Abstract 1918: Gene characterization of lung-tumorspheres for their usage as an in vitro screening platform for testing new therapeutic strategies
Authors: Herreros-Pomares Alejandro , Munera-Maravilla Ester , Calabuig-Fariñas Silvia , Lucas Rut , Farràs Rosa , Blasco Ana , Torres Susana , Ferri Jose , Guijarro Ricardo , Martorell Miguel , Jantus-Lewintre Eloísa , Carlos Camps 
Published: July 2017

Abstract
Background: Lung cancer features like treatment resistance or tumor relapse have been linked to cancer stem cells (CSCs), a population 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 tumorspheres from lung cancer cell lines and tumor tissue from resectable non-small cell lung cancer (NSCLC) patients and to use them as an in vitro platform for drug screening.
Methods: This study was performed on cells from seven NSCLC tumor samples and five cell lines (H1650, H1993, H1395, A549 and PC9) 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 analyzed by RTqPCR. Drugs commonly used in clinical guidelines (Cisplatin, Paclitaxel, Gefitinib, Erlotinib, Afatinib, Pemetrexed and Vinorelbine) and specific inhibitors of Wnt and Hedgehog pathways (Iwp2, Xav939, LDE225 and Vismodegib and Salynomicin) were tested in triplicates at 4 different concentrations. Cell viability was measured after 48h and 72h using MTS Assay, normalized to the respective mock-treated control cells and presented as percentage of control. Statistical analyses were considered significant at p&lt;0.05.
Results: Lung tumorspheres had significant increased expression of CSC-related genes EPCAM1, CD44, CCND1, KLF4 and CDKN1A, compared to their paired-adherent cells. Likewise, epithelial to mesenquimal transition (EMT) inducer SNAI1 and integrin ITGA6 were overexpressed in spheroids too. Regarding stemness pathway, Notch pathway ligands JAG1 and DLL4 and receptors NOTCH1, NOTCH2 and NOTCH3 showed higher expression in lungspheres. In Wnt pathway, higher expression levels of WNT3, CTNNB1 and GSK3B were found in tumorspheres. No significant differences were found for the rest of genes. Drug screening showed classical anticancer drugs, such as Cisplatin, Vinorelbine or Pemetrexed, had mild cytotoxic effects on lungspheres, obtaining significant differences with the adherent-cultured cells. In contrast, the stemness pathways inhibitors IWP2, XAV939, Salinomycin and Vismodegib showed higher cytotoxic effects on spheroids than in cells grown in monolayers.
Conclusions: Lung-tumorspheres derived from cancer cell lines and primary tumor tissues show increased levels of CSC markers and components of Notch and Wnt signaling pathways compared to the cells grown in adherence. Spheroids showed resistance to classical anticancer drugs, and a greater response to inhibitors from Notch, Wnt and Hedgehog pathways, strengthening its possible use as a short-term culture platform for a simple, and cost-effective screening to investigate novel therapeutic approaches. Supported by grants RD12/0036/0025 from RTICC-FEDER, PI12-02838/PI15-00753 from ISCIII and SEOM/2012.
Citation Format: Alejandro Herreros-Pomares, Ester Munera-Maravilla, Silvia Calabuig-Fariñas, Rut Lucas, Rosa Farràs, Ana Blasco, Susana Torres, Jose Ferri, Ricardo Guijarro, Miguel Martorell, Eloísa Jantus-Lewintre, Camps Carlos. Gene characterization of lung-tumorspheres for their usage as an in vitro screening platform for testing new therapeutic strategies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1918. doi:10.1158/1538-7445.AM2017-1918