#METABOLOMICS WORKBENCH juliehaines_20250807_085445 DATATRACK_ID:6260 STUDY_ID:ST004109 ANALYSIS_ID:AN006812 PROJECT_ID:PR002584 VERSION 1 CREATED_ON August 8, 2025, 3:55 pm #PROJECT PR:PROJECT_TITLE Serum metabolomics from WT and KRAS knockout mice PR:PROJECT_SUMMARY We generated a clinically relevant animal model of cancer cachexia using an PR:PROJECT_SUMMARY inducible, lung epithelial cell specific KrasG12D/+ (G12D) mice. Serum PR:PROJECT_SUMMARY metabolomics was used alongside analyses of tumor, cellular, mutational, and PR:PROJECT_SUMMARY transcriptomic characteristics to assess the degree to which this mouse model PR:PROJECT_SUMMARY phenocopies human lung cancer cachexia. PR:INSTITUTE University of Colorado Anschutz Medical Campus PR:LABORATORY Lab of Angelo D'Alessandro in collaboration with lab of Michael Toth PR:LAST_NAME Haines PR:FIRST_NAME Julie PR:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA PR:EMAIL julie.haines@cuanschutz.edu PR:PHONE 3037243339 #STUDY ST:STUDY_TITLE Early adipose tissue wasting in a preclinical model of human lung cancer ST:STUDY_TITLE cachexia - Serum metabolomics from WT and KRAS knockout mice ST:STUDY_SUMMARY Cancer cachexia (CC), a syndrome of skeletal muscle and adipose wasting, reduces ST:STUDY_SUMMARY responsiveness to therapies and increases mortality. There are no approved ST:STUDY_SUMMARY treatments for CC, which may relate to discordance between pre-clinical models ST:STUDY_SUMMARY and human CC. To address the need for clinically relevant models of lung CC, we ST:STUDY_SUMMARY generated inducible, lung epithelial cell specific KrasG12D/+ (G12D) mice. G12D ST:STUDY_SUMMARY mice develop CC over a protracted time course and phenocopy tissue and tumor, ST:STUDY_SUMMARY cellular, mutational, transcriptomic, and metabolic characteristics of human ST:STUDY_SUMMARY lung CC. G12D mice demonstrate early loss of adipose, a phenotype that was ST:STUDY_SUMMARY apparent across numerous models of CC and translates to patients with lung ST:STUDY_SUMMARY cancer. Tumor-released factors promote adipocyte lipolysis, a driver of adipose ST:STUDY_SUMMARY wasting in CC, and adipose wasting was inversely related to tumor burden. Thus, ST:STUDY_SUMMARY G12D mice model key features of human lung CC and highlight a role for early ST:STUDY_SUMMARY tumor metabolic reprogramming of adipose tissue in CC. ST:INSTITUTE University of Colorado Anschutz Medical Campus ST:LAST_NAME Haines ST:FIRST_NAME Julie ST:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA ST:EMAIL julie.haines@cuanschutz.edu ST:PHONE 3037243339 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:GENDER Not applicable #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 - 9 Sample source:serum | Genotype:WT RAW_FILE_NAME(Raw file name)=DS1-68-001 SUBJECT_SAMPLE_FACTORS - 11 Sample source:serum | Genotype:WT RAW_FILE_NAME(Raw file name)=DS1-68-002 SUBJECT_SAMPLE_FACTORS - 12 Sample source:serum | Genotype:WT RAW_FILE_NAME(Raw file name)=DS1-68-003 SUBJECT_SAMPLE_FACTORS - 13 Sample source:serum | Genotype:WT RAW_FILE_NAME(Raw file name)=DS1-68-004 SUBJECT_SAMPLE_FACTORS - 14 Sample source:serum | Genotype:WT RAW_FILE_NAME(Raw file name)=DS1-68-005 SUBJECT_SAMPLE_FACTORS - 15 Sample source:serum | Genotype:WT RAW_FILE_NAME(Raw file name)=DS1-68-006 SUBJECT_SAMPLE_FACTORS - 16 Sample source:serum | Genotype:KRAS RAW_FILE_NAME(Raw file name)=DS1-68-007 SUBJECT_SAMPLE_FACTORS - 17 Sample source:serum | Genotype:KRAS RAW_FILE_NAME(Raw file name)=DS1-68-008 SUBJECT_SAMPLE_FACTORS - 18 Sample source:serum | Genotype:KRAS