Effect Of Hypoxia-induced Pancreatic Cancer Cells On Tumor Angiogenesis Via Exosome Pathway

Pancreatic cancer is one of the most common gastrointestinal cancers in the world,and also a deadly disease with a very high degree of malignancy.The median survival rate is about 3-6 months,and the 5-year survival rate is less than 5%.Currently,the best treatment for pancreatic cancer has not been treated by surgery.However,based on the clinical characteristics of pancreatic cancer,the optimal surgical period is often missed.Meanwhile,radiotherapy and chemotherapy are not good for pancreatic cancer treatment,resulting in a very poor prognosis of pancreatic cancer.Therefore,to further explore and understand the molecular mechanism of the development of pancreatic cancer,and to explore new therapeutic directions and targets are crucial to improve the prognosis of pancreatic cancer.Exosomes is a composed of double membrane with cell secretion of 30-100 nm in diameter utricle bubble,its internal contains a protein,nucleic acids,lipids,participate in the cells in the process of communication,such as immune response,in recent years found that its development is closely related with the incidence of pancreatic cancer,through influence the information transfer between cells involved,to participate in the regulation of tumor microenvironment,impact resistant tumor cells,such as adjusting the EMT process.Hypoxia microenvironment has been proved to be one of the characteristics of most solid tumors and plays an important role in the occurrence and development of tumors.Hypoxia in local microenvironment promotes the proliferation,invasion and metastasis of tumor cells by changing the metabolism of cells.Objective: Extraction and identification of exosomes from pancreatic cancer and compare differences of exosomes from different sources of pancreatic cancer.To compare the effects of exosomes from different sources on proliferation and migration of vascular endothelial cells.To compare the effects of exosomes from different sources on tumor angiogenesis in pancreatic cancer and identifying mechanisms.It provides a basis for the study of pancreatic cancer angiogenesis and provides a new direction for the anti-angiogenesis therapy.Methods: Under the same cell density,PaTu-8988 cells of pancreatic cancer were cultured for 48 h under hypoxic(94%N2?5%CO2?1%O2)and normoxic conditions in the Serum medium without exosomes.The supernatant of cell culture was collected,Exosomes in the medium were separated by exosome extraction kit.The proliferation of HUVECs cells was detected by CCK-8 assay under the influence of exosomes from different sources.Themigration ability of HUVECs cells was detected by Transwell experiment and cell scratch.The angiogenic ability of HUVECs cells under different conditions was detected by angiogenesis experiments.The expression of VEGF-A and the activation of AKT/m-TOR signaling pathway were detected by western-blot.Results: 1.The size of Patu-8988 exosomes was 30~100 nm and expressed CD9,CD63,CD81 exosomes surface markers.2.Hypoxia-induced pancreatic cancer cells secrete more exosomes than those in the normoxic environment.3.Patu-8988 exosomes promoted the proliferation,migration and angiogenesis of HUVECs cells,and hypoxia-induced patu-8988 exosomes had a stronger promoting effect.4.Meanwhile,wstren-blot results showed that exosomes of PaTu-8988 cells could promote the up-regulated expression of VEGF-A in HUVECs cells,and the phosphorylation of AKT and m-TOR,among which hypoxia-induced exosomes PaTu-8988 cells have stronger promoting effect.Conclusion: 1.The concentration and protein expression of exosomes secreted by hypoxia-induced pancreatic cancer cells was higher than that secreted by Normoxia;2.Hypoxia-induced pancreatic cancer cells can promote the proliferation,migration and angiogenesis of HUVECs cells through exosomes;3.Hypoxia-induced pancreatic cancer exosomes may up-regulate the protein expression of VEGF-A to promote tumor angiogenesis by activating the phosphorylation of AKT/m-TOR.