Exosomes Derived From HeLa Cells Break Down Vascular Integrity By Triggering Endoplasmicreticulum Stress In Endothelial Cells

Background and ObjectiveTumor cells derived exosomes accelerate the progression of tumor via promoting tumor angiogenesis,inducing the differentiation of carcinoma-associated fibroblasts,participating immunoregulation which results in immune evasion,as well as educating the micro-environment of metastasis organs.Tumor angiogenesis,however,is always mingled with the break down of integrity of tumor vessels.Tumor cells can cause endothelial cell necroptosis and break down the vascular endothelial tight junction by means of exosomes secretion,leading to metastasis.However,the effects of tumor-derived exosomes on cancer metastasis still remain unclear.Here we explored the effect of tumor-derived exosomes on endothelial cells(ECs)and the related mechanisms.Methods:HeLa exosomes were collected using ultra-centrifugation method,and the characters of exosome were determined by particle size analysis,Western bloting and electron microscope.Primary human umbilical vein endothelial cells(HUVECs)were isolated form the umbilical vein and passaged till 3rd-6th passage.HUVECs were treated with the exosomes derived from HeLa cells(ExoHela)or human cervical epithelial cells(ExoHCEC).The permeability of endothelial monolayer and expression levels of tight junction proteins zonula occludens-1(ZO-1)and Claudin-5(CDLN5)were assessed.The profiles of microRNA in ExoHela and mRNA in HUVECs after treated with ExoHela were analyzed.Effect of ExoHela on vascular permeability in vivo was assessed by detecting the exudation of i.v.injected FITC-Dextran in lung,liver and ear.Expression of endothelial ZO-1 and CLND5 in pulmonary vessel were analyzed by immunofluorescent stain after the injection of exosomes via tail vein.Luciferase-labeled 4T1 tumor cells were implanted into nude mice,followed with injection of the exosomes.Tumor metastasis was assessed by bioluminescence imaging and target tissue sectioning for H&E staining.Results:Substance collected form the HeLa culture medium using ultra-centrifugation carried all the essential characters of exosome.Uptake of exosome by HUVECs took place 6 hours later after mixing ExoHela with HUVEC.Protein levels of ZO-1 and CLND5 in HUVEC were significantly reduced,and the permeability of endothelial monolayer was increased when HUVECs were treated with ExoHela,while mRNA levels of ZO-1 and CLDN5 remained unchanged.Injection of ExoHela also caused the increase of vascular permeability and tumor metastasis in vivo.The microRNA sequencing of ExoHela showed that microRNAs targeting at mRNAs of ZO-1 and CLDN5 were in low level.Corresponding microRNA inhibitors did not block the inhibitory effect of ExoHela on ZO-1 and CLDN5.The mRNA sequencing of ExoHela-treated HUVECs showed significantly increased expression of genes involved in endoplasmic reticulum(ER)stress pathway,which was further confirmed by real time-PCR and western blot.Inhibition of ER stress by knocking down PERK pathway restored the protein level of ZO-1 and CLDN5.Conclusion:Exosomes derived from HeLa cells break down endothelial integrity and promote tumor metastasis by triggering ER stress in ECs.Such effect is microRNA-independent.