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Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages
Cellular Immunology xxx (2016) xxx–xxx
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Research paper
Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages Liwei Sun 1, Bonian Chen 1, Rong Jiang ⇑, Jinduo Li, Bin Wang Tianjin Key Laboratory of Cerebral Vascular and Neurodegenerative Disease, Department of Intervention, Tianjin Huanhu Hospital, Tianjin 300060, PR China
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Article history: Received 12 September 2016 Revised 30 October 2016 Accepted 1 November 2016 Available online xxxx Keywords: Resveratrol Lung cancer STAT3 Macrophages
a b s t r a c t In cancer, tumor associated macrophages (TAMs) play an important role in the cancer progression, evasion of immunity and dissemination of cancer cells. Inhibition of the activation or the M2 polarization of TAMs is an effective therapy for cancer. In the present study, we investigated the ability of resveratrol (RES) to inhibit lung cancer growth using in vitro and in vivo studies, and examined the underlying mechanisms. We demonstrated that M2 polarization of human monocyte derived macrophage (HMDMs) induced by the lung cancer cells conditioned medium was inhibited by RES. Additionally, RES exhibited inhibitory function in lung cancer cells co-cultured with human macrophages. The activity of signal transducer and activator of transcription 3 (STAT3) was significantly decreased by RES. Moreover, in a mouse lung cancer xenograft model, RES significantly inhibited the tumor growth, which was associated with inhibition of cell proliferation and decreased expression of p-STAT3 in tumor tissues. Further, RES inhibits F4/80 positive expressing cells and M2 polarization in the tumors. These results suggest that RES can effectively inhibit lung cancer progression by suppressing the protumor activation of TAMs. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Despite advances in the clinical management of lung cancer, lung cancer remains as the leading cause of cancer-related deaths worldwide [1]. It is increasingly recognized that the interaction between tumor and immune effector cells enhances tumor growth, invasion and local immunosuppression, to evade from antitumor immune responses, which is also essential to achieve effective extravasation at prospective metastatic sites [2]. Thus primary tumors, and possibly, circulating tumor cells (CTCs), are capable of recruiting immunosuppressive cells particularly of the myeloid-macrophage lineage. These immunosuppressive cells undergo functional polarization driven by tumor-derived factors [3]. These ‘‘tumor-educated” macrophages are involved in all stages of cancer metastasis: cancer invasion, intravasation, survival in the circulation, and sustainable growth at secondary lesions are all promoted by these macrophages [4]. Because of their existence as a major cellular component of murine and human tumors, they are commonly termed tumor-associated macrophages (TAMs). Abbreviations: TAMs, tumor associated macrophages; RES, resveratrol; STAT3, signal transducer and activator of transcription 3. ⇑ Corresponding author. E-mail address: [email protected] (R. Jiang). 1 Contributed equally.
Originally, TAMs are proposed to be involved in anti-tumor immunity, whereas recent evidences unveiled the tumor-promoting effect of TAMs [5,6]. Epidemiological evidences indicate a strong link between increased macrophage infiltration and poor prognosis in lung cancer [7]. TAM density was found to be negatively correlated with poor patient survival and frequent recurrence [8]. The median survival of patients with high macrophages density was significantly shorter than those who had a lower macrophages density [7]. These studies suggested that TAMs play a crucial role in cancer development. Plasticity and flexibility are key indicators of the activation states of macrophages. Whether TAMs assume tumor-preventing or tumor-promoting role is dependent on their polarization statues [9]. The activation states, M1 and M2, denote classical activation and alternative activation of macrophages, respectively. In benign or regressive tumors, the majority of TAMs are classically activated macrophages (M1-like), which exert pro-inflammatory activity by antigen-presenting and promoting tumor lysis [10]. Conversely, TAMs in malignant tumors resemble alternatively activated macrophages (M2-like), enhancing tumor angiogenesis, migration and invasion. M2-like macrophages suppress antitumor immune responses. Thus, M2-like TAMs are promising targets for adjuvant anticancer therapies. Encouraging anti-tumor efficacy has been achieved by recent M2-TAM-targeting approaches. For example,
http://dx.doi.org/10.1016/j.cellimm.2016.11.002 0008-8749/Ó 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: L. Sun et al., Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.11.002
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targeted delivery of peptide to M2-like TAM demonstrated improvement in the survival of tumor bearing mouse [11]. In another study, in vitro co-culturing of cancer cells with M2 macrophages showed activation of signal transducer and activator of transcription 3 (STAT3), which is an important modulator of tumor progression and chemo-resistance in cancer cells [12]. M2 macrophages activated by cancer cells secrete several cytokines, such as IL-6 and IL-10, which in turn promote cancer progression [13,14]. Therefore, inhibiting macrophage polarization into the M2 phenotype, and blocking the tumor-macrophage interaction could be a viable approach toward efficient cancer therapy. Resveratrol (RES) possess multiple pharmacological activities in extending longevity and treatment of cardiovascular disease, diabetes and cancer [15]. RES is a polyphenol that naturally exists in grapes and red wine. The investigation of RES originated from studies on ‘French Paradox’, which describes beneficial cardiovascular outcomes of a high-fat diet in French cuisine [16]. Its anti-cancer effects have been well documented in a variety of cancers, whereby RES regulated cell division, growth, angiogenesis and metastasis [17]. In lung cancer, RES was shown to promote premature senescence in cancer cells (A549 and H460 cells) via inducing NAPDH oxidase-5 (Nox5) [18], which resulted in inhibition of cancer cell proliferation and survival [19]. Recent studies showed that RES exerts anti-tumor effects by regulating the release of cytokines. RES selectively inhibits or activates the release of cytokines. RESsuppressed cytokines include IL-6, IL-12 and TNF-a [20,21]. However, the beneficial effects of RES in lung cancer remain to be clarified. In the present study, we investigated the ability of resveratrol to inhibit lung cancer growth using in vitro and in vivo models. Given that M2 macrophages are closely associated in cancer progression, we also investigated the relevance between macrophage