Clinical Significance And Specific Mechanism Of Tumor-associated Macrophages In Mesenchymal Circulating Tumor Cell-mediated Colorectal Cancer Metastasis

Tumor-associated macrophage(TAM)are major components of tumor microenvironment that frequently associated with tumor metastasis in human cancers.Circulating tumor cell(CTC),originating from primary tumor sites,is considered to be the precursors of tumor metastasis,and its role in the development of colorectal cancer has attracted more and more attention.However,the regulatory mechanism of TAM in CTC-mediated tumor metastasis still remains unclear.This topic mainly includes the three parts,as the following:Part 1.TAM at invasive front is correlated with EMT,~MCTC ratio,and poor prognosis in colorectal cancer patientsBackground Colorectal cancer is the third most common malignant cancer in the world and one of the leading causes of cancer-related death.Metastasis,a multi-step complex process involving multiple factors,is still the main cause for colorectal cancer-related deaths.Circulating tumor cell(CTC),originating from primary tumor or metastatic sites,is considered to be the precursors of metastases.EMT in cancer,as known to increase cell motility and invasive potential,has been proposed to play the critical role in CTC generation.CTC,which gains more mesenchymal traits by EMT,is easy to survive and metastasize.Activation of EMT process typically requires interaction between tumor cells and the local tumor microenvironment,which involves TAM.However,the relationship between TAM,EMT and CTC in colorectal cancer has not been elucidated.Methods Immunohistochemical staining was used to detect the macrophages infiltration(CD68 and CD163),epithelial–mesenchymal transition(EMT)markers(E-cadherin and Vimentin)expression in serial sections of human colorectal cancer specimens.The correlations between macrophages infiltration and clinicopathologic features,mesenchymal CTC ratio,and patients'prognosis were analyzed.Results Near tumor invasive front,high level of CD163 was associated with less E-cadherin and more Vimentin,an indication of EMT.At the same time,the level of CD68 was not associated with the EMT program.However,at tumor stroma neither CD163 nor CD68 expression was associated with the EMT program.Interestingly,further analyses found that ~MCTC ratio was significantly associated with the expression of CD163,but not with CD68 at tumor invasive front.By contrast,either CD163 or CD68 expression at tumor stroma was insignificantly associated with ~MCTC ratio.Further prognostic analysis revealed that,at the invasive front of colorectal cancer,high level of CD68 expression was insignificantly associated with worse RFS and OS,however,high level of CD163 expression was significantly correlated with poor RFS and OS.Either CD68 or CD163 expression at non-invasive front was not associated with the prognosis of colorectal cancer patients.Univariate and multivariate analyses showed that CD163 expression at the invasive front was an independent prognostic factor associated with poor RFS and OS.Conclusions CD163~+TAMs at invasive front promote the release of ~MCTC by mediating the EMT program of primary tumor cells,thereby promoting tumor progression and affecting the prognosis of colorectal cancer patients.Part ?: TAM induce EMT to promote migration and invasion of colorectal cancer cells and its specific mechanismBackground Noteworthy,emerging studies have suggested that TAMs play important roles in tumor metastases by regulating EMT of cancer cells.However,the roles and mechanisms of the crosstalk between TAMs and cancer cells in EMT of colorectal cancer are still unclear.Methods A co-culture assay in vitro was used to evaluate the role of TAMs on colorectal cancer EMT,proliferation,migration and invasion,and ELISA,luciferase reporter assay and CHIP were performed to uncover the underlying mechanism.Results To determine the above clinical results,we utilized an in vitro model of tumorassociated macrophages,and tumor cells induced TAMs of a mixed M1/M2 phenotype.colorectal cancer–conditioned macrophages regulated EMT program to enhance colorectal cancer cells migration and invasion by secreting IL6.TAMs-derived IL6 activated the JAK2/STAT3 pathway,and activated STAT3 transcriptionally inhibited the tumor suppressor mi R-506-3p in colorectal cancer cells.mi R-506-3p,a key mi RNA regulating Fox Q1,was downregulated in colorectal cancer cells,resulting in increased Fox Q1 expression,which in turn led to the production of CCL2 that promoted macrophage recruitment.Conclusion TAM induce EMT to promote migration and invasion of colorectal cancer cells via IL6,and STAT3-mi R-506-3p-Fox Q1 axis is critical for TAM-induced colorectal cancer cell growth,migration,and invasion.Part ?: TAM enhanced colorectal cancer tumorigenesis in vivoBackground In previous in vitro experiments,we have demonstrated that TAMinduced EMT to promote proliferation,migration and invasion of colorectal cancer cells.Then,we used the method of subcutaneous tumor formation and tail vein injection in nude mice to verify the effect of TAM on the subcutaneous tumor formation and liver/lung metastasis of colorectal cancer cells.Methods The effect of TAM on the subtumor tumorigenic ability of colorectal tumors was evaluated by subcutaneous tumor formation model in nude mice.The effect of TAM on EMT status of colorectal tumor cells was further evaluated by RT-PCR and immunohistochemical staining.Ki-67 staining and CTC collection and identification analysis was used to verify the effect of TAM on tumor proliferation and ~MCTC ratio;tail vein injection was used to verify the effect of TAM on liver and lung metastasis of colorectal cancer.Results TAM regulates the EMT status of colorectal cancer cells by secreting IL6 and promotes the growth of subcutaneous tumor formation in nude mice;It also enhances tumor proliferation and ~MCTC ratio;TAM can promote liver and lung metastasis of colorectal cancer cells by secreting IL6.Conclusion TAMs enhanced colorectal cancer tumorigenesis in vivo.