#METABOLOMICS WORKBENCH dmukha_20240309_152224 DATATRACK_ID:4697 STUDY_ID:ST003152 ANALYSIS_ID:AN005171 PROJECT_ID:PR001941 VERSION 1 CREATED_ON March 27, 2024, 1:21 pm #PROJECT PR:PROJECT_TITLE The shutdown of NADH oxidation via Respiratory Complex I mimics fatty acid PR:PROJECT_TITLE biosynthesis inhibition PR:PROJECT_TYPE LC-MS Quantitative Analysis PR:PROJECT_SUMMARY Proliferating cancer cells actively utilize anabolic processes for biomass PR:PROJECT_SUMMARY production, including de novo biosynthesis of amino acids, nucleotides, and PR:PROJECT_SUMMARY fatty acids. The key enzyme of the fatty acid biosynthesis pathway, fatty acid PR:PROJECT_SUMMARY synthase (FASN), is widely recognized as a promising therapeutic target in PR:PROJECT_SUMMARY cancer and other health conditions. Here, we establish a metabolic signature of PR:PROJECT_SUMMARY FASN inhibition using a panel of pharmacological inhibitors (GSK2194069, PR:PROJECT_SUMMARY TVB-2640, TVB-3166, C75, cerulenin, and Fasnall). We find that the activity of PR:PROJECT_SUMMARY some commonly used FASN inhibitors is inconsistent with the metabolic signature PR:PROJECT_SUMMARY of FASN inhibition (accumulation of malonate, succinate, malonyl coenzyme A, PR:PROJECT_SUMMARY succinyl coenzyme A, and other metabolic perturbations). Moreover, we show that PR:PROJECT_SUMMARY one of these putative FASN inhibitors, Fasnall, is a respiratory Complex I PR:PROJECT_SUMMARY inhibitor that mimics FASN inhibition through NADH accumulation and consequent PR:PROJECT_SUMMARY depletion of the tricarboxylic acid cycle metabolites. We demonstrate that PR:PROJECT_SUMMARY Fasnall impairs tumor growth in several oxidative phosphorylation-dependent PR:PROJECT_SUMMARY cancer models, including combination therapy-resistant melanoma patient-derived PR:PROJECT_SUMMARY xenografts. Fasnall administration does not reproduce neurological side effects PR:PROJECT_SUMMARY in mice reported for other Complex I inhibitors. Our results have significant PR:PROJECT_SUMMARY implications for understanding the FASN role in human health and disease and PR:PROJECT_SUMMARY provide evidence of therapeutic potential for Complex I inhibitors with fast PR:PROJECT_SUMMARY systemic clearance. Our findings also highlight the continuing need for PR:PROJECT_SUMMARY validation of small molecule inhibitors to distinguish high-quality chemical PR:PROJECT_SUMMARY probes and to expand the understanding of their application. PR:INSTITUTE Wistar Institute PR:DEPARTMENT Molecular and Cellular Oncogenesis Program, Ellen and Ronald Caplan Cancer PR:DEPARTMENT Center PR:LABORATORY Schug's Lab PR:LAST_NAME Mukha PR:FIRST_NAME Dzmitry PR:ADDRESS 3601 Spruce St., Philadelphia, Pennsylvania 19104, USA PR:EMAIL dmukha@wistar.org PR:PHONE +12154956903 PR:FUNDING_SOURCE This work was supported by grants from the National Institutes of Health (NIH) PR:FUNDING_SOURCE National Cancer Institute (NCI) DP2 CA249950-01 (Z.T.S.), NIH NCI P01 CA114046 PR:FUNDING_SOURCE (Z.T.S.), Melanoma Research Foundation 717173 (Z.T.S.), and Susan G. Komen PR:FUNDING_SOURCE CCR19608782 (Z.T.S.). PR:PUBLICATIONS Submission Pending PR:CONTRIBUTORS Dzmitry Mukha, Jena Dessain, Seamus O’Connor, Katherine Pniewski, Fabrizio PR:CONTRIBUTORS Bertolazzi, Jeet Patel, Mary Mullins, Zachary T. Schug #STUDY ST:STUDY_TITLE Metabolomics analysis of murine xenograft tumors derived from human breast ST:STUDY_TITLE cancer cell line MCF7 1 h after isotopic glucose bolus ST:STUDY_TYPE Intracellular metabolomics, [U-13C6] glucose