polarization and the antitumor effects of RES. 2. Materials and methods 2.1. Cell culture Human lung cancer cells, A549 and H1299, were acquired from the American Type Culture Collection (Manassas, VA, USA). Lewis lung cancer (LLC) cells were obtained from the Cell Bank of the China Science Academy (Shanghai, China). All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY) and regularly checked for Mycoplasma contamination. Cells were incubated in a humidified atmosphere with 5% CO2 at 37 °C. Peripheral blood mononuclear cells were collected from healthy volunteer donors, with written informed consent filed for all sample collection. Subsequent analysis was performed on all healthy donors. Experiments regarding human studies were performed according to protocols approved by the Tianjin Huanhu Hospital Review Board. CD14+ monocytes were isolated from the peripheral blood mononuclear cells using magnetic-activated cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) based on positive selection, followed by co-culturing with GM-CSF (10 ng/ml, Wako, Tokyo, Japan) or M-CSF (50 ng/ml, WAKO) for a week for macrophage differentiation. The differentiated macrophages were then used as human monocyte-derived macrophages (HMDMs) in the present study, as described previously [22]. 2.2. Preparation of the tumor conditioned medium (TCM) The tumor conditioned medium was acquired from the supernatant of A549 cultured medium after 72 h of incubation. The conditioned media was collected from the Transwell insert systems,
followed by filtration through 0.2 lm pores (Sartorius Stedim Biotech, Germany). The TCM was stored at 80 °C until further usage in the ELISA assays. 2.3. Cytokine ELISA kit HMDMs were seeded in 12-well plates at the density of 3 105 cells/well. HMDM cells were subjected to lipopolysaccharide (LPS) (100 ng/mL, Sigma, Louis, MO, USA) stimulation for 24 h, followed by incubation with TCM or with RES-containing TCM (Sigma, Louis, MO, USA) for 24 h. ELISA was used to determine IL-10, IL-12 and TNF-a secretion according to manufacturer’s guidelines (eBioscience, San Diego, CA, USA). 2.4. Cell proliferation assay Viability assay was performed on A549 and H1299 cells using the Cell-Light EdU Apollo488 In Vitro Imaging Kit (RiboBio) in accordance to the manufacturer’s protocol. Briefly, cells incubated with 10 lM EdU for 2 h were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.3% Triton X-100 and stained with EdU. Cell nucleus were stained by 10-min-incubation with 5 lg/ mL DAPI (40 ,6-diamidino-2-phenylindole). Number counting of Edu-positive cells was counted under a light microscope based on five random fields (100). All assays were performed in triplicate independently. 2.5. MTT assay MTT assay was used to determine the effect of RES on the viability of A549 and H1299 cells. Briefly, cells were treated with RES or STAT3 inhibitor (WP1066) (Sigma, Louis, MO, USA) for 48 h. At the end of treatment, 50 lL of 5 mg/mL MTT were added and the resulting formazan crystals were dissolved in 100 lL of DMSO, followed by measuring absorbance at 570 nm by an Automated Microplate Reader (Bio-Tek, USA). The cell viability was calculated using the following equation: cell viability (%) = (OD of treated cells/OD of control cells) 100. All assays were performed in triplicate with at least 3 independent experiments. 2.6. RNA isolation, reverse transcription and qRT-PCR Total RNA from BMDM was extracted and purified using the Easy Pure RNA Kit (Transgen Biotech Co., Ltd), followed by cDNA synthesis. Quantitative real time-PCR (qRT-PCR) was carried out using SYBR Premix ExTaq (TaKaRa, Dalian, China) on a Stratagene Mx3000P real-time PCR system (Agilent Technologies, Inc., CA, USA). All mRNA levels were normalized to the GAPDH level. The quantification of mRNA levels were performed using the 2DDCT method and normalized to GADPH. PCR reactions were performed in triplicates independently. Primers used in PCR reaction were as follows: MRC1, forward primer: 50 -AGG GAC CTG GAT GGA TGA CA-30 ; and reverse primer: 50 -TGT ACC GCA CCC TCC ATC TA-30 ; CCL24, forward primer: 50 -TGT CTG CAG TTG AGC CTA CG-30 ; and reverse primer: 50 -GTT CGG GAC CCT GGA GTT AG-30 ; chil3, forward primer: 50 -CAT GAG CAA GAC TTG CGT GAC-30 ; and reverse primer: 50 -GGT CCA AAC TTC CAT CCT CCA-30 ; Retnla, forward primer: 50 -CCC TGC TGG GAT GAC TGC TA-30 ; and reverse primer: 50 TGC AAG TAT CTC CAC TCT GGA TCT-30 ; IL-10, forward primer: 50 ACT GGC ATG AGG ATC AGC AG-30 and reverse primer: 50 -CTC CTT GAT TTC TGG GCC AT-30 ; Arg1, forward primer: 50 -GAT TAT CGG AGC GCC TTT CT-30 and reverse primer: 50 -CCA CAC TGA CTC TTC CAT TCT-30 ; CD206, forward primer: 50 -CTG CAG ATG GGT GGG TTA TT-30 and reverse primer: 50 -GGC ATT GAT GCT GCT GTT ATG-30 ; GAPDH, forward primer: 50 -CAG CCT CAA GAT CAT CAG CA-30 and reverse primer: 50 -TGT GGT CAT GAG TCC TTC CA-30 .
Please cite this article in press as: L. Sun et al., Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.11.002
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2.7. Western blot analysis
3. Results
Cells were harvested by trypsinization, washed with cold PBS, and lysed with ice-cold lysis buffer supplemented with protease inhibitors (PI) as described previously. Equivalent amounts of protein (30 lg) were loaded onto precast gels (Biorad) and resolved using SDS-PAGE. Proteins were blotted onto nitrocellulose membranes (0.45 lM; Bio-Rad). Membranes were incubated in blocking solution (1 PBS, 0.1% Tween-20, and 5% non-fat dry milk powder) at room temperature for 1 h. Primary antibodies against anti-pSTAT3, anti-STAT3 or anti-b-Actin (Cell signaling Technology, MA, USA) were used to incubate with the membrane at 4 °C overnight with gentle shaking. After extensive washing with TBST, membranes were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad) for 1 h at room temperature and protein bands were detected with the enhanced chemiluminescence method (SuperSignal West Pico substrate; Pierce; Rockford, IL).
3.1. RES inhibits M2 polarization of HMDMs
2.8. Tumor models Animal studies were performed strictly according to the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and a protocol approved by the Committee on the Ethics of Animal Experiments of Tianjin Huanhu Hospital. All surgeries were performed under anesthesia with sodium pentobarbital, with precautions to minimize animal suffering. C57BL/6 mice, at the age of 4–5 weeks, were acquired from the National Rodent Laboratory Animal Resource (Shanghai, China). LLCs model was constructed by injecting 1 106 cells subcutaneously to the flanks of C57BL/6. RES, at the dose of 100 mg/kg, or vehicle control were administered i.p. once daily from the 10th day tumor inoculation (5 mice per group). Tumors were monitored twice a week. Tumor volumes were calculated after measuring length and width of the tumor with calipers and tumor volumes were calculated as (Length Width2)/2. After 4 weeks, animals were sacrificed, with tumors collected.
