The Role Of Wnt2b In Polarization Of HCC-Associated Macrophages And Related Mechanisms

Object:Liver is an organ with immunological properties.The occurrence and development of hepatocellular carcinoma(HCC)is associated with the immunosuppressive tumor microenvironment(TME)in the liver.In addition to tumor cells,the TME mainly consists of tumor-associated stromal cells and non-cellular components such as cytokines and growth factors.Macrophages,a type of tumor-associated stromal cell,are a major component of most solid tumors,including HCC.These infiltrated macrophages are known as tumor-associated macrophages(TAMs),which are mainly differentiated from peripheral circulating monocytes rather than the expansion of resident macrophages in tumor tissue.TAMs can interact with HCC cells to promote the proliferation,metastasis,stemness formation,drug resistance and angiogenesis of HCC,and inhibit adaptive immunity,suggestting that targeting TAMs may be a potential therapeutic strategy for HCC.It has been reported that,TAMs with tumor killing effect show the similar phenotypic characteristics with M1-type macrophages,while TAMs with tumor promoting effect show the similar phenotypic characteristics with M2-type macrophages.Ample evidences indicate that TAMs are primarily M2 polarized which can be induced by tumor-derived signals.Various soluble factors(IL-10,TGF-?,IL-4,etc)in the TME play an important role in the polarization and functional regulation of TAMs.As an important member of pattern recognition receptor,the toll-like receptors(TLRs)are mainly expressed in macrophages and other innate immune cells.Current studies have also found that some TLR agonists can reverse M2 polarization of macrophages when used alone or in combination with other drugs,indicating the role of TLR agonists in regulating the polarization of TAMs.As a secreted protein,Wnt ligands mediate a highly conserved signaling pathway during biological evolution,playing an essential role in many physiological and pathological processes,especially in the development of tumors.Aberrant activation of the Wnt/?-catenin signalling pathway has been observed in HCC patients,which is closely related to the occurrence and development of HCC,the formation of stemness and drug resistance.In addition to the effect on tumor cells themselves,Wnt signaling has recently been found to play an important role in the formation of a tumor immunosuppressive microenvironment by mediating cell interactions.As an important member of the Wnt family,the expression of Wnt2b in tumor cells has been found to promote the proliferation and stemness formation and inhibit the apoptosis of HCC cells in our previous study.However,the role of Wnt2b and it mediated signal activation in macrophage polarization in the TME,especially in the HCC microenvironment,is still unclear.In recent years,reprograming of cell metabolism has been considered as an important mechanism in the development of malignant tumors.In addition to tumor cells,a variety of immune cells in the microenvironment also undergo metabolic reprograming during the tumor process,which is closely related to the changes in their cellular functions.In the tumor microenvironment,although TAMs have similar phenotypic characteristics to M2-type macrophages,there are differences in their selection of metabolic patterns.In the microenvironment,enhanced glycolysis promotes the occurrence and development of tumors.However,the effect of tumor cells on the metabolic phenotype of TAMs and the regulatory effect of metabolic remodeling on TAMs polarization in HCC microenvironment have not been fully clarified.It has been reported that activation of Wnt signaling is involved in regulating the enhancement of glycolysis in tumor cells.However,the regulatory effect between Wnt and glycolysis in TAMs and its mechanisms have not been described.In this study,we tried to determine the polarized phenotype of HCC-associated macrophages(HCC-TAMs)and the role of HCC-TAMs in promoting the development of EMT in HCC.Furthermore,during this process,we clarified the mechanism of the Wnt2b/?-catenin signalling pathway.Our findings reveal a novel mechanism in the complex regulatory network between HCC cells and macrophages as well as the formation of an immunosuppressive microenvironment in tumor tissues.Methods:1.THP-1-induced macrophages(THP-1-M)were treated with conditional medium(HCC-TCM)from different HCC cell lines to obtain HCC-associated macrophages(HCC-TAMs).Flow cytometry(FCM)and quantitative real-time PCR(qPCR)were used to detect the expression of polarization related markers in/on HCC-TAMs.2.The effects of HCC-TAMs on the proliferation and migration of HCC cells were detected by MTT and scratch assay.3.The expression of Wnt2b in HCC-TAMs was detected by qPCR.4.The expression levels of Wnt2b and CD68+ macrophages were determined by immunofluorescence using tissue microarrays(TMA)containing pairs of tumors and matched para-carcinoma tissues of HCC patients.5.FCM and qPCR were used to detect the effect of over-expressing Wnt2b on the expression of polarization related markers in/on THP-1-M.6.FCM and qPCR were used to detect the effect of silencing the expression of Wnt2b or ?