The Effect Of Macrophage Polarization On Metastasis Of Ovarian Cancer

Objective:To investigate the effects of macrophages with different polarization states on the metastasis of epithelial ovarian cancer and its possible regulatory mechanisms and related signaling pathways.Methods:1.The BALB/C mice were sacrificed by cervical dislocation.Bone Marrow-derived Macrophage(BMDM)was extracted from bilateral tibia and fibula bone marrow.The cells were cultured using macrophage colony stimulating factor(M-CSF).They were stimulated with lipopolysaccharides(LPS),interferon-γ(IFN-γ)and interleukin-4(IL-4),respectively,and cultured to obtain M1 and M2 type macrophages.2.Flow cytometry was used to identify M1 and M2 macrophages.Macrophages were identified by F4/80 and CD11b surface markers.M1 macrophages utilized CD11c marker.M2 macrophages utilized CD206 marker.Designing 9 pairs of qRT-PCR primers,identifying M1 and M2 type macrophages by qRT-PCR method,in which M1 macrophages IL-1β,TNFα,IL6 and iNOS expression increased,and Arg-1,IL-10,TGF,Pparg and MCP-1 expression increased in M2 macrophages.3.Establishing the 0.4μM Transwell co-culture system,co-cultured with ovarian cancer cells in different macrophage polarization systems M1 and M2.The research contents include:(1)Scratch healing test,Transwell observation of ovarian cancer cell migration ability;(2)Western blot,Real-time PCR and other methods to detect E-cadherin,Vimentin and other EMT molecular phenotype changes,(3)Western blot and other methods to detect EMT-related transcription factors such as Twist,Snail1 protein.4.Using poly-HEMA coated 6-well plates,ovarian cancer cells co-cultured with different macrophage polarization systems were cultured in suspension(ovarian cancer cells in the lower chamber and macrophages in the upper chamber).The cells were collected at 24-48 hours after culture.The apoptosis rate was measured by Annexin V method.The macrophages polarization state was compared to the anoikis of ovarian cancer cells.5.Establishing the 0.4μM Transwell co-culture system,co-cultured with ovarian cancer cells in different macrophage polarization system.The migration ability of ovarian cancer cells was analyzed.Transwell-Matrigel was used to observe the invasiveness of ovarian cancer cells and the changes of focal adhesion kinase (FAK)and matrix metalloproteinases(MMPs)were observed..FAK focuses on the phosphorylation status of p-FAK(Try397).Gelatin zymography and Western blot were used to detect changes in the expression and function of matrix metalloproteinases(MMPs),including MMP2,MMP7,and MMP9.6.Establishment of nude mouse ovarian cancer xenograft model:SK-OV-3 ovarian cancer cells were used,and the concentration was adjusted to 1.0 x 10~7/ml.The cell suspension was injected intraperitoneally with 0.5 ml.Macrophages obtained by the M1 system and the M2 system were induced twice at 1,3 days after the injection.Observe the growth of the tumor and observe the formation of ascites.The nude mice were sacrificed at the appropriate time(about 4-6 weeks),the counts of metastases were observed and ovarian cancer metastasis was evaluated according to the number and quality of metastases.Results:1.BMDM were successfully extracted and cultured to obtain M1 and M2 type macrophages,respectively,and were used in the following experiments.2.Through flow cytometry experiments,the surface markers of CD11c in M1 macrophages induced by differentiation were elevated,and the CD206 markers in M2 macrophages were elevated.The above two methods fully demonstrate that bone marrow macrophages differentiate M1 and M2 macrophages successfully.The results of qRT-PCR showed that the expression of IL-1β,TNFα,IL-6 and iNOS in M1 macrophages increased,and the expression of Arg-1,IL-10,TGF-β, Pparg and MCP-1 in M2 macrophages increased.3.The M1 and M2 type macrophages were co-cultured with ovarian cancer cells and found that the migration of ovarian cancer cells co-cultured with M1 macrophages decreased;the expression of EMT-related molecules E-cadherin,N-cadherin and Vimentin expression decreased;The transcription factors of Twist,Snail1 expression was partially reduced.The migration ability of ovarian cancer cells co-cultured with M2 type macrophages was enhanced;the expression of E-cadherin-related molecules was decreased,the expression of N-cadherin and Vimentin was increased,and the transcription factors Twist and Snail1 was increased.4.There was no difference in the apoptosis rate of ovarian cancer cells in the ovarian cancer cells co-cultured with M1 macrophages.And the apoptosis rate of the ovarian cancer cells co-cultured with M2 macrophages was extent inhibit.5.The M1 and M2 macrophages were co-cultured with ovarian cancer cells,respectively.The results showed that the expression levels of FAK,p-FAK (Tyr397),and MMP7 in ovarian cancer cells co-cultured with M1 macrophages were comparable.Compared with a certain reduction but not all cell lines,the difference in MMP9 was not statistically significant.The expression of FAK, p-FAK(Tyr397)and MMPs in ovarian cancer cells co-cultured with M2 macrophages increased significantly,and the invasion ability of ovarian cancer cells increased.6.Nude mouse ovarian cancer xenograft model results:The control group and M1 macrophage injected nude mice body weight did not change significantly,a small amount of ascites formation,fewer metastases;nude mice injected with M2 macrophages have more ascites formation,more metastases.Conclusions:1.Mice bone marrow macrophages extracted,cultured and differentiated to obtain the experimental needs of M1 and M2 macrophages.2.It has been confirmed that the metastatic ability of non-contacted co-cultured ovarian cancer cells with M1 macrophages has a certain degree of inhibition,but it is not very significant.It is proved that there is a certain relationship with EMT and FAK.The non-contact co-culture of ovarian cancer cells with M2 macrophages showed a significant increase in metastasis capacity.It is shown that the enhancement of ovarian cancer metastasis has certain correlation with EMT, apoptosis inhibition and FAK,MMPs,etc.