Mechanism Study Of Breast Cancer Cell Exosomal TSP1 Facilitating Transendothelial Metastasis Of Carcinoma Cells

Tumor cells metastasis is one of the causes of death in cancer patients.The main steps of tumor metastasis include: escaping from site in situ,infiltration into blood vessels,vascular cavity transporting,localization and proliferation at novel distant organs.Among these processes,tumor cell infiltration and breakthrough of the vascular barrier into the humoral circulation system is an important step during tumor metastasis,in which tumor cells build their own tumor microenvironment to facilitate tumor cell metastasis.The tumor microenvironment is basically composed of vascular endothelial cells,fibroblasts,lymphocytes,extracellular vesicles and extracellular matrix composed of proteoglycan and collagen,and plays vital roles in tumor proliferation,invasion and metastasis.Exosome derived from tumor cells is one kind of extracellular vesicles in the tumor microenvironment,which contains cell metabolism products and cellular component molecules such as proteins and nucleic acids.Exosome takes part in shaping the tumor microenvironment,especially for the remodeling of intercellular connections of vascular endothelial cells,which is a key step affecting the penetration of tumor cells into blood vessels.In this study,we found tumor-derives exsomes could facilitate the breast cancer cell matastasis and explored the molecular mechanism of exosomes in affecting transendothelial migration of breast cancer cell by using cellular,biochemical and molecular biology methods,and in vitro,in vivo cell metastatic models.The breast cancer cells and their secretion materials are important components in the tumor microenvironment.In order to study the effect of tumor microenvironment on the transendothelial migration of breast cancer cells,the tumor cell culture supernatant was firstly prepared,and then seperated into exosome-rich and exosome-free fractions.The exosome-rich fractions can significantly promote the transendothelial migration of breast cancer cells more significantly by transwell.Next,we extracted and examined wheather the breast cancer cell-derived exosome could be able to act on vascular endothelial cells and the intercellular junction molecules VE-cadherin and ZO-1 of vascular endothelial cells.We found that after treating HUVECs with the breast cancer cell-derived exosomes,these intercellular junction molecules were significantly down-regulated,and the ability to form tubes of HUVECs was weakened.It is suggested that exosomes derived from breast cancer cells may promote the transendothelial migration of tumor cells by destroying the integrity of vascular endothelial cells.Which protein or parts of the exosomes play a key role in this process? Exosomal mass spectrometry was explored and data analysis identified a wide range of adhesion-related proteins component by Gene Ontology analysis.Among the candidate molecules related to biological processes such as intercellular junction and adhesion,Thrombospondin-1(TSP1)was identified the candidate molecule with the highest enrichment scores,suggesting that TSP1 may be a key protein that regulated the main biological process of tumor cell metastasis.Next,we found that TSP1 is highly expressed in the metastatic breast cancer cell line MDA-MB-231 compared to the non-metastatic breast cancer cell line MCF7,and the TSP1 amount was significantly higher in exosomes than that in cell lysates,suggesting that TSP1 may contribute to the breast cancer metastasis.We overexpressed TSP1 in MCF7 cells,and found that TSP1 amount in exosomes derived from TSP1 overexpression MCF7 cells was increased and these exosomes could promoted transendothelial migration of tumor cells.On the other hand,we knocked down TSP1 in MDA-MB-231 cells,and found that for the exosomes derived from the knocked-down TSP1 MDA-MB-231 cells,the ability to promote tumor cell transendothelial migration was decreased.These results revealed that exosomal TSP1 is the key protein promoting transendothelial migration of breast cancer cells.How does TSP1 in breast cancer exosomes promote transendothelial migration of tumor cells? We treasted HUVECs with different TSP1 expression exosomes.Our results showed that exosomes with higher TSP1 level decreased the intercellular junction protein VE-cadherin and ZO-1 in HUVECs,and further destroyed the integrity of vascular endothelial intercellular connections.Exosomes derived from overexpressed TSP1 breast cancer cells significantly inhibited the expression of VE-cadherin and ZO-1,while knocking down THBS1 or TSP1 inhibitor LSKL-treated breast cancer cell derived exosomes increased the VE-cadherin and ZO-1 expression of HUVECs.These results indicated that TSP1 in breast cancer derived exosomes could down-regulate vascular endothelial cell intercellular junction proteins.In order to verify if TSP1 in breast cancer derived exosomes could reduce the integrity of vascular endothelial cell connections and promote transendothelial migration of breast cancer cells,we adopted the zebrafish cancer cell migration system for exploration.The results showed that exosomes derived from breast cancer cells that overexpress TSP1 reduced the expression of VE-cadherin,ZO-1,and CD146 in zebrafish vascular endothelial cells.Compared to low-expressing TSP1 breast cancer cells,the migtation of highexpressing TSP1 breast cancer cells in zebrafish was increased.These data confirms that TSP1 inhibited the intercellular junction molecules in zebrafish and increased the migration capacity of breast cancer cells in vivo,indicating that breast cancer cells could migrate more easily after the treatmen of exsomes derived from TSP1 high-expression breast cancer cells.In summary,this thesis reveals the important role of tumor cell-derived exosomal TSP1 in promoting metastasis of breast cancer cell,and clarifies the important transendothelial migration mechanism of tumor cells,and provides new targets for effective intervention in breast cancer clinical treatment.