#METABOLOMICS WORKBENCH juliehaines_20230221_084725 DATATRACK_ID:3753 STUDY_ID:ST002487 ANALYSIS_ID:AN004060 PROJECT_ID:PR001607 VERSION 1 CREATED_ON February 21, 2023, 9:03 am #PROJECT PR:PROJECT_TITLE Simultaneous targeting of PD-1 and IL2Rβγ with radiation therapy to inhibit PR:PROJECT_TITLE pancreatic cancer growth and metastasis PR:PROJECT_SUMMARY In pancreatic ductal adenocarcinoma (PDAC) patients, we show that response to PR:PROJECT_SUMMARY radiation therapy (RT) is characterized by increased IL2Rβ and IL2Rγ and PR:PROJECT_SUMMARY decreased ILR2α expression. The bispecific aPD1-IL2v is a PD-1-targeted IL-2 PR:PROJECT_SUMMARY variant (IL2v) immunocytokine with engineered IL-2 cis-targeted to PD-1 and PR:PROJECT_SUMMARY abolished IL2Rα binding, which enhances tumor-antigen specific T cell PR:PROJECT_SUMMARY activation while reducing regulatory T cell (Treg) suppression. Using aPD1-IL2v PR:PROJECT_SUMMARY in orthotopic PDAC KPC-driven tumor models, we show marked improvement in local PR:PROJECT_SUMMARY and metastatic survival along with profound increase in tumor-infiltrating PR:PROJECT_SUMMARY polyfunctional CD8+ T cell subsets with a transcriptionally and metabolically PR:PROJECT_SUMMARY active phenotype, and preferential activation of antigen-specific CD8+ T cells. PR:PROJECT_SUMMARY In combination with single dose RT, aPD1-IL2v treatment results in a robust, PR:PROJECT_SUMMARY durable expansion of polyfunctional CD8+ T cells, T cell stemness, PR:PROJECT_SUMMARY tumor-specific memory immune response, natural killer (NK) cell activation, and PR:PROJECT_SUMMARY decreased Tregs. These data show that aPD1-IL2v leads to profound local and PR:PROJECT_SUMMARY distant response in PDAC. PR:INSTITUTE University of Colorado Denver PR:LABORATORY Lab of Angelo D'Alessandro in collaboration with lab of Sana Karam 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 Simultaneous targeting of PD-1 and IL2Rβγ with radiation therapy to inhibit ST:STUDY_TITLE pancreatic cancer growth and metastasis - mouse spleen and lymph node T-cell ST:STUDY_TITLE metabolomics ST:STUDY_SUMMARY Mice were orthotopically implanted with PK5L1940 cells then radiation therapy (8 ST:STUDY_SUMMARY Gy) administered 7 days post-implantation. PD1-IL2v and aCD25 dosed once per ST:STUDY_SUMMARY week beginning day 7 post-implantation. Spleen and associated lymph nodes were ST:STUDY_SUMMARY collected at time of sacrifice and sorted for CD8+ T-cells. The obtained T-cells ST:STUDY_SUMMARY were profiled by mass spectrometry-based metabolomics. ST:INSTITUTE University of Colorado Denver 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 #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 - MSL-1 factor:Untrx RAW_FILE_NAME=AG-23-MSL-1-r4 SUBJECT_SAMPLE_FACTORS - MSL-2 factor:Untrx RAW_FILE_NAME=AG-23-MSL-2-r5 SUBJECT_SAMPLE_FACTORS - MSL-3 factor:Untrx RAW_FILE_NAME=AG-23-MSL-3-r6 SUBJECT_SAMPLE_FACTORS - MSL-4 factor:RT RAW_FILE_NAME=AG-23-MSL-4-r13 SUBJECT_SAMPLE_FACTORS - MSL-5 