2.9. Immunohistochemistry Tumors sections at the thickness of 5 lm were obtained by cryosection. These sections were then deparaffinized and rehydrated according to standard histological protocols, followed by antigen retrieval with 10 mM sodium citrate buffer (pH 6.0), using a pressure cooker maintained at 95° C for 20 min. The sections were then cooled at room temperature for 20 min, washed with PBS and blocked with 1% BSA/2% goat serum before incubation with either anti-Ki-67, anti-pSTAT3 (Cell signaling Technology, MA, USA) and anti-F4/80 (Abcam, Cambridge, MA, USA). After extensive washing, the sections were incubated with biotinylated secondary antibody, followed by horseradish peroxidaseconjugated streptavidin. Subsequently, 2,4-diaminobenzidine substrates were added and sections were counterstained with hematoxylin. The positively stained cells was counted using the BZ9000 analysis software program (Keyence, Osaka, Japan).
2.10. Statistical analysis Data were presented as mean ± SEM unless otherwise stated. The statistical significance of differences between two groups was analyzed by either Student t test or simple one-way ANOVA using Prism version 5 (GraphPad Software, Inc.). Differences with p < 0.05 were considered statistically significant.
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First, we evaluated whether RES was able to inhibit the lung cancer cell conditioned medium induced M2 polarization of human monocyte-derived macrophages (HMDMs). As shown in Fig. 1A, tumor conditioned medium (TCM) increased the secretion of IL10, one of the M2 phenotype markers. This is accompanied by the decrease in the secretion of IL-12 and TNF-a, which are M1 phenotype markers. RES induced a significant decrease in TCMinduced IL-10 secretion, and increased IL-12 and TNF-a secretion. To further confirm the role of RES in M2-like polarization, changes of M2 markers, including MRC1, CCL24, chil3 and Retnla, were evaluated by real-time PCR. It was found that all these genes were downregulated by 20 lM RES compared with untreated group (Fig. 1B). These data suggested that RES exerted inhibitory function by reducing the M2 polarization of HMDMs. 3.2. RES inhibits the proliferation of lung cancer cells by abrogating HMDM activation As it is widely accepted that activated M2 HMDMs accelerate cancer cell proliferation, we hypothesized that RES disrupts the communication between cancer cells and macrophages. To test this hypothesis, we examined the proliferation of A549 and H1299 cells when they were co-cultured with macrophages. As expected, the proliferation of both A549 and H1200 was increased. Whereas, this pro-tumor effect was abrogated by RES treatment (p < 0.01, Fig. 2A). We next assessed cytokine production to test the effect of RES on HMDM activation. Since most cancer cell lines secrete no or negligible amount of IL-10 and IL-10 is preferentially produced by activated HMDMs, IL-10 can serve as a biomarker to evaluate HMDM activation. As shown in Fig. 2B, IL-10 production was substantially induced by the co-culture condition, and IL-10 production was greatly suppressed by RES (p < 0.01). These data indicated that RES suppresses lung cancer cell proliferation, at least in part, through inhibiting soluble factors that communicate between the TAMs and cancer cells. 3.3. RES decreases STAT3 activation in lung cancer cells Due to the important role of STAT3 in both M2 polarization and cancer cell proliferation [12], we reasoned that RES could affect STAT3 activation. As shown in Fig. 3A, STAT3 activation was significantly suppressed by RES treatment in A549 and H1299 cells. A similar inhibition of STAT3 activation and cell growth were observed when lung cancer cells were treated with a STAT3 inhibitor (WP1066) (Fig. 3A and B), suggesting that STAT3 activation is an important regulator of lung cancer cell proliferation. 3.4. RES inhibits lung cancer tumor growth and STAT3 activation in vivo To validate the anti-tumor role of RES in vivo, we used RES to treat a mouse model of LLC. A clear difference was seen in the growth of primary tumors between RES treated mice and control mice after subcutaneous injection of LLC: RES treatment led to a significant reduction of primary tumor growth rate (Fig. 4A). Consistently, the tumor weight in RES treated group was significantly lower (Fig. 4B). To determine if RES functioned primarily through inhibition of macrophages, we used clodronate liposomes to deplete macrophages in circulation. While RES significantly inhibited tumor growth in mice without macrophage depletion, the effects were partially compromised in mice with macrophage
Please cite this article in press as: L. Sun et al., Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.11.002
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Fig. 1. Effect of RES on surface markers in HMDMs. Human monocyte-derived macrophages (HMDMs) were cultured with tumor conditioned medium (TCM group) or without TCM (Control group). HMDMs were treated with resveratrol (RES) (20 lM) with TCM for 24 h (TCM + RES group). The levels of IL-10, IL-12 and TNF-a secretion were determined with ELISA (A). qRT-PCR was performed to characterize change of mRNA levels of M2-markers (B). Data are presented as mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01 versus control.
Fig. 2. Effect of RES on lung cancer cell proliferation and HMDM activation. (A) Lung cancer cells (A549 and H1299) treated with 20 lM RES were incubated with HMDMs for 24 h. Cell proliferation were subsequently determined by EdU incorporation assay. (B) ELISA was used to measure IL-10 production. Control: cancer cells without HMDMs; HMDMs: cancer cells with HMDMs; HMDMs + RES: cancer cells with HMDMs + RES. Data are presented as mean ± SEM from three independent experiments. *p < 0.05, ** p < 0.01 versus control.
Please cite this article in press as: L. Sun et al., Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.11.002
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Fig. 3. Effect of RES on STAT3 activation in lung cancer cells. A549 and H1299 cells were incubated with RES (20 lM) or WP1066 (5 lM) for 48 h, followed by Western blot analysis (A) and MTT assay (B). Data are presented as mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01 versus control.
