| Background Hematogenous metastasis is the main cause of death in malignant tumor.The integrity of the vascular endothelium barrier is the key to affecting the transendothelial migration of tumor cells and leading to the blood metastasis of tumor.Tumor-derived Extracellular Vesicles(TEVs),as nanoscale lipid bilayer membranes released by tumor cells,regulate tumor progression by participating in tumor microenvironment,immune escape,and angiogenesis.Clinical studies have found that the level of TEVs in peripheral blood of cancer patients increases with tumor progression,but whether it can promote tumor metastasis by affecting vascular endothelial permeability is still unclear.Previous collaborative studies have found that brain-derived microparticles can break the blood-brain barrier and enter the peripheral circulation during brain injury,induce high vascular endothelial permeability in vivo and in vitro experiments,and promote EVs clearance to protect the endothelial integrity damage caused by ischemic injury.Therefore,we speculated that TEVs might promote blood metastasis of gastric cancer by inducing vascular endothelial permeability.The study of tumor metastasis mechanism is closely related to tumor therapy.Astragalus polysaccharides(APS),as the main component of Astragalus,played an anti-tumor role by inhibiting the proliferation of tumor cells,promoting apoptosis and enhancing immune function.It is not known whether Astragalus polysaccharides have other anti-tumor mechanisms.Researchers have found that APS has a protective effect on vascular endothelial damage in cardiovascular and cerebrovascular diseases.In the process of tumor metastasis,some factors in plasma,such as TEVs,may cause vascular endothelial barrier damage of target organs.APS may play a role in endothelial protection,which provides a new direction for the study of the anti-tumor metastasis mechanism of APS.Objective This research project intends to use high-risk gastric cancer in western China as a research object to investigate whether TEVs activate endothelial cells,increase vascular endothelial permeability,enhance gastric cancer cell adhesion and transendothelial migration,and promote tumor metastasis.It is elucidated the relationship between VEGFA-VEGFR2-Src-VE-cadherin signaling pathway and TEVs mediating endothelial barrier injury,the role and possible mechanism of APS in TEVs mediating hematogenous metastasis of gastric cancer is clarifying.Methods1.Peripheral blood was collected from patients with gastric cancer and healthy subjects.Plasma EVs and TEVs were detected by flow cytometry,plasma VWF levels were measured by ELISA.The correlation of the levels of plasma TEVs with differentiation,metastasis,and VWF levels in gastric cancer were analyzed by correlation analysis.2.Mouse gastric cancer subcutaneously transplanted and metastasis model were established.Flow cytometry was used to detect plasma TEVs content in tumor-bearing mice,and the relationship between TEVs level with tumor size and metastasis was analyzed by correlation analysis.TEVs were injected into tail vein of tumor-bearing mice to increase the content,and Lactadherin were injected to reduce plasma TEVs levels.Pulmonary surface nodules,weights,and lung metastasis areas were observed.VWF was detected in plasma and lung tissues.We explored the role of TEVs in hematogenous metastasis of gastric cancer.3.TEVs were isolated and obtained in vitro,which phenotype and morphology were identified by flow cytometry and electron microscopy.Laser confocal and flow cytometry were used to examine the relationship of TEVs and endothelial cells.Transendothelial electrical resistance assay,Transwell model and Mouse transplanted tumor were used to detect the effects of TEVs on endothelial barrier permeability and tumor cell transendothelial migration.Scanning electron microscopy,ELISA and immunofluorescence were used to detect the morphology of endothelial cells,VWF release,and the expression of adhesion molecules CD31 and F-actin on the endothelial surface after TEVs treatment.The effect of TEVs on the VEGFA-VEGFR2-Src-VE-cadherin pathway were detected by In-Cell Western,ELISA and immunofluorescence methods.4.Mouse metastasis model was established.The changes of lung metastasis and immune organs in tumor-bearing mice were detected by intraperitoneal injection of APS,and the related role of APS in TEVs-mediated hematogenous metastasis was analyzed.5.By means of in vitro experiments,we examined the effects of APS on gastric cancer cells releasing TEVs.We also researched the effects of APS on TEVs mediated endothelial