#METABOLOMICS WORKBENCH estancliffe_20230324_124549 DATATRACK_ID:3819 STUDY_ID:ST002534 ANALYSIS_ID:AN004169 PROJECT_ID:PR001630 VERSION 1 CREATED_ON March 29, 2023, 12:31 pm #PROJECT PR:PROJECT_TITLE Using Mass Spectrometry Imaging to Map Fluxes Quantitatively in the Tumor PR:PROJECT_TITLE Ecosystem PR:PROJECT_SUMMARY Tumors are comprised of a multitude of cell types spanning different PR:PROJECT_SUMMARY microenvironments. Mass spectrometry imaging (MSI) has the potential to identify PR:PROJECT_SUMMARY metabolic patterns within the tumor ecosystem and surrounding tissues, but PR:PROJECT_SUMMARY conventional workflows have not yet fully integrated the breadth of experimental PR:PROJECT_SUMMARY techniques in metabolomics. Here, we combine MSI, stable isotope labeling, and a PR:PROJECT_SUMMARY spatial variant of Isotopologue Spectral Analysis to map distributions of PR:PROJECT_SUMMARY metabolite abundances, nutrient contributions, and metabolic turnover fluxes PR:PROJECT_SUMMARY across the brains of mice harboring GL261 glioma, a widely used model for PR:PROJECT_SUMMARY glioblastoma. When integrated with MSI, the combination of ion mobility, PR:PROJECT_SUMMARY Desorption Electrospray Ionization, and Matrix Assisted Laser Desorption PR:PROJECT_SUMMARY revealed disruption in multiple anabolic pathways. De novo fatty acid synthesis PR:PROJECT_SUMMARY flux was determined to be increased by approximately 3-fold in glioma relative PR:PROJECT_SUMMARY to surrounding healthy tissue. Fatty acid elongation flux was elevated even PR:PROJECT_SUMMARY higher at 8-fold and highlights the importance of elongase activity in glioma. PR:PROJECT_SUMMARY The fluxes we examined were uniformly increased throughout the entire tumor PR:PROJECT_SUMMARY region, revealing a high degree of metabolic homogeneity in our model of PR:PROJECT_SUMMARY glioblastoma. PR:INSTITUTE Washington University in St. Louis PR:LAST_NAME Stancliffe PR:FIRST_NAME Ethan PR:ADDRESS 1 Brookings Dr. Campus Box 1134, St. Louis, MO 63105 PR:EMAIL estancliffe@wustl.edu PR:PHONE 3194644881 #STUDY ST:STUDY_TITLE Using Mass Spectrometry Imaging to Map Fluxes Quantitatively in the Tumor ST:STUDY_TITLE Ecosystem ST:STUDY_SUMMARY Tumors are comprised of a multitude of cell types spanning different ST:STUDY_SUMMARY microenvironments. Mass spectrometry imaging (MSI) has the potential to identify ST:STUDY_SUMMARY metabolic patterns within the tumor ecosystem and surrounding tissues, but ST:STUDY_SUMMARY conventional workflows have not yet fully integrated the breadth of experimental ST:STUDY_SUMMARY techniques in metabolomics. Here, we combine MSI, stable isotope labeling, and a ST:STUDY_SUMMARY spatial variant of Isotopologue Spectral Analysis to map distributions of ST:STUDY_SUMMARY metabolite abundances, nutrient contributions, and metabolic turnover fluxes ST:STUDY_SUMMARY across the brains of mice harboring GL261 glioma, a widely used model for ST:STUDY_SUMMARY glioblastoma. When integrated with MSI, the combination of ion mobility, ST:STUDY_SUMMARY Desorption Electrospray Ionization, and Matrix Assisted Laser Desorption ST:STUDY_SUMMARY revealed disruption in multiple anabolic pathways. De novo fatty acid synthesis ST:STUDY_SUMMARY flux was determined to be increased by approximately 3-fold in glioma relative ST:STUDY_SUMMARY to surrounding healthy tissue. Fatty acid elongation flux was elevated even ST:STUDY_SUMMARY higher at 8-fold and highlights the importance of elongase activity in glioma. ST:STUDY_SUMMARY The fluxes we examined were uniformly increased throughout the entire tumor ST:STUDY_SUMMARY region, revealing a high degree of metabolic homogeneity in our model of ST:STUDY_SUMMARY glioblastoma. ST:INSTITUTE Washington University in St. Louis ST:DEPARTMENT Chemistry ST:LABORATORY