| BackgroundHepatocellular carcinoma (HCC) is one of the most common cancers worldwide that accounts for half a million deaths per year. Despite the new progress in diagnosis and treatment, HCC is still characterized by high incidence of metastasis and frequent recurrence. Recent studies have highlighted the important roles of tumor microenvironments in the progression, invasion and metastasis of HCC.Tumor associated macrophages (TAMs) constitute a major component in the tumor microenvironment and contribute greatly to the HCC growth and metastasis. Macrophages are highly versatile and multifunctional cells. At least two states of macrophage activation have been identified:the classically activated (M1) macrophages and the alternatively activated (M2) macrophages. M1macrophages that are characterized by high production of pro-inflammatory molecules (like IL-12, TNF-α, iNOS), promotion of Th1response, powerful microbicidal and tumoricidal activity, M2macrophages produce high amounts of IL-10, express scavenger receptors, mannose receptors (or CD206), IL-6and arginase1(Arg-1), and exhibit anti-inflammatory, tissue remodeling and tumor progression functions.Recent studies have indicated that tumors "educate" monocytes as they are recruited to exhibit an M2-like phenotype by producing cytokines, such as IL-10, transforming growth factor (TGF)-β and others. The upregulation of M2-associated genes in TAMs in HCC have been recently confirmed by gene expression profile. Clinical studies have shown that high number of TAMs correlate with angiogenesis, metastasis, and poor prognosis of many cancers including HCC. Indeed, specific macrophage-targeted therapies are now taking the first step into clinical arena. Therefore, identifying molecules supporting the activation and the protumoral function of TAMs are of great importance.T cell immunoglobulin and mucin-domain containing protein-3(Tim-3) was initially discovered as a marker of differentiated IFN-y-producing Thl and Tel cells. As an immunosuppressive molecule, Tim-3has been shown to promote immunological tolerance by inhibition on T cell-mediated responses. Dysregulated Tim-3expression has been identified in many clinical diseases and blockage of Tim-3rescued the exhausted T cells in chronic virus infection. A strong correlation between Tim-3expression and immunosuppression in TILs has been recently confirmed in tumors. Blockade of the Tim-3pathway enhanced T cells mediated antitumor immunity in HCC. Abundant Tim-3expression has been demonstrated in macrophage. Previous studies suggested that Tim-3regulates macrophages in different ways under distinct stimulations. Tim-3negatively regulate macrophage activation in response to TLR-4signaling, while on the other hand, Tim-3induce activation of macrophages and facilitate the production of H2O2, NO for the clearance of invaded pathogens during pregnancy. However, the roles of Tim-3in the activation and proneoplastic role of TAMs promoted by tumor-derived signals remain completely unknown. Our previous data show that Tim-3is upregulated on monocytes/macrophages related to the HCC. In this study, we found that tumor microenvironment promote the expression of Tim-3on TAMs which in turn foster HCC progression.Methods and Results1. Tim-3expression is significantly increased on TAMs of HCC patients and significantly correlates with the poor suvival of HCC pateintsTo evaluate the role of Tim-3in TAMs in HCC, we first analyzed the expression of Tim-3on macrophages from tumor and nontumor tissues. IHC double stain and confucal analysis were performed to analyze Tim-3expression in TAMs in a HCC tissue array. In order to estimate the correlation of Tim-3expression in TAMs with the patients’survival,69pairs of tissue were divided into two groups according to the density and intensity of Tim-3staining. The survive curve was drawed according to Tim-3score and patients lifetime. Immunofluorescent and double immunohistochemical staining confirmed the co-localization of Tim-3and CD68(stand for macrophages) in HCC tissues. Confocal analysis demonstrated significantly increased Tim-3+CD68+cells in tumor sections compared with paratumor sections(p=0.0220). More interestingly, the Tim-3expression on CD68+macrophages analyzed by both immunofluorescent and double immunohistochemical staining was significantly correlated with the poor survival of HCC patients(p＜0.001). All these suggested that Tim-3expression is selectively upregulated on macrophages infiltrating in HCC that might promote the progress of diseases.2. TGF-β in tumor microenvironment promotes Tim-3expression on macrophages and facilitates M2-like