNOSlow) polarized during LLC development. This mirrored the M2 activation state of macrophages after EtOH treatment. M2 macrophages, another immune-suppressing leukocyte population (Marttila-Ichihara et al., 2009), had poor antigen-presenting capacity, prevented T cell activation, and promoted the recruitment of Tregs (Fig. 4).
Temporal Changes in TAM Activation States
Overexpression of IL-10 in Serum and Tumor
Because the stage of tumor progression affected the activation state of macrophages, we hypothesized that EtOH consumption changed the tumor microenvironment to promote macrophage activation. Macrophages were stained with anti-CD11b to readily distinguish them from other lymphocytes. For all samples, monocytes displaying no detectable arginase I or iNOS were designated as ArgIlow/iNOSlow. M2 macrophages were stained as ArgIhigh/iNOSlow, and M1 macrophages were stained as ArgIlow/iNOShigh. Colocalization of the binding of different antibodies to the same cell was detected with multiple fluorescent stains. These activation state changes in macrophages were shown in control and EtOH-treated tumors. Immunoblots were not different with regard to iNOS expression between control and EtOH-treated tumors, but arginase I expression was significantly increased in the EtOH-treated group.
Overexpression of IL-10 in serum has been reported in several other malignancies: melanoma and ovarian carcinoma (Wang et al., 2011). The product of tumor cells, including extracellular matrix components, IL-10, and chemokines such as MCP-1 can activate macrophages and cause M2 polarization, which is a cancer-promoting mode. We assayed IL-10 contents in serum by ELISA and expression of IL-10 in the tumor by Western blot and immunohistochemistry. Data show that IL-10 in the EtOH-treated group increased significantly (Fig. 5A–D), suggesting that EtOH promoted MCP-1 and IL-10 expression in the tumor. Effect of EtOH on In Vitro Tumor Angiogenesis To test the hypothesis that TAMs may mediate EtOHinduced tumor angiogenesis, we used a 3D angiogenic
Fig. 4. Temporal changes in tumor-associated macrophage (TAM) activation states. (A) Two-color immunohistochemical detection of CD11b+ (red) and arginase I+ (green) or inducible nitric oxide synthase (iNOS)+ (green) cells in tumors with/without 1% ethanol (EtOH) treatment in a Lewis lung cancer (LLC) C57BL/6 mice model 22 days after tumor implantation. (B) Fluorescent intensity was scanned, and the ratio of ArgI/iNOS was calculated and analyzed statistically. The results showed that EtOH-treated macrophages displayed polarized M2 (ArgIhigh/iNOSlow) TAMs during LLC development.
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EtOH Supported Recruitment of Immune Cells Especially TAMs into Tumors Immunohistochemical staining revealed that CD11b+ TAMs accumulated at tumor sites (Fig. 3A). Surface expression of CD11b and F4/80 in cell populations obtained from tumors by fluorescence-activated cell sorting (Fig. 3B) indicated that the number and percentage of CD11b+/F4/80+ TAMs increased significantly in the tumor from the EtOHtreated group.
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Fluorescent intensity was scanned, and the ratio of ArgI/ iNOS was calculated and statistically analyzed. EtOH supports macrophages displayed M2 (ArgIhigh/iNOSlow) polarized during LLC development. This mirrored the M2 activation state of macrophages after EtOH treatment. M2 macrophages, another immune-suppressing leukocyte population (Marttila-Ichihara et al., 2009), had poor antigen-presenting capacity, prevented T cell activation, and promoted the recruitment of Tregs (Fig. 4).
Temporal Changes in TAM Activation States
Overexpression of IL-10 in Serum and Tumor
Because the stage of tumor progression affected the activation state of macrophages, we hypothesized that EtOH consumption changed the tumor microenvironment to promote macrophage activation. Macrophages were stained with anti-CD11b to readily distinguish them from other lymphocytes. For all samples, monocytes displaying no detectable arginase I or iNOS were designated as ArgIlow/iNOSlow. M2 macrophages were stained as ArgIhigh/iNOSlow, and M1 macrophages were stained as ArgIlow/iNOShigh. Colocalization of the binding of different antibodies to the same cell was detected with multiple fluorescent stains. These activation state changes in macrophages were shown in control and EtOH-treated tumors. Immunoblots were not different with regard to iNOS expression between control and EtOH-treated tumors, but arginase I expression was significantly increased in the EtOH-treated group.
