{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002534","ANALYSIS_ID":"AN004169","VERSION":"1","CREATED_ON":"March 29, 2023, 12:31 pm"},

"PROJECT":{"PROJECT_TITLE":"Using Mass Spectrometry Imaging to Map Fluxes Quantitatively in the Tumor Ecosystem","PROJECT_SUMMARY":"Tumors are comprised of a multitude of cell types spanning different microenvironments. Mass spectrometry imaging (MSI) has the potential to identify metabolic patterns within the tumor ecosystem and surrounding tissues, but conventional workflows have not yet fully integrated the breadth of experimental techniques in metabolomics. Here, we combine MSI, stable isotope labeling, and a spatial variant of Isotopologue Spectral Analysis to map distributions of metabolite abundances, nutrient contributions, and metabolic turnover fluxes across the brains of mice harboring GL261 glioma, a widely used model for glioblastoma. When integrated with MSI, the combination of ion mobility, Desorption Electrospray Ionization, and Matrix Assisted Laser Desorption revealed disruption in multiple anabolic pathways. De novo fatty acid synthesis flux was determined to be increased by approximately 3-fold in glioma relative to surrounding healthy tissue. Fatty acid elongation flux was elevated even higher at 8-fold and highlights the importance of elongase activity in glioma. The fluxes we examined were uniformly increased throughout the entire tumor region, revealing a high degree of metabolic homogeneity in our model of glioblastoma.","INSTITUTE":"Washington University in St. Louis","LAST_NAME":"Stancliffe","FIRST_NAME":"Ethan","ADDRESS":"1 Brookings Dr. Campus Box 1134, St. Louis, MO 63105","EMAIL":"estancliffe@wustl.edu","PHONE":"3194644881"},

"STUDY":{"STUDY_TITLE":"Using Mass Spectrometry Imaging to Map Fluxes Quantitatively in the Tumor Ecosystem","STUDY_SUMMARY":"Tumors are comprised of a multitude of cell types spanning different microenvironments. Mass spectrometry imaging (MSI) has the potential to identify metabolic patterns within the tumor ecosystem and surrounding tissues, but conventional workflows have not yet fully integrated the breadth of experimental techniques in metabolomics. Here, we combine MSI, stable isotope labeling, and a spatial variant of Isotopologue Spectral Analysis to map distributions of metabolite abundances, nutrient contributions, and metabolic turnover fluxes across the brains of mice harboring GL261 glioma, a widely used model for glioblastoma. When integrated with MSI, the combination of ion mobility, Desorption Electrospray Ionization, and Matrix Assisted Laser Desorption revealed disruption in multiple anabolic pathways. De novo fatty acid synthesis flux was determined to be increased by approximately 3-fold in glioma relative to surrounding healthy tissue. Fatty acid elongation flux was elevated even higher at 8-fold and highlights the importance of elongase activity in glioma. The fluxes we examined were uniformly increased throughout the entire tumor region, revealing a high degree of metabolic homogeneity in our model of glioblastoma.","INSTITUTE":"Washington University in St. Louis","DEPARTMENT":"Chemistry","LABORATORY":"Patti","LAST_NAME":"Stancliffe","FIRST_NAME":"Ethan","ADDRESS":"1 Brookings Dr. Campus Box 1134, St. Louis, MO 63105","EMAIL":"estancliffe@wustl.edu","PHONE":"3194644881","NUM_GROUPS":"2","TOTAL_SUBJECTS":"8","NUM_FEMALES":"8"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"4-1_serum",
"Factors":{"Sample type":"serum","Isotopically labeled":"yes","Group":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"4-1_serum_pns.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"4-2_serum",
"Factors":{"Sample type":"serum","Isotopically labeled":"yes","Group":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"4-2_serum_pns.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"4-3_serum",
"Factors":{"Sample type":"serum","Isotopically labeled":"yes","Group":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"4-3_serum_pns.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"4-4_serum",
"Factors":{"Sample type":"serum","Isotopically labeled":"yes","Group":"serum"},
"Additional sample data":{"RAW_FILE_NAME":"4-4_serum_pns.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1-1_tumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"no","Group":"brain_tumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain1-1_LH5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1-1_nontumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"no","Group":"brain_nontumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain1-1_RH5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1-3_tumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"no","Group":"brain_tumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain1-3_LH5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1-3_nontumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"no","Group":"brain_nontumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain1-3_RH5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1-5_tumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"no","Group":"brain_tumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain1-5_LH5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"1-5_nontumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"no","Group":"brain_nontumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain1-5_RH5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"2-3_tumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"no","Group":"brain_tumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain2-3_LH5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"2-3_nontumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"no","Group":"brain_nontumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain2-3_RH5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"2-5_tumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"no","Group":"brain_tumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain2-5_LH5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"2-5_nontumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"no","Group":"brain_nontumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain2-5_RH5.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"4-1_tumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"yes","Group":"brain_tumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain4-1_LH2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"4-1_nontumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"yes","Group":"brain_nontumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain4-1_RH2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"4-2_tumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"yes","Group":"brain_tumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain4-2_LH1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"4-2_nontumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"yes","Group":"brain_nontumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain4-2_RH1.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"4-3_tumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"yes","Group":"brain_tumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain4-3_LH2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"4-3_nontumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"yes","Group":"brain_nontumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain4-3_RH2.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"4-4_tumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"yes","Group":"brain_tumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain4-4_LH4.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"4-4_nontumor",
"Factors":{"Sample type":"brain tissue","Isotopically labeled":"yes","Group":"brain_nontumor"},
"Additional sample data":{"RAW_FILE_NAME":"pHILIC_Brain4-4_RH4.mzML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Brains were embedded in 5% wt. carboxymethyl cellulose (Millipore Sigma) in water and stored at -80 °C. Next, 20 µm thick sections were collected at -20 °C by using a CM1860 cryostat (Leica Biosystems). Superfrost Plus slides (Thermo Fisher Scientific) were used after cleaning with ethanol. Sections were dried under vacuum, stored at -80 °C until use, and thawed under vacuum immediately prior to analysis. For MALDI, 10 µm thick sections were collected on SiO2 passivated, indium tin oxide coated polished float glass slides (Delta Technologies Limited, Loveland, CO, USA). Serial 50 µm thick sections were collected for extraction and LC/MS analysis after dividing between tumor-containing and non-tumor hemispheres (as described below).","SAMPLE_TYPE":"Brain"},

