The Functional Effects Of Hepatic Stellate Cells-derived CCL5 On Breast Cancer Cells In Hepatic Stellate Cell And Breast Cancer Cell Co-culture System

Objective: With the improvement of diagnostic techniques and comprehensive treatments including surgery,chemotherapy,radiotherapy,endocrine therapy,targeted therapy and immunotherapy in recent years,the mortality rates of patients with breast cancer has been significantly reduced.However,distant metastasis is still the main cause of death in breast cancer patients.The liver is the third most metastasissusceptible organ for breast cancer,after the bone and the lung.Due to insensitivity to treatment,the prognosis of patients with breast cancer liver metastasis(BCLM)is worse than the former two.Therefore,it is urgent to explore the underlying molecular mechanism of occurrence and development of BCLM,which will provide theoretical basis for a better understanding of this malignancy and also filter the potential therapeutic targets.Both the original "seed and soil" hypothesis postulated by Paget in 1989 and the revised "seed and soil" hypothesis modified by Fidler in 2003 have clearly emphasized the important role of tumor microenvironment in tumor metastasis.Therefore,as one of the highly metastasis-permissive organs,liver provides prometastatic microenvironment for the survival and progress of tumor cells.The hepatic microenvironment is extremely complex which is comporised of cellular and noncellular components.Hepatic stellate cells(HSCs)is a type of hepatic nonparenchymal cells,and quiescent HSCs(q HSCs)play an important role in maintaining the normal structure and functions of liver.However,q HSCs can be activated by liver injury and inflammatory stimuli and then transdifferentiate into myofibroblast(α-SMA positive).This type of activated HSCs(a HSCs)remold the liver microenvironment by recruit inflammatory/immune cells through releasing chemokines and cytokines and also by producing ECM(Extracellular matrix)that rich in collagen I and IV.In fact,previous studies have demonstrated that HSC play crucial roles in the hepatic tumor microenvironment.Similarly,q HSCs obtain dramatic phenotypic transformation upon activation by tumor cells,with changes in expression profile of secreted protein and increased production of ECM constituents.Meanwhile,a HSCs affect tumor cell adhesion,proliferation,invasion,migration,and angiogenesis by providing prometastatic microenvironment.However,there is still a lack of researches about the effect of HSCs on breast cancer cells.Therefore,from the perspective of tumor microenvironment and cell interaction,the purpose of our study is to explore where the cross-talk exist between HSC and breast cancer,and more important,to investigate the effects of HSC on the breast cancer in vitro co-culture system.Method: 1.In vitro co-culture system of HSCs and breast cancer cells were established by using Transwell permeable support insets.Base on the different purpose of experiment,we also collected conditioned medium(CM)from HSCs to co-culture breast cancer cells.2.Inverted phase contrast microscope was applied to observe morphological changes of co-cultured cells.3.CCK-8 assay,Ed U assay,clone formation assay and vivo xenografts were used to detect proliferation ability of breast cancer cells.4.Migration ability of breast cancer cells was measured by Wound-healing assay.5.Invasion ability of breast cancer cells was measured by Transwell matrigel invasion assay.6.m RNA expression was detected by RT-PCR.7.Protein expression was detected by Western bolt.8.The concentration of cytokines was detected by Elisa.9.Differential secreted cytokines were detected by QAH-CAA-1000 cytokine antibody array from Raybiotech.10.Specific si-RNA was used to interfere gene expression.11.Gene Ontology(GO)analysis of elevated cytokines were conducted by using software 6.8 of DAVID Bioinformatics Resources.12.The elevated cytokines involved potential signal pathways were analyzed by KEGG analysis.13.Kaplan-Meier Plotter database was used to perform survival analysis,and online GEPIA database was used to conduct correlation analysis.14.Statistical analysis: all data were presented as Mean ±SD,SPSS 22.0 and Graphpad Prizm 6 was used for statistical analysis and image production respectively.A Student t test for unpaired data to compare the values between the two groups and one-way ANOVA was used to compare variance among multiple groups.P < 0.05 was considered statistically significant.Results: 1.Effects of breast cancer cells on HSC.LX-2 cells experienced a myofibroblastlike morphology change after co-cultured with breast cancer cells.The α-SMA expression of LX-2 cells was elevated,which is known as a hallmark of myofibroblasts.These results suggested that breast cancer cells enhance activation of HSCs and induce myofibroblast-like transformation of HSCs.2.Effects of a HSC on biological behavior of breast cancer cells.First,CCK-8 assay,Ed U assay,and clone formation assay revealed that the proliferation ability of breast cancer cells was significant increased