Overexpression of IL-10 in serum has been reported in several other malignancies: melanoma and ovarian carcinoma (Wang et al., 2011). The product of tumor cells, including extracellular matrix components, IL-10, and chemokines such as MCP-1 can activate macrophages and cause M2 polarization, which is a cancer-promoting mode. We assayed IL-10 contents in serum by ELISA and expression of IL-10 in the tumor by Western blot and immunohistochemistry. Data show that IL-10 in the EtOH-treated group increased significantly (Fig. 5A–D), suggesting that EtOH promoted MCP-1 and IL-10 expression in the tumor. Effect of EtOH on In Vitro Tumor Angiogenesis To test the hypothesis that TAMs may mediate EtOHinduced tumor angiogenesis, we used a 3D angiogenic
Fig. 4. Temporal changes in tumor-associated macrophage (TAM) activation states. (A) Two-color immunohistochemical detection of CD11b+ (red) and arginase I+ (green) or inducible nitric oxide synthase (iNOS)+ (green) cells in tumors with/without 1% ethanol (EtOH) treatment in a Lewis lung cancer (LLC) C57BL/6 mice model 22 days after tumor implantation. (B) Fluorescent intensity was scanned, and the ratio of ArgI/iNOS was calculated and analyzed statistically. The results showed that EtOH-treated macrophages displayed polarized M2 (ArgIhigh/iNOSlow) TAMs during LLC development.
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model in which endothelial cells and macrophages from mouse bone marrow or mouse TAMs (CD11b+) were cultured together. Endothelial cells attached to Cytodex beads were formed a 3D capillary tube-like network indicative of angiogenesis. As shown in Fig. 6, SVEC attached, sprouted, and formed narrow cordlike structures. The inclusion of macrophages in this system modestly increased the number and the length of spouts from SVEC, but EtOH significantly increased the spouts (spouts were 4.8% in SVEC cultures alone; 11.3% in cocultured with macrophages; 68.6% in cocultured with macrophages treated with EtOH). Spout percentages were 22.1% in SVECs cocultured with CD11b+ TAMs and 70.8% with EtOH treatment.
and chemokines, such as VEGF and MMP-9, which directly promote angiogenesis and tumor metastasis (Mantovani and Sica, 2010). In EtOH-treated groups, after tumor implantation, expression of intracellular MMP-9 protein increased (Fig. 8). MMP-9 expression significantly increased after EtOH exposure, especially in hypoxic areas of tumor necrosis. EtOH Suppressed Cytotoxicity of TAMs Macrophages in the tumor can produce cytokines which regulate the influx of Tregs to suppress responses of cytotoxic T cells and TAMs. Our data revealed cytotoxicity of TAMs in the EtOH-treated group decreased more than controls as measured by MTT (Fig. 9).
EtOH Promoted Tumor Angiogenesis in a LLC Model Vascular endothelial cells were stained with PECAM-1 (CD31). Tumor MVDs were calculated by new vessel formation using CD31 staining, and microvessels appeared as brown linear fragments. With EtOH treatment, tumor MVDs increased markedly (Fig. 7). Macrophages in the tumor microenvironment facilitate tumor invasion and angiogenesis by secreting several inflammatory cytokines
EtOH Induced RAW264.7 Cells to M2 Polarized To confirm that EtOH induced M2 macrophage polarization, we treated RAW264.7 macrophage cells with 0.1% EtOH and unstimulated cells were used as controls. Slight arginase I staining was consistently seen in unstimulated RAW264.7 cells. After cell stimulation for 24, 48, and 72 hours, cells were probed for membrane protein CD11b
Fig. 5. Overexpression of interleukin 10 (IL-10) in serum and tumor. IL-10 in serum and in tumors was assayed by ELISA, Western blot, and immunohistochemistry. (A) Serum IL-10 concentration histogram and statistical analysis. (B) IL-10 expression in tumor detected by Western blot. (C) Comparison of IL-10 in tumors from mice with/without ethanol (EtOH) treatment was detected by immunohistochemistry of IL-10 antibody. (D) Quantitation was calculated as the IL-10 positive area/total area. Columns represent means of 10 samples in each group n = 10 per group, *p < 0.05. IL-10 in serum and tumor increased significantly after 1% EtOH treatment.