depletion, suggesting that the inhibitory effect of RES was partially mediated by TAMs (Fig. 4A and B). Since dysregulated tumor cell proliferation is a hallmark of cancers, we analyzed the antiproliferative effects of RES in LLC tumor xenografts with immunohistochemical detection of Ki-67-positive cells. As shown in Fig. 4C, RES significantly decreased the expression of Ki-67. Moreover, RES significantly decreased p-STAT3 staining in LLC tumors compared with control group (Fig. 4C). These results suggested that RES inhibits lung cancer tumor growth associated with suppressing cell proliferation and STAT3 activation in vivo. 3.5. RES reduces F4/80 positive expressing cells and M2-like activation in LLC tumors To investigate whether RES acted through macrophages in the inhibition of primary lung cancer growth, we first examined macrophage tumor infiltration with immunohistochemical (IHC) analysis, which revealed that RES treatment resulted in a significant reduction in the percentages of F4/80+ macrophages (Fig. 5A). RT-PCR was used to evaluate the expression levels of M2 macrophage markers (IL-10, Arg1 and CD206) in LLC tumors, which were significantly lower in RES-treated mice (Fig. 5B). These results confirmed that RES inhibited lung cancer tumor growth by reducing F4/80 positive expressing cells and M2 polarization in the tumors. 4. Discussion In the present study, we demonstrated the anti-tumor effect of RES. RES exhibited inhibitory function in lung cancer cell partly through suppressing soluble factors that communicate between
the TAMs and cancer cells. Moreover, RES was shown to significantly inhibit the growth of LLC tumor xenografts. Concomitantly, decreased expression of p-STAT3 was seen in tumor tissues. Furthermore, F4/80 positive expressing cells and M2 polarization in the LLC tumors were inhibited by RES. Collectively, these observations suggested the anti-cancer effect of RES was attributed to the inhibition of macrophage activation and STAT3 activation. Given macrophages are involved in multiple pathways of tumorigenesis, the potential treatment strategies could be targeting macrophage recruitment, differentiation and activation [23]. Previous studies indicated that down-regulation of the prostaglandin E2/IL-6/STAT3 activation loop is essential for inhibiting macrophage functions [24]. Here, we demonstrated that EdU incorporation was increased in cancer cells after co-culturing with control M2 macrophages rather than RES-treated macrophages. The level of cancer-related cytokines secreted by macrophages was lower after RES treatment, indicating that the activation of M2 macrophages was attenuated by RES. Co-culture experiments showed that RES reduced EdU incorporation in cancer cells cocultured with M2 macrophages, suggesting that the tumormacrophage interaction was abrogated by RES. Given that STAT3 is involved in macrophage differentiation and cancer progression [25]. We demonstrated that cancer cell growth could be reduced by RES and STAT3-activation was also inhibited. These results confirmed that RES assumes an anti-cancer role possibly by inhibiting STAT3 activation. Depletion or reversal of M2 polarization of TAMs in late-stage tumors is a promising therapeutic strategy. Previously, clodronate-encapsulated liposomes was used for macrophage depletion in mice bearing urethane-induced lung tumors. It was
Please cite this article in press as: L. Sun et al., Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.11.002
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Fig. 4. Effect of RES on growth of lung tumors and STAT3 activation in vivo. C57BL/6 mice were subcutaneously injected with LLCs in the flank. RES (100 mg/kg) or vehicle control were administered i.p. once daily from day 10 after tumor cell injection. (A) tumor size (n = 5) was measured twice one week. (B) weight of tumors (n = 5). (C) LCC tumors were processed for immunostaining for Ki-67 and p-STAT3. Scale bars are 50 lm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 versus control.
Fig. 5. Effect of RES on TAM infiltration and polarization. (A) immunostaining images showing the infiltration of macrophages in the tumor tissues. The percentage of F4/80 positive macrophages was presented. Scale bar, 50 lm. (B) The mRNA expression of IL-10, Arg1 and CD206 in LLC tumors tissues were detected using RT-qPCR. Data are presented as mean ± SEM. **p < 0.01 compared with the control group.
Please cite this article in press as: L. Sun et al., Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.11.002
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shown that, after 4 to 6 weeks of treatment, macrophage population in the lungs were reduced to less than 50% of normal levels [26], whereby tumor burden was also decreased by 50%. In the present study, RES was shown to significantly suppress tumor growth in mice this indicates that TAMs are important for the inhibition effects of RES. In addition, c-myc silencing increased immature TAMs, characterized by reduced vascular endothelial growth factor (VEGF), hypoxia inducible factor-1 (HIF-1), and matrix metalloprotease-9 expression (MMP-9) levels [27]. Conversely, treatment with the neuropeptide methionine enkephalin (MENK) induced the polarization of macrophages to an M1 phenotype (CD206 and Arg-1 low, MHCII and iNOS high), along with tumoricidal responses [28]. As indicated by all these studies, polarization into the M1 phenotype was linked to reduced tumor burden and improved survival, which qualified the repolarization of TAMs as another therapeutic strategy. Strategies of M2 polarization reversal can also potentially enhance T cell–mediated anti-tumoral responses and improve immunotherapy efficacy [29], which makes them one of the most promising therapeutic approaches. Herein, we observed that RES drastically reduced the percentages of F4/80+ macrophages. In RES-treated mice, M2-like had significantly lower expression of M2 markers, namely IL-10, Arg1 and CD206. However, the percentage of F4/80 positive cells was low in LLC tumors. The reduction in IL-10, Arg1 and CD206 with RES treatment would show very little difference in expression on a per cell basis, if normalized to the low percentage of F4/80 positive cells. These results suggested that RES induced sluggish growth of lung cancer tumors by decreasing F4/80 positive expressing cells and M2 polarization. As recently reviewed by Olopade et al., TAMs play important roles in tumor progression and include the promotion of neo-angiogenesis, tumor immune evasion and metastatic behavior [30]. Recent studies also showed that decreasing macrophage recruitment is connected with apoptosis and metastasis signal pathway in cancers [31,32]. Thus, a reduction in F4/80 positive cells could mean a reduced recruitment but could also mean induced apoptosis and metastasis by RES presence. Notably, the remaining F4/80 positive cells might be M1 in phenotype. Further preclinical studies are warranted evaluating RES treatment for inhibiting the recruitment and function of M1 and M2 tumor infiltrating macrophages. Considering that current cancer therapies are forfeited by the ability of tumors to evade immune attack with the assistance of TAMs and other stromal cells [33,34], and TAMtargeting therapies has been complicated by the diverse TAM phenotypes within tumors [35], our findings provided another approach to enhance cancer therapy efficacy by linking TAM to the anti-tumor effect of RES. In conclusion, we demonstrated that RES is a potential tumor suppressor in lung cancer. The anti-tumor efficacy of RES stems from the reversal of STAT3 activation in TAMs. The ability of RES inhibiting soluble factors that communicate between tumor cells and TAMs may block the feedforward loop between the lung cancer cells and TAMs. Therefore, RES can potentially be developed as a potent therapy for lung cancer. Funding None. Conflict of interests The authors declare that there is no conflict of interests. Acknowledgments