phenotypeAccumulated evidence has shown that cancer cells "educate" macrophages in the tumor tissues. To investigate whether tumor microenvironment in liver cancer induce Tim-3expression on macrophages, we prepared HCC conditioned medium (HCM) by culturing mice HCC cell line H22in serum free DMEM for24hours. Peritoneal macrophages (PMs) isolated from BALB/c mice were treated with HCM at indicated dose and different time period. HCM increased Tim-3expression on peritoneal macrophages in a HCM-dose and-time dependent manner. Consistent with the M2-like phenotype of TAM in HCC, HCM promoted Tim-3expression as well as the macrophages alternative activation. After stimulation with HCM, macrophages displayed enhanced expression of M2markers CD206and Arg-1. Both the expression of CD206and Arg-1showed the similar dynamic change to Tim-3expression in HCM-treated macrophages. In contrast, HCM decreased the expression of M1marker, TNF-α. Similar results were got from peritoneal macrophages stimulated with HCM derived from another murine liver cancer cell line Hepal-6. All these data suggest that tumor microenvironment promote Tim-3expression on macrophages and facilitated M2-like phenotype.The tumor microenvironment induced Tim-3expression was further confirmed by in vivo assay with BALB/c mice bearing with hepatoma cells H22. The percentage of CDllb+Tim-3+cells isolated from tumors were much higher than that from spleens (n=10, p＜0.0001). More importantly, compared with healthy serum, serum isolated from HCC patients greatly enhanced Tim-3expression and CD206expression on human monocyte cell line THP-1cells.It has been well known that TGF-β is upregulated in patients with HCC and plays a tumor-promoting role in liver cancer. Facilitating the alternative activation of macrophages has been identified as one of the critical biological roles for TGF-β. Most hepatocarcinoma cells are able to constitutively synthesize and secrete TGF-β. We therefore hypothesized that TGF-P might be responsible for the HCM meidated up-regulation of Tim-3on TAMs. To test this hypothesis, peritoneal macrophages were preincubated with the TGF-P receptor inhibitor (SB431542) before stimulation with HCM. SB431542significantly decreased the number of Tim-3positive macrophages promoted by HCM. Consistently, exogenous TGF-P significantly increased Tim-3expression in cultured PMs. Western blot results also confirmed the critical roles of TGF-P in the augmenting Tim-3expression on the macrophages promoted by tumor microenvironment. In addition, preincubation of HCM with TGF-P neutralizing antibody decreased the Tim-3production in peritoneal macrophages. Moreover, results of luciferase reporter assay showed that TGF-β enhanced the activity of Tim-3promoter both in HCC cell line Huh7and mouse macrophage cell line RAW264.7in a dose dependant manner, indicating that TGF-β increase the transcription of Tim-3. This is in line with the previous report in mast cells.In line with the decreased expression of Tim-3in macrophages, phenotype analysis showed that SB431542significantly repressed HCM-induced M2activation (downregulated CD206expression, Arg-1expression, IL-10secretion but upregulated IL-12production and TNF-a expression). Collectively, these results suggest that TGF-P in the HCC tumor microenviroment promotes the Tim-3expression and M2polarization of macrophages. Consistently, preincubation of THP-1cells with SB431542greatly inhibited Tim-3and CD206expression promoted by serum from HCC patients.3. Interference of Tim-3reverses TGF-β mediated M2-like phenotypes of macrophagesSince the high consistence of Tim-3expression with M2phenotypes of macrophages, we subsequently explored the role of Tim-3in macrophage alternative activation. We firstly compared Tim-3expression level in M1and M2macrophages. Both bone marrow derived macrophages (BMDMs) and peritoneal macrophages were induced into M1or M2differentiation by the well-established approaches described in the methods. Our results showed that although Tim-3is consistently expressed in macrophages, Tim-3expression in M2was significantly higher than that in Ml macrophages. Polarization of M1and M2from BMDMs and peritoneal macrophages was further confirmed by phenotypic analysis including CD206, IL-10, IL-12, Arg-1, iNOS and TNF-a.To evaluate the role of Tim-3in HCM or TGF-β-induced M2-like phenotypes, we interfered Tim-3pathway with three different approaches:Tim-3blocking antibody (anti-Tim-3), Tim-3siRNA (siTim-3) and Tim-3shRNA expressing lentivirus (shTim-3-Lv). The peritoneal macrophages freshly isoloated form BALB/c were knockdown for Tim-3followed by stimulation with HCM or TGF-β. We