factor:RT RAW_FILE_NAME=AG-23-MSL-5-r9 SUBJECT_SAMPLE_FACTORS - MSL-6 factor:RT RAW_FILE_NAME=AG-23-MSL-6-r7 SUBJECT_SAMPLE_FACTORS - MSL-7 factor:IL2v RAW_FILE_NAME=AG-23-MSL-7-r12 SUBJECT_SAMPLE_FACTORS - MSL-8 factor:IL2v RAW_FILE_NAME=AG-23-MSL-8-r15 SUBJECT_SAMPLE_FACTORS - MSL-9 factor:IL2v RAW_FILE_NAME=AG-23-MSL-9-r14 SUBJECT_SAMPLE_FACTORS - MSL-10 factor:RT+IL2v RAW_FILE_NAME=AG-23-MSL-10-r1 SUBJECT_SAMPLE_FACTORS - MSL-11 factor:RT+IL2v RAW_FILE_NAME=AG-23-MSL-11-r8 SUBJECT_SAMPLE_FACTORS - MSL-12 factor:RT+IL2v RAW_FILE_NAME=AG-23-MSL-12-r11 SUBJECT_SAMPLE_FACTORS - MSL-13 factor:aCD25 RAW_FILE_NAME=AG-23-MSL-13-r10 SUBJECT_SAMPLE_FACTORS - MSL-14 factor:aCD25 RAW_FILE_NAME=AG-23-MSL-14-r3 SUBJECT_SAMPLE_FACTORS - MSL-15 factor:aCD25 RAW_FILE_NAME=AG-23-MSL-15-r2 #COLLECTION CO:COLLECTION_SUMMARY Spleens of C57BL/6 mice were homogenized to a single cell suspension by mashing CO:COLLECTION_SUMMARY the spleen through a 70 uM cell strainer, and the erythrocytes were lysed with CO:COLLECTION_SUMMARY ACK (ammonium-chloride-potassium) lysis buffer for 3 min at RT. CD8 T cells were CO:COLLECTION_SUMMARY sorted with a CD8-negative selection Stemcell isolation kit following CO:COLLECTION_SUMMARY manufacturer instructions. Cells were washed with PBS at 1200RPM, 4degC for 10 CO:COLLECTION_SUMMARY minutes and pellets were flash frozen. CO:SAMPLE_TYPE Spleen; T-cells #TREATMENT TR:TREATMENT_SUMMARY Mice were orthotopically implanted with PK5L1940 cells. RT administered 7 days TR:TREATMENT_SUMMARY post-implantation. PD1-IL2v and aCD25 dosed once per week beginning day 7 TR:TREATMENT_SUMMARY post-implantation. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites from frozen T-cell pellets were extracted with cold 5:3:2 SP:SAMPLEPREP_SUMMARY MeOH:acetonitrile:water at a concentration of 4 million cells/mL. Samples were SP:SAMPLEPREP_SUMMARY vortexed 30 min at 4 degrees C then supernatants clarified by centrifugation (10 SP:SAMPLEPREP_SUMMARY min, 10,000 g, 4 degrees C) and transferred to autosampler vials. SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Negative C18 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 2.1 x 150 mm, 1.7 um CH:SOLVENT_A 95% water 5% acetonitrile 1 mM ammonium acetate CH:SOLVENT_B 95% acetonitrile 5% water 1 mM ammonium acetate CH:FLOW_GRADIENT 0-0.5 min 0% B, 0.5-1.1 min 0-100% B, 1.1-2.75 min hold at 100% B, 2.75-3 min CH:FLOW_GRADIENT 100-0% B, 3-5 min hold at 0% B CH:FLOW_RATE 450 uL/min CH:COLUMN_TEMPERATURE 45 CH:SAMPLE_INJECTION 10 uL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 480 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS We use a Thermo Orbitrap Exploris 120 (not available in the drop down box). MS:MS_COMMENTS Resolution 120,000, scan range 65-975 m/z, maximum injection time 100 ms, MS:MS_COMMENTS microscans 1, automatic gain control (AGC) detection duration 20 msec, source MS:MS_COMMENTS voltage 2.0 kV, capillary temperature 320 C, vaporizer temp 200 C, and sheath MS:MS_COMMENTS gas 50, auxiliary gas 10, and sweep gas 1 (all nitrogen). Data converted to MS:MS_COMMENTS mzXML using RawConverter. Metabolites were annotated and integrated using Maven MS:MS_COMMENTS in conjunction with the KEGG database. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples MSL-1 MSL-2 MSL-3 MSL-4 MSL-5 MSL-6 MSL-7 MSL-8 MSL-9 MSL-10 MSL-11 MSL-12 MSL-13 MSL-14 MSL-15 Factors factor:Untrx factor:Untrx factor:Untrx factor:RT factor:RT factor:RT factor:IL2v factor:IL2v factor:IL2v factor:RT+IL2v factor:RT+IL2v factor:RT+IL2v factor:aCD25 factor:aCD25 factor:aCD25 L-cystine 43963 1650 4752 3545 6025 3670 12223 4893 3792 4950 10809 15111 7941 2203 0 ADP 268482 30510 93663 14227 79423 12500 230306 92940 105986 123161 50742 83275 145680 64482 263940 GDP 35639 6734 36295 360 26530 12109 22063 24500 16242 30445 9147 20965 40322 25215 37779 CDP 107886 49467 66513 88669 90670 146967 5490 27948 17564 23735 18211 12614 10113 26497 48640 Cytidine 158019 9365 6463 8801 19624 5067 114368 59088 79671 61754 28903 79336 25621 50802 8065 UDP 994415 200388 330875 368497 808117 411942 627753 724402 743880 549227 535269 591840 632855 537286 368416 Xanthine 854929 2233 3804 3970 83823 18958 294676 40276 152937 51494 29625 235279 3276 42807 15782 5-Hydroxyisourate 3550 3689 6030 6810 7391 11411 5249 15853 8350 6091 14096 6199 7325 7867 6219 Urate 460284 148057 14623 171208 81371 313290 185330 89638 138233 203689 34998 384746 16744 80784 0 Adenylosuccinic acid 78239 0 312 0 7909 0 47606 2734 11053 10171 2608 17572 0 2478 1274 UDP-glucose 171044 0 20854 0 70996 7839 46399 33558 47870 41442 19143 20154 20514 24934 34668 Phosphate 22142932 16221733 37146324 3111541 43834328 25736296 14884719 33527174 27821616 36012456 39138028 35079608 40429132 34556316 46475004 Diphosphate 3667087 2550076 7267859 66317 8337971 5144761 2694636 6821682 4359119 7878210 7766020 5942359 6332894 7616070 11751784 D-Glucose 423444 3386060 2333913 7882769 1783717 2982387 142759 378147 180006 538100 279463 107138 192408 180171 422578 D-Glucose 6-phosphate 1382984 18496 93627 2643 303839 39850 253166 306171 228304 251532 141755 197013 192631 247020 239756 D-Fructose 1-6-bisphosphate 1732566 2373 81177 400 165035 15680 289306 20398 102296 86691 31209 263823 15678 47718 32423 D-Glyceraldehyde 3-phosphate/Glycerone phosphate 521648 1042 65176 461 293329 28174 169459 31419 143359 200704 99253 278310 9236 94816 9090 1-3-Bisphosphoglycerate 1170615 3928 252538 2201 664318 12833 154847 15398 270714 88973 47941 198234 20541 61160 206121 2/3-Phospho-D-glycerate 123872 1120 81885 2185 290003 12164 29772 102983 102710 222862 104448 62357 23951 135145 58093 Lactate 1187587 643679 516314 1434072 699642 808649 626420 200982 230343 515729 180679 565893 216768 266606 195455 Mannitol 104343 79164 140639 186710 165222 56986 31674 25624 23295 30585 52353 13994 64459 41092 94737 Citrate 5169674 140011 704377 158148 2492591 456076 1441253 441889 2236255 1187263 1487048 1974994 394735 2528073 1012813 