#METABOLOMICS WORKBENCH japizsaab_20220805_152657 DATATRACK_ID:3390 STUDY_ID:ST002527 ANALYSIS_ID:AN004161 PROJECT_ID:PR001627 VERSION 1 CREATED_ON March 28, 2023, 10:05 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 HPLC-MS-MS analysis amino acid levels in PDAC IF samples upon arginase ST:STUDY_TITLE inhibition ST:STUDY_SUMMARY To test the hypothesis that myeloid arginase-1 activity could be responsible for ST:STUDY_SUMMARY pancreatic ductal adenocarninoma microenvironmental arginine starvation (PMID: ST:STUDY_SUMMARY 30990168), we generated orthotopic allograft mPDAC tumors in a mouse model with ST:STUDY_SUMMARY myeloid specific Arg1 knockout (LysM-Cre+/+-; Arg1fl/fl) and control animals ST:STUDY_SUMMARY (Arg1fl/fl). We also tested this with pharmacological inhibition of arginase-1 ST:STUDY_SUMMARY with the small-molecule inhibitor CB-1158. We then isolated IF from these tumors ST:STUDY_SUMMARY at end-stage and measured the levels of amino acids including arginine and ST:STUDY_SUMMARY ornithine in these samples. 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 - T245 Genotype:NA | Treatment:CB1158 RAW_FILE_NAME=Mui-Lin-20210408-02-2.raw SUBJECT_SAMPLE_FACTORS - T244 Genotype:NA | Treatment:CB1158 RAW_FILE_NAME=Mui-Lin-20210408-04-2.raw SUBJECT_SAMPLE_FACTORS - T241 Genotype:NA | Treatment:CB1158 RAW_FILE_NAME=Mui-Lin-20210408-06-2.raw SUBJECT_SAMPLE_FACTORS - T242 Genotype:NA | Treatment:CB1158 RAW_FILE_NAME=Mui-Lin-20210408-08-2.raw SUBJECT_SAMPLE_FACTORS - T231 Genotype:NA | Treatment:CB1158 RAW_FILE_NAME=Mui-Lin-20210408-33-2.raw SUBJECT_SAMPLE_FACTORS - T278 Genotype:NA | Treatment:Vehicle RAW_FILE_NAME=Mui-Lin-20210408-01-2.raw SUBJECT_SAMPLE_FACTORS - T238 Genotype:NA | Treatment:Vehicle RAW_FILE_NAME=Mui-Lin-20210408-03-2.raw SUBJECT_SAMPLE_FACTORS - T282 Genotype:NA | Treatment:Vehicle RAW_FILE_NAME=Mui-Lin-20210408-05-2.raw SUBJECT_SAMPLE_FACTORS - T400 Genotype:NA | Treatment:Vehicle RAW_FILE_NAME=Mui-Lin-20210408-07-2.raw SUBJECT_SAMPLE_FACTORS - T239 Genotype:NA | Treatment:Vehicle RAW_FILE_NAME=Mui-Lin-20210408-09-2.raw SUBJECT_SAMPLE_FACTORS - T234 Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA RAW_FILE_NAME=Mui-Lin-20210408-10-2.raw SUBJECT_SAMPLE_FACTORS - T214 Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA RAW_FILE_NAME=Mui-Lin-20210408-30-2.raw SUBJECT_SAMPLE_FACTORS - T203 Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA RAW_FILE_NAME=Mui-Lin-20210408-23-2.raw SUBJECT_SAMPLE_FACTORS - T236 Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA RAW_FILE_NAME=Mui-Lin-20210408-25-2.raw SUBJECT_SAMPLE_FACTORS - T215 Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA RAW_FILE_NAME=Mui-Lin-20210408-28-2.raw SUBJECT_SAMPLE_FACTORS - T298 Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA RAW_FILE_NAME=Mui-Lin-20210408-29-2.raw SUBJECT_SAMPLE_FACTORS - T233 Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA RAW_FILE_NAME=Mui-Lin-20210408-34-2.raw SUBJECT_SAMPLE_FACTORS - T111 Genotype:Arg1fl/fl | Treatment:NA RAW_FILE_NAME=Mui-Lin-20210408-24-2.raw SUBJECT_SAMPLE_FACTORS - T202 Genotype:Arg1fl/fl | Treatment:NA RAW_FILE_NAME=Mui-Lin-20210408-27-2.raw SUBJECT_SAMPLE_FACTORS - T237 Genotype:Arg1fl/fl | Treatment:NA RAW_FILE_NAME=Mui-Lin-20210408-31-2.raw SUBJECT_SAMPLE_FACTORS - T217 Genotype:Arg1fl/fl | Treatment:NA RAW_FILE_NAME=Mui-Lin-20210408-32-2.raw #COLLECTION CO:COLLECTION_SUMMARY Tumors were rapidly dissected after euthanizing animals. Tumors were weighed and CO:COLLECTION_SUMMARY rinsed in blood bank saline solution (150 mM NaCl) and blotted on filter paper CO:COLLECTION_SUMMARY (VWR, Radnor, PA, 28298–020). The process of dissection and tumor preparation CO:COLLECTION_SUMMARY took < 3min. Tumors were cut in half and put onto 20µm nylon mesh filters CO:COLLECTION_SUMMARY (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. CO:SAMPLE_TYPE Interstitial Fluid #TREATMENT TR:TREATMENT_SUMMARY For genetically modified mice, LysM-Cre and Arg1fl/fl C57BL6J mice were bred to