#METABOLOMICS WORKBENCH pbjonker_20250209_162831 DATATRACK_ID:5611 STUDY_ID:ST003718 ANALYSIS_ID:AN006099 PROJECT_ID:PR002307 VERSION 1 CREATED_ON February 12, 2025, 7:40 pm #PROJECT PR:PROJECT_TITLE Microenvironmental arginine restriction sensitizes pancreatic cancers to PR:PROJECT_TITLE polyunsaturated fatty acids by suppression of lipid synthesis PR:PROJECT_TYPE Metabolomics PR:PROJECT_SUMMARY Nutrient limitation is a characteristic feature of poorly perfused tumors. These PR:PROJECT_SUMMARY changes in nutrient availability impose metabolic constraints and perturb PR:PROJECT_SUMMARY metabolic pathways in cancer cells, in contrast to cells in well-perfused PR:PROJECT_SUMMARY tissues. Consequently, targeting the metabolic dependencies created by tumor PR:PROJECT_SUMMARY microenvironmental constraints may be a promising antineoplastic therapeutic PR:PROJECT_SUMMARY approach. To identify these adaptations, we challenged pancreatic cancer cell PR:PROJECT_SUMMARY lines (mouse Pancreatic Ductal Adenocarcinoma - PDAC) with pathophysiological PR:PROJECT_SUMMARY nutrient levels and analyzed changes to cell metabolism. Here, we report that PR:PROJECT_SUMMARY arginine limitation in pancreatic cancer perturbs saturated and monounsaturated PR:PROJECT_SUMMARY fatty acid synthesis by suppressing the lipogenic transcription factor SREBP1. PR:PROJECT_SUMMARY Synthesis of these acyl species is critical to maintaining a balance of PR:PROJECT_SUMMARY saturated, monounsaturated, and polyunsaturated fatty acids in cellular PR:PROJECT_SUMMARY membranes. We found that, as a consequence of the loss of fatty acid synthesis, PR:PROJECT_SUMMARY pancreatic cancer cells were unable to maintain balanced lipidomes when exposed PR:PROJECT_SUMMARY to polyunsaturated fatty acids, leading to cell death by ferroptosis. PR:PROJECT_SUMMARY Importantly, we found orally administering oils rich in polyunsaturated fats PR:PROJECT_SUMMARY reduces tumor burden in mice with pancreatic cancer. In sum, this study PR:PROJECT_SUMMARY illustrates that arginine restriction in the tumor microenvironment alters PR:PROJECT_SUMMARY pancreatic cancer cells by perturbing lipid synthesis, making them sensitive to PR:PROJECT_SUMMARY supplementation with polyunsaturated fats. PR:INSTITUTE University of Chicago PR:DEPARTMENT Ben May Department of Cancer Research PR:LABORATORY Muir Lab PR:LAST_NAME Jonker PR:FIRST_NAME Patrick PR:ADDRESS 929 E 57th st. Chicago IL, 60637 PR:EMAIL pbjonker@uchicago.edu PR:PHONE 6162884547 #STUDY ST:STUDY_TITLE Metabolomics of plasma and tumor interstitial fluid (TIF) of tumors from ST:STUDY_TITLE Lyz2-Cre(+/+);Arg1(fl/fl) mice. ST:STUDY_TYPE Metabolomics ST:STUDY_SUMMARY Mice or the genotype Lyz2-Cre(-/-);Arg1(fl/fl) or Lyz2-Cre(+/+);Arg1(fl/fl) were ST:STUDY_SUMMARY implanted with PDAC tumors and allowed to grow for 4 weeks. After 3 weeks of ST:STUDY_SUMMARY growth, mice were placed on 8% deuterated water for fatty acid synthesis ST:STUDY_SUMMARY analysis. To confirm that Lyz2-Cre(+/+);Arg1(fl/fl) had plasma concentrations of ST:STUDY_SUMMARY arginine, we performed metabolomics analysis on the plasma and tumor ST:STUDY_SUMMARY interstitial fluid of tumors from each mouse genotype. We found that arginine ST:STUDY_SUMMARY concentrations rose to plasma levels in Lyz2-Cre(+/+);Arg1(fl/fl) mice. This ST:STUDY_SUMMARY confirmed that this mouse model allows us to assay arginine deprivation in vivo. ST:INSTITUTE University of Chicago ST:DEPARTMENT Ben May Department for Cancer Research ST:LABORATORY Muir Lab ST:LAST_NAME Jonker ST:FIRST_NAME Patrick ST:ADDRESS 929 E 57th St. Chicago IL, 60637 ST:EMAIL pbjonker@uchicago.edu ST:PHONE 6162884547 ST:NUM_GROUPS 10 #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 - Plasma_1 Sample source:Plasma | Genotype:Lyz2-Cre-/- Arg1fl/fl RAW_FILE_NAME(Raw File name)=Plasma_1.mzML SUBJECT_SAMPLE_FACTORS - Plasma_10 Sample