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MB Sample ID: SA300941
Local Sample ID: | 16 |
Subject ID: | SU002913 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Species Group: | Mammals |
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Subject:
Subject ID: | SU002913 |
Subject Type: | Human |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Species Group: | Mammals |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
16 | SA300941 | FL036549 | Orthotopic | Treatment |
Collection:
Collection ID: | CO002906 |
Collection Summary: | 1. Cell Culture The patient-derived medulloblastoma cell line D425MED, first established at Duke University, Durham, NC, USA, was grown in MEM media (Gibco, Waltham, MA, USA) supplemented with 5% FBS (Gibco, Waltham, MA, USA) and 1% NEAA (Gibco, Waltham, MA, USA). The MED211 patient-derived xenograft was obtained from the Brain Tumor Resource Lab, Seattle, WA, USA and has been previously described. We developed a cell line from the MED211 PDX model by removing tumor tissue from the tumor as described. MED211 cells were grown in neurobasal media with EGF/FGF (Peprotech, Rocky Hill, NJ, USA). In vitro metabolic flux experiments involved the media in confluent cells being changed just before the experiment. Three biological replicate samples of each cell line were pulsed with 10 μM U-glucose (13C6 99% purity) label from Cambridge Isotope (No. CLM-1396-1) or 4 μM U-glutamine (13C5, 15N2, 99% purity) label from Cambridge Isotope (No. CNLM-1275-H-0.5) for 2 h. Following the pulse, cells were spun down and washed with PBS. 1 mL of 80% UPLC-grade ice cold methanol was added to each pellet. Pellets were vortexed for 1 min and incubated at −80 °C to extract metabolites. Analysis of metabolites is described below. 2. Animal Studies Orthotopic xenografting D425MED and MED211 involved the following process. After induction of general anesthesia with ketamine/xylazine in Nu/Nu mice, a burr hole was made in the skull of female Nu/Nu mice Charles River (Wilmington, MA, USA) 1 mm to the right of and 2 mm posterior to the lambdoid suture with an 18 gauge needle. The needle of a Hamilton syringe was inserted to a depth of 2.5 mm into the cerebellum using a needle guard, and 100,000 D425MED cells or MED211 cells in 3 μL of media were injected. MED211 tumors were established by serial transplantation of the patient-derived xenograft and not from cells in culture. All animals were monitored daily until they became symptomatic, exhibiting weight loss, hunching and ataxia. Mice were sacrificed to harvest tumor and uninvolved cerebellum and cortex in the same mouse for histology and metabolic studies. Prior to tumor implantation, flank xenografting of D425MED and MED211 involved, animals being anesthetized with a mixture of 10% ketamine and 5% xylazine. One million cells of D425MED or MED211 suspended in 200 μL of a 50:50 mix of Matrigel (Corning) and media were injected for each flank tumor. Cells were injected using an 18 gauge needle. One tumor was implanted behind each flank, so each mouse carried four flank tumors. In Vivo Stable Isotope Labeling and Metabolite Extraction and Analyses Uniformly labeled glutamine was prepared at a 100 μM concentration in PBS and uniformly labeled glucose was prepared as a 20% solution in PBS. Three animals per group were given three 100 μL IP injections of isotope spaced 15 min apart. Euthanasia occurred two hours after the second isotope injection. Tumors were visually identified in the right cerebellar hemisphere due to their more grey/white appearance compared to the normal cerebellum and were dissected and immediately removed and flash frozen in liquid nitrogen. All uniformly labeled isotopes were obtained from Cambridge Isotope Labs, Tewksbury, MA, USA. Frozen tumors were manually homogenized in liquid nitrogen using a mortar and pestle chilled by dry ice and liquid nitrogen. As the flank tumors were very large, an aliquot of tumor powder was weighed and incubated at −80 °C with 5 volumes of 80% ice-cold HPLC grade methanol to extract metabolites. |
Sample Type: | Tumor cells |
Treatment:
Treatment ID: | TR002922 |
