#METABOLOMICS WORKBENCH KhoaD_Pham_20230729_172530 DATATRACK_ID:4192 STUDY_ID:ST002806 ANALYSIS_ID:AN004562 PROJECT_ID:PR001753
VERSION             	1
CREATED_ON             	August 3, 2023, 10:55 am
#PROJECT
PR:PROJECT_TITLE                 	Comprehensive Metabolic Profiling of MYC-Amplified Medulloblastoma Tumors
PR:PROJECT_TITLE                 	Reveals Key Dependencies on Amino Acid, Tricarboxylic Acid and Hexosamine
PR:PROJECT_TITLE                 	Pathways
PR:PROJECT_SUMMARY               	Reprogramming of cellular metabolism is a hallmark of cancer. Altering
PR:PROJECT_SUMMARY               	metabolism allows cancer cells to overcome unfavorable microenvironment
PR:PROJECT_SUMMARY               	conditions and to increase and invade. Medulloblastoma is the most common
PR:PROJECT_SUMMARY               	malignant brain tumor in children. Genomic amplification of MYC defines a subset
PR:PROJECT_SUMMARY               	of poor-prognosis medulloblastoma. We performed comprehensive metabolic studies
PR:PROJECT_SUMMARY               	of human MYC-amplified medulloblastoma by comparing the metabolic profiles of
PR:PROJECT_SUMMARY               	tumor cells in three different conditions—in vitro, in flank xenografts, and
PR:PROJECT_SUMMARY               	orthotopic xenografts in the cerebellum. Principal component analysis showed
PR:PROJECT_SUMMARY               	that the metabolic profiles of brain and flank high-MYC medulloblastoma tumors
PR:PROJECT_SUMMARY               	clustered closely together and separated away from the normal brain and in vitro
PR:PROJECT_SUMMARY               	MYC-amplified cells. Compared to typical brains, MYC-amplified medulloblastoma
PR:PROJECT_SUMMARY               	orthotopic xenograft tumors showed upregulation of the TCA cycle and the
PR:PROJECT_SUMMARY               	synthesis of nucleotides, hexosamines, amino acids, and glutathione. There was
PR:PROJECT_SUMMARY               	significantly higher glucose uptake and usage in orthotopic xenograft tumors
PR:PROJECT_SUMMARY               	compared to flank xenograft tumors and cells in culture. In orthotopic tumors,
PR:PROJECT_SUMMARY               	glucose was the primary carbon source for the de novo synthesis of glutamate,
PR:PROJECT_SUMMARY               	glutamine, and glutathione through the TCA cycle. In vivo, the glutaminase II
PR:PROJECT_SUMMARY               	pathway was the main pathway utilizing glutamine. Glutathione was the most
PR:PROJECT_SUMMARY               	abundant upregulated metabolite in orthotopic tumors compared to normal brains.
PR:PROJECT_SUMMARY               	Glutamine-derived glutathione was synthesized through the glutamine transaminase
PR:PROJECT_SUMMARY               	K (GTK) enzyme in vivo. In conclusion, high MYC medulloblastoma cells have
PR:PROJECT_SUMMARY               	different metabolic profiles in vitro compared to in vivo, and critical
PR:PROJECT_SUMMARY               	vulnerabilities may be missed by not performing in vivo metabolic analyses.
