Wnt5a-induced M2 Polarization Of Tumorassociated Macrophages Via IL-10 Promotes Colorectal Cancer Progression

Tumor-associated macrophage(TAM),as one of the important components of tumor microenvironment,is closely related to the occurrence,invasion and metastasis of malignant tumors.TAM is mainly divided into two types: M1 and M2 phenotype.Among them,M2-like TAM is generally considered to play an important role in tumor support.Several studies have shown that Wnt5 a is mainly expressed in the tumor stroma,especially in TAMs.However,whether Wnt5 a regulates the M2 polarization and biological function of TAMs in colorectal cancer is incompletely understood.This study mainly includes the following three parts:Part 1.The ratio of Wnt5a~+ TAM/TAM is correlated with clinicopathological characteristics and poor prognosis in colorectal cancer patientsBackground Metastasis is a major cause of cancer-related deaths.TAM in the tumor microenvironment can secrete various mediators such as cytokines or chemokines to suppress anti-tumor immune response,stimulate angiogenesis and finally enhance cancer cells proliferation,invasion,intravasation and dissemination for metastasis.Although TAMs exert an important role in cancer metastasis,not all types of TAM are involved in tumor progression and metastasis.Several studies have shown that Wnt5 a is mainly expressed in the tumor stroma,especially in TAMs.However,the clinical significance of Wnt5a~+ TAM subtype in colorectal cancer is unclear.Methods Immunofluorescence staining was performed to detect CD68 and Wnt5 a expression in colorectal tissues from patients(63 CRC specimens VS 20 normal tissues).The correlations between the number of TAM,Wnt5a~+ TAM,the ratio of Wnt5a~+ TAM/TAM and clinicopathologic features,patients' prognosis were analyzed.Results In colorectal cancer patients,high expression of Wn5a~+ TAM is associated with lymphatic invasion and TNM stage,and high ratio of Wn5a~+ TAM/TAM is associated with tumor invasion,lymphatic invasion,TNM stage,and lymph node metastasis.Further prognostic analysis showed that high expression of Wn5a~+ TAM and high ratio of Wnt5a~+ TAM/TAM were both significantly correlated with poor disease-free progression(RFS)and overall survival(OS).The expression of CD68+ TAM has no correlation with RFS and OS.Univariate and multivariate analysis showed that the ratio of Wnt5a~+ TAM/TAM was an independent prognostic factor for poor RFS and OS in colorectal cancer patients.In addition,Wnt5 a was mainly co-localized with the M2 marker CD163.Conclusions The ratio of Wnt5a~+ TAM/TAM is significantly associated with the clinicopathological characteristics and poor prognosis in colorectal cancer patients,and the Wnt5a~+ TAM subtype is an M2-like TAM.Part II: The specific mechanism study of Wnt5 a regulating M2 polarization of TAM and promoting colorectal cancer cells proliferation,migration and invasionBackground M2 polarization of TAMs is a multi-factor,multi-step and complex pathological process.Recent studies have shown that Wnt signaling plays a major role in M2-like TAMs polarization.However,whether Wnt5 a regulates the polarization and biological function of TAMs in colorectal cancer is incompletely understood.Methods A co-culture system was used to construct a TAM model in vitro and a series of cell and molecular biology experiments were performed to study the effect of Wnt5 a on TAM polarization and the underlying mechanism of Wnt5 a regulating TAM polarization.The effects of Wnt5 a on the biological functions of TAM were further evaluated by experiments such as clone formation,migration and invasion.Results TAM induced by co-culture with colorectal cancer cells was an M2 phenotype macrophage and highly expressed Wnt5 a.Subsequently,we found that Wnt5 a induced macrophages to secrete IL-10,which then acted as an autocrine cytokine to induce M2 polarization of these macrophages.IL-10 neutralizing antibody completely reversed the pro-M2 effect of Wnt5 a.Mechanistically,the Ca KMII-ERK1/2-STAT3 pathway was required for Wnt5a-mediated IL-10 expression in macrophages.Furthermore,Wnt5 ainduced M2 macrophages promoted CRC cells proliferation,migration and invasion;knockdown of Wnt5 a in TAMs significantly impaired the pro-tumor functions of TAMs.Conclusion Wnt5 a could induce M2 polarization of TAMs by regulating CaKMII-ERK1/2-STAT3 pathway–mediated IL-10 secretion,ultimately promoting tumor growth and metastasis of CRC.Part III: Silencing Wnt5 a inhibits the pro-tumor effect of TAMs in vivoBackground We further performed the experiment of subcutaneous tumor formation in nude mice to verify the effect of TAM on the ability of subcutaneous tumor formation and cancer metastasis,and the regulatory effect of Wnt5 a on the biological functions of TAM.Methods To evaluate the role of TAM in tumor growth using a model of subcutaneous tumor formation in nude mice,the tumor size was recorded at different time points and the effect of TAM on tumor proliferation was further analyzed by Ki-67 staining.The heart blood of nude mice was collected to isolate and count circulating tumor cell(CTC),and liver and lung tissues were harvested for HE staining to assess the effect of TAM on liver and lung metastasis of tumors.Results Wnt5a~+ TAM promoted tumor growth,enhanced the proliferation of tumor cells,increased the number of CTC,and promoted liver or lung metastasis of colorectal cancer;while knockdown of Wnt5 a in TAM significantly impaired the cancerpromoting functions of Wnt5a~+ TAM.Conclusion Knockdown of Wnt5 a significantly inhibits the pro-tumor effect of TAMs in vivo.