The Roles And Mechanisms Of Tumor-associated Macrophages In Promoting Motility Of HCC Cells

ObjectiveHepatocellular carcinoma(HCC) is a typical inflammation-related tumor, in which the existence of chronic inflammation promotes the origin and development of tumor. TAM is a prominent component of inflammatory microenvironment in tumor. TAMs infiltration is associated with metastasis and recurrence in HCC.TAMs are heterogeneous and exhibit different phenotypes according the various kinds of cell signals in tumor tissue. Two distinct polarization states have been described for macrophages:M1and M2macrophages. It is traditionally believed that M1macrophages play a role in promoting the development of tumors while M2macrophages have a role of anti-tumor. And previous studies have revealed that TAMs incline to express an M2-like phenotype in most established malignant tumors. However, Both M1-like (CD68+HLA-DR+) and M2-like (CD68+HLA-DR-) macrophages are found in HCC tissues. So what’s the function and mechanism of M1-like macrophages in HCC tissues? There is not a widely acknowledged conclusion yet.TIPE2is one of the members of the tumor necrosis factor-α-induced protein-8family. It exerts important functions in maintaining immune homeostasis. The deletion of TIPE2in mice leads to severe multi-organ inflammation and the abnormal expression of TEPE2in human is associated with systemic autoimmunity. More and more evidences indicate that TIPE2play an important role in the progress of tumor. For example, TIPE2is downregulated in human primary HCC compared with the paired adjacent non-tumor tissues. As a newly described immune negative regulator, could TIPE2inhibit the cell metastasis promoted by macrophages in HCC? Something further should be done to explore it.By analyzing the relationship between CD68+HLA-DR+M1-like macrophages and cell metastasis in HCC tissues and inducing THP-1cells to differentiate into M1-polarized macrophages in vitro, the supernatant of which was collected to treat HCC cells, we demonstrate that M1-like macrophages could accelerate the process of metastasis in HCC. Further more, by blocking the NF-κB pathway in HCC cells we can come to a conclusion that the M1-like macrophages may promote cell metastasis through NF-κB/FAK pathways. What’s more, in our experiments, HCC cells which highly express TIPE2were cocultured with THP-1-derived macrophages or treated by TNF-a. According to the results, we demonstrate that TIPE2may inhibit the cell metastasis promoted by tumor-related inflammation by blocking the FAK pathways in HCC.Methods1Detection of the ratio of CD68+HLA-DR+macrophages in HCC tissues by immunohistochemistry(1) HCC clinical specimensSeventy-five cases of HCC specimens were obtained from Shanghai Outdo Biotech Company. All the patients gave their written informed consent. The medical ethical committee of Shandong University approved this study. All the cases consist of20cases with metastasis (vascular invasion, portal vein tumor thrombosis or distant metastasis) and55cases without metastasis.(2) Immunohistochemistry for CD68and HLA-DRImmunohistochemisty for CD68and HLA-DR was performed on a4mm thick section with a double-staining kit. Briefly, paraffin sections were dewaxed and hydrated before heat-mediated antigen retrieval in EDTA buffer. The endogenous peroxidase was blocked by incubation with3%H2O2, and nonspecific binding sites were blocked with1%BSA. After incubation with the primary antibodies (mouse anti-human CD68monoclonal antibody,1:400dilution; rabbit anti-human HLA-DR monoclonal antibody,1:200dilution) at4℃overnight, then the secondary antibodies (goat anti-mouse IgG antibody conjugated to horseradish peroxidase and goat anti-rabbit IgG antibody conjugated to alkaline phosphatase) at room temperature for1h, the sections were developed in diaminobenzidine solution and AP-red solution. The section was then counterstained with hematoxylin and mounted in an aqueous mounting medium. Negative controls were performed by omitting the primary antibodies.(3) Analysis of the correlation The CD68+TAM and CD68+HLA-DR+M1-like TAM were counted manually at high-power field (magnification of x400) by microscopy. The number of TAM or M1-like TAM in each sample was determined by averaging the number of TAM or M1-like TAM in3high-power fields where the most density of CD68staining was observed. The data was analyzed with software GraphPad Prism5.0.2Macrophage polarization and preparation of conditional medium(1) The polarization of macrophages in vitroHuman monocytic cell line THP-1was obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science. Cells were maintained in RPMI1640supplemented with10%heat-inactivated fetal bovine serum at37℃C with5%CO2.To generate M1-polarized macrophages, THP-1cells were treated with LPS and IFN-y. And to generate M2-polarized macrophages, THP-1cells were treated with IL-4and IL-13. The M1and M2macrophages were collected and their RNA or protein was extracted for their phenotype analysis by RT-PCR or Westein blot assay.