Patti ST:LAST_NAME Stancliffe ST:FIRST_NAME Ethan ST:ADDRESS 1 Brookings Dr. Campus Box 1134, St. Louis, MO 63105 ST:EMAIL estancliffe@wustl.edu ST:PHONE 3194644881 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 8 ST:NUM_FEMALES 8 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - 4-1_serum Sample type:serum | Isotopically labeled:yes | Group:serum RAW_FILE_NAME=4-1_serum_pns.mzML SUBJECT_SAMPLE_FACTORS - 4-2_serum Sample type:serum | Isotopically labeled:yes | Group:serum RAW_FILE_NAME=4-2_serum_pns.mzML SUBJECT_SAMPLE_FACTORS - 4-3_serum Sample type:serum | Isotopically labeled:yes | Group:serum RAW_FILE_NAME=4-3_serum_pns.mzML SUBJECT_SAMPLE_FACTORS - 4-4_serum Sample type:serum | Isotopically labeled:yes | Group:serum RAW_FILE_NAME=4-4_serum_pns.mzML SUBJECT_SAMPLE_FACTORS - 1-1_tumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor RAW_FILE_NAME=pHILIC_Brain1-1_LH5.mzML SUBJECT_SAMPLE_FACTORS - 1-1_nontumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor RAW_FILE_NAME=pHILIC_Brain1-1_RH5.mzML SUBJECT_SAMPLE_FACTORS - 1-3_tumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor RAW_FILE_NAME=pHILIC_Brain1-3_LH5.mzML SUBJECT_SAMPLE_FACTORS - 1-3_nontumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor RAW_FILE_NAME=pHILIC_Brain1-3_RH5.mzML SUBJECT_SAMPLE_FACTORS - 1-5_tumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor RAW_FILE_NAME=pHILIC_Brain1-5_LH5.mzML SUBJECT_SAMPLE_FACTORS - 1-5_nontumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor RAW_FILE_NAME=pHILIC_Brain1-5_RH5.mzML SUBJECT_SAMPLE_FACTORS - 2-3_tumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor RAW_FILE_NAME=pHILIC_Brain2-3_LH5.mzML SUBJECT_SAMPLE_FACTORS - 2-3_nontumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor RAW_FILE_NAME=pHILIC_Brain2-3_RH5.mzML SUBJECT_SAMPLE_FACTORS - 2-5_tumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor RAW_FILE_NAME=pHILIC_Brain2-5_LH5.mzML SUBJECT_SAMPLE_FACTORS - 2-5_nontumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor RAW_FILE_NAME=pHILIC_Brain2-5_RH5.mzML SUBJECT_SAMPLE_FACTORS - 4-1_tumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor RAW_FILE_NAME=pHILIC_Brain4-1_LH2.mzML SUBJECT_SAMPLE_FACTORS - 4-1_nontumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor RAW_FILE_NAME=pHILIC_Brain4-1_RH2.mzML SUBJECT_SAMPLE_FACTORS - 4-2_tumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor RAW_FILE_NAME=pHILIC_Brain4-2_LH1.mzML SUBJECT_SAMPLE_FACTORS - 4-2_nontumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor RAW_FILE_NAME=pHILIC_Brain4-2_RH1.mzML SUBJECT_SAMPLE_FACTORS - 4-3_tumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor RAW_FILE_NAME=pHILIC_Brain4-3_LH2.mzML SUBJECT_SAMPLE_FACTORS - 4-3_nontumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor RAW_FILE_NAME=pHILIC_Brain4-3_RH2.mzML SUBJECT_SAMPLE_FACTORS - 4-4_tumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor RAW_FILE_NAME=pHILIC_Brain4-4_LH4.mzML SUBJECT_SAMPLE_FACTORS - 4-4_nontumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor RAW_FILE_NAME=pHILIC_Brain4-4_RH4.mzML #COLLECTION CO:COLLECTION_SUMMARY Brains were embedded in 5% wt. carboxymethyl cellulose (Millipore Sigma) in CO:COLLECTION_SUMMARY water and stored at -80 °C. Next, 20 µm thick sections were collected at -20 CO:COLLECTION_SUMMARY °C by using a CM1860 cryostat (Leica Biosystems). Superfrost Plus slides CO:COLLECTION_SUMMARY (Thermo Fisher Scientific) were used after cleaning with ethanol. Sections were CO:COLLECTION_SUMMARY dried under vacuum, stored at -80 °C until use, and thawed under vacuum CO:COLLECTION_SUMMARY immediately prior to analysis. For MALDI, 10 µm thick sections were collected CO:COLLECTION_SUMMARY on SiO2 passivated, indium tin oxide coated polished float glass slides (Delta CO:COLLECTION_SUMMARY Technologies Limited, Loveland, CO, USA). Serial 50 µm thick sections were CO:COLLECTION_SUMMARY collected for extraction and LC/MS analysis after dividing between CO:COLLECTION_SUMMARY tumor-containing and non-tumor hemispheres (as described below). CO:SAMPLE_TYPE Brain #TREATMENT TR:TREATMENT_SUMMARY No treatments were applied. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Sections of 50 µm brain tissue were cut into left (tumor) and right (non-tumor) SP:SAMPLEPREP_SUMMARY hemispheres with a razor blade and collected into Eppendorf tubes while being SP:SAMPLEPREP_SUMMARY kept frozen in the cryostat. The samples were then extracted with 2:2:1 SP:SAMPLEPREP_SUMMARY acetonitrile, methanol, water at a ratio of 80 µL per mg wet weight. The weight SP:SAMPLEPREP_SUMMARY was calculated based on area and thickness of the sections. The solvent was SP:SAMPLEPREP_SUMMARY added to the tissue and vigorously vortexed. For serum analysis, 5 µL serum was SP:SAMPLEPREP_SUMMARY mixed with 45 µL of a 4:4:1 mixture of acetonitrile, methanol, water and SP:SAMPLEPREP_SUMMARY vortexed. Extraction blanks were prepared with 5 µL water instead of serum. All SP:SAMPLEPREP_SUMMARY samples were kept at -20 °C overnight. After centrifugation at 14,000 g for 10 SP:SAMPLEPREP_SUMMARY min at 4 °C, the supernatant was transferred to an LC/MS vial. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Metabolites were separated via hydrophilic interaction liquid chromatography CH:CHROMATOGRAPHY_SUMMARY (HILIC) by using a SeQuant ZIC-pHILIC column (100 x 2.1 mm, 5 µm, polymer, CH:CHROMATOGRAPHY_SUMMARY Merck-Millipore) with a ZIC-pHILIC guard column (20 mm x 2.1 mm, 5 µm). The CH:CHROMATOGRAPHY_SUMMARY column compartment temperature was 40 °C and the flow rate was set to 250 µL CH:CHROMATOGRAPHY_SUMMARY min-1. The mobile phases consisted of A: 95% water, 5% acetonitrile, 20 mM CH:CHROMATOGRAPHY_SUMMARY ammonium bicarbonate, 0.1% ammonium hydroxide solution (25% ammonia in water), 5 CH:CHROMATOGRAPHY_SUMMARY µM medronic acid; and B: 95% acetonitrile, 5% water. The following linear CH:CHROMATOGRAPHY_SUMMARY gradient was applied: 0 to 1 min, 90% B; 12 min, 35% B; 12.5 to 14.5 min, 25% B; CH:CHROMATOGRAPHY_SUMMARY 15 min, 90% B followed by a re-equilibration phase of 4 min at 400 µL min-1 and CH:CHROMATOGRAPHY_SUMMARY 2 min at 250 µL min-1. The samples were kept at 6 °C in the autosampler. The CH:CHROMATOGRAPHY_SUMMARY injection volume was 5 µL. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME SeQuant ZIC-HILIC (150 x 2.1mm,5um) CH:SOLVENT_A 95% water, 5% acetonitrile, 20 mM ammonium bicarbonate, 0.1% ammonium hydroxide CH:SOLVENT_A solution (25% ammonia in water), 5 µM medronic acid CH:SOLVENT_B 95% acetonitrile, 5% water CH:FLOW_GRADIENT 0 to 1 min, 90% B; 12 min, 35% B; 12.5 to 14.5 min, 25% B; 15 min, 90% B CH:FLOW_GRADIENT followed by a re-equilibration phase of 4 min at 400 µL min-1 and 2 min at 250 CH:FLOW_GRADIENT µL min-1 CH:FLOW_RATE 250 µL min-1 CH:COLUMN_TEMPERATURE 40 °C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap ID-X tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS A Vanquish UHPLC system was coupled to an Orbitrap ID-X Tribrid mass MS:MS_COMMENTS spectrometer (Thermo Fisher Scientific) via electrospray ionization with the MS:MS_COMMENTS following source conditions: sheath gas flow 50 arbitrary units (Arb), auxiliary MS:MS_COMMENTS gas flow 10 Arb, sweep gas flow 1 Arb, ion transfer tube temperature 300 °C, MS:MS_COMMENTS vaporizer temperature 200 °C respectively. The RF lens value was 60%. Data were MS:MS_COMMENTS acquired in negative and positive polarity with a spray voltage of 2.8 kV and MS:MS_COMMENTS 3.5 kV, respectively. MS1 data were acquired from 67-900 m/z at a resolution of MS:MS_COMMENTS 120,000 with an automatic gain control (AGC) target of 2e5 and a maximum MS:MS_COMMENTS injection time of 200 ms in polarity switching mode. MS2 data for metabolite MS:MS_COMMENTS identification were acquired at a resolution of 15,000 with an AGC target of MS:MS_COMMENTS 2.5e4 and a maximum injection time of 70 ms in negative and positive mode MS:MS_COMMENTS separately. A 5 ppm mass error and 10 s dynamic exclusion were applied. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak areas MS_METABOLITE_DATA_START Samples 1-1_tumor 1-1_nontumor 1-3_tumor 1-3_nontumor 1-5_tumor 1-5_nontumor 2-3_tumor 2-3_nontumor 2-5_tumor 2-5_nontumor 4-1_tumor 4-1_nontumor 4-2_tumor 4-2_nontumor 4-3_tumor 4-3_nontumor 4-4_tumor 4-4_nontumor 4-1_serum 4-2_serum 4-3_serum 4-4_serum Factors Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_tumor Sample type:brain tissue | Isotopically labeled:no | Group:brain_nontumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_tumor Sample type:brain tissue | Isotopically labeled:yes | Group:brain_nontumor Sample type:serum | Isotopically labeled:yes | Group:serum Sample type:serum | Isotopically labeled:yes | Group:serum Sample type:serum | Isotopically labeled:yes | Group:serum Sample type:serum | Isotopically labeled:yes | Group:serum Aspartate 49890591 56680348 65606901 72920147 41265543 40551848 41293916 47634054 50768538 52252654 82450479 87193499 54112204 110856241 75801717 88420280 61787641 68207425 19546771 18791006 26469805 31244547 Citrate 61614858 39917573 59244619 58022323 66913864 51475556 54675293 43015171 58566533 48840907 57248390 73794138 55286581 84133615 59217951 73234446 58776580 49245436 191645105 293822408 264927111 183926962 N-actylasparate 713745151 865968393 949131996 969317235 387901312 586116361 546622245 677592843 607496898 721316986 1507728368 1566929856 872821112 1387742400 1290416954 1460698880 915698200 1347663584 1568528 1785828 5810129 2267476 Ribose 5-phosphate 5889548 4242044 4292110 4158670 4169047 3231357 3171181 2885488 5429669 3823249 4433524 4633286 4597684 6734628 5255441 6911781 3809251 2748154 918343 379433 7154108 7764144 AMP 116078523 97172267 140770516 122900184 82970541 64317993 74392530 78910510 112758371 108837245 78649251 77427287 69657050 68769341 77934278 96937396 89056441 70235821 21803 9913 13176 14023 Palmitoylcarnitine 129252520 57000622 109944414 82565954 56464902 15983096 43975388 16543022 65160272 32027846 76696240 39120816 168718168 78183848 51214926 33307574 56097685 22358310 28822824 342064279 38083719 22209631 16:0 Palmitic acid 675917839 709497980 569425556 580512216 427159031 495218216 368844684 396012211 515519443 484230053 606916307 549043485 646765543 656912662 503275228 579309850 436937319 434141818 4018102178 8434115568 4199479608 2708062872 20:4 Arachidonic acid 938710833 1247880860 1036237671 1216912072 578554233 886139045 625089095 797721822 737684200 1013508593 1092652642 1215744662 602540159 1037715278 960991392 1309349390 632443194 964167652 715070530 489263085 656292695 538638771 18:0 Stearic acid 668831865 825063254 667338069 737700691 416582900 577562001 385020868 493976483 540141128 626967477 690614001 724541041 483399398 648820390 575850686 775448588 416174192 559151385 1666013118 1641766468 1184250539 1211909887 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Standardized name Formula Exact mass Aspartate Aspartic acid C4H7NO4 133.0375 Citrate Citric acid C6H8O7 192.027 N-actylasparate N-Acetylaspartic acid C6H9NO5 175.0481 Ribose 5-phosphate Ribose 5-phosphate C5H11O8P 230.0192 AMP AMP C10H14N5O7P 347.0631 Palmitoylcarnitine CAR 16:0 C23H45NO4 399.3349 16:0 Palmitic acid Palmitic acid C16H32O2 256.2402 20:4 Arachidonic acid Arachidonic acid C20H32O2 304.2402 18:0 Stearic acid Stearic acid C18H36O2 284.2715 METABOLITES_END #END