tracing ST:STUDY_SUMMARY Metabolomics analysis of murine xenograft tumors derived from human breast ST:STUDY_SUMMARY cancer cell line MCF7 1 h after isotopic glucose bolus. The NSG female mice were ST:STUDY_SUMMARY ~55 days after grafting tumors. [U-13C6] D-Glucose bolus was delivered ST:STUDY_SUMMARY intraperitoneally with a 10 mg/kg dose of Fasnall. ST:INSTITUTE Wistar Institute ST:DEPARTMENT Molecular and Cellular Oncogenesis Program, Ellen and Ronald Caplan Cancer ST:DEPARTMENT Center ST:LABORATORY Schug's Lab ST:LAST_NAME Mukha ST:FIRST_NAME Dzmitry ST:ADDRESS 3601 Spruce St, Philadelphia, PA 19104, USA ST:EMAIL dmukha@wistar.org ST:PHONE 2154956903 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 12 ST:NUM_FEMALES 12 ST:PUBLICATIONS Submission Pending #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN NOD Scid Gamma (NSG) SU:AGE_OR_AGE_RANGE ~4.5 months SU:WEIGHT_OR_WEIGHT_RANGE 24.3 +/- 1.9 g (mean +/- SD) SU:GENDER Female SU:ANIMAL_HOUSING Wistar Animal Facility SU:SPECIES_GROUP Rodents (order Rodentia) #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 - 01_Fasnall_MCF7_Tum_Gluc_1h_F_1066 Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall RAW_FILE_NAME(Raw File Name)=01_Fasnall_MCF7_Tum_Gluc_1h_F_1066.raw; RAW_FILE_NAME(Raw File Name)=NEG_01_Fasnall_MCF7_Tum_Gluc_1h_F_1066.mzXML; RAW_FILE_NAME(Raw File Name )=POS_01_Fasnall_MCF7_Tum_Gluc_1h_F_1066.mzXML SUBJECT_SAMPLE_FACTORS - 02_Fasnall_MCF7_Tum_Gluc_1h_F_1068 Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall RAW_FILE_NAME(Raw File Name)=02_Fasnall_MCF7_Tum_Gluc_1h_F_1068.raw; RAW_FILE_NAME(Raw File Name)=NEG_02_Fasnall_MCF7_Tum_Gluc_1h_F_1068.mzXML; RAW_FILE_NAME(Raw File Name )=POS_02_Fasnall_MCF7_Tum_Gluc_1h_F_1068.mzXML SUBJECT_SAMPLE_FACTORS - 03_Fasnall_MCF7_Tum_Gluc_1h_F_1069 Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall RAW_FILE_NAME(Raw File Name)=03_Fasnall_MCF7_Tum_Gluc_1h_F_1069.raw; RAW_FILE_NAME(Raw File Name)=NEG_03_Fasnall_MCF7_Tum_Gluc_1h_F_1069.mzXML; RAW_FILE_NAME(Raw File Name )=POS_03_Fasnall_MCF7_Tum_Gluc_1h_F_1069.mzXML SUBJECT_SAMPLE_FACTORS - 04_Fasnall_MCF7_Tum_Gluc_1h_F_1070 Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall RAW_FILE_NAME(Raw File Name)=04_Fasnall_MCF7_Tum_Gluc_1h_F_1070.raw; RAW_FILE_NAME(Raw File Name)=NEG_04_Fasnall_MCF7_Tum_Gluc_1h_F_1070.mzXML; RAW_FILE_NAME(Raw File Name )=POS_04_Fasnall_MCF7_Tum_Gluc_1h_F_1070.mzXML SUBJECT_SAMPLE_FACTORS - 05_Fasnall_MCF7_Tum_Gluc_1h_F_1071 Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall RAW_FILE_NAME(Raw File Name)=05_Fasnall_MCF7_Tum_Gluc_1h_F_1071.raw; RAW_FILE_NAME(Raw File Name)=NEG_05_Fasnall_MCF7_Tum_Gluc_1h_F_1071.mzXML; RAW_FILE_NAME(Raw File Name )=POS_05_Fasnall_MCF7_Tum_Gluc_1h_F_1071.mzXML SUBJECT_SAMPLE_FACTORS - 06_Fasnall_MCF7_Tum_Gluc_1h_F_1072 Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall RAW_FILE_NAME(Raw File Name)=06_Fasnall_MCF7_Tum_Gluc_1h_F_1072.raw; RAW_FILE_NAME(Raw File Name)=NEG_06_Fasnall_MCF7_Tum_Gluc_1h_F_1072.mzXML; RAW_FILE_NAME(Raw File Name )=POS_06_Fasnall_MCF7_Tum_Gluc_1h_F_1072.mzXML SUBJECT_SAMPLE_FACTORS - 07_Fasnall_MCF7_Tum_Gluc_1h_F_1073 Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall RAW_FILE_NAME(Raw File Name)=07_Fasnall_MCF7_Tum_Gluc_1h_F_1073.raw; RAW_FILE_NAME(Raw File Name)=NEG_07_Fasnall_MCF7_Tum_Gluc_1h_F_1073.mzXML; RAW_FILE_NAME(Raw File Name )=POS_07_Fasnall_MCF7_Tum_Gluc_1h_F_1073.mzXML SUBJECT_SAMPLE_FACTORS - 08_Fasnall_MCF7_Tum_Gluc_1h_D_1074 Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:Vehicle RAW_FILE_NAME(Raw File Name)=08_Fasnall_MCF7_Tum_Gluc_1h_D_1074.raw; RAW_FILE_NAME(Raw File Name)=NEG_08_Fasnall_MCF7_Tum_Gluc_1h_D_1074.mzXML; RAW_FILE_NAME(Raw File Name )=POS_08_Fasnall_MCF7_Tum_Gluc_1h_D_1074.mzXML SUBJECT_SAMPLE_FACTORS - 09_Fasnall_MCF7_Tum_Gluc_1h_D_1075 Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:Vehicle RAW_FILE_NAME(Raw File Name)=09_Fasnall_MCF7_Tum_Gluc_1h_D_1075.raw; RAW_FILE_NAME(Raw File Name)=NEG_09_Fasnall_MCF7_Tum_Gluc_1h_D_1075.mzXML; RAW_FILE_NAME(Raw File Name )=POS_09_Fasnall_MCF7_Tum_Gluc_1h_D_1075.mzXML SUBJECT_SAMPLE_FACTORS - 10_Fasnall_MCF7_Tum_Gluc_1h_D_1077 Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:Vehicle RAW_FILE_NAME(Raw File Name)=10_Fasnall_MCF7_Tum_Gluc_1h_D_1077.raw; RAW_FILE_NAME(Raw File Name)=NEG_10_Fasnall_MCF7_Tum_Gluc_1h_D_1077.mzXML; RAW_FILE_NAME(Raw File Name )=POS_10_Fasnall_MCF7_Tum_Gluc_1h_D_1077.mzXML SUBJECT_SAMPLE_FACTORS - 11_Fasnall_MCF7_Tum_Gluc_1h_D_1079 Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:Vehicle RAW_FILE_NAME(Raw File Name)=11_Fasnall_MCF7_Tum_Gluc_1h_D_1079.raw; RAW_FILE_NAME(Raw File Name)=NEG_11_Fasnall_MCF7_Tum_Gluc_1h_D_1079.mzXML; RAW_FILE_NAME(Raw File Name )=POS_11_Fasnall_MCF7_Tum_Gluc_1h_D_1079.mzXML SUBJECT_SAMPLE_FACTORS - 12_Fasnall_MCF7_Tum_Gluc_1h_D_1080 Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:Vehicle RAW_FILE_NAME(Raw File Name)=12_Fasnall_MCF7_Tum_Gluc_1h_D_1080.raw; RAW_FILE_NAME(Raw File Name)=NEG_12_Fasnall_MCF7_Tum_Gluc_1h_D_1080.mzXML; RAW_FILE_NAME(Raw File Name )=POS_12_Fasnall_MCF7_Tum_Gluc_1h_D_1080.mzXML SUBJECT_SAMPLE_FACTORS - 135standard_091823_1 Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA RAW_FILE_NAME(Raw File Name)=135standard_091823_1.raw; RAW_FILE_NAME(Raw File Name)=NEG_135standard_091823_1.mzXML; RAW_FILE_NAME(Raw File Name )=POS_135standard_091823_1.mzXML SUBJECT_SAMPLE_FACTORS - 135standard_091823_2 Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA RAW_FILE_NAME(Raw File Name)=135standard_091823_2.raw; RAW_FILE_NAME(Raw File Name)=NEG_135standard_091823_2.mzXML; RAW_FILE_NAME(Raw File Name )=POS_135standard_091823_2.mzXML SUBJECT_SAMPLE_FACTORS - 80MeOH_Blank Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA RAW_FILE_NAME(Raw File Name)=80MeOH_Blank.raw; RAW_FILE_NAME(Raw File Name)=NEG_80MeOH_Blank.mzXML; RAW_FILE_NAME(Raw File Name )=POS_80MeOH_Blank.mzXML SUBJECT_SAMPLE_FACTORS - 80MeOH_Blank_20230918145946 Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA RAW_FILE_NAME(Raw File Name)=80MeOH_Blank_20230918145946.raw; RAW_FILE_NAME(Raw File Name)=NEG_80MeOH_Blank_20230918145946.mzXML; RAW_FILE_NAME(Raw File Name )=POS_80MeOH_Blank_20230918145946.mzXML SUBJECT_SAMPLE_FACTORS - 80MeOH_Blank_20230918155328 Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA RAW_FILE_NAME(Raw File Name)=80MeOH_Blank_20230918155328.raw; RAW_FILE_NAME(Raw File Name)=NEG_80MeOH_Blank_20230918155328.mzXML; RAW_FILE_NAME(Raw File Name )=POS_80MeOH_Blank_20230918155328.mzXML SUBJECT_SAMPLE_FACTORS - 80MeOH_Blank_20230918220912 Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA RAW_FILE_NAME(Raw File Name)=80MeOH_Blank_20230918220912.raw; RAW_FILE_NAME(Raw File Name)=NEG_80MeOH_Blank_20230918220912.mzXML; RAW_FILE_NAME(Raw File Name )=POS_80MeOH_Blank_20230918220912.mzXML SUBJECT_SAMPLE_FACTORS - 80MeOH_Blank_20230918223604 Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA RAW_FILE_NAME(Raw File Name)=80MeOH_Blank_20230918223604.raw; RAW_FILE_NAME(Raw