Active and inactive metabolic pathways in tumor spheroids: determination by GC-MS
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Updated: Jun 17, 2026
Advancements in the Metabolic Profiling of Three-Dimensional Brain Tumor Spheroids for Drug Screening
Published on: September 5, 2025
Michael G Hunnewell1, Neil S Forbes
1Dept. of Chemical Engineering, University of Massachusetts Amherst, Amherst, MA 01003, USA.
Abstract:
Active metabolic pathways in three-dimensional cancer-cell cultures are potential chemotherapeutic targets that would be effective throughout tumors. Chaotic vasculature creates cellular regions in tumors with distinct metabolic behavior that are only present in aggregate cell masses. To quantify cancer cell metabolism, transformed mouse fibroblasts were grown as spheroids and fed isotopically labeled culture medium. Metabolite uptake and production rates were measured as functions of time. Gas chromatography-mass spectrometry was used to quantify the extent of labeling on amino acids present in cytoplasmic extracts. The labeling pattern identified several active and inactive metabolic pathways: Glutaminolysis was found to be active, and malic enzyme and gluconeogenesis were inactive. Transformed cells in spheroids were also found to actively synthesize serine, cysteine, alanine, aspartate, glutamate, and proline; and not synthesize glutamine. The activities of these pathways suggest that cancer cells consume glutamine for biosynthesis and not to provide cellular energy. Determining active metabolic pathways indicates how cells direct carbon flow and may lead to the discovery of novel molecular targets for anticancer therapy.
Insights
Cancer cells in 3D cultures show active glutaminolysis and serine synthesis, indicating glutamine is used for biosynthesis, not energy. This reveals potential targets for novel anticancer therapies.
Area of Science:
- Biochemistry
- Cancer Biology
- Metabolomics
Background:
- Tumor heterogeneity creates distinct metabolic zones.
- Three-dimensional (3D) cancer cell cultures mimic in vivo tumor microenvironments.
- Metabolic pathways are crucial for cancer cell survival and proliferation.
Purpose of the Study:
- To quantify cancer cell metabolism in 3D spheroids.
- To identify active and inactive metabolic pathways in transformed mouse fibroblasts.
- To explore potential therapeutic targets based on metabolic activity.
Main Methods:
- Culturing transformed mouse fibroblasts as multicellular spheroids.
- Utilizing isotopically labeled culture medium to trace metabolic flux.
- Employing gas chromatography-mass spectrometry (GC-MS) for metabolite quantification.
Main Results:
- Glutaminolysis was identified as an active metabolic pathway.
- Malic enzyme and gluconeogenesis were found to be inactive.
- Active synthesis of serine, cysteine, alanine, aspartate, glutamate, and proline was observed, while glutamine synthesis was inactive.
Conclusions:
- Cancer cells in 3D cultures utilize glutamine primarily for biosynthesis rather than energy production.
- Identifying active metabolic pathways provides insight into cellular carbon flow.
- These findings suggest novel molecular targets for anticancer drug development.
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