Abstract

     Glioblastoma is an aggressive tumor that is associated with distinctive infiltrating microglia/macrophages populations. Previous studies demonstrated that chlorogenic acid (5-caffeoylquinic acid, CHA), a phenolic compound with low molecular weight, has an anti-tumor effect in multiple malignant tumors. In the present study, we focused on the macrophage polarization to investigate the molecular mechanisms behind the anti-glioma response of CHA in vitro and in vivo. We found that CHA treatment increased the expression of M1 markers induced by LPS/IFNγ, including iNOS, MHC II (I-A/I-E subregions) and CD11c, and reduced the expression of M2 markers Arg and CD206 induced by IL-4, resulting in promoting the production of apoptotic-like cancer cells and inhibiting the growth of tumor cells by co-culture experiments. The activations of STAT1 and STAT6, which are two crucial signaling events in M1 and M2-polarization, were significantly promoted and suppressed by CHA in macrophages, respectively. Furthermore, In G422 xenograft mice, CHA increased the proportion of CD11c-positive M1 macrophages and decreased the distribution of CD206-positive M2 macrophages in tumor tissue, consistent with the reduction of tumor weight observed in CHA-treated mice. Overall these findings indicated CHA as a potential therapeutic approach to reduce glioma growth through promoting M1-polarized macrophage and inhibiting M2 phenotypic macrophage.

Introduction

     Glioblastoma multiforme (GBM) is the most aggressive pattern among brain tumors. GBM has an extremely poor prognosis status: patient survival remains less than 15 months1, even after surgical ablation and routine treatments with chemo- and radio-therapeutic agents. It has been widely shown that the immune microenvironment has a central role in GBM. Microglia/macrophages represent the largest tumor-infiltrating immune cell population, contributing to tumor progression and metastasis2. Thus, a novel research field is focused on immunotherapy-based drug discovery.

    Macrophage is broadly categorized as “classically activated” pro-inflammatory M1 macrophages and “alternatively activated” anti-inflammatory M2 macrophages3,4. Macrophages can differentiate into various activation states owing to the cytokine balance in the microenvironment. M1 phenotypic macrophage activation, in response to interferon γ (IFNγ) or lipopolysaccharide (LPS), is characterized by up-regulation of inducible nitric oxide synthase (iNOS) and interleukins IL6 or IL12, and enhancement of the Th1 immune response5,6,7. Alternatively, M2 phenotypic macrophage activation is induced by IL-4 and IL-13 through binding to a common receptor subunit, IL-4Rα, and characterized by increased expression of arginase 1 (Arg1), IL-10 and mannose receptor. In nonmalignant or regressing tumors, the majority of tumor-associated macrophage (TAM) is M1-like, which represents pro-inflammatory activity to kill microorganisms and promote tumor lysis5,7. In malignant tumors, TAM has been shown to be of predominant in M2 phenotype, which has a pro-tumor function by secretion of proangiogenic factors8 and immunosuppressive cytokines9. Increased proportion of M2-type TAM has been associated with poor prognosis in multiple tumors including Hodgkin’s lymphoma, cholangiocarcinoma, breast carcinoma, as well as cerebral glioma10,11,12. Thus, several research groups focused on M2-like TAMs as a potential targets for anticancer therapies and have gained encouraging results. In addition, skewing TAM differentiation away from the M2 to M1 phenotype promoted antitumor immune responses and reduced tumor growth and metastasis in hepatocellular carcinoma (HCC), prostate cancer, breast cancer and glioblastoma13,14,15,16. Furthermore, M1-type macrophages have been reported to be associated with prolonged survival time in patients with non small-cell lung cancer (NSCLC)17,18.

     Some natural compounds and therapeutic drugs have been reported to affect macrophage phenotypic differentiation to exert their antitumor effect. These compounds include the natural products zoledronic acid19 and emodin20, therapeutic monoclonal antibodies including anti-CD4713,21 and the phosphatidylserine-targeting antibody 2aG414, and chemotherapy agents, doxycycline and metformin22,23. Chlorogenic acid (5-caffeoylquinic acid, CHA), the ester formed between caffeic acid and quinic acid24, is a phenolic compound found in the human diet, in coffee, apples, pears and in green tea. It has been shown to have several beneficial biological properties, including anti-bacterial effects, anti-inflammatory effects, and anti-oxidant effects, as well as anti-tumor activities25,26. It has been found to exert anti-inflammatory effects through NF-κB inhibition of prostaglandin E synthesis and cyclo-oxygenase-2 and by suppressing JNK/AP-1 activation27. It has been shown to activate calcineurin and enhance macrophage function in vivo and in vitro28. Furthermore, CHA has been proposed to inhibit tumor growth and angiogenesis by inhibiting tyrosinase and matrix metalloproteinase (MMP)-929,30, or by the suppression of HIF-1α stabilizationand AKT phosphorylation31. Recently, published findings from pre-clinical experimental and phase I clinical studies have shown that treatment with CHA has shown therapeutic effects in breast cancer32, brain tumors33, lung cancer34, colon cancer35 and chronic myelogenous leukemia29. However, the molecular mechanisms behind the anti-cancer response of CHA and its effects on macrophage polarization, remains yet largely unclear and need to be clarified.