PR:INSTITUTE                     	Johns Hopkins
PR:LAST_NAME                     	Pham
PR:FIRST_NAME                    	Khoa
PR:ADDRESS                       	600 N. Wolfe Street, Pathology Bldg., Rm. 401, Baltimore, Maryland, 21287, USA
PR:EMAIL                         	kpham8@jhmi.edu
PR:PHONE                         	4109553439
#STUDY
ST:STUDY_TITLE                   	Comprehensive Metabolic Profiling of MYC-Amplified Medulloblastoma Tumors
ST:STUDY_TITLE                   	Reveals Key Dependencies on Amino Acid, Tricarboxylic Acid and Hexosamine
ST:STUDY_TITLE                   	Pathways
ST:STUDY_SUMMARY                 	Reprogramming of cellular metabolism is a hallmark of cancer. Altering
ST:STUDY_SUMMARY                 	metabolism allows cancer cells to overcome unfavorable microenvironment
ST:STUDY_SUMMARY                 	conditions and to increase and invade. Medulloblastoma is the most common
ST:STUDY_SUMMARY                 	malignant brain tumor in children. Genomic amplification of MYC defines a subset
ST:STUDY_SUMMARY                 	of poor-prognosis medulloblastoma. We performed comprehensive metabolic studies
ST:STUDY_SUMMARY                 	of human MYC-amplified medulloblastoma by comparing the metabolic profiles of
ST:STUDY_SUMMARY                 	tumor cells in three different conditions—in vitro, in flank xenografts, and
ST:STUDY_SUMMARY                 	orthotopic xenografts in the cerebellum. Principal component analysis showed
ST:STUDY_SUMMARY                 	that the metabolic profiles of brain and flank high-MYC medulloblastoma tumors
ST:STUDY_SUMMARY                 	clustered closely together and separated away from the normal brain and in vitro
ST:STUDY_SUMMARY                 	MYC-amplified cells. Compared to typical brains, MYC-amplified medulloblastoma
ST:STUDY_SUMMARY                 	orthotopic xenograft tumors showed upregulation of the TCA cycle and the
ST:STUDY_SUMMARY                 	synthesis of nucleotides, hexosamines, amino acids, and glutathione. There was
ST:STUDY_SUMMARY                 	significantly higher glucose uptake and usage in orthotopic xenograft tumors
ST:STUDY_SUMMARY                 	compared to flank xenograft tumors and cells in culture. In orthotopic tumors,
ST:STUDY_SUMMARY                 	glucose was the primary carbon source for the de novo synthesis of glutamate,
ST:STUDY_SUMMARY                 	glutamine, and glutathione through the TCA cycle. In vivo, the glutaminase II
ST:STUDY_SUMMARY                 	pathway was the main pathway utilizing glutamine. Glutathione was the most
ST:STUDY_SUMMARY                 	abundant upregulated metabolite in orthotopic tumors compared to normal brains.
ST:STUDY_SUMMARY                 	Glutamine-derived glutathione was synthesized through the glutamine transaminase
ST:STUDY_SUMMARY                 	K (GTK) enzyme in vivo. In conclusion, high MYC medulloblastoma cells have
ST:STUDY_SUMMARY                 	different metabolic profiles in vitro compared to in vivo; critical
ST:STUDY_SUMMARY                 	vulnerabilities may be missed by not performing in vivo metabolic analyses.
ST:INSTITUTE                     	Johns Hopkins
ST:LAST_NAME                     	Pham
ST:FIRST_NAME                    	Khoa
ST:ADDRESS                       	600 N. Wolfe Street, Pathology Bldg., Rm. 401, Baltimore, Maryland, 21287, USA
ST:EMAIL                         	kpham8@jhmi.edu
ST:PHONE                         	4109553439
#SUBJECT
SU:SUBJECT_TYPE                  	Human
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#FACTORS
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	CB D425 GLC1	1	Treatment:Orthotopic	RAW_FILE_NAME=CB D425 GLC1.mzXML
SUBJECT_SAMPLE_FACTORS           	CB D425 GLC2	2	Treatment:Orthotopic	RAW_FILE_NAME=CB D425 GLC2.mzXML
SUBJECT_SAMPLE_FACTORS           	CB D425 GLC3	3	Treatment:Orthotopic	RAW_FILE_NAME=CB D425 GLC3.mzXML
SUBJECT_SAMPLE_FACTORS           	CB D425 GLC4	4	Treatment:Orthotopic	RAW_FILE_NAME=CB D425 GLC4.mzXML
SUBJECT_SAMPLE_FACTORS           	CB D425 GLC5	5	Treatment:Orthotopic	RAW_FILE_NAME=CB D425 GLC5.mzXML
SUBJECT_SAMPLE_FACTORS           	CB D425 GLN1	6	Treatment:Orthotopic	RAW_FILE_NAME=CB D425 GLN1.mzXML
SUBJECT_SAMPLE_FACTORS           	CB D425 GLN2	7	Treatment:Orthotopic	RAW_FILE_NAME=CB D425 GLN2.mzXML
SUBJECT_SAMPLE_FACTORS           	CB D425 GLN3	8	Treatment:Orthotopic	RAW_FILE_NAME=CB D425 GLN3.mzXML
