RESUMO
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 O2 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, HIF1α) 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.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Humanos , Carcinoma Pulmonar de Células não Pequenas/patologia , Transição Epitelial-Mesenquimal/genética , Vimentina/metabolismo , Neoplasias Pulmonares/patologia , Células Neoplásicas Circulantes/patologia , RNA Mensageiro/genética , Queratinas , Receptores ErbB , Hipóxia , Oxigênio , Biomarcadores , Linhagem Celular , Linhagem Celular Tumoral , Biomarcadores Tumorais/genética , Microambiente TumoralRESUMO
Background: Non-small cell lung cancer (NSCLC) often presents at an incurable stage, and majority of patients will be considered for palliative treatment at some point in their disease. Despite recent advances, the prognosis remains poor, with a median overall survival of 12-18 months. Liquid biopsy-based biomarkers have emerged as potential candidates for predicting prognosis and response to therapy in NSCLC patients. This pilot study evaluated whether combining circulating tumour cells and clusters (CTCs) and cell-free DNA (cfDNA) can predict progression-free survival (PFS) in NSCLC patients. Methods: CTC and cfDNA/ctDNA from advanced stage NSCLC patients were measured at study entry (T0) and 3-months post-treatment (T1). CTCs were enriched using a spiral microfluidic chip and characterised by immunofluorescence. ctDNA was assessed using an UltraSEEK® Lung Panel. Kaplan-Meier plots were generated to investigate the contribution of the presence of CTC/CTC clusters and cfDNA for PFS. Cox proportional hazards analysis compared time to progression versus CTC/CTC cluster counts and cfDNA levels. Results: Single CTCs were found in 14 out of 25 patients, while CTC clusters were found in 8 out of the 25 patients at T0. At T1, CTCs were found in 7 out of 18 patients, and CTC clusters in 1 out of the 18 patients. At T0, CTC presence and the combination of CTC cluster counts with cfDNA levels were associated with shorter PFS, p = 0.0261, p = 0.0022, respectively. Conclusions: Combining CTC cluster counts and cfDNA levels could improve PFS assessment in NSCLC patients. Our results encourage further investigation on the combined effect of CTC/cfDNA as a prognostic biomarker in a large cohort of advanced stage NSCLC patients.
