{ "METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002806","ANALYSIS_ID":"AN004563","VERSION":"1","CREATED_ON":"August 3, 2023, 10:55 am"}, "PROJECT":{"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"}, "STUDY":{"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","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_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"}, "SUBJECT_SAMPLE_FACTORS":[ { "Subject ID":"CB D425 GLC1", "Sample ID":"1", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB D425 GLC1.mzXML"} }, { "Subject ID":"CB D425 GLC2", "Sample ID":"2", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB D425 GLC2.mzXML"} }, { "Subject ID":"CB D425 GLC3", "Sample ID":"3", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB D425 GLC3.mzXML"} }, { "Subject ID":"CB D425 GLC4", "Sample ID":"4", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB D425 GLC4.mzXML"} }, { "Subject ID":"CB D425 GLC5", "Sample ID":"5", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB D425 GLC5.mzXML"} }, { "Subject ID":"CB D425 GLN1", "Sample ID":"6", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB D425 GLN1.mzXML"} }, { "Subject ID":"CB D425 GLN2", "Sample ID":"7", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB D425 GLN2.mzXML"} }, { "Subject ID":"CB D425 GLN3", "Sample ID":"8", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB D425 GLN3.mzXML"} }, { "Subject ID":"CB D425 GLN4", "Sample ID":"9", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB D425 GLN4.mzXML"} }, { "Subject ID":"CB D425 GLN5", "Sample ID":"10", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB D425 GLN5.mzXML"} }, { "Subject ID":"CB D425 NL1", "Sample ID":"11", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB D425 NL1.mzXML"} }, { "Subject ID":"CB D425 NL2", "Sample ID":"12", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB D425 NL2.mzXML"} }, { "Subject ID":"CB D425 NL3", "Sample ID":"13", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB D425 NL3.mzXML"} }, { "Subject ID":"CB MED211 NL1", "Sample ID":"14", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB MED211 NL1.mzXML"} }, { "Subject ID":"CB MED211 NL2", "Sample ID":"15", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB MED211 NL2.mzXML"} }, { "Subject ID":"CB MED211 NL3", "Sample ID":"16", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CB MED211 NL3.mzXML"} }, { "Subject ID":"CTX D425 GLC1", "Sample ID":"17", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX D425 GLC1.mzXML"} }, { "Subject ID":"CTX D425 GLC2", "Sample ID":"18", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX D425 GLC2.mzXML"} }, { "Subject ID":"CTX D425 GLC3", "Sample ID":"19", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX D425 GLC3.mzXML"} }, { "Subject ID":"CTX D425 GLC4", "Sample ID":"20", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX D425 GLC4.mzXML"} }, { "Subject ID":"CTX D425 GLC5", "Sample ID":"21", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX D425 GLC5.mzXML"} }, { "Subject ID":"CTX D425 GLN1", "Sample ID":"22", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX D425 GLN1.mzXML"} }, { "Subject ID":"CTX D425 GLN2", "Sample ID":"23", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX D425 GLN2.mzXML"} }, { "Subject ID":"CTX D425 GLN3", "Sample ID":"24", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX D425 GLN3.mzXML"} }, { "Subject ID":"CTX D425 GLN4", "Sample ID":"25", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX D425 GLN4.mzXML"} }, { "Subject ID":"CTX D425 GLN5", "Sample ID":"26", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX D425 GLN5.mzXML"} }, { "Subject ID":"CTX D425 NL1", "Sample ID":"27", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX D425 NL1.mzXML"} }, { "Subject ID":"CTX D425 NL2", "Sample ID":"28", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX D425 NL2.mzXML"} }, { "Subject ID":"CTX D425 NL3", "Sample ID":"29", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX D425 NL3.mzXML"} }, { "Subject ID":"CTX MED211 NL1", "Sample ID":"30", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX MED211 NL1.mzXML"} }, { "Subject ID":"CTX MED211 NL2", "Sample ID":"31", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX MED211 NL2.mzXML"} }, { "Subject ID":"CTX MED211 NL3", "Sample ID":"32", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"CTX MED211 NL3.mzXML"} }, { "Subject ID":"D425 O-Tumor GLC1", "Sample ID":"33", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"D425 O-Tumor GLC1.mzXML"} }, { "Subject ID":"D425 O-Tumor GLC2", "Sample ID":"34", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"D425 