Proteomic Analysis And Metastasis Mechanism Of Breast Cancer-derived Exosomes

The recurrence and metastasis of breast cancer is the main cause of death of breast cancer patients.The main metastatic sites of breast cancer are lung,liver,bone and brain.Before metastasis,a series of molecular and cellular changes occur in distant organs,forming a pre-metastatic niche,providing a suitable growth environment for tumor cells in blood circulation,promoting the colonization and growth of tumor cells,and forming metastatic lesions.Exosome is a subtype of extracellular vesicles,which has important biological functions.For example,exosome secreted by tumor cells will be transported to distant organs through blood in which lipids,proteins or nucleic acids will directly or indirectly affect the phenotype of receptor cells.The purpose of this study is to identify and analyze the proteomics of exosomes from breast cancer cell lines and plasma of breast cancer patients based on mass spectrometry,and to further explore the mechanism of highly invasive breast cancer cell line exocrine bodies in metastasis.We mainly used electrospray mass spectrometry(ESI-MS)technology,based on the research strategy of "bottom-up"(bottom-up),to hydrolyze the exosomes extracted by ultracentrifugation into peptides.The ion information of peptides and fragments were collected by mass spectrometry,and the peptide signals were compared with the known sequences in the database to get protein information.We identified and analyzed the exosome proteomic data of triple negative breast cancer cell line MDA-MB-231,estrogen receptor positive breast cancer cell line MCF-7 and normal breast epithelial cell line MCF-10A,screened the candidate protein EphA2,and preliminarily explored the effect of EphA2 in breast cancer exosome on vascular endothelial cells.The increase of vascular endothelial barrier permeability promoted the occurrence of tumor metastasis.Our results showed that breast cancer exosome EphA2 protein promoted the migration,permeability of human umbilical vein endothelial cell line and angiogenesis in vitro.Western Blot showed that after incubation with EphA2-rich exosomes,tight junction related proteins ZO-1 and occludin in whole cell lysate of HUVEC cells were down-regulated,while RhoA protein was up-regulated.Western Blot also showed that the expression of EphA2 in exosome from plasma of breast cancer patients was higher than that in healthy controls.Therefore,we believe that highly invasive breast cancer cell lines regulate vascular endothelial cell permeability by secreting exosomes carrying EphA2 protein,which is a potential molecular mechanism for regulating breast cancer metastasis.