File Name)=NEG_80MeOH_Blank_20230918223604.mzXML; RAW_FILE_NAME(Raw File Name )=POS_80MeOH_Blank_20230918223604.mzXML #COLLECTION CO:COLLECTION_SUMMARY Tissue metabolite extracts were prepared from ~50 mg snap-frozen tissue samples. CO:COLLECTION_SUMMARY Each sample was weighed while frozen. Frozen samples were ground at the CO:COLLECTION_SUMMARY temperature of liquid nitrogen by Retch Cryomill. Then, 80% methanol was added CO:COLLECTION_SUMMARY to each tube at a ratio of 1 ml per 50 mg of tissue. CO:COLLECTION_PROTOCOL_FILENAME DM_metabolomics_samples.txt CO:SAMPLE_TYPE Tumor cells CO:COLLECTION_METHOD 80% methanol extraction CO:VOLUMEORAMOUNT_COLLECTED ~1 ml CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_VIALS 1.5 ml plastic centrifuge tubes CO:STORAGE_VIALS 1.5 ml plastic centrifuge tubes #TREATMENT TR:TREATMENT_SUMMARY Animals received 1 g/kg [U-13C6] D-glucose bolus intraperitoneally with a 10 TR:TREATMENT_SUMMARY mg/kg dose of Fasnall. TR:TREATMENT_COMPOUND Fasnall TR:TREATMENT_ROUTE Intraperitoneal injection TR:TREATMENT_DOSE 10 mg/kg TR:TREATMENT_DOSEVOLUME 50 ul TR:TREATMENT_DOSEDURATION 1 h TR:TREATMENT_VEHICLE 1:1 DMSO/PBS TR:ANIMAL_ANESTHESIA None TR:ANIMAL_FASTING None TR:ANIMAL_ENDP_EUTHANASIA CO2 TR:ANIMAL_ENDP_TISSUE_COLL_LIST 50 mg tumor samples #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Tissue metabolite extracts were prepared from ~50 mg snap-frozen tissue samples. SP:SAMPLEPREP_SUMMARY Each sample was weighed while frozen. Frozen samples were ground at the SP:SAMPLEPREP_SUMMARY temperature of liquid nitrogen by Retch Cryomill. Then, 80% methanol was added SP:SAMPLEPREP_SUMMARY to each tube at a ratio of 1 ml per 50 mg of tissue. All samples were SP:SAMPLEPREP_SUMMARY centrifuged at 18,000 g and 4 °C for 20 min. The supernatant was then SP:SAMPLEPREP_SUMMARY transferred to new Eppendorf tubes and centrifuged again with the same SP:SAMPLEPREP_SUMMARY parameters. After centrifugation, the extracts were transferred to glass LC-MS SP:SAMPLEPREP_SUMMARY vials. SP:SAMPLEPREP_PROTOCOL_FILENAME DM_metabolomics_samples.txt SP:EXTRACTION_METHOD 80% methanol SP:EXTRACT_ENRICHMENT None SP:EXTRACT_CLEANUP None SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION None SP:SAMPLE_DERIVATIZATION None SP:SAMPLE_SPIKING None SP:SUBCELLULAR_LOCATION Intracellular metabolites #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 RS CH:COLUMN_NAME Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um) CH:SOLVENT_A Water with 0.01% ammonium hydroxide and 20 mM ammonium bicarbonate CH:SOLVENT_B Acetonitrile CH:FLOW_GRADIENT A linear solvent gradient of a total duration 22.5 min was starting with 0.2 CH:FLOW_GRADIENT ml/min flow rate of 80% solvent B, 12.5 min – 30%, 15 min – 30%, 15.2 min CH:FLOW_GRADIENT – 80%, 20 min – 80%, 21 min – flow rate 0.3 ml/min, 22 min – flow rate CH:FLOW_GRADIENT 0.3 ml/min, 22.1 min – flow rate 0.2 ml/min. CH:FLOW_RATE 0.2 ml/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Wistar Institute Proteomics & Metabolomic Core AN:OPERATOR_NAME Nicole Gorman AN:DETECTOR_TYPE Orbitrap #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The HESI ion source voltage was set to the following parameters: 3,600 V in both MS:MS_COMMENTS polarity modes, sheath gas 30, auxiliary gas 5, spare gas 0, probe heater 200 MS:MS_COMMENTS °C, capillary temperature 325 °C, S-Lens RF level 65. The mass spectrometer MS:MS_COMMENTS was set to acquire data in the polarity-switching mode averaging two microscans