source:Plasma | Genotype:Lyz2-Cre+/+ Arg1fl/fl RAW_FILE_NAME(Raw File name)=Plasma_10.mzML SUBJECT_SAMPLE_FACTORS - Plasma_11 Sample source:Plasma | Genotype:Lyz2-Cre-/- Arg1fl/fl RAW_FILE_NAME(Raw File name)=Plasma_11.mzML SUBJECT_SAMPLE_FACTORS - Plasma_14 Sample source:Plasma | Genotype:Lyz2-Cre+/+ Arg1fl/fl RAW_FILE_NAME(Raw File name)=Plasma_14.mzML SUBJECT_SAMPLE_FACTORS - Plasma_2 Sample source:Plasma | Genotype:Lyz2-Cre-/- Arg1fl/fl RAW_FILE_NAME(Raw File name)=Plasma_2.mzML SUBJECT_SAMPLE_FACTORS - Plasma_4 Sample source:Plasma | Genotype:Lyz2-Cre-/- Arg1fl/fl RAW_FILE_NAME(Raw File name)=Plasma_4.mzML SUBJECT_SAMPLE_FACTORS - Plasma_5 Sample source:Plasma | Genotype:Lyz2-Cre+/+ Arg1fl/fl RAW_FILE_NAME(Raw File name)=Plasma_5.mzML SUBJECT_SAMPLE_FACTORS - Plasma_6 Sample source:Plasma | Genotype:Lyz2-Cre+/+ Arg1fl/fl RAW_FILE_NAME(Raw File name)=Plasma_6.mzML SUBJECT_SAMPLE_FACTORS - Plasma_7 Sample source:Plasma | Genotype:Lyz2-Cre+/+ Arg1fl/fl RAW_FILE_NAME(Raw File name)=Plasma_7.mzML SUBJECT_SAMPLE_FACTORS - Plasma_8 Sample source:Plasma | Genotype:Lyz2-Cre-/- Arg1fl/fl RAW_FILE_NAME(Raw File name)=Plasma_8.mzML SUBJECT_SAMPLE_FACTORS - Plasma_9 Sample source:Plasma | Genotype:Lyz2-Cre-/- Arg1fl/fl RAW_FILE_NAME(Raw File name)=Plasma_9.mzML SUBJECT_SAMPLE_FACTORS - TIF_01 Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre-/- Arg1fl/fl RAW_FILE_NAME(Raw File name)=TIF_01.mzML SUBJECT_SAMPLE_FACTORS - TIF_02 Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre-/- Arg1fl/fl RAW_FILE_NAME(Raw File name)=TIF_02.mzML SUBJECT_SAMPLE_FACTORS - TIF_04 Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre-/- Arg1fl/fl RAW_FILE_NAME(Raw File name)=TIF_04.mzML SUBJECT_SAMPLE_FACTORS - TIF_05 Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre+/+ Arg1fl/fl RAW_FILE_NAME(Raw File name)=TIF_05.mzML SUBJECT_SAMPLE_FACTORS - TIF_06 Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre+/+ Arg1fl/fl RAW_FILE_NAME(Raw File name)=TIF_06.mzML SUBJECT_SAMPLE_FACTORS - TIF_07 Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre+/+ Arg1fl/fl RAW_FILE_NAME(Raw File name)=TIF_07.mzML SUBJECT_SAMPLE_FACTORS - TIF_08 Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre-/- Arg1fl/fl RAW_FILE_NAME(Raw File name)=TIF_08.mzML SUBJECT_SAMPLE_FACTORS - TIF_09 Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre-/- Arg1fl/fl RAW_FILE_NAME(Raw File name)=TIF_09.mzML SUBJECT_SAMPLE_FACTORS - TIF_10 Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre+/+ Arg1fl/fl RAW_FILE_NAME(Raw File name)=TIF_10.mzML SUBJECT_SAMPLE_FACTORS - TIF_11 Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre-/- Arg1fl/fl RAW_FILE_NAME(Raw File name)=TIF_11.mzML SUBJECT_SAMPLE_FACTORS - TIF_14 Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre+/+ Arg1fl/fl RAW_FILE_NAME(Raw File name)=TIF_14.mzML #COLLECTION CO:COLLECTION_SUMMARY 5 µL of TIF or plasma sample was mixed with 45 µL of ice-cold acetonitrile CO:COLLECTION_SUMMARY containing stable isotope labeled amino acid mix from Cambridge Isotope CO:COLLECTION_SUMMARY Labs(MSK-A1-1.2) as internal standards. Only 3 uL were analyzed for sample CO:COLLECTION_SUMMARY "TIF_07" due to low sample volume from TIF extraction. Samples were vortexed, CO:COLLECTION_SUMMARY incubated on ice for 20 minutes, centrifuged at 20,000g for 20mins at 4°C and CO:COLLECTION_SUMMARY the supernatant was transferred to an autosampler vial for LC-MS analysis. CO:COLLECTION_SUMMARY Arginine calibration curves was prepared from 0.1µ-500 µM levels. CO:SAMPLE_TYPE Blood (plasma), Tumor interstitial fluid #TREATMENT TR:TREATMENT_SUMMARY Mice were injected with mPDAC3 orthotopic tumors. After 4 weeks of tumor growth, TR:TREATMENT_SUMMARY tumor interstitial fluid and plasma were extracted for analysis of arginine TR:TREATMENT_SUMMARY levels. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 5 µL of TIF or plasma sample was mixed with 45 µL of ice-cold acetonitrile SP:SAMPLEPREP_SUMMARY containing stable isotope labeled amino acid mix from Cambridge Isotope SP:SAMPLEPREP_SUMMARY Labs(MSK-A1-1.2) as internal standards. Samples were vortexed, incubated on ice SP:SAMPLEPREP_SUMMARY for 20 minutes, centrifuged at 20,000g for 20mins at 4°C and the supernatant SP:SAMPLEPREP_SUMMARY was transferred to an autosampler vial for LC-MS analysis. Arginine calibration SP:SAMPLEPREP_SUMMARY curves was prepared from 0.1µ-500 µM levels. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Fisher IQ-X CH:COLUMN_NAME Waters XBridge BEH Amide (150 x 2.1mm, 2.5um) CH:SOLVENT_A 90% water/5% acetonitrile/5% methanol; 0.2% acetic acid; 20mM ammonium acetate CH:SOLVENT_B 90% acetonitrile/10% water; 0.2% acetic acid; 10mM ammonium acetate CH:FLOW_GRADIENT 0min: 95% B, 9min: 70% B, 9.75min: 40% B, 12min: 40% B, 13min: 30% B, 14min: CH:FLOW_GRADIENT 30%B, 14.1min: 10% B,17min: 10% B, 17.5min: 95% B, 22min: 95% B CH:FLOW_RATE 0.3 mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME University of Chicago Metabolomics Platform AN:OPERATOR_NAME Hardik Shah #MS MS:INSTRUMENT_NAME Thermo Orbitrap IQ-X Tribrid MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS IQX orbitrap high resolution MS parameters were: sheath gas flow = 40, aux gas MS:MS_COMMENTS flow = 7, sweep gas flow = 1, spray voltage = 2800 for negative, 3600 for MS:MS_COMMENTS positive, ion transfer tube = 250°C, vaporizer temp=350°C, REF lens=60%. Data MS:MS_COMMENTS acquisition was done using Xcalibur 4.1 (ThermoFisher Scientific) and performed MS:MS_COMMENTS in switch polarity mode with a range of 70-1000 m/z, a resolving power of MS:MS_COMMENTS 60,000, an AGC target=100%, and a maximum injection time of 118ms. Tracefinder MS:MS_COMMENTS 4.1 was used for quantitation analysis. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples Plasma_1 Plasma_10 Plasma_11 Plasma_14 Plasma_2 Plasma_4 Plasma_5 Plasma_6 Plasma_7 Plasma_8 Plasma_9 TIF_01 TIF_02 TIF_04 TIF_05 TIF_06 TIF_07 TIF_08 TIF_09 TIF_10 TIF_11 TIF_14 Factors Sample source:Plasma | Genotype:Lyz2-Cre-/- Arg1fl/fl Sample source:Plasma | Genotype:Lyz2-Cre+/+ Arg1fl/fl Sample source:Plasma | Genotype:Lyz2-Cre-/- Arg1fl/fl Sample source:Plasma | Genotype:Lyz2-Cre+/+ Arg1fl/fl Sample source:Plasma | Genotype:Lyz2-Cre-/- Arg1fl/fl Sample source:Plasma | Genotype:Lyz2-Cre-/- Arg1fl/fl Sample source:Plasma | Genotype:Lyz2-Cre+/+ Arg1fl/fl Sample source:Plasma | Genotype:Lyz2-Cre+/+ Arg1fl/fl Sample source:Plasma | Genotype:Lyz2-Cre+/+ Arg1fl/fl Sample source:Plasma | Genotype:Lyz2-Cre-/- Arg1fl/fl Sample source:Plasma | Genotype:Lyz2-Cre-/- Arg1fl/fl Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre-/- Arg1fl/fl Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre-/- Arg1fl/fl Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre-/- Arg1fl/fl Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre+/+ Arg1fl/fl Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre+/+ Arg1fl/fl Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre+/+ Arg1fl/fl Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre-/- Arg1fl/fl Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre-/- Arg1fl/fl Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre+/+ Arg1fl/fl Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre-/- Arg1fl/fl Sample source:Tumor interstitial fluid | Genotype:Lyz2-Cre+/+ Arg1fl/fl Arginine 1594263 1936971 1502279 844009 1404713 656054 1730766 2561984 1968170 2096487 1741259 768107 563093 330932 1457609 4019277 1049330 2412945 305871 2777994 311827 2201279 Arginine_13C6_15N4 4541290 4544158 4533726 2055202 3085187 2021229 2072691 4387478 3664918 4473189 4195646 6564309 8078259 4640306 3625785 9450923 4538091 11268090 8421393 5202421 2486197 4052144 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Arginine Arginine_13C6_15N4 METABOLITES_END #END