Study On The Biological Effect And Mechanism Of Tumor-associated Macrophage Exosomes On Breast Cancer Cells

As one of the most abundant immune cells in the tumor microenvironment,the information exchange between macrophages and tumor cells has become the focus of basic research in cancer.However,research on the effects of tumor-associated macrophage-derived exosomes on breast cancer cells is rare.To this end,we focused on the biological behavioral regulation of tumor-associated macrophageexosome-breast cancer cells,and explored the effects of tumor-associated macrophages derived exosomes on proliferation,migration,invasion and apoptosis of breast cancer cells.The research can not only enrich the understanding of exosome as a cell communication carrier and its important role in the shaping of tumor microenvironment,but also lay a theoretical foundation for screening related molecular targets for early diagnosis,drug efficacy evaluation and prognosis of clinical tumor patients.ObjectiveTo analyze the effects of tumor-associated macrophages and their derived exosomes on the biological behavior of breast cancer cells;To bioinformatically analyze the mechanism of tumor-associated macrophage exosomes on the biological behavior of breast cancer cells.Methods1.The culture supernatant of breast cancer cells(BT549,MDA-MB-231)was used to treat PMA-activated THP-1 cells for 48 h to obtain tumor-associated macrophages;Flow cytometry was used to detect the expression of tumor-associated macrophage markers CD68 and CD206;CCK-8 method,cell scratch,transwell and flow cytometry were used to detect the effects of tumor-associated macrophage culture supernatant on proliferation,migration,invasion and apoptosis of breast cancer cells(BT549,MDA-MB-231).2.Extraction of tumor-associated macrophage exosomes by ultracentrifugation;Western blotting,transmission electron microscopy,nanoparticle tracking analysis(NTA)and other methods were used to determine exosome markers,morphology and particle diameter;Tumor-associated macrophage exosomes were marked with PKH67,and inverted fluorescence microscopy was used to observe the uptake of exosomes in breast cancer cells(BT549,MDA-MB-231);CCK-8 method,cell scratch,transwell,flow cytometry and other experimental methods to detect the impact of tumor-associated macrophage exosomes on proliferation,migration,invasion and apoptosis of breast cancer cells(BT549,MDA-MB-231).The effects of exosome release inhibitor(GW4869)on the biological behavior of breast cancer cells(BT549,MDA-MB-231)were examined.3.High-throughput sequencing detection were used to detect the m RNA expression in breast cancer cells(BT549,MDA-MB-231)treated with tumor-associated macrophage exosomes;GSEA analysis analyzed enrichment pathways in two breast cancer cell lines;Co-differentially expressed genes in two breast cancer cell lines were screened by R language;m RNA and mi RNA expression data in breast cancer and paracancerous specimens were downloaded from the TCGA breast cancer clinical specimen database;R language shows the difference in m RNA expression of co-differentially expressed genes between cancer and adjacent cancer in breast cancer clinical specimens;The K-M Plotter database presented the survival analysis curves of co-differentially expressed genes;q-PCR further verified and screened the differentially expressed genes;Target Scan and mi RWalk databases screened upstream regulatory mi RNAs for co-differentially expressed genes;R language calculates the correlation between m RNA and mi RNA expression of TCGA breast cancer specimens.Results1.Flow cytometry showed that the number of CD68 and CD206 double positive cells in tumor-associated macrophages was significantly increased compared with the control group(P<0.01).The CCK-8 method showed that tumor-associated macrophages had no significant effect on the proliferation of breast cancer cells(BT549,MDA-MB-231)compared with the control group;Cell scratches,transwell migration and invasion experiments showed that tumor-associated macrophages could promote the migration and invasion of breast cancer cells(BT549,MDA-MB-231)compared with the control group(P<0.01).Flow cytometry showed that tumor-associated macrophages inhibited apoptosis of breast cancer cells(BT549,MDA-MB-231)compared with the control group(P<0.05).2.After extracting tumor-associated macrophage exosomes by ultracentrifugation,immunoblotting revealed that CD63 and TSG101 were expressed in tumor-associated macrophage exosomes;Transmission electron microscopy and nanoparticle tracking analysis showed that the tumor-associated macrophage exosomes exhibited disc-like and bilayer membrane structure and the particle diameters were mostly distributed between 140 and 160 nm.Exosome fluorescent labeling uptake experiments showed that PKH67-labeled tumor-associated macrophage exosomes were significantly uptaken by breast cancer cells and distributed in the cytoplasm;CCK-8 assay showed that tumor-associated macrophage exosomes had no significant effect on the proliferation of breast cancer cells(BT549,MDA-MB-231)compared with the control group;Cell scratches,transwell migration and invasion experiments showed that tumor-associated macrophage exosomes significantly promoted migration and invasion of breast cancer cells(BT549,MDA-MB-231)compared with the control group(P<0.01).Moreover,this effect can be eliminated with the use of exosome release inhibitor(GW4869)(P<0.01);Flow cytometry showed that tumor-associated macrophage exosomes had no significant effect on apoptosis of breast cancer cells(BT549,MDA-MB-231)compared with the control group.3.High-throughput sequencing results show that the available data of the original data are as high as 96%,and the matching rate with the human genome is over 98%.The control group and the experimental group are basically separated.Compared with the control group,there were 347 up-regulated genes and 222down-regulated genes in the experimental group of BT549 cell line;173 up-regulated genes and 157 down-regulated genes in the MDA-MB-231 cell experimental group;GSEA analysis of two breast cancer co-enrichment pathways are interferon-αresponse and interferon-γ response markers;There were 10 co-differentiated genes in the two breast cancer cell lines,2 of which were non-protein-encoding genes,and 8protein-encoding-genes: PCDHGA1,CDK2AP1,PROSER1,ITGβ4,SLC25A28,WDR6,DUSP19 and GMFB.The TCGA database showed that except for WDR6,the other seven co-differentiated genes were significantly different in breast cancer and adjacent tissues(P<0.01).K-M Plotter database analysis shows that there was a significant correlation between the expression of 8 co-differentiated genes and the survival rate of patients(P<0.01).The results of q-PCR showed that PCDHGA1,ITGβ4 and GMFB were significantly different in BT549 cells,while ITGβ4,GMFB and SLC25A28 were significantly different in MDA-MB-231 cells.Based on the results of the above data analysis combined with Target Scan and mi RWalk database information,Two co-differentiated genes and their possible upstream regulatory negatively related mi RNA molecules,including ITGβ4/mi R-190 b and GMFB/mi R-660,were identified for TCGA analysis,patient survival analysis,and q-PCR.Conclusion1.Tumor-associated macrophages induced by breast cancer cells exhibit an M2 polarization phenotype.2.Tumor-associated macrophages can promote the migration,invasion and inhibition of apoptosis of breast cancer cells BT549 and MDA-MB-231.3.Tumor-associated macrophage exosomes can promote the migration and invasion of breast cancer cells BT549 and MDA-MB-231.4.The results of high-throughput sequencing and q-PCR showed that the promotion of tumor-associated macrophages on breast cancer cell migration and invasion was closely related to the dysregulation of genes such as ITGβ4 and GMFB.