Treatment resistance in cancer has been linked to a population of tumor cells with self-renewal properties called cancer stem cells (CSCs). The aim of this study was to isolate and characterize CSCs from lung cancer cell lines and tumor tissue from resected non-small cell lung cancer (NSCLC) patients.
The study was performed on tumor cells from 8 resected NSCLC patients and 12 NSCLC cell lines. Suspension cultures were established for CSC isolation (3D tumorspheres), whereas differentiated tumor cells were cultured under adherent conditions (2D). In vitro differentiation, proliferation and chemotherapy resistance and in vivo tumor initiation capacity were tested. 60 CSC-related genes were evaluated by RTqPCR. Gene expression results were validated at protein level by immunoblot and IF.
8 out of 20 primary NSCLC cultures were successfully established, forming 3D tight spheroids or loose aggregates. Tumorspheres showed proper differentiation capacity, unlimited exponential growth, high resistance to cisplatin, paclitaxel, vinorelbine and pemetrexed, and great tumor initiation potential. Gene expression analysis revealed high variability between cell lines and patient cultures and that 2D cultures were more homogenous than 3D. Tight tumorspheres expressed more ECAD than loose ones. Lung tumorspheres had significantly higher expression of CSC-related genes (ALDH1A1, KLF4, NANOG, CD44, CD90, CDKN1A, JUNB, MDM2), invasion promoters (MMP9, SNAI1, ITGA6), ligands and receptors of Notch (NOTCH1, NOTCH3, DLL4, JAG1) and Wnt components (CTNNB1 and GSK3B) than their corresponding 2D cultures cells. Based on their significant and consistent overexpression in all tumorspheres: CD44, NANOG, CDKN1A, SNAI1, ITGA6 and NOTCH3 were selected to constitute a gene signature. Protein expression analyses showed overexpression of proteins encoded by the gene signature on tumorspheres from ADC patients.
Lung tumorspheres are a useful platform for CSCs characterization. The expression signature proposed could provide the basis for developing novel therapies for the treatment of lung ADC. Supported by grants RD12/0036/0025 from RTICC-FEDER, PI12-02838 and PI15-00753 from ISCIII.
Clinical trial identification
Legal entity responsible for the study
Laboratorio de Oncología Molecular, Fundación de Investigación Hospital General Universitario de Valencia.
RTICC-FEDER and Instituto de Salud Carlos III.
All authors have declared no conflicts of interest.