Formation Of Ovarian Cancer Pre-metastatic Niches And Related Molecule Mechanisms

Objective:(1)Through the pre-metastatic niches model of mice,the omental tissues at different time points were selected,and the differential genes related to the microenvironment before ovarian cancer metastasis were screened by RNA-seq method;(2)the collagen triple helix in ovarian cancer tissues Correlation analysis of protein 1(CTHRC1)and M2 macrophage infiltration density on clinical data;(3)cytologically verifying the mechanism of CTHRC1 promoting tumor-associated macrophage polarization and its biological behavior in ovarian cancer influences.Methods:(1)C57BL/6 female ovarian cancer cell ID8 cells were used,lentivirus was stably transfected with tdtomato/luciferas,C57BL/6 female mice were intraperitoneally injected,and 24 hours,3 days,10 days,and 21 days were selected for injection.In vivo imaging of animals,the mice in the 24-hour group were sacrificed,the omental tissue was dissected,DAPI staining,confocal microscopy imaging;anatomical control group,24-hour group,3-day group,21-day group omental tissue,fixed paraffin embedding,HE staining,observation under microscope;(2)RNA-seq of mouse omental tissue at different time points,heat map and KEGG analysis for differential gene analysis;(3)detection of normal ovarian tissue by immunohistochemistry The relationship between the expression of CTHRC1 in ovarian cancer tissues and the tumor-associated macrophage infiltration density of CD68+CD163+ in cancer tissues;(4)Density gradient centrifugation of human peripheral venous blood using lymphocyte separation solution,extracting the tunica layer,and then applying Macrophage colony-stimulating factor(M-CSF)was induced to obtain macrophage Mφ;(5)ovarian cancer cell line SKOV3 was selected,and lentivirus stably transfected to construct CTHRC1 knockdown and overexpression group cell lines,and recombinant protein r CTHRC was applied.1 and tumor cell culture supernatant was co-cultured with M-CSF-inducedhuman peripheral blood mononuclear cells;Western blot,flow cytometry and RT-PCR were used to detect the expression of surface markers CD163 and CD206 on M2 macrophages;(6)Western blot analysis was used to detect the expression of βcatenin and p STAT6 in Mφ induced by M-CSF;(7)Co-culture of macrophages with tumor cells SKOV3 and HO8910,and detection of proliferation of ovarian cancer cells by CCK8,Transwell and cell adhesion assays Changes in invasiveness,migration,and adhesion.Results:(1)After intraperitoneal injection of tumor cells,in vivo imaging of small animals showed that the fluorescence signal in mice increased with the increase of days.On the third day,the fluorescent signal position in the body began to concentrate in the upper abdomen;(2)Transcriptome sequencing There were 22 differential genes in the formation of microenvironment before ovarian cancer metastasis.The expression of CTHRC1 increased in 24 h vs con group and day3 vs 24 h group,indicating that CTHRC1 plays a role in the formation of microenvironment before metastasis.;(3)CTHRC1 expression and CD68+CD163+ tumor-associated macrophage infiltration density It was closely related to tumor stage(P=0.002).At the same time,the expression of CTHRC1 was positively correlated with the infiltration density of CD68+ macrophages and CD163+M2 tumor-associated macrophages(P=0.029,P=0.004);recombinant protein r CTHRC1 After co-culture with tumor cell supernatant and MCSF-induced macrophages,Western blot,flow cytometry and RT-PCR were used to detect the expression of surface markers of M2 macrophages.Compared with the control group,r CTHRC1 group and The expression levels of CD163 and CD206 in the overexpressed CTHRC1 group increased significantly(P<0.05).(4)Western blot analysis of macrophages after treatment with r CTHRC1 and culture supernatant,compared with the control group,overexpression group and recombinant protein group The expression of βcatenin(P<0.05)and STAT6 phosphorylation(P<0.05)were significantly increased.(5)The invasiveness and migration ability of ovarian cancer cells were significantly enhanced after co-culture with Mφ(P<0.05).Conclude: By establishing a model of pre-metastatic niches in C57BL/6 mice,the differentially expressed genes associated with the pre-metastatic microenvironment of ovarian cancer were screened,and CTHRC1 with a large difference in expression was further explored;CTHRC1 was highly expressed in ovarian cancer,and The expression of CTHRC1 is closely related to the infiltration density of CD68+CD163+ tumorassociated macrophages,suggesting that the expression of CTHRC1 is consistent with tumor-associated macrophage infiltration,which is closely related to the staging of ovarian cancer,and at the molecular level,CTHRC1 overexpression promotes tumorassociated macrophage polarization,possibly by further enhancing STAT6 phosphorylation by interacting with Wnt/βcatenin,and is involved in enhancing the invasion and metastasis of ovarian cancer cells.