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Research paper
Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages Liwei Sun 1, Bonian Chen 1, Rong Jiang ⇑, Jinduo Li, Bin Wang Tianjin Key Laboratory of Cerebral Vascular and Neurodegenerative Disease, Department of Intervention, Tianjin Huanhu Hospital, Tianjin 300060, PR China
a r t i c l e
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Article history: Received 12 September 2016 Revised 30 October 2016 Accepted 1 November 2016 Available online xxxx Keywords: Resveratrol Lung cancer STAT3 Macrophages
a b s t r a c t In cancer, tumor associated macrophages (TAMs) play an important role in the cancer progression, evasion of immunity and dissemination of cancer cells. Inhibition of the activation or the M2 polarization of TAMs is an effective therapy for cancer. In the present study, we investigated the ability of resveratrol (RES) to inhibit lung cancer growth using in vitro and in vivo studies, and examined the underlying mechanisms. We demonstrated that M2 polarization of human monocyte derived macrophage (HMDMs) induced by the lung cancer cells conditioned medium was inhibited by RES. Additionally, RES exhibited inhibitory function in lung cancer cells co-cultured with human macrophages. The activity of signal transducer and activator of transcription 3 (STAT3) was significantly decreased by RES. Moreover, in a mouse lung cancer xenograft model, RES significantly inhibited the tumor growth, which was associated with inhibition of cell proliferation and decreased expression of p-STAT3 in tumor tissues. Further, RES inhibits F4/80 positive expressing cells and M2 polarization in the tumors. These results suggest that RES can effectively inhibit lung cancer progression by suppressing the protumor activation of TAMs. Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction Despite advances in the clinical management of lung cancer, lung cancer remains as the leading cause of cancer-related deaths worldwide [1]. It is increasingly recognized that the interaction between tumor and immune effector cells enhances tumor growth, invasion and local immunosuppression, to evade from antitumor immune responses, which is also essential to achieve effective extravasation at prospective metastatic sites [2]. Thus primary tumors, and possibly, circulating tumor cells (CTCs), are capable of recruiting immunosuppressive cells particularly of the myeloid-macrophage lineage. These immunosuppressive cells undergo functional polarization driven by tumor-derived factors [3]. These ‘‘tumor-educated” macrophages are involved in all stages of cancer metastasis: cancer invasion, intravasation, survival in the circulation, and sustainable growth at secondary lesions are all promoted by these macrophages [4]. Because of their existence as a major cellular component of murine and human tumors, they are commonly termed tumor-associated macrophages (TAMs). Abbreviations: TAMs, tumor associated macrophages; RES, resveratrol; STAT3, signal transducer and activator of transcription 3. ⇑ Corresponding author. E-mail address: [email protected] (R. Jiang). 1 Contributed equally.
Originally, TAMs are proposed to be involved in anti-tumor immunity, whereas recent evidences unveiled the tumor-promoting effect of TAMs [5,6]. Epidemiological evidences indicate a strong link between increased macrophage infiltration and poor prognosis in lung cancer [7]. TAM density was found to be negatively correlated with poor patient survival and frequent recurrence [8]. The median survival of patients with high macrophages density was significantly shorter than those who had a lower macrophages density [7]. These studies suggested that TAMs play a crucial role in cancer development. Plasticity and flexibility are key indicators of the activation states of macrophages. Whether TAMs assume tumor-preventing or tumor-promoting role is dependent on their polarization statues [9]. The activation states, M1 and M2, denote classical activation and alternative activation of macrophages, respectively. In benign or regressive tumors, the majority of TAMs are classically activated macrophages (M1-like), which exert pro-inflammatory activity by antigen-presenting and promoting tumor lysis [10]. Conversely, TAMs in malignant tumors resemble alternatively activated macrophages (M2-like), enhancing tumor angiogenesis, migration and invasion. M2-like macrophages suppress antitumor immune responses. Thus, M2-like TAMs are promising targets for adjuvant anticancer therapies. Encouraging anti-tumor efficacy has been achieved by recent M2-TAM-targeting approaches. For example,
http://dx.doi.org/10.1016/j.cellimm.2016.11.002 0008-8749/Ó 2016 Elsevier Inc. All rights reserved.
Please cite this article in press as: L. Sun et al., Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.11.002
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targeted delivery of peptide to M2-like TAM demonstrated improvement in the survival of tumor bearing mouse [11]. In another study, in vitro co-culturing of cancer cells with M2 macrophages showed activation of signal transducer and activator of transcription 3 (STAT3), which is an important modulator of tumor progression and chemo-resistance in cancer cells [12]. M2 macrophages activated by cancer cells secrete several cytokines, such as IL-6 and IL-10, which in turn promote cancer progression [13,14]. Therefore, inhibiting macrophage polarization into the M2 phenotype, and blocking the tumor-macrophage interaction could be a viable approach toward efficient cancer therapy. Resveratrol (RES) possess multiple pharmacological activities in extending longevity and treatment of cardiovascular disease, diabetes and cancer [15]. RES is a polyphenol that naturally exists in grapes and red wine. The investigation of RES originated from studies on ‘French Paradox’, which describes beneficial cardiovascular outcomes of a high-fat diet in French cuisine [16]. Its anti-cancer effects have been well documented in a variety of cancers, whereby RES regulated cell division, growth, angiogenesis and metastasis [17]. In lung cancer, RES was shown to promote premature senescence in cancer cells (A549 and H460 cells) via inducing NAPDH oxidase-5 (Nox5) [18], which resulted in inhibition of cancer cell proliferation and survival [19]. Recent studies showed that RES exerts anti-tumor effects by regulating the release of cytokines. RES selectively inhibits or activates the release of cytokines. RESsuppressed cytokines include IL-6, IL-12 and TNF-a [20,21]. However, the beneficial effects of RES in lung cancer remain to be clarified. In the