SUBJECT_SAMPLE_FACTORS           	CB D425 GLN4	9	Treatment:Orthotopic	RAW_FILE_NAME=CB D425 GLN4.mzXML
SUBJECT_SAMPLE_FACTORS           	CB D425 GLN5	10	Treatment:Orthotopic	RAW_FILE_NAME=CB D425 GLN5.mzXML
SUBJECT_SAMPLE_FACTORS           	CB D425 NL1	11	Treatment:Orthotopic	RAW_FILE_NAME=CB D425 NL1.mzXML
SUBJECT_SAMPLE_FACTORS           	CB D425 NL2	12	Treatment:Orthotopic	RAW_FILE_NAME=CB D425 NL2.mzXML
SUBJECT_SAMPLE_FACTORS           	CB D425 NL3	13	Treatment:Orthotopic	RAW_FILE_NAME=CB D425 NL3.mzXML
SUBJECT_SAMPLE_FACTORS           	CB MED211 NL1	14	Treatment:Orthotopic	RAW_FILE_NAME=CB MED211 NL1.mzXML
SUBJECT_SAMPLE_FACTORS           	CB MED211 NL2	15	Treatment:Orthotopic	RAW_FILE_NAME=CB MED211 NL2.mzXML
SUBJECT_SAMPLE_FACTORS           	CB MED211 NL3	16	Treatment:Orthotopic	RAW_FILE_NAME=CB MED211 NL3.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX D425 GLC1	17	Treatment:Orthotopic	RAW_FILE_NAME=CTX D425 GLC1.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX D425 GLC2	18	Treatment:Orthotopic	RAW_FILE_NAME=CTX D425 GLC2.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX D425 GLC3	19	Treatment:Orthotopic	RAW_FILE_NAME=CTX D425 GLC3.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX D425 GLC4	20	Treatment:Orthotopic	RAW_FILE_NAME=CTX D425 GLC4.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX D425 GLC5	21	Treatment:Orthotopic	RAW_FILE_NAME=CTX D425 GLC5.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX D425 GLN1	22	Treatment:Orthotopic	RAW_FILE_NAME=CTX D425 GLN1.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX D425 GLN2	23	Treatment:Orthotopic	RAW_FILE_NAME=CTX D425 GLN2.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX D425 GLN3	24	Treatment:Orthotopic	RAW_FILE_NAME=CTX D425 GLN3.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX D425 GLN4	25	Treatment:Orthotopic	RAW_FILE_NAME=CTX D425 GLN4.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX D425 GLN5	26	Treatment:Orthotopic	RAW_FILE_NAME=CTX D425 GLN5.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX D425 NL1	27	Treatment:Orthotopic	RAW_FILE_NAME=CTX D425 NL1.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX D425 NL2	28	Treatment:Orthotopic	RAW_FILE_NAME=CTX D425 NL2.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX D425 NL3	29	Treatment:Orthotopic	RAW_FILE_NAME=CTX D425 NL3.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX MED211 NL1	30	Treatment:Orthotopic	RAW_FILE_NAME=CTX MED211 NL1.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX MED211 NL2	31	Treatment:Orthotopic	RAW_FILE_NAME=CTX MED211 NL2.mzXML
SUBJECT_SAMPLE_FACTORS           	CTX MED211 NL3	32	Treatment:Orthotopic	RAW_FILE_NAME=CTX MED211 NL3.mzXML
SUBJECT_SAMPLE_FACTORS           	D425 O-Tumor GLC1	33	Treatment:Orthotopic	RAW_FILE_NAME=D425 O-Tumor GLC1.mzXML
SUBJECT_SAMPLE_FACTORS           	D425 O-Tumor GLC2	34	Treatment:Orthotopic	RAW_FILE_NAME=D425 O-Tumor GLC2.mzXML
SUBJECT_SAMPLE_FACTORS           	D425 O-Tumor GLC3	35	Treatment:Orthotopic	RAW_FILE_NAME=D425 O-Tumor GLC3.mzXML
SUBJECT_SAMPLE_FACTORS           	D425 O-Tumor GLC4	36	Treatment:Orthotopic	RAW_FILE_NAME=D425 O-Tumor GLC4.mzXML
SUBJECT_SAMPLE_FACTORS           	D425 O-Tumor GLC5	37	Treatment:Orthotopic	RAW_FILE_NAME=D425 O-Tumor GLC5.mzXML
SUBJECT_SAMPLE_FACTORS           	D425 O-Tumor GLN1	38	Treatment:Orthotopic	RAW_FILE_NAME=D425 O-Tumor GLN1.mzXML
SUBJECT_SAMPLE_FACTORS           	D425 O-Tumor GLN2	39	Treatment:Orthotopic	RAW_FILE_NAME=D425 O-Tumor GLN2.mzXML
SUBJECT_SAMPLE_FACTORS           	D425 O-Tumor GLN3	40	Treatment:Orthotopic	RAW_FILE_NAME=D425 O-Tumor GLN3.mzXML
SUBJECT_SAMPLE_FACTORS           	D425 O-Tumor GLN4	41	Treatment:Orthotopic	RAW_FILE_NAME=D425 O-Tumor GLN4.mzXML
SUBJECT_SAMPLE_FACTORS           	D425 O-Tumor GLN5	42	Treatment:Orthotopic	RAW_FILE_NAME=D425 O-Tumor GLN5.mzXML
SUBJECT_SAMPLE_FACTORS           	D425 O-Tumor NL1	43	Treatment:Orthotopic	RAW_FILE_NAME=D425 O-Tumor NL1.mzXML
SUBJECT_SAMPLE_FACTORS           	D425 O-Tumor NL2	44	Treatment:Orthotopic	RAW_FILE_NAME=D425 O-Tumor NL2.mzXML
