Role And Mechanism Of CD68 Positive Macrophages In The Migration And Invasion Of Endometrial Cancer Cells

BackgroundSubstantial studies have revealed that chronic inflammation promotes tumor growth,invasion and angiogenesis via tissue remodeling of the tumor microenvironment.The periodic damage and repair process of the endometrium is identified as chronic inflammation response.Thus,there is an evident and persistent inflammatory microenvironment in endometrial cancer,which plays critical roles in cancer progression.Tumor associated macrophages(TAMs)account for more than half of the immune cells infiltrated in tumor microenvironment.Macrophages are originated from monocytes and exhibit two distinct phenotypes:M1(activated by IFN-r and LPS)and M2 phenotype(activated by IL-4 and IL-13).M1 macrophages mainly play roles in antigen presentation and killing of pathogenic microorganisms,while the M2 phenotype exerts pro-tumoral effects.TAMs participate in tumor development via the production of an array of cytokines and chemokines.Currently,it remains unclear how TAMs play roles in endometrial cancer progression.Recent discoveries have identified that estrogen has a potential immunoregulatory effect on the tumor microenvironment.It has been reported that estrogen receptors(ERs)are expressed on the infiltrating macrophages of several cancers.Ovarian cancer macrophages express ERa and ER?,and estrogen promotes tumor progression by inducing polarization and infiltration of macrophages.In mouse lung cancer model,expression of ER? in macrophages is also detected,and estrogen can enhance the secretion of VEGF in macrophages and promote the recruitment of macrophages.In addition,ERs are expressed on macrophages of eutopic endometrium in endometriosis patients.Estrogen promotes the secretion of VEGF and HGF in macrophages and promote the angiogenesis of eutopic lesions.In conclusion,in some tumors,estrogen can interact with macrophages in the tumor microenvironment to jointly regulate the malignant tumor development.However,the effect of macrophage ERs have not been well clarified.So far,the expression of ERs on macrophages and its role in the progression of endometrial cancer have not been reported.Yeh CR et al.'s research discovered that there was a relationship between ER?and chemokine CCL.CCL18 is predominantly secreted by M2 macrophages.The overexpression of CCL18 in M2 macrophages has been discovered in several tumors,including ovarian cancer,gastric cancer and glioma.Furthermore,CCL 18 could promote epithelial mesenchymal transition(EMT)-like features of breast cancer cells.Epithelial characters can transform into mesenchymal phenotypes,allowing the epithelium to acquire motile and invasive abilities,which is defined as EMT.The metastasis of tumor cells are often accompanied by changes in cell morphology and cytoskeletal status.Kinesin superfamily(KIFs)are motor molecules and are mainly involved in cell motion-related functions,such as the transport of biomacromolecules.Substantial evidence verifies that KIFs participate in the development of cancers.Numerous studies have revealed that estrogen not only plays a direct role via ER expressed on tumor cells,but also indirectly regulates the biological behavior of tumor via ERs expressed on microenvironment macrophages.Interestingly,we discovered for the first time that ERa was expressed on partial of macrophages in the microenvironment of endometrial cancer.We assume that estrogen regualtes the interaction between tumor cells and tumor microenvironment macrophages to participate in the endometrial cancer progression.In this research,we will initially explore the role of ERa-positive macrophages in the progression of endometrial cancer by immunohistochemistry.We also perform in vitro experiments to analyze whether macrophages could regulate the invasion and migration of endometrial cancer cells via ERa and explore its potential molecular mechanisms.The research provides a new insight into the effect of estrogen on endometrial cancer microenvironment.PART ?Expression of ERa on the macrophages of endometrial cancer and its clinical significanceObjectiveTo detect the expression of ERa on the macrophages of endometrial cancer tissue,and to investigate the clinical significance of ERa-positive macrophages in endometrial cancer development.Methods1.The tissue microarray(TMA)containing 85 endometrial cancer