Treatment resistance and relapse have been associated to cancer stem cells (CSCs), a highly tumorigenic subpopulation of cells with self-renewal properties and the ability to grow forming tumorspheres in non-adherent conditions. The aim of this study was to isolate and characterize CSCs from lung cancer cell lines and tumor-tissue from resectable non-small cell lung cancer (NSCLC).
The study was performed on tumour cells from 8 resected NSCLC patients and 12 NSCLC cell lines grown in monolayer and as spheroids. The expression of 60 genes, including CSC-markers, pluripotency inducers, cell cycle regulators, invasion promoters and components of Notch, Wnt and Hedgehog pathways was analysed by RTqPCR. In addition, protein levels of CSC-markers (ALDH1A1, CD133, CD166, CD44 and EpCAM), pluripotency inducers (Nanog, Oct-4 and Sox2), Wnt components (Wnt3 and β-catenin) and Notch1 were assessed by western blot and immunofluorescence.
Lung tumorspheres had significantly higher expression levels of CSC-related genes EPCAM1, CD44, ALDH1A1, CDKN1A, CCND1, and KLF4 compared to their paired-adherent cells. Similarly, epithelial to mesenquimal transition (EMT) inducer SNAI1 and integrins ITGA2, ITGA6 and ITGB1 were overexpressed in lungspheres. Notch pathway ligands, JAG1 and DLL4, receptors, NOTCH1, NOTCH2 and NOTCH3, and the effector factor HES1 showed increased expression in spheroids. In Wnt, higher expression levels of WNT3, CTNNB1 and GSK3B were found in tumorspheres. No significant differences were found for the rest of genes analyzed. Western blot and immunofluorescence analyses revealed that CD44, CD133, Sox2 and β-catenin were highly expressed in spheroids from cell lines and patients’ samples. The expression of the rest of proteins evaluated differed among specimens.
Our results suggest four molecules which could act as markers for CSCs in NSCLC. Genes related to Notch and Wnt signaling pathways were more expressed in spheroids compared to the cells grown in adherence, suggesting that both pathways could be interesting targets against lung CSCs. Supported by grants RD12/0036/0025 from RTICC-FEDER, and PI12-02838 and PI15-00753 from ISCIII.
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Legal entity responsible for the study
Fundación de Investigación Hospital General Universitario de Valencia.
Supported by grants RD12/0036/0025 from RTICC-FEDER, and PI12-02838 and PI15-00753 from ISCIII.
All authors have declared no conflicts of interest.