Objective To investigate the role of Notch signaling pathway to maintain the stem cell-like characteristics of osteosarcoma and its underlying mechanism.Methods Lentiviral NICD1 or Numb-shRNA was transduced into MG63 osteosarcoma cells to activate Notch activity in vitro.The impact of Notch on osteosarcoma stem cells were assessed by the tumor sphere formation assay and flow cytometry analysis of cell surface markers STRO-1/CD117.The expression of stem cell related genes (Sox2,Oct4) were evaluated by Western blot and qPCR.The nude mice were randomly divided into 3 groups:the NICD1 overexpression (NICD-OE) group,the DAPT group and the control (CON) group.The tumor growth was monitored for 8 weeks and the tumor volume and weight were recorded weekly.To investigate whether Notch regulates Eph pathway,Eph pathway related protein EphB,pEphB was measured by Western blot.The impact of ephrinB 1 on NICD overexpression cell were assessed by tumor sphere formation assay.The expression of Sox2 and Oct4 was evaluated by Western blot.Results NICD1 overexpression or Numb-shRNA increased the activity of Notch pathway.The Notch-activated osteosarcoma showed enhanced in vitro tumor spheroid formation capacity,increased Stro-1/CD117double positive ratio,and upregulated expression of Sox2 and Oct4 in vitro.In animal experiments,it was found that activation of Notch pathway promoted tumor formation in vivo and Notch inhibition decreased it.The primary osteosarcoma cells were obtained from mice xenograft treated with DAPT and its tumor sphere formation capacity was significantly reduced.Finally,The Notch pathway inhibits the phosphorylation of EphB,as well as the downstream signal pathway of EphB,but there is no significant change in total EphB.The activation of Eph pathway inhibited Notch induced up-regulation of tumor sphere formation and Sox2 and Oct4 expression.Conclusion Notch signaling pathway maintains the stem cell-like characteristics of osteosarcoma probably by inhibiting the Eph pathway.