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Metabolic Drug Response Phenotyping in Colorectal Cancer Organoids by LC-QTOF-MS
Sylvia K Neef1, Nicole Janssen1, Stefan Winter1
1Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany and University of Tuebingen, 70376 Tuebingen, Germany.
Abstract:
As metabolic rewiring is crucial for cancer cell proliferation, metabolic phenotyping of patient-derived organoids is desirable to identify drug-induced changes and trace metabolic vulnerabilities of tumor subtypes. We established a novel protocol for metabolomic and lipidomic profiling of colorectal cancer organoids by liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) facing the challenge of capturing metabolic information from a minimal sample amount (<500 cells/injection) in the presence of an extracellular matrix (ECM). The best procedure of the tested protocols included ultrasonic metabolite extraction with acetonitrile/methanol/water (2:2:1, v/v/v) without ECM removal. To eliminate ECM-derived background signals, we implemented a data filtering procedure based on the p-value and fold change cut-offs, which retained features with signal intensities >120% compared to matrix-derived signals present in blank samples. As a proof-of-concept, the method was applied to examine the early metabolic response of colorectal cancer organoids to 5-fluorouracil treatment. Statistical analysis revealed dose-dependent changes in the metabolic profiles of treated organoids including elevated levels of 2'-deoxyuridine, 2'-O-methylcytidine, inosine and 1-methyladenosine and depletion of 2'-deoxyadenosine and specific phospholipids. In accordance with the mechanism of action of 5-fluorouracil, changed metabolites are mainly involved in purine and pyrimidine metabolism. The novel protocol provides a first basis for the assessment of metabolic drug response phenotypes in 3D organoid models.
Insights
We developed a new method for metabolic profiling of colorectal cancer organoids using mass spectrometry. This technique identifies drug responses and metabolic vulnerabilities in tumors, aiding personalized cancer treatment strategies.
Area of Science:
- Oncology
- Metabolomics
- Biochemistry
Background:
- Metabolic reprogramming is vital for cancer cell growth.
- Patient-derived organoids are valuable models for studying tumor biology and drug responses.
- Current metabolomic techniques face challenges in analyzing minimal sample amounts and complex matrices like extracellular matrix (ECM).
Purpose of the Study:
- To establish a robust protocol for metabolomic and lipidomic profiling of colorectal cancer organoids.
- To enable the identification of drug-induced metabolic changes and vulnerabilities in minimal samples.
- To apply the developed method for assessing early drug responses in 3D organoid models.
Main Methods:
- Developed a novel liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) protocol for colorectal cancer organoid metabolomics and lipidomics.
- Optimized metabolite extraction using ultrasonication without ECM removal.
- Implemented a data filtering strategy to mitigate ECM-derived background signals based on p-value and fold change.
Main Results:
- Successfully profiled minimal samples (<500 cells/injection) of colorectal cancer organoids.
- Identified dose-dependent metabolic alterations in organoids treated with 5-fluorouracil, including changes in purine and pyrimidine metabolism.
- Detected elevated levels of specific nucleosides and depleted phospholipids, consistent with 5-fluorouracil's mechanism of action.
Conclusions:
- The novel LC-QTOF-MS protocol enables sensitive metabolic and lipidomic analysis of colorectal cancer organoids.
- This method facilitates the study of metabolic drug response phenotypes in 3D organoid models.
- Provides a foundation for uncovering metabolic vulnerabilities and guiding therapeutic strategies in colorectal cancer.
