| Objective:Compared with2D cell culture, this study highlight the development process of3D cell culture technology system which would be more suitable for the isolation and identification of lung cancer stem-like cells. To explore the application of3D cell culture for the evaluation of proliferation, apoptosis, invasion, and drug resistance of lung cancer.Methods:1. The establishment of3D cell cultureMade with104/well of the human lung adenocarcinoma cell line A549and RPMI1640complete medium containing10%fetal bovine serum. Cell suspension were cultured in BME (basal medium). Growth curve was drawed after7days of culture. Stem-like cell was identified through methods including mammosphere culture, drug resistance, invasion assay and Flow Cytometry. And then compare the data between A549cells were cultured in3D systems and2D systems. Collect each group of cells which cultured3,7,11,14,20days were made of cell suspension. Adjust the cell concentration of1×105cells/100μl, were labeled with a marker antibody respectively, and labeled FITC-IgG2b, APC-IgGl, PE-IgGl as a control for flow cytometric immunophenotyping.2.The functional detection of lung cancer stem cellComparison of balling rate, drug resistance, tumor growth in vivo and identification of stem-like cell between2D and3D cultured cells. And then compare the data between A549cells were cultured in3D systems and2D systems The cells were cultured in each group5000cells/3ml MFM seeded into6-well plates, the cells were observed for balling rate after7days. Inoculate with5×104cells/mouse into SPF mice rspectively,N=4. Measure tumor size of each block every three days and paint growth curve. Build invasion chambers by Transwell and Matrigel to separate high-invasive and minimally invasive cell populations, and assay cell migration by scratch repair. Detect changes in stem cells-like lung cancer related gene and expression levels of protein by quantitative real-time PCR and Western blot in3D and2D cell culture.3. The applications of3D cell culture in cancer drug pharmacodynamicsThese cells between two groups were collected to detecte their sensitivity to cisplatin by MTT assay.Results:1. The establishment of3D cell cultureThe cell growth after3D culture following2D cultured is signifcantly higher than only2D cultured. Phenotype experiments demonstrate that these cells are CD44and CD326double-positive and CD24negative.2.The functional detection of lung cancer stem cellCompared with2D, cells from3D have higher rate of tumor formation (20.75±0.85d vs60.25±1.49d,P<0.01)and tumor sphere formation (28.50±1.17%vs8.67±0.80%, P<0.01), and more ability of invasiveness and wound healing (125.67±6.02vs64.67±11.37,83.75±5.84%vs35.63±3.05%, P<0.01). The expression levels of stem cells-associated genes by3D culture cells is significantly higher than2D.3. The applications of3D cell culture in cancer drug pharmacodynamics3D cultured cells are more invasive and resistant to therapy (58.17±2.19%vs33.27±5.76%in48h, P<0.01),(41.70±5.81%vs27.30±4.25%in72h, P<0.01) than2D cultured cells with different concentrations of cisplatin and pemetrexed. It is found that the expression of β-catenin protein by3D cultured cells is much higher than2D cell culture. Combined with MTT assay results that the IC50of cisplatin and pemetrexed are6.58±1.25μg/ml and7.86±1.60μg/ml through3D cultured cells.Conclusion:The proportion of stem-like cells in A549cell line after3D cell culture significantly increased compared with that of cultured in traditional2D cell culture system, indicating that the3D cell culture system can enrich lung cancer stem cells. It is proved that the stem cells isolated from human lung adenocarcinoma cell line A549by3D system culture techniques has a stronger proliferative capacity, reduced apoptosis, increased metastatic potential and worse drugs reactivity than2D cell cultured. The expression of the protein of stem cells significantly increased and the amount of drug half inhibition concentration is greatly improved. |
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