SUBJECT_SAMPLE_FACTORS           	D425 O-Tumor NL3	45	Treatment:Orthotopic	RAW_FILE_NAME=D425 O-Tumor NL3.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 GLC1	46	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 GLC1.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 GLC2	47	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 GLC2.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 GLC3	48	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 GLC3.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 GLC4	49	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 GLC4.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 GLN1	50	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 GLN1.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 GLN2	51	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 GLN2.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 GLN3	52	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 GLN3.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 GLN4	53	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 GLN4.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 GLN5	54	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 GLN5.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 GLN6	55	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 GLN6.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 GLN7	56	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 GLN7.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 GLN8	57	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 GLN8.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 NL1	58	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 NL1.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 NL2	59	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 NL2.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 NL3	60	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 NL3.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor D425 NL4	61	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor D425 NL4.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 GLC1	62	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 GLC1.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 GLC2	63	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 GLC2.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 GLC3	64	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 GLC3.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 GLC4	65	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 GLC4.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 GLN1	66	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 GLN1.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 GLN2	67	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 GLN2.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 GLN3	68	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 GLN3.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 GLN4	69	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 GLN4.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 GLN5	70	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 GLN5.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 GLN6	71	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 GLN6.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 GLN7	72	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 GLN7.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 GLN8	73	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 GLN8.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 NL1	74	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 NL1.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 NL2	75	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 NL2.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 NL3	76	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 NL3.mzXML
SUBJECT_SAMPLE_FACTORS           	Flank Tumor MED211 NL4	77	Treatment:Flank Tumor	RAW_FILE_NAME=Flank Tumor MED211 NL4.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro D425 GLN1	78	Treatment:Invitro	RAW_FILE_NAME=Invitro D425 GLN1.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro D425 GLN2	79	Treatment:Invitro	RAW_FILE_NAME=Invitro D425 GLN2.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro D425 GLN3	80	Treatment:Invitro	RAW_FILE_NAME=Invitro D425 GLN3.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro D425 GLUC1	81	Treatment:Invitro	RAW_FILE_NAME=Invitro D425 GLUC1.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro D425 GLUC2	82	Treatment:Invitro	RAW_FILE_NAME=Invitro D425 GLUC2.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro D425 GLUC3	83	Treatment:Invitro	RAW_FILE_NAME=Invitro D425 GLUC3.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro D425 NL1	84	Treatment:Invitro	RAW_FILE_NAME=Invitro D425 NL1.