Conventional two-dimensional (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 three-dimensional (3D) cell culture systems such as Matrigel, hydrogels, micropatterned plates, and hanging drop that overcome these drawbacks but they suffer from technical challenges including long spheroid formation times, difficult handling for high throughput assays, and/or matrix contamination for metabolic studies. Magnetic 3D bioprinting (M3DB) can circumvent these issues by utilizing nanoparticles that enable spheroid formation and growth via magnetizing cells. M3DB spheroids have been shown to emulate tissue and tumor microenvironments while exhibiting higher resistance to toxic agents than their 2D counterparts. It is, however, unclear if and how such 3D systems impact cellular metabolic networks, which may determine altered 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 comparable between 2D and M3DB culture systems for both cell lines. The exceptions were the reduced capacity for de novo synthesis of pyrimidine and sugar nucleotides in M3DB than 2D cultures of A549 and PANC1 cells as well as the presence of gluconeogenic activity in M3DB spheroids of PANC1 cells but not in the 2D counterpart. More strikingly, selenite induced much less perturbation of these pathways in the spheroids relative to the 2D counterparts in both cell lines, which is consistent with the corresponding lesser effects on 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.
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This work was supported in part by NIH grants 1P01CA163223-01A1 (to ANL and TWMF), 1U24DK097215-01A1 (to RMH, TWMF, and ANL), and Redox Metabolism Shared Resource(s) of the University of Kentucky Markey Cancer Center (P30CA177558).
The following are available online at https://www.mdpi.com/2218-1989/8/3/40/s1, Figure S1: Higher selenite resistance of A549 or PANC1 spheroids is not due to lower Se accumulation than the 2D counterparts; Figure S2: A549 spheroids respond less to selenite than 2D cultures in terms of morphology, protein content, mitotic index, and necrosis; Figure S3: PANC1 spheroids respond less to selenite than 2D cultures in terms of morphology, protein content, mitotic index, and necrosis.; Figure S4: 13C atom-resolved tracing of the synthesis of 13C2-acetyl CoA and 13C1-isotopologues of Krebs cycle metabolites from 13C6-glucose.
Fan, Teresa W. -M.; El-Amouri, Salim S.; Macedo, Jessica K. A.; Wang, Qing Jun; Song, Huan; Cassel, Teresa; and Lane, Andrew N., "Stable Isotope-Resolved Metabolomics Shows Metabolic Resistance to Anti-Cancer Selenite in 3D Spheroids Versus 2D Cell Cultures" (2018). Center for Environmental and Systems Biochemistry Faculty Publications. 5.