Effect Of Exosomal Protein IGFBP3 On Biological Behavior And Prostate Cancer Cell Tumor Microenvironment

Objective:The treatment of prostate cancer after bone metastasis is one of the thorny challenges facing urologists today.Exploring the underlying mechanisms of prostate cancer bone metastasis would be beneficial in reducing the risk of prostate cancer progression.It has been found that tumor cell-derived exosomes,as a key mediator in the development of tumorigenesis,also show a considerable role in prostate cancer bone metastasis.This project aims to investigate the differential expression of serum exosomal proteins before and after bone metastasis and verify their effects on the biological behavior of prostate cancer cells and tumor microenvironment,providing new clues and ideas for the study of the mechanism and treatment of prostate cancer bone metastasis.Methods:1.Detection and validation of differential expression of serum exosomal proteins in patients with prostate cancer.1)Serum exosomes from 5 patients with bone metastases and 5 patients with prostate cancer without bone metastases were extracted,identified and analyzed by protein mass spectrometry to look for differential proteins.2)Ten patients with unmetastatic and metastatic prostate cancer,including those in 1),were selected to extract their serum exosomes and verify the expression of insulin-like growth factor binding protein 3(IGFBP3)by ELISA.3)The relationship between IGFBP3 and the malignancy of prostate cancer was determined by comparing the expression of IGFBP3 in the mass spectrometry results with the Gleason score of the corresponding patients.2.To investigate the effect of exosomal protein IGFBP3 on the biological behavior of prostate cancer cells.1)Osteotriol(IGFBP3 inducer)promoted the production of exosomes from PC3 cells overexpressing IGFBP3,and lentiviral constructs of RM-1 cell line(RM-1-CMV-IGFBP3)overexpressing IGFBP3 and producing the corresponding exosomes.2)The effects of IGFBP3 on the migration,proliferation and invasion of prostate cancer cells were verified by cell scratch assay,cell proliferation assay and cell invasion assay.3.To investigate the effect of exosomal protein IGFBP3 on the microenvironment of prostate cancer tumors.1)To investigate the effect of exosomal protein IGFBP3 on tumor vascular microenvironment by using osteotriol-induced IGFBP3 overexpressing exosomes on human umbilical vein endothelial cells(HUVEC)in the process of tube-formation.2)To investigate the effect of exosomal IGFBP3 on the immune microenvironment of tumors by verifying the effect of exosomes of IGFBP3 induced by osteopontin overexpression on macrophage polarization process.3)To investigate the effect of exosomal protein IGFBP3 on the osteogenic ecological niche of prostate cancer by verifying the interference of RM-1-CMV-IGFBP3 cell exosomes on the interaction between prostate cancer cells and osteoblasts.Results:1)There were 27 differentially expressed proteins in the serum exosomes of patients with and without bone metastases,of which 20 differentially expressed proteins were low after bone metastases and the opposite.The expression of IGFBP3 in serum exosomes was decreased in patients without bone metastases compared with those with bone metastases,and the expression of IGFBP3 was negatively correlated with the malignancy of prostate cancer.2)Osteopontin promoted the expression of IGFBP3 in PC3 cell-derived exosomes,and the promotion effect was most significant at a concentration of 150 nM.After co-culture of osteotriol-induced exosomes with PC3 cells,their ability to promote migration and proliferation of PC3 cells decreased.The lentiviral construct RM-1-CMV-IGFBP3 cell line was found to have decreased migratory,proliferative and invasive abilities after culture.After co-culture with PC3 cells using exosomes derived from the RM-1-CMV-IGFBP3 cell line,the proliferation,migration and invasion ability of PC3 cells were found to be decreased.3)Interference with the tube-forming process of HUVEC by exosomes revealed that the pro-angiogenic ability of induced exosomes was significantly reduced compared to that of uninduced exosomes.In terms of immune microenvironment,RM-1-CMV-IGFBP3 cellular exosomes had a reduced ability to promote the conversion of macrophages(M0)into tumor-associated macrophages(M2)compared to RM-1 cellular exosomes.In terms of osteogenic ecotopes,the detection of cell proliferation marker protein Ki67 after co-culture of RM-1 cells with pro-osteoblasts(MC-3T3-E1)showed increased Ki67 expression in RM-1 cells;compared with RM-1 cell exosomes,RM-1-CMV-IGFBP3 cell exosomes promoted the co-culture of MC-3T3-E1 with RM-1 cells after RM-1 cells Ki67 expression was decreased.Conclusion:1.Decreased expression of serum exosomal protein IGFBP3 in patients with bone metastases from prostate cancer.2.The exosomes of prostate cancer cells overexpressing IGFBP3 have reduced ability to induce migration,proliferation and invasion of prostate cancer cells.3.Exosomes overexpressing IGFBP3 have a reduced ability to construct vascular,immune and osteogenic tumor microenvironments.