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and changes in arginase I and iNOS expression. Colocalization experiments indicated that the same cells stained for both proteins. Arginase I expression was significantly higher in RAW264.7 cells after EtOH exposure compared with control cells. Polarization was more apparent with a longer induction time (Fig. 10). DISCUSSION Alcohol is a known dietary component and is usually only of concern when it is abused, at which point it becomes immunosuppressive. Nonetheless, immune response regulation by EtOH is complex and dose-dependent as well as dependent on the exposure duration and the cell type targeted (Crews et al., 2006). Little is known about how EtOH exposure induces or alters immune cells in a lung cancer microenvironment during tumor progression and metastasis.
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Tumors consist of both tumor and stromal cells and create a unique microenvironment essential for tumor progression and invasion. The tumor microenvironment can actively program immune dysfunction allowing the tumor cell to escape immunologic attack (Rabinovich et al., 2007). TAMs, CD4+CD25+ Tregs, and myeloid-derived suppressor cells are known to inhibit host antitumor immune activity (Finn, 2008; Zhang and Meadows, 2008). TAMs have 2 activation phenotypes that are regulated by varied cytokines. M1 is classically polarized by lipopolysaccharide, interferon-c and releases IL-6 and IL-12. M1 up-regulate iNOS. M2, an alternative polarized phenotype by IL-4, IL-13, releases anti-inflammatory IL-10. M2 up-regulate arginase I and mannose receptors. During wound healing, M2 can facilitate angiogenesis and tissue remodeling. Arginase I catalyzes the formation of ornithine, which leads to synthesis of polyamines necessary for DNA synthesis and the stimulation of cell
Fig. 6. Effect of ethanol (EtOH) on in vitro tumor angiogenesis. (A) In vitro tumor angiogenesis in mouse bone marrow macrophages after EtOH treatment. (B) In vitro tumor angiogenesis in mouse tumor-associated macrophages (CD11b+) after EtOH treatment.
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Fig. 7. Ethanol (EtOH) promoted tumor angiogenesis in a Lewis lung cancer model. (A) Tumor microvessel densities (MVDs) were calculated by new vessel formation using CD31 staining. Microvessels appeared as brown linear fragments. Comparison of MVDs by CD31+ tumor endothelial cells by hematoxylin and eosin staining (1,2) and immunostaining (3,4) between mice with/without EtOH treatment. (B) Comparison of MVDs between control and EtOH treatment groups, *p < 0.05.
Fig. 9. Ethanol (EtOH) suppressed cytotoxicity of tumor-associated macrophages (TAMs). TAM cytotoxicity was assayed by MTT and EtOH increased cytotoxicity.
Fig. 8. Ethanol (EtOH) promoted tumor angiogenesis in a Lewis lung cancer model. Matrix metalloproteinase 9 (MMP-9) was detected by immunostaining. (A) MMP-9 expression-promoted tumor metastasis markedly increased in EtOH-treated mice, especially in hypoxic areas of tumor necrosis. (B) Quantitation was calculated as the MMP-9 positive area/total area. Columns represent means of 10 samples in each group, *p < 0.05 compared with the control group.
