Prostate Cancer Cell Derived Exosomes Promote Metastasis By Regulating The Function Of Stromal Cells In Tumor Microenvironment

Prostate cancer is a serious disease threatening men's health worldwide and it is the second and third cause of cancer death in the United States and Europe respectively.In the late stage,prostate cancer develops into metastatic castration resistant prostate cancer(m CRPC)and it is the major death cause of prostate cancer.Therefore,studying the mechanism of prostate cancer metastasis is of great significance for the prevention and treatment of prostate cancer.Exosomes are extracellular vesicles with a diameter of 30-150 nm,carrying information such as DNA,RNA and proteins from host cells,which can mediate signal exchange between local and distant organs.Exosomes can comprehensively regulate tumor development by promoting tumor cell proliferation and invasion,regulating the function of stromal cells and immune cells,and promoting the formation of distant pre-metastasis niche,while the most important part is that exosomes can promote the stromal cell differentiating into cancer associated fibroblast.Studies have found that exosomes derived from hepatoma cells ? pancreatic carcinoma cells and lymphoid leukemia cells can promote the transformation of stromal cells into cancer-associated fibroblasts and regulate the formation of tumor microenvironment.Therefore,our research focuses on elucidating the role of prostate cancer cell-derived exosomes in the conversion of normal prostate stromal cell into CAF and its mechanisms.1.Methods: The LNCa P-AI+F cell-derived exosomes were extracted by ultracentrifugation and QIAGEN membrane affinity column method.The morphological structure,Zetaview particle size analysis and protein identification were used to compare the characteristics of the two methods.The exosomes derived from PC3 M and DU145 prostate cancer cells were extracted by ultracentrifugation.The morphology of exosomes was observed by electron microscopy.The diameter distribution and concentration of the extracted particles were detected by Zetaview particle size analysis.The expression of exosome marker proteins(CD63 and ALIX)and the target proteins(EGFR and SRC)were identified by Western blot.Results: Typical structure of exosomes isolated by ultracentrifugation and membrane affinity column were both observed by electron microscope.But the membrane affinity column method co-precipitated some protein polymers which can be captured by electron microscope.The Zetaview particle size distribution results of both methods are consistent with the literature description.Western blot showed that the bands of ALIX,EGFR and ?-actin of membrane affinity column were weaker than those of ultracentrifugation.Exosomes of PC3 M and DU145 revealed a typical double membrane structure with a diameter of 100 nm which is consistent with the morphological characteristics of exosomes,Western blot results showed that the exosomes extracted by the three prostate cancer cells all expressed CD63 and ALIX exosome marker proteins.In addition,EGFR and SRC were enriched in exosomes from three prostate cancer cells.Zetaview particle size analysis showed that the size distribution of the extracted exosomes was consistent with the diameter distribution range of exosomes(30-150 nm).2.Methods: The exosomes extracted from three cancer cells were stained with PKH67 and incubated with the stromal cell WPMY-1 at a concentration of 40?g/ml.Confocal microscopy was used to observe the uptake of exosomes by stromal cells;Transwell experiments were used to detect the change of migration and invasion ability of the stromal cell WPMY-1;q PCR was used to detect the expression change of CAF-related molecules secreted by the stromal cell WPMY-1.Results: Green fluorescence of PKH67 was observed in the cytoplasm of WPMY-1 cells after incubation with exosomes which confirmed that three prostate cancer cell derived exosomes could be ingested by WPMY-1 cells.Transwell experiments showed that exosomes promote the migration and invasion ability of WPMY-1 cells significantly;q PCR results showed that the three prostate cancer cell derived exosomes promote the expression of CAF-related molecules IL-8,PDGFB and MMP9 apparently,and the increased expression of MMP9 and PDGFB by exosomes of PC3 M and DU145 could be reversed by the EGFR inhibitor gefitinib.3.Methods:Western blot was used to detect the expression of CAF marker proteins and the phosphorylation of EGFR signaling pathway related proteins while the expression of ?-SMA was also detected by immunofluorescence.Results: LNCa P-AI+F cell derived exosomes had no significant effect on the expression of ?-SMA and Vimentin,but LNCa P-AI+F derived exosomes significantly promoted the phosphorylation of EGFR and ERK1/2 of WPMY-1 cells;Exosome derived from PC3 M could enhance the expression of Vimentin while exosomes derived from DU145 cells could promote the expression of ?-SMA of WPMY-1 cells.Besides,PC3 M and DU145 cell derived exosomes could promote the phosphorylation of EGFR and ERK1/2 of the stromal cell WPMY-1.Conclusion: Prostate cancer cell derived exosomes enriched of EGFR and SRC proteins could enhance the migration and invasion ability and increase the expression of CAF marker proteins ?-SMA and Vimentin of the stromal cell WPMY-1 by activating EGFR-ERK signaling pathway,which means exosomes could convert the stromal cell into CAF.