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro D425 NL2	85	Treatment:Invitro	RAW_FILE_NAME=Invitro D425 NL2.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro D425 NL3	86	Treatment:Invitro	RAW_FILE_NAME=Invitro D425 NL3.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro MED211 GLC1	87	Treatment:Invitro	RAW_FILE_NAME=Invitro MED211 GLC1.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro MED211 GLC2	88	Treatment:Invitro	RAW_FILE_NAME=Invitro MED211 GLC2.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro MED211 GLC3	89	Treatment:Invitro	RAW_FILE_NAME=Invitro MED211 GLC3.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro MED211 GLN1	90	Treatment:Invitro	RAW_FILE_NAME=Invitro MED211 GLN1.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro MED211 GLN2	91	Treatment:Invitro	RAW_FILE_NAME=Invitro MED211 GLN2.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro MED211 GLN3	92	Treatment:Invitro	RAW_FILE_NAME=Invitro MED211 GLN3.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro MED211 NL1	93	Treatment:Invitro	RAW_FILE_NAME=Invitro MED211 NL1.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro MED211 NL2	94	Treatment:Invitro	RAW_FILE_NAME=Invitro MED211 NL2.mzXML
SUBJECT_SAMPLE_FACTORS           	Invitro MED211 NL3	95	Treatment:Invitro	RAW_FILE_NAME=Invitro MED211 NL3.mzXML
SUBJECT_SAMPLE_FACTORS           	MED211 O-Tumor NL1	96	Treatment:Orthotopic	RAW_FILE_NAME=MED211 O-Tumor NL1.mzXML
SUBJECT_SAMPLE_FACTORS           	MED211 O-Tumor NL2	97	Treatment:Orthotopic	RAW_FILE_NAME=MED211 O-Tumor NL2.mzXML
SUBJECT_SAMPLE_FACTORS           	MED211 O-Tumor NL3	98	Treatment:Orthotopic	RAW_FILE_NAME=MED211 O-Tumor NL3.mzXML
#COLLECTION
CO:COLLECTION_SUMMARY            	1. Cell Culture The patient-derived medulloblastoma cell line D425MED, first
CO:COLLECTION_SUMMARY            	established at Duke University, Durham, NC, USA, was grown in MEM media (Gibco,
CO:COLLECTION_SUMMARY            	Waltham, MA, USA) supplemented with 5% FBS (Gibco, Waltham, MA, USA) and 1% NEAA
CO:COLLECTION_SUMMARY            	(Gibco, Waltham, MA, USA). The MED211 patient-derived xenograft was obtained
CO:COLLECTION_SUMMARY            	from the Brain Tumor Resource Lab, Seattle, WA, USA and has been previously
CO:COLLECTION_SUMMARY            	described. We developed a cell line from the MED211 PDX model by removing tumor
CO:COLLECTION_SUMMARY            	tissue from the tumor as described. MED211 cells were grown in neurobasal media
CO:COLLECTION_SUMMARY            	with EGF/FGF (Peprotech, Rocky Hill, NJ, USA). In vitro metabolic flux
CO:COLLECTION_SUMMARY            	experiments involved the media in confluent cells being changed just before the
CO:COLLECTION_SUMMARY            	experiment. Three biological replicate samples of each cell line were pulsed
CO:COLLECTION_SUMMARY            	with 10 μM U-glucose (13C6 99% purity) label from Cambridge Isotope (No.
CO:COLLECTION_SUMMARY            	CLM-1396-1) or 4 μM U-glutamine (13C5, 15N2, 99% purity) label from Cambridge
CO:COLLECTION_SUMMARY            	Isotope (No. CNLM-1275-H-0.5) for 2 h. Following the pulse, cells were spun down
CO:COLLECTION_SUMMARY            	and washed with PBS. 1 mL of 80% UPLC-grade ice cold methanol was added to each
CO:COLLECTION_SUMMARY            	pellet. Pellets were vortexed for 1 min and incubated at −80 °C to extract
CO:COLLECTION_SUMMARY            	metabolites. Analysis of metabolites is described below. 2. Animal Studies
CO:COLLECTION_SUMMARY            	Orthotopic xenografting D425MED and MED211 involved the following process. After
CO:COLLECTION_SUMMARY            	induction of general anesthesia with ketamine/xylazine in Nu/Nu mice, a burr
CO:COLLECTION_SUMMARY            	hole was made in the skull of female Nu/Nu mice Charles River (Wilmington, MA,
CO:COLLECTION_SUMMARY            	USA) 1 mm to the right of and 2 mm posterior to the lambdoid suture with an 18
CO:COLLECTION_SUMMARY            	gauge needle. The needle of a Hamilton syringe was inserted to a depth of 2.5 mm
CO:COLLECTION_SUMMARY            	into the cerebellum using a needle guard, and 100,000 D425MED cells or MED211
CO:COLLECTION_SUMMARY            	cells in 3 μL of media were injected. MED211 tumors were established by serial
CO:COLLECTION_SUMMARY            	transplantation of the patient-derived xenograft and not from cells in culture.
