Isolation, Screening And Verification Analysis Of Serum Exosomal Differential Proteins Related To Breast Cancer Metastasis

Objective: To isolate,screen,and validate differentially expressed proteins in serum exosomes of patients with metastatic and non-metastatic breast cancer.Methods: 25 serum samples from patients with lymph nodes or distant metastasis(M(+)group)and 25 samples without lymph nodes or distant metastasis(M(-)group)were collected.Serum exosomes were isolated using the EIQ3 kit.The presence of CD9 and CD63 markers of 8serum exosomes samples selected from two groups were confirmed by western blotting,while the size of these serum exosomes samples were meaured by Nanosight Tracking Analysis.Label free quantitative proteomic mass spectrometry and bioinformatics analysis were used to identify the differentially expressed proteins in serum exosomes of breast cancer patients,which were also quantified by Western blot.Results: The average particle size of the serum extract is between40-150 nm and the expression of CD9 and CD63 were positive.There is a large amount of protein expression between 35 and 75 KD,among which the difference is obvious in the range of 35-48 KD.A total of 301 proteins were identified by mass spectrometry,and 16 differential proteins were screened.GO analysis was performed on 16 differential proteins.The major functional categories emerged in exosomes are complement activity and protease inhibitor activity.Majority of serum exosomal proteins were found to be from exosomes.We found that the most differential proteins are contribute to protein metabolism.The levels of exosomal vitronectin(VN)and Kallistatin(KAL)was increased in M+ compared to those serum exosomes in M-,while the levels of exosomal Obscurins(OBSCN)was decreased.By expanding the sample size,three proteins were significantly different by Western blotting.Conclusion: There are differential proteins in the serum exosomes of metastatic breast cancer and non-metastatic patients.The exosomal proteins VN,KAL and OBSCN have significant differences,which may provide a basis for the diagnosis and treatment of breast cancer metastasis.