#METABOLOMICS WORKBENCH japizsaab_20230323_132310 DATATRACK_ID:3814 STUDY_ID:ST002528 ANALYSIS_ID:AN004162 PROJECT_ID:PR001627 VERSION 1 CREATED_ON March 28, 2023, 10:09 am #PROJECT PR:PROJECT_TITLE Pancreatic tumors activate arginine biosynthesis to adapt to myeloid-driven PR:PROJECT_TITLE amino acid stress PR:PROJECT_SUMMARY Nutrient stress in the tumor microenvironment requires cancer cells to adopt PR:PROJECT_SUMMARY adaptive metabolic programs to maintain survival and proliferation. Therefore, PR:PROJECT_SUMMARY knowledge of microenvironmental nutrient levels and how cancer cells cope with PR:PROJECT_SUMMARY such nutrition is critical to understand the metabolism underpinning cancer cell PR:PROJECT_SUMMARY biology. Previously, we performed quantitative metabolomics of the interstitial PR:PROJECT_SUMMARY fluid (the local perfusate) of murine pancreatic ductal adenocarcinoma (PDAC) PR:PROJECT_SUMMARY tumors to comprehensively characterize nutrient availability in the PR:PROJECT_SUMMARY microenvironment of these tumors (Sullivan et al., 2019a). Here, we develop PR:PROJECT_SUMMARY Tumor Interstitial Fluid Medium (TIFM), a cell culture medium that contains PR:PROJECT_SUMMARY nutrient levels representative of the PDAC microenvironment, enabling study of PR:PROJECT_SUMMARY PDAC metabolism under physiological nutrition. We show that PDAC cells cultured PR:PROJECT_SUMMARY in TIFM, compared to standard laboratory models, adopt a cellular state more PR:PROJECT_SUMMARY similar to PDAC cells in tumors. Further, using the TIFM model we identified PR:PROJECT_SUMMARY arginine biosynthesis as a metabolic adaptation PDAC cells engage to cope with PR:PROJECT_SUMMARY microenvironmental arginine starvation driven by myeloid cells in PDAC tumors. PR:PROJECT_SUMMARY Altogether, these data show that nutrient availability in tumors is an important PR:PROJECT_SUMMARY determinant of cancer cell metabolism and behavior, and cell culture models that PR:PROJECT_SUMMARY incorporate physiological nutrient availability have improved fidelity and PR:PROJECT_SUMMARY enable the discovery of novel cancer metabolic phenotypes. PR:INSTITUTE University of Chicago PR:LAST_NAME Apiz Saab PR:FIRST_NAME Juan PR:ADDRESS 929 E. 57th St. PR:EMAIL japizsaab@uchicago.edu PR:PHONE 7738346506 #STUDY ST:STUDY_TITLE Concentrations of amino acids in interstitial fluid and whole tumor samples of a ST:STUDY_TITLE murine PDAC orthotopic tumor model, measured by LC-MS ST:STUDY_SUMMARY We extracted polar metabolites from the interstitial fluid and whole tumor ST:STUDY_SUMMARY samples of orthotopic murine PDAC tumors. Weused LC-MS to measure the ST:STUDY_SUMMARY concentration of amino acids in the interstitial fluid of orthotopic murine PDAC ST:STUDY_SUMMARY tumors and compared this to the intratumoral concentrations. ST:INSTITUTE University of Chicago ST:LAST_NAME Apiz Saab ST:FIRST_NAME Juan ST:ADDRESS 929 E. 57th St. ST:EMAIL japizsaab@uchicago.edu ST:PHONE 7738346506 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #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 - TIF_01 Sample type:PDAC_TIF RAW_FILE_NAME=20220131_QE1_JAB_Job2501_TIF_01.raw SUBJECT_SAMPLE_FACTORS - TIF_02 Sample type:PDAC_TIF RAW_FILE_NAME=20220131_QE1_JAB_Job2501_TIF_02.raw SUBJECT_SAMPLE_FACTORS - TIF_03 Sample type:PDAC_TIF RAW_FILE_NAME=20220131_QE1_JAB_Job2501_TIF_03.raw SUBJECT_SAMPLE_FACTORS - Tumor_01 Sample type:PDAC_Tumor RAW_FILE_NAME=20220202_QE1_JAB_Job2501_Tissue_01.raw SUBJECT_SAMPLE_FACTORS - Tumor_02 Sample type:PDAC_Tumor RAW_FILE_NAME=20220202_QE1_JAB_Job2501_Tissue_02.raw SUBJECT_SAMPLE_FACTORS - Tumor_03 Sample type:PDAC_Tumor RAW_FILE_NAME=20220202_QE1_JAB_Job2501_Tissue_03.raw SUBJECT_SAMPLE_FACTORS - Tumor_04 Sample type:PDAC_Tumor RAW_FILE_NAME=20220202_QE1_JAB_Job2501_Tissue_04.raw #COLLECTION CO:COLLECTION_SUMMARY Briefly, tumors were rapidly dissected after euthanizing animals. Tumors were CO:COLLECTION_SUMMARY weighed and rinsed in blood bank saline solution (150 mM NaCl) and blotted on CO:COLLECTION_SUMMARY filter paper (VWR, Radnor, PA, 28298–020). The process of dissection and tumor CO:COLLECTION_SUMMARY preparation took < 3min. Tumors were cut in half and put onto 20µm nylon mesh CO:COLLECTION_SUMMARY filters (Spectrum Labs, Waltham, MA, 148134) on top of 50 mL conical tubes, and CO:COLLECTION_SUMMARY centrifuged for 10min. at 4°C at 400xg. IF was then collected, snap-frozen in CO:COLLECTION_SUMMARY liquid nitrogen and stored at -80°C until further analysis. Tumors were then CO:COLLECTION_SUMMARY immediately snap frozen using a BioSqueezer (BioSpec) cooled with liquid CO:COLLECTION_SUMMARY nitrogen and stored at -80°F until further analysis. CO:SAMPLE_TYPE Tumor, Interstitial fluid #TREATMENT TR:TREATMENT_SUMMARY Orthotopic tumors were implanted in C57BL6J mice at 8-12 weeks of age. 4 weeks TR:TREATMENT_SUMMARY after induction interstitial fluid and tumors were collected. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cryogenically frozen tumor pieces were ground to a fine homogenous powder with a SP:SAMPLEPREP_SUMMARY liquid nitrogen cooled mortar and pestle. ~30mg of tissue powder was weighed SP:SAMPLEPREP_SUMMARY into sample tubes, and metabolites were extracted with 600µL HPLC grade SP:SAMPLEPREP_SUMMARY methanol, 300µL HPLC grade water, and 400µL chloroform. Samples were vortexed SP:SAMPLEPREP_SUMMARY for 10min at 4°C, centrifuged 21,000xg at 4°C for 10 min. 400µL of the SP:SAMPLEPREP_SUMMARY aqueous top layer was removed into a new tube and dried under nitrogen. Dried SP:SAMPLEPREP_SUMMARY tumor extracts were resuspended in 100µL HPLC grade water and LC-MS analysis SP:SAMPLEPREP_SUMMARY was performed. For TIF samples, we extracted polar metabolites from 5µL of SP:SAMPLEPREP_SUMMARY sample using 45µL of a 75:25:0.1 HPLC grade acetonitrile:methanol:formic acid SP:SAMPLEPREP_SUMMARY extraction mix. Samples in extraction mix were vortexed for 10 min at 4°C and SP:SAMPLEPREP_SUMMARY centrifugated at 15,000x rpm for 10 min at 4°C to pellet insoluble material. SP:SAMPLEPREP_SUMMARY 20µL of the soluble polar metabolite supernatant was moved to sample vials for SP:SAMPLEPREP_SUMMARY analysis by LC-MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME SeQuant ZIC-HILIC (100 x 2.1mm, 3.5um) CH:SOLVENT_A 20 mM ammonium carbonate, 0.1% ammonium hydroxide CH:SOLVENT_B acetonitrile CH:FLOW_GRADIENT linear gradient from 80% to 20% B; 20–20.5 min: linear gradient from 20% to CH:FLOW_GRADIENT 80% B; 20.5–28 min: hold at 80% B CH:FLOW_RATE 0.150 mL/min CH:COLUMN_TEMPERATURE 25 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS XCalibur 965 2.2 software (Thermo 966 Fisher Scientific) was used for MS:MS_COMMENTS identification and quantification of metabolites. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area (m/z) MS_METABOLITE_DATA_START Samples TIF_01 TIF_02 TIF_03 Tumor_01 Tumor_02 Tumor_03 Tumor_04 Factors Sample type:PDAC_TIF Sample type:PDAC_TIF Sample type:PDAC_TIF Sample type:PDAC_Tumor Sample type:PDAC_Tumor Sample type:PDAC_Tumor Sample type:PDAC_Tumor alanine 873643406.1 300033478.8 437592864.6 2487879405 2088008449 2892498109 3211396346 arginine 13632462.61 7850610.231 7788971.36 663801656.8 457965182.3 713032399.7 952896429.1 aspartate 157205296.9 35249787.54 36808070.15 698380583.4 508557351.1 541641452.2 683959979.3 citrulline 185927072.7 57903362.26 116580604.6 470656618.3 356883299.1 851658464 608591575.2 glutamate 587289803.3 179433233 293888405.1 2666382720 2260218373 2690867316 2780140686 glutamine 636826627.4 266699065.9 242041168.2 3881811349 2045465512 4611460745 3128982704 histidine 126268136.9 89674333.21 117725298 332995695.6 245867488.9 433578485.6 341319430.6 lysine 68606224.9 33774891.55 50279290.92 450448648.5 364558732.5 540667040 616471448.3 methionine 138586002.9 38922361.09 65384158.02 1082379458 714251397.4 1190624536 1599749049 ornithine 26021252.71 14590149.32 18818687.74 79951183.15 61119173.03 55140924.15 98211195.03 phenylalanine 240506144.9 69328661.11 97181611.23 2793575848 749753882.5 1620792060 2072956014 proline 3717738424 432391643.5 1284861845 18849505357 15895818565 23520582877 24256372258 serine 36087779.79 5401935.656 10843766.69 202804788.3 123109682.8 176916320.1 217025790.7 threonine 148105928.8 42999938.68 73230999.21 710526494.2 492165852.6 1126531197 1260479053 tyrosine 116193556.9 35056233.9 57047836.54 735802531.3 448408056.9 557984996.6 705870085.4 valine 219686202.9 64838946.44 NA 2449504808 1350786174 2048903669 1855178365 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name alanine arginine aspartate citrulline glutamate glutamine histidine lysine methionine ornithine phenylalanine proline serine threonine tyrosine valine METABOLITES_END #END