#METABOLOMICS WORKBENCH juliehaines_20230221_120146 DATATRACK_ID:3757 STUDY_ID:ST002489 ANALYSIS_ID:AN004063 PROJECT_ID:PR001607 VERSION 1 CREATED_ON February 21, 2023, 12:18 pm #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 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. T-cells from spleen were obtained ST:STUDY_SUMMARY through sorting, cultured in the presence of 13C6 glucose for 6h, and glucose ST:STUDY_SUMMARY fluxes profiled via 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 - AG-23-TC-1 factor:unx RAW_FILE_NAME=AG-23-TC-1_r6- SUBJECT_SAMPLE_FACTORS - AG-23-TC-2 factor:unx RAW_FILE_NAME=AG-23-TC-2_r4- SUBJECT_SAMPLE_FACTORS - AG-23-TC-3 factor:unx RAW_FILE_NAME=AG-23-TC-3_r3- SUBJECT_SAMPLE_FACTORS - AG-23-TC-4 factor:RT RAW_FILE_NAME=AG-23-TC-4_r8- SUBJECT_SAMPLE_FACTORS - AG-23-TC-5 factor:RT RAW_FILE_NAME=AG-23-TC-5_r12- SUBJECT_SAMPLE_FACTORS - AG-23-TC-6 factor:RT RAW_FILE_NAME=AG-23-TC-6_r11- SUBJECT_SAMPLE_FACTORS - AG-23-TC-7 factor:IL2v RAW_FILE_NAME=AG-23-TC-7_r14- SUBJECT_SAMPLE_FACTORS - AG-23-TC-8 factor:IL2v RAW_FILE_NAME=AG-23-TC-8_r1- SUBJECT_SAMPLE_FACTORS - AG-23-TC-9 factor:IL2v RAW_FILE_NAME=AG-23-TC-9_r5- SUBJECT_SAMPLE_FACTORS - AG-23-TC-10 factor:RT+IL2v RAW_FILE_NAME=AG-23-TC-10_r2- SUBJECT_SAMPLE_FACTORS - AG-23-TC-11 factor:RT+IL2v RAW_FILE_NAME=AG-23-TC-11_r9- SUBJECT_SAMPLE_FACTORS - AG-23-TC-12 factor:RT+IL2v RAW_FILE_NAME=AG-23-TC-12_r15- SUBJECT_SAMPLE_FACTORS - AG-23-TC-13 factor:aCD25 RAW_FILE_NAME=AG-23-TC-13_r7- SUBJECT_SAMPLE_FACTORS - AG-23-TC-14 factor:aCD25 RAW_FILE_NAME=AG-23-TC-14_r13- SUBJECT_SAMPLE_FACTORS - AG-23-TC-15 factor:aCD25 RAW_FILE_NAME=AG-23-TC-15_r10- #COLLECTION CO:COLLECTION_SUMMARY C57BL/6 mice were orthotopically implanted with PK5L1940 cells. 8 Gy RT was CO:COLLECTION_SUMMARY administered 7 days post-implantation. PD1-IL2v and aCD25 dosed once per week CO:COLLECTION_SUMMARY beginning day 7 post-implantation. Spleens and lymph nodes of C57BL/6 mice were CO:COLLECTION_SUMMARY harvested 17 days post implantation and homogenized to a single cell suspension CO:COLLECTION_SUMMARY by mashing through a 70 uM cells strainer. For spleen processing, and the CO:COLLECTION_SUMMARY erythrocytes were lysed with ACK (ammonium-chloride-potassium) lysis buffer for CO:COLLECTION_SUMMARY 5 min at RT. CD8 T cells were sorted with a CD8-negative selection Stemcell CO:COLLECTION_SUMMARY isolation kit following manufacturer instructions. CO:SAMPLE_TYPE T-cells #TREATMENT TR:TREATMENT_SUMMARY CD8+ T cells were cultured for 6h in the presence of 25 mM U-13C-glucose in TR:TREATMENT_SUMMARY glucose-free RPMI. Cells were washed in PBS at 1200RPM, 4degC for 10 minutes and TR:TREATMENT_SUMMARY pellets were flash frozen. #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 4e6 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 Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE 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 AG-23-TC-1 AG-23-TC-2 AG-23-TC-3 AG-23-TC-4 AG-23-TC-5 AG-23-TC-6 AG-23-TC-7 AG-23-TC-8 AG-23-TC-9 AG-23-TC-10 AG-23-TC-11 AG-23-TC-12 AG-23-TC-13 AG-23-TC-14 AG-23-TC-15 Factors factor:unx factor:unx factor:unx 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 Glucose_M+0 1989593 572482.3 1483604 2388277 1042860 1753610 1873150 2008127 2005074 932287.3 1873801 2422508 1578506 1490044 1514221 Glucose_M+6 2.20E+07 9776315 2.08E+07 2.34E+07 1.37E+07 1.60E+07 2.02E+07 2.00E+07 2.21E+07 1.54E+07 2.14E+07 1.96E+07 2.14E+07 1.94E+07 1.90E+07 Hexose-6-phosphate_M+0 11817.44 3017.994 10235.61 6972.78 