samples and 85 normal samples was immunohistochemically stained with CD68 and ER?.CD68-positive(CD68+)macrophages in tumor specimens were classified into three groups:nest macrophages,stroma macrophages and margin macrophages.The correlation between CD68+ macrophages density at different tissue sites and clinicopathological parameters of endometrial cancer was analyzed by chi-square test.We examined the association between ERa expression in the epithelial and stromal compartments and FIGO stage.2.To further verify the expression of ERa on the macrophages of endometrial cancer tissue,double immunohistochemistry staining(CD68 and ER?)was performed.The relationship between ER?+ CD68+ macrophages density and FIGO stage was also examined.Results1.CD68+ macrophages were detected in 56 out of the 85(65.8%)endometrial cancer specimens.The mean mount of CD68+ macrophages in normal tissue was lower than that of endometrial cancer tissue(P<0.001).The number of stroma or margin CD68+macrophages was remarkably larger than that of nest macrophages(P<0.001,P<0.001,respectively).There was no discrepancy in the CD68+ macrophage density between stroma and margin group(P>0.05).Furthermore,the number of CD68+ macrophages in the tumoral stroma was correlated with lymph node metastasis and FIGO stage.2.The ER? expression score of the epithelial and stromal compartments was lower in the advanced stage(FIGO ?-IVstage)group than in the early stage(FIGO ?-? stage)group(P<0.001).3.Double immunohistochemistry staining showed that ERa was expressed on the endometrial cancer macrophages.The samples with ER?+TCD68+ macrophages accounted for 46.4%of those with CD68+ macrophages in tumor specimens,and only 21.4%of those in normal tissues(P<0.05).Unexpectedly,the percentage of tissues with ERa CD68+ macrophages in the advanced stage group(73.9%)was apparently higher than early stage group(27.3%)(P<0.01).Conclusions1.CD68+ macrophages density in the endometrial cancer tumoral stroma was positively correlated with lymph node metastasis and FIGO stage.2.There is a negative association between ERa expression in the epithelial and stromal compartments and FIGO stage of endometrial cancer.3.We are the first to report that ERa was expressed on partial of the macrophages of endometrial cancer tissue,and ERa CD68+ macrophages were positively correlated with FIGO stage of endometrial cancer,indicating ER?+CD68+ macrophages may participant in endometrial cancer progression.PART?M2 macrophages ERa induced epithelial to mesenchymal transition of endometrial cancer cells by CCL18 secretionObjectiveTo investigate the role of macrophages ERa in the progression of endometrial cancer via an array of in vitro assay.Methods1.The human monocyte line THP1 cells and human peripheral blood monocytes(PBMCs)were induced into M2 phenotype,and the M2 markers were examined by qRT-PCR.2.M2 macrophages were treated with ERa agonist(PPT)/antagonist(MPP),and then conditioned medium was harvested.Transwell,wound healing and 3D invasion assay were performed to detect the effect of CM from PPT-stimulated M2 cells(M2-PPT-CM)on the migration and invasion of endometrial cancer cells Ishikawa and KLE.Cytoskeleton staining assay was employed to investigate the effect of M2-PPT-CM on cell morphology and cytoskeleton status.The effect of M2-PPT-CM on EMT markers was detected by Western blotting and qRT-PCR.3.We performed ELISA and qRT-PCR to investigate the ERa-mediated chemokine profiles in M2.Western blotting was employed to explore the underlying mechanism of ER?-mediated CCL18 secretion in M2 macrophages.4.The effect of recombinant CCL18(rCCL18)on the EMT of endometrial cancer cells was examined by above assays.Anti-CCL18 neutralizing antibody was employed to identify that PPT-stimulated M2 cells induced EMT of endometrial cancer cells by CCL18 secretion.Results1.The mRNA level of M2 markers were increased in THP1 or PBMCs-derived M2 macrophages compared with M1 macrophages.2.Transwell and wound healing assay showed that M2-PPT-CM enhanced the migration of Ishikawa cells.Ishikawa cells treated with M2-PPT-CM formed longer invasive projections compared with control cells.The cell morphology of Ishikawa treated with M2-PPT-CM appeared irregular and longer,and the microtubule and actin fiber appeared more organized.The expression of the epithelial marker(E-cadherin)in Ishikawa treated by M2-PPT-CM were significantly