Treatment Summary: | In vitro metabolic flux experiments involved the media in confluent cells being changed just before the experiment. Three biological replicate samples of each cell line were pulsed with 10 μM U-glucose (13C6 99% purity) label from Cambridge Isotope (No. CLM-1396-1) or 4 μM U-glutamine (13C5, 15N2, 99% purity) label from Cambridge Isotope (No. CNLM-1275-H-0.5) for 2 h. Following the pulse, cells were spun down and washed with PBS. 1 mL of 80% UPLC-grade ice cold methanol was added to each pellet. Pellets were vortexed for 1 min and incubated at −80 °C to extract metabolites. Analysis of metabolites is described below. In Vivo Stable Isotope Labeling and Metabolite Extraction and Analyses Uniformly labeled glutamine was prepared at a 100 μM concentration in PBS and uniformly labeled glucose was prepared as a 20% solution in PBS. Three animals per group were given three 100 μL IP injections of isotope spaced 15 min apart. Euthanasia occurred two hours after the second isotope injection. Tumors were visually identified in the right cerebellar hemisphere due to their more grey/white appearance compared to the normal cerebellum and were dissected and immediately removed and flash frozen in liquid nitrogen. All uniformly labeled isotopes were obtained from Cambridge Isotope Labs, Tewksbury, MA, USA. |
Sample Preparation:
Sampleprep ID: | SP002919 |
Sampleprep Summary: | In vitro metabolic flux experiments involved the media in confluent cells being changed just prior to the experiment. Three biological replicate samples of each cell line were pulsed with 10 μM U-glucose (13C6 99% purity) label from Cambridge Isotope (No. CLM-1396-1) or 4 μM U-glutamine (13C5, 15N2, 99% purity) label from Cambridge Isotope (No. CNLM-1275-H-0.5) for 2 h. Following the pulse, cells were spun down and washed with PBS. 1 mL of 80% UPLC-grade ice cold methanol was added to each pellet. Pellets were vortexed for 1 min and incubated at −80 °C to extract metabolites. Analysis of metabolites is described below. Frozen tumors were manually homogenized in liquid nitrogen using a mortar and pestle chilled by dry ice and liquid nitrogen. As the flank tumors were very large, an aliquot of tumor powder was weighed and incubated at −80 °C with 5 volumes of 80% ice-cold HPLC grade methanol to extract metabolites. Samples (both in vivo and in vitro) were centrifuged at 14,000× g rpm for 10 min at 4 °C, and the supernatants were transferred to glass insert liquid chromatography vials. |
Combined analysis:
Analysis ID | AN004562 | AN004563 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | HILIC | HILIC |
Chromatography system | Agilent 1290 | Agilent 1290 |
Column | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) |
MS Type | ESI | ESI |
MS instrument type | QTOF | QTOF |
MS instrument name | Agilent 6520 QTOF | Agilent 6520 QTOF |
Ion Mode | POSITIVE | NEGATIVE |
Units | Peak area | Peak area |
Chromatography:
Chromatography ID: | CH003428 |
Instrument Name: | Agilent 1290 |
Column Name: | Waters ACQUITY UPLC BEH Amide (100 x 2.1mm,1.7um) |
Column Temperature: | 45 |
Flow Gradient: | 0.3 mL/minute. Mobile phases consisted of A (water + 0.1% formic acid) and B (acetonitrile + 0.1% formic acid). The column was equilibrated at 2.5/97.5 (A/B) and maintained for 1 min post injection. Mobile-phase A increased in a linear gradient from 2.5% to 65% from 1 to 9 min post injection then stepped to 97.5% A from 9 to 11 min to wash the column. |
Flow Rate: | 0.3 mL/minute |
Solvent A: | 100% water; 0.1% formic acid |
Solvent B: | 100% acetonitrile; 0.1% formic acid |
Chromatography Type: | HILIC |
MS:
MS ID: | MS004308 |
Analysis ID: | AN004562 |
Instrument Name: | Agilent 6520 QTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | Liquid chromatography–mass spectrometry data were analyzed using Agilent Qualitative Analysis B.07.00 and Elucidata Metabolomic Analysis and Visualization ENgine (El-MAVEN) |
Ion Mode: | POSITIVE |
MS ID: | MS004309 |
Analysis ID: | AN004563 |
Instrument Name: | Agilent 6520 QTOF |
Instrument Type: | QTOF |
MS Type: | ESI |
MS Comments: | Liquid chromatography–mass spectrometry data were analyzed using Agilent Qualitative Analysis B.07.00 and Elucidata Metabolomic Analysis and Visualization ENgine (El-MAVEN) |
Ion Mode: | NEGATIVE |