Illuminating the Cellular and Molecular Response to Drug Treatment by Combining Bioenergetic Measurements with LC/MS Omics

Significance of the topic

Cancer cells often reprogram their metabolism to support proliferation and survival. Assessing drug-induced changes across bioenergetic and molecular levels provides a comprehensive view of mechanism of action and potential vulnerabilities.

Objectives and study overview

This study aimed to integrate cellular bioenergetic measurements with untargeted metabolomic and lipidomic profiling to characterize metabolic adaptations in THP-1 leukemia cells treated with two tyrosine kinase inhibitors, AG-879 and SU1498.

Methodology

• THP-1 cells were incubated with AG-879 or SU1498 for 2 h and 18 h at their respective IC50 concentrations.
• Bioenergetic assays using the Agilent Seahorse XF Pro analyzer measured basal mitochondrial (mitoATP) and glycolytic (glycoATP) ATP production, proton leak, and spare respiratory capacity (SRC).
• Cells were lysed and quenched with trifluoroethanol; polar metabolites and lipids were fractionated on Captiva EMR–Lipid plates using the Agilent Bravo Metabolomics Sample Prep Platform.
• Polar metabolites were analyzed by HILIC LC/Q-TOF; lipids were profiled by reversed-phase LC/Q-TOF.
• Data processing and feature extraction were performed with Agilent MassHunter Explorer; statistical analysis included volcano plots and PCA. Lipid annotations used MassHunter Lipid Annotator and Mass Profiler Professional.

Instrumentation

• Agilent Seahorse XF Pro analyzer
• Agilent NovoCyte flow cytometer with NovoSampler Q
• Agilent Bravo Metabolomics Sample Prep Platform
• Agilent 1290 Infinity II Bio LC system
• Agilent Revident LC/Q-TOF with Dual Jet Stream source
• Agilent MassHunter Acquisition, Explorer, Lipid Annotator, and Mass Profiler Professional software

Key results and discussion

• Both inhibitors decreased mitochondrial ATP production and increased glycolytic ATP output; SU1498 induced a faster and stronger uncoupling effect and reduced SRC, indicating impaired mitochondrial fitness.
• Proton leak measurements confirmed mitochondrial uncoupling for both treatments, with distinct kinetics for AG-879 (gradual) versus SU1498 (rapid).
• Metabolomic profiling revealed depletion of glycolytic and pentose phosphate intermediates, accumulation of pantothenic acid, and SU1498-specific increases in purine metabolites (uridine, inosine, hypoxanthine, guanine) accompanied by glutamine depletion.
• Lipidomic analysis showed that SU1498 treatment led to a marked elevation of triglyceride species and selective enrichment of polyunsaturated phosphatidylinositols, suggesting altered lipid storage and signaling pools.
• Integration of bioenergetic, metabolomic, and lipidomic data supports a new hypothesis: SU1498-treated cells engage anaplerotic pathways to replenish TCA cycle intermediates and fuel lipid biosynthesis under mitochondrial stress.

Benefits and practical applications

This multi-technique workflow enables deep characterization of drug mechanisms, identification of off-target metabolic effects, discovery of potential biomarkers, and rational design of combination therapies targeting altered metabolic pathways.

Future trends and potential uses

• Integration of 13C-labelled flux analysis to quantify pathway activities.
• Use of substrate oxidation modules to define fuel dependencies under treatment.
• Comparison with healthy cell models to assess selectivity and toxicity.
• Scaling to higher throughput and application of machine learning for pattern recognition in complex datasets.

Conclusion

Combining Seahorse bioenergetics with untargeted LC/MS metabolomics and lipidomics uncovers complementary insights into cellular responses to inhibitors, exemplified by anaplerotic compensation in drug-treated leukemia cells.

References

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