Summary of Study ST002806

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, where it has been assigned Project ID PR001753. The data can be accessed directly via it's Project DOI: 10.21228/M8413M This work is supported by NIH grant, U2C- DK119886.


This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002806
Study TitleComprehensive Metabolic Profiling of MYC-Amplified Medulloblastoma Tumors Reveals Key Dependencies on Amino Acid, Tricarboxylic Acid and Hexosamine Pathways
Study SummaryReprogramming of cellular metabolism is a hallmark of cancer. Altering metabolism allows cancer cells to overcome unfavorable microenvironment conditions and to increase and invade. Medulloblastoma is the most common malignant brain tumor in children. Genomic amplification of MYC defines a subset of poor-prognosis medulloblastoma. We performed comprehensive metabolic studies of human MYC-amplified medulloblastoma by comparing the metabolic profiles of tumor cells in three different conditions—in vitro, in flank xenografts, and orthotopic xenografts in the cerebellum. Principal component analysis showed that the metabolic profiles of brain and flank high-MYC medulloblastoma tumors clustered closely together and separated away from the normal brain and in vitro MYC-amplified cells. Compared to typical brains, MYC-amplified medulloblastoma orthotopic xenograft tumors showed upregulation of the TCA cycle and the synthesis of nucleotides, hexosamines, amino acids, and glutathione. There was significantly higher glucose uptake and usage in orthotopic xenograft tumors compared to flank xenograft tumors and cells in culture. In orthotopic tumors, glucose was the primary carbon source for the de novo synthesis of glutamate, glutamine, and glutathione through the TCA cycle. In vivo, the glutaminase II pathway was the main pathway utilizing glutamine. Glutathione was the most abundant upregulated metabolite in orthotopic tumors compared to normal brains. Glutamine-derived glutathione was synthesized through the glutamine transaminase K (GTK) enzyme in vivo. In conclusion, high MYC medulloblastoma cells have different metabolic profiles in vitro compared to in vivo; critical vulnerabilities may be missed by not performing in vivo metabolic analyses.
Johns Hopkins University
Last NamePham
First NameKhoa
Address600 N. Wolfe Street, Pathology Bldg., Rm. 401, Baltimore, Maryland, 21287, USA
Submit Date2023-07-29
Raw Data AvailableYes
Raw Data File Type(s)mzXML
Analysis Type DetailLC-MS
Release Date2023-08-20
Release Version1
Khoa Pham Khoa Pham application/zip

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Collection ID:CO002906
Collection Summary:1. Cell Culture The patient-derived medulloblastoma cell line D425MED, first established at Duke University, Durham, NC, USA, was grown in MEM media (Gibco, Waltham, MA, USA) supplemented with 5% FBS (Gibco, Waltham, MA, USA) and 1% NEAA (Gibco, Waltham, MA, USA). The MED211 patient-derived xenograft was obtained from the Brain Tumor Resource Lab, Seattle, WA, USA and has been previously described. We developed a cell line from the MED211 PDX model by removing tumor tissue from the tumor as described. MED211 cells were grown in neurobasal media with EGF/FGF (Peprotech, Rocky Hill, NJ, USA). In vitro metabolic flux experiments involved the media in confluent cells being changed just before the experiment. Three biological replicate samples of each cell line were pulsed with 10 μM U-glucose (13C6 99% purity) label from Cambridge Isotope (No. CLM-1396-1) or 4 μM U-glutamine (13C5, 15N2, 99% purity) label from Cambridge Isotope (No. CNLM-1275-H-0.5) for 2 h. Following the pulse, cells were spun down and washed with PBS. 1 mL of 80% UPLC-grade ice cold methanol was added to each pellet. Pellets were vortexed for 1 min and incubated at −80 °C to extract metabolites. Analysis of metabolites is described below. 2. Animal Studies Orthotopic xenografting D425MED and MED211 involved the following process. After induction of general anesthesia with ketamine/xylazine in Nu/Nu mice, a burr hole was made in the skull of female Nu/Nu mice Charles River (Wilmington, MA, USA) 1 mm to the right of and 2 mm posterior to the lambdoid suture with an 18 gauge needle. The needle of a Hamilton syringe was inserted to a depth of 2.5 mm into the cerebellum using a needle guard, and 100,000 D425MED cells or MED211 cells in 3 μL of media were injected. MED211 tumors were established by serial transplantation of the patient-derived xenograft and not from cells in culture. All animals were monitored daily until they became symptomatic, exhibiting weight loss, hunching and ataxia. Mice were sacrificed to harvest tumor and uninvolved cerebellum and cortex in the same mouse for histology and metabolic studies. Prior to tumor implantation, flank xenografting of D425MED and MED211 involved, animals being anesthetized with a mixture of 10% ketamine and 5% xylazine. One million cells of D425MED or MED211 suspended in 200 μL of a 50:50 mix of Matrigel (Corning) and media were injected for each flank tumor. Cells were injected using an 18 gauge needle. One tumor was implanted behind each flank, so each mouse carried four flank tumors. In Vivo Stable Isotope Labeling and Metabolite Extraction and Analyses Uniformly labeled glutamine was prepared at a 100 μM concentration in PBS and uniformly labeled glucose was prepared as a 20% solution in PBS. Three animals per group were given three 100 μL IP injections of isotope spaced 15 min apart. Euthanasia occurred two hours after the second isotope injection. Tumors were visually identified in the right cerebellar hemisphere due to their more grey/white appearance compared to the normal cerebellum and were dissected and immediately removed and flash frozen in liquid nitrogen. All uniformly labeled isotopes were obtained from Cambridge Isotope Labs, Tewksbury, MA, USA. Frozen tumors were manually homogenized in liquid nitrogen using a mortar and pestle chilled by dry ice and liquid nitrogen. As the flank tumors were very large, an aliquot of tumor powder was weighed and incubated at −80 °C with 5 volumes of 80% ice-cold HPLC grade methanol to extract metabolites.
Sample Type:Tumor cells