Antitumor Effect And Mechanism Of Human Umbilical Cord Mesenchymal Stem Cell Conditioned Medium On KGN Cells

Umbilical cord mesenchymalstem cells(UCMSCs)are a class of stem cells capable of self-renewal and multi-differentiation.They have the advantages of wide source,simple collection,rapid expansion,strong migration ability,synthesis of various cytokines and regulation of immunity.It shows a unique tendency to tumor tissues,which promotes the targeted migration of MSCs to the tumor site and aggregation in tumor tissues,which has high potential and clinical application prospect in the aspects of cell therapy and targeted therapy of tumor.However,the relationship between MSCs and tumors has been controversial.Some reports have shown that MSCs have anti-tumor effects,while others have reported that MSCs have cancer-promoting effects that promote the occurrence and development of tumors.On the one hand,this contradictory result may be related to the different sensitivities of different tumor cells to different tissue sources of MSCs;on the other hand,current in vitro studies have not taken into account the inhibition of tumor cell loss of contact,an important mechanism of tumor occurrence and development,resulting in contradictions among various reports.Ovarian granulosa cell tumor(OGCT)is a malignant tumor derived from ovarian sex cord stromal cells.It is subject to recurrence several years after operation and is a potential metastatic malignancy.More effective treatments are needed for advanced and recurrent GCT.Many studies have shown that UCMSCs mainly play their biological roles in a paracrine way.Umbilical cord mesenchymal stem cells conditional medium(UCMSCs-CM)contained a large number of paracrine products of UCMSCs,including exosomes and cytokines.A key biological characteristic of tumor cells is loss of contact inhibition.Although there are many reports on the effect of UCMSCs-CM on tumor cells,few people have paid attention to the relationship between UCMSCs-CM and contact inhibition of tumor cells.This study evaluated the effects of UCMSCs-CM on proliferation,apoptosis,migration and invasion of granulosa tumor cell line(KGN)cells at the cellular level,and further screened out the anti-tumor components of UCMSCs-CM.In addition,the mechanism of UCMSCs was explored from the perspective of tumor cell loss of contact inhibition,so as to provide theoretical and experimental basis for the development of cell-free biological products for clinical treatment of ovarian granulosa cell tumor.(1)Human umbilical cord mesenchymal stem cells were successfully isolated.The isolated cells expressed mesenchymal stem cell surface markers CD29,CD44,CD90,CD105,but did not express hematopoietic stem cell surface markers CD34 and CD45,the isolated h UCMSCs also have differentiation potential into adipocytes,chondrocytes,and osteoblasts.Exosomes derived from umbilical cord mesenchymal stem cells(UCMSCs-EXOs)were extracted,and the cytokine array assay was conducted to compare the different cytokine levels in UCMSCs-CM and control medium.Among 104 cytokines evaluated,the contents were Adiponectin,Serpin E1,IL-8,Pentraxin 3,Apolipoprotein A-1,Dkk-1,Vitamin D BP,Chitinase 3-like 1,MCP-3,MCP-1 etc.(2)When KGN cells at high density(1.5×10~5cells/cm~2),UCMSCs-CM significantly inhibited the proliferation,migration and invasion of KGN cells,induced cell cycle arrest at G1 phase,and promoted cell apoptosis.However,UCMSCs-CM had no significant effects on proliferation,cell cycle,apoptosis,migration and invasion of KGN cells when the KGN cells was at low density(3×10~3 cells/cm~2).(3)When KGN cells at high density(1.5×10~5 cells/cm~2),UCMSCs-CM activated intracellular Hippo signaling pathway,and Hippo signaling pathway inhibitor XMU-MP-1could reverse UCMSCs-CM-induced activation of Hippo signaling pathway in KGN cells,as well as changes in cell viability,cell cycle,apoptosis and cell invasion.However,UCMSCs-CM did not activate Hippo signaling pathway when the KGN cells was at low density(3×10~3cells/cm~2).(4)UCMSCs-EXOs had no significant effect on KGN cell viability,migration ability and the key protein expression of Hippo signaling pathway regardless of KGN at high(1.5×10~5cells/cm~2)or low density(3×10~3 cells/cm~2).(5)The highest content of adiponectin in UCMSCs-CM significantly inhibited the proliferation of KGN cells,promoted cell apoptosis and activated intracellular Hippo signaling pathway when KGN cells were at high density(1.5×10~5 cells/cm~2).Exogenous adiponectin and UCMSCs-CM had the same antitumor effect.In conclusion,UCMSCs-CM could significantly inhibit cell viability,cell migration and invasion,induce cell cycle arrest at G1 phase and promote apoptosis of KGN cells at high density through restoration of contact inhibition of KGN cells by activating the Hippo pathway.Further studies showed that adiponectin in UCMSCs-CM mediated its antitumor effect in KGN cells.