Joanna Kapeleris1,2, Juliana Müller Bark1,2,3, Shanon Ranjit1, Derek Richard4, Ian Vela5, Kenneth O'Byrne4,6, Chamindie Punyadeera7,8,9,10. 1. Centre for Biomedical Technologies, School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, 60 Musk Avenue, GPO Box 2434, Kelvin Grove, QLD, 4059, Australia. 2. Translational Research Institute, Woolloongabba, Brisbane, Australia. 3. Saliva and Liquid Biopsy Translational Laboratory, Griffith Institute for Drug Discovery, Griffith University, 46 Don Yong Road, Nathan, Brisbane, Australia. 4. Cancer & Ageing Research Program, Queensland University of Technology, Translational Research Institute, Woolloongabba, Brisbane, Australia. 5. Australian Prostate Cancer Research Centre, Queensland University of Technology, Princess Alexandra Hospital, Translational Research Institute, Brisbane, QLD, Australia. 6. Princess Alexandra Hospital, Woolloongabba, QLD, Australia. 7. Centre for Biomedical Technologies, School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, 60 Musk Avenue, GPO Box 2434, Kelvin Grove, QLD, 4059, Australia. c.punyadeera@griffith.edu.au. 8. Translational Research Institute, Woolloongabba, Brisbane, Australia. c.punyadeera@griffith.edu.au. 9. Saliva and Liquid Biopsy Translational Laboratory, Griffith Institute for Drug Discovery, Griffith University, 46 Don Yong Road, Nathan, Brisbane, Australia. c.punyadeera@griffith.edu.au. 10. Menzies Health Institute Queensland, Griffith University, Gold Coast, Australia. c.punyadeera@griffith.edu.au.
Abstract
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.
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.
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