Heparan Sulfate Analogues Regulate Tumor-derived Exosomes Formation That Attenuate Exosomes Functions In Tumor Processes

At present,malignant tumor is a difficult problem that puzzles the whole human being.The characteristics of tumors are high invasiveness and metastasis.Exosomes were considered as the waste of cell metabolism previously,however,it was found that exosomes played an important role in the development of tumors in later studies.Exosomes will make a significant contribution for cancer treatment in the future,and will be important in solving the tumor problem.Heparan sulfate(HS)is a kind of linear polysaccharide,which exists in cell membrane and extracellular matrix.It has been found that HS is involved in the production and uptake of exosomes,particularly in the migration and invasion of tumor cells.Studies have shown that HS is significant in the biosynthesis and secretion of exosomes,but the relationship between its structure and function is still unclear.Therefore,the purpose of this study is to synthesize HS analogues with definite structure,and to study the effects of different sulfated sites and HS analogues with different molecular weights on the size,quantity and composition of exosomes synthesized by tumor cells.Furthermore,the influence of exosomes stimulated by HS analogues on the biological functions of tumor cells was discussed,thus providing scientific basis and new strategies for the regulation of tumor microenvironment and tumor intervention research.The main work is as follows:(1)Preparation and characterization of heparan sulfate analogues.Based on heparan sulfate analogues,6-O-DeSH was obtained by chemical derivatization.The main reagent is N,O-(trimethyl-silyl)-trifluoroacetamide(BTSA).The structure of the product was characterized by FT-IR,1H-NMR and 13C-NMR,and the molecular weight of the sample was determined by GPC-MALLS.The Zeta potential of the sample was measured by Zetasizer Nano analyzer.The element content of the sample was determined by element analyzer,and after a series of characterization,6-O-DeSH was confirmed to be successfully prepared.(2)Effect of heparan sulfate analogue structure on exosomes formation.Exosomes with different structures are obtained from mouse melanoma cells which were stimulated by HS analogues.The exosomes were characterized by observing the morphology of exosomes by transmission electron microscope(TEM),and measuring the size of exosomes by nanoparticle tracking technology(NTA)and dynamic light scattering(DLS).Exosome marker proteins were detected by western blot,and it was confirmed that exosome was successfully extracted.Different methods were used to investigate the effects of different HS analogues on the size of exosomes,the number and composition of exosomes.The results showed that the change of sulfation site of HS analogues had a significant effect on exosomes secretion and composition,but the particle size had no obvious effect.(3)Effect of heparan sulfate analogue structure on exosome function.The effects of different HS analogues on tumor cell proliferation,migration and invasion were studied by cytotoxicity test,wound healing,transwell and other experiments.The results showed that HS analogues with different molecular weights had no significant effect on exosomes function.However,the HS analogues which modified by different sulfation sites,the exosomes of heparin group have a significant inhibitory effect on the proliferation,migration and invasion of tumor cells.After the removal of 6-O sulfation group,the inhibitory function is obviously weakened,indicating that sulfation sites have a significant effect on its function.In the measurement of migration and invasion function,similar experimental results were found in the proliferation experiment.In order to explore the influence mechanism of different HS analogues on function,the expression of heparanase in exosomes was determined by western blot.The expression of exosomes cytokines was determined by ELISA,the results showed that the heparanase content of exosome secreted by tumor cells stimulated by heparin decreased significantly.The detection of cytokines showed that the expression of IL-6cytokines in heparin and low molecular weight heparin group was lower,while the expression of IL-10 and TGF-β cytokines was higher.After removal of sulfate at 6-O position,the cytokines of IL-10 and TGF-β did not change significantly,but the expression of IL-6 was higher.It indicated that sulfation of this site played a key role in mediating the expression of exosome cytokines.To sum up,this study discussed the influence of HS analogues with different structures on exosomes production and function.The molecular weight change of HS analogue has significant influence on exosomes production and function.At the same time,the 6-O sulfate group in HS analogues plays an important role in this process.The possible mechanism is that HS analogues with different structures affect the biological function of tumor exosomes by regulating the expression of tumor exosomes load-related proteins.