RAW_FILE_NAME(Raw file name)=DS1-68-009 SUBJECT_SAMPLE_FACTORS - 19 Sample source:serum | Genotype:KRAS RAW_FILE_NAME(Raw file name)=DS1-68-010 SUBJECT_SAMPLE_FACTORS - 20 Sample source:serum | Genotype:KRAS RAW_FILE_NAME(Raw file name)=DS1-68-011 #COLLECTION CO:COLLECTION_SUMMARY All animal experiments were approved by the University of Vermont Institutional CO:COLLECTION_SUMMARY Animal Care and Use Committee, in compliance with the National Institutes of CO:COLLECTION_SUMMARY Health Guidelines for Use and Care of Laboratory Animals. Mice were housed with CO:COLLECTION_SUMMARY sex-matched litter mates and given ad libitum access to standard chow and water, CO:COLLECTION_SUMMARY except for a subset of mice that were housed singly to monitor food intake as CO:COLLECTION_SUMMARY specified. Animals were allocated by genotype. G12D mice CO:COLLECTION_SUMMARY (Scgb1a1-CreERTM-KrasLSL-G12D/+) were generated by crossing Scgb1a1-CreERTM mice CO:COLLECTION_SUMMARY with KrasLSL-G12D/+ mice and were maintained on a mixed background. At 4-5 CO:COLLECTION_SUMMARY months of age, G12D or wild-type Scgb1a1-CreERTM (WT) littermates were CO:COLLECTION_SUMMARY administered five consecutive daily intraperitoneal (IP) injections of 20 mg/ml CO:COLLECTION_SUMMARY tamoxifen (Sigma-Aldrich, St. Louis, MO, USA) dissolved in corn oil at a dose of CO:COLLECTION_SUMMARY 1 mg tamoxifen/10 g body weight. Body weight was monitored 2-3 times weekly and CO:COLLECTION_SUMMARY food intake was measured in a sub-group of individually housed mice for 6 weeks. CO:COLLECTION_SUMMARY At 6- or 12-weeks post-tamoxifen injections, mice were euthanized by cervical CO:COLLECTION_SUMMARY dislocation under pentobarbital anesthesia. Blood was collected by cardiac CO:COLLECTION_SUMMARY puncture in serum separator tubes and centrifuged to isolate serum, aliquoted, CO:COLLECTION_SUMMARY and stored at -80°C. CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY No treatment, baseline (steady state) characterization only. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Serum specimens were thawed on ice then a 20 uL aliquot was mixed with 480 uL of SP:SAMPLEPREP_SUMMARY ice cold 5:3:2 methanol:acetonitrile:water (v/v/v). Extraction was performed SP:SAMPLEPREP_SUMMARY vigorous vortexing at 4°C followed by centrifugation for 10 min at 18,000 g at SP:SAMPLEPREP_SUMMARY 4°C. Extracts were transferred to autosampler vials (on ice) and analyzed SP:SAMPLEPREP_SUMMARY immediately. SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACT_STORAGE 4℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Positive C18 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1 mm,1.7 µm) CH:SOLVENT_A 100% Water; 0.1% formic acid CH:SOLVENT_B 100% Acetonitrile; 0.1% formic acid CH:FLOW_GRADIENT 0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min CH:FLOW_GRADIENT 95-5% B, 3-5 min hold at 5% B CH:FLOW_RATE 450 µL/min CH:COLUMN_TEMPERATURE 45°C CH:SAMPLE_INJECTION 6 µL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, MS:MS_COMMENTS microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, MS:MS_COMMENTS capillary temperature 320°C, and sheath gas 45, auxiliary gas 15, and sweep gas MS:MS_COMMENTS 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were MS:MS_COMMENTS annotated and integrated using Maven in conjunction with the KEGG database. