Identifying the Metabolic “Achilles Heel” of Adult and Childhood Brain Cancers Using LC-MS-based Metabolite Profiling Dong-Hyun Kim (Presenter) University of Nottingham Presenter Bio: Dr Dong-Hyun Kim is an Assistant Professor and Co-Director of Centre for Analytical Bioscience in the School of Pharmacy, the University of Nottingham. His research career has focused on the advancement and application of mass spectrometry (MS)-based metabolomics methods in order to investigate complex biological and/or clinical problems. His role includes coordination of internal and external project work as well as fostering interdisciplinary applications of MS-based analytical techniques. He is also Co-Director of Metabolic Profiling Forum which is a not-for-profit organisation that runs the International Metabomeeting conference series.

Authors: James A. Wood (1), Richard Grundy (1), David A. Barrett (2), Ruman Rahman (1), Dong-Hyun Kim (2)
(1) Children’s Brain Tumour Research Centre, School of Medicine, University of Nottingham, UK. (2) Centre for Analytical Bioscience, Advanced Materials and Healthcare Technologies Division, School of Pharmacy, University of Nottingham, UK.

Glioblastoma multiforme (GBM) was recently shown to be dependent on extracellular sources of lipids to meet cellular requirements as opposed to de novo biosynthesis. We conducted LC-MS-based metabolomics to characterise the cell metabolome of GBM cell lines cultured under lipoprotein-deficient conditions to replicate nutrient stresses seen in vivo. As a result, cell line-specific metabolite changes were observed calling for the need for integration with transcriptomics data to elucidate genetic drivers of cell line-specific stress responses. This study has highlighted the metabolic dependencies of GBM under nutrient-limiting conditions which will inform on the development of new therapeutic strategies targeting stress responses.

Introduction

Glioblastoma multiforme (GBM) is a highly malignant brain tumour with dismal outcomes across all age groups as a result of resistance mechanisms following conventional surgery, chemotherapy, and radiotherapy regimens [1]. Trials investigating the therapeutic efficacy of agents targeting cell signalling pathways have demonstrated no clinical benefit to overall survival in GBM due to several redundant survival signalling pathways, stem-like quiescence, and intratumour heterogeneity [2]. As a result, there has been much interest in targeting the metabolic requirements of GBM as an alternative strategy as all tumour cells regardless of genetic make-up require energy and building blocks to proliferate.

Lipoproteins are multi-molecular assemblies consisting of proteins as well as cholesterol and lipid species. They function to transport nutrients from the gut and liver to the periphery. Although the blood-brain barrier blocks the passage of low-density lipoproteins (LDLs), small high-density lipoproteins can traverse into the central nervous system (CNS). The role of lipoproteins within the CNS is to transfer phospholipids and cholesterol between cells but they are also implicated in the pathogenesis of Alzheimer’s disease [3]. Interestingly, recent work has highlighted a non-oncogene addiction of cellular and mouse models of adult GBM to lipoproteins in the maintenance of cholesterol homeostasis and tumour growth [4-5]. In our study, we conducted liquid chromatography-mass spectrometry (LC-MS)-based metabolomics [6-7] and transcriptomics to understand the consequences of growth under lipoprotein-deficient conditions at the metabolic and gene transcript level.

Methods

Six GBM cell lines were chosen to study lipoprotein dependence across age group as well as tumour grade: U87 (adult grade IV), KNS42 (paediatric grade IV), SF188 (paediatric grade IV), UW479 (paediatric grade III), Res259 (paediatric grade II), and Res186 (paediatric grade I). All cell lines were cultured in the same medium consisting of 1 g/L glucose Dulbecco’s modified eagle medium (DMEM) with 10% foetal bovine serum (FBS), penicillin/streptomycin, and added non-essential amino acid supplements (normal medium). Growth under lipoprotein-deplete conditions was conducted through the replacement of FBS component within the normal medium with lipoprotein-deficient calf serum (LCS).