present study, we investigated the ability of resveratrol to inhibit lung cancer growth using in vitro and in vivo models. Given that M2 macrophages are closely associated in cancer progression, we also investigated the relevance between macrophage polarization and the antitumor effects of RES. 2. Materials and methods 2.1. Cell culture Human lung cancer cells, A549 and H1299, were acquired from the American Type Culture Collection (Manassas, VA, USA). Lewis lung cancer (LLC) cells were obtained from the Cell Bank of the China Science Academy (Shanghai, China). All cell lines were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (Gibco, Grand Island, NY) and regularly checked for Mycoplasma contamination. Cells were incubated in a humidified atmosphere with 5% CO2 at 37 °C. Peripheral blood mononuclear cells were collected from healthy volunteer donors, with written informed consent filed for all sample collection. Subsequent analysis was performed on all healthy donors. Experiments regarding human studies were performed according to protocols approved by the Tianjin Huanhu Hospital Review Board. CD14+ monocytes were isolated from the peripheral blood mononuclear cells using magnetic-activated cell sorting (Miltenyi Biotec, Bergisch Gladbach, Germany) based on positive selection, followed by co-culturing with GM-CSF (10 ng/ml, Wako, Tokyo, Japan) or M-CSF (50 ng/ml, WAKO) for a week for macrophage differentiation. The differentiated macrophages were then used as human monocyte-derived macrophages (HMDMs) in the present study, as described previously [22]. 2.2. Preparation of the tumor conditioned medium (TCM) The tumor conditioned medium was acquired from the supernatant of A549 cultured medium after 72 h of incubation. The conditioned media was collected from the Transwell insert systems,
followed by filtration through 0.2 lm pores (Sartorius Stedim Biotech, Germany). The TCM was stored at 80 °C until further usage in the ELISA assays. 2.3. Cytokine ELISA kit HMDMs were seeded in 12-well plates at the density of 3 105 cells/well. HMDM cells were subjected to lipopolysaccharide (LPS) (100 ng/mL, Sigma, Louis, MO, USA) stimulation for 24 h, followed by incubation with TCM or with RES-containing TCM (Sigma, Louis, MO, USA) for 24 h. ELISA was used to determine IL-10, IL-12 and TNF-a secretion according to manufacturer’s guidelines (eBioscience, San Diego, CA, USA). 2.4. Cell proliferation assay Viability assay was performed on A549 and H1299 cells using the Cell-Light EdU Apollo488 In Vitro Imaging Kit (RiboBio) in accordance to the manufacturer’s protocol. Briefly, cells incubated with 10 lM EdU for 2 h were fixed with 4% paraformaldehyde (PFA), permeabilized with 0.3% Triton X-100 and stained with EdU. Cell nucleus were stained by 10-min-incubation with 5 lg/ mL DAPI (40 ,6-diamidino-2-phenylindole). Number counting of Edu-positive cells was counted under a light microscope based on five random fields (100). All assays were performed in triplicate independently. 2.5. MTT assay MTT assay was used to determine the effect of RES on the viability of A549 and H1299 cells. Briefly, cells were treated with RES or STAT3 inhibitor (WP1066) (Sigma, Louis, MO, USA) for 48 h. At the end of treatment, 50 lL of 5 mg/mL MTT were added and the resulting formazan crystals were dissolved in 100 lL of DMSO, followed by measuring absorbance at 570 nm by an Automated Microplate Reader (Bio-Tek, USA). The cell viability was calculated using the following equation: cell viability (%) = (OD of treated cells/OD of control cells) 100. All assays were performed in triplicate with at least 3 independent experiments. 2.6. RNA isolation, reverse transcription and qRT-PCR Total RNA from BMDM was extracted and purified using the Easy Pure RNA Kit (Transgen Biotech Co., Ltd), followed by cDNA synthesis. Quantitative real time-PCR (qRT-PCR) was carried out using SYBR Premix ExTaq (TaKaRa, Dalian, China) on a Stratagene Mx3000P real-time PCR system (Agilent Technologies, Inc., CA, USA). All mRNA levels were normalized to the GAPDH level. The quantification of mRNA levels were performed using the 2DDCT method and normalized to GADPH. PCR reactions were performed in triplicates independently. Primers used in PCR reaction were as follows: MRC1, forward primer: 50 -AGG GAC CTG GAT GGA TGA CA-30 ; and reverse primer: 50 -TGT ACC GCA CCC TCC ATC TA-30 ; CCL24, forward primer: 50 -TGT CTG CAG TTG AGC CTA CG-30 ; and reverse primer: 50 -GTT CGG GAC CCT GGA GTT AG-30 ; chil3, forward primer: 50 -CAT GAG CAA GAC TTG CGT GAC-30 ; and reverse primer: 50 -GGT CCA AAC TTC CAT CCT CCA-30 ; Retnla, forward primer: 50 -CCC TGC TGG GAT GAC TGC TA-30 ; and reverse primer: 50 TGC AAG TAT CTC CAC TCT GGA TCT-30 ; IL-10, forward primer: 50 ACT GGC ATG AGG ATC AGC AG-30 and reverse primer: 50 -CTC CTT GAT TTC TGG GCC AT-30 ; Arg1, forward primer: 50 -GAT TAT CGG AGC GCC TTT CT-30 and reverse primer: 50 -CCA CAC TGA CTC TTC CAT TCT-30 ; CD206, forward primer: 50 -CTG CAG ATG GGT GGG TTA TT-30 and reverse primer: 50 -GGC ATT GAT GCT GCT GTT ATG-30 ; GAPDH, forward primer: 50 -CAG CCT CAA GAT CAT CAG CA-30 and reverse primer: 50 -TGT GGT CAT GAG TCC TTC CA-30 .
Please cite this article in press as: L. Sun et al., Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.11.002
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2.7. Western blot analysis
3. Results
Cells were harvested by trypsinization, washed with cold PBS, and lysed with ice-cold lysis buffer supplemented with protease inhibitors (PI) as described previously. Equivalent amounts of protein (30 lg) were loaded onto precast gels (Biorad) and resolved using SDS-PAGE. Proteins were blotted onto nitrocellulose membranes (0.45 lM; Bio-Rad). Membranes were incubated in blocking solution (1 PBS, 0.1% Tween-20, and 5% non-fat dry milk powder) at room temperature for 1 h. Primary antibodies against anti-pSTAT3, anti-STAT3 or anti-b-Actin (Cell signaling Technology, MA, USA) were used to incubate with the membrane at 4 °C overnight with gentle shaking. After extensive washing with TBST, membranes were incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies (Bio-Rad) for 1 h at room temperature and protein bands were detected with the enhanced chemiluminescence method (SuperSignal West Pico substrate; Pierce; Rockford, IL).
3.1. RES inhibits M2 polarization of HMDMs
2.8. Tumor models Animal studies were performed strictly according to the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health, and a protocol approved by the Committee on the Ethics of Animal Experiments of Tianjin Huanhu Hospital. All surgeries were performed under anesthesia with sodium pentobarbital, with precautions to minimize animal suffering. C57BL/6 mice, at the age of 4–5 weeks, were acquired from the National Rodent Laboratory Animal Resource (Shanghai, China). LLCs model was constructed by injecting 1 106 cells subcutaneously to the flanks of C57BL/6. RES, at the dose of 100 mg/kg, or vehicle control were administered i.p. once daily from the 10th day tumor inoculation (5 mice per group). Tumors were monitored twice a week. Tumor volumes were calculated after measuring length and width of the tumor with calipers and tumor volumes were calculated as (Length Width2)/2. After 4 weeks, animals were sacrificed, with tumors collected.
