Wnt/β-catenin Signaling Regulates M2-like Macrophage Polarization And Reinforces The Abilities To Promote The Progression Of Hepatocellular Carcinoma

【Background】 China is the severely afflicted area by hepatocellular carcinoma,with prevalence and death rates accounting for nearly 70 percent of Asia,as the highest area in Asia.Primary liver cancer is also the fourth most frequently diagnosed cancer and has already ranked the third place for death related cancer in our country.Due to the concealment of the cancer development,most of the patients is diagnosed at the late stage,which is not efficiently response to any treatment,and prognosis is poor.As a result,hepatocellular carcinoma is such a disease that has already posed a serious threat to the life and health of our people.The etiology of liver cancer in China is closely related to hepatitis B or C virus infection,and the liver fibrosis cirrhosis induced by the late stage of chronic inflammatory disease is considered as the main cause of hepatocellular carcinogenesis.At present,the treatment of liver cancer in the world is not ideal.For early patients,surgery is the preferred treatment,while patients at the middle and late stage can only rely on some palliative care.And because the liver itself is a digestive organ with detoxification and metabolic function,it shows insensitive to chemotherapy.In recent years,the development and apply of small molecule inhibitors have failed to change the embarrassment of liver cancer treatment and bring a great economic burden on patients.In order to improve the prognosis of patients,Many arduous works is still needed to explore the comprehensive pathogenesis mechanism of hepatocellular carcinoma.Recently,inflammation has been highlighted as the seventh hallmark of cancer,which establishes the relationship between tumor cells and tumor microenvironment(TME).As a major component of TME,tumor-associated macrophages(TAMs)play a pivotal role in the progression of inflammation-related cancers,including HCC.Many studies have indicated that TAMs promote tumor initiation,angiogenesis,metastasis and suppression of adaptive immunity through production of large amount of cytokines,chemokines,growth factors and matrix metalloproteases in TME.Indeed,infiltrated TAMs are associated with poor prognosis of HCC patients.These studies suggest that TAMs can be a potential target for HCC therapy.TAMs possess high heterogeneity,which can ascribe to their origin and activation status and function.Under inflammatory stimulation,monocytes were recruited to injured tissue and differentiated into macrophages with different polarized activation state.The growing evidence shows that crosstalk between tumor cells and macrophages are involved in tumor progression.Many kinds of soluble factors are important message carriers on regulating cell-cell interaction,such as Wnts.Wnt ligands are secreted proteins,which not only participate in cellular proliferation,migration and tissue patterning during embryonic development,but are also involved in many diseases,especially tumorgenesis.Generally,Wnt proteins can be secreted into extracellular milieu through Wntless,and then bind to frizzled receptor at the signaling-competent cells to induce canonical Wnt/β-catenin pathway or noncanonical Wnt/Ca2+ pathway in paracrine/autocrine manners.Several studies have shown that an autocrine mechanism for constitutive Wnt pathway activation in human cancer cells including breast cancer,ovarian cancer,non-small cell lung carcinoma and so on.However,whether the tumor derived Wnts ligands could regulate the differentiation of TAMs through the activation of Wnt/β-catenin signaling in macrophage is still unknown.【Aims】 Wnt signalling has been reported to regulate hemeotopoictic cells differentiation,especially play a critical role in T-cell development and dendritic-cell maturation.Our preliminary experiments found that there were significant differences in the expression of Wnt/β-catenin signal in macrophages of different polarization states.We plan to study the function and polarization of macrophages by silencing the expression of β-catenin in macrophages as well as its effect on the biological behaviors of tumor cells.After that we attempt to explore the manner of crosstalk between tumor cells and macrophage.Finally,we further testified our observation on the clinical HCC specimen by staining CD68+β-catenin+Arg-1/MR.Based on our findings,we expect to provide some theoretical foundations for hepatocellular carcinoma therapy with immuno-biological method in the futher.【Methods】 1.Firstly,we sorted CD11b+ cells from bone marrow of C57BL/6 mice.At the same time,we induced macrophage with MCSF.We detected downstream genes of the Wnt/β-catenin signaling between CD11b+ monocyte and macrophage using QPCR.After polarized macrophage with LPS+IFN-γ or IL-4,we further detected the different expression of Wnts ligand,receptors and downstream genes of the Wnt/β-catenin signaling with different method,such as QPCR,Western-blot,IF and FACS.What’more,we established mouse liver cancer model by inoculating hepa1-6 cells in order to sorted TAMs in the tumor tissue and detected the relationship between M2 macrophage markers and Wnt/β-catenin signaling molecules.2.We added Wnt3 a to M1 and M2-type BMDMs to activate Wnt/β-catenin signaling.After 24 h,we analyzed the M1 and M2 markers of macrophage polarization.On the other hand,we added ICG001 or si RNA against β-catenin in M2-type BMDMs to inhibit Wnt/β-catenin signaling,and performed experiments as above mentioned.Beside QPCR,we also applied IF and FACS to examine these molecules expression.Moreover,we blocked c-Myc expression in M2-type BMDMs with or without Wnt3a to explore the mechanism by which Wnt/β-catenin promoted M2-polarization in macrophage.3.We applied si RNA to block down β-catenin in M2-type BMDMs.On the one side,we collected the conditional medium of BMDMs to incubate the tumor cells.Plate clone formation assay was used to detect the rate of clone formation between silencing and negative control groups.Wound healing assay was used to