Studies On The Mechanism Of The Extracellular Vesicles Promoting The Progression Of Lung Adenocarcinoma And Regulating The Mechanical Properties Of Cancer Cells

Background: Lung adenocarcinoma is a common malignancy with poor prognosis.Recent studies have demonstrated that extracellular vesicle(EV)secreted by cancer cells can promote the cancer progression.In recent years,some researches shown that cancer cells’ mechanical properties changed in cancer progression,which indicates the cancer-promoting functions of EVs may be related to the mechanical changes in cancer cells.However,there is no report about the relationship between the cancer promotion through the EVs and regulating the mechanical properties in lung adenocarcinoma cells.This thesis studies the mechanisms of correlation between EVs released by lung adenocarcinoma cells in promoting cancer progression and regulating cancer cells’ mechanical properties,laying a novel insight for the treatment of lung adenocarcinoma.Methods: EVs were extracted fromthe supernatant of lung adenocarcinoma cells(H1299 cells and A549 cells)via ultracentrifugation combined with ultrafiltration.The EVs were defined as H1299-EVs and A549-EVs,respectively.The morphologies and sizes of EVs were identified by scanning electron microscopy(SEM)and atomic force microscopy(AFM).The markers of EVs were determined by Western blot(WB).AFM was used to measure the young’s modulus and adhesion of H1299 cells,A549 cells and BEAS-2B cells.Based on co-cultured EVs and cancer cells,the EVs promoting cancer activities and changed in the cancer cells’ mechanics were analyzed.MTT assay,colony formation assay,wound healing assay,and cell invasion assay were performed to evaluate the cellar viability,clone formation,migration and invasion ability,respectively.AFM was used to measure the Young’s modulus and adhesion of cells.SEM and immunofluorescence were used to observe filopodia and F-actin,respectively.The differential gene expression of H1299 cells before and after co-culture with H1299-EVs was compared by mRNA sequencing.The possible signal pathways were analyzed and the protein expression was detected by bioinformatics analysis and WB,respectively.After treatment with LY294002,the effects of H1299-EVs on cellular activity,clone formation,migration,invasion of H1299 cells and the expression level of the proteins were detected again.The young’s modulus,adhesion,filopodia,and distribution of F-actin were measured.A subcutaneous xenograft tumor model in nude mice was established by H1299 cells,and H1299-EVs and LY294002 were injected,respectively.The weight and volume of the tumors in each group were assessed.Histological sections of the tumors were stained with HE.The target protein expression levels were examined by extracting primary cancer cells fromprimary tumor tissue.Results: H1299-EVs and A549-EVs were extracted via ultracentrifugation combined with ultrafiltration.The experiment on the mechanical properties of the H1299 cells,A549 cells and BEAS-2B cells showed that H1299 cells had the lowest Young’s modulus and adhesion,while BEAS-2B cells had the highest Young’s modulus and adhesion.The experimental results of EVs induced cells showed that H1299-EVs and A549-EVs promoted the proliferation,migration,and invasion of H1299 cells and A549 cells,respectively.H1299-EVs and A549-EVs induced cytoskeleton rearrangement and promoted the formation of cells’ filopodia.H1299-EVs and A549-EVs reduced Young’s modulus and adhesion of the cells.The mRNA sequencing and bioinformatics analysis results showed that it was enriched in AKT/mTOR pathway.H1299-EVs increased the expression of p-AKT,p-mTOR,and p-CDC42 in H1299 cells and LY294002 inhibited the effect of H1299-EVs.LY294002 also inhibited H1299-EVs to promote the progression of lung adenocarcinoma and regulate the mechanical properties of cells.The results obtained in vivo were similar to those observed in vitro.Conclusion: This study revealed the mechanismthat the EVs promoted the progression of lung adenocarcinoma and regulated the mechanical properties of cancer cells.In vitro and in vivo experiments verified that EVs induced cancer promotion and regulated the lung adenocarcinoma cells’ mechanical properties by activating AKT/mTOR pathway and CDC42.The study provides new ideas for the treatment of lung adenocarcinoma.