Macrophage Promotes The Migration And Invasion Of Ovarian Carcinoama Cells Via Promoting The Secretion Of CXCL16/CXCR6

Background:Ovarian cancer is one of the most common gynecologic malignant tumors worldwide,and China is one of the most severly afflicted area owing to the poor hygiene and life condition.With the development of medical technology,although the diagnosis and treatment of ovarian cancer have been highly improved,the prognosis of patients are still unsatisfied,and the 5-year survival for the advanced patients is only 40%aroud.Metastasis is an important procedure during the pathogenesis of cancer,and is the first cause for tumor associated-death.Recent studies have demonstrated that tumor microenvironment has played important roles during the metastasis of tumors.Scholars have doucmented that ovarain cancer cells could interacted with the tumor microenvironment to promote the processes of development,immunosuppression,metastasis,relapse and so on.Tumor-associated macrophages(TAMs)is an important part of tumor microenvironment.In ovarian cancer,TAMs accounts for approximately 40%of tumor microenvironment.According to their biological function,TAMs could be divided into two types:M1 and M2.Specifically,M1type could be induced by IFN?and TNFa to secrete the inflammatory cytokines,such as IL-6,IL-12,and IL23-to kill the carcinoma cells and pathogene.M2 type of TAMs could induced by TGF?,IL4,and IL13 to secrete severla growth-associated factors,which could promote the development of tumors and formation of tumor vessels.During the interaction bewteen TAMs and tumor cells,ovarian cancer cells could secrete amount of active substance to regulate the tumor microenvironment,resulting in promotion effects on the proliferation,invasion,and migration of tumor cells.Therefore,it is important to explore the interaction of tumor microenvironment and tumor cells,so that could provide some new insights in understanding the molecular mechanism,optimizing the strategy of diganosis and treatment,and exploring new targeting drugs of ovarian cancer.CXCL16 is one of members in CXC chemokine family,and usually presented two types under physiological state:membrane binding type and secreting type.Commonly,CXCL16 can be detected in CD8+T cells,Th cells,nature killer cells,and macrophages,and its unique receptor CXCR6 usually can be detected in the undifferentiated CD8+T cells,nature killer T cells,CD8+T cells and CD4+T cells.Previou studies reported that CXCL16/CXCR6 axis has played critical roles in the cell adhesion,inflammatory reaction,development of Thymus T cells,and immune response,and most of them focused on inflammation and immnue associated disease,such asrheumatic arthritis,atherosclerosis,HIV infection,hepatitis,and pneumonia.Studies in recent years have revealed that abnoraml high expression of CXCL16 and CXCR6 have been detected in several malignant tumors,and this abnormal expression is usually associated with the proliferation,migration,invasion,and prognosis of patients.All of these findings indicated that CXCL16/CXCR6 axis might play critical roles in the development of carcinoma.However,the molecular mechanism of interaction between ovarian cancer cells and macrophage is still rarely reported.Considering of this,this study aimed to explore the expression of CXCL16 and CXCR6,and activation of macriphage in ovarian cancer tissues.Then,a co-culture system was conducted using SKOV3 cell and macrophage to mimic the microenvironment of ovarian cancer.Following this,the molecular mechanism of CXCL16/CXCR6 in the interacation of SKOV3 and macrophage,as well as the underlying mechanism have been investigated,so that could provide some new insights and spports for in-depth investigation,optimizing treatment,and exploration of new targetting drugs.Objectives1.To explore the expression of CXCL16 and CXCR6,and the activation of macrophages,as well as the association between the expression of CXCR6 and actibation of macrophages.2.To explore the biofunction of CXCL16 and CXCR6 during the interaction between ovarian cancer cells and macrophage.Moreover,to explore the bioeffects of CXCL16 and CXCR6 on the biological behaviors of ovarian cancer cells.3.To explore the changes of CXCL16 and its downstream signaling pathway after knocked down CXCR6 in ovarian cells,so that clarify the biological significance of CXCL16/CXCR6 in the migration and invasion of ovarian cancer.4.Regulating the potential downstream signaling pathways using specific inhibitors to clarify the pathways that CXCL16/CXCR6 axis involved in to regulate the migraion and invasion of ovarian cancer.Methods1.Association between the expression of CXCR6 and actibation of macrophages in ovarian cancer.Firstly,we have selected 20 cases of ovarian cancer patients as the objectives,and collected their carcinoma and para-carcinoma tisses to extract total RNA using TRIzol method.Then,the expression levels of CXCL16,CXCR6,TNF-?,and IL-6(the last two are the biomarker for macrophages)were determined using quantitative real-time PCR.The protein expression levels of CXCL16,CXCR6,and NF-?B,the downstream of TNF-?,were determined using western blotting.Finally,correlations between the expression of CXCR6 and CXCL16,TNF-?,as well as IL-6 were analyzed using the Pearson method.2.Biofunction of CXCL16 and CXCR6 during the interaction between ovarian cancer cells and macrophageFirstly,we have conducted an in vitro co-cultured system using