Mass Spectrometry-Based Metabolomics of JJN-3 Cells: Establishment of Sampling Method and Metabolic Profiling of Stress Response

Abstract

Cancer is a leading cause of death worldwide, and in the search of improved treatment efficacy and novel cancer treatments, the field of metabolomics has achieved much attention recent years. Metabolomics provides analysis of changing metabolite levels in biological samples. As changed metabolism is one of the hallmarks of cancer cells, metabolomics can be used to get a better understanding of the biological mechanisms in cancer cells. The scope of this study was divided in three parts. The aim of the first part was to establish a sampling method for human suspension cells, applicable for metabolic analysis. Two different sampling methods, centrifugation and fast filtration, were applied. GC-Q-MS was applied to obtain semi-quantitative measurements of metabolites, and a comparison between the two sampling methods were made. Fast filtration showed to be an applicable sampling method for suspension cells in metabolic analysis. Fast filtration was superior to centrifugation in terms of sampling time, metabolite recovery and number of metabolites extracted. By applying fast filtration, a sampling method was concluded to have been established.The second aim of this study was to investigate if JJN-3 cells could be synchronized. Double thymidine block and serum starvation was used in the attempt to synchronize the cells. It was concluded that synchronization of JJN-3 cells could be achieved by thymidine block. For the third part, the aim was to do a pilot study of metabolic stress response in JJN-3 cells. Two different anti-cancer drugs, cisplatin and 5-fluorouracil, were used either alone or in combination with an APIM-containing peptide, ATX-101. Both target and non-target metabolic approaches were performed, applying GC-QqQ-MS and UPLC-QTOF-MS, respectively. Cells stressed with either cisplatin or 5-fluorouracil in combination with APIM were concluded impact the cell metabolism, compared to untreated cells.