"TREATMENT":{"TREATMENT_SUMMARY":"No treatments were applied."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Sections of 50 µm brain tissue were cut into left (tumor) and right (non-tumor) hemispheres with a razor blade and collected into Eppendorf tubes while being kept frozen in the cryostat. The samples were then extracted with 2:2:1 acetonitrile, methanol, water at a ratio of 80 µL per mg wet weight. The weight was calculated based on area and thickness of the sections. The solvent was added to the tissue and vigorously vortexed. For serum analysis, 5 µL serum was mixed with 45 µL of a 4:4:1 mixture of acetonitrile, methanol, water and vortexed. Extraction blanks were prepared with 5 µL water instead of serum. All samples were kept at -20 °C overnight. After centrifugation at 14,000 g for 10 min at 4 °C, the supernatant was transferred to an LC/MS vial."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Metabolites were separated via hydrophilic interaction liquid chromatography (HILIC) by using a SeQuant ZIC-pHILIC column (100 x 2.1 mm, 5 µm, polymer, Merck-Millipore) with a ZIC-pHILIC guard column (20 mm x 2.1 mm, 5 µm). The column compartment temperature was 40 °C and the flow rate was set to 250 µL min-1. The mobile phases consisted of A: 95% water, 5% acetonitrile, 20 mM ammonium bicarbonate, 0.1% ammonium hydroxide solution (25% ammonia in water), 5 µM medronic acid; and B: 95% acetonitrile, 5% water. The following linear gradient was applied: 0 to 1 min, 90% B; 12 min, 35% B; 12.5 to 14.5 min, 25% B; 15 min, 90% B followed by a re-equilibration phase of 4 min at 400 µL min-1 and 2 min at 250 µL min-1. The samples were kept at 6 °C in the autosampler. The injection volume was 5 µL.","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"SeQuant ZIC-HILIC (150 x 2.1mm,5um)","SOLVENT_A":"95% water, 5% acetonitrile, 20 mM ammonium bicarbonate, 0.1% ammonium hydroxide solution (25% ammonia in water), 5 µM medronic acid","SOLVENT_B":"95% acetonitrile, 5% water","FLOW_GRADIENT":"0 to 1 min, 90% B; 12 min, 35% B; 12.5 to 14.5 min, 25% B; 15 min, 90% B followed by a re-equilibration phase of 4 min at 400 µL min-1 and 2 min at 250 µL min-1","FLOW_RATE":"250 µL min-1","COLUMN_TEMPERATURE":"40 °C"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Orbitrap ID-X tribrid","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"UNSPECIFIED","MS_COMMENTS":"A Vanquish UHPLC system was coupled to an Orbitrap ID-X Tribrid mass spectrometer (Thermo Fisher Scientific) via electrospray ionization with the following source conditions: sheath gas flow 50 arbitrary units (Arb), auxiliary gas flow 10 Arb, sweep gas flow 1 Arb, ion transfer tube temperature 300 °C, vaporizer temperature 200 °C respectively. The RF lens value was 60%. Data were acquired in negative and positive polarity with a spray voltage of 2.8 kV and 3.5 kV, respectively. MS1 data were acquired from 67-900 m/z at a resolution of 120,000 with an automatic gain control (AGC) target of 2e5 and a maximum injection time of 200 ms in polarity switching mode. MS2 data for metabolite identification were acquired at a resolution of 15,000 with an AGC target of 2.5e4 and a maximum injection time of 70 ms in negative and positive mode separately. A 5 ppm mass error and 10 s dynamic exclusion were applied."},

