Autophagy Induced VEGFR-2 Activation Promotes The Formation Of Vasculogenic Mimicry By Glioma Stem Cells

Tumor growth and metastasis depend on tumor neovascularization.At present,there are many reports about tumor angiogenesis and mechanism,and the new method of antiangiogenesis in the treatment of tumor has been applied in clinic.However,the current antiangiogenesis treatment methods and efficacy are limited,the main reason is the lack of understanding of the mechanisms beyond the classical angiogenesis,and lack of treatment strategies.Angiogenesis and vasculogenesis are classical tumor angiogenic pathways.In recent years,it was also found that tumor cells can construct tumor microcirculation through itself deformation,and formed an independent on endothelial cell dependent vascular mode(vasculogenic mimicry,VM).Our previous studies have found that glioma stem cells(GSCs)has the ability to form VM,which is the key cell in the formation of VM.VEGFR-2 plays a key role in the formation of VM mediated by GSCs.Currently,VM are found lack of response to antangiogenesis treatment of VEGF(such as bevacizumab,BEV).we also observed that without the VEGF(BEV)GSCs can still form a tube like structure(VM),and accompanied by VEGFR-2-Y1175 phosphorylation sites and increasing of autophagosome.This suggests that VEGFR-2 activation pathway may exist in VEGF dependent and independent manner,which autophagy may be through the role of the latter.Autophagy is the process of lysosomal degradation of damaged organelles and macromolecules,which relate to cell survival,growth,differentiation and internal environment stability,and participate in all aspects of life.In recent years,the research progress of autophagy has put forward a new point of view on the pathogenesis and prevention strategy of many diseases,including cancer.Previous studies have shown that autophagy is complex and controversial in tumor.But at present,most scholars believe that without oxygen,nutrients and growth factors in the microenvironment,the tumor cells through the formation of autophagy to degradate their own folding proteins and damaged cells to survive.Current research shows that there are similarities in the formation conditions and mechanism of autophagy and VM.At first,VM formed by tumor cells themselves deformation,and emerged from rapid growth and insufficient blood supply of early and advanced stages of lack of blood vessels in the area;similarly,tumor cells in the lack of nutrition and oxygen supply,through its intracellular monolayer or bilayer membrane wrapped to form autophagosomes,and maintain cell survival.Secondly,autophagy and VM are both adapted to the gene regulation and the process of reforming the microenvironment.Again,recently there have been reported in the literature that the autophagy related protein,microtubule associated protein-1 light chain 3(LC3),is highly expressed in malignant melanoma VM cells.All this suggested that autophagy may be involved in the formation of tumor VM.In order to further understand the mechanism and the role of autophagy in VM,and VM formation caused by BEV in the process of by anti-angiogenic therapy,we first establish and validate the new three-dimensional(3D)culture model of VM formed by glioma stem cell(GSCs),and on the basis of the model,observed the formation of autophagy in VM-formed cells;then observed the effects of the formation of VM by up and down regulation of autophagy;and explored the autophagy regulation in VM formation mechanism and BEV for the treatment of glioma stem cell transplantation tumor caused increase of VM and anti autophagy role in it;Finally,we detected the expression of autophagy related gene Atg5 and p-VEGFR-2 in human glioblastoma tissue,and analyzed their prognosis and relationship between them.The main results and conclusions are as follows:1.Establishment of VM model for in vitro 3D scaffold culture.Glioblastoma multiforme(GBM)cell lines U87 and primary cells were cultured in 3D collagen scaffold,and were cultured in DMEM+10% fetal bovine serum,stem cell culture medium(STM),or endothelial cell culture medium(EBM)three days respectively.Hematoxylin and eosin(HE)staining,immunofluorescence staining,scanning electron microscope and Western blotting(WB)experiment were used to observed the scaffold.The results showed that:(1)Compared to the other two groups,the tube like structures induced by EBM expressed high level of VM related proteins such as VE-Cadherin and Laminin B2,phosphorylation of VEGFR-2 expression was significantly increased at the same time.(2)Only in the culture of EBM can induce the formation of tube like structures.The above results indicated that the new 3D model of VM in vitro was a good culture model.2.Autophagy promotes phosphorylation of VEGFR-2 to form VM by GSCs independent of VEGF.(1)Using the GSCs containing a GFP-LC3 reporter,we found that the number of green dots in the cytoplasm of the tumor cells was significantly higher than that of the other two groups under the culture of EBM(P<0.001).WB detection showed high expression of autophagy related protein Atg7,ATG5 and LC3 under the culture of EBM.The expression level of p62 protein which is in inverse proportion to the level of autophagy,was lower in the culture of EBM,indicating the level of autophagy was higher.(2)VM induced by EBM coexpressed LC3 and VM related proteins under the immunofluorescence double staning.(3)Autophagy inducer rapamycin(RAPA)increased the number of autophagosomes in GSCs,which were significantly higher than those in the control group(P<0.01).Meanwhile,VM in RAPA group was significantly higher than that in the control group.WB detection showed high expression of LC3,low level of p62,and VM related proteins,such as VE-Cadherin and Laminin B2,and VEGFR-2 and its phosphorylation level increased together.