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 ID	ST002806
Study Title	Comprehensive Metabolic Profiling of MYC-Amplified Medulloblastoma Tumors Reveals Key Dependencies on Amino Acid, Tricarboxylic Acid and Hexosamine Pathways
Study 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; critical vulnerabilities may be missed by not performing in vivo metabolic analyses.
Institute	Johns Hopkins University
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
Submit Date	2023-07-29
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2023-08-20
Release Version	1

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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
SA300853	57	Flank Tumor
SA300854	56	Flank Tumor
SA300855	58	Flank Tumor
SA300856	60	Flank Tumor
SA300857	61	Flank Tumor
SA300858	55	Flank Tumor
SA300859	59	Flank Tumor
SA300860	53	Flank Tumor
SA300861	48	Flank Tumor
SA300862	47	Flank Tumor
SA300863	49	Flank Tumor
SA300864	51	Flank Tumor
SA300865	62	Flank Tumor
SA300866	52	Flank Tumor
SA300867	54	Flank Tumor
SA300868	64	Flank Tumor
SA300869	73	Flank Tumor
SA300870	72	Flank Tumor
SA300871	74	Flank Tumor
SA300872	75	Flank Tumor
SA300873	77	Flank Tumor
SA300874	76	Flank Tumor
SA300875	71	Flank Tumor
SA300876	70	Flank Tumor
SA300877	65	Flank Tumor
SA300878	46	Flank Tumor
SA300879	66	Flank Tumor
SA300880	67	Flank Tumor
SA300881	69	Flank Tumor
SA300882	68	Flank Tumor
SA300883	63	Flank Tumor
SA300884	50	Flank Tumor
SA300885	85	Invitro
SA300886	86	Invitro
SA300887	87	Invitro
SA300888	88	Invitro
SA300889	83	Invitro
SA300890	82	Invitro
SA300891	78	Invitro
SA300892	79	Invitro
SA300893	80	Invitro
SA300894	81	Invitro
SA300895	89	Invitro
SA300896	84	Invitro
SA300897	94	Invitro
SA300898	90	Invitro
SA300899	93	Invitro
SA300900	95	Invitro
SA300901	91	Invitro
SA300902	92	Invitro
SA300903	8	Orthotopic
SA300904	7	Orthotopic
SA300905	9	Orthotopic
SA300906	10	Orthotopic
SA300907	6	Orthotopic
SA300908	11	Orthotopic
SA300909	12	Orthotopic
SA300910	2	Orthotopic
SA300911	96	Orthotopic
SA300912	13	Orthotopic
SA300913	97	Orthotopic
SA300914	98	Orthotopic
SA300915	4	Orthotopic
SA300916	3	Orthotopic
SA300917	5	Orthotopic
SA300918	14	Orthotopic
SA300919	45	Orthotopic
SA300920	44	Orthotopic
SA300921	43	Orthotopic
SA300922	33	Orthotopic
SA300923	32	Orthotopic
SA300924	30	Orthotopic
SA300925	31	Orthotopic
SA300926	42	Orthotopic
SA300927	41	Orthotopic
SA300928	36	Orthotopic
SA300929	35	Orthotopic
SA300930	37	Orthotopic
SA300931	38	Orthotopic
SA300932	40	Orthotopic
SA300933	39	Orthotopic
SA300934	1	Orthotopic
SA300935	29	Orthotopic
SA300936	19	Orthotopic
SA300937	20	Orthotopic
SA300938	18	Orthotopic
SA300939	17	Orthotopic
SA300940	15	Orthotopic
SA300941	16	Orthotopic
SA300942	21	Orthotopic
SA300943	22	Orthotopic
SA300944	27	Orthotopic
SA300945	28	Orthotopic
SA300946	26	Orthotopic
SA300947	25	Orthotopic
SA300948	23	Orthotopic
SA300949	24	Orthotopic
SA300950	34	Orthotopic

Showing results 1 to 98 of 98

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.

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.

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