when co-cultured with LX-2 CM.Then,breast cancer cells mixed with a HSCs were inoculated into nude mice and the result showed that a HSCs promoted the growth of the breast cancer xenograft tumors.The above results indicated that a HSCs promoted the proliferation ability of breast cancer cells.Next,Wound-healing assay and Transwell invasion assay found that the migration and invasion ability of breast cancer cells in co-culture group were significant increased compared with the group cultured alone.Moreover,luminal breast cancer cell line MCF-7 and T47 D in co-culture system exprienced mesenchymal morphology change and accompanied with N-cadherin and Vimentin overexpression,and decreased expression of E-cadherin,which suggested that epithelial-mesenchymal transition(EMT)occurred within the breast cancer cells of co-culture group.In summary,the above results indicated that a HSC could promote the proliferation,migration,invasion ability and EMT of breast cancer cells.3.CCL5 is a key cytokine released by a HSCs in co-culture system.To investigate which molecular(s)in co-culture system that might be involved in the effects of a HSC on biological function of breast cancer cells,cytokines microarrays that containing antibodies against 80 cytokines were used to identify the differentially expressed cytokines between co-culture group and breast cancer cells cultured alone.Bioinformatics analysis was performed according to the results from cytokine arrays screening.The results showed that the level of CCL5 increased significantly in the coculture group.Enzyme-linked immune sorbent assay(Elisa)verified that CCL5 concentration in co-culture group and LX-2 group was significantly higher than that in the tumor cell group,and the co-culture group had the highest concentration.Furthermore,Gene Ontology analysis showed that the molecular functions of CCL5 mainly enriched in protein binding,cytokine activity and protein kinase activity;its biological processes mainly enriched in the positive regulation of ERK1 and ERK2 cascade reaction,positive regulation of cell migration.It signaling pathways mainly enriched in cytokine-cytokine receptor interaction signaling pathway and PI3K-Akt signaling pathway.In general,these results indicated that the differential expression cytokine CCL5 in co-culture microenvironment was mainly secreted by HSCs and might involve in many crucial biological functions of breast cancer cells.4.CCL5 paracrine by HSCs affect the biological behavior of breast cancer cells.First,si-RNA was used to knock down the expression of CCL5 in LX-2 cells and breast cancer cells.CCK-8 assay,Wound-healing assay and Transwell invasion assay were used to measure the proliferation,migration and invasion ability of breast cancer cells respectively.Our results showed that these aggressive characteristics of breast cancer cells significantly repressed when co-cultured with si-CCL5/LX-2.Meanwhile,we found that the inhibitory effect was recovered by adding exogenous CCL5 into siCCL5/LX-2 group(positive control).In addition,we co-cultured si-CCL5/MDA-MB-231 cells with different LX-2 CM to explore the role of CCL5 secreted by breast cancer cells.The results showed that CM from si-NC/LX-2 and si-CCL5/LX-2+CCL5 still confer a growth advantage to si-CCL5/MDA-MB-231 cells.Furthermore,to clarify whether CCL5 could exert its role via CCR5,we added CCR5 antagonists Maraviroc into si-NC/LX-2 group(negative control).We found that application of Maraviroc resulted in suppression of CCL5-induced proliferation,migration and invasion ability of breast cancer cells.These results indicated that HSC-derived CCL5 could enhance proliferation,migration and invasion ability of breast cancer cells through CCR5.5.Mechanism of CCL5/CCR5 axis affecting breast cancer cells in co-culture system.The KM-plotter was used to analyses the prognostic effect of CCR5 in breast cancer patients.The result showed that CCR5 was associated with unfavorable prognosis in breast cancer patients.Meanwhile,the result from GEPIA database found that there was a positive correlation between CCL5 and CCR5.According to the results from our previous bioinformatics analysis,CCL5 involved in many crucial biological functions,and its pathway enrichment mainly in PI3K-Akt pathway.Here,the results indicated that HSC-derived CCL5 could form CCL5/CCR5 axis through binding to CCR5 of breast cancer cells,and the CCL5/CCR5 axis might affect the malignant behavior of breast cancer cells via PI3K/Akt pathway.Conclusions: 1.Breast cancer cells enhance activation of HSCs and induce myofibroblast-like transformation of HSCs.2.Activated HSCs enhance malignant behavior of breast cancer cells,including proliferation,migration,invasion ability and EMT.3.CCL5 is a crucial cytokine secreted by a HSCs and affect the malignant biological behavior of breast cancer.4.a HSC-derived CCL5 affect the biological behavior of breast cancer cells via CCR5 of breast cancer cells.5.In vitro co-culture system,CCL5/CCR5 axis may affect the biological behavior of breast cancer cells through PI3K/Akt pathway.