-catenin on the expression of polarization related markers in/on HCC-TAMs.7.Western blotting and immunofluorescence were used to detect the effect of silencing Wnt2b on the expression and nuclear translocation of ?-catenin in HCC-TAMs.8.Western blotting was used to detect the effect of over-expressing Wnt2b in THP-1-M on the epithelial-mesenchymal transformation(EMT)of HCC cells.9.Western blotting was used to detect the effect of silencing Wnt2b or ?-catenin in HCC-TAMs on the EMT of HCC cells.10.MTT and wound healing assay were used to detect the effect of silencing Wnt2b or?-catenin in HCC-TAMs on the proliferation and migration of HCC cells.11.qPCR were used to detect the effect of silencing Wnt2b or ?-catenin on the expression of key glycolytic enzymes in HCC-TAMs.12.The effect of silencing Wnt2b or ?-catenin on extracellular acidification rate(ECAR)in HCC-TAMs were detected by Seahorse analyser.13.The effect of glycolysis inhibitor 2DG treatment on the expression of polarization related markers in HCC-TAMs was detected by qPCR.14.Western blotting was used to detect the effect of 2DG treatment on the EMT-promoting role of HCC-TAMs.15.FCM and qPCR were used to detect the effect of TLR9 agonist CpG ODN treatment on the expression of polarization related markers in/on HCC-TAMs.16.qPCR was used to detect the effect of CpG ODN treatment on the expression of Wnt2b in HCC-TAMs.17.Western blotting and immunofluorescence were used to detect the effect of CpG ODN treatment on the expression and nuclear translocation of ?-catenin in HCC-TAMs.18.Western blotting was used to detect the effect of CpG ODN treatment in HCC-TAMs on the EMT of HCC cells.19.qPCR were used to detect the effect of CpG ODN treatment on the expression of key glycolytic enzymes in HCC-TAMs.20.The effect of CpG ODN treatment on ECAR level in HCC-TAMs was detected by Seahorse analyser.21.qPCR was used to detect the effect of CpG ODN treatment on the expression of c-Myc in HCC-TAMs.22.SMMC-7721 cells with or without the different TAMs were mixed with matrigel and subcutaneously injected into the right subaxillary of immunodeficient mice.The effects of different HCC-TAMs on the growth of subcutaneous tumors and average tumor weight of each group in mice at the time of euthanisation were investigated.The expression of vimentin,E-cadherin,?-catenin,c-Myc in tumor tissues were detected by immunohistochemistry staining.23.Overall survival of HCC patients related to the expression levels of Wnt2b,N-cadherin and Snail was generated by the GEPIA2 database.24.The effect of IL-10 or TGF-? treatment on the expression of Wnt2b,?-catenin and c-myc in THP-1-M was detected by qPCR.Results:1.HCC-TCM can induce M2 polarization of macrophagesTHP-1-M were treated with HCC-TCM and found that TCM derived from different HCC cell lines up-regulated the expression of M1-type macrophage markers CD163,IL-10 and CCR2.Meanwhile,we also found that the mRNA levels of Ml-type macrophage markers IL-12,NOS2,and TNF-? were downregulated.These results indicate that the supernatant secreted by HCC could induce the polarization of macrophages to M2-like TAMs.2.HCC-TCM-induced HCC-TAMs play a tumor-promoting roleIncubating HCC cells with the secretion supernatant of HCC-TAMs above promoted the epithelial-mesenchymal transformation of HCC cells and enhanced the proliferation and migration ability of HCC cells.3.HCC-TCM promotes M2 polarization via Wnt2b/?-catenin signallingTCM from different HCC cell lines could upregulate the expression of Wnt2b in macrophages to different degrees under the condition of M2 polarization of THP-1-M.In addition,fluorescence colocalization staining analysis for tumor tissues of HCC patients also showed that the expression of Wnt2b in macrophages within the tumor tissues was higher than that in adjacent tissues.Western blotting and immunofluorescence results both showed that the expression of ?-catenin in the cytoplasm and nucleus of macrophages were up-regulated after HCC-TCM treatment.Silencing Wnt2b could inhibit this phenomenon.Overexpression of Wnt2b promoted M2-type polarization of macrophages.Silencing Wnt2b by the lentivirus carrying targeted Wnt2b-shRNA could inhibit HCC-TCM-induced M2-type polarization of macrophages.Furthermore,silencing ?-catenin also inhibited HCC-TCM-induced M2-type polarization of macrophages.4.The activation of Wnt2b/?-catenin signalling enhances TAMs-induced tumor-promoting effectsHCC cells were cultured in the presence of culture medium from control or Wnt2b-overexpressed THP-1-M for 48 h and found that the supernant from Wnt2b-overexpressed THP-1-M could up-regulate the expression of mesenchymal marker N-cadherin and Vimentin in HCC cells to varying degrees,and meanwhile up-regulated the expression of EMT-related transcription factors Snailin and Twist.Consistently,compared with the HCC cells treated with the culture medium of control HCC-TAMs,the expression of the mesenchymal markers and the EMT-related transcription factors was downregulated in HCC cells treated with the culture medium of Wnt2b or ?-catenin-silenced HCC-TAMs.In addition,the proliferation and migration of HCC cells were obviously suppressed by the silencing of Wnt2b or ?