interaction,permeability,tumor cell adhesion and transmigration,endothelial cell activation,and VEGFA-VEGFR2 pathway.In addition,APS was used on macrophages in vitro to detect the effects on macrophage cytoskeleton protein,phagocytosis and TEVs clearance.6.The main active components and potential targets of Astragalus polysaccharide on gastric cancer metastasis were screened by network pharmacology,and its core genes were analyzed.Results1.The levels of total EVs and TEVs in peripheral blood of gastric cancer patients were significantly higher than those in healthy subjects.The levels of peripheral blood TEVs in patients with poorly differentiated gastric cancer were significantly higher than those in moderately differentiated,well-differentiated,intraepithelial tumors,and healthy subjects.TEVs levels in peripheral blood of gastric cancer patients with metastasis were significantly higher than those without metastasis.TEVs levels in peripheral blood of gastric cancer patients were not correlated with gender and age,but were negatively correlated with tumor differentiation,and were positively correlated with tumor metastasis and plasma VWF levels.2.The levels of plasma TEVs in tumor-bearing mice increased with the increase of tumor size.And the levels of plasma TEVs in metastatic tumor-bearing mice were significantly higher than those in non-metastatic group.The surface pulmonary nodule,lung weight,and tumor-bearing areas in TEVs group were significantly higher than control group,and VWF expression in the plasma and lung tissues was increased.The level of TEVs in peripheral blood,tumor-bearing areas of lung tissue,and the expression of VWF decreased after Lactadherin treatment.3.TEVs were isolated and acted on endothelial cells for 24h in vitro,then TEVs were intaked by endothelial cells.Dynasore partially inhibited the action of endothelial cells and TEVs.The permeability of endothelial barrier and the transendothelial migration of gastric cancer cells increased after TEVs were treated.Endothelial activation,VWF release,cytoskeleton contraction,cell morphology changed,and CD31 expression decreased after TEVs were treated.TEVs could activate the VEGFA-VEGFR2-Src-VE-cadherin pathway.4.APS could reduce the increase of lung metastasis mediated by TEVs,and reduce plasma TEVs and VWF levels.APS could also partially improve the weight loss caused by MFC and TEVs,increase the spleen index and thymus index,increase the infiltration of CD8~+T cells,CD20~+B cells and CD68~+macrophages to metastasis,and increase the number of liver macrophages.5.APS could reduce the production of TEVs and TEVs-mediated increase of endothelial barrier permeability and increased transendothelial migration of gastric cancer cells,partially inhibited the activation of TEVs-mediated endothelial cells and VEGFA-VEGFR2 pathway.In addition,APS promoted the cytoskeletal protein rearrangement of mouse macrophages,enhanced their phagocytosis and increased the phagocytosis of TEVs.6.The network pharmacology of traditional Chinese medicine found 11 active components of APS,299 potential targets,9875 targets related to gastric cancer metastasis,and 130 core targets of APS for gastric cancer metastasis.GO and KEGG analysis confirmed that APS could play an anti-metastasis role in gastric cancer through extracellular vesicles and VEGFA/VEGFR2 pathway.Conclusion1.Level of plasma TEVs in patients with gastric cancer increased with tumor progression,suggesting that TEVs was involved in gastric cancer progression.TEVs can promote the hematogenous metastasis by activating vascular endothelial cells,increasing the permeability of endothelial barrier,enhancing the adherence of tumor cells to the endothelium and transendothelial migration.TEVs-mediated endothelial barrier damage is closely related to activation of VEGFA-VEGFR2-Src-VE-cadherin pathway and decrease of VE-cadherin expression.2.Astragalus polysaccharides plays a protective role in TEVs-mediated tumor metastasis.Enhancing anti-tumor immunity,reducing the production of TEVs,promoting the clearance of TEVs by macrophages,inhibiting TEVs-mediated endothelial cell activation and VEGFA-VEGFR2 pathway activation,which may be new mechanisms of its anti-tumor metastasis.3.Traditional Chinese medicine network pharmacological studies have confirmed that APS can play an anti-metastasis role in gastric cancer through extracellular vesicles and VEGFA/VEGFR2 pathway.The regulation of APS on gastric cancer metastasis is a multitarget-multipathway-multieffect molecular network model. |
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