TR:TREATMENT_SUMMARY generate LysM-Cre +/+; Arg1fl/fl and litter mate control Arg1fl/fl mice. TR:TREATMENT_SUMMARY Orthotopic pancreatic cancer tumors were implanted in C57BL6J mice at 8-12 weeks TR:TREATMENT_SUMMARY of age. At endpoint, mice were euthanized by cervical dislocation, and tumors TR:TREATMENT_SUMMARY were harvested for TIF extraction. For Pharmacological inhibition, orthotopic TR:TREATMENT_SUMMARY tumors were implanted in C57BL6J mice at 8-12 weeks of age. 4 weeks after TR:TREATMENT_SUMMARY induction, once tumors were close end stage disease, CB-1158 (MedChem Express) TR:TREATMENT_SUMMARY dissolved in sterile water was administered by oral gavage at 100mg/kg. The TR:TREATMENT_SUMMARY acidity caused by the HCl in the drug solution was neutralized by adding TR:TREATMENT_SUMMARY equivalent amount of NaOH and an equivalent NaCl in sterile water solution was TR:TREATMENT_SUMMARY prepared as vehicle. 2hrs after treatment with CB-1158 or vehicle, mice were TR:TREATMENT_SUMMARY euthanized by cervical dislocation, and tumors were harvested for TIF TR:TREATMENT_SUMMARY extraction. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY polar metabolites were extracted from 5µL IF samples using 45µL 75:25:0.1 HPLC SP:SAMPLEPREP_SUMMARY grade acetonitrile:methanol:formic acid extraction mix into which a mixture of SP:SAMPLEPREP_SUMMARY isotopically labeled amino acids of known concentrations (Cambridge Isotope SP:SAMPLEPREP_SUMMARY Laboratories, MSK-A2-1.2) was added. Samples were vortexed for 10 min, and SP:SAMPLEPREP_SUMMARY centrifuged at maximum speed for 10 min. 30 μL of each extract was removed and SP:SAMPLEPREP_SUMMARY dried under nitrogen gas and stored −80°C. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters XBridge Amide (100 x 4.6mm,3.5um) CH:SOLVENT_A 95% (vol/vol) water, 5% (vol/vol) acetonitrile, 10 mM ammonium hydroxide, 10 mM CH:SOLVENT_A ammonium acetate, pH = 9.0 CH:SOLVENT_B 100% Acetonitrile CH:FLOW_GRADIENT 0 min, 15% A; 2.5 min, 64% A; 12.4 min, 40% A; 12.5 min, 30% A; 12.5-14 min, 30% CH:FLOW_GRADIENT A; 14-21 min, 15% A CH:FLOW_RATE 150 μL/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 In positive/negative polarity switching mode, an m/z scan range from 60 to 900 MS:MS_COMMENTS was chosen and MS1 data was collected at a resolution of 70,000. The automatic MS:MS_COMMENTS gain control (AGC) target was set at 1 × 106 and the maximum injection time was MS:MS_COMMENTS 200 ms. The targeted ions were subsequently fragmented, using the higher MS:MS_COMMENTS energy collisional dissociation (HCD) cell set to 30% normalized collision MS:MS_COMMENTS energy in MS2 at a resolution power of 17,500. Besides matching m/z, target MS:MS_COMMENTS metabolites are identified by matching either retention time with analytical MS:MS_COMMENTS standards and/or MS2 fragmentation pattern. Data acquisition and analysis were MS:MS_COMMENTS carried out by Xcalibur 4.1 software and Tracefinder 4.1 software, respectively MS:MS_COMMENTS (both from Thermo Fisher Scientific). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area (m/z) MS_METABOLITE_DATA_START Samples T278 T245 T238 T244 T282 T241 T400 T242 T239 T234 T203 T111 T236 T202 T215 T298 T214 T237 T217 T231 T233 Factors Genotype:NA | Treatment:Vehicle Genotype:NA | Treatment:CB1158 Genotype:NA | Treatment:Vehicle Genotype:NA | Treatment:CB1158 Genotype:NA | Treatment:Vehicle Genotype:NA | Treatment:CB1158 Genotype:NA | Treatment:Vehicle Genotype:NA | Treatment:CB1158 Genotype:NA | Treatment:Vehicle Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA Genotype:Arg1fl/fl | Treatment:NA Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA Genotype:Arg1fl/fl | Treatment:NA Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA Genotype:Arg1fl/fl | Treatment:NA Genotype:Arg1fl/fl | Treatment:NA Genotype:NA | Treatment:CB1158 Genotype:LysM-Cre+/+; Arg1fl/fl | Treatment:NA alanine 523882213 665366442 553355746 659426901 855120451 683908395 720401203 811402985 596203035 1030701024 