"MS_METABOLITE_DATA":{
"Units":"Peak areas",

"Data":[{"Metabolite":"Aspartate","1-1_tumor":"49890591","1-1_nontumor":"56680348","1-3_tumor":"65606901","1-3_nontumor":"72920147","1-5_tumor":"41265543","1-5_nontumor":"40551848","2-3_tumor":"41293916","2-3_nontumor":"47634054","2-5_tumor":"50768538","2-5_nontumor":"52252654","4-1_tumor":"82450479","4-1_nontumor":"87193499","4-2_tumor":"54112204","4-2_nontumor":"110856241","4-3_tumor":"75801717","4-3_nontumor":"88420280","4-4_tumor":"61787641","4-4_nontumor":"68207425","4-1_serum":"19546771","4-2_serum":"18791006","4-3_serum":"26469805","4-4_serum":"31244547"},{"Metabolite":"Citrate","1-1_tumor":"61614858","1-1_nontumor":"39917573","1-3_tumor":"59244619","1-3_nontumor":"58022323","1-5_tumor":"66913864","1-5_nontumor":"51475556","2-3_tumor":"54675293","2-3_nontumor":"43015171","2-5_tumor":"58566533","2-5_nontumor":"48840907","4-1_tumor":"57248390","4-1_nontumor":"73794138","4-2_tumor":"55286581","4-2_nontumor":"84133615","4-3_tumor":"59217951","4-3_nontumor":"73234446","4-4_tumor":"58776580","4-4_nontumor":"49245436","4-1_serum":"191645105","4-2_serum":"293822408","4-3_serum":"264927111","4-4_serum":"183926962"},{"Metabolite":"N-actylasparate","1-1_tumor":"713745151","1-1_nontumor":"865968393","1-3_tumor":"949131996","1-3_nontumor":"969317235","1-5_tumor":"387901312","1-5_nontumor":"586116361","2-3_tumor":"546622245","2-3_nontumor":"677592843","2-5_tumor":"607496898","2-5_nontumor":"721316986","4-1_tumor":"1507728368","4-1_nontumor":"1566929856","4-2_tumor":"872821112","4-2_nontumor":"1387742400","4-3_tumor":"1290416954","4-3_nontumor":"1460698880","4-4_tumor":"915698200","4-4_nontumor":"1347663584","4-1_serum":"1568528","4-2_serum":"1785828","4-3_serum":"5810129","4-4_serum":"2267476"},{"Metabolite":"Ribose 5-phosphate","1-1_tumor":"5889548","1-1_nontumor":"4242044","1-3_tumor":"4292110","1-3_nontumor":"4158670","1-5_tumor":"4169047","1-5_nontumor":"3231357","2-3_tumor":"3171181","2-3_nontumor":"2885488","2-5_tumor":"5429669","2-5_nontumor":"3823249","4-1_tumor":"4433524","4-1_nontumor":"4633286","4-2_tumor":"4597684","4-2_nontumor":"6734628","4-3_tumor":"5255441","4-3_nontumor":"6911781","4-4_tumor":"3809251","4-4_nontumor":"2748154","4-1_serum":"918343","4-2_serum":"379433","4-3_serum":"7154108","4-4_serum":"7764144"},{"Metabolite":"AMP","1-1_tumor":"116078523","1-1_nontumor":"97172267","1-3_tumor":"140770516","1-3_nontumor":"122900184","1-5_tumor":"82970541","1-5_nontumor":"64317993","2-3_tumor":"74392530","2-3_nontumor":"78910510","2-5_tumor":"112758371","2-5_nontumor":"108837245","4-1_tumor":"78649251","4-1_nontumor":"77427287","4-2_tumor":"69657050","4-2_nontumor":"68769341","4-3_tumor":"77934278","4-3_nontumor":"96937396","4-4_tumor":"89056441","4-4_nontumor":"70235821","4-1_serum":"21803","4-2_serum":"9913","4-3_serum":"13176","4-4_serum":"14023"},{"Metabolite":"Palmitoylcarnitine","1-1_tumor":"129252520","1-1_nontumor":"57000622","1-3_tumor":"109944414","1-3_nontumor":"82565954","1-5_tumor":"56464902","1-5_nontumor":"15983096","2-3_tumor":"43975388","2-3_nontumor":"16543022","2-5_tumor":"65160272","2-5_nontumor":"32027846","4-1_tumor":"76696240","4-1_nontumor":"39120816","4-2_tumor":"168718168","4-2_nontumor":"78183848","4-3_tumor":"51214926","4-3_nontumor":"33307574","4-4_tumor":"56097685","4-4_nontumor":"22358310","4-1_serum":"28822824","4-2_serum":"342064279","4-3_serum":"38083719","4-4_serum":"22209631"},{"Metabolite":"16:0 Palmitic acid","1-1_tumor":"675917839","1-1_nontumor":"709497980","1-3_tumor":"569425556","1-3_nontumor":"580512216","1-5_tumor":"427159031","1-5_nontumor":"495218216","2-3_tumor":"368844684","2-3_nontumor":"396012211","2-5_tumor":"515519443","2-5_nontumor":"484230053","4-1_tumor":"606916307","4-1_nontumor":"549043485","4-2_tumor":"646765543","4-2_nontumor":"656912662","4-3_tumor":"503275228","4-3_nontumor":"579309850","4-4_tumor":"436937319","4-4_nontumor":"434141818","4-1_serum":"4018102178","4-2_serum":"8434115568","4-3_serum":"4199479608","4-4_serum":"2708062872"},{"Metabolite":"20:4 Arachidonic acid","1-1_tumor":"938710833","1-1_nontumor":"1247880860","1-3_tumor":"1036237671","1-3_nontumor":"1216912072","1-5_tumor":"578554233","1-5_nontumor":"886139045","2-3_tumor":"625089095","2-3_nontumor":"797721822","2-5_tumor":"737684200","2-5_nontumor":"1013508593","4-1_tumor":"1092652642","4-1_nontumor":"1215744662","4-2_tumor":"602540159","4-2_nontumor":"1037715278","4-3_tumor":"960991392","4-3_nontumor":"1309349390","4-4_tumor":"632443194","4-4_nontumor":"964167652","4-1_serum":"715070530","4-2_serum":"489263085","4-3_serum":"656292695","4-4_serum":"538638771"},{"Metabolite":"18:0 Stearic acid","1-1_tumor":"668831865","1-1_nontumor":"825063254","1-3_tumor":"667338069","1-3_nontumor":"737700691","1-5_tumor":"416582900","1-5_nontumor":"577562001","2-3_tumor":"385020868","2-3_nontumor":"493976483","2-5_tumor":"540141128","2-5_nontumor":"626967477","4-1_tumor":"690614001","4-1_nontumor":"724541041","4-2_tumor":"483399398","4-2_nontumor":"648820390","4-3_tumor":"575850686","4-3_nontumor":"775448588","4-4_tumor":"416174192","4-4_nontumor":"559151385","4-1_serum":"1666013118","4-2_serum":"1641766468","4-3_serum":"1184250539","4-4_serum":"1211909887"}],