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples 9 11 12 13 14 15 16 17 18 19 20 Factors Sample source:serum | Genotype:WT Sample source:serum | Genotype:WT Sample source:serum | Genotype:WT Sample source:serum | Genotype:WT Sample source:serum | Genotype:WT Sample source:serum | Genotype:WT Sample source:serum | Genotype:KRAS Sample source:serum | Genotype:KRAS Sample source:serum | Genotype:KRAS Sample source:serum | Genotype:KRAS Sample source:serum | Genotype:KRAS L-alanine 12378889 10214370 10771785 17083576 13035261 6434284 10316001 13111701 10061215 11949218 11081308 L-arginine 59524748 50403880 43099156 41966976 44744740 38874044 42709236 44321028 24231690 36773060 42364952 L-asparagine 1041051 769186 978258 2322707 859895 726279 906091 1046097 840767 883077 554519 L-aspartate 539608 537012 367503 954949 502698 422079 632533 544879 556452 537483 487122 L-histidine 11068810 9052075 8702756 13939928 16604308 10376550 11348607 4784323 11054504 12614333 13642274 L-leucine 98651872 89292064 86599648 124972032 96477824 90417704 107338104 80581312 101082944 94395488 82597976 L-methionine 21732342 16246054 14382379 23561924 16336417 12509802 19357202 13308456 17201740 18954674 12786967 L-phenylalanine 57837780 49668684 55583604 52372724 53863700 49003780 69890432 57681672 58478076 56967388 47746192 L-proline 48455772 40025336 34884384 63529972 49616912 37492616 50577720 44670224 42228676 49992680 38928648 L-serine 2240610 1528415 1378679 2126832 1628543 1535903 1765095 1558095 1727603 1746379 1423142 L-threonine 8060341 5901732 6045961 7683385 5631421 4618086 7122368 6644062 6523981 5949879 4864557 L-tryptophan 38967248 31398934 31473624 28658328 23364680 22399500 34264016 40527820 24295558 26250368 29180512 L-tyrosine 17560666 15200544 14899343 24007634 16717872 14186635 19629720 14536153 15597606 16798510 12271725 L-valine 164103872 141650528 118924152 172833216 181854432 187515728 143671568 120762000 187243312 174529648 159264784 Guanine 11130 10580 21483 882284 986578 1455156 12182 7737 350168 27422 51504 Cytosine 2623081 2133406 2380102 3050964 3008777 2981366 2261942 2433572 2327122 2410581 1749255 Thymine 279132 314773 367366 1296399 295671 359638 348792 366268 191813 306289 206016 Uracil 2952876 1876474 5308921 1771075 5281233 4103717 4568014 3000664 3724894 3796507 2028348 Hypoxanthine 20026 70617 823411 827716 5223022 10499367 102852 47234 126302 71283 66969 Xanthine 63931 46767 2256545 245185 2342237 4383452 105725 47416 21527 41298 23318 Allantoate 3884775 3237415 2827007 4361103 3860983 2165829 6025607 2388706 5599447 7210743 3583602 (S)(+)-Allantoin 692876 685754 635812 3050772 621471 833453 458236 705380 371470 346166 358784 5-Hydroxyisourate 371474 281110 600298 1113145 1090186 561173 757203 489715 627722 675068 820722 5-6-Dihydrothymine 813097 504870 614940 2063748 642161 545577 677399 621385 391655 662318 384143 Nicotinamide 7583256 5271769 7055778 5746041 7871283 5320867 5477728 7151763 6957581 11030380 8312012 Phosphate 26268256 23873696 22769608 38917280 22508396 27371188 32718528 39915164 38679044 43039568 47153868 2-Oxoglutaramate 1739 3726 3692 156822 4495 0 9980 4973 2311 2310 0 Glutathione 1676980 1331522 1708812 1395323 1677754 1222637 1818256 1886815 1173060 1356887 1192462 5-Oxoproline 36245264 31024450 32779178 34046368 42189060 28612114 37102664 34574160 29054926 35856120 36756932 Cys-Gly 110615 116548 146028 101663 77146 51924 195155 175417 92337 132901 82136 gamma-L-Glutamyl-D-alanine 436711 424491 458709 745374 668677 398949 249084 416441 165550 304345 284591 (5-L-Glutamyl)-L-glutamine 1540435 1263529 1087928 1926122 1687958 1035062 1179230 1307009 993306 962697 821245 Ornithine 2854367 1777638 2092182 3714797 2574864 1498923 3337566 2381467 4438792 3219497 2103373 