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Mapping Metabolic Networks in 3D spheroids using Stable Isotope-Resolved Metabolomics
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1  Center for Environmental and Systems Biochemistry, Dept. of Toxicology and Cancer Biology, Markey Cancer Center, University of Kentucky


Conventional 2D cell cultures are grown on rigid plastic substrates with unrealistic concentration gradients of O2, nutrients, and treatment agents. More importantly, 2D cultures lack cell-cell and cell-extracellular matrix (ECM) interactions, which are critical for regulating cell behavior and functions. There are several 3D cell culture systems such as Matrigel, hydrogels, hanging drop, and micropatterned plates that overcome these drawbacks but they suffer from technical challenges including long spheroid formation times with variable efficiency and difficult handling for high throughput assays, especially for SIRM studies. Magnetic 3D bioprinting (M3DB) can circumvent these issues by utilizing cells magnetized with magnetic nanoparticles that enable spheroid formation. M3DB spheroids have been shown to emulate tissue and tumor microenvironments while exhibiting higher resistance to toxic agents than their 2D counterparts (1). It is, however, unclear if and how such 3D systems impact cellular metabolic networks, which may determine toxic responses in cells. We employed a Stable Isotope-Resolved Metabolomics (SIRM) approach with 13C6-glucose as tracer to map central metabolic networks both in 2D cells and M3DB spheroids formed from lung (A549) and pancreatic (PANC1) adenocarcinoma cells without or with an anti-cancer agent (sodium selenite). We found that the extent of 13C label incorporation into metabolites of glycolysis, the Krebs cycle, the pentose phosphate pathway, and purine/pyrimidine nucleotide synthesis was largely similar between 2D and M3DB culture systems for both cell lines. The exceptions were the higher 13C-ribose incorporation into uracil nucleotides in 2D than M3DB cultures of A549 cells and the presence of gluconeogenic activity in M3DB spheroids of PANC1 cells but not in the 2D counterpart. Selenite induced insignificant perturbations in these pathways in the spheroids relative to the 2D counterparts in both cell lines, which is consistent with the corresponding changes in morphology and growth. Thus, the increased resistance of cancer cell spheroids to selenite may be linked to the reduced capacity of selenite to perturb these metabolic pathways necessary for growth and survival.

  1. Tseng, H., Gage, J.A., Shen, T., Haisler, W.L., Neeley, S.K., Shiao, S., Chen, J., Desai, P.K., Liao, A., Hebel, C. et al. (2015) A spheroid toxicity assay using magnetic 3D bioprinting and real-time mobile device-based imaging. Scientific reports, 5, 13987.
Keywords: 3D spheroids, SIRM, 13C6-glucose, selenite, glycolysis, the Krebs cycle, purine and nucleotide synthesis, A549, PANC1