decreased,while mesenchymal markers(N-cadherin,Vimentin)and transcription factor Twistl were evidently increased.3.The mRNA level of CCL18 increased nearly 12-fold in the PPT-treated group compared with that in the control,and blocking ERa with MPP reversed the PPT-induced CCL18 upregulation.The secretion of CCL18 protein in the PPT-treated group significantly increased nearly 2-fold compared with the control,and MPP inhibited PPT-induced CCL18 upregulation.PPT increased the p-ERK1/2 protein levels of M2 macrophages.The inhibitor of ERK1/2 apparently reversed PPT-induced CCL18 secretion.4.Treatment with rCCL18 for 12 h promoted the migration and invasion of Ishikawa cells.The morphology of Ishikawa treated with rCCL18 appeared irregular and longer,and the microtubule and actin fiber appeared organized.E-cadherin was significantly decreased in Ishikawa treated with rCCL18 and N-cadherin,Vimentin and Twistl were significantly increased.Furthermore,these effects of M2-PPT-CM could be inhibited by an anti-CCL 18 antibody.Similarly,these data could be replicated in another endometrial cancer cell line KLE cells.The data that used CM from PBMCs-derived M2 macrophages were in accordance with that using THP-1 condition medium.Conclusions1.ERa agonist induced CCL18 secretion by activating the ERK1/2 pathway.2.rCCL18 induced EMT of endometrial cancer cells.3.ERa agonist-treated THP1-derived M2 induced EMT of endometrial cancer cells via CCL18 secretion.4.Human peripheral blood monocytes-derived M2 macrophages treated with ERa agonist induced EMT of endometrial cancer cells.PAR?M2 macrophages ERa promoted migration and invasion of endometrial cancer cells by CCL18/mTOR/KIF5B-mediated epithelial to mesenchymalObjectiveTo explore the underlying mechanism of macrophages ERa-mediated EMT in endometrial cancerMethods1.We performed qRT-PCR to examine the mRNA level of KIFs in Ishikawa cultured with rCCL18 or M2-PPT-CM.Western blotting and qRT-PCR were employed to examine the expression of EMT markers in Ishikawa with KIF5B silencing.Transwell assay was employed to investigate the effect of rCCL18 or M2-PPT-CM on the migration of endometrial cancer cells with KIF5B knockdown.Cytoskeleton staining assay was employed to investigate the effect of rCCL18 or M2-PPT-CM on the cell morphology and the microtubule and actin fiber status of endometrial cancer cells with KIF5B knockdown.2.Immunohistochemistry was performed to investigate the relationship between KIF5B level and clinicopathological parameters.We also analyse the relationship between KIF5B expression score and the number of CD68+ macrophages.3.Western blotting was performed to examine the protein phosphorylation level of the PI3K/AKT/mTOR signaling pathway in endometrial cancer cells treated with rCCL18 or M2-PPT-CM.We detected the mRNA level of KIFs in endometrial cancer cell Ishikawa treated with rCCL18 or M2-PPT-CM with or without mTOR inhibitor We employed transwell assay to examine the migration of Ishikawa treated with rCCL18 or M2-PPT-CM with or without mTOR inhibitorResults1.Treatment of Ishikawa cells with rCCL18 or M2-PPT-CM for 24 h dramatically increased KIF5B expression.E-cadherin was increased and N-cadherin,Vimentin and Twistl were significantly decreased in Ishikawa cells with KIF5B silencing than control.M2-PPT-CM or rCCL18 did not influence the migration of Ishikawa cells transfected with KIF5B-siRNA.The effect of M2-PPT-CM or rCCL18 on cell morphology and cytoskeleton status was diminished in Ishikawa with KIF5B silencing.KIF5B expression in tumor tissue was apparently higher than control(P<0.001).Furthermore,the KIF5B staining intensity of tumor tissue was positively correlated with FIGO stage(P<0.01).The counts of CD68+ macrophages was positively correlated with KIF5B expression scores in the epithelial compartment of endometrialcancer(r=0.442,P<0.001).2.The p-AKT and p-mTOR protein levels in Ishikawa cells cultured with rCCL18 or M2-PPT-CM for 2 h were upregulated.The mTOR inhibitor significantly counteracted KIF5B upregulation induced by rCCL18 or M2-PPT-CM.Conclusions1.We are the first to discover the correlation between KIF5B and EMT.CCL18 from M2-PPT-CM induced EMT in Ishikawa cells by KIF5B.2.KIF5B expression was positively correlated with the progression of endometrial cancer.3.ERa agonist-treated M2 cells induced KIF5B upregulation and EMT in Ishikawa by the PI3K/AKT/mTOR pathway.