Liquid chromatography-mass spectrometry-based metabolomics and fluxomics reveals the metabolic alterations in glioma U87MG multicellular tumor spheroids versus two-dimensional cell cultures.

RATIONALE: Multicellular tumor spheroids (MCTSs) that reconstitute the metabolic characteristics of in vivo tumor tissue may facilitate the discovery of molecular biomarkers and effective anticancer therapies. However, little is known about how cancer cells adapt their metabolic changes in complex three-dimensional (3D) microenvironments. Here, using the two-dimensional (2D) cell model as control, the metabolic phenotypes of glioma U87MG multicellular tumor spheroids were systematically investigated based on static metabolomics and dynamic fluxomics analysis. METHODS: A liquid chromatography-mass spectrometry-based global metabolomics and lipidomics approach was adopted to survey the cellular samples from 2D and 3D culture systems, revealing marked molecular differences between them. Then, by means of metabolomic pathway analysis, the metabolic pathways altered in glioma MCTSs were found using 13 C6 -glucose as a tracer to map the metabolic flux of glycolysis, the tricarboxylic acid (TCA) cycle, de novo nucleotide synthesis, and de novo lipid biosynthesis in the MCTS model. RESULTS: We found nine metabolic pathways as well as glycerolipid, glycerophospholipid and sphingolipid metabolism to be predominantly altered in glioma MCTSs. The reduced nucleotide metabolism, amino acid metabolism and glutathione metabolism indicated an overall lower cellular activity in MCTSs. Through dynamic fluxomics analysis in the MCTS model, we found that cells cultured in MCTSs exhibited increased glycolysis activity and de novo lipid biosynthesis activity, and decreased the TCA cycle and de novo purine nucleotide biosynthesis activity. CONCLUSIONS: Our study highlights specific, altered biochemical pathways in MCTSs, emphasizing dysregulation of energy metabolism and lipid metabolism, and offering novel insight into metabolic events in glioma MCTSs.

Spatiotemporal pharmacometabolomics based on ambient mass spectrometry imaging to evaluate the metabolism and hepatotoxicity of amiodarone in HepG2 spheroids.

Three-dimensional (3D) cell spheroid models combined with mass spectrometry imaging (MSI) enables innovative investigation of in vivo-like biological processes under different physiological and pathological conditions. Herein, airflow-assisted desorption electrospray ionization-MSI (AFADESI-MSI) was coupled with 3D HepG2 spheroids to assess the metabolism and hepatotoxicity of amiodarone (AMI). High-coverage imaging of >1100 endogenous metabolites in hepatocyte spheroids was achieved using AFADESI-MSI. Following AMI treatment at different times, 15 metabolites of AMI involved in N-desethylation, hydroxylation, deiodination, and desaturation metabolic reactions were identified, and according to their spatiotemporal dynamics features, the metabolic pathways of AMI were proposed. Subsequently, the temporal and spatial changes in metabolic disturbance within spheroids caused by drug exposure were obtained via metabolomic analysis. The main dysregulated metabolic pathways included arachidonic acid and glycerophospholipid metabolism, providing considerable evidence for the mechanism of AMI hepatotoxicity. In addition, a biomarker group of eight fatty acids was selected that provided improved indication of cell viability and could characterize the hepatotoxicity of AMI. The combination of AFADESI-MSI and HepG2 spheroids can simultaneously obtain spatiotemporal information for drugs, drug metabolites, and endogenous metabolites after AMI treatment, providing an effective tool for in vitro drug hepatotoxicity evaluation.