(2) Preparation of conditional mediumIn order to prepare the conditioned medium, THP-1cells (in a six-well plate,5X105cells per well) were treated with LPS and IFN-y. After a thorough wash to remove all PMA, LPS and IFN-y, the M1-polarized macrophages derived from the THP-1cells were further cultured in RPMI1640medium for24h. The supernatant was collected and filtered to be used as conditional medium in the following experiments.3The changes of migration ability and related pathways of HCC cells(1) Detection of migration ability of HCC cells by transwell cell migration assayHuman hepatocellular carcinoma cell lines SMMC-7721and HepG2were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science. All the cells were maintained in RPMI1640supplemented with10%heat-inactivated fetal bovine serum at37℃with5%CO2HCC cells were seeded at the concentration of1×105per well into the upper chamber of a transwell apparatus with an8um pore size membrane in regular DMEM medium (RM) or conditioned medium (CM) derived from M1macrophages. After culture of24h, cells that had not penetrated the filter were wiped out with cotton swabs, and cells that had migrated to the lower surface of the filter were stained with crystal violet, examined by microscopy, and photographed. Values for migration were expressed as the average number of migrated cells per microscopic field (X100) over five fields.(2) Expression of related genes in HCC by Western-blot methodsAs for analysis of cell signaling, HCC cells were seeded into six-well plates. After that, the HCC cells were incubated in CM for12h or24h. The cells cultured in plate were harvest for Western-blot assay.4Transwell cell migration assay after blockade of NF-kB signaling pathways(1) Blockade of NF-KB signaling pathways with Bay11-7082HCC cells were pretreated with Bay11-7082at20μM for1h in RM before they were seeded at the concentration of1X105per well into the upper chamber of the transwell apparatus in conditional medium (CM) containing20μM of Bay11-7082for24h. Then the HCC cells cultured in the transwells were further analyzed for their migration.(2) Blockade of NF-KB signaling pathways by transfected P65-SiRNAThe P65-SiRNA or NC SiRNA was transfected into HCC cells with lipofectamineTM2000Transfection Reagent. The HCC cells at the concentration of1×105per well were subjected to transwell migration assay24h after transfection.5Western-blot assay after blockade of NF-KB signaling pathways (1) Blockade of NF-KB signaling pathways with Bay11-7082As for analysis of cell signaling, HCC cells were seeded into six-well plates and pretreated with Bay11-7082at20μM for1h in RM. After that, the HCC cells were incubated in CM containing20uM of Bay11-7082for12h or24h. The cells cultured in plates were harvest for Western-blot assay.(2) Blockade of NF-KB signaling pathways by transfected P65-SiRNAThe P65-SiRNA or NC SiRNA was transfected into HCC cells with lipofectamineTM2000Transfection Reagent. After that, the HCC cells were incubated in CM for12h or24h. Then, all the cells were harvested for Western blot assay.6Detection of cell migration ability and change of cell pathways in HCC cells which highly express TIPE2and cocultured with THPl-derived macrophages(1) Coculture of HCC cells with THP-1-derived macrophagesHepatocellular carcinoma cell line SMMC-772、HuH-7and human monocytic cell line THP-1were obtained from the Cell Bank of Type Culture Collection of Chinese Academy of Science. Cells were maintained in RPMI1640or DMEM supplemented with10%FBS at37℃with5%CO2.Our experiments include two groups:the control group and the coculture group. HCC cells were transfected with TIPE2plasmid or pRK-5plasmid, respectively. And THP-1cells treated with320nM PMA were seeded into the upper chamber of a transwell apparatus with an0.4um pore size membrane. Next day, after washing chambers to wipe out PMA, THP-1cells were cocultured with HCC cells. HCC cells which were transfected with TIPE2plasmid or pRK-5plasmid and not cocultured with THP-1cells were classified into control group.(2) Detection of cell migration ability by transwell migration assayAfter cocultured for24h, HCC cells were seeded at the concentration of1×105per well into the upper chamber of a transwell apparatus with an8um pore size membrane. After24h, cells that had migrated to the lower surface of the filter were stained with crystal violet, examined by microscopy, and photographed. Values for migration were expressed as the average number of migrated cells permicroscopic field over five fields.