#METABOLOMICS WORKBENCH japizsaab_20230324_032803 DATATRACK_ID:3816 STUDY_ID:ST002530 ANALYSIS_ID:AN004164 PROJECT_ID:PR001627 VERSION 1 CREATED_ON March 28, 2023, 10:13 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 LCMS analysis of amino acid levels in PDAC interstitial fluid samples upon ass1 ST:STUDY_TITLE cancer cell knock out ST:STUDY_SUMMARY We used CRISPR-Cas9 to knockout (KO) Ass1 in mPDAC cells. We tested if ST:STUDY_SUMMARY inhibiting arginine de novo synthesis would impair PDAC tumor growth. To test ST:STUDY_SUMMARY this, we generated orthotopic PDAC tumors with mPDAC-Ass1 KO cells and control ST:STUDY_SUMMARY cells where Ass1 was re-expressed (Ass1KO; mASS1). 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 - 20210804_01 Sample type:ASS1KO RAW_FILE_NAME=Mui-Ale-20210804-01-.raw SUBJECT_SAMPLE_FACTORS - 20210804_02 Sample type:ASS1KO RAW_FILE_NAME=Mui-Ale-20210804-02-.raw SUBJECT_SAMPLE_FACTORS - 20210804_05 Sample type:ASS1KO RAW_FILE_NAME=Mui-Ale-20210804-05-.raw SUBJECT_SAMPLE_FACTORS - 20210804_08 Sample type:ASS1KO RAW_FILE_NAME=Mui-Ale-20210804-08-.raw SUBJECT_SAMPLE_FACTORS - 20210804_10 Sample type:ASS1KO RAW_FILE_NAME=Mui-Ale-20210804-10-.raw SUBJECT_SAMPLE_FACTORS - 20210804_11 Sample type:ASS1KO RAW_FILE_NAME=Mui-Ale-20210804-11-.raw SUBJECT_SAMPLE_FACTORS - 20210804_13 Sample type:ASS1KO RAW_FILE_NAME=Mui-Ale-20210804-13-.raw SUBJECT_SAMPLE_FACTORS - 20210804_17 Sample type:ASS1KO RAW_FILE_NAME=Mui-Ale-20210804-17-.raw SUBJECT_SAMPLE_FACTORS - 20210804_19 Sample type:ASS1KO RAW_FILE_NAME=Mui-Ale-20210804-19-.raw SUBJECT_SAMPLE_FACTORS - 20210804_20 Sample type:ASS1KO RAW_FILE_NAME=Mui-Ale-20210804-20-.raw SUBJECT_SAMPLE_FACTORS - 20210804_22 Sample type:ASS1KO RAW_FILE_NAME=Mui-Ale-20210804-22-.raw SUBJECT_SAMPLE_FACTORS - 20210804_03 Sample type:ASS1KO;mASS1 RAW_FILE_NAME=Mui-Ale-20210804-03-.raw SUBJECT_SAMPLE_FACTORS - 20210804_04 Sample type:ASS1KO;mASS1 RAW_FILE_NAME=Mui-Ale-20210804-04-.raw SUBJECT_SAMPLE_FACTORS - 20210804_06 Sample type:ASS1KO;mASS1 RAW_FILE_NAME=Mui-Ale-20210804-06-.raw SUBJECT_SAMPLE_FACTORS - 20210804_07 Sample type:ASS1KO;mASS1 RAW_FILE_NAME=Mui-Ale-20210804-07-.raw SUBJECT_SAMPLE_FACTORS - 20210804_09 Sample type:ASS1KO;mASS1 RAW_FILE_NAME=Mui-Ale-20210804-09-.raw SUBJECT_SAMPLE_FACTORS - 20210804_12 Sample type:ASS1KO;mASS1 RAW_FILE_NAME=Mui-Ale-20210804-12-.raw SUBJECT_SAMPLE_FACTORS - 20210804_14 Sample type:ASS1KO;mASS1 RAW_FILE_NAME=Mui-Ale-20210804-14-.raw SUBJECT_SAMPLE_FACTORS - 20210804_15 Sample type:ASS1KO;mASS1 RAW_FILE_NAME=Mui-Ale-20210804-15-.raw SUBJECT_SAMPLE_FACTORS - 20210804_16 Sample type:ASS1KO;mASS1 RAW_FILE_NAME=Mui-Ale-20210804-16-.raw SUBJECT_SAMPLE_FACTORS - 20210804_18 Sample type:ASS1KO;mASS1 RAW_FILE_NAME=Mui-Ale-20210804-18-.raw SUBJECT_SAMPLE_FACTORS - 20210804_21 Sample type:ASS1KO;mASS1 RAW_FILE_NAME=Mui-Ale-20210804-21-.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 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 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_SUMMARY HPLC 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% water/5% acetonitrile; 10 mM ammonium hydroxide; 10 mM ammonium acetate, pH CH:SOLVENT_A = 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 20210804_01 20210804_02 20210804_05 20210804_08 20210804_10 20210804_11 20210804_13 20210804_17 20210804_19 20210804_20 20210804_22 20210804_03 20210804_04 20210804_06 20210804_07 20210804_09 20210804_12 20210804_14 20210804_15 20210804_16 20210804_18 20210804_21 Factors Sample type:ASS1KO Sample type:ASS1KO Sample type:ASS1KO Sample type:ASS1KO Sample type:ASS1KO Sample type:ASS1KO Sample type:ASS1KO Sample type:ASS1KO Sample type:ASS1KO Sample type:ASS1KO Sample type:ASS1KO Sample type:ASS1KO;mASS1 Sample type:ASS1KO;mASS1 Sample type:ASS1KO;mASS1 Sample type:ASS1KO;mASS1 Sample type:ASS1KO;mASS1 Sample type:ASS1KO;mASS1 Sample type:ASS1KO;mASS1 Sample type:ASS1KO;mASS1 Sample type:ASS1KO;mASS1 Sample type:ASS1KO;mASS1 Sample type:ASS1KO;mASS1 alanine 767374489 777249191 756971607 437041064 481277536 890966100 925359610 679885821 339932299 896799432 979407383 917387748 