2.9. Immunohistochemistry Tumors sections at the thickness of 5 lm were obtained by cryosection. These sections were then deparaffinized and rehydrated according to standard histological protocols, followed by antigen retrieval with 10 mM sodium citrate buffer (pH 6.0), using a pressure cooker maintained at 95° C for 20 min. The sections were then cooled at room temperature for 20 min, washed with PBS and blocked with 1% BSA/2% goat serum before incubation with either anti-Ki-67, anti-pSTAT3 (Cell signaling Technology, MA, USA) and anti-F4/80 (Abcam, Cambridge, MA, USA). After extensive washing, the sections were incubated with biotinylated secondary antibody, followed by horseradish peroxidaseconjugated streptavidin. Subsequently, 2,4-diaminobenzidine substrates were added and sections were counterstained with hematoxylin. The positively stained cells was counted using the BZ9000 analysis software program (Keyence, Osaka, Japan).
2.10. Statistical analysis Data were presented as mean ± SEM unless otherwise stated. The statistical significance of differences between two groups was analyzed by either Student t test or simple one-way ANOVA using Prism version 5 (GraphPad Software, Inc.). Differences with p < 0.05 were considered statistically significant.
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First, we evaluated whether RES was able to inhibit the lung cancer cell conditioned medium induced M2 polarization of human monocyte-derived macrophages (HMDMs). As shown in Fig. 1A, tumor conditioned medium (TCM) increased the secretion of IL10, one of the M2 phenotype markers. This is accompanied by the decrease in the secretion of IL-12 and TNF-a, which are M1 phenotype markers. RES induced a significant decrease in TCMinduced IL-10 secretion, and increased IL-12 and TNF-a secretion. To further confirm the role of RES in M2-like polarization, changes of M2 markers, including MRC1, CCL24, chil3 and Retnla, were evaluated by real-time PCR. It was found that all these genes were downregulated by 20 lM RES compared with untreated group (Fig. 1B). These data suggested that RES exerted inhibitory function by reducing the M2 polarization of HMDMs. 3.2. RES inhibits the proliferation of lung cancer cells by abrogating HMDM activation As it is widely accepted that activated M2 HMDMs accelerate cancer cell proliferation, we hypothesized that RES disrupts the communication between cancer cells and macrophages. To test this hypothesis, we examined the proliferation of A549 and H1299 cells when they were co-cultured with macrophages. As expected, the proliferation of both A549 and H1200 was increased. Whereas, this pro-tumor effect was abrogated by RES treatment (p < 0.01, Fig. 2A). We next assessed cytokine production to test the effect of RES on HMDM activation. Since most cancer cell lines secrete no or negligible amount of IL-10 and IL-10 is preferentially produced by activated HMDMs, IL-10 can serve as a biomarker to evaluate HMDM activation. As shown in Fig. 2B, IL-10 production was substantially induced by the co-culture condition, and IL-10 production was greatly suppressed by RES (p < 0.01). These data indicated that RES suppresses lung cancer cell proliferation, at least in part, through inhibiting soluble factors that communicate between the TAMs and cancer cells. 3.3. RES decreases STAT3 activation in lung cancer cells Due to the important role of STAT3 in both M2 polarization and cancer cell proliferation [12], we reasoned that RES could affect STAT3 activation. As shown in Fig. 3A, STAT3 activation was significantly suppressed by RES treatment in A549 and H1299 cells. A similar inhibition of STAT3 activation and cell growth were observed when lung cancer cells were treated with a STAT3 inhibitor (WP1066) (Fig. 3A and B), suggesting that STAT3 activation is an important regulator of lung cancer cell proliferation. 3.4. RES inhibits lung cancer tumor growth and STAT3 activation in vivo To validate the anti-tumor role of RES in vivo, we used RES to treat a mouse model of LLC. A clear difference was seen in the growth of primary tumors between RES treated mice and control mice after subcutaneous injection of LLC: RES treatment led to a significant reduction of primary tumor growth rate (Fig. 4A). Consistently, the tumor weight in RES treated group was significantly lower (Fig. 4B). To determine if RES functioned primarily through inhibition of macrophages, we used clodronate liposomes to deplete macrophages in circulation. While RES significantly inhibited tumor growth in mice without macrophage depletion, the effects were partially compromised in mice with macrophage
Please cite this article in press as: L. Sun et al., Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.11.002
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Fig. 1. Effect of RES on surface markers in HMDMs. Human monocyte-derived macrophages (HMDMs) were cultured with tumor conditioned medium (TCM group) or without TCM (Control group). HMDMs were treated with resveratrol (RES) (20 lM) with TCM for 24 h (TCM + RES group). The levels of IL-10, IL-12 and TNF-a secretion were determined with ELISA (A). qRT-PCR was performed to characterize change of mRNA levels of M2-markers (B). Data are presented as mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01 versus control.
Fig. 2. Effect of RES on lung cancer cell proliferation and HMDM activation. (A) Lung cancer cells (A549 and H1299) treated with 20 lM RES were incubated with HMDMs for 24 h. Cell proliferation were subsequently determined by EdU incorporation assay. (B) ELISA was used to measure IL-10 production. Control: cancer cells without HMDMs; HMDMs: cancer cells with HMDMs; HMDMs + RES: cancer cells with HMDMs + RES. Data are presented as mean ± SEM from three independent experiments. *p < 0.05, ** p < 0.01 versus control.
Please cite this article in press as: L. Sun et al., Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.11.002
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Fig. 3. Effect of RES on STAT3 activation in lung cancer cells. A549 and H1299 cells were incubated with RES (20 lM) or WP1066 (5 lM) for 48 h, followed by Western blot analysis (A) and MTT assay (B). Data are presented as mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01 versus control.
