Logo: to the web site of Uppsala University

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common form of cancer worldwide. Radiotherapy, with or without surgery, represents the major approach to curative treatment. However, not all tumors are equally sensitive to irradiation. It is therefore of interest to apply newer system biology approaches (e.g., metabolic profiling) in squamous cancer cells with different radiosensitivities in order to provide new insights on the mechanisms of radiation response. In this study, two cultured HNSCC cell lines from the same donor, UM-SCC-74A and UM-SCC-74B, were first genotyped using Short Tandem Repeat (STR), and assessed for radiation response by the means of clonogenic survival and growth inhibition assays. Thereafter, cells were cultured, irradiated and collected for subsequent metabolic profiling analyses using liquid chromatography-mass spectrometry (LC-MS). STR verified the similarity of UM-SCC-74A and UM-SCC-74B cells, and three independent assays proved UM-SCC-74B to be clearly more radioresistant than UM-SCC-74A. The LC-MS metabolic profiling demonstrated significant differences in the intracellular metabolome of the two cell lines before irradiation, as well as significant alterations after irradiation. The most important differences between the two cell lines before irradiation were connected to nicotinic acid and nicotinamide metabolism and purine metabolism. In the more radiosensitive UM-SCC-74A cells, the most significant alterations after irradiation were linked to tryptophan metabolism. In the more radioresistant UM-SCC-74B cells, the major alterations after irradiation were connected to nicotinic acid and nicotinamide metabolism, purine metabolism, the methionine cycle as well as the serine, and glycine metabolism. The data suggest that the more radioresistant cell line UM-SCC-74B altered the metabolism to control redox-status, manage DNA-repair, and change DNA methylation after irradiation. This provides new insights on the mechanisms of radiation response, which may aid future identification of biomarkers associated with radioresistance of cancer cells.

Geodia barretti is a marine sponge common in the north Atlantic and waters outside of Norway and Sweden. The sampling and subsequent treatment as well as storage of sponges for metabolomics analyses can be performed in different ways, the most commonly used being freezing (directly upon collection or later) or by storage in solvent, commonly ethanol, followed by freeze-drying. In this study we therefore investigated different sampling protocols and their effects on the detected metabolite profiles in LC-MS. Sponges (G. barretti) were collected outside the Swedish west coast and pieces from three sponge specimens were either flash frozen in liquid nitrogen, frozen later after the collection cruise, stored in ethanol or stored in methanol. The storage solvents as well as the actual sponge pieces were analyzed, all samples were analyzed with hydrophilic interaction liquid chromatography (HILIC) as well as reversed phase liquid chromatography with high resolution mass spectrometry (HRMS) in positive and negative ionization mode. The data were evaluated using multivariate data analysis. The highest metabolite intensities were found in the frozen samples (flash frozen and frozen after sampling cruise) as well as in the storage solvents (methanol and ethanol). Metabolites extracted from the sponge pieces that had been stored in solvent were found in very low intensity, since the majority of metabolites were extracted to the solvents to a high degree. The exception being larger peptides and some lipids. The lowest variation between replicates were found in the flash frozen samples. In conclusion, the preferred method for sampling of sponges for metabolomics was found to be immediate freezing in liquid nitrogen. However, freezing the sponge samples after some time proved to be a reliable method as well, albeit with higher variation between the replicates. Thus, the study highlights the importance of saving ethanol extracts after preservation of specimens; these valuable extracts could be further used in studies of natural products, chemosystematics or metabolomics.

Hydrophilic interaction liquid chromatography (HILIC)/electrospray ionisation-mass spectrometry (ESI-MS) has gained interest for the analysis of polar analytes in bioanalytical applications in recent years. However, ESI-MS is prone to adduct formation of analytes. In contrast to reversed phase chromatography, small inorganic ions have retention in HILIC, i.e. analytes and inorganic ions may co-elute, which could influence the adduct formation. In the present paper, it was demonstrated that the co-elution of sodium ions or potassium ions and analytes in HILIC/ESI-MS affect the adduct formation and that different concentrations of sodium ions and potassium ions in biological samples could have an impact on the quantitative response of the respective adducts as well as the quantitative response of the protonated adduct. The co-elution also lead to cluster formation of analytes and sodium formate or potassium formate, causing extremely complicated spectra. In analytical applications using HILIC/ESI-MS where internal standards are rarely used or not properly matched, great care needs to be taken to ensure minimal variation of inorganic ion concentration between samples. Moreover, the use of alkali metal ion adducts as quantitative target ions in relative quantitative applications should be made with caution if proper internal standards are not used.

Inorganic ions, such as sodium and potassium, are present in all biological matrices and are sometimes also added during sample preparation. However, these inorganic ions are known to hamper electrospray ionization -mass spectrometry (ESI-MS) applications, especially in hydrophilic interaction liquid chromatography (HILIC) where they are retained and can be detected as adducts and clusters with mobile phase components or analytes. The retention of inorganic ions leads to co-elution with analytes and as a result ion-suppression, extensive adduct formation and problems with reproducibility. In the presented work, a sample preparation method using cation exchange solid phase extraction (SPE) was developed to trap Na⁺ and K⁺ ions from human blood plasma and head and neck cancer cells for the analysis of small cationic, anionic as well as neutral organic analytes. The investigated analytes were small, hydrophilic compounds typically in focus in metabolomics studies. The samples were analyzed using full-scan HILIC-ESI-quadrupole time of flight (QTOF)-MS with an untargeted, screening approach. Method performance was evaluated using multivariate data analysis as well as relative quantifications, spiking of standards to evaluate linearity of response and post-column infusion to study ion-suppression. In blood plasma, the reduction of sodium and potassium ion concentration resulted in improved sensitivity increased signal intensity for 19 out of 28 investigated analytes, improved linearity of response, reduced ion-suppression and reduced cluster formation as well as adduct formation. Thus, the presented method has significant potential to improve data quality in metabolomics studies.