Author of

• 16751

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  P1.05 - Poster Session with Presenters Present (ID 457)

  • Event: WCLC 2016
  • Type: Poster Presenters Present
  • Track: Early Stage NSCLC
  • Presentations: 2
  • Moderators:
  • Coordinates: 12/05/2016, 14:30 - 15:45, Hall B (Poster Area)

  • 16764

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    P1.05-013 - Lung Tumorspheres as a Platform for Testing New Therapeutic Strategies in Non-Small Cell Lung Cancer (ID 4848)

    14:30 - 14:30  |  Author(s): S. Calabuig Fariñas
    • Abstract

    Background:
    Resistance to treatment is one of the causes influencing the high mortality of lung cancer. This feature seems to be linked to a subpopulation known as Cancer Stem Cells (CSCs), which are able to grow as spheroids (suspension culture). The aim of the study was to obtain tumorspheres from lung cancer cell lines and to use them as an in vitro platform for drug screening.
    
    Methods:
    Cells from lung cancer cell lines (A549, H1650, PC9, H460 and H358) were grown in monolayer and as spheroids. Cultured cells were used: (i) to compare the cytotoxic effect of anticancer drugs in adherent vs lung-tumorspheres (ii) to perform a high-throughput screening with a commercial chemical library (Prestwick) and (iii) to analyze the citotoxicity of specific inhibitors of Wnt, Hedgehog and Notch pathways. Briefly, cells were plated at the desired density in 200 μl of medium in 96-well plates and compounds were added at 4 different concentrations (n=3). Cell viability was measured after 48 and 72h, using MTS Assay. Cell viability was normalized to the respective mock-treated control cells and presented as percentage of control.
    
    Results:
    Cells cultured in serum-free conditions were able to form spheroids, such as stem-like cells. Under these culture conditions, classical anticancer drugs (cisplatin, paclitaxel, vinorelbine and pemetrexed) exhibited mild or null cytotoxic effects on A549, H1650, PC9, H460 and H358 spheroids. Moreover, we performed a high-throughput screening with Prestwick library and remarkably, three compounds reduced the number of viable cancer cells. As regards ‘stemness’ inhibitors, Wnt (IWP2 and XAV939) and Hedgehog inhibitors (Vismodegib) show high activity against tumorspheres (p<0.05), suggesting them as possible therapeutic strategies in NSCLC
    
    Conclusion:
    Our data suggest that lung-tumorspheres showed resistance to classical anticancer drugs, strengthening its possible use as a short-term culture platform for a simple, and cost- effective screening to investigate novel therapeutic approaches. In this setting, some compounds were identified as promising therapeutic agents on lung tumorspheres, but confirmatory data are still necessary. This project was supported by [RD12/0036/0025] from RTICC, SEOM 2012, [PI12-02838 and PI15-00753] from ISCIII
    
    

  • 16765

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    P1.05-014 - Stemness Gene Expression Profile of Tumorspheres from Non-Small Cell Lung Cancer (ID 4748)

    14:30 - 14:30  |  Author(s): S. Calabuig Fariñas
    • Abstract

    Background:
    Lung cancer features like chemoresistance, tumor progression or metastasis have consolidated lung cancer as the first cause of death cancer-related worldwide. Cancer stem cells (CSCs) are small subpopulations of stem-like cells that have been associated to these traits, constituting a promising target, but remaining largely unknown. In this study, we isolated CSCs from lung cancer cell lines and tumor tissue of resectable NSCLC patients using a sphere-forming assay and analyzed their gene expression profile.
    
    Methods:
    The investigation was carried out on cells from seven NSCLC tumor samples and six cell lines (H1650, H1993, H358, A549, PC9, H460) grown in monolayer and as spheroids. The expression of CSC-markers (CD133, EPCAM1, ALDH1A1, CD166, ABCG2, CD44, MUC1, BMI1, THY1), pluripotency promoters (KLF4, OCT4, NANOG, SOX2, MYC, CCND1), cell cycle regulators (CDKN1A, CDKN2A, MDM2, WEE1), invasiveness-related genes (CDH1, VIM, SNAI1, MMP2, MMP9, CEACAM5, ITGA2, ITGA6, ITGB1), Notch pathway (NOTCH1, NOTCH2, NOTCH3, JAG1, DLL1, DLL4, NUMB, HEY1, HES1), Wnt pathway (WNT1, WNT2, WNT3, WNT5A, CTNBB1, DKK1, FZD7) and Hedgehog pathway (SMO, PTCH1, SHH, GLI1) components were analyzed by quantitative real time PCR (RTqPCR). ACTB, CDKN1B and GUSB genes were used as housekeeping controls for the relative expression calculation.
    
    Results:
    Lungspheres showed significantly higher expression of the CSC-markers EPCAM1, CD44 and ALDH1A1 (p= 0.028, p= 0.021 and p= 0.043, respectively) and the quiescence promoter CDKN1A (p= 0.021) in comparison with their paired-monolayer cells. The epithelial to mesenquimal transition (EMT) inducer, SNAI1, as well as integrins ITGA2, ITGA6 and ITGB1 were overexpressed in tumorspheres (p= 0.011, p= 0.018, p= 0.016 and p= 0.013, respectively). Regarding the Notch signaling pathway, most ligands (JAG1 and DLL4) and receptors (NOTCH1 and NOTCH2) analyzed had increased expression in spheroids (p= 0.021, p= 0.028, p= 0.038 and p= 0.036, respectively). In Wnt pathway, we found higher expression levels of WNT3, CTNBB1 and GSK3B (p= 0.021, p= 0.008 and p= 0.021, respectively) in tumorspheres. No significant results were found for the rest of genes analyzed.
    
    Conclusion:
    Lung cancer spheroids from primary tumors and cell lines showed increased levels of genes related to CSCs properties. Genes belonging to Notch and Wnt signaling pathways were found to be more expressed in tumorspheres, suggesting that these pathways could be interesting lung-CSC targets. This work was supported in part, by grants RD12/0036/0025 from RTICC, and PI12-02838/PI15-0753 from ISCIII.