Succinate 43891 87244 102929 188573 115919 49054 30868 34480 28222 54458 49256 28789 42687 79875 39469 Fumarate 216076 9425 11784 9401 38558 13803 116732 18750 39907 47923 29651 46433 14178 31946 16545 Malate 1519210 52980 285720 82742 791836 176143 820943 341185 571117 616442 555192 1371193 243279 563212 416602 2-Hydroxyglutarate/Citramalate*** (%CV=31.25) 55351 7973 13491 3580 51255 10951 18012 7546 28285 19374 26368 18626 9671 16142 11668 alpha-D-Ribose 1-phosphate 447637 3992 50025 1246 437049 79087 188967 140696 319761 312659 173006 272401 44127 330837 95685 5-Phospho-alpha-D-ribose 1-diphosphate 93406 1561 10482 0 44796 2801 55655 46207 132169 97751 84420 15374 4298 67861 34253 Dehydroascorbate 263050 12095 26703 10733 108426 39754 68821 31611 103567 52077 63534 63367 15317 106263 25836 UDP-N-acetyl-D-glucosamine 452523 3026 34350 0 165200 14082 272366 109719 184396 175684 117416 159171 32455 99584 68432 Glycolate 109868 137976 235394 138131 208824 142567 134789 207014 121138 100005 155292 197305 173847 125993 161497 N-Acetyl-L-citrulline 42022 34469 28490 71228 20316 25999 58906 28378 55933 37477 22991 32792 34980 32969 7466 L-Cysteate 13409 12226 5532 9531 6456 8655 10071 6226 10712 7839 9364 14256 6741 5910 18764 Glycerol 3-phosphate 46830 2456 15972 1428 51215 23981 20126 43401 57300 106154 34001 38191 2655 57549 19904 Ethanolamine phosphate 2559881 35574 252671 13404 941046 64999 1582107 698503 1046622 1189261 710176 1308192 314610 601378 370270 Octanoic acid (caprylate) 77843 131529 95118 114379 78266 85022 127971 133138 87083 78605 81493 115745 73490 106209 67534 Nonanoic acid (pelargonate) 922756 1075030 654945 1285880 1098707 930976 977252 1363611 1251925 1052921 1349510 937572 762380 976898 795306 Decanoic acid (caprate) 323268 483444 467808 668549 619130 475015 610371 683007 714245 291091 433444 676287 631825 488500 523059 Dodecanoic acid (lauric acid) 301332 436007 293821 364273 365145 391439 387215 467962 401798 288683 284638 333689 313480 348170 238048 Tetradecanoic acid (myristic acid) 704732 1320614 1112186 1320130 1665798 1628543 975353 1368791 1057957 997143 775041 1616647 1098642 704700 926216 Hexadecanoic acid (palmitic acid) 15198033 29100870 20738458 27146342 37355700 24802094 13409557 33964160 13580519 15771707 14503209 29664570 11176117 15528353 14597779 Hexadecenoic acid (Palmitoleic acid) 537994 510838 362585 376660 1063939 585439 454392 502132 448454 372645 313579 679807 308250 364913 523875 Octadecenoic acid (Oleic acid) 6926603 2361502 2047029 2379165 10650884 6421995 3116907 5633439 3122430 4365384 2404150 4803618 1324841 2002717 2614855 Linoleic acid ((9Z,12Z)-Octadecadienoic acid) 5268920 616418 1362268 1391346 9321381 7764527 2722649 3583602 4300749 3037445 2616824 5210467 770324 1495486 2181361 a-Linolenic acid (Octadecatrienoic acid) 244431 33667 50737 59122 315451 352006 111096 111680 96167 101475 50465 96665 24450 61578 87814 Arachidonic acid (Eicosatetraenoic acid) 11127387 714745 1936727 1266878 