CO:COLLECTION_SUMMARY            	All animals were monitored daily until they became symptomatic, exhibiting
CO:COLLECTION_SUMMARY            	weight loss, hunching and ataxia. Mice were sacrificed to harvest tumor and
CO:COLLECTION_SUMMARY            	uninvolved cerebellum and cortex in the same mouse for histology and metabolic
CO:COLLECTION_SUMMARY            	studies. Prior to tumor implantation, flank xenografting of D425MED and MED211
CO:COLLECTION_SUMMARY            	involved, animals being anesthetized with a mixture of 10% ketamine and 5%
CO:COLLECTION_SUMMARY            	xylazine. One million cells of D425MED or MED211 suspended in 200 μL of a 50:50
CO:COLLECTION_SUMMARY            	mix of Matrigel (Corning) and media were injected for each flank tumor. Cells
CO:COLLECTION_SUMMARY            	were injected using an 18 gauge needle. One tumor was implanted behind each
CO:COLLECTION_SUMMARY            	flank, so each mouse carried four flank tumors. In Vivo Stable Isotope Labeling
CO:COLLECTION_SUMMARY            	and Metabolite Extraction and Analyses Uniformly labeled glutamine was prepared
CO:COLLECTION_SUMMARY            	at a 100 μM concentration in PBS and uniformly labeled glucose was prepared as
CO:COLLECTION_SUMMARY            	a 20% solution in PBS. Three animals per group were given three 100 μL IP
CO:COLLECTION_SUMMARY            	injections of isotope spaced 15 min apart. Euthanasia occurred two hours after
CO:COLLECTION_SUMMARY            	the second isotope injection. Tumors were visually identified in the right
CO:COLLECTION_SUMMARY            	cerebellar hemisphere due to their more grey/white appearance compared to the
CO:COLLECTION_SUMMARY            	normal cerebellum and were dissected and immediately removed and flash frozen in
CO:COLLECTION_SUMMARY            	liquid nitrogen. All uniformly labeled isotopes were obtained from Cambridge
CO:COLLECTION_SUMMARY            	Isotope Labs, Tewksbury, MA, USA. Frozen tumors were manually homogenized in
CO:COLLECTION_SUMMARY            	liquid nitrogen using a mortar and pestle chilled by dry ice and liquid
CO:COLLECTION_SUMMARY            	nitrogen. As the flank tumors were very large, an aliquot of tumor powder was
CO:COLLECTION_SUMMARY            	weighed and incubated at −80 °C with 5 volumes of 80% ice-cold HPLC grade
CO:COLLECTION_SUMMARY            	methanol to extract metabolites.
CO:SAMPLE_TYPE                   	Tumor cells
#TREATMENT
TR:TREATMENT_SUMMARY             	In vitro metabolic flux experiments involved the media in confluent cells being
TR:TREATMENT_SUMMARY             	changed just before the experiment. Three biological replicate samples of each
TR:TREATMENT_SUMMARY             	cell line were pulsed with 10 μM U-glucose (13C6 99% purity) label from
TR:TREATMENT_SUMMARY             	Cambridge Isotope (No. CLM-1396-1) or 4 μM U-glutamine (13C5, 15N2, 99% purity)
TR:TREATMENT_SUMMARY             	label from Cambridge Isotope (No. CNLM-1275-H-0.5) for 2 h. Following the pulse,
TR:TREATMENT_SUMMARY             	cells were spun down and washed with PBS. 1 mL of 80% UPLC-grade ice cold
TR:TREATMENT_SUMMARY             	methanol was added to each pellet. Pellets were vortexed for 1 min and incubated
TR:TREATMENT_SUMMARY             	at −80 °C to extract metabolites. Analysis of metabolites is described below.
