Stable Isotope-Resolved Metabolomics Shows Metabolic Resistance to Anti-Cancer Selenite in 3D Spheroids versus 2D Cell Cultures

Abstract

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.

Single Spheroid Metabolomics: Optimizing Sample Preparation of Three-Dimensional Multicellular Tumor Spheroids

“…The sample preparation strategy is limited to spheroids with diameters >400-500 µm and with maximum diameters of 900-1000 µm due to potential error-prone handling and growth limitation, respectively. Other studies in tumor spheroid analysis were performed using various culturing and extraction protocols such as (1) gelatinous cultivation of spheroids and methanol-water extraction [21], (2) magnetized cells to form 3D cultures and cold acetonitrile, 70% methanol or 80% acetone extraction [22], (3) gel-based ultra-low attachment plates of 20-25 spheroids per sample followed by derivatization and chloroform/methanol extraction [23]. Overall, most -omics mass spectrometry-based studies on tumor spheroid analysis rely on extraction strategies with cold organic solvents such as methanol, ethanol or isopropanol [21][22][23][24].…”

“…Other studies in tumor spheroid analysis were performed using various culturing and extraction protocols such as (1) gelatinous cultivation of spheroids and methanol-water extraction [21], (2) magnetized cells to form 3D cultures and cold acetonitrile, 70% methanol or 80% acetone extraction [22], (3) gel-based ultra-low attachment plates of 20-25 spheroids per sample followed by derivatization and chloroform/methanol extraction [23]. Overall, most -omics mass spectrometry-based studies on tumor spheroid analysis rely on extraction strategies with cold organic solvents such as methanol, ethanol or isopropanol [21][22][23][24]. This is consistent with our tumor spheroid workflow based on non-scaffold based cultivation on ultra-low attachment plates and the optimized cold 80% methanol extraction with a single washing step.…”

“…The already-mentioned studies [18][19][20][21][22] with 3D cultures involving LC-MS-based metabolomics workflows, including lipidomics and fluxomics applications utilized washing of the samples and organic solvents (methanol, acetonitrile) with an aqueous proportion for extraction, mostly in cold state. To the best of our knowledge, a thorough evaluation of 3D cancer cell models is still lacking.…”

“…Up to date, only a few reports on metabolomics in 3D MTS models exist. The studies involved a range of LC-MS-based metabolomics workflows including lipidomics and fluxomics applications [21][22][23][24][25]. Despite showing the power of combining advanced models such as 3D MTSs and metabolomics, the validation of sample preparation was not addressed comprehensively.…”

Mass spectrometry imaging of endogenous metabolites in response to doxorubicin in a novel 3D osteosarcoma cell culture model

“…The study of drug distributions and effects on downstream pathways via metabolomics approaches has successfully been used to predict antitumour cytotoxic responses by the analysis of extracellular metabolites in cancer patients . These metabolomic studies have been extended to three‐dimensional (3D) cell culture studies, whereby the environment recapitulates the drug resistance seen in vivo , and have further shown that metabolomic profiles may predict the malignant potential of cells grown in 3D cell culture …”