The Role Of Prostate Cancer-Derived Exosomesroe Macrophage Polarization

Objective: In this article,we aim to investigate the role of prostate cancer cell-derived exosomes in inducing macrophage polarization and try to find out its principles.Methods:(1)Isolation and identifications of exosomes(PCa-exo)derived from prostate cancer cells : two prostate cancer cells,PC-3M-2B4 and PC-3M-IE8.Exosomes were extracted from conditioned medium by Millipore ultrafiltration.PCa-exos were stained with phosphotungstic acid and observed by transmission electron microscopy;then the particle size of the extracted exosomes were identified by nanoparticle size analysis;specific molecular markers on the surface of exosomes were detected by Western blotting.(2)Detections of macrophage polarization induced by PCa-exos:THP-1 was cultured in a complete medium which contained 10 ng/ml PMA and inoculated into 24-well cell culture plate.After 6 hours,LPS(15 ng/ml)and IFN-?(50 ng/ml)were used to stimulate macrophages to M1 subtype and IL-4(25 ng/ml)was added to stimulate macrophages to M2 subtype in 48 h.At the same time,the PCa-exos(100 ug/ml)were added into the macrophages and cultured for 48 hour.After that,PCa-exos labeled with green fluorescent PKH67 were added to the macrophages and observed whether exosomes can be absorbed by macrophages;the subtype of macrophages were identified by fluorescent staining with CD206 antibody;the expression of IL-10?IL-1? and other factors in macrophages and their supernatants were detected by q-PCR and ELISA respectively;Western blotting was used to detect the level of phosphorylated protein of mTOR,AKT and STAT3 in macrophage cells after cells treated with different treatments.(3)Analysis of miRNAs in PCa-exos: the expression of microRNAs such as let-7b in the extracted exosomes were detected by q-PCR.(4)The functional experiments of macrophages induced by PCa-exos:transwell and angiogenesis experiment in vitro were used to detect the invasive and proangiogenic ability of macrophages induced by PCa-exos.Results:(1)The results of electron microscopy and nano-particle size analysis showed that exosomes could be proposed by ultrafiltration.The morphology was mostly round and typical tea-like structure with a diameter of 40-160 nm.The surface-specific molecular markers of exosomes,CD63 and TGS101 were detected on the surface of PCa-exos by western blotting;(2)The exosomes could be largely absorbed by macrophages within 24 hours,the uptake rate of 2B4-exos and IE8-exos by macrophages were about 70%,and 64% respectively;the expression of CD206 in macrophages induced by PCa-exos were similar to M2 subtype macrophage;the expression of inflammatory factors such as IL-10 and IL-1? in macrophages induced by PCa-exos were consistent with the cytokine expression profile of M2 subtype macrophages;the level of phosphorylated protein of AKT and STAT3 were significantly higher in macrophages induced by PCa-exos than the control group;(3)the results of q-PCR showed that the exosomes were rich in microRNAs such as miR-23 c,miR-451 a and let-7b;(4)the cell supernatant from macrophages induced by PCa-exos could promote the invasion and angiogenesis ability of prostate cancer cells by transwell and angiogenesis assay in vitro.Conclusions: Millipore ultrafiltration could be used to extract the PCa-exos that conformed to the experimental requirements;PCa-exos could induce macrophages into M2 subtype;2B4-exo and IE8-exo were rich in microRNAs that may induce macrophage polarization to the M2 subtype by activating the expression of P-AKT/P-STAT3 protein in macrophages;the cell supernatant from macrophages induced by PCa-exos promoted the invasion and angiogenesis of prostate cancer cells PC-3M-2B4 and PC-3M-IE8 in vitro.