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.
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Abbreviations
- AUC:
-
Area under the curve
- BC:
-
Bladder cancer
- ES:
-
Effect size
- GC:
-
Gas chromatography
- GC-SQ:
-
Gas chromatography-single quadrupole
- HMDB:
-
Human metabolome database
- HS-SPME:
-
Headspace solid-phase microextraction
- MS:
-
Mass spectrometry
- OPLS-DA:
-
Orthogonal partial least squares-discriminant analysis
- PCA:
-
Principal component analysis
- PLS-DA:
-
Partial least squares-discriminant analysis
- QCs:
-
Quality controls
- RI:
-
Retention index
- SCC:
-
Squamous cell carcinoma
- TA:
-
Total area
- TCC:
-
Transitional cell carcinoma
- VOCs:
-
Volatile organic compounds
- VIP:
-
Variable importance in projection
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Acknowledgements
This work received financial support from the European Union (FEDER funds POCI/01/0145/FEDER/007728) and National Funds (FCT/MEC, Fundação para a Ciência e Tecnologia and Ministério da Educação e Ciência) under the Partnership Agreement PT2020 UID/MULTI/04378/2013. This study is a result of the project NORTE-01-0145-FEDER-000024, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement (DESignBIOtecHealth - New Technologies for three Health Challenges of Modern Societies: Diabetes, Drug Abuse and Kidney Diseases), through the European Regional Development Fund (ERDF). C.J.’s research is funded by a research grant from Research Center of Portuguese Oncology Institute of Porto (FB-GEBC-27) and S.M.-R. is a PhD fellow from Fundação para a Ciência e Tecnología (FCT SFRH/BD/112673/2015). M.C. acknowledges FCT through the project UID/Multi/04546/2013.
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DR was responsible for the execution of the experimental work and data analysis. AMA supported cell culture and data analysis. JP helped with the statistical analysis of the data. SM-R, CJ and RH kindly provided the cell lines used in the study and gave conceptual advice. PGP, MLB and MC designed and supervised the study. DR wrote the manuscript with input from MC. All authors critically commented on and approved the final submitted version of the paper.
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Rodrigues, D., Pinto, J., Araújo, A.M. et al. Volatile metabolomic signature of bladder cancer cell lines based on gas chromatography–mass spectrometry. Metabolomics 14, 62 (2018). https://doi.org/10.1007/s11306-018-1361-9
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DOI: https://doi.org/10.1007/s11306-018-1361-9