1064349344 595038393 868296015 1207982150 685347221 816675164 522068620 603741202 756460459 757752305 953702346 alanine_13C3_15N 69444297 121026475 92751805 117597621 117476696 126949939 113590585 124493375 101535044 111900612 98340772 103612981 91253941 99587109 111175217 91535427 46896091 117799638 93160154 101514396 90772111 arginine 15340384 509477213 15963464 448670179 44070934 696484257 303242696 485936625 77999505 852856228 902790825 21933398 846566956 418282234 901672616 940740561 345309030 30916052 17567574 39064591 789413918 arginine_13C6_15N4 706974619 1429784641 1132933084 1199365402 1322819866 1378726057 1242799318 1264095985 1074413296 1041782009 1007811620 1071493892 863984865 1131312438 1067893988 813697156 407899696 1174649819 822954194 1060947605 908468868 aspartic acid 254788767 218634105 230325424 358848958 245178714 291241713 193027292 354647087 494417951 592926273 856042597 411280238 371268591 793994914 382494001 596844261 520963586 345515529 558556008 577551365 826085193 aspartic acid_13C4_15N 54229900 53794414 69040857 65801128 71426321 76315972 82266092 91842409 91407961 84619374 106556099 116994749 99447919 109348747 108163057 99296601 63022828 107076488 94254069 108528709 112941511 citrulline 55871932 29410641 54428610 41575757 56707942 43066219 73939072 50142728 75197723 96428149 122261165 100433915 163589561 124525152 135188307 179648859 139503479 132171268 124775827 143139516 140653201 citrulline_13C5 560411090 465568905 637520284 565469074 633329215 604233533 700218353 693677878 767840460 686817792 900839473 1188608663 1183661206 1040862386 1299168219 1211403944 1075223383 1687433573 1315583245 1261980165 1010161149 cystine 5356958 4647092 3763879 5379871 7101641 10970674 7050325 7599334 4491406 4298805 279455 6699636 2408775 2567491 5216990 4691150 2598554 1956840 5801144 2481165 1153881 cystine_13C6_15N2 7321265 10331815 8020501 9103151 8453714 9942144 7687599 7876234 7082849 5784516 4833916 5678798 5062870 4793232 5082532 4241024 2267928 5983144 4117765 4070646 2658445 glutamic acid 1107396968 1280321186 1152785862 1350888795 1094317187 1230369405 979933376 1418571862 1329095089 1729172619 1960935093 1083229104 1181242183 1928478064 1174231526 1277827839 976715048 908308737 1205186985 1261827361 1803091610 glutamic acid_13C5_15N 101603642 144488619 135878426 134251602 134620922 147562177 162349643 160460589 128308808 123556208 103530454 126449216 108885588 103445784 128776672 96052809 54211955 134085224 100629170 116441195 104144761 glycine 197994169 290848314 230333923 328864536 256881737 305004116 307732749 371705708 357183086 586692237 592746348 263504115 278710501 466066675 284691688 395800791 344438394 274460954 360446999 361927672 524736536 glycine_13C2_15N 37848110 63526592 51565373 57957822 52187872 59458847 59000656 62815113 50586044 52247948 45252136 50139503 45005392 46446157 55051041 43021577 25688791 60875725 43745023 49714086 45704051 histidine 143681431 100762317 163989976 114134595 156504931 138422665 209737160 151375524 164326379 208934198 432969277 439214786 490594125 490482695 507247821 490445989 439844244 492919265 433382708 535761667 395721989 histidine_13C6_15N3 154924402 131022286 171157245 153500179 170879212 184373608 208001923 190266659 219963303 184819124 416359625 517486803 473052341 441920042 524645023 479076591 376958016 720005922 477289783 531467594 374930029 lysine 599020398 889287351 850866765 753268994 1099507577 844543145 1053961046 720013605 638197267 771478489 558321484 735641476 615972102 937901226 922388423 644167810 333460576 662295469 557172878 866361612 692760257 lysine_13C6_15N2 229382409 454039178 347025278 406756393 395823755 454422313 397990810 416370683 350389129 357008628 338831096 328552540 291735404 363452867 359736319 287830147 150877594 375883675 270743258 376135487 317559472 methionine 259719150 343500193 391370623 312556785 560894477 425575258 577366548 447294356 329858652 648268972 493236529 353482928 557923616 681197733 493416454 576801119 197477407 