5430224 3336222 4888341 4674585 3110253 6195247 2332813 8718033 1699648 1742870 3483754 Docosahexaenoic acid 3440235 216377 969481 408371 2634631 1832777 2249247 1888406 2134761 1510435 1263660 2439894 542649 961734 1436957 Eicosapentaenoic acid 181174 23087 36536 37366 106035 199346 82512 67285 35909 39964 26269 56373 11611 19960 45186 (8Z-11Z-14Z)-Icosatrienoic acid 902185 41722 136933 59659 712681 296866 500543 369977 313612 312544 257551 649019 116159 141875 290132 (7Z-10Z-13Z-16Z-19Z)-Docosa-7-10-13-16-19-pentaenoic acid 642325 55219 129934 99537 456159 433277 354249 338696 325429 316011 221894 471053 93531 169707 303623 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID parent medRt L-cystine C00491 239.0150 0.6830 ADP C00008 426.020447 0.57 GDP C00035 442.015167 0.538 CDP C00112 402.004822 2.145 Cytidine C00475 242.077789 0.656 UDP C00015 402.997437 0.777 Xanthine C00385 151.025558 0.696 5-Hydroxyisourate C11821 183.013046 0.772 Urate C00366 167.020386 0.645 Adenylosuccinic acid C03794 462.064911 0.516 UDP-glucose C00029 565.045288 0.541 Phosphate C00009 96.969231 0.596 Diphosphate C00013 176.935074 0.587 D-Glucose C00031 179.055389 0.666 D-Glucose 6-phosphate C02965 259.021118 0.57 D-Fructose 1-6-bisphosphate C00354 338.987335 0.53 D-Glyceraldehyde 3-phosphate/Glycerone phosphate C00118 168.989685 0.576 1-3-Bisphosphoglycerate C00236 264.950928 0.528 2/3-Phospho-D-glycerate C00631 184.984894 0.535 Lactate C01432 89.024048 0.644 Mannitol C00392 181.070953 0.684 Citrate C00158 191.018982 0.573 Succinate C00042 117.018814 0.575 Fumarate C00122 115.003181 0.546 Malate C00149 133.013672 0.567 2-Hydroxyglutarate/Citramalate*** (%CV=31.25) C02630 147.029297 0.593 alpha-D-Ribose 1-phosphate C00620 229.010925 0.578 5-Phospho-alpha-D-ribose 1-diphosphate C00119 388.942932 0.516 Dehydroascorbate C05422 173.008423 0.556 UDP-N-acetyl-D-glucosamine C00043 606.071838 0.54 Glycolate C00160 75.008469 0.724 N-Acetyl-L-citrulline C15532 216.095154 0.704 L-Cysteate C00506 167.996643 0.818 Glycerol 3-phosphate C00093 171.005554 0.579 Ethanolamine phosphate C00346 140.011261 0.617 Octanoic acid (caprylate) C06423 143.107178 2.087 Nonanoic acid (pelargonate) C01601 157.122787 2.184 Decanoic acid (caprate) C01571 171.138336 2.273 Dodecanoic acid (lauric acid) C02679 199.169525 2.481 Tetradecanoic acid (myristic acid) C06424 227.200729 2.775 Hexadecanoic acid (palmitic acid) C00249 255.231918 3.207 Hexadecenoic acid (Palmitoleic acid) C08362 253.216293 2.84 Octadecenoic acid (Oleic acid) C00712 281.247528 3.249 Linoleic acid ((9Z,12Z)-Octadecadienoic acid) C01595 279.231903 2.885 a-Linolenic acid (Octadecatrienoic acid) C06427 277.216217 2.645 Arachidonic acid (Eicosatetraenoic acid) C00219 303.231842 2.769 Docosahexaenoic acid C06429 327.231781 2.66 Eicosapentaenoic acid C06428 301.216156 2.574 (8Z-11Z-14Z)-Icosatrienoic acid C03242 305.247437 2.975 (7Z-10Z-13Z-16Z-19Z)-Docosa-7-10-13-16-19-pentaenoic acid C16513 329.247803 2.802 METABOLITES_END #END