#METABOLOMICS WORKBENCH KhoaD_Pham_20230729_172530 DATATRACK_ID:4192 STUDY_ID:ST002806 ANALYSIS_ID:AN004563 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 NEGATIVE 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_AN004563_Results.txt UNITS:Peak area Has m/z:Yes Has RT:Yes RT units:Minutes #END