Summary of Study ST002806

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, 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.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

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.
Institute
Johns Hopkins University
Last NamePham
First NameKhoa
Address600 N. Wolfe Street, Pathology Bldg., Rm. 401, Baltimore, Maryland, 21287, USA
Emailkpham8@jhmi.edu
Phone4109553439
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
https://dx.doi.org/10.21228/M8413M
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001753
Project DOI:doi: 10.21228/M8413M
Project Title:Comprehensive Metabolic Profiling of MYC-Amplified Medulloblastoma Tumors Reveals Key Dependencies on Amino Acid, Tricarboxylic Acid and Hexosamine Pathways
Project Summary:Reprogramming 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, and critical vulnerabilities may be missed by not performing in vivo metabolic analyses.
Institute:Johns Hopkins
Last Name:Pham
First Name:Khoa
Address:600 N. Wolfe Street, Pathology Bldg., Rm. 401, Baltimore, Maryland, 21287, USA
Email:kpham8@jhmi.edu
Phone:4109553439

Subject:

Subject ID:SU002913
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Treatment
SA30085357Flank Tumor
SA30085456Flank Tumor
SA30085558Flank Tumor
SA30085660Flank Tumor
SA30085761Flank Tumor
SA30085855Flank Tumor
SA30085959Flank Tumor
SA30086053Flank Tumor
SA30086148Flank Tumor
SA30086247Flank Tumor
SA30086349Flank Tumor
SA30086451Flank Tumor
SA30086562Flank Tumor
SA30086652Flank Tumor
SA30086754Flank Tumor
SA30086864Flank Tumor
SA30086973Flank Tumor
SA30087072Flank Tumor
SA30087174Flank Tumor
SA30087275Flank Tumor
SA30087377Flank Tumor
SA30087476Flank Tumor
SA30087571Flank Tumor
SA30087670Flank Tumor
SA30087765Flank Tumor
SA30087846Flank Tumor
SA30087966Flank Tumor
SA30088067Flank Tumor
SA30088169Flank Tumor
SA30088268Flank Tumor
SA30088363Flank Tumor
SA30088450Flank Tumor
SA30088585Invitro
SA30088686Invitro
SA30088787Invitro
SA30088888Invitro
SA30088983Invitro
SA30089082Invitro
SA30089178Invitro
SA30089279Invitro
SA30089380Invitro
SA30089481Invitro
SA30089589Invitro
SA30089684Invitro
SA30089794Invitro
SA30089890Invitro
SA30089993Invitro
SA30090095Invitro
SA30090191Invitro
SA30090292Invitro
SA3009038Orthotopic
SA3009047Orthotopic
SA3009059Orthotopic
SA30090610Orthotopic
SA3009076Orthotopic
SA30090811Orthotopic
SA30090912Orthotopic
SA3009102Orthotopic
SA30091196Orthotopic
SA30091213Orthotopic
SA30091397Orthotopic
SA30091498Orthotopic
SA3009154Orthotopic
SA3009163Orthotopic
SA3009175Orthotopic
SA30091814Orthotopic
SA30091945Orthotopic
SA30092044Orthotopic
SA30092143Orthotopic
SA30092233Orthotopic
SA30092332Orthotopic
SA30092430Orthotopic
SA30092531Orthotopic
SA30092642Orthotopic
SA30092741Orthotopic
SA30092836Orthotopic
SA30092935Orthotopic
SA30093037Orthotopic
SA30093138Orthotopic
SA30093240Orthotopic
SA30093339Orthotopic
SA3009341Orthotopic
SA30093529Orthotopic
SA30093619Orthotopic
SA30093720Orthotopic
SA30093818Orthotopic
SA30093917Orthotopic
SA30094015Orthotopic
SA30094116Orthotopic
SA30094221Orthotopic
SA30094322Orthotopic
SA30094427Orthotopic
SA30094528Orthotopic
SA30094626Orthotopic
SA30094725Orthotopic
SA30094823Orthotopic
SA30094924Orthotopic
SA30095034Orthotopic
Showing results 1 to 98 of 98

Collection:

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

Treatment:

Treatment ID:TR002922
Treatment Summary: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. 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.

Sample Preparation:

Sampleprep ID:SP002919
Sampleprep Summary:In vitro metabolic flux experiments involved the media in confluent cells being changed just prior to 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. 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. Samples (both in vivo and in vitro) were centrifuged at 14,000× g rpm for 10 min at 4 °C, and the supernatants were transferred to glass insert liquid chromatography vials.

Combined analysis:

Analysis ID AN004562 AN004563
Analysis type MS MS
Chromatography type HILIC HILIC
Chromatography system Agilent 1290 Agilent 1290
Column Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)
MS Type ESI ESI
MS instrument type QTOF QTOF
MS instrument name Agilent 6520 QTOF Agilent 6520 QTOF
Ion Mode POSITIVE NEGATIVE
Units Peak area Peak area

Chromatography:

Chromatography ID:CH003428
Instrument Name:Agilent 1290
Column Name:Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)
Column Temperature:45
Flow Gradient:0.3 mL/minute. Mobile phases consisted of A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid). The column was equilibrated at 2.5/97.5 (A/B) and maintained for 1 min post injection. Mobile-phase A increased in a linear gradient from 2.5% to 65% from 1 to 9 min post injection then stepped to 97.5% A from 9 to 11 min to wash the column.
Flow Rate:0.3 mL/minute
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:HILIC

MS:

MS ID:MS004308
Analysis ID:AN004562
Instrument Name:Agilent 6520 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:Liquid chromatography–mass spectrometry data were analyzed using Agilent Qualitative Analysis B.07.00 and Elucidata Metabolomic Analysis and Visualization ENgine (El-MAVEN)
Ion Mode:POSITIVE
  
MS ID:MS004309
Analysis ID:AN004563
Instrument Name:Agilent 6520 QTOF
Instrument Type:QTOF
MS Type:ESI
MS Comments:Liquid chromatography–mass spectrometry data were analyzed using Agilent Qualitative Analysis B.07.00 and Elucidata Metabolomic Analysis and Visualization ENgine (El-MAVEN)
Ion Mode:NEGATIVE
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