Sorting Of Osteosarcoma Stem Cell And Identification Of Malignant Phenotype

BackgroundAccording to the theory of cancer stem cells, Tumor consists of numerous subpopulations with heterogeneity. Cancer stem cells are the subpopulations, which have the ability of self-renew and proliferation. CSCs also maintain the integrity of the genome, and have the same ability of self-renew and differentiation. Contrary to normal stem cells, the spread and proliferation of the CSCs is out of control.With the developing of CSCs theory, CSCs have been identified from many kinds of tumor. The surface markers, growth pattern, biological properties are different with normal cells.CSCs also exist in osteosarcoma.143-B cell lines have great metastatic potential and tumorigenesis abilities, but the CSCs have not been sorted from 143-B. To further study the biological behavior of osteosarcoma CSCs, we take 143-B as the source of CSCs, sorting and identify the CSCs from 143-B, study the biological characteristics of osteosarcoma CSCs.Part I Sorting CSCs from 143 B with limited dilution methodObjective:Explore the method of CSCs sorting from 143-B for further study on the biological characteristics of osteosarcoma.Method:Soft-agar, side polulation, limited dilution, were used for sorting. Then use the second generation cells, evaluate the expression of Nanog and OCT4 with PCR.Result: Soft-agar sorting subpopulation cells exhibit similar exrepssion of Nanong and OCT4 with parental generation. SP and limited dilution sorting subpopulation cells expressed higher Nanog and OCT4 than parental.Conclusion:SP and limited dilution could effectively used for 143-B CSCs sorting.Part II: study of biological characteristic of 143-B CSCsObjective: To study the biological characteristic of 143-B CSCs. Provide the preliminary theoretical support for the treatment of osteosarcoma. Osteosarcoma.Methods: 1.Sphere formation in soft agar. Use the soft agar for sphere formation test, observe the sphere formation ability.2.Western-blotting. Comparing the Nanog and OCT4 protein expression of 143-B, 143-L(limited dilution) and 143-SP(SP sorting) with WB.3. Immunofluorescence test the expression of stem cells markers. Paraformaldehyde fix 143-B and 143-L cells. IF test the expression of Nanog and OCT4.4. Detection of multi-directional differentiation potential ability. Test the potential ability of osteoblast differentiation, chondrogenic differentiation and adipogenic differentiation.5.Drug resistant test.(CIS-platinum、 Doxorubicin)Cells were plated in 96-well plate and exposed to a series of concentrations of DOX for 24 h. cell viability was measured with CCK-8.IC50 were calculated to measure the drug resistant ability.6. Tumorigenesis test. Nude mices were assigned to 6 group. 143-B and 143-L with 3 different cell concentration(1×106,1×105,1×104). Tumor volumes and body weight were recorded periodically.7. Statistical analysis. SPSS 17.0 were used for analysis.Results: 1.143-L have greater sphere formation ability than parental. 2. In WB and IF test, Nanog and OCT4 protein expressed higher in 143-L and 143SP than 143 B. 143-L and 143-SP had no significant differences in Nanog and OCT4 protein expression.3.143-L have osteoblast, chondrogenic and adipogenic differentiation ability. 143-B exhibit slight osteoblast differentiation ability, and have no chondrogenic and adipogenic differentiation ability.4. IC50 of DOX on 143-B is 0.83ug/ml while 143-L is 2.5ug/ml. IC50 of CIS on 143-B is 0.11ug/ml while 143-L is 0.25ug/ml.143-L exhibit higher DOX resistance.5. After 5 weeks, visible tumor were see in the 143-L 1x105 and 143-L 1x106 groupsAll the other group can not see obvious visible tumor.Conclution: limited dilution sorting 143-B CSCs expressed higher Nanog and OCT4, and exhibit greater self-renewal ability. And 143-L cell have multi-differentiation potential. 143-L have greater DOX resistance and Tumorigenesis ability than parental.