Lung cancer stem cells (CSCs) are a small subpopulation of cells with self-renewal, tumorigenic properties and the ability to grow to form tumorspheres in non-adherent conditions. Recently, the study on soluble molecules has been a new focus to understand the interaction between CSCs and tumor microenvironment. The aim of this work is to compare the release of cytokines between monolayer cells and tumorspheres of NSCLC cultures.
The study was performed on medium supernatant of cells from 8 NSCLC tumour patients samples and 10 cell lines (A549, H1650, H460, H23, H358, H2228, HCC827, PC9, H1993, SW900) grown in monolayer and tumorspheres at 2 different densities (104 and 105 cells/ml); supernatant was recollected at 12h and 24h. We analyzed 8 soluble factors with immunosuppressive (IL-4, IL-10, IL-13), and immunoregulatory (IL-6, IL-8, IL-17A, TNFa, IFN) capacity through sensitivity bead-based multiplex assay using the Millipore kit from the Luminex 100/200.
IL-4, IL-6, IL-8, TNFa levels were detected in all samples while IFN, IL-10, IL-13, IL-17A were beyond the detectable range in adherents for most of cultures (Detection range [pg/ml] of IL-4: 1.83-7497.38, IL-6: 0.18-813.66, IL-8: 0.31-1042.75, TNFa: 0.43-1758.47, IFN: 0.61-2468.39, IL-10: 1.46-6056.85, IL-13: 0.24-1002.76, IL-17A: 0.73-3074.88). We observed significant differences in levels of IL-6, IL-13, IL-17A, TNFa, IFN between adherent cells and tumorspheres. Moreover, IL-4, IL-10, IL-13, IL-17A, IFN levels in tumorspheres were higher than monolayer. Otherwise, IL-6 and TNFa were secreted more in monolayer. IL-8 is the most secreted molecule in both adherents and tumorspheres by all cultures.
Our preliminary results suggest that high levels of IL-6 and IL-8 were detected in all samples. IL-8, IL-13, IL-17A and IFN secreted by lung CSCs could be involved in the modulation of the immune microenvironment. Additionally, adherent cells show increased levels of IL-6 and TNFa compared to tumorspheres, and IL-8, IL-4, IFN, IL-10, IL-13, IL-17A show the opposite. The next is to extend the cohort to validate our results and to study others immunoregulatory factors. Supported by grants from FEDER and PI12-02838 and PI15-00753 from ISCIII.
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Fundación de Investigación del Hospital General Universitario de Valencia.
Supported by grants from FEDER and PI12-02838 and PI15-00753 from ISCIII.
All authors have declared no conflicts of interest.