Dahuang Zhechong Pill Suppresses Colorectal Cancer Liver Metastasis Via Ameliorating Exosomal CCL2 Primed Pre-metastatic Niche

Background and aim:Colorectal cancer(CRC)is one of the most major gastrointestinal malignancies,which incidence and mortality has increased in China in recent years.Dahuang Zhechong Pill(DZP)is a classical formula from'"Synopsis of Prescriptions of the Golden Chamber".It has been used for treatment of abdominal masses(including tumorous diseases)for thousands of years.This study aimed to test if DZP suppresses the metastasis of colorectal cancer(CRC)by ameliorating the fibrosis status of the future metastatic organ.Methods:Liver metastasis was observed by injection of MC38-EGFP cells with stably expressing enhanced green fluorescence protein beneath the splenic capsule of C57BL/6J mice.MC38-EGFP-derived exosomes were analyzed by Label-free comparative proteomics.mRNA expression was determined by Quantitative PCR.Protein expression was determined by immunohistochemistry,immunofluorescence and Western blot.Collagen deposition was determined by Masson staining.All data were statistically analyzed using SPSS.Results:1.DZP suppresses the growth and liver metastasis of CRC.Mice CRC liver metastasis model was established by MC38-EGFP spleen injection.Compared with Model group,the tumor size,weight and fluorescence intensity in the spleen was significantly reduced by DZP.And the fluorescence intensity in the liver and the number of liver metastases foci was also significantly reduced by DZP.2.DZP suppresses the pre-metastatic niche formation by regulating liver fibrosis.Compared with Control group,Masson staining and immunohistochemistry staining showed that collagen deposition and Fibronectin(FN)expression were significantly increased in Model group,which were down-regulated by DZP.Spearrman correlation analysis showed that the expression of FN which represents liver ECM reconstruction was positively correlated with liver metastasis.3.DZP regulates exosomal CCL2-mediated macrophage infiltration and phenotype.Exosome proteomics showed that the up-regualted CC chemokine ligand-2(CCL2)in Model group was repressed by DZP treatment.QPCR and Western blot showed that DZP also markedly lowered the expression of CCL2 and its receptor CCR2 which up-regualted in Model group in the liver.Spearman correlation analysis indicated that the relative expression level of CCL2 mRNA was positively correlated with liver metastases.CCL2 induces macrophage recruitment.Immunohistochemical staining showed that compared with Control group,the expression of macrophage marker F4/80 in the liver was significantly increased,and the ratio of CCR2+ F4/80+macrophages was significantly increased in Model group,both of them were significantly decreased after DZP treatment.Western blot showed that compared with Control group,the expression of cleaved-TGF-?1 and FN were markedly up-regulated in Model group,which were down-regulated by DZP.Immunofluorescence staining showed that,compared with Control group,the ratio of M1 macrophages in the model group was significantly decreased,while that of M2 macrophages was significantly increased,and DZP treatment significantly reversed the M1/M2 paradigm shifted in Model group.4.Inhibition of fibrosis and CCR2 reduce CRC liver metastasis.Anti-fibrosis agent Pirfenidone and CCR2 inhibitor CCR2-RA-[R]reduce CRC liver metastasis and ameliorate TGF-?1 mediated liver fibrosis which up-regualted in Model group.Conclusions:DZP inhibits the liver metastasis of CRC by suppressing CCL2 mediated M2-skewing paradigm and ameliorating the pro-fibrotic microenvironment.