Background: Treatment resistance is related to cancer stem cells (CSCs), a highly tumorigenic subpopulation of cells capable of growing and forming tumor spheres under non-adherent conditions. This study aimed to isolate and characterise CSCs from resected non-small cell lung cancer (NSCLC) patients' tumor-tissue and cell lines like tumor spheres and to use them as an in vitro platform for drug screening.

Methods: The study was performed on tumour cells from 8 resected NSCLC patients and 12 NSCLC cell lines grown in monolayer and as spheres. The expression of 60 genes, including CSC-markers, pluripotency inducers, cell cycle regulators and components of the Notch, Wnt and Hedgehog pathways was analysed by RTqPCR. Protein levels of relevant markers were assessed by WB and IF. Based on CSCs' characterization, two new-synthetized compounds, called compound A and compound B, were tested in the 12 cell lines and on tumour cells from 4 resected NSCLC patients, in 8 different concentrations. Cell viability was measured after 48 hours using MTS assay normalized to the respective mock-treated control cells and presented as a percentage of the control. Statistical analyses were considered significant at p<0.05.

Results: Patients´ median age was 65 years [56-76], 73.3% were male and 45% were adenocarcinomas. Lung tumor spheres had a significantly higher expression of CSC-related genes CD44, ALDH1A1, CD90, CDKN1A, JUNB, NANOG, KLF4 and MDM2 than their paired-adherent cells. Invasion promoters SNAI1, ITGA6, and MMP9 were overexpressed in tumor spheres. Notch pathway ligands, JAG1 and DLL4, and receptors, NOTCH1 and NOTCH3, showed increased expression in spheroids than in differentiated cells. In Wnt, higher expression levels of CTNNB1 and GSK3B were found in spheres; when comparing the expression for both conditions. WB and IF revealed that EpCAM, CD44, ALDH1A1, Sox2 and β-catenin were overexpressed in spheroids whereas E-cadherin was overexpressed in the adherent cells. Drug screening showed that classical anticancer drugs had mild cytotoxic effects on both conditions. Stemness pathways inhibitors IWP2, XAV939, Salinomycin and Vismodegib had stronger cytotoxic effects on spheroids than on the cells grown in monolayers. Cytotoxic effect was stronger in the tumor spheres of the lines and patients treated with compounds A and B. Currently, these compounds are being tested in vivo, in tumors induced in xenografts with cells of patients tested in vitro, to determine the mechanism of action and cytotoxic effect on cancer stem cells in the future.

Conclusions: Our data suggest that lung tumor spheres provide a useful short-term culture platform for the simple and cost-effective characterization of CSCs. We found molecules that could be used as novel therapeutic approach in NSCLC. Supported by grants RD12/0036/0025 from RTICC-FEDER, and PI12-02838 and PI15-00753 from ISCIII.

Citation Format: Alejandro Herreros Pomares, Cristóbal Aguilar-Gallardo, Hector Amado, Eva Escorihuela, Ana Blasco, Silvia Calabuig-Fariñas, Juan Murga-Clausell, Eloísa Jantus-Lewintre, Carlos Camps. Lung tumor spheres as in vitro platform for testing new therapeutic strategies against cancer stem cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3056.