     In this study, we investigate the role of CHA-mediated macrophage polarization in glioblastoma progression. We found that, in vitro, CHA induced LPS/IFNγ- but inhibited IL4-responsive genes through promoting STAT1 signaling and inhibiting STAT6 signaling, respectively. Thereby, CHA promoted the production of apoptotic-like cancer cells and inhibited tumor growth by affecting the interaction between macrophages and cancer cells. We also found that CHA inhibits growth of G422 glioma in vivo, and this effect was associated with a decrease of M2-like TAMs and recruitment of M1-like TAMs into tumor tissue.

Results

CHA promoted M1 polarization of macrophages induced by lipopolysaccharide (LPS) and interferon (IFN)-γ

      We detected the effect of CHA on macrophages polarization at the concentrations of 0.5 μM to 5 μM, and the proliferation of macrophages was not affected under these doses of CHA (Supplementary Fig. 1A). Initially, the M1/M2 macrophage phenotype marker genes induced by IFNγ and LPS (M1 inducer) were analyzed using RT-PCR analysis. Treatment with IFNγ/LPS significantly up-regulated the mRNA levels of the M1 marker gene iNOS or IL12 in Ana-1 and RAW264.7 macrophages (P ≤ 0.01). Meanwhile, IFNγ/LPS treatment significantly reduced the mRNA levels of the M2 marker gene Arg1 in Ana-1 cells, but that of RAW264.7 cells was increased (P ≤ 0.01) (Supplementary Fig. 2A,B). Then, at exposure to CHA, IFNγ/LPS-induced iNOS mRNA level was further up-regulated and the Arg1 mRNA level was significantly down-regulated in Ana-1 cells in a concentration-dependent manner (P ≤ 0.05, P ≤ 0.01) (Fig. 1A). Similarly, in RAW264.7 cells, combination with 1 μM of CHA significantly increased the iNOS mRNA level and decreased the Arg1 mRNA level when compared with IFNγ/LPS-treated alnoe (P ≤ 0.05) (Fig. 1B). To further investigate the role of CHA in M1 macrophage differentiation, cell surface markers were assessed by flow cytometry. As shown in Fig. 1C and D, treatment with IFNγ/LPS increased the expression of CD11c and MHC Class II subunits IA/IE, and those were dose and time-dependently increased after combination with CHA in RAW264.7 cells, especially in the expression of CD11c. These results indicated that CHA could promote M1 phenotypic differentiation in macrophages in vitro.

Figure 1: Effect of CHA on macrophage marker expression induced by lipopolysaccharide (LPS) and interferon (IFN)-γ.

Macrophages were treated with LPS (10 ng/ml) and IFNγ (20 ng/ml) with or without different concentrations of CHA for 24 h or 48 h. The mRNA levels of M1-marker gene iNOS and M2-marker gene Arg in Ana-1 (A) and RAW264.7 cells (B) were measured by real-time RT-PCR. The expression of mRNAs was normalized to GAPDH. The expressions of CD11c and MHC Class II IA-IE in RAW264.7 cells for 24 h (C) or 48 h (D) were evaluated by flow cytometry. The histogram bars represent three independent experiments. The data are presented as the mean ± SD. *p-value < 0.05, **p-value < 0.01 vs. LPS/IFNγ.