(3) Detection of the change of cell pathways by western blot assayAfter cocultured for24h, the transwell chambers were abandoned and total protein was extracted from HCC cells to detect the change of P-FAK576and P-FAK861by Western-blot assay.7Detection of cell migration ability and change of cell pathways in HCC cells which highly express TIPE2(1) HCC cells which highly express TIPE2were treated with TNF-aOur experiments were devided into two groups:the TNF-a-treated group and the control group. HCC cells were transfected with TIPE2plasmid or pRK-5plasmid, respectively. After24h, TNF-a was added into the supernatant of HCC cells. HCC cells which were transfected with TIPE2plasmid or pRK-5plasmid and not treated with TNF-a were classified into control group.(2) Detection of cell migration ability by transwell migration assayAfter treated by TNF-a for24h, HCC cells were were seeded at the concentration of1×105per well into the upper chamber of a transwell apparatus with an8um pore size membrane. After24h, cells that had migrated to the lower surface of the filter were stained with crystal violet, examined by microscopy, and photographed. Values for migration were expressed as the average number of migrated cells per microscopic field over five fields.(3) Detection of the change of cell pathways by western blot assayAfter treated with TNF-a for24h, HCC cells were collected and total protein was extracted to detect the change of P-FAK576and P-FAK861by Western-blot assay.Results1Infiltration of CD68+HLA-DR+macrophages was associated with metastasis in HCCThe infiltration of M1-like TAM in HCC tissues was evaluated by examination of co-expression of CD68and HLA-DR. Among all HCC specimens, the expression of CD68was shown in cell membrane and cytoplasm and developed into a brown color. The expression of HLA-DR was shown in cell membrane and cytoplasm and developed into a red color. The M1-like macrophages were shown in mixed color of brown and red because they expressed both CD68and HLA-DR.All the HCC cases were classified into group with metastasis and group without metastasis. In the metastasis group, the average number of TAM was57per high-power field and the average number of Ml-like TAM was45per high-power field. In the group without metastasis, the average number of TAM was43per high-power field and the average number of M1-like TAM was30per high-power field. The average number of TAM or Ml-like TAM was significantly different between the group with metastasis and group without metastasis (P<0.05). These results clearly revealed a significant correlation between TAM infiltration and metastasis in HCC.2Polarization of macrophages derived from THP-1cell lineWe generated M1-like or M2-like macrophages with IFN-y and LPS or IL-4and IL-13, respectively. Then related genes were analyzed by RT-PCR and Western-blot methods. Compared to M2macrophages, the Ml macrophages expressed high HLA-DR, IL-6and TNF-a.3Ml-like macrophages promoted motility of HCC cellsThe results of transwell cell migration assay show that cells in the CM group showed a significant increase in the number of migrating cells compared to the RM group. It is indicated that the CM from the M1macrophages enhanced the migratory abilities of HCC cells.4M1-like macrophages promoted the motility of HCC cells via activation of NF-κB/FAK pathwayHCC cells (SMMC-7721and HepG2) were subject to Western-blot assay after12h or24h of culture in RM or CM derived from M1macrophage. The CM induced upregulated expression of phosphorylated P-65, indicating the activation of NF-κB signaling in HCC cells. The CM also activated FAK by upregulating expression of phosphorylated FAK compared to the RM. Enhanced activation of FAK is a hallmark of cell migration.5The change of HCC cell migration after blockade of NF-κB signaling pathwaysIn cell migration assay, the number of cells migrating to lower surface of the transwell membrane in CM was significantly greater than that in RM. However, the number of migrating cells in CM containing Bay11-7802was significantly less than that in CM. Technique of siRNA was used to confirm the above results further. In the transwell migration assay, the number of HCC cells transfected with NC siRNA migrating to lower surface of the Transwell membrane was significantly greater in CM than that in RM. However, the number of HCC cells transfected with P65siRNA migrating into membrane surface of the Transwell was significantly less than that of the HCC cells transfected with NC siRNA in CM. The experiment was repeated twice. The results indicate that the enhanced migration of HCC cells by M1-like macrophages was abrogated by transient silencing P65gene.6The change of cell pathways after blockade of NF-κBThe activation of NF-κB and FAK signaling in HCC cells cultured in RM, CM or CM containing Bay11-7802for12h or24h was analyzed by Western-blot assay. The phosphor-P65and phosphor-FAK was increased in the HCC cells cultured in CM compared to RM. By contrast, the phosphorylation of P-65and FAK was decreased in the HCC cells cultured in CM containing Bay11-7802compared to in CM.In order to confirm the above results further, P65siRNA was used to block the NF-κB signaling pathway. The phosphorylation of P-65and FAK in the HCC cells transfected with NC siRNA was increased in CM compared to that in RM. By contrast, the phosphorylation of P-65and FAK in the HCC cells transfected with P65siRNA was decreased compared to that in the HCC cells transfected with NC siRNA in CM.7TIPE2inhibited cell metastasis and activation of FAK pathway promoted by THP-1-derived macrophages in HCC.