Roles And Mechanisms Study Of Lgr4 Mediated Macrophage Polarization In Tumor Formation And Progression

Tumor associated macrophages(TAM)is a kind of tumor-resident monocytes which secret large amounts of cytokines,growth factors and functional intermediates to regulate tumor microenvironment.TAMs are closely correlated with immunosuppression in the tumor microenvironment which facilitate the escaping of tumor cells from surveilliance of the immune system and even the proliferation and metastasis of tumor cells.Meanwhile,tumor microenvirenment results in the heterogeneity and plasticity of TAMs which plays very important roles in tumor formation.Thus,TAMs has become the most populor target in cancer immunotherapy.As the richest immune cells in the tumor microenvironment,TAMs transduce extracellular signals through cell membrane receptors and lead to diverse functional regulations of macrophages.As the largest receptor family,G protein coupled-receptors(GPCRs)are crucial sensors of extracellular signals in cells.Although 826 GPCRs have been found,their roles in regulation of macrophage polarization remain largely unknown.In order to explore their contributions in the TAMs polarization,we conducted the RNA-Seq to clarified specific genes especially GPCRs that differentially expressed in M1/M2 polarized macrophage.Our studies demonstrated that Leucine-rich repeat containing G protein-couple receptor 4(Lgr4)was up-regulated in IL-4 primed M2 type bone marrow-derived macrophages(BMMs)while was down-regulated in IFN-?primed M1 type BMMs.And Lgr4-/-BMMs prefer to obtain a bias Ml and impaired M2 feature in macrophage polarization.When compared with Lgr4+/+ BMMs,Lgr4-/-BMMs has significantly upregulated M1 marker genes including IL-1?,TNF-?,iNOS or CXCL10 and down-regulated M2 marker gens such as CD206,Arg1 or Ym1,indicating that Lgr4 is involved in the polarization of macrophages and may play potential roles in pro-tumor capacity of TAMs.Since the contribution of TAMs to tumorigenesis have been well elucidated,we seek to investigate potential role of Lgr4 in tumor formation and progression via regulation of TAMs.Then we collected clinical information of LGR4&cancer by searching the TCGA and Oncomine database and found that LGR4 was abnormally expressed in various human cancers,including lung and breast cancer and are correlated with poor prognosis of patients.Of note,we found that both LGR4 and it's ligands Rspondins are well co-localized or correlative with M2 TAM marker genes,indicating the intrinsic relationship of LGR4 mediated TAM polarization with tumor formation and progression.To further illucidate the correlation of Lgr4 and TAMs with tumor development,we established disparate mouse tumor models for further investigation.In our study,subcutaneous co-injection of Lewis lung cancer cells(LLCs)with Lgr4-/-macrophages resulted in distinct tumor inhibition compared with wild type counterparts.And urethane-induced lung carcinogenesis or inoculation of LLCs in Lgr4 macrophage conditional.knockout mice(Lgr4flox/flox,Lysm-Cre)also displayed severer retardation of tumor growth compared with the control groups.Immunofluorescence and flow cytometry analysis showed an intense decrease of M2 TAM(CD206+),but increase of M1 TAM(CD 16/32+)that characterized by IL-12high and TNF-? high in the tumors of Lgr4 conditional knockout mice.Simultaneously,more CD8+ T cells were found infiltrated in the tumors of Lgr4 conditional knockout mice.And similar phenotypes were also observed in the B16F10 melanoma model.Our data showed that Lgr4 deficiency in TAMs rebalance their polarization,lead to the remodeling of tumor microenvironment and the severe inhibition of tumors.Mechanically,we confirmed that both Rspondin1 and Rspondin3 are able to polarize macrophages into an M2 state.Then,we verified the Rspondin1-Lgr4/Erk/Stat3 axis in the regulation of macrophage polarization,as showed by the reduced phosphorylation of Stat3 in Lgr4-/-macrophages treated with recombinant R-spondins1 and IL-4.And Stat3 agonist can rescue the deficient M2 state of Lgr4-/-macrophages both in vitro and in vivo.Accordingly,we blocked Rspo1-Lgr4 signaling by treating LLC tumor bearing mice with Lgr4 extracllular domain or anti-Rspodinl antibody and found that tumor growth in these mice was significantly inhibited.What's more,both LGR4-ECD and anti-Rspol antibody can synergistically improve the tumoricidal efficiency of anti-PD1 antibody in the mouse B16F10 melanoma model.Likewise,LGR4-ECD and anti-Rspo1 imbalanced the M1/M2 state of TAM in the tumor microenvironment with a simultaneously increase of activated CD8+ T cells and,to our surprise,severe decrease of MDSCs were observed.Taken together,these data established Lgr4 as an important regulator in macrophage polarization and clarified the role of Lgr4 mediated TAMs polarization in the tumor formation and progression,suggesting that Lgr4 might emerge as a novel immune checkpiont and potential drug target in cancer therapy.