CHA inhibited M2 polarization of macrophages induced by interleukin (IL)-4

      We also analyzed the impact of CHA on M2 polarization of macrophages induced by IL-4. As shown in Supplementary Fig. 2A,B, IL-4 significantly increased the mRNA levels of the M2 marker gene Arg1 or IL10 in Ana-1 and RAW264.7 cells, and reduced the mRNA levels of the M1 marker gene iNOS in Ana-1 cells (P ≤ 0.05, P ≤ 0.01). CHA decreased the expression of the IL-4-induced Arg1 mRNA levels in a dose-dependent manner, but with a significant increase in the iNOS mRNA level at 1 μM of CHA in IL-4 treated Ana-1 cells (P ≤ 0.05) (Fig. 2A). IL-10, another M2 marker gene, was up-regulated by IL-4, which was also found to be dose-dependently inhibited by treatment with CHA in Ana-1 cells (supplementary Fig. 2C). A similar pattern of iNOS mRNA levels was also observed in IL-4-treated RAW264.7 cells at 0.1 or 1 μM of CHA (P ≤ 0.05, P ≤ 0.01) (Fig. 2B). Furthermore, IL-4-induced CD206 expression was decreased by 10% at 2 μM of CHA in Ana-1 cells, but remained unchanged in RAW264.7 cells (Fig. 2C,D). These findings indicated that CHA effectively suppressed M2 phenotypic differentiation in Ana-1 macrophages.

Figure 2: Effect of CHA on macrophage marker expression induced by interleukin (IL)-4.Ana-1 and RAW264.7 cells were treated with interleukin (IL)-4 (20 ng/ml) in the presence of DMSO or different concentrations of CHA for 24 h. The mRNA levels of M1-marker gene iNOS and M2-marker gene Arg in Ana-1 (A) and RAW264.7 cells (B) were measured by real-time RT-PCR. The expression of mRNAs was normalized to GAPDH. The expressions of CD206 in Ana-1 (C) and RAW264.7 cells (D) were evaluated by flow cytometry. The histogram bars represent three independent experiments. The data are presented as the mean ± SD. *p-value < 0.05, **p-value < 0.01 vs.IL-4.CHA regulated macrophage polarization by promoting STAT1 activation and suppressing STAT6 activation

We next sought to investigate the molecular mechanisms of CHA on macrophage polarization. Signal transducer and activator of transcription 1 (STAT1) and STAT6 signaling pathways play critical roles in the M1 and M2 macrophage polarization, respectively36,37. We detected the STAT1 and STAT6 activation after treatment with CHA in M1/M2 inducer stimulated-macrophage. As shown in Fig. 3A,B, the protein expressions of p(Ser727)-STAT1, p(Tyr701)-STAT1 and total STAT1 were significantly upregulated when Ana-1 and RAW264.7 cells were treated with M1-inducer (LPS/IFNγ) (P ≤ 0.01), and phosphor-STAT1 expression was further increased by the administration of CHA, as shown by significant increases in expressions of p(Tyr701)-STAT1 and p(Tyr701)-STAT1/total STAT1 at 1 μM of CHA in Ana-1 cells and by significant induction of p(Ser727)-STAT1 and p(Ser727)-STAT1/total STAT1 in RAW264.7 cells at high dose of CHA (2, 4 μM). Meanwhile, upregulations of p(Tyr641)-STAT6 and p(Tyr641)-STAT6/total STAT6 were observed when Ana-1 or RAW264.7 cells were treated with IL-4 for 24 h, which were significantly reduced by combination with CHA in a dose-dependent manner, particularly in RAW264.7 cells (P ≤ 0.05, P ≤ 0.01) (Fig. 3C,D). These results indicated that CHA could have a regulating effect on macrophage polarization mainly via STAT1 and STAT6 signaling pathways.

Figure 3: Effect of CHA on the STAT signaling pathways in macrophages.

Ana-1 (A) and RAW264.7 (B) cells were treated with lipopolysaccharide (LPS) (10 ng/ml) and interferon (IFN)-γ (20 ng/ml) with or without different concentrations of CHA for 24 h. The expressions of Ser727-phosphorylated STAT1 (p-STAT1ser727), Tyr701-phosphorylated STAT1 (p-STAT1Tyr701) and STAT1were evaluated by a Western blot analysis. Ana-1 (C) and RAW264.7 (D) cells were treated with interleukin (IL)-4 (20 ng/ml) in the presence of DMSO or indicated concentrations of CHA for 24 h. The expressions of Tyr641-phosphorylated STAT6 (p-STAT6Tyr641) and STAT6 were evaluated by a Western blot analysis. The histogram bars represent three independent experiments. The data are presented as the mean ± SD. #p-value < 0.05, ##p-value < 0.01 vs. Con. *p-value < 0.05, **p-value < 0.01 vs. LPS/IFNγ or IL-4.

