| Background and Objective:Colorectal cancer(CRC)is the third most common malignant tumor in the world.The tumor microenvironment plays an important role in the progression of CRC,especially in distant metastasis.As an important component of the microenvironment,the specific mechanism of the imbalance of the gut microbiota in the initiation and progression of CRC remains unknown.The aim of this study was to investigate the association between intestinal flora imbalance and invasion and metastasis of colorectal cancer,and to explore potential mechanisms between gut flora and tumors and tumor microenvironment.Methods:1,Antibiotics to construct a mouse model of imbalance of flora;RT-PCR and mRNA chip analysis to screen key proteins related to tumor metastasis caused by imbalance of flora;2,Western Blot and immunohistochemical staining were used to detect the expression of this protein in human colorectal cancer tissues.And analyze the relationship between its expression and clinical parameters;3,Transwell,cell 3D culture,CCK8,plate clone,nude mouse subcutaneous tumor formation and tail vein lung metastasis model were used to observe the effect of endogenous protein changes on the proliferation and metastasis ability of CRC cells in vitro and in vivo;4,Macrophage-like cells and mouse primary macrophages were used to establish a co-culture system with tumor cells;Transwell assay was used to detect changes in cell migration and invasion ability with or without knocking out the secreted peptides;5,Flow cytometry and immunofluorescence were used to detect the changes of macrophage morphology and typing after addition of exogenous recombinant protein,and the changes of macrophage signal pathway were detected by Western Blot and immunofluorescence;6,Co-immunoprecipitation(CO-IP)and immunofluorescence co-localization screen the target of secreted proteins and macrophages;7,The protein chip further detects changes in the secretion of cytokines by macrophages after the addition of exogenous recombinant proteins,and the signaling pathway that promote tumor cell invasion phenotype.Results:The expression of lipopolysaccharide(LPS)in human colorectal tumor tissues was increased(X2=32.091,P<0.001).The imbalance of E.coli in mouse model can promote the in situ proliferation(t=1.538,P<0.001)and liver metastasis(t=1.800,P<0.001)of colorectal cancer cells.mRNA chip analysis and RT-PCR were screened for secreted protein-cathepsin K(CTSK)as a key protein associated with bacterial imbalance and tumor metastasis.The expression of CTSK in human colorectal tumors was significantly higher than that in adjacent tissues(x2=32.136,P<0.001),and it was positively correlated with poor prognosis.In vitro and in vivo functional experiments indicate that CTSK promotes the invasive phenotype of tumor cells and can be reversed by the CTSK-specific inhibitor—Odanacatib.In the co-culture system,macrophages can promote the migration and invasion of tumor cells,and can be reversed by knocking out CTSK secreted peptides.the tumor-secreted CTSK could bind to toll-like receptor 4(TLR4)to stimulate the M2 polarization of tumor-associated macrophages(TAMs)via an mTOR-dependent pathway.Neither rCTSK stimulated CRC growth and metastasis,nor rCTSK induced M2 macrophage polarization could take place in TRL4-/-mice.Meanwhile,CTSK could stimulate the secretion of cytokines secreted by M2 TAMs including IL1α、IL4、IL10 and IL17,which,in turn,promote the invasion and metastasis of CRC cells through NFκB pathway.Clinically,overexpression of CTSK in human CRC tissues is always accompanied with high M2 TAMs in the stroma(t=0.256,p<0.001),and correlated with CRC metastasis and poor prognosis.Conclusion:Our current research identifies CTSK as a mediator between the imbalance of gut microbiota and CRC metastasis.More importantly,we illustrated a CTSK-mediated positive feedback loop between CRC cells and TAMs during metastasis,prompting CTSK as a novel predictive biomarker and feasible therapeutic target for CRC. |
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