540282191 342851057 652198568 400248967 methionine_13C5_15N 461969136 851758798 700130234 831353163 815643951 878650097 772495142 826633064 705702735 835967885 760060777 794769438 787991168 895759291 882820610 728477236 359260597 955268513 698774174 790608522 786620323 ornithine 246638195 368608046 344633432 320366816 520033523 497448736 443303858 424975832 294889136 213207447 110343431 318171255 155737275 379101601 204601614 138302802 61451324 293990744 239505867 395350334 142431552 ornithine_13C5_15N2 3259039860 6449344416 5097888885 5641227533 6092477903 6717172996 5628841379 6257266246 5108721046 4866226712 4531488368 4913379074 3961907035 4979794268 4971953680 4358008105 2107771269 5279343718 3984258015 4945883421 4185480921 phenylalanine 481063815 619653047 816117574 676611838 753463567 658273338 776675162 627838935 446843738 866173051 636087991 667670645 1226832222 1048374046 900342785 813873969 397895669 873842720 572242459 983666985 755194547 phenylalanine_13C9_15N 674632629 1144814058 1079020231 1176253221 1058133706 1149717206 1068190705 1034154803 1050736995 1114993388 950221749 1009666587 1041716717 1097408843 1146608429 938170901 488658318 1352740754 889074964 1092759213 930319961 proline 1539070312 2024281834 2089896459 1893168193 2510895879 2034279972 2263437358 2589289942 1730662510 3792062760 3113572337 2181254993 3250435361 3776112969 2462875049 2818341949 1497235151 2451749225 2290957550 2690030074 2603121989 proline_13C5_15N 851487273 1715048635 1383417421 1520787006 1502133218 1575203467 1294506206 1565234496 1290530871 1497881583 1256201202 1366733889 1260290311 1249040436 1475517155 1127802397 665083564 1644617394 1119093489 1265265220 1057990747 serine 74192789 56920384 76994820 74275118 85182186 68078823 90130405 91421481 86856922 128367637 156938846 92544491 141744813 158942604 111111435 126574735 101289459 113733767 107684073 132603230 136274951 serine_13C3_15N 63525403 59696331 75417215 72415486 76256971 75049490 76771182 85732363 82137915 78685261 87305921 92815343 85498977 87068366 98150371 77472975 45981687 116664933 84756007 90053654 84902177 threonine_13C4_15N 179581416 285715922 217928682 253247466 243474699 273099406 270499379 267698711 230556505 221609342 175215792 220768025 201809317 192431302 243574396 197410680 103621613 261755716 195877961 200184325 172712676 tryptophan 216994791 303231125 343047027 353496774 340597676 401508527 281716102 308339384 274396429 392044589 346625413 297428121 398630838 430936044 279960655 307589430 147538809 246935993 222817919 293504194 341369823 tyrosine 150409617 196790303 232004348 177540543 245265482 221486944 294516756 237292820 164522997 277624919 210613525 174772362 391303162 279271800 256597945 255830778 138672286 241238349 152694265 329513176 226189112 tyrosine_13C9_15N 270374403 472737416 380382330 429670558 376964675 429723918 385885497 428387458 375482051 398246992 348177501 386398316 352341182 371228552 434357576 320797828 195868215 400893682 316216948 372369212 334477001 valine 451721379 637144368 623678862 666322623 614348743 578157575 665016681 611980158 419937829 1026131146 627650668 591857445 738986109 857053599 757004829 672482744 449938234 734438928 562337709 1023647891 757782676 valine_13C5_15N 258360853 467474662 377353367 427291938 385873177 469401707 383578388 431614870 369559787 433427360 314977884 395950441 344847179 376660648 410687924 315284580 185073526 433850799 345573325 404641262 355715375 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name alanine alanine_13C3_15N arginine arginine_13C6_15N4 aspartic acid aspartic acid_13C4_15N citrulline citrulline_13C5 cystine cystine_13C6_15N2 glutamic acid glutamic acid_13C5_15N glycine glycine_13C2_15N histidine histidine_13C6_15N3 lysine lysine_13C6_15N2 methionine methionine_13C5_15N ornithine ornithine_13C5_15N2 phenylalanine phenylalanine_13C9_15N proline proline_13C5_15N serine serine_13C3_15N threonine_13C4_15N tryptophan tyrosine tyrosine_13C9_15N valine valine_13C5_15N METABOLITES_END #END