"Metabolites":[{"Metabolite":"Aspartate","Standardized name":"Aspartic acid","Formula":"C4H7NO4","Exact mass":"133.0375"},{"Metabolite":"Citrate","Standardized name":"Citric acid","Formula":"C6H8O7","Exact mass":"192.027"},{"Metabolite":"N-actylasparate","Standardized name":"N-Acetylaspartic acid","Formula":"C6H9NO5","Exact mass":"175.0481"},{"Metabolite":"Ribose 5-phosphate","Standardized name":"Ribose 5-phosphate","Formula":"C5H11O8P","Exact mass":"230.0192"},{"Metabolite":"AMP","Standardized name":"AMP","Formula":"C10H14N5O7P","Exact mass":"347.0631"},{"Metabolite":"Palmitoylcarnitine","Standardized name":"CAR 16:0","Formula":"C23H45NO4","Exact mass":"399.3349"},{"Metabolite":"16:0 Palmitic acid","Standardized name":"Palmitic acid","Formula":"C16H32O2","Exact mass":"256.2402"},{"Metabolite":"20:4 Arachidonic acid","Standardized name":"Arachidonic acid","Formula":"C20H32O2","Exact mass":"304.2402"},{"Metabolite":"18:0 Stearic acid","Standardized name":"Stearic acid","Formula":"C18H36O2","Exact mass":"284.2715"}]
}

}