The In Vitro Study Of Serum-free Culture Combined DDP Enriching Human Lung Cancer Stem Cells

Lung cancer is one of the most harmful tumors to the human's health and life. So far, the existing lung cancer treatments can only prolong patients'survival and improve the quality of life, almost all of the patients eventually die of original disease. Recently a theory proposes that there is a small subset within a tumor having the characteristics of stem cells, such as infinite proliferation potential and self-renewal which can initiate and maintain the tumor. Target therapy of the CSCs may significantly improve the tumor treatment. The scarcity in quantity and improper approaches for isolation and purification of CSCs become the major obstacles for great development in CSCs field. Therefore establishing a more efficient method to isolate, enrich and culture CSCs is necessary for the further research.Objective:①To prove whether there exist cancer stem cells in A549 human lung cancer cell line.②According to the chemotherapy-resistant feature of CSCs, adopt suspension culture combined with anticancer regimens for screening high purity lung cancer stem cell in A549 human lung cancer cell line.Methodology:Groups: Parental A549 cell line group; Serum-free cultured cells, CSCs group; Serum-free cultured combined with Drug-selected cells, DSCs group. ①An equal number of cells from 3 groups were plated at a concentration of 5000 cell/well in 6-well plates containing SFM. Inverted microscope was used for observation.②The cells in 3 groups limit diluted at a concentration of 10 cell/well in 96-well plates containing SFM to examine the capability of single cell to form lung cancer sphere.③Cell Counting Kit-8 (CCK-8) assay were done to compare the ability of self-renewal, differentiation, and resistance to DDP among 3 groups.④3 groups cells were stained by Hoechst 33342 fluorescent dye, Hoechst blue-Red graph was established by FASC BD Aria II, and the side population (SP) phenotype cell content was analyzed,after adding reserpine, the Hoechst 33342 transport blocker, SP"gate"was set as the fluorescent decreased region. The SP content in the"gate"was compared.Results:①The morphology of drug-selected A549 cells: After 3-cycle-selection by 7μg/ml DDP, the morphology of parental A549 cell line was changed obviously: the polygon cells which lined with dense became enlarged and flattened, and the number of particles on the cells'surface was increased.②The cells'self-renewal and differentiation capability: the tumor sphere formation of single cell had been observed by inverted microscope for 2 weeks. We found that DSCs could form lung cancer sphere at day 3, while CSCs at day 5, both the size and the number of sphere by DCSs were larger.③Resistance to DDP: CCK-8 assay was used to determine the cell apoptosis after 24h DDP treatment, and the finding was analyzed by SPSS 17.0. DSCs showed higher resistance capability to DDP than CSCs(IC50 value is 66.52±2.49μg/ml vs. 37.82±0.39μg/ml),DSCs and CSCs showed～6 fold and～3.5 fold resistance increase compared with parental A549 cell line(IC50 value is 10.81±0.39μg/ml) (P<0.01).④Analysis of SP phenotype: Hoechst blue-Red graphs revealed SP fractions were substantially higher in DSCs than CSCs(12.32±1.51% vs.7.93±1.44%), the fraction in DSCs and CSCs were～12 fold and～8 fold to that in parental A549(1.29±0.48%)(P<0.01).Conclusion: ①In human lung cancer A549 cell line, there existed the lung cancer stem cells with higher capability of self-renewal, differentiation and drug resistance.②Serum-free culture combined with DDP regimens could screen more purified lung cancer stem cells economically and feasibly.