MS:MS_COMMENTS with 60,000 resolution, automatic gain control (AGC) target 5e6, scan range MS:MS_COMMENTS 72-1080 m/z, and maximum orbitrap injection time (IT) 200 ms. Data were MS:MS_COMMENTS converted into the mzXML format by ProteoWizard and analyzed in MAVEN. MS:ION_SOURCE_TEMPERATURE 200 MS:ION_SPRAY_VOLTAGE 3600 MS:IONIZATION Both MS:SOURCE_TEMPERATURE 200 MS:AUTOMATIC_GAIN_CONTROL 5e6 MS:DATAFORMAT RAW #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AU MS_METABOLITE_DATA_START Samples 01_Fasnall_MCF7_Tum_Gluc_1h_F_1066 02_Fasnall_MCF7_Tum_Gluc_1h_F_1068 03_Fasnall_MCF7_Tum_Gluc_1h_F_1069 04_Fasnall_MCF7_Tum_Gluc_1h_F_1070 05_Fasnall_MCF7_Tum_Gluc_1h_F_1071 06_Fasnall_MCF7_Tum_Gluc_1h_F_1072 07_Fasnall_MCF7_Tum_Gluc_1h_F_1073 08_Fasnall_MCF7_Tum_Gluc_1h_D_1074 09_Fasnall_MCF7_Tum_Gluc_1h_D_1075 10_Fasnall_MCF7_Tum_Gluc_1h_D_1077 11_Fasnall_MCF7_Tum_Gluc_1h_D_1079 12_Fasnall_MCF7_Tum_Gluc_1h_D_1080 135standard_091823_1 135standard_091823_2 80MeOH_Blank 80MeOH_Blank_20230918145946 80MeOH_Blank_20230918155328 80MeOH_Blank_20230918220912 80MeOH_Blank_20230918223604 Factors Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:10 mg/kg Fasnall Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:Vehicle Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:Vehicle Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:Vehicle Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:Vehicle Sample source:Xenograft tumors | Isotopic Tracer:1 g/kg [U-13C6] D-glucose in PBS | Drug Treatment:Vehicle Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA Sample source:NA | Isotopic Tracer:NA | Drug Treatment:NA Lactate C12 PARENT 1.04E+09 4.90E+08 6.49E+08 5.42E+08 1.22E+09 9.57E+08 1.32E+09 8.09E+08 1.51E+09 1.23E+09 9.72E+08 1.01E+09 2607360 1856740 2144238 2024791 2374520 3.91E+07 4.05E+07 Lactate C13-label-1 1.18E+08 7.18E+07 8.35E+07 7.26E+07 1.46E+08 1.07E+08 1.42E+08 6.53E+07 9.27E+07 9.75E+07 6.86E+07 8.29E+07 73109.91 55165.14 58438.14 65520.28 73310.12 1387041 1376190 Lactate C13-label-2 7.40E+07 5.75E+07 6.43E+07 6.01E+07 9.81E+07 8.12E+07 8.19E+07 2.90E+07 3.34E+07 4.10E+07 2.31E+07 3.40E+07 0 0 0 0 0 0 0 Lactate C13-label-3 2.59E+08 2.80E+08 2.66E+08 2.84E+08 3.24E+08 3.75E+08 3.46E+08 1.62E+08 2.89E+08 2.66E+08 1.30E+08 1.62E+08 0 0 0 21942.22 0 0 0 Pyruvate C12 PARENT 3116095 1557424 1324684 2584175 2626931 1759294 2128900 1492025 1.23E+07 2024434 2259355 1604336 4001277 317929.8 274600.4 232495.6 256079.6 817393.8 743917.1 Pyruvate C13-label-1 263180 160753.4 118836.9 257111.2 218636.5 137659.5 145568.6 78647.88 520428.5 116263 110861.3 91406.22 103248.7 0 0 5723.053 0 23903.31 29404.51 Pyruvate C13-label-2 117729.6 104131.4 64375.66 207341.5 107065.3 67199.38 75001.52 25478.76 117580.6 35635 29918.94 21629.89 0 0 0 0 0 0 0 Pyruvate C13-label-3 348614.8 552186.8 234603.1 927805 323733.5 331836.3 250054.2 140384.3 987501.8 178242.1 128344 96039.53 0 0 0 0 0 0 3607.141 Acetyl-CoA C12 PARENT 189027.3 59398.17 97454.16 41579.04 179876.7 107060.5 203920.4 149607.6 223410.3 210180.3 105005.2 175782.4 0 0 0 0 0 1699318 1858351 Acetyl-CoA C13-label-1 58406.16 12709.68 28464.62 12310.07 47776.73 29221.78 77380.2 45004.95 59224.64 59464.97 27661.63 62221.94 0 0 0 0 0 489229.5 537359.9 Acetyl-CoA C13-label-2 24987.81 23481.79 5707.416 10058.18 29906.94 27640.26 50326.55 7304.326 43548.19 46985.21 5486.941 14621.21 0 0 0 0 0 171318.6 181838.6 Acetyl-CoA C13-label-3 0 0 0 0 0 0 6648.216 0 0 9299.972 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-10 