Characterization Of Lung Tumorspheres By Gene Expression And Flow Cytometry: Differential Expression In Csc-Related Markers And Signaling Pathways

Chemoresistance, progression and metastasis have made of lung cancer the first cause of cancer mortality. These features were linked to a subpopulation of cells, named cancer stem cells (CSCs), which remain largely unknown. 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). Methods: Tumor cells from resected NSCLC and cell lines (H1650, H1993, A549, and PC9) were grown in monolayer and as spheroids. RTqPCR was performed to analyze the mRNA expression of CSCs-related genes: CSC-markers (EPCAM1, ALDH1A1, CD166, ABCG2, CD44, CD133); pluripotency genes (KLF4, OCT4, NANOG, SOX2, MYC, CCND1); Notch pathway (NOTCH1, NOTCH3, HEY1); Wnt pathway (WNT1, WNT5A, DKK1, FZD7) and Hedgehog pathway (SMO, PTCH1, SHH, GLI1). ACTB and CDKN1B were used as endogenous controls for relative expression calculation. The expression of lung stem cell markers EpCAM, CD166, E-cadherin, CD90, CD44, CD34, CD133 and ABCG2 was assessed by flow cytometry. The tumor-initiating cell capacity of selected lung-spheres was tested in vivo to confirm tumorigenicity. Results: Lung-tumorspheres had increased expression of EPCAM, CD44 and ALDH1A1 (p = 0.028, p = 0.021 and p = 0.043, respectively) when compared to cells grown in adherence. Likewise, NANOG, KLF4 and OCT4 tended to be more expressed in tumorspheres. Relative gene expression of NOTCH1 was also higher in spheroids than in monolayer cells (p = 0.028), in concordance with the same tendency observed in NOTCH3. Similarly, RTqPCR analysis revealed a possible activation of the canonical Wnt pathway in tumorspheres, with high expression levels of the downstream effector gene CCND1 (p = 0.05), along with a repression of DKK1 inhibitor. Regarding to the non-canonical Wnt pathway, its activator WNT5A showed lower expression levels in spheroids compared to monolayer-culture cells. Concerning the expression levels of Hedgehog pathway9s genes, we found that SMO and PTCH1 were underexpressed in lung-tumorspheres compared with their paired adherent-cultured cells (p = 0.028 and p = 0.069). Flow cytometry revealed that EpCAM and CD44 were highly expressed in lungspheres obtained from cell lines and primary tumors. The expression of CD166 differed among the cell lines. Furthermore, EpCAM+/CD90- subpopulation were the ones able to induce tumor in xenotrasplant mouse model demostrating tumor-initiating capacity in vivo. Conclusions: Lung-tumorspheres derived from cancer cell lines and primary tumor tissues show increased levels of EpCAM and others CSC markers. Genes related to Notch and Wnt signaling pathways were more expressed in spheroids compared to the cells grown in adherence, suggesting both pathways as interesting lung-CSC targets. Supported by grants RD12/0036/0025 from RTICC, PI12-02838 and PI12-0956 from ISCIII and SEOM/2012.[E.M.-M., A.H.-P. and A.M.-R. contributed equally to this work.] Citation Format: Ester Munera-Maravilla, Alejandro Herreros-Pomares, Alicia Martinez-Romero, Sandra Tejedor, Silvia Calabuig-Farinas, Eloisa Jantus-Lewintre, Rut Lucas, Eva Escorihuela, Rosa Farras, Carlos Camps. Characterization of lung tumorspheres by gene expression and flow cytometry: differential expression in CSC-related markers and signaling pathways. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3354.