Isolation Of Lung Cancer Stem Cells And Its Drug Resistance-related Features

Background:Non-small cell lung cancer (NSCLC) was the leading cause of cancer-related mortality and morbidity treatment failure. The chemoresistance of lung cancer remains a major unresolved clinical and scientific problem. In recent years, increasing evidence indicates that cancer stem cells (CSCs) are initiators of the occurrence, development and recurrence of malignant tumors. In addition, CSCs proposed may responsible for the failure of chemotherapy.Cancer stem cell was a small proportion of cells which had the stem cell-like characteristics reside in tumor, which had the ability of self-renewal, multi-potent differentiation, tumorigenicity. Many kinds of cancer stem cells were isolated from a variety of tumors, such as malignant glioma, breast cancer, leukemia and lung cancer. Research showed that cancer stem cells had higher capacity of the natural resistance than the differentiated. For instance, MRP1 was high expression in glioma stem cells and ABCG2 in breast cancer stem cells. The researches of the human lung cancer stem cell were still known poorly. However, chemoresistance studied comprehensively and deeply. In order to provide experimental basis for targeted therapy of the cancer, we isolated and identified lung stem cell which from A549 cell line under serum-free floating stem cell medium, and explored the biological character and mechanisms of drug resistance of the cancer stem cell.Objective:1,Isolation of lung cancer stem cell spheres from human lung cancer cell line A549; detect the expression of stem cell markers2,Explore the biological characteristics of drug resistanceMaterials:Human lung cancer cell line A549 were preserved in Institute of Respiratory Diseases, Xinqiao Hospital of Third Military Medical University. NOD/SCID mice were purchased from the animal institute of the Xinqiao Hospital of Third Military Medical University,3-4weeks. All experiments were approved in the Institute of Respiratory Diseases, Xinqiao Hospital of Third Military Medical University.Methods:1. Cell culture:A549 Lung cancer cells culture in DMEM supplemented with 10% fetal bovine serum with 5%CO2 at 37℃, A549 cancer spheres cells grow in serum-free DMEM-F12 supplemented with 4u/L insulin, 1:1 B27,20ng/mL epidermal growth factor and 20ng/mL basic fibroblast growth factor with 5%CO2 at 37℃.2. Flow cytometry analysis of the expression of CD133 of A549 cancer spheres and its parental cell line A549.3. After immunofluorescence staining, the expression of stem cell makers (CD133 and CD44s) were detected by laser confocal microscopy (LCM).4. MTT assay was used to detect the cell proliferation,1C50 of A549 cancer spheres and its parental cell line A549.5. Use Flow cytometry and Immunofluorescence to analyze cell cycle and apoptosis.6. Use Western blot and Immunofluorescence to analyze the expresstion of GST-πprotein.7. RT-PCR was used to detect the levels of MRP 1, LRP mRN A.8. Tumor formation in an animal model NOD/SCID mice.Results:1. Serum-free floating culture system could be used to culture A549 cancer spheres. Observed under microscope:spheres clusters of different sizes, floated in culture medium, each small spheres/well contained 10-20 cells, structure is more dense and difficult to disperse, bright and strong refraction were observed under microscope.2. Our results showed that the percentage of CD 133+expresstion in A549 cancer spheres cell was significantly higher than A549 cells line:sphere,66±1.23%; cell line,10.2±0.83%; P<0.05.3. Fluorescent immunostaining revealed that some stem cell-related markers, such as CD133 and CD44s were positive in these secondary tumor spheres.4. To evaluate A549 cancer spheres self-renewal capability, We examined the growth of A549 cancer spheres cells and A549 cells line by the MTT assay. The doubling time of A549 cancer spheres cells in serum-free stem cell culture medium was 53.96h. However, A549 cells line more quiescent in DMEM with 10% FBS was 75.6h. Spheres cell are more resistant to Cisplatin and Gemcitabine than A549 cells line, IC50 of Cisplatin in spheres and cell line is 18.25±0.66 mg/L and 3.8±0.36 mg/L, P<0.05. IC50 of Gemcitabine in spheres and cells line is 280.38±8.2 mg/L and 189.47±5.65 mg/L, P<0.05.5. Spheres have no difference in cell cycle distribution (G0/G1 and S) with its parental cell line. However, an increase in the number of A549 spheres cell in the G2/M phase was apparent, the proportion of cells in G2/M cell cycle of spheres and cell line is (17.2±0.54)% and (4.75±0.35)%, P<0.05. Both the DDP and GME treated A549 cell line (A549 cell line/DDP and A549 cell line/GME) at the S phase significantly decreased compared with control group, P<0.05. The DDP and GME treated groups (A549 spheres/DDP and A549 spheres/GME) had no significant changes in the G0/G1 phase and S phase were seen as compared with the control group. Meanwhile, under the effect of DDP or GEM, the apoptotic percentages of A549 cancer spheres cells and A549 cells line had increase compared with control, and A549 cells line was more significantly increased the apoptotic percentages than A549 cancer spheres cells:sphere/DDP,14.64±1.23%; cell line/DDP,72.16±5.63%; sphere/GME,34.38±2.94%; cell line/GEM. 77.32±4.73%; sphere control,4.16±1.12%; cell line control,11.83±1.56%; P<0.05.6. RT-PCR analysis showed that MRP1 and LRP mRNA expression of A549 cancer spheres were higher than A549 cells line, but not found in HBE cells. GST-πprotein were expressed significantly higher levels in the A549 cancer spheres than in the A549 cells line, but still not expressed in the HBE cell. Fluorescent immunostaining revealed that strong red fluorescence was observed in the cytoplasm.GST-πprotein were expressed significantly higher levels in the A549 cancer spheres than in the A549 cells line. 7. After subcutaneous tumor, In the first 45 days remove the subcutaneous tumors were to Pathological examination. These results were further confirmed by analyzing the Immunofluorescence staining of GST-πin A549 cancer spheres and A549 cells line, strong red fluorescence was observed in the cytoplasm, the fluorescence intensity(mean) of spheres(18.53±0.77) shown higher to cells line (12.54±0.49), P<0.05.Conclusions:1. A549 cancer spheres could be cultured successfully under serum-free foating-culture system, in which CD133+ and CD44s were highly expressed.2. A549 cancer spheres possess stronger drug-resistance characteristics than A549 cells line.