-catenin.These results suggest that the expression of Wnt2b/?-catenin in macrophages may affect the tumor-promoting effect of HCC-TAMs.5.Wnt2b/?-catenin signaling augments the glycolysis of HCC-TAMsThe expression of key glycolytic enzymes and ECAR level were obviously up-regulated in HCC-TAMs.The expression of key glycolytic enzymes and ECAR level of HCC-TAMs was remarkably decreased by the silencing of Wnt2b or ?-catenin compared with the control group.Next,we further examined the effect of inhibiting macrophage glycolysis on the polarization of HCC-TAMs.Notably,we found that the glycolytic inhibitor 2DG(2-deoxy-D-glucose)upregulated the expression of IL-12 while downregulating the expression of IL-10 in HCC-TAMs,and blocked the ability of HCC-TAMs to induce HCC EMT.These findings indicate the involvement of Wnt2b/?-catenin signalling in glycolysis regulation and the tumor-promoting effects of HCC-TAMs.6.Wnt2b/?-catenin signalling in TCM-induced M2 polarization can be inhibited by the TLR9 agonistThe expression of CD 163,IL-10 and CCR2 on/in TAMs treated with TLR9 agonist CpG ODN was lower than that of TAMs treated with TCM alone.This was accompanied by the upregulation of IL-12,TNF-?,and NOS2.Furthermore,we found that CpG ODN treatment decreased the expression of Wnt2b,the expression and nuclear translocation of ?-catenin,and the expression of Axin-2 in HCC-TAMs.Notably,CpG ODN suppressed the ability of TAMs in promoting HCC EMT.These findings suggest that TLR9 agonists can inhibit the activation of Wnt2b/?-catenin signalling in HCC-TAMs,thereby inhibiting M2 polarization.7.The TLR9 agonist suppresses the glycolysis of HCC-TAMs via c-MycTCM upregulated the expression of key glycolytic enzymes(GLUT1,HK2,PKM2,TPI,LDHA)and ECAR level of macrophages,which was downregulated by CpG OND treatment.The expression of c-Myc in HCC-TAMs was inhibited by either the silencing of Wnt2b expression or by CpG ODN treatment.These results indicate that c-Myc mediates Wnt2b/?-catenin signal-regulated glycolysis of HCC-TAMs,which can be suppressed by TLR9 agonists.8.Inhibition of Wnt2b/?-catenin signaling reduces the tumor-promoting effect of HCC-TAMs in vivoSMMC-7721 cells alone or in combination with different TAMs were inoculated subcutaneously into the right subaxillary of mice to establish the subcutaneous tumor bearing model in immunodeficient mice.We observed that the tumor growth rate was faster in mice inoculated with a mixture of SMMC-7721 cells and TAMs than in mice inoculated with SMMC-7721 cells alone,indicating that TAMs could promote HCC growth in vivo.However,TAMs-induced tumor-promoting effects could be suppressed by CpG ODN pre-treatment.Meanwhile,compared with tumor tissues from the inoculation of SMMC-7721 cells alone,the expression of E-cadherin was significantly downregulated,but the expression of vimentin,?-catenin and c-Myc was upregulated in tumor tissues that were inoculated from the mixture of SMMC-7721 and TAMs,which was reversed by CpG ODN pre-treatment of TAMs.Subsequently,compared with mice inoculated with a mixture of SMMC-7721 cells and TAMs that were transfected with a vector,we found that tumor growth rate and tumor load were remarkably reduced in mice inoculated with a mixture of SMMC-7721 cells and TAMs that had either silenced Wnt2b or ?-catenin.Meanwhile,compared with tumor tissues from SMMC-7721 combined with TAMs transfected with a vector,the expression of E-cadherin was remarkably upregulated,but the expression of vimentin,?-catenin and c-Myc was downregulated in tumor tissues from SMMC-7721 mixed with silenced Wnt2b or ?-catenin in TAMs.These results suggest that the Wnt2b/?-catenin signaling mediates TAMs-induced HCC-promoting effects in vivo,and TLR9 agonists might be a potential therapeutic agent.9.Expression of Wnt2b,N-cadherin,and Snail in tumor tissues is associated with poor prognosis in HCC patientsGEPIA2 database was used to analyse the influence of Wnt2b expression on the prognosis of HCC patients.The overall survival of HCC patients with high expression of Wnt2b was significantly lower than that of the low expression group.In addition,the expression of EMT-related markers N-cadherin and Snail was also associated with poor prognosis of patients.Conclusions:Our current study illustrated that polarization-promoting factors in the HCC TME can activate ?-catenin by upregulating the expression of Wnt2b in macrophages,which can promote the polarization of TAMs to M2-like macrophages,and then M2-polarized TAMs promote the EMT of tumors and promote tumor progression,a process associated with the activation of HCC-TAMs glycolysis.TLR9 agonist CpG ODG can act as a blocker of Wnt2b signal which can inhibit M2 polarization of HCC-TAMs induced by HCC-TCM.As a potential target of tumor therapy,Wnt2b can not only promote malignant formation of tumor cells,but also play an important role in the formation of immunosuppressive tumor microenvironment.Targeting Wnt2b in TAMs might be a great potential treatment strategy in immune therapy of HCC,and TLR9 agonist CpG ODN might act as a Wnt2b signal inhibitor for HCC therapy.