Facilitating Effect And Underlying Mechanisms Of Bone Marrow Mesenchymal Stem Cells On Tumor Angiogenesis In Gastric Cancer Associated With Chronic Helicobacter Pylori Infection

Part ?: BM-MSCs promote angiogenesis in gastric cancer associated with chronic Helicobacter pylori infectionObjective: Gastric cancer is the fifth most common cancer and the third most common cause of cancer death globally.Many factors are implicated in the carcinogenesis and progression of gastric cancer,including prevalence of Helicobacter pylori(H.pylori)infection,the main cause of gastric cancer.BM-derived mesenchymal stem cells(BM-MSCs)have been shown to be recruited to gastric cancers and to promote tumor growth.Several studies have pointed to BM-MSCs promote tumor growth by enhancing angiogenesis.However,whether BM-MSCs can promote tumor angiogenesis in chronic H.pylori infection-related gastric cancer is still unclear.This aim of this study is to investigate the effect of BM-MSCs on angiogenesis in gastric cancer associated with chronic H.pylori infection.Methods:Mice were lethally irradiated and injected i.v.with unfractionated bone marrow cells harvested aseptically from age-matched LSL-tdtomato-female mice.After three months of chronic H.pylori infection,engraftment of LSL-tdtomato marrow-derived cells was tracked with LSL-tdtomato staining.The pathology of the stomach tissue was analyzed by HE staining.GFP fluorescentlabeled BM-MSCs transplantation into the serosal layer of mice gastric antrum model was established after three months of chronic H.pylori infection.After 3 months,the level of BMMSCs engraftment and migration was assessed by confocal microscope observation of GFP fluorescence.The pathology of the stomach tissue was analyzed by HE staining.Immunohistochemistry and Immunofluorescence were performed to detect the expression of vascular markers(CD31,NG2,VWF and LAMININ-1)in the stomach of different groups of mice.Results:1.In BM transplantation mice,engraftment of LSL-tdtomato marrow-derived cells were detectable in mice gastric at 3 months of H.pylori infection.HE staining revealed that intraepithelial neoplasia was detected.2.In BM-MSCs transplantation mice,BM-MSCs can be transplanted into the stomach for a long time under chronic H.pylori-infection,and migrate to the site of chronic mucosal injury.Engraftment of BM-MSCs leads to more dysplasia and gastric carcinoma.3.Engraftment of BM-MSCs increased vasculogenic markers,such as CD31,NG2,VWF and LAMININ-1 in stomach tissues in mice with H.pylori-infection.Conclusion: BM-MSCs engraftment in the tumor microenvironment of chronic H.pylori infection inflammation-induced gastric cancer and contributes to tumor tumorigenicity and angiogenesis.Part ?:BM-MSCs promotes GC angiogenesis in vivo and the tube formation and migration of HUVEC following H.pylori stimulationObjective: In the previous study,we found that in the microenvironment of chronic H.pylori infection,BM-MSCs migration and engraftment in the stomach promote gastric cancer and angiogenesis.However,the direct effect of BM-MSCs on gastric cancer angiogenesis is still unknown,so the purpose of this experiment is to clarify the effect of BM-MSCs on gastric cancer angiogenesis.Methods: We established an in vitro co-culture system,in which Human umbilical vein endothelial cells(HUVEC)was indirectly cocultured with SGC cells and separated from SGC cells by a semipermeable membrane.Conditioned medium(CM)from BM-MSCs stimulated by H.pylori was add to co-culture system.The effect of CM from BM-MSCs on the proliferation,migration,apoptosis,adhesion,tubule formation of HUVEC in the microenvironment of gastric cancer was observed.The Chicken chorioallantoic membrane assay(CAM)assay was performed to evaluate in vivo angiogenic activity.BM-MSCs and gastric cancer cells were mixed and injected into CAM to detect its role in tumor angiogenesis.In addition,we subcutaneously transplanted SGC cells and mixed with BM-MSCs into BALB/c nude mice and tumor volumes were monitored every two days.HE staining,IHC staining against CD31 and Ki67 was performed to detect the tumor growth and angiogenesis in tumors.The angiogenesis fluorescence imaging in nude mice was detected by Bruker In-Vivo Xtreme(Germany)and quantified by measuring the tumor uptake of the probe Angio Sense750 EX.Results: In vitro co-culture system,CM from BM-MSCs Promoted HUVEC proliferation,adhesion,migration and tubule formation,while alleviated apoptosis of HUVEC.Compared with the control,BM-MSCs