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-11 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-12 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-13 0 0 0 0 0 0 0 0 3694.506 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-14 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-15 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-16 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-17 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-18 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-19 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-20 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-21 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-22 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Acetyl-CoA C13-label-23 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Citrate C12 PARENT 3.94E+07 2.81E+07 2.83E+07 1.97E+07 3.37E+07 5.23E+07 3.10E+07 3.06E+07 3.04E+07 2.85E+07 3.47E+07 3.68E+07 1081692 1371217 1626160 2366708 1796453 1.18E+07 1.22E+07 Citrate C13-label-1 8076712 6753702 6363084 4203986 6226928 9251377 6531156 4886630 6545448 5630346 8756875 7763134 74563.23 91494.76 118900.1 246132.2 167280.7 702995.2 901891.3 Citrate C13-label-2 6733044 6168826 6217270 3227525 4710418 7775826 5235740 3355983 5418466 4339460 5815264 5550022 0 0 0 56923.57 19190.65 0 111592.5 Citrate C13-label-3 1706486 1916823 1679139 988972.3 1114159 2005316 1354914 889421.7 1427507 1177508 1669690 1368256 0 0 0 0 0 0 21604.88 Citrate C13-label-4 426493.2 643281.2 500493.5 314629.2 314608.2 522731.6 367351.2 237672.7 380786.9 257697.3 433500.9 321134.4 0 0 0 0 0 0 0 Citrate C13-label-5 85841.73 181630.9 150558.9 82074.16 74905.95 129998.4 107953.6 19186.83 95324.76 83747.65 96282.35 72121.12 0 0 0 0 0 0 0 Citrate C13-label-6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Succinate C12 PARENT 8431491 4987498 7293202 6102866 1.77E+07 1.19E+07 1.79E+07 1.10E+07 7496316 2.04E+07 1.30E+07 2.10E+07 681427.3 293310.8 278506.1 309122.9 337160.5 1227154 1337873 Succinate C13-label-1 1160258 661883.2 889472.8 810712.3 2204766 1300548 2859612 1496185 1138881 3314463 2393260 3261469 31256.45 11750.97 5638.453 11764.31 0 47577.27 52898.97 Succinate C13-label-2 918983.7 626971.2 934048.9 711254.3 1522810 1245643 1974301 1130700 847936.8 2331192 1505612 2053293 3581.744 0 0 0 0 0 0 Succinate C13-label-3 157017.6 129484.1 162823.1 150188.4 254392 185062.9 441950.6 197954.4 231905.4 459198.5 304940.4 311374 0 0 0 0 0 0 0 Succinate C13-label-4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Glycerol 3-phosphate C12 PARENT 1.84E+07 3875093 5523946 3215721 2.16E+07 1.19E+07 2.34E+07 1.80E+07 3.23E+07 2.92E+07 2.02E+07 2.96E+07 9843.078 0 0 0 0 1.27E+07 1.40E+07 Glycerol 3-phosphate C13-label-1 1218454 277566 349510.7 211440.1 1193600 713100.9 1204913 868775.8 1156403 1315748 758250.8 1300293 0 0 0 0 0 413102.9 410604 Glycerol 3-phosphate C13-label-2 342655.8 113556.8 133318.8 82292.3 288904.8 185745.5 194040.5 118033.7 0 93060.78 11101.43 120552.4 0 0 0 0 0 0 0 Glycerol 3-phosphate C13-label-3 912211.2 493008.9 398497.6 240176.2 803887.6 410238.3 548637.2 430554.2 1554586 542297.9 355712.7 475075.9 0 0 0 0 0 0 0 Dihydroxyacetone phosphate (DHAP) C12 PARENT 2507468 743572.2 940614.8 277031 1109148 794017.2 1126752 805745 4115239 1239198 8799527 1286122 0 0 0 0 0 157206 176217.1 Dihydroxyacetone phosphate (DHAP) C13-label-1 177327.5 80384.57 70874.65 18849.54 73419.45 76082.79 68200.88 44011.37 141823.7 65519.3 352310.3 