(1) The effection of TIPE2on migration ability of control group cellsFrom the results of transwell migration assay, we can see that both HCC cells transfected with pRK-5plasmid and cells transfected with TIPE2plasmid in control group have a low migration ability, and there is no obvious relation between two groups (P>0.05)(2) The effection of TIPE2on migration ability of coculture group cellsIn cell migration assay, the number of migrating cells which transfected with pRK-5plasmid in coculture group was significantly greater than that in control group. The results indicated that macrophages could increase the migration ability of HCC cells. However, the number of HCC cells transfected with TIPE2plasmid migrating into membrane surface of the transwell was significantly less than that of the HCC cells transfected with pRK-5plasmid in coculture group. These results indicated that the enhanced migration of HCC cells promoted by macrophages was abrogated by TIPE2gene.(3) The effection of TIPE2on the activation of FAK pathway in control group cellsThe activation of FAK signaling of HCC cells was analyzed by Western blot assay. The expression of P-FAK576and P-FAK861is low in both HCC cells transfected with TIPE2plasmid and cells transfected with pRK-5plasmid in control group, and there is no obvious between two groups (P>0.05)(4) The effection of TIPE2on the activation of FAK pathway in coculture group cellsThe expression of P-FAK576and P-FAK861was increased in the HCC cells which transfected with pRK-5plasmid in coculture group compared to cells in control group. The results indicated that macrophages could activate the FAK pathways in HCC cells. By contrast, the expression of P-FAK576and P-FAK861was decreased in HCC cells transfected with TIPE2plasmid compared to that in the HCC cells transfected with pRK-5plasmid in coculture group. These results indicated that the the activaton of FAK pathways in HCC cells promoted by macrophages was abrogated by TIPE2gene.8TIPE2inhibited cell metastasis and activation of FAK pathway promoted by TNF-a in HCC.(1) The effection of TIPE2on migration ability of control group cellsIn order to confirm the above results further, TNF-a was used to examine the effects of TIPE2on HCC cells. Our experiments were classified into TNF-a-treated group and control group. In cell migration assay, both HCC cells transfected with pRK-5plasmid and cells transfected with TIPE2plasmid in control group have a low migration ability, and there is no obvious relation between two groups (P>0.05)(2) The effection of TIPE2on migration ability of cells treated with TNF-αIn the transwell migration assay, the number of HCC cells transfected with pRK-5plasmid migrating to lower surface of the transwell membrane was significantly greater in TNF-α-treated group than that in control group. The results indicate that TNF-α could play a role like macrophages and increase the migration ability of HCC cells. However, the number of HCC cells transfected with TIPE2plasmid migrating into membrane surface of the transwell was significantly less than that of the HCC cells transfected with pRK-5plasmid in TNF-α-treated group. These results indicate that the enhanced migration of HCC cells by TNF-awas abrogated by TIPE2gene.(3) The effection of TIPE2on the activation of FAK pathway in control group cellsThe activation of FAK signaling of HCC cells was analyzed by Western-blot assay. The expression of P-FAK576and P-FAK861is low in both HCC cells transfected with TIPE2plasmid and cells transfected with pRK-5plasmid in control group, and there is no obvious relation between two groups (P>0.05)(4) The effection of TIPE2on the activation of FAK pathway in TNF-α-treated group cellsThe expression of P-FAK576and P-FAK861was increased in the HCC cells which transfected with pRK-5plasmid in TNF-α-treated group compared to cells in control group. The results indicated that TNF-α could activate the FAK pathways in HCC cells. By contrast, the expression of P-FAK576and P-FAK861was decreased in HCC cells transfected with TIPE2plasmid compared to that in the HCC cells transfected with pRK-5plasmid in TNF-α-treated group. These results indicate that the the activaton of FAK pathways in HCC cells promoted by TNF-α was abrogated by TIPE2gene.Conclusion1M1-like macrophages may increase cell metastasis through NF-κB/FAK pathway in HCC. 2TIPE2inhibits cell metastasis promoted by macrophages or TNF-a through blocking the FAK pathways in HCC.