significantly increased vascular density of cancer xenografts on the CAM.The SGC cells mixed with BM-MSCs group obtains the subcutaneous tumors with larger volume and weight,compared with that of SGC cells group.HE staining,Ki-67 and CD31 IHC staining supports BM-MSCs obtained a stimulatory role in gastric tumor growth and formation of neovascularization in tumors.The tumor vascular permeability fluorescence imaging in vivo demonstrated that BM-MSCs significantly promoted the degree of tumor neovascularization in the SGC-induced tumor xenografts.Treatment of H.pylori supernatant enhances these effects of BMMSCs.Conclusion: BM-MSCs promoted angiogenesis induced by gastric cancer cells in vitro and in vivo.Pretreatment of H.pylori supernatant can enhance the angiogenic ability of BM-MSCs.Part ? THBS4/integrin ?2 axis mediate BM-MSCs induced angiogenesis in GCObjective: The purpose of this study was to further clarify the specific mechanism of BM-MSCs promote angiogenesis of chronic Helicobacter pylori infection-associated gastric cancer.Methods: To explore the specific mechanism of BM-MSCs promoting chronic H.pylori infectionrelated gastric cancer angiogenesis,we performed RNA-SEQ Analysis to screen for differential genes.Genes related to angiogenesis were obtained through enrichment analysis(Gene Ontology,GO).The gastric tissues were verified at the m RNA and protein level to verify the differential gene THBS4.IHC and IF were performed to verify the differential gene.The effect of H.pylori supernatant intervention on the expression of THBS4 in BM-MSCs was analyzed.In order to investigate the biological behaviors of THBS4 in BM-MSCs promoting angiogenesis,we constructed the Lentivirus vector(sh-THBS4 and sh-NC).CAM,xenograft tumor model,and HUVEC tube formation assay were used for in vivo and in vitro angiogenesis study to investigate the effect of knockdown THBS4 in BM-MSCs on GC angiogenesis.Results: RNA-SEQ and GO showed that BM-MSCs transplantation caused the up-regulation of many differential gene expressions related to angiogenesis.The results of q RT-PCR experiments demonstrated that compared with the chronic H.pylori-infection group,the expression of Kng1,THBS4,Prodh2,Serpina1 d,Myl3 and Angptl3 genes increased significantly by BM-MSCs transplantation.Western Blot,IHC and IF of THBS4 in stomach tissue showed that THBS4 is the most expressed gene.It was found that H.pylori pretreatment increased the expression of THBS4 protein of BM-MSCs.H.pylori-pretreated BM-MSCs strongly accelerated gastric cancer angiogenesis in CAM assay and xenograft tumor model,while these effects were dramatically inhibited when THBS4 was silenced.We then performed THBS4-knockdown MSCs and NCknockdown MSCs transplantation into mice gastric serosa layer with chronic H.pylori-infection.IF of vasculogenic markers revealed that NC-knockdown BM-MSCs were more efficient in inducing angiogenesis.The ?2-specific antibody was able to alleviate r THBS4 induced migration and tube formation capacities of HUVEC.Conclusion: THBS4/ Integrin ?2 axis mediates BM-MSCs promote angiogenesis of gastric cancer associated with chronic Helicobacter pylori infection.Part ? Targeting PI3K/AKT pathway abrogate THBS4/integrin ?2 axis induced promotion of angiogenesisObjective: The purpose of this part of the experiment is to further explore the specific mechanism of THBS4 on endothelial cells.Methods: The THBS4/integrin ?2 axis is known to activate a number of downstream signaling.Then we evaluated these downstream signaling molecules of the THBS4/integrin ?2 axis in HUVEC.Next,we investigate the role and requirement of AKT signaling in r THBS4 facilitating the migration and tube formation of HUVEC.In addition,we investigate the role and requirement of AKT signaling in r THBS4 promoting GC angiogenesis in xenograft tumor model.Results: Western Blot demonstrated that p-AKT level was positively correlated with r THBS4 treatment.r THBS4 treatment enhanced AKT phosphorylation,while this effect was dramatically restrained by pretreatment with integrin ?2-specific antibody or the PI3 K inhibitor LY294002.In addition,the promotion effects on migration and tube formation of HUVEC mediated by r THBS4/integrin ?2 axis were partly counteracted by PI3 K inhibition.In xenograft tumor model,the promotion effects on GC angiogenesis mediated by r THBS4 were partly counteracted by PI3 K inhibition.Conclusion: Inhibition of PI3K/ AKT signaling pathway partly counteracted r THBS4-induced gastric cancer angiogenesis.