TR:TREATMENT_SUMMARY             	In Vivo Stable Isotope Labeling and Metabolite Extraction and Analyses Uniformly
TR:TREATMENT_SUMMARY             	labeled glutamine was prepared at a 100 μM concentration in PBS and uniformly
TR:TREATMENT_SUMMARY             	labeled glucose was prepared as a 20% solution in PBS. Three animals per group
TR:TREATMENT_SUMMARY             	were given three 100 μL IP injections of isotope spaced 15 min apart.
TR:TREATMENT_SUMMARY             	Euthanasia occurred two hours after the second isotope injection. Tumors were
TR:TREATMENT_SUMMARY             	visually identified in the right cerebellar hemisphere due to their more
TR:TREATMENT_SUMMARY             	grey/white appearance compared to the normal cerebellum and were dissected and
TR:TREATMENT_SUMMARY             	immediately removed and flash frozen in liquid nitrogen. All uniformly labeled
TR:TREATMENT_SUMMARY             	isotopes were obtained from Cambridge Isotope Labs, Tewksbury, MA, USA.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	In vitro metabolic flux experiments involved the media in confluent cells being
SP:SAMPLEPREP_SUMMARY            	changed just prior to the experiment. Three biological replicate samples of each
SP:SAMPLEPREP_SUMMARY            	cell line were pulsed with 10 μM U-glucose (13C6 99% purity) label from
SP:SAMPLEPREP_SUMMARY            	Cambridge Isotope (No. CLM-1396-1) or 4 μM U-glutamine (13C5, 15N2, 99% purity)
SP:SAMPLEPREP_SUMMARY            	label from Cambridge Isotope (No. CNLM-1275-H-0.5) for 2 h. Following the pulse,
SP:SAMPLEPREP_SUMMARY            	cells were spun down and washed with PBS. 1 mL of 80% UPLC-grade ice cold
SP:SAMPLEPREP_SUMMARY            	methanol was added to each pellet. Pellets were vortexed for 1 min and incubated
SP:SAMPLEPREP_SUMMARY            	at −80 °C to extract metabolites. Analysis of metabolites is described below.
SP:SAMPLEPREP_SUMMARY            	Frozen tumors were manually homogenized in liquid nitrogen using a mortar and
SP:SAMPLEPREP_SUMMARY            	pestle chilled by dry ice and liquid nitrogen. As the flank tumors were very
SP:SAMPLEPREP_SUMMARY            	large, an aliquot of tumor powder was weighed and incubated at −80 °C with 5
SP:SAMPLEPREP_SUMMARY            	volumes of 80% ice-cold HPLC grade methanol to extract metabolites. Samples
SP:SAMPLEPREP_SUMMARY            	(both in vivo and in vitro) were centrifuged at 14,000× g rpm for 10 min at 4
SP:SAMPLEPREP_SUMMARY            	°C, and the supernatants were transferred to glass insert liquid chromatography
SP:SAMPLEPREP_SUMMARY            	vials.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Agilent 1290
CH:COLUMN_NAME                   	Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)
CH:SOLVENT_A                     	100% water; 0.1% formic acid
CH:SOLVENT_B                     	100% acetonitrile; 0.1% formic acid
CH:FLOW_GRADIENT                 	0.3 mL/minute. Mobile phases consisted of A (water + 0.1% formic acid) and B
CH:FLOW_GRADIENT                 	(acetonitrile + 0.1% formic acid). The column was equilibrated at 2.5/97.5 (A/B)
CH:FLOW_GRADIENT                 	and maintained for 1 min post injection. Mobile-phase A increased in a linear
CH:FLOW_GRADIENT                 	gradient from 2.5% to 65% from 1 to 9 min post injection then stepped to 97.5% A
CH:FLOW_GRADIENT                 	from 9 to 11 min to wash the column.
CH:FLOW_RATE                     	0.3 mL/minute
CH:COLUMN_TEMPERATURE            	45
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6520 QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Liquid chromatography–mass spectrometry data were analyzed using Agilent
MS:MS_COMMENTS                   	Qualitative Analysis B.07.00 and Elucidata Metabolomic Analysis and
MS:MS_COMMENTS                   	Visualization ENgine (El-MAVEN)
MS:MS_RESULTS_FILE               	ST002806_AN004562_Results.txt	UNITS:Peak area	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END