80983.95 0 0 0 0 0 0 0 Dihydroxyacetone phosphate (DHAP) C13-label-2 96549 90606.75 88854.21 32628.22 48078.61 60460.97 38962.21 43263.16 0 32341.3 47515.09 46210.71 10913.73 0 38037.56 13992.76 31242.23 37097.33 27797.92 Dihydroxyacetone phosphate (DHAP) C13-label-3 150314.9 163894.8 78343.71 23469.59 54485.97 40811.82 31919.27 12942.58 65255.55 10228.59 195261.2 21223.03 0 0 0 0 0 0 0 D-Sedoheptulose 7-phosphate C12 PARENT 2139294 967580.9 1290080 607103.2 1275198 3301989 1378117 905378.5 897059.5 1701395 6920630 1658804 0 0 5695.613 5967.424 3141.899 1805585 1749383 D-Sedoheptulose 7-phosphate C13-label-1 315407.5 213414.2 231349.5 135365.8 186241.3 532352.1 184146.5 109957.9 83307.05 211738 769408.2 195013 0 0 0 0 0 124498.3 134169.4 D-Sedoheptulose 7-phosphate C13-label-2 235345.4 254225.1 191095.5 133004 103029.9 321745.1 86073.69 55702.53 54409.67 91641.71 427894.7 75219.3 0 0 0 0 0 0 0 D-Sedoheptulose 7-phosphate C13-label-3 90922.9 115306.9 62605.49 49624.54 38470.04 119812.5 36502.9 31755.77 11437.41 28498.76 170017.2 35558.55 0 0 0 0 0 0 0 D-Sedoheptulose 7-phosphate C13-label-4 36186.82 56010.02 27025.08 24809.78 16778.94 62653.07 14876.08 4507.662 0 22873.92 50498.39 7859.015 0 0 0 0 0 0 0 D-Sedoheptulose 7-phosphate C13-label-5 42341.89 81265.94 41656.07 40425.82 23751.21 64812.16 19669.01 5637.551 0 12157.33 56306.26 12466.06 0 0 0 0 0 0 0 D-Sedoheptulose 7-phosphate C13-label-6 0 18501.96 0 4350.509 4078.059 0 0 0 0 0 0 0 0 0 0 0 0 0 0 D-Sedoheptulose 7-phosphate C13-label-7 6818.995 26595.87 15385.01 6720.401 5108.047 10745.97 4953.435 6382.723 0 5828.414 0 0 4441.408 0 0 0 0 0 0 Malate C12 PARENT 1.15E+08 4.98E+07 6.27E+07 4.37E+07 1.29E+08 8.11E+07 1.09E+08 6.92E+07 8.55E+07 1.05E+08 1.02E+08 1.06E+08 492436.9 94879.77 94843.52 141197.7 136328.1 1.02E+07 1.21E+07 Malate C13-label-1 1.91E+07 8420733 9966138 7341680 2.00E+07 1.06E+07 1.92E+07 1.19E+07 1.49E+07 1.84E+07 2.00E+07 1.73E+07 11969.09 2643.006 0 3761.722 0 376368.7 444363.5 Malate C13-label-2 1.21E+07 6686504 7723764 4752038 1.08E+07 7833182 1.10E+07 6256136 9597771 1.02E+07 1.08E+07 9802972 0 0 0 0 0 0 5235.26 Malate C13-label-3 3205173 2497396 2429891 1779212 3697000 2651725 3654576 1793068 3119126 3291837 2874605 2474932 0 0 0 0 0 0 0 Malate C13-label-4 401562.4 406365.9 369423.1 307555.7 439534.5 309970.4 435556.3 220877.2 453212.7 409270.8 453067.8 327303 10629.14 0 0 5020.491 8075.684 0 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention time quantified m/z parent pubchem id kegg id regno comments Lactate C12 PARENT 4.624223 89.02434 89.02434 107689 C00186 37125 Lactate C13-label-1 4.614271 90.02765 89.02434 107689 C00186 37125 Lactate C13-label-2 4.554017 91.03088 89.02434 107689 C00186 37125 Lactate C13-label-3 4.610717 92.03435 89.02434 107689 C00186 37125 Pyruvate C12 PARENT 3.17425 87.00868 87.00867 1060 C00022 1584 Pyruvate C13-label-1 3.187129 88.01204 87.00867 1060 C00022 1584 Pyruvate C13-label-2 3.222198 89.01542 87.00867 1060 C00022 1584 Pyruvate C13-label-3 3.240418 90.01871 87.00867 1060 C00022 1584 Acetyl-CoA C12 PARENT 8.0807 808.1188 808.1187 444493 C00024 50043 Acetyl-CoA C13-label-1 8.082333 809.1214 808.1187 444493 C00024 50043 Acetyl-CoA C13-label-2 8.087103 810.123 808.1187 444493 C00024 50043 Acetyl-CoA C13-label-3 8.096434 811.1272 808.1187 444493 C00024 50043 Acetyl-CoA C13-label-4 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-5 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-6 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-7 