Modelling reoxygenation effects in non-small cell lung cancer cell lines and showing epithelial-mesenchymal transition

Purpose Circulating tumour cells (CTCs) are a rare cell subpopulation regulated by the tumour microenvironment. In hypoxic conditions, CTCs are able to invade the lymphatic and circulatory systems leading to metastasis at distant sites. Methods To mimic in vivo oxygen variations and effects on CTCs, we have cultured five non-small cell lung cancer (NSCLC) cell lines under normoxic and hypoxic conditions, followed by a pulse of reoxygenation for 4 h. Results Proliferation, spheroid-formation and colony formation ability under varying O-2 levels were investigated. Proliferation rate was not altered when cells were cultured in 2D models under hypoxic conditions. However, we observed that hypoxia enhanced in vitro formation of tumour-spheres and accelerated clonogenicity of NSCLC cell lines. In addition, cells exposed to hypoxia and reoxygenation conditions showed altered expression of epithelial-mesenchymal transition (EMT) related genes in NSCLC cell lines both at mRNA (AKT1, CAMK2NH1, DESI1, VIM, MAP1B, EGFR, ZEB1, HIF1 alpha) and protein levels (Vimentin, Pan-cytokeratin). Conclusion Our data suggest that when investigating CTCs as a prognostic biomarker in NSCLC, it is also essential to take into consideration EMT status to obtain a comprehensive overview of CTCs in circulation.