division while depleting the arginine substrate needed to produce NO. It has been hypothesized that immune cells produce a mutagenic environment by generating both reactive nitrogen and oxygen species (Cho et al., 2013; Redente et al., 2007). In our study, 4 weeks of EtOH exposure significantly increased tumor size and weight. EtOH exposure promoted tumor metastasis to the lungs; the metastatic rate increased
from 35% in the control to 75% in the EtOH-exposed group. MCP-1 is a chemoattractant for macrophages to the inflammatory areas, is related to a variety of inflammatory diseases, (Melgarejo et al., 2009) and is correlated with tumor angiogenesis (Wang et al., 2012). We confirmed that in a LLC mouse model, MCP-1 expression increased after EtOH treatment and CD11b+/F4/80+ TAMs significantly increased and infiltrated around the tumor stroma. Also, TAMs accumulate at tumor sites to maintain immune tolerance which circumvents tumor immunity (Zou, 2005). We report that after EtOH exposure, tumors progressed to malignancy, and macrophages increased and were polarized to M2 displaying ArgIhigh/iNOSlow. Classical M1 may be phenotypically characterized with expression of iNOS and alternative M2 with expression of arginase I. Macrophages stained with arginase I or iNOS had a temporal dependence on the stage of tumor development. RAW264.7 cells treated with EtOH stained more intensely for arginase I than controls, but iNOS
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expression diminished. These results were in accordance with the features of M2 macrophages and indicated that EtOH could polarize macrophages to the M2 phenotype. Using an in vitro 3D angiogenic model, we saw that macrophages and CD11b+ TAMs could promote tumor angiogenesis, and this ability was significantly enhanced with EtOH. Growth and expansion of tumor masses are strictly dependent on angiogenesis. Thus, we calculated tumor MVDs by new vessel formation using CD31 staining and found that tumor MVDs increased after EtOH treatment. In contrast, TAMs had high pro-angiogenic activity and can secrete MMP-9 actively, facilitating endothelial cell migration. Consistent with the phenotype of tumor growth and distant metastasis, MMP-9 expression also increased more markedly after EtOH exposure compared to control. The evidence suggested the effects of EtOH intake on MMP-9 expression may be linked to the mechanisms of EtOHinduced LLC tumor progression. After EtOH treatment, obvious tumor necrosis was observed. Macrophage accumulation and increased MMP-9 expression in hypoxic areas of the tumor were particularly associated with necrotic tissue. Tumor cells may be activated by TAMs and secrete angiogenic factors. The interaction between them was, in part, mediated through soluble factors involving the IL-10 pathway.
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An inflammatory microenvironment may increase mutation rates: activated inflammatory cells can serve as reactive nitrogen intermediate sources which can induce DNA damage and genomic instability (Grivennikov et al., 2010). TAM phenotype analysis suggests that transcriptional factors NFjB and HIF-1 are master regulators of their transcriptional programs and that these factors centrally regulate tumor progression and metastasis (Sica and Bronte, 2007). In this study, tumors in control samples invaded to fat and fascia, but in the EtOH-treated group, tumors invaded into deep tissue and skeletal muscle. In addition to TAMs, IL-10 increased in serum and tumors. Production of IL-10 may provide a mechanism for tumor evasion of T cell–mediated immune responses. Our results revealed cytotoxicity of TAMs as evidenced by decreased MTT in the EtOH-treated group. All of these changes could promote immunosuppression, tumor progression, and metastasis. In summary, our work suggests that chronic alcohol consumption promotes TAMs recruitment into microdomains, leading to immunosuppression. TAMs dysfunction was consistent with increased immunosuppression in the LLC EtOH-treated mice. TAMs especially M2 phenotypes constituted the major components of leukocyte infiltration. Progression and metastasis of LLC may be mediated by recruitment TAMs, especially under the influence of EtOH.
Fig. 10. Ethanol (EtOH) induced RAW264.7 cells to M2 polarized. (A) Two-color immunofluorescent detection of CD11b+ (red) and arginase I+ (green) or inducible nitric oxide synthase (iNOS)+ (green) in RAW264.7 cells with/without 1% EtOH treatment. RAW264.7 cells lost their M1 (iNOS-expressing) phenotype and immunostained exclusively for arginase I. RAW264.7 cells’ M2 polarization was more apparent after a longer period of induction. (B) Fluorescent intensity was scanned, and the ratio of ArgI/iNOS was calculated and statistically analyzed. The results showed that EtOH-treated macrophages displayed a polarized M2 phenotype (ArgIhigh/iNOSlow). (C) Immunoblotting of iNOS and arginase I in control and EtOH-treated RAW264.7 cells. iNOS decreased but arginase I increased after EtOH treatment. b-actin used as a loading control for the gel.
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