Daniela Rodrigues1, Joana Pinto2, Ana Margarida Araújo2, Sara Monteiro-Reis3, Carmen Jerónimo3,4, Rui Henrique3,4,5, Maria de Lourdes Bastos2, Paula Guedes de Pinho2, Márcia Carvalho6,7. 1. UCIBIO/REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal. daniela.fgrodrigues@gmail.com. 2. UCIBIO/REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal. 3. Cancer Biology & Epigenetics Group, Research Center (CI-IPOP), Portuguese Oncology Institute of Porto (IPO Porto), Porto, Portugal. 4. Department of Pathology and Molecular Immunology-Biomedical Sciences Institute (ICBAS), University of Porto, Porto, Portugal. 5. Department of Pathology, Portuguese Oncology Institute of Porto (IPO Porto), Porto, Portugal. 6. UCIBIO/REQUIMTE, Laboratory of Toxicology, Department of Biological Sciences, Faculty of Pharmacy, University of Porto, Porto, Portugal. mcarv@ufp.edu.pt. 7. UFP Energy, Environment and Health Research Unit (FP-ENAS), University Fernando Pessoa, Porto, Portugal. mcarv@ufp.edu.pt.
Abstract
INTRODUCTION: Recent studies provide a convincing support that the presence of cancer cells in the body leads to the alteration of volatile organic compounds (VOCs) emanating from biological samples, particularly of those closely related with tumoral tissues. Thus, a great interest emerged for the study of cancer volatilome and subsequent attempts to confirm VOCs as potential diagnostic biomarkers. OBJECTIVES: The aim of this study was to determine the volatile metabolomic signature of bladder cancer (BC) cell lines and provide an in vitro proof-of-principle that VOCs emanated into the extracellular medium may discriminate BC cells from normal bladder epithelial cells. METHODS: VOCs in the culture media of three BC cell lines (Scaber, J82, 5637) and one normal bladder cell line (SV-HUC-1) were extracted by headspace-solid phase microextraction and analysed by gas chromatography-mass spectrometry (HS-SPME/GC-MS). Two different pH (pH 2 and 7) were used for VOCs extraction to infer the best pH to be used in in vitro metabolomic studies. RESULTS: Multivariate analysis revealed a panel of volatile metabolites that discriminated cancerous from normal bladder cells, at both pHs, although a higher number of discriminative VOCs was obtained at neutral pH. Most of the altered metabolites were ketones and alkanes, which were generally increased in BC compared to normal cells, and alcohols, which were significantly decreased in BC cells. Among them, three metabolites, namely 2-pentadecanone, dodecanal and γ-dodecalactone (the latter only tentatively identified), stood out as particularly important metabolites and promising volatile biomarkers for BC detection. Furthermore, our results also showed the potential of VOCs in discriminating BC cell lines according to tumour grade and histological subtype. CONCLUSIONS: We demonstrate that a GC-MS metabolomics-based approach for analysis of VOCs is a valuable strategy for identifying new and specific biomarkers that may improve BC diagnosis. Future studies should entail the validation of volatile signature found for BC cell lines in biofluids from BC patients.
INTRODUCTION: Recent studies provide a convincing support that the presence of cancer cells in the body leads to the alteration of volatile organic compounds (VOCs) emanating from biological samples, particularly of those closely related with tumoral tissues. Thus, a great interest emerged for the study of cancer volatilome and subsequent attempts to confirm VOCs as potential diagnostic biomarkers. OBJECTIVES: The aim of this study was to determine the volatile metabolomic signature of bladder cancer (BC) cell lines and provide an in vitro proof-of-principle that VOCs emanated into the extracellular medium may discriminate BC cells from normal bladder epithelial cells. METHODS: VOCs in the culture media of three BC cell lines (Scaber, J82, 5637) and one normal bladder cell line (SV-HUC-1) were extracted by headspace-solid phase microextraction and analysed by gas chromatography-mass spectrometry (HS-SPME/GC-MS). Two different pH (pH 2 and 7) were used for VOCs extraction to infer the best pH to be used in in vitro metabolomic studies. RESULTS: Multivariate analysis revealed a panel of volatile metabolites that discriminated cancerous from normal bladder cells, at both pHs, although a higher number of discriminative VOCs was obtained at neutral pH. Most of the altered metabolites were ketones and alkanes, which were generally increased in BC compared to normal cells, and alcohols, which were significantly decreased in BC cells. Among them, three metabolites, namely 2-pentadecanone, dodecanal and γ-dodecalactone (the latter only tentatively identified), stood out as particularly important metabolites and promising volatile biomarkers for BC detection. Furthermore, our results also showed the potential of VOCs in discriminating BC cell lines according to tumour grade and histological subtype. CONCLUSIONS: We demonstrate that a GC-MS metabolomics-based approach for analysis of VOCs is a valuable strategy for identifying new and specific biomarkers that may improve BC diagnosis. Future studies should entail the validation of volatile signature found for BC cell lines in biofluids from BC patients.
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