Identification And Characterization Of Tumor Cell Line (K3) Mutated From Bone Marrow Mesenchymal Stem Ells And It-Derived Cancer Stem Cells

Objective To investigate the biological characteristics of the cancer cell line(K3) transformed from rat bone marrow mesenchymal stem cell in rat tail.To establish the method to isolate and identify the cancer stem cells(K3-SP) in K3 cell line,and explore the biological characteristics and gene expression difference between K3-SP cells and K3-NSP cells by cell biology and molecular biology technology,and analyze the enrichment of K3 CSC by serum-free cultrue.Methods The biological characteristics of K3 cells were analyzed by growth curve,FCM,PCR,immunohistochemistry staining et al.K3 cells were cultrued in10%,5%FBS DMEM,serum-free cultrue condition and serum-free cultrue condition with bFGF,EGF,LIF and K3 cells were injected into BALB/c nude mice.After the cell number was enough,the cellls or tumor tissues were collected and dissociated to get the single cells.K3-SP and K3-NSP cells were isolated by flow cytometry from K3 cells or K3 tumor cells.K3-SP cells and K3-NSP cells were injected subcutaneously into the right or left back of the same BALB/c nude mouse to determine their tumorigenic capacity respectively.To determine their self-renewal and multi-linkage differentiation capacity, Histopathological analyses were performed.Microarray was used to search for different expression genes.The results of microarray were confirmed by Real-time quantitative PCR.K3 cells were cultrued in serum-free cultrue media to enrich K3-SP cells.Results The K3 cells expressed the similar surface antigens as rat-MSC such as positive for CD29,CD44,CD90,negative for CD45.The K3 cells expressed the similar genes and have the similar growth characteristics as tumor cells,and lead to tumor in BALB/c.K3 cells contain SP cells.There was different quantity of SP cells in different K3 cultrue condition.The percentage of K3-SP cells in K3 cells cultrued in 10%FBS and 5%FBS was 1.01%and 0.73%,respectively.K3-SP cells had stronger tumorigenic capacity,5×10~3 K3-SP cells could form the tumor.The tumor tissue type was fibroma sarcomatosum.The microarray analysis revealed that the number of different expression genes beween K3-SP and K3-NSP was 57,up-regulated genes were 41,down-regulated genes were 16.Real-time PCR confirmed up-regulation genes T-Bx3, Mafb,Plk2 and one down-regulation gene Vegf.The histopathological analysis showed that P53,PCNA,ABCG2,OCT4 of K3-SP of tumor tissue were positive,but K3-NSP were negative.K3 cells both form suspended tumor spheres in serum-free and serum-free with bFGF,EGF, LIF.Real-time PCR showed that the expression of OCT4 significantly increased in K3 cells cultrued in two serum-free condition.After cultrued in serum-free with bFGF,EGF,LIF condition for 15 days,the percentage of K3-SP reached 7.73%. Conclusions K3 cells derive from tumor cells mutate form Rat-MSC in vivo,and contain K3-SP.There are apparently difference between K3-SP and K3-NSP in tumorigenic capacity and gene expreesion.K3-SP cells have stronger tumorigenic capacity.The serum-free cultrue condition apparently enrich K3-SP.Our findings provide the experiment basement for further study of the specific molecular marker of K3-SP.