L-Citrulline 4473284 2991108 3633845 4092564 2933097 1835490 3459014 4119202 2640194 3150544 4451471 Spermidine 114370 84631 74349 324502 152051 248671 388249 327797 629114 654180 342019 Spermine 6172 9059 2534 38812 16946 43437 38239 31112 138652 82155 30471 D-Glucosamine 4437775 4014091 6413310 2487746 5395989 5806464 2875009 3334681 3795388 2035200 2208677 Homocarnosine 143590 166508 66689 999637 321119 400524 166284 141709 338841 183624 1118440 Carnosine 12168 26656 37255 444989 111725 162223 11637 24509 150191 52586 279309 5-Guanidino-2-oxopentanoate 51635 119652 82165 1556867 100608 32270 96663 120372 89795 73787 63184 Phosphocreatine 237902 89003 21952 662995 26524 508368 161743 212745 500956 360300 161150 Creatine 128028776 93140592 91855024 253874272 152595168 205388240 104791768 127880472 142826992 100421424 152814768 Creatinine 6992760 6600379 7227091 54401076 11224970 8869431 6683702 7372033 5795094 5658337 6464417 4-Acetamidobutanoate 71589 71486 103652 1110114 187384 152286 104885 111221 139864 95888 35284 N-Acetylornithine 1451006 1266194 886133 1478058 1144338 895334 1438793 1169131 976022 1052659 881663 Guanidinoacetate 227246 264150 202234 1870287 252092 175850 332627 273394 366006 387749 298354 trans-4-Hydroxy-L-proline 1520022 955614 1012100 815737 1304710 597286 938932 722222 1455535 912596 867482 5-6-Dihydrouracil 984051 672718 1448580 4598646 1024564 794596 718901 849920 610765 738147 507713 Taurine 16831840 16069070 13692034 37436036 16580360 19110754 14872808 16260759 16348854 15823649 16589172 L-Methionine S-oxide 404544 381023 390922 626914 226619 210883 427331 351767 173240 328775 176989 5-Hydroxyindoleacetate 210784 332813 303836 1804300 158576 199581 263487 485270 296188 299046 380810 Indole-3-acetaldehyde 34166900 38607520 38735868 35641356 40922600 44502644 32184016 30688828 33697680 27303266 27565322 Indoxyl 3887297 5219981 4806618 15290081 2259404 2340934 9080562 8929855 2269222 6443183 5039777 6-Hydroxykynurenic acid 175701 214496 367905 13906575 238157 192450 230066 162106 128171 114029 137175 Anthranilate 12210862 14649299 13622017 43824196 8762729 7532688 12287266 11443303 9492620 11192396 9848560 Dopamine 41895 151200 82610 365546 65549 43644 96719 120929 61295 64003 72576 Serotonin 2575754 2603460 2620854 2857314 3409179 3735684 2184388 2032573 2529635 1881572 1842284 Ectoine 2113497 2743400 2597538 13020219 1775813 1396926 2296645 2223627 1611745 2090065 1209048 Inositol 1-2-3-5-6-pentakisphosphate 59847 40981 22618 48156 81116 72844 53538 43059 32491 91571 47017 Ethanolamine phosphate 15446 41994 8751 55291 29258 25976 34207 5576 79020 113689 24187 N-Methylethanolamine phosphate 965141 913392 1606141 2111781 3725775 2969913 909594 714329 1674565 2263487 1607245 Sphingosine 1-phosphate 643234 551658 547539 568820 625355 414219 491243 698423 778910 408210 627198 Acetylcholine 5161506 4915886 4257118 7861701 4860274 5389137 5945782 6324598 6947496 6047447 7496132 Choline 91290280 99147912 86682720 103341160 113923904 113929904 104347992 100388664 118413248 119219976 111166872 L-Carnitine 91301792 77138248 67776424 169160320 117304416 123601024 79567528 92769408 113390872 82717000 122588576 acetyl-carnitine (acyl-C2) 119426528 78758624 66367620 118879144 120835656 116900656 64477424 90227016 101683056 79026104 99644152 propionyl-carnitine (acyl-C3) 1906116 2368420 1667274 3061029 2666976 2395496 2860366 3654896 2578125 2893886 3360541 butanoyl-l-carnitine (acyl-C4) 1468098 1075397 1891268 1716380 2104538 1752079 1743923 2362902 3158111 2981914 3036055 acyl-C4-OH 716120 