592429379 271084133 639805345 582879208 627972132 562873140 830653814 650058827 565222900 827250875 alanine_13C3_15N 8227648 7200302 6812217 7305998 7467993 6176263 8194685 7773241 5315795 7416957 7049154 7464871 7182348 6064572 7860594 8017385 8647310 8407094 7201381 7695638 7233349 7050010 arginine 81989938 66850506 28865877 110069587 128783026 24994825 66688818 51331729 68021772 17865837 39521174 12937081 140817272 63115237 23921488 179588936 20145457 243521867 17488566 211092965 70433054 45587325 arginine_13C6_15N4 59641608 49500616 47269338 46308858 52635004 44727387 68037628 50178754 31196140 60300655 73640739 60093131 49649465 35258506 66899604 66686739 66422093 68769173 64579238 68148217 57728209 68016636 asparagine 158356777 158793383 160737583 117051994 111206422 159868501 243095881 126495741 67150072 166903355 194740389 176357268 98842701 51675369 129591794 123829444 131294086 121613953 154801571 159302848 127607888 168701764 aspartic acid 1058903669 1178271988 950346836 582432711 704721429 1293121037 1022487024 659243027 307907579 957984386 1161130884 925925343 345700722 264615119 819066331 633871136 960623537 1028534491 787542371 658194420 681341466 1241699776 aspartic acid_13C4_15N 17861097 16495433 13418834 15218716 15812075 13040890 19308219 16098502 15902237 17305253 19257902 18365892 24942456 21054078 19046173 18572755 17819072 17866812 17031000 17609876 17493472 17511446 citrulline 434160244 451211838 334277516 318132271 319599544 467560253 508496936 487574578 145650595 405229547 458786670 510055698 272648925 130792028 483748287 347203174 345957212 344214118 316692381 486577963 404622783 366660581 citrulline_13C5 + 707293716 635392200 561400710 658577354 635139981 525681331 714355095 641361781 520855974 668238478 725340255 685725240 733171833 624271005 691170847 720421123 722544907 694330868 681075723 654173445 631921481 696846282 cystine 43463873 50741996 16486629 45561476 37592582 40091139 61275753 31785533 38168985 27071865 56774411 40313555 80052763 32205240 21505037 53549142 30437918 35751753 14764784 48628578 44637089 34123583 cystine_13C6_15N2 3295062 3533474 2417374 2252577 3133842 2425230 3793357 2889080 3406214 3124499 2656054 3241417 4337961 4990347 3397875 3658613 3057951 3551214 3004190 3383612 4001408 3792137 glutamic acid 2176687381 2602829630 2241916112 1570876744 1877302903 2969555188 2311092023 1900238216 1134770086 1934313232 3404580926 2555560194 1207667750 812687498 1832403848 1643563380 2173646674 1804135261 2199887254 1852133364 1802442731 3098947794 glutamic acid_13C5_15N 21481136 18893476 17394365 19697712 21805801 16156430 21742438 19983059 14307445 19343219 21889037 20630462 22753916 18271991 21982937 21943627 21672619 22021204 18977262 20957839 22002150 21317179 glutamine 2621948598 3240579377 2649704102 2495443646 2604684733 3024182927 2973354848 2341958776 1308809255 2823937544 3301662716 2443083724 1391149137 986892220 1895706972 2182028150 2720834379 2840919830 1896605110 2507892988 2810647530 3438032883 glycine 404078671 545582047 439084645 356370457 311582979 587449859 547807782 398386689 193039385 499623264 692233478 636904161 309398350 144072866 387301578 333213196 384787813 308576063 450859098 398956997 380154458 584583591 glycine_13C2_15N 4951755 4621906 4282822 5109129 4735150 3517857 4641575 4448224 3342368 4377903 4758208 4504134 4595453 4127363 4684684 4979845 4743248 4874513 4316118 4608146 4665638 4501671 histidine 795150427 892728542 690704586 533485670 689273890 717736819 1413799542 698704108 235491567 908150407 1069929790 906951261 346730881 234102157 809950629 795182597 796217280 826212027 821614276 900246273 779320617 997620557 histidine_13C6_15N3 88855279 84437945 68197188 79286278 82817898 61832389 113743667 75392924 59862305 93652279 115834034 93857596 87850010 78419129 99324166 97599390 92579823 99368862 93393161 92873205 91496373 109775323 lysine 602004888 425602880 445339930 357231866 470100698 426084917 840052 412850 438695 501499 442300 477592203 241194498 106626497 496341372 537534277 611255 555962 460440 571007 539097 602635 lysine_13C6_15N2 27793797 23218304 21007201 22943037 