detect the mobility of tumor cells after the above treatment.On the other side,coculture system was used to detect the migration and invasion ability of tumor cells in the presence of BMDMs with or without si-β-catenin.Finally,we observed the proliferation percentage of CD8+ cytotoxic T lymphocyte after co-cultured with si-β-catenin or si R transfected different polarized macrophages by mixed lymphocyte reaction assay.4.We exposed mature BMDMs to the cultural medium of tumor cells with or without ICG001.After 24 h incubation,we analyzed the expression of MR,Arg-1 and c-Myc,β-catenin by Western-Blot.We established the tumor cell line in which WLS was downregulation by sh-RNA virus infection.And then collected the cultural medium to incubate the BMDMs as above to testify whether tumor derived Wnts ligand could promote the M2 polarization through Wnt/β-catenin signaling.At last,we used the WLS-knockdown hepa1-6 cell to inoclulate mouse liver cancer model.After 3 weeks,tumors were weighed,the percentage of TAMs,CD3+T cells(CD4+ /CD8+),Treg cells were compared between two groups.The IL-12 and IL-10 secretion of TAMs were analyzed by FACS intracellular staining.5.To further dissect the relationship between activated Wnt/β-catenin signaling and M2 macrophages in hepatocarcinoma patients,we observed the expression of CD68(a pan macrophage marker),β-catenin and M2-related markers MR or Arg-1 in 25 frozen tumor sections by immunofluoresence staining.The mean density of MR/Arg-1 and β-catenin in CD68+ cells were evaluated and counted.【Results】 1.The purity of monocytes and macrophages was about 97% by FACS assay.The QRT-PCR results showed that the expression of β-catenin,c-Myc and cyclin D1 was significantly increased in mature macrophages,suggesting that Wnt signaling was activated during monocytes differentiation into macrophages.We also found that the expression of Wnt receptors,such as Fzd7 and Fzd9,were only higher in M2 macrophages but not in M1 macrophages,it is the same with the expression of β-catenin,c-Myc and Axin2.The QRT-PCR results showed that TAMs in hepatic tumor indeed expressed higher M2-related markers.Meanwhile,we found that Wnt/β-catenin signaling was activated in TAMs too when compared with the common kuffer cells.2.In the M1-type BMDMs,the result showed that the expression of M1 macrophage markers,such as TNF-α and IL-12,was significantly reduced,and the functional marker i NOS was only reduce slightly under M1 status with Wnt3 a or Li Cl treatment.Meanwhile,the expression of Arg1,MR and IL-10 was significantly increased in the same situation.After adding Wnt3 a to M2 macrophages,we found that the expression of M2-related molecules was increased significantly compared with that under IL-4 stimulation alone.Consistently,Wnt/β-catenin signaling was activated in M2 macrophages with Wnt3 a treatment.ICG001 treatment dramatically reduced the expression of M2 macrophage-related molecules following IL-4 stimulation.Meanwhile,the activated Wnt/β-catenin signaling was successfully inhibited in the ICG001 plus IL-4 group.In the data from IF,the MR+F4/80+ or Arg1+F4/80+ macrophages increased obviously in IL-4 plus Wnt3 a treatment,compared with IL-4 treatment alone.Whereas,these M2 macrophages decreased markedly with ICG001 treatment.Then,c-Myc si RNA(sic-Myc)or control si RNA(si R)were transfected into BMDMs 24 h before IL-4 stimulation,the expression of MR,Arg1 and Ym1,was reduced significantly in sic-Myc plus IL-4 group compared with that in control group.Similarly,promotion of M2 macrophages by Wnt3 a was abrogated after sic-Myc treatment,demonstrating that the c-Myc was responsible for M2 macrophage polarization mediated by Wnt/β-catenin signaling.3.The result showed that the colony number of Hepa1-6 cells cultured in conditional medium(CM)from M2 macrophages with β-catenin knockdown was significantly less than that cultured in CM from the control.And the results showed that si-β-catenin in M2 macrophages induced a remarkably slower migration than the control and IL-4 alone group after co-cultured for 16 h.In the coculture system,the results showed that si-β-catenin in M2 macrophages significantly restrained the migrated cell numbers,as well as the invaded cells number.The FACS assay showed that si-β-catenin transfected M0 or M2 macrophages promoted CD8+T cell proliferation stronger than si R transfected M0 or M2.4.The result showed that the expression of MR,Arg1,β-catenin and c-Myc were all increased significantly in macrophages after CM treatment,but this effect was reversed after blocking Wnt/β-catenin signaling by ICG001 or WLS knocked down.By IF staining,we found that WLS silenced tumor cell could result in reducing tumor growth.What’s more,there was an obvious decline for the percentage of TAMs/Tregs and IL-10 production.However,the percentage of CD3+T cells was improved,as well as the IL-12 secretion from TAMs.The results showed that the number of F4/80+ MR/Arg-1+ cells was reduced by immunofluoresence staining.5.As the immunofluoresence staining showed that there were many CD68+Arg1+ or CD68+MR+ M2-like TAMs infiltrated in liver tumor.The quantification assay showed that the level of nuclear β-catenin was positively correlated with the Arg1 or MR expression in CD68+ macrophages in HCC patient biopsies.【Conclusion】 1.Wnt/β-catenin signaling is involved in monocytes differentiation and can promote macrophages into M2 polarization which is through the regulation of c-Myc.2.Blocking of Wnt/β-catenin signaling in M2 macrophages abrogat TAMs phenotypes in vitro which can inhibit the malignant biological behavior of tumor cells and improve the growth of CD3+ T cells.It provides the theoretical and experimental basis of a new therapy strategy on targeting Wnt signaling in TAMs for liver cancer treatment.3.Wnt ligands secreted from tumor cells initiates Wnt/β-catenin signaling activation in M2 macrophages through paracrine manner leading to more Tregs and less CD3+ T cells infiltrated.Therefore,our study bring forth the crosstalk between tumor cells and tumor associated macrophages via Wnt/β-catenin signaling.