702748 1078982 1729020 1456217 1485059 554578 728521 786040 769151 678546 acyl-C4-DC 32174 15842 63430 218298 13641 1699 19996 93223 112918 52389 110665 acyl-C5-OH 207496 192007 240634 285041 220297 231421 191886 161275 197250 185571 213270 hexanoyl-L-carnitine (acyl-C6) 872021 583429 949470 833854 690606 595606 620271 703352 800460 629691 506617 L-octanoylcarnitine (acyl-C8) 189074 131492 164826 124870 142166 137423 107213 146982 153397 134389 90433 octenoyl-l-carnitine (acyl-C8:1) 168473 123550 98326 164683 136096 134969 83742 129347 100846 69213 66872 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z r.t. L-alanine C00041 90.055321 0.677 L-arginine C00062 175.118408 0.672 L-asparagine C00152 133.060455 0.717 L-aspartate C00049 134.044403 0.673 L-histidine C00135 156.076294 0.655 L-leucine C00123 132.101578 0.804 L-methionine C00073 150.057877 0.712 L-phenylalanine C00079 166.085785 1.46 L-proline C00148 116.070564 0.695 L-serine C00065 106.050056 0.671 L-threonine C00188 120.06543 0.716 L-tryptophan C00078 205.096558 1.76 L-tyrosine C00082 182.080673 0.72 L-valine C00183 118.086159 0.685 Guanine C00242 152.056259 0.751 Cytosine C00380 112.050583 0.709 Thymine C00178 127.04995 0.695 Uracil C00106 113.034607 0.739 Hypoxanthine C00262 137.045349 0.708 Xanthine C00385 153.040237 0.705 Allantoate C00499 177.060425 0.653 (S)(+)-Allantoin C02350 159.05069 0.697 5-Hydroxyisourate C11821 185.031448 0.683 5-6-Dihydrothymine C00906 129.065567 0.666 Nicotinamide C00153 123.055122 0.808 Phosphate C00009 98.984383 0.775 2-Oxoglutaramate C00940 146.044281 0.79 Glutathione C00051 308.089935 0.689 5-Oxoproline C01879 130.04953 0.673 Cys-Gly C01419 179.047943 0.721 gamma-L-Glutamyl-D-alanine C03738 219.09671 0.698 (5-L-Glutamyl)-L-glutamine C05283 276.11795 0.692 Ornithine C01602 133.096817 0.676 L-Citrulline C00327 176.102402 0.677 Spermidine C00315 146.164688 0.624 Spermine C00750 203.222427 0.638 D-Glucosamine C00329 180.085999 0.739 Homocarnosine C00884 241.128571 0.659 Carnosine C00386 227.113037 0.658 5-Guanidino-2-oxopentanoate C03771 174.086761 0.758 Phosphocreatine C02305 212.042358 0.721 Creatine C00300 132.07634 0.691 Creatinine C00791 114.066162 0.712 4-Acetamidobutanoate C02946 146.080688 0.745 N-Acetylornithine C00437 175.107315 0.706 Guanidinoacetate C00581 118.060989 0.678 trans-4-Hydroxy-L-proline C01157 132.065201 0.673 5-6-Dihydrouracil C00429 115.050179 0.727 Taurine C00245 126.021729 0.691 L-Methionine S-oxide C02989 166.052658 0.679 5-Hydroxyindoleacetate C05635 192.06485 1.808 Indole-3-acetaldehyde C00637 160.07518 0.928 Indoxyl C05658 134.059708 1.818 6-Hydroxykynurenic acid C08480 206.044235 1.771 Anthranilate C00108 138.054535 0.695 Dopamine C03758 154.085815 1.782 Serotonin C00780 177.101715 0.917 Ectoine C06231 143.08107 0.734 Inositol 1-2-3-5-6-pentakisphosphate C04579 580.901917 0.664 Ethanolamine phosphate C00346 142.025894 0.706 N-Methylethanolamine phosphate C01210 156.041595 0.677 Sphingosine 1-phosphate C06124 380.254425 3.869 Acetylcholine C01996 146.117081 0.707 Choline C00114 104.107101 0.684 L-Carnitine C00318 162.111908 0.682 acetyl-carnitine (acyl-C2) C02571 204.12236 0.708 propionyl-carnitine (acyl-C3) C03017 218.137924 0.713 butanoyl-l-carnitine (acyl-C4) C02862 232.153519 0.715 acyl-C4-OH HMDB13127 248.14827 0.706 acyl-C4-DC HMDB13133 262.127411 0.713 acyl-C5-OH ac107 262.164154 1.779 hexanoyl-L-carnitine (acyl-C6) HMDB00756 260.184753 1.849 L-octanoylcarnitine (acyl-C8) HMDB00791 288.21579 1.932 octenoyl-l-carnitine (acyl-C8:1) ac111 286.200287 1.904 METABOLITES_END #END