Exosomal CAV1 Discovered In Human Lung Carcinoma Tissues As A Potential Tool For Diagnosis Of Lung Cancer

BACKGROUND&OBJECTIVELung cancer is the highest morbidity and mortality disease among malignant tumors.However,the current diagnostic methods for lung cancer have such disadvantages as low sensitivity,poor specificity and invasive,so it is urgent to establish convenient,sensitive and non-invasive methods for the early diagnosis and differential diagnosis of lung cancer.The exosomes released by the cells are 30-200nm vesicles containing specific biologically active molecules such as proteins,nucleic acids and lipids.Studies have shown that under various physiological conditions,exosomes can accurately reflect the pathological state of secretory cells and are easy to sample.Therefore,exosomes have become a new type of clinical diagnostic markers with great potential.Tissue-derived exosomes respond more specifically to the pathological status of the source cells,which has broader prospects in screening exosomal biomarkers.In this study,we isolated lung cancer tissue-derived exosomes and used proteomics to screen for differential proteins in exosomes,and nanoflow cytometry was used to verify the serum exosomal protein of lung cancer patients in order to find diagnostic markers for lung cancer.METHODS1.Isolation of tissue derived exosomes1.1 Establish a method for isolating tissue derived exosomes.1.2 Identification of exosomes using transmission electron microscopy,NTA,and Western blot experiments.2.Quantitative results of tissue derived exosomal proteomics obtained by LC-MS/MS.3.Validation of serum exosomal proteins by nanoflow cytometry.4.Statistical analysis.5.Cell experiments5.1 Cells and exosomes overexpression,co-culture,cell proliferation,migration experiments.5.2 Exosome uptake experiment.5.3 R18 exosomal membrane fusion experiment.RESULTS1.Tissue-derived exosomes were identified using transmission electron microscopy,NTA,and Western blot.Lung tissue-derived exosomes were obtained with high purity.2.Quantitative results of proteomics were obtained using LC-MS/MS technology,and signal pathway prediction and function analysis were performed on some proteins with different expressions.Combining the results of the TCGA database and HPA database,preliminary screening had potential CAV1 is a protein molecule used in the early diagnosis of lung cancer and the differential diagnosis of lung cancer and benign lung disease.3.371 Serum samples were verified using nanoflow cytometry.Compared with patients with benign lung disease and healthy control,exosomal CAV1 expression was significantly down-regulated in patients with lung cancer.The AUC was 0.957 and 0.879(p<0.05).4.The results showed that the diagnostic efficacy of exosomal CAV1 is higher than traditional lung cancer markers and imaging diagnostic methods,and the specific expression of exosomal CAV1 in lung cancer patients is significantly reduced,while detecting free CAV1 protein in serum alone has no ability to distinguish lung cancer.It reflects the sensitivity and specificity of exosomal CAV1 in the diagnosis of lung cancer.5.The results show that CAV1 is highly expressed in tumor cell exosomes.Exosomes overexpressing CAV1 can inhibit cell proliferation and migration after co-culture with tumor cells.Immunofluorescence experiments and membrane fusion experiments with R18 dyes showed that tumor cells can take up exosomal CAV1 and retain their membrane surface protein CAV1 on the tumor cell membrane through membrane fusion,and can affect the expression of EGFR on the cell surface.CONCLUSION1.There are differences in the expression of exosomal protein from lung carcinoma tissues and para-carcinoma tissues,the CAV1 membrane protein was significant differences.2.Serum derived exosomal CAV1 is significantly reduced in patients with lung cancer,and its diagnostic efficacy is superior to existing traditional biomarkers and imaging methods.3.The exosomal CAV1 secreted by lung cancer cells is significantly reduced.Exosomal CAV1 fuse with cell membrane to enter into tumor cells,which in turn affects the CAV1 level on the cell membrane surface.4.CAV1 can regulate the expression of EGFR on cell membrane surface.