depletion, suggesting that the inhibitory effect of RES was partially mediated by TAMs (Fig. 4A and B). Since dysregulated tumor cell proliferation is a hallmark of cancers, we analyzed the antiproliferative effects of RES in LLC tumor xenografts with immunohistochemical detection of Ki-67-positive cells. As shown in Fig. 4C, RES significantly decreased the expression of Ki-67. Moreover, RES significantly decreased p-STAT3 staining in LLC tumors compared with control group (Fig. 4C). These results suggested that RES inhibits lung cancer tumor growth associated with suppressing cell proliferation and STAT3 activation in vivo. 3.5. RES reduces F4/80 positive expressing cells and M2-like activation in LLC tumors To investigate whether RES acted through macrophages in the inhibition of primary lung cancer growth, we first examined macrophage tumor infiltration with immunohistochemical (IHC) analysis, which revealed that RES treatment resulted in a significant reduction in the percentages of F4/80+ macrophages (Fig. 5A). RT-PCR was used to evaluate the expression levels of M2 macrophage markers (IL-10, Arg1 and CD206) in LLC tumors, which were significantly lower in RES-treated mice (Fig. 5B). These results confirmed that RES inhibited lung cancer tumor growth by reducing F4/80 positive expressing cells and M2 polarization in the tumors. 4. Discussion In the present study, we demonstrated the anti-tumor effect of RES. RES exhibited inhibitory function in lung cancer cell partly through suppressing soluble factors that communicate between
the TAMs and cancer cells. Moreover, RES was shown to significantly inhibit the growth of LLC tumor xenografts. Concomitantly, decreased expression of p-STAT3 was seen in tumor tissues. Furthermore, F4/80 positive expressing cells and M2 polarization in the LLC tumors were inhibited by RES. Collectively, these observations suggested the anti-cancer effect of RES was attributed to the inhibition of macrophage activation and STAT3 activation. Given macrophages are involved in multiple pathways of tumorigenesis, the potential treatment strategies could be targeting macrophage recruitment, differentiation and activation [23]. Previous studies indicated that down-regulation of the prostaglandin E2/IL-6/STAT3 activation loop is essential for inhibiting macrophage functions [24]. Here, we demonstrated that EdU incorporation was increased in cancer cells after co-culturing with control M2 macrophages rather than RES-treated macrophages. The level of cancer-related cytokines secreted by macrophages was lower after RES treatment, indicating that the activation of M2 macrophages was attenuated by RES. Co-culture experiments showed that RES reduced EdU incorporation in cancer cells cocultured with M2 macrophages, suggesting that the tumormacrophage interaction was abrogated by RES. Given that STAT3 is involved in macrophage differentiation and cancer progression [25]. We demonstrated that cancer cell growth could be reduced by RES and STAT3-activation was also inhibited. These results confirmed that RES assumes an anti-cancer role possibly by inhibiting STAT3 activation. Depletion or reversal of M2 polarization of TAMs in late-stage tumors is a promising therapeutic strategy. Previously, clodronate-encapsulated liposomes was used for macrophage depletion in mice bearing urethane-induced lung tumors. It was
Please cite this article in press as: L. Sun et al., Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.11.002
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Fig. 4. Effect of RES on growth of lung tumors and STAT3 activation in vivo. C57BL/6 mice were subcutaneously injected with LLCs in the flank. RES (100 mg/kg) or vehicle control were administered i.p. once daily from day 10 after tumor cell injection. (A) tumor size (n = 5) was measured twice one week. (B) weight of tumors (n = 5). (C) LCC tumors were processed for immunostaining for Ki-67 and p-STAT3. Scale bars are 50 lm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01 versus control.
Fig. 5. Effect of RES on TAM infiltration and polarization. (A) immunostaining images showing the infiltration of macrophages in the tumor tissues. The percentage of F4/80 positive macrophages was presented. Scale bar, 50 lm. (B) The mRNA expression of IL-10, Arg1 and CD206 in LLC tumors tissues were detected using RT-qPCR. Data are presented as mean ± SEM. **p < 0.01 compared with the control group.
Please cite this article in press as: L. Sun et al., Resveratrol inhibits lung cancer growth by suppressing M2-like polarization of tumor associated macrophages, Cell. Immunol. (2016), http://dx.doi.org/10.1016/j.cellimm.2016.11.002
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shown that, after 4 to 6 weeks of treatment, macrophage population in the lungs were reduced to less than 50% of normal levels [26], whereby tumor burden was also decreased by 50%. In the present study, RES was shown to significantly suppress tumor growth in mice this indicates that TAMs are important for the inhibition effects of RES. In addition, c-myc silencing increased immature TAMs, characterized by reduced vascular endothelial growth factor (VEGF), hypoxia inducible factor-1 (HIF-1), and matrix metalloprotease-9 expression (MMP-9) levels [27]. Conversely, treatment with the neuropeptide methionine enkephalin (MENK) induced the polarization of macrophages to an M1 phenotype (CD206 and Arg-1 low, MHCII and iNOS high), along with tumoricidal responses [28]. As indicated by all these studies, polarization into the M1 phenotype was linked to reduced tumor burden and improved survival, which qualified the repolarization of TAMs as another therapeutic strategy. Strategies of M2 polarization reversal can also potentially enhance T cell–mediated anti-tumoral responses and improve immunotherapy efficacy [29], which makes them one of the most promising therapeutic approaches. Herein, we observed that RES drastically reduced the percentages of F4/80+ macrophages. In RES-treated mice, M2-like had significantly lower expression of M2 markers, namely IL-10, Arg1 and CD206. However, the percentage of F4/80 positive cells was low in LLC tumors. The reduction in IL-10, Arg1 and CD206 with RES treatment would show very little difference in expression on a per cell basis, if normalized to the low percentage of F4/80 positive cells. These results suggested that RES induced sluggish growth of lung cancer tumors by decreasing F4/80 positive expressing cells and M2 polarization. As recently reviewed by Olopade et al., TAMs play important roles in tumor progression and include the promotion of neo-angiogenesis, tumor immune evasion and metastatic behavior [30]. Recent studies also showed that decreasing macrophage recruitment is connected with apoptosis and metastasis signal pathway in cancers [31,32]. Thus, a reduction in F4/80 positive cells could mean a reduced recruitment but could also mean induced apoptosis and metastasis by RES presence. Notably, the remaining F4/80 positive cells might be M1 in phenotype. Further preclinical studies are warranted evaluating RES treatment for inhibiting the recruitment and function of M1 and M2 tumor infiltrating macrophages. Considering that current cancer therapies are forfeited by the ability of tumors to evade immune attack with the assistance of TAMs and other stromal cells [33,34], and TAMtargeting therapies has been complicated by the diverse TAM phenotypes within tumors [35], our findings provided another approach to enhance cancer therapy efficacy by linking TAM to the anti-tumor effect of RES. In conclusion, we demonstrated that RES is a potential tumor suppressor in lung cancer. The anti-tumor efficacy of RES stems from the reversal of STAT3 activation in TAMs. The ability of RES inhibiting soluble factors that communicate between tumor cells and TAMs may block the feedforward loop between the lung cancer cells and TAMs. Therefore, RES can potentially be developed as a potent therapy for lung cancer. Funding None. Conflict of interests The authors declare that there is no conflict of interests. Acknowledgments
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