23795530 19596337 27609094 22135969 16016727 25457603 28451945 24804678 23505410 18916110 26989178 27601829 28408238 28241856 25655041 27239345 24650625 28820545 methionine 413771899 391583915 374643593 253196882 282206437 385706724 474463709 315735894 101554304 395475144 383668399 536695932 220306242 103490165 367410573 339131865 289476245 355979112 370924166 399008533 336964321 305014743 methionine_13C5_15N 59416067 56235892 52340073 59554436 56336136 52818864 59481996 55814171 41295503 58148761 60589009 55013574 52883700 42752276 58984530 56791623 62922646 62461439 58091309 56636952 56352400 59799973 ornithine 284996900 215492272 233801808 139951519 210340143 213341058 298108833 220503584 36694217 253046295 241646050 295445533 68620238 39125461 278904612 239305356 286689878 267194508 291780575 299798747 258985345 251735199 ornithine_13C5_15N2 + 654045320 545755778 499420131 530748567 566852491 450414640 694433451 524604030 384296765 623157101 721435337 612915734 570293831 455669207 670690493 674830145 707817900 678312778 629286530 672188955 600133649 715194696 phenylalanine 1232146773 960813057 1094820422 674260648 826973994 1092059637 1583334414 970359537 83728903 1048603264 794966540 1271657342 277943888 98884788 1167724778 992669273 1050871750 1063489504 1129425714 1024264168 919094433 1094490465 phenylalanine_13C9_15N 185544810 164778605 162863228 180312278 173702010 158168438 188297968 175424162 83074581 179194311 200352048 173468451 153796483 95124919 185954797 187359072 187937135 179244243 183607631 183781235 169137162 196403741 proline 4546214025 4519391424 4560345230 3131174808 3502472117 5184309394 5663267162 4609871690 1648534167 5061257389 4677202646 5490471969 2512646019 1353253978 4776479613 3977495777 4317555651 3813571567 5566703491 3943321879 3699300400 4992335968 proline_13C5_15N 315818516 291750559 289086695 370422938 343694753 275511551 321253439 328787734 324106729 319646856 319736265 287553961 404657518 411357362 316278818 356438652 351420830 333541928 312909583 340319422 343250282 325268654 serine 200225615 183098666 177275503 125214117 133944679 178259713 228285361 149721894 79123707 188869587 197208662 208028842 126784365 68924544 149423994 141783088 147806708 152914010 189835843 171100738 143870907 180508770 serine_13C3_15N 11884538 10545961 10343501 12309097 11568996 8567074 11051171 10559155 11238247 11307075 10289005 10794525 13173507 13598181 11583995 13549323 12218837 12618688 10481214 12120818 11296200 11201566 threonine_13C4_15N 37165661 33477407 32179578 37125456 35262465 29525905 39791799 33506586 24633622 37288935 38778802 34298330 32098320 28635242 36185658 37116328 41078269 39103088 36233869 35212000 36233419 35989949 tryptophan 715976592 575436918 511261984 619769759 776170861 621143570 763229746 546545705 70703379 476484602 784390948 662328055 129744408 71094897 684668864 594312900 572193791 707793077 653794657 819598005 599817883 588925618 tyrosine 461343728 500509879 433553775 352543424 373947205 414148939 809045527 446709083 85227032 519408375 452912181 909856799 349263951 149016965 465081081 506537328 359433870 353395972 441354010 575870256 461741970 364255814 tyrosine_13C9_15N 59670706 55907020 47757158 56875157 54744984 49472130 61480976 56762827 39709388 60035322 60653343 55701022 56728041 47778460 58314096 58814577 62046027 60929777 58124823 58926913 58008620 59469305 valine 354549419 337471952 320975746 256842735 237679608 421608805 390605846 342743063 55824599 336357871 267430779 434141961 106203545 64316270 365023244 328333335 324068098 345837661 325025675 329996862 306626650 326738056 valine_13C5_15N 22674823 21377475 19750447 23699495 22269225 20031837 24524905 21959467 15291636 22035869 23095095 22137047 21662186 18457071 22770916 23015323 22715768 21680073 22270685 22464901 22283556 21209891 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name alanine alanine_13C3_15N arginine arginine_13C6_15N4 asparagine aspartic acid aspartic acid_13C4_15N citrulline citrulline_13C5 + cystine cystine_13C6_15N2 glutamic acid glutamic acid_13C5_15N glutamine 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