The Role Of Pancreatic Cancer-Derived Exosomes In Liver Metastasis

Objective The aim of this study were to extract and purify the exosomes derived from isogenic pancreatic cancer cells with different metastatic potential and investigate the effect of exosomes derived from highly metastatic potential pancreatic cancer cells on the invasion and metastasis of low metastatic potential pancreatic cancer cells in vitro.We established a liver pre-metastasis niche(PMN) and surgical orthotopic implantation(SOI) metastatic model and investigate the cellular and molecular mechanisms of pancreatic cancer-derived exosomes in inducing liver PMN and promoting hepatic metastasis in vivo.We screened the liver metastasis-associated proteins to provide a theoretical basis for the invasion and metastasis and find a new target for the diagnosis and treatment of pancreatic cancer in the future.Methods Purified exosomes were isolated using ultracentrifugation combined with sucrose density gradient centrifugation.The morphological and structural proteins of exosomes were identified by transmission electron microscopy(TEM) and Western Blot.The BCA assay was used to determine the protein concentration of both exosomes.The MTT cell adhesion,wound-healing,and transwell invasion assays were used to observe the characteristics of invasion and metastasis affected by exosomes derived from high metastatic potential pancreatic cancer cells.C57BL/6 mice were injected intravenously with two types of cell-derived exosomes,the confocal microscopy,immunofluorescence,western blot and flow cytometry were used to compare the distribution of the two exosomes in mice and the effects of inducing liver PMN.C57BL/6(SOI) metastatic mice were injected intravenously with two types of cell-derived exosomes,the H&E staining,immunohistochemistry and masson staining were used to observe the cellular and molecular mechanisms of exosomes promoting hepatic metastasis of pancreatic cancer in vivo.We extracted the proteins of exosomes from two different metastatic potential cells and identified the protein components by iTRAQ quantitative proteomics.We screened the proteins differentially expressed between high-and low-metastatic exosomes,and further performed bioinformatic analysis and validated by Western Blot.Results We successfully extracted and purified exosomes from pancreatic cancer cell culture supernatant by ultracentrifugation combined with sucrose density gradient centrifugation.Sizes and morphological characteristics of exosomes derived from two pancreatic cancer cell lines were assessed via TEM.Exosomes from two cell lines were cup-shaped and ranged from 50~150 nm in diameter.Western Blot confirmed the presence of classical common exosome markers TSG101,CD9,and MHC-I.Our results in vitro show that Panc02-H7 EXO are easily taken up by Panc02 cells.We found that Panc02-H7 EXO decreased adhesion,increased migration,invasion and upregulated the expression of CXCR4and MMP-9 in recipient cells.Pancreatic cancer-derived exosomes accumulated in the lung,liver,and spleen,with less accumulation in the brain and bone marrow,Panc02-H7 EXO accumulated at higher levels in the lung,liver,and bone marrow than Panc02 EXO.Panc02-H7 EXO and Panc02 EXO can mobilize and recruit CD11b~+ and CD45~+ hematopoietic progenitor cells,activateα-SMA~+ hepatic stellate cells,and induce liver S100A8 and S100A9 expression in liver PMN,but the effect of Panc02-H7 EXO is stronger than Panc02 EXO.Panc02-H7 EXO and Panc02 EXO can promote the invasion and metastasis of Panc02 cells in vivo,by recruitting F4/80~+ macrophages,α-SMA~+ hepatic stellate cells and neutrophils,and inducing inflammatory cytokines Fibronection,S100A8 and S100A9 upregulation and collagen deposition in the liver microenvironment.4,517 proteins were identified via iTRAQ-based proteomic analysis,79 proteins were screened as differentially expressed proteins,among them 33 proteins were up-regulated and 46 were down-regulated.Bioinformatics analyses showed that most of the differentially expressed proteins were involved in pancreatic cancer growth,invasion,and metastasis,and that metabolism-related signaling pathways were involved in exosome-mediated intracellular communication.Conclusion The methods of extracted and purified exosomes from pancreatic cancer cell culture supernatant by ultracentrifugation combined with sucrose density gradient centrifugation are simple and effective.Highly metastatic potential pancreatic cancer-derived exosomes are easily taken up by the recipient cells and can significantly reduce the adhesion properties and increase their ability to migrate and invade.Pancreatic cancer cell-derived exosomes have the characteristics of organophilic distribution and are effective in inducing hepatic inflammation and fibro-metastasis microenvironment formation.High metastatic potential pancreatic cancer-derived exosomes promote the growth,invasion,and metastasis of SOI pancreatic cancer in vivo.Metastasis-associated proteins screened by iTRAQ quantitative proteomics,which involved in the molecular functions and signaling pathways may play an important role in the invasion and metastasis of pancreatic cancer.The specific mechanism and clinical value need further study.