After the cell culture, the culture medium was removed and intracellular metabolites were extracted by adding methanol:chloroform:water (1:3:1), then cells were scraped and centrifuged at 13,200 g at 4 °C for 10 min. The extracts were transferred to pre-cooled tubes and stored at -80 °C until LC-MS analysis. After the metabolite extraction, chromatographic separation was performed on HILIC and C18 columns and detection was carried out on an Exactive Orbitrap mass spectrometer (Thermo Fisher Scientific) [8-10].

Results

We hypothesised that cellular metabolism under lipoprotein-deficient conditions would be alter to meet the lipid and cholesterol needs of proliferating tumour cells. As such, metabolites from all six cell lines under lipoprotein-replete and –deplete conditions were extracted and profiled using LC-MS. Approximately 805 human metabolites including lipids were identified putatively following data pre-processing. Multivariate analysis demonstrated a distinct metabolomic and lipidomic profiles under lipoprotein-deficient conditions in all cell lines. Univariate analysis highlighted several metabolites and lipids with increased or decreased abundance under lipoprotein-deficient conditions. Many metabolites demonstrated similar changes across several cell lines indicating a common metabolic response under lipoprotein-deficient conditions. Interestingly, the KNS42 and SF188 cell lines displayed a lower abundance of a particular lipid species, hinting at dysregulated specific lipid metabolism under lipoprotein-deficient conditions. Several membrane lipids were observed to be altered providing evidence of altered plasma membrane dynamics which needs further investigation in terms of changes to membrane fluidity and the consequences on cell motility, a feature highly pertinent to the invasive nature of GBM cells.

Transcriptomics analysis was also performed to determine gene transcripts with altered expression after 72 hr growth under lipoprotein deficient conditions. We hypothesised that the expression of several metabolic genes would be upregulated to compensate for the lack of lipoproteins to sustain tumour cell proliferation. Bioinformatics analysis revealed an enrichment of genes associated with the synthesis of the specific lipid species identified by the metabolite profiling. In combination with the metabolomics analysis, this highlighted the importance of lipoproteins to the maintenance of tumour growth via the regulation of the specific lipid synthesis. More detailed results with the specific lipid species will be discussed in this presentation.

Conclusions & Discussion

This work has highlighted a metabolic vulnerability within both adult and paediatric glioma cells in terms of lipoproteins and their association with lipid metabolism. The findings presented thus far call for studies examining the therapeutic feasibility and efficacy of drugs targeting a specific lipid metabolism. This is particularly pertinent within the brain as some of lipids are an essential component of brain tissue and fundamental to normal functional activity. We have tested an existing drug on all six cell lines based on the metabolomics and transcriptomics results and these results will be discussed at this talk. Finally, this study has generated several questions requiring downstream investigation, including biological mechanism studies, animal studies, and drug development. The scope of these future studies entails years of investigation before the impact of this research goes from bench to bedside.

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8. Surrati, A., Linforth, R., Fisk, I. D, Sottile, V., Kim, D. H. Non-destructive characterisation of mesenchymal stem cell differentiation using LC-MS-based metabolite footpriting. Analyst 141, 3776-3787 (2016).

9. Steketee, P. C., Vincent, I. M., Achcar, F., Giordani, F., Kim, D. H., Creek, D. J., Freund, Y., Jacobs, R., Rattigan, K., Horn, D., Field, M. C., MacLeod, A., Barrett, M. P. Benzoxaborole treatment perturbs S-adenosyl-L-methionine metabolism in Trypanosoma brucei. PLOS Neglected Tropical Diseases, 12(5), e0006450 (2018).

10. Schatschneider, S., Abdelrazig, S., Safo, L., Henstra, A. M., Millat, T., Kim, D. H., Winzer, K., Minton, N. P., Barrett, D. A. Quantitative isotope dilution high-resolution mass spectrometry analysis of multiple intracellular metabolites in Clostridium autoethanogenum using uniformly 13C-labelled standards derived from Spirulina. Analytical Chemistry 90, 4470-4477 (2018).