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Objective:To highlight the developmental process of 3D cell culture technology system, which is more suitable for isolating and identifying lung cancer stem-like cells than 2D cell culture technology system, and to explore the application of 3D cell cultures in the evaluation of proliferation, apoptosis, invasion, and drug resistance of lung cancer. Methods:Cells (104/well) from the human lung adenocarcinoma cell lines A549 and RPMI 1640 were cultured in complete medium containing 10%fetal bovine serum. Cell suspension was cultured in a BME basal medium. A growth curve was drawn after 7 d of culture. The stem-like cell was identified through a mammosphere culture, drug resistance and invasion assay, and flow cytometry. Data of A549 cells cultured in 3D and 2D tra-ditional cell culture technologies were compared. Results: Cells from the 3D cell culture had higher tumor formation rates [(20.75 ± 0.85) d vs. (60.25 ± 1.49) d, P<0.01)] and tumor sphere formation (28.50%± 1.17%vs. 8.67%± 0.80%, P<0.01) than those from the 2D cell culture. Moreover, cells from 3D cell culture were more invasive and resistant to therapy (58.17%± 2.19%vs. 41.70%±5.81%in 48 h, P<0.01;33.27%±5.76%vs. 27.30%±4.25%in 72 h, P<0.01). Phenotype experimental results demonstrated that the CD44 and CD326 cells were double-positive, whereas the CD24 cell was negative. Conclusion:The proportion of stem-like cells in A549 cell line after 3D cell culture significantly increased compared with 2D cell culture. The 3D cell culture can promote the proliferation of lung cancer stem cells.
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Objective:This work aims determine the expression of the neurotensin (NTS) gene in hepatocellular carcinoma (HCC) subgrouping using immunohistochemical staining (IHC) as well as to evaluate the correlation between the activation of NTS/IL-8 pathway in HCC and inflammatory response in microenvironment and epithelial mesenchymal transition (EMT) in cancer and in the prognosis of patients. Methods:Tumor tissues and corresponding adjacent normal tissue were collected from 64 cases of HCC patients. The expression levels of NTS protein and multiple inflammation and EMT-related proteins, including IL-8, VEGF, MMP9, CD68, E-Cadherin,β-Catenin, and Vimentin, were examined in 64 cases of paraffin-embedded HCC tissues using the immunohistochemistry (IHC) staining method. The clinical outcome and overall survival (OS) among 64 cases of HCC patients were compared. Results:We found that the frequency of NTS-expressing tissues among all HCC samples was 17.19%(11/64). Significantly increased IL-8 protein was confirmed in 90.91%of NTS+HCC samples and was positively correlated with the levels of NTS protein in cancer tissues (P=0.036), which implied the dysfunctional activation of NTS/IL-8 pathway in HCC. The levels of VEGF and MMP9 were significantly correlated with the co-expression of NTS and IL-8 in HCC. Evident features of EMT, including decreased membrane expression of E-Cadherin and increased accumulation of cytoplasmicβ-Catemin and Vimentin, were found in NTS+IL-8+samples. The co-expression of NTS and IL-8 in cancer was significantly correlated with the clinical outcomes of patients, as the mortality rate of NTS+IL-8+HCC patients is 2.5-fold higher than that of others after surgery (P=0.022).Accordingly, the OS of NTS+IL-8+HCC patients significantly decreased (24.65±4.45 m vs. 75.79±16.32 m, P=0.013), and these patients are at a higher risk of death at an expected hazard ratio (HR) of 3.457. Conclusion:The NTS/IL-8 pathway is dysfunctionally activated in a subgroup of HCC samples. Highly expressed NTS is associated with increased inflammatory response in microenvironment, enhanced EMT in cancer, and worse prognosis in HCC patients.