O-Tumor GLC2.mzXML"} }, { "Subject ID":"D425 O-Tumor GLC3", "Sample ID":"35", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"D425 O-Tumor GLC3.mzXML"} }, { "Subject ID":"D425 O-Tumor GLC4", "Sample ID":"36", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"D425 O-Tumor GLC4.mzXML"} }, { "Subject ID":"D425 O-Tumor GLC5", "Sample ID":"37", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"D425 O-Tumor GLC5.mzXML"} }, { "Subject ID":"D425 O-Tumor GLN1", "Sample ID":"38", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"D425 O-Tumor GLN1.mzXML"} }, { "Subject ID":"D425 O-Tumor GLN2", "Sample ID":"39", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"D425 O-Tumor GLN2.mzXML"} }, { "Subject ID":"D425 O-Tumor GLN3", "Sample ID":"40", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"D425 O-Tumor GLN3.mzXML"} }, { "Subject ID":"D425 O-Tumor GLN4", "Sample ID":"41", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"D425 O-Tumor GLN4.mzXML"} }, { "Subject ID":"D425 O-Tumor GLN5", "Sample ID":"42", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"D425 O-Tumor GLN5.mzXML"} }, { "Subject ID":"D425 O-Tumor NL1", "Sample ID":"43", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"D425 O-Tumor NL1.mzXML"} }, { "Subject ID":"D425 O-Tumor NL2", "Sample ID":"44", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"D425 O-Tumor NL2.mzXML"} }, { "Subject ID":"D425 O-Tumor NL3", "Sample ID":"45", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"D425 O-Tumor NL3.mzXML"} }, { "Subject ID":"Flank Tumor D425 GLC1", "Sample ID":"46", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 GLC1.mzXML"} }, { "Subject ID":"Flank Tumor D425 GLC2", "Sample ID":"47", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 GLC2.mzXML"} }, { "Subject ID":"Flank Tumor D425 GLC3", "Sample ID":"48", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 GLC3.mzXML"} }, { "Subject ID":"Flank Tumor D425 GLC4", "Sample ID":"49", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 GLC4.mzXML"} }, { "Subject ID":"Flank Tumor D425 GLN1", "Sample ID":"50", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 GLN1.mzXML"} }, { "Subject ID":"Flank Tumor D425 GLN2", "Sample ID":"51", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 GLN2.mzXML"} }, { "Subject ID":"Flank Tumor D425 GLN3", "Sample ID":"52", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 GLN3.mzXML"} }, { "Subject ID":"Flank Tumor D425 GLN4", "Sample ID":"53", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 GLN4.mzXML"} }, { "Subject ID":"Flank Tumor D425 GLN5", "Sample ID":"54", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 GLN5.mzXML"} }, { "Subject ID":"Flank Tumor D425 GLN6", "Sample ID":"55", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 GLN6.mzXML"} }, { "Subject ID":"Flank Tumor D425 GLN7", "Sample ID":"56", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 GLN7.mzXML"} }, { "Subject ID":"Flank Tumor D425 GLN8", "Sample ID":"57", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 GLN8.mzXML"} }, { "Subject ID":"Flank Tumor D425 NL1", "Sample ID":"58", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 NL1.mzXML"} }, { "Subject ID":"Flank Tumor D425 NL2", "Sample ID":"59", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 NL2.mzXML"} }, { "Subject ID":"Flank Tumor D425 NL3", "Sample ID":"60", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 NL3.mzXML"} }, { "Subject ID":"Flank Tumor D425 NL4", "Sample ID":"61", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor D425 NL4.mzXML"} }, { "Subject ID":"Flank Tumor MED211 GLC1", "Sample ID":"62", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 GLC1.mzXML"} }, { "Subject ID":"Flank Tumor MED211 GLC2", "Sample ID":"63", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 GLC2.mzXML"} }, { "Subject ID":"Flank Tumor MED211 GLC3", "Sample ID":"64", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 GLC3.mzXML"} }, { "Subject ID":"Flank Tumor MED211 GLC4", "Sample ID":"65", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 GLC4.mzXML"} }, { "Subject ID":"Flank Tumor MED211 GLN1", "Sample ID":"66", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 GLN1.mzXML"} }, { "Subject ID":"Flank Tumor MED211 GLN2", "Sample ID":"67", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 GLN2.mzXML"} }, { "Subject ID":"Flank Tumor MED211 GLN3", "Sample ID":"68", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 GLN3.mzXML"} }, { "Subject ID":"Flank Tumor MED211 GLN4", "Sample ID":"69", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 GLN4.mzXML"} }, { "Subject ID":"Flank Tumor MED211 GLN5", "Sample