CHA inhibited the tumor cell growth by affecting the interactions between macrophages and cancer cells

     Considering that the highest concentration of CHA used in macrophages polarization did not directly affect the proliferation of tumor cell lines and rodent nerve cells including primary neurons and glial cell lines (CTX and BV2, etc) that could serve as origin of gliomas (Supplementary Fig. 1A–C), we examined whether CHA regulates the interactions between macrophages and brain cancer cells. The proliferation and morphological feature of U87 cells were almost unaffected by treatment with indicated concentrations of CHA, IFNγ/LPS (M1 inducer), or IL-4 (M2 inducer) alone (Supplementary Fig. 3). In the condition of co-culture experiments, the proliferation of U87 cells was increased by co-cultured with M2 inducer-treated macrophages cell lines (RAW264.7 and Ana-1), and this pro-tumor function of the M2 phenotypic macrophages was significantly suppressed by CHA treatment (Fig. 4A–F). Furthermore, we found that the growth of U87 cells was significantly decreased in co-cultured with M1 inducer-treated macrophages (P ≤ 0.05), and the anti-tumor effect of CHA under the assay conditions used was performance in the induction of U87 cells to be as an apoptotic-like shape (Fig. 4A–F). A similar phenomenon was observed when murine forestomach cancer cell MFC-GFP was co-cultured with M1/M2-inducer stimulated macrophages, treatment with 1 μM or 5 μM CHA also decreased the stimulatory role of M2 phenotypic macrophage on tumor growth (Fig. 4G–J). These data indicated that CHA exhibited antitumor effect through intervention of the cell-cell interactions between macrophages and cancer cells.

Figure 4: Effect of CHA on tumor cells growth in co-culture of tumor cells with macrophages.Tumor cells (1 × 103) were mono-cultured in lipopolysaccharide (LPS) (10 ng/ml) and interferon (IFN)-γ (20 ng/ml) (M1 stimulator) or interleukin (IL)-4 (20 ng/ml) (M2 stimulator) alone, or in combination with indicated concentration of CHA for 48 h, or were co-cultured with M1 stimulator or M2 stimulator-treated macrophages in the presence of CHA for 48 h. U87-RFP-Luc glioma cells were co-cultured with RAW264.7 (A) or Ana-1 (D) cells, and then tumor cellular morphology was visualized by fluorescence microscopy. The proliferation of U87-RFP-Luc cell was represented as fluorescent area (B,E) or overall photon counts of cells (C,F) by using software of ImageProPlus and EnSpire Multimode Plate Reader, respectively. MFC-GFP forestomach cancer cells were co-cultured with RAW264.7 (G) or Ana-1 (I) cells. The morphology of MFC-GFP cells were visualized by fluorescence microscopy and the proliferation of MFC-GFP cells were assessed by overall fluorescent area of cells using ImageProPlus software (H,J). The histogram bars represent three independent experiments. The data are presented as the mean ± SD. *p-value < 0.05, **p-value < 0.01; as evaluated using Student’s t-test.

CHA inhibited glioma growth in murine model

     We next evaluated the anti-cancer effect of CHA using an in vivo xenograft G422 glioma murine model. As shown in Fig. 5A,B, CHA was able to inhibit the tumor growth of G422-bearing mice in a dose-dependent manner without loss of body weight. Tumor inhibition rates were 30% and 48% in the groups treated with 20 mg/kg and 40 mg/kg CHA, respectively (P ≤ 0.05). In addition, flow cytometry analysis was performed to investigate the effects of different phenotypic macrophages infiltrating in tumor tissue after the administration of CHA. Triple immune-staining of CD11c, MHC-II IA/IE and F4/80 results showed that CHA treated G422-bearing mice contained more CD11c+ and MHC-II IA/IE+ double positive cells in F4/80+ macrophages infiltrating in tumor tissue than the control group (Fig. 5C,D). Co-immunostaining of CD206, CD11c and F4/80 showed that the percentages of macrophages expressed CD206 in tumor tissue of CHA-treated mice were fewer than the control group (26.6% in 20 mg/kg group; 8.33% in 40 mg/kg group Vs 63.8% in control group) (Fig. 5E,F). Furthermore, the results from immunofluorescence staining further confirmed the above findings, M1-type macrophages that showed positive staining with antibodies to CD11c and F4/80 were increased in tumor sections from CHA-treated mice (Fig. 5G,I), while M2 macrophages infiltrating into tumor sections that immunostaining with CD206 and F4/80 were significantly reduced by CHA treatment (P ≤ 0.05, P ≤ 0.01) (Fig. 5H,J). Taken together, these results indicated that treatment with CHA significantly reduced the infiltrating M2-type macrophages but increased the M1-type macrophages accumulation in tumor tissue from G422-bearing mice. In addition, in G422 orthotopic glioma model, we found that the slices of intracranial tumor and tumor volume visualized in magnetic resonance imaging (MRI) were decreased after CHA administration (Supplementary Fig. 4A,B).

Figure 5: Effect of CHA on glioblastoma progression in G422 xenograft model.