demonstrated that reduction of Tim-3expression in macrophages significantly weakened IL-10secretion and enhanced IL-12secretion caused by TGF-0and HCM. Similar results were obtained from flow cytometry analysis of IL-10and IL-12intracellular staining in HCM treated PMs. RT-PCR data further verified the HCM induced macrophages alternative activation (Arg-1high and TNF-α low). Moreover, knockdown of Tim-3reversed the HCM induced M2phenotype of macrophages identified as decreased Arg-1expression and increased TNF-a production. In accordance, Tim-3konckdown increased the activation of STAT1which is crucial for M1activation, but decreased the activation of STAT6, one of the critical transcription factor for M2activation. These data together support the idea that Tim-3is crucial for tumor induced macrophage alternative activation.4. Tim-3expressed in the macrophages promotes HCC progression both in vitro and in vivoIt has been well documented that M2-like TAMs promote the HCC progression and metastasis. We therefore explored the role of Tim-3in the protumoral effects of alternative activated macrophages. Murine hepatoma cell line Hepal-6cells were cultured with supernatants of M2macrophages infected with shTim-3-Lv or control lentivirus. Tim-3knockdown in M2macrophages significantly reduced M2-promoted cell growth and colony formation of Hepal-6. Transwell assays further demonstrated that interference of Tim-3in the alternative activated macrophages greatly inhibited both the migration and invasion of Hepal-6cells.To further evaluate the role of augmented Tim-3expressed by alternative activated macrophages in the HCC progression in vivo, BALB/c mice were subcutaneous injected with H22cells together with Tim-3-knockdown M2cells derived from BMDMs (BALB/c) at the ration of1:4. Compared to control M2cells, shTim-3-Lv infected M2cells significantly inhibited the growth of H22homograft in BALB/c mice. Consistently, Ki67immunochemical staining in the shTim-3-Lv treated tumor section was significantly less than that from control mice, supporting that Tim-3expressed on M2promote the proliferation of HCC cells. Taken together, our data suggests that Tim-3expressed in the TAMs promotes HCC growth both in vitro and in5. Tim-3promotes IL-6production of macrophages via NF-κB pathwayGiven that the macrophages perform their functions mostly by secreting cytokines, Cytometric Bead Array (CBA) was involved to compare the secretion of cytokines in the supernatant of M2macrophages derived from BMDMs which had been infected with shTim-3-Lv and control lentivirus. As expected, the knockdown of Tim-3significantly increased IL-12but decreased IL-10production. Moreover, Tim-3significantly enhanced the production of IL-6and GM-CSF which have been reported as the major protumoral cytokines produced by TAMs. However, IL-1(3secretion was not affected by Tim-3interference.NF-κB is a crucial transcription factor contributing to many biological and pathological processes including cancer-related inflammation and malignant progression. Recent studies have highlighted the key role of NF-κB in maintaining the immunosuppressive phenotype of TAMs. We therefore aim to investigate the activation of NF-κB in Tim-3-silenced M2cells. As expected, the phosphorylated p65which is the functional subunit of NF-κB was down-regulated by shTim-3-Lv in macrophages no matter whether macrophages were alternatively activated by IL-4or TGF-β.Suppression of NF-κB activity with specific inhibitor restored the inhibitory effects of shTim-3-Lv on IL-6production in IL-4or TGF-β stimulated macrophages, supporting that NF-κB plays a key role in Tim-3-promoted IL-6production of M2like macrophages. Significantly, neutralization of IL-6with its blocking antibody greatly reversed the cell growth suppression in Hepal-6cells mediated by shTim-3-Lv infected M2like macrophage. Taken together, our results here suggest that Tim-3promotes IL-6production by activating NF-κB and therefore enhances liver cancer cell growth.Conclusion:our work suggests that Tim-3is upregulated on TAMs and indicates poor prognosis; TGF-β in liver tumor microenvironment enhances the transcription of Tim-3in TAMs which in turn promotes the alternative activation of macrophages; Tim-3on macrophages activated TAMs produce high level of inflammatory cytokines foster the HCC growth, including IL-6via NF-κB pathway.Innovation and significance:Our finding here highlight the potential role of Tim-3in the alternative activation and protumoral functions of TAMs in HCC and provide a new target aiming tumor associated macrophages in HCC therapy. |
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