ID":"70", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 GLN5.mzXML"} }, { "Subject ID":"Flank Tumor MED211 GLN6", "Sample ID":"71", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 GLN6.mzXML"} }, { "Subject ID":"Flank Tumor MED211 GLN7", "Sample ID":"72", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 GLN7.mzXML"} }, { "Subject ID":"Flank Tumor MED211 GLN8", "Sample ID":"73", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 GLN8.mzXML"} }, { "Subject ID":"Flank Tumor MED211 NL1", "Sample ID":"74", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 NL1.mzXML"} }, { "Subject ID":"Flank Tumor MED211 NL2", "Sample ID":"75", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 NL2.mzXML"} }, { "Subject ID":"Flank Tumor MED211 NL3", "Sample ID":"76", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 NL3.mzXML"} }, { "Subject ID":"Flank Tumor MED211 NL4", "Sample ID":"77", "Factors":{"Treatment":"Flank Tumor"}, "Additional sample data":{"RAW_FILE_NAME":"Flank Tumor MED211 NL4.mzXML"} }, { "Subject ID":"Invitro D425 GLN1", "Sample ID":"78", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro D425 GLN1.mzXML"} }, { "Subject ID":"Invitro D425 GLN2", "Sample ID":"79", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro D425 GLN2.mzXML"} }, { "Subject ID":"Invitro D425 GLN3", "Sample ID":"80", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro D425 GLN3.mzXML"} }, { "Subject ID":"Invitro D425 GLUC1", "Sample ID":"81", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro D425 GLUC1.mzXML"} }, { "Subject ID":"Invitro D425 GLUC2", "Sample ID":"82", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro D425 GLUC2.mzXML"} }, { "Subject ID":"Invitro D425 GLUC3", "Sample ID":"83", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro D425 GLUC3.mzXML"} }, { "Subject ID":"Invitro D425 NL1", "Sample ID":"84", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro D425 NL1.mzXML"} }, { "Subject ID":"Invitro D425 NL2", "Sample ID":"85", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro D425 NL2.mzXML"} }, { "Subject ID":"Invitro D425 NL3", "Sample ID":"86", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro D425 NL3.mzXML"} }, { "Subject ID":"Invitro MED211 GLC1", "Sample ID":"87", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro MED211 GLC1.mzXML"} }, { "Subject ID":"Invitro MED211 GLC2", "Sample ID":"88", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro MED211 GLC2.mzXML"} }, { "Subject ID":"Invitro MED211 GLC3", "Sample ID":"89", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro MED211 GLC3.mzXML"} }, { "Subject ID":"Invitro MED211 GLN1", "Sample ID":"90", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro MED211 GLN1.mzXML"} }, { "Subject ID":"Invitro MED211 GLN2", "Sample ID":"91", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro MED211 GLN2.mzXML"} }, { "Subject ID":"Invitro MED211 GLN3", "Sample ID":"92", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro MED211 GLN3.mzXML"} }, { "Subject ID":"Invitro MED211 NL1", "Sample ID":"93", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro MED211 NL1.mzXML"} }, { "Subject ID":"Invitro MED211 NL2", "Sample ID":"94", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro MED211 NL2.mzXML"} }, { "Subject ID":"Invitro MED211 NL3", "Sample ID":"95", "Factors":{"Treatment":"Invitro"}, "Additional sample data":{"RAW_FILE_NAME":"Invitro MED211 NL3.mzXML"} }, { "Subject ID":"MED211 O-Tumor NL1", "Sample ID":"96", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"MED211 O-Tumor NL1.mzXML"} }, { "Subject ID":"MED211 O-Tumor NL2", "Sample ID":"97", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"MED211 O-Tumor NL2.mzXML"} }, { "Subject ID":"MED211 O-Tumor NL3", "Sample ID":"98", "Factors":{"Treatment":"Orthotopic"}, "Additional sample data":{"RAW_FILE_NAME":"MED211 O-Tumor NL3.mzXML"} } ], "COLLECTION":{"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_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."}, "SAMPLEPREP":{"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."}, "CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Agilent 1290","COLUMN_NAME":"Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um)","SOLVENT_A":"100% water; 0.1% formic acid","SOLVENT_B":"100% acetonitrile; 0.1% formic acid","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","COLUMN_TEMPERATURE":"45"}, "ANALYSIS":{"ANALYSIS_TYPE":"MS"}, "MS":{"INSTRUMENT_NAME":"Agilent 6520 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","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)","MS_RESULTS_FILE":"ST002806_AN004563_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes"} }