SKOV3 cells,THP-1 cells,and TNF-a to mimic the interaction between ovarian cancer cells and macrophage in vivo.Then,cells were divided into three groups:?SKOV3;?SKOV3+low concentration of TNF-a;?SKOV3+macrophage+low concentration of TNF-a,and the expression levels of CXCL16,CXCR6,and NF-?B,the phosphorylation of PI3K and Akt,proliferation,migration,and invasion of SKOV3 cells were investigated.3.Changes of CXCL16 and its downstream signaling pathway after knocked down CXCR6 in ovarian cellsFirstly,we have koncked down CXCR6 in SKOV3 cells using siRNA method,and cells were divided into three groups:?SKOV3+macrophage+low concentration of TNF-a;?SKOV3+siRNAl+macrophage+low concentration of TNF-a;?SKOV3+siRNA2+macrophage+low concentration of TNF-a.Then,the expression levels of CXCL16,CXCR6,and NF-?B,the phosphorylation of PI3K and Akt,proliferation,migration,and invasion of SKOV3 cells were investigated.4.The pathways that CXCL16/CXCR6 axis involved in to regulate the migration and invasion of ovarian cancerThe PI3K/Akt signaling pathway was inhibited using PI3K and Akt specific inhibitor LY294002 and MK-2206,and cells were divided into:?SKOV3s+macrophage+low concentration of TNF-a;?SKOV3+PI3K inhibitor(LY294002)+macrophage+low concentration of TNF-a;?SKOV3+AKT inhibitor(MK-22062HC1)+macrophage+low concentration of TNF-a.Then,the expression levels of CXCL16,CXCR6,and NF-?B,the phosphorylation of PI3K and Akt,proliferation,migration,and invasion of SKOV3 cells were investigated.Results1.Association between the expression of CXCR6 and actibation of macrophages in ovarian cancer.(1)The mRNA expression levels of CXCL16,CXCR6,TNF-a,and IL-6 in ovarian cancer tissues were significantly higher than that in the adjacent tissues;(2)Protein expression levels of CXCL16,CXCR6,and NF-?B p65 in ovarian cancer tissues were significantly higher than that in the adjacent tissues;(3)Expression of CXCR6 was significantly positively correlated with the expression of CXCL16,TNF-a,and IL-6.2.Biofunction of CXCL16 and CXCR6 during the interaction between ovarian cancer cells and macrophage(1)SKOV3 co-cultured with macrophage could promote the secretion of CXCL16 in SKOV3 cells;(2)SKOV3 co-cultured with macrophage could upregulate the mRNA expression levels of CXCL16 and CXCR6;(3)SKOV3 co-cultured with macrophage could significantly elevated the protein expression levels CXCL16,CXCR6,and NF-?B p65;(4)SKOV3 co-cultured with macrophage for 96 could significantly inhibit the proliferation of SKOV3 cells;(5)SKOV3 co-cultured with macrophage could significantly promote the phosphorylation of PI3K and Akt in SKOV3 cells;(6)SKOV3 co-cultured with macrophage could significantly promote the migration and invasion of SKOV3 cells.3.Changes of CXCL16 and its downstream signaling pathway after knocked down CXCR6 in ovarian cells(1)In the co-culture system of SKOV3 and macrophage,knocked down CXCR6 had no significant effect on the secretion and mRNA expression of CXCL16;(2)In the co-culture system of SKOV3 and macrophage,knocked down CXCR6 could significantly downregulated the expression level of NF-?B p65,and the phosphorylation of PI3K and Akt in SKOV3 cells;(3)In the co-culture system of SKOV3 and macrophage,knocked down CXCR6 could significantly reduced the migration and invasion ability of SKOV3 cells.4.The pathways that CXCL16/CXCR6 axis involved in to regulate the migraion and invasion of ovarian cancer(1)In the co-culture system of SKOV3 and macrophage,inhibiting the activation of PI3K and Akt could significantly downregulate the expression level of CXCR6,but had no effect on the expression of CXCL16 in SKOV3 cells;(2)In the co-culture system of SKOV3 and macrophage,inhibiting the activation of PI3K and Akt could significantly downregulate the protein expression levels of CXCR6 and NF-?B p65 in SKOV3 cells;(3)In the co-culture system of SKOV3 and macrophage,inhibiting the activation of PI3K and Akt could significantly downregulate the phosphorylation levels of PI3K and Akt in SKOV3 cells;(4)In the co-culture system of SKOV3 and macrophage,inhibiting the activation of PI3K and Akt could significantly downregulate the migration and invasion ability of SKOV3 cells.Conclusion:1.Compared with the adjacent tissues,expression levles of CXCL16 and CXCR6 were higher expressed in the ovarian cancer tissues,and expression of CXCR6 was positively correlated with the activation of macrophages;2.Tumor-associated macrophages could improve the expression levels of CXCL16 and CXCR6,and phosphorylation levels of PI3K and Akt to impair the invasion and migration abilities of SKOV3 cells;3.Inhibiting PI3K or Akt could significantly downregulated the expression level of CXCR6,and reverse the effect of macrophages on the invasion and migration abilities of SKOV3 cells.New points1.This study was first to investigate the expression levels of chemokine CXCL16 and its receptor CXCR6 in ovarian cancer,and their association with the activation of macrophage.These finding might provide some data support for ovarain cancer research in the future;2.This study had constructed an in vitro co-cultured system to mimic tumor microenvironment in ovarian cancer to explore the molecular mechanism of CXCL16/CXCR6 in the interaction between ovarian cancer cells and macrophages.These findings have provided some foundation for the in-depth molecular mechanism investigations and the exploration of new targeting drugs for ovarian cancer in the furture.