(4)Autophagy inhibitor chloroquine(CQ)increased the number of autophagosomes in GSCs,which were significantly higher than those in the control group(P<0.01).However,although the expression of LC3 in CQ group was up-regulated by WB detection,there was an increase in the level of p62,which showed that the whole level of autopghagy was decreased.Meanwhile,the number of VM in CQ group was significantly lower than that of the control group(P<0.01).In the other,WB detection showed low expression VM related proteins,such as VE-Cadherin and Laminin B2,and VEGFR-2 and its phosphorylation level decreased together.(5)We additionally knocked down(KD)Atg5(Atg5-KD)with sh RNA in GSCs.This resulted in a marked decrease in intracellular green fluorescent spots in GSCs cultured with EBM(P<0.01)together with significantly reduced numbers of tubules(P<0.01).WB detection showed low expression VM related proteins,such as VE-Cadherin and Laminin B2,and VEGFR-2 and its phosphorylation level decreased together.(6)WB detection showed high level of VM related proteins in BEV treated GSCs,and VEGFR-2 and its phosphorylation level increased together,but the level of p62 decreased,and the effect was reversed by CQ or Atg5-KD.(7)Confocal microscopy showed an increased ROS level released by GSCs cultured in EBM than that of the other two groups,which was reduced by addition of CQ.In contrast,RAPA treatment of GSCs increased the levels of ROS.After BEV treatment,the levels of ROS produced by GSCs in EBM were significantly elevated,which were inhibited by CQ.Combined treatment of GSCs in EBM with BEV and an antioxidant N-acetylcysteine(NAC)significantly decreased the phosphorylation of VEGFR-2 without changes of both LC3-I to LC3-II conversion and degradation of p62.These results suggest that autophagy induces VEGFR-2 and its phosphorylation levels by regulating ROS levels to regulate VM formation,which is not dependent on VEGF-A.BEV can induce the level of autophagy and VM formation,through combined with the autophagy inhibitor CQ,we can reduce the generation of autophagy and VM.3.VM induced by BEV can be reduced by down-regulation of autophagy in orthotopic transplantation tumor.(1)Double staining of CD34 and PAS showed that BEV can inhibit the formation of orthotopic implantation of NOD / SCID mouse xenograft vascular,but increased the number of VM,the effect was reversed by Atg5-KD.(2)Chloroquine and BEV each can make the NOD-SCID mouse GSCs orthotopic transplantation tumor volume reduction,the effect was more obvious in the mice receiving both CQ and BEV.In contrast,the number of VM channels increased in the tumors of mice treated with BEV.CQ reduced the channels of VM,but little difference in vessel density as compared with tumors grown in the mice without any treatment.Immunohistochemical staining revealed that Atg5 was potently upregulated by the BEV treatment,whereas CQ can significantly lower level of ATG5 and the number of VM.These results suggest that BEV can induce the formation of VM,and the expression of ATG5 is increased simultaneously.Inhibition of autophagy can reduce the formation of VM,and improve the efficacy of BEV.4.High expression of Atg5 is correlated with VM formation and the survival of GBM patients.(1)Immunofluorescence showed coexpression of stemness markers CD133,autophagy-related protein LC3 and VM-related proteins VE-Cadherin and laminin B2 in human glioblastoma tissue,namely the expression of CD133 cells with high expression of VM related proteins and LC3;CD133,VM related protein and autophagy-related proteins were highly expressed in tumor tissues comparing to tumor adjacent tissues using the WB detection,which is consistent with immunofluorescence results.(2)We studied the tumor specimens from 95 patients by double staining of Atg5 and PAS,patients with VM-positive GBM had shorter progression-free survival(PFS)and overall survival(OS)than those with VM-negative tumors(P <0.001).(3)Patients with high Atg5 levels in tumors had shorter PFS and OS than those with low Atg5 levels in tumors(P <0.001).There was a significant correlation between VM and Atg5 expression in GBM specimens(r = 0.6727,P <0.001).(4)Patients with high p-VEGFR2 levels in tumors had shorter PFS(P <0.001)and OS(P <0.01)than those with low p-VEGFR2 levels in tumors.Analysis showed a markedly correlation between Atg5 and p-VEGFR2 expression in GBM specimens(r = 0.7743,P <0.001).(5)Through immunohistochemistry staining of EGFR,PDGFRA and p53,GBM can be divided into two molecular subtypes,which is the classical-like(CL)type and proneural-like(PNL)type.PNL type had better PFS and OS than CL type(P <0.001).There was a significant correlation between the two subtypes and Atg5 expression in GBM specimens(P=0.002).The results show that human GBM have high level of VM and autophagy related proteins.VM is an important factor in GBM patients with poor prognosis.VM and the expression of ATG5 and p-VEGFR-2 showed significantly positive correlation,indicating that autophagy may be an important reason for the formation of VM and lead to poor clinical prognosis by the activation of VEGFR-2.The expression of Atg5 is higher in the poor prognosis of CL subtype,and lower in the better prognosis of PNL subtype,indicating that Atg5 is not only an indicator of poor prognosis,but also a major clue to predict the subtypes of glioblastoma.Therefore,Atg5 can be used as an important target for the treatment of GBM in the future.In summary,the present results suggest that(1)autophagy mediated VEGFR-2 activation to promote VM formation by ROS,the pathway is independent of VEGF-A;BEV induced VM can be reduced by inhibition of autophagy.(2)VM is an important factor of poor prognosis of GBM,and the expression of ATG5 and p-VEGFR-2 are closely related to VM.The expression of Atg5 was a significant correlation with molecular subtypes.These results provide a new target and theoretical basis for the treatment of VM of GBM.