Preliminary Analysis Of Exosomes Differential Proteome On Non-small Cell Lung Cancer From Cell Lines And Serum

BACKGROUND&OBJECTIVELung cancer is one of the most common and the deadliest forms of cancers worldwide.Non-small cell lung cancer(NSCLC)is 85%of lung cancer.For lacking an effective and sensitive tool for early detection,most patients already have progressed to middle-late cancer at the time of diagnosis,leaving few if any viable therapeutic options available for effective intervention,so it is important for early detection in NSCLC.Exosomes are nano-sized structures surrounded by a lipid bilayer and they are secreted by most cell types which contain a variety of cell-specific nucleic acids,proteins and lipids.Exosomes function by protecting and transporting biomacromolecule inside them to target cells,and exosomes protein is involved in an important part of the development of disease.The protein in exosomes may be less influenced by high abundance ratios of blood proteins and more stable than serum.Exosomes also carry some specific information,mediated disease development,has the potential for biomarkers.In our study,we will establish a series of isolation purification and identification method for exosomes.Then analyzing exosomes differential proteome on non-small cell lung cancer from cell lines and serum preliminary,and forecasting candidate biomarkers for NSCLC.METHODWe employed a mass spectrometry(LC-MS/MS)quantitative proteomics strategy to examine the different exosomal proteins expression in non-small cell lung cancer(NSCLC).Exosomes,isolated from not only the pooled serum of 8 patients with NSCLC(stages Ⅰ and Ⅱ),8 patients with NSCLC(stage IV)and 12 normal volunteers,but also the cell culture medium of an immortalized normal bronchial epithelial cell line 16HBE and a NSCLC cell line A549,were separated by ultracentrifugation.Written informed consents were obtained from all patients and normal volunteers.RESULT1.Separation and identification of exosomes derived from serum and cell culture mediumTo demonstrate the presence of exosomes,the vesicles isolated from cell culture medium(CCM)of A549 cell lines and serum were determined by Nanosight,TEM and western blot.Nanoparticle tracking analysis(NTA)was used to calculate the total number of and the overall sizes distribution of exosomes.Most of particles about 30-150nm in size,lipid bilayer structure were also found by TEM and the size of particles was also consistent with the description of exosomes.To investigate whether the particles were,at least in part,exosomal in origin,the presence of established exosomal markers was determined using western blot for CD63 and TSG101 and negative control for calnexin.2.Qualitative and quantitative analysis of exosomes derived from serum and cell culture medium3172 exosomal proteins were identified in three pooled serum and two cell lines.The screening standard was Ratio>2 or<0.5,P value<0.05.Ddifferential protein included protein differential expression and protein unique expression.Compared with the serum purified exosomes(SPEs)of normal volunteers,we found 25 proteins upregulated and 33 proteins downregulated in the NSCLC patients(stages Ⅰ and Ⅱ),23proteins upregulated and 27 proteins downregulated in the NSCLC patients(stagesⅢ and Ⅳ).Then 25proteins were unregulated and 26 proteins were downregulated in the NSCLC(stagesⅢ and Ⅳ)patients compared with the SPEs of NSCLC(stages Ⅰand Ⅱ)patients.Next,we found 65 proteins upregulated and 62 proteins downregulated in exosomes derived from two cell lines,507 proteins were only identified in one cell line.3.Differential proteome analysis of exosomes derived from serum and cell culture mediumExosomal protein FGA,FGB,FN1 were different between three pooled serum and two cell lines,and the difference up to Hundreds of times.The differential proteins profile of NSCLC exosomes that suggested exosomal proteins which were related to cell adhesion molecules(CAMs)had an association with tumor metastasis,like MHC2、MHC1、VCAN、CNTN1、ICAM1 and so on.CONCLUSION1.Exosomes were successfully isolated from A549 CCM and serum by ultracentrifugation were identified by Nanosight,TEM and western blot.2.Compared with SPEs of NSCLC patients and healthy volunteers,particles,size and spread distribution had significant differences(p<0.001).SPEs of NSCLC patients had more particles higher spread distribution and bigger size than healthy volunteers.3.The investigation of the NSCLC exosomal proteome has identified enriched protein cargo that may contribute to the metabolic process,ECM-receptor interaction and Cell adhesion molecules(CAMs),which may have potential clinical implications in lung cancer progression.4.We identified several novel candidates that could be utilized as a multi-marker protein panel in next verification,such as FGA,FGB,FN1,SLC2A1,MHC2,VCAN,CNTN1,ICAM1,which may contribute to lung cancer progression.