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-8 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-9 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-10 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-11 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-12 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-13 8.1447 821.1583 808.1187 444493 C00024 50043 Acetyl-CoA C13-label-14 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-15 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-16 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-17 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-18 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-19 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-20 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-21 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-22 NA NA 808.1187 444493 C00024 50043 Peak was not detected Acetyl-CoA C13-label-23 NA NA 808.1187 444493 C00024 50043 Peak was not detected Citrate C12 PARENT 10.71224 191.0195 191.0195 311 C00158 37071 Citrate C13-label-1 10.7147 192.0226 191.0195 311 C00158 37071 Citrate C13-label-2 10.67262 193.0262 191.0195 311 C00158 37071 Citrate C13-label-3 10.68299 194.0294 191.0195 311 C00158 37071 Citrate C13-label-4 10.67679 195.0327 191.0195 311 C00158 37071 Citrate C13-label-5 10.68454 196.0359 191.0195 311 C00158 37071 Citrate C13-label-6 NA NA 191.0195 311 C00158 37071 Peak was not detected Succinate C12 PARENT 9.439565 117.0191 117.0191 1110 C00042 1966 Succinate C13-label-1 9.451841 118.0225 117.0191 1110 C00042 1966 Succinate C13-label-2 9.461269 119.0257 117.0191 1110 C00042 1966 Succinate C13-label-3 9.463387 120.0292 117.0191 1110 C00042 1966 Succinate C13-label-4 NA NA 117.0191 1110 C00042 1966 Peak was not detected Glycerol 3-phosphate C12 PARENT 9.350879 171.0062 171.0062 439162 C00093 37088 Glycerol 3-phosphate C13-label-1 9.348742 172.0096 171.0062 439162 C00093 37088 Glycerol 3-phosphate C13-label-2 9.36029 173.013 171.0062 439162 C00093 37088 Glycerol 3-phosphate C13-label-3 9.357393 174.0163 171.0062 439162 C00093 37088 Dihydroxyacetone phosphate (DHAP) C12 PARENT 9.54091 168.9905 168.9905 668 C00111 37815 Dihydroxyacetone phosphate (DHAP) C13-label-1 9.537518 169.9938 168.9905 668 C00111 37815 Dihydroxyacetone phosphate (DHAP) C13-label-2 9.522561 170.9969 168.9905 668 C00111 37815 Dihydroxyacetone phosphate (DHAP) C13-label-3 9.537522 172.0005 168.9905 668 C00111 37815 D-Sedoheptulose 7-phosphate C12 PARENT 10.01168 289.0326 289.0326 165007 C05382 37585 D-Sedoheptulose 7-phosphate C13-label-1 10.01735 290.0359 289.0326 165007 C05382 37585 D-Sedoheptulose 7-phosphate C13-label-2 10.01087 291.0389 289.0326 165007 C05382 37585 D-Sedoheptulose 7-phosphate C13-label-3 10.00945 292.0427 289.0326 165007 C05382 37585 D-Sedoheptulose 7-phosphate C13-label-4 10.01733 293.046 289.0326 165007 C05382 37585 D-Sedoheptulose 7-phosphate C13-label-5 10.01958 294.0494 289.0326 165007 C05382 37585 D-Sedoheptulose 7-phosphate C13-label-6 10.00315 295.0526 289.0326 165007 C05382 37585 D-Sedoheptulose 7-phosphate C13-label-7 10.0382 296.0556 289.0326 165007 C05382 37585 Malate C12 PARENT 9.885712 133.014 133.014 222656 C00149 37105 Malate C13-label-1 9.888937 134.0174 133.014 222656 C00149 37105 Malate C13-label-2 9.88649 135.0208 133.014 222656 C00149 37105 Malate C13-label-3 9.882583 136.024 133.014 222656 C00149 37105 Malate C13-label-4 9.843167 137.0273 133.014 222656 C00149 37105 METABOLITES_END #END