6931.506 13089.78 13132.64 6147.406 10689.89 6541.762 10195.31 9501.78 10402.26 6550.748 4972.391 Hexose-6-phosphate_M+6 18339.7 6564.603 14239.47 16004.5 42505.54 18478.29 15194.23 10376.95 30355.51 18428.38 19328 21523.23 10927.41 19819.15 14362.22 D-Fructose 1-6-bisphosphate_M+0 29005.78 42160.6 50077.98 20851.47 22811.56 32691.87 29122.48 51798.53 34634.48 88245.56 30589.52 16783.07 51781.52 26417.01 50672.77 D-Fructose 1-6-bisphosphate_M+6 74868.02 34503.06 36220.23 88910.82 82829.97 104284.4 132515 25655.94 68316.24 30866.15 89825.59 163956.4 72033.23 109534.6 93840.87 Pyruvate_M+0 253504.9 70396.09 195864.6 186249.1 246236.8 227265.2 213839.1 217614.8 268285.4 181844.2 291092.4 317661.8 172339.3 195143.9 230005 Pyruvate_M+3 867057.2 295017.5 600439.6 891858.3 431945.9 652068.3 809411.3 651391 858204.9 479635.5 862021.7 961003.3 735680.2 600770.6 619295.1 lactate_M+0 3.33E+07 8701968 2.45E+07 3.40E+07 1.40E+07 3.55E+07 3.34E+07 2.86E+07 3.31E+07 2.01E+07 4.16E+07 4.62E+07 2.24E+07 2.50E+07 3.18E+07 lactate_M+3 1.13E+07 4876626 1.02E+07 1.24E+07 6772302 8400465 1.10E+07 9021960 1.15E+07 7576858 1.09E+07 1.07E+07 1.09E+07 1.05E+07 1.02E+07 Citrate_M+0 4322024 2387251 3486168 4486138 3314521 4447914 4164268 3412389 5608790 3121328 5032368 5654732 3286321 3496223 5185366 Citrate_M+2 41334.71346 24409.25464 16999.41219 43119.70287 14021.38652 39520.82662 52772.53859 32910.70352 49584.45076 27368.45523 45545.53499 51793.50741 33955.06572 35092.06261 55454.3457 Citrate_M+3 0 0 1736.637 0 4036.231 3042.417 2081.638 996.9238 2734.607 5402.378 3194.026 6793.652 898.3865 2560.271 2532.431 2-Oxoglutarate_M+0 41745.66 23811.24 45577.12 60906.93 35287.09 53644.08 37536.49 45845.44 56597.59 38905.49 71041.93 70264.05 43323.93 36463.47 46480.68 Itaconate_M+0 110021.6 47055.34 172893.6 130283.4 87914 129447.2 109752.9 82803.31 126574.8 82220.13 143106.8 160825.4 95895.06 83796.79 107848.5 Succinate_M+0 2380294 1363054 1975404 2895479 1210312 2727923 2841507 2559157 2953298 1959703 2943533 3293913 1934168 1964487 2362002 Succinate_M+4 126634.7 17383.96 65931.29 109571.1 29743.43 100422.4 105798.9 88562.69 108490.6 50398.93 117784.9 148287.1 73222.62 49376 68930.62 Fumarate_M+0 203550.6 119487 151006.3 229831.5 201390.3 241334.1 276938 175679.6 252646.6 150138 249266.8 311073.9 155308 194863.6 222716.1 Fumarate_M+4 30672.02 3073.481 5383.873 22718.25 0 8983.027 17406.44 9186.634 24636.99 0 13084.33 38824.85 5829.133 11739.7 8511.388 (S)-Malate_M+0 1018787 543024.6 867056 1145905 760168.3 1316830 1390578 786693.5 1499621 872919.7 1229825 1536751 874115.6 956706.7 1142126 L-Aspartate_M+0 2733399 1971289 1885170 2554098 4074295 2398746 2568417 2165505 3866648 2393941 2630006 2702801 2855902 2946113 2215134 Aspartate_M+2 142820.0395 20621.3745 62513.61088 151966.6663 62544.75488 121027.2277 138685.7447 99554.18232 104850.5695 67698.58022 134267.0084 135028.3773 105983.2737 72619.01523 102851.0006 L-Glutamate_M+0 3382096 2508285 2756622 3282401 3789698 3581658 3625937 2842410 5075388 3328219 4512590 4160467 3663769 3643569 3134800 Glutamate_M+2 97678.2596 33411.08788 122284.2185 129771.837 71094.47355 83665.48455 121300.9406 62996.54975 99262.8513 55479.53753 109612.6153 115150.6873 108223.6988 105168.6038 96315.43 Glutamate_M+3 2742224 1182770 2625640 2897717 1609743 1906638 2560316 2315206 2695735 1783191 2538450 2427194 2709748 2440292 2287754 2-Hydroxyglutarate_M+0 136666.5 68668.09 106922.4 157885.2 92983.73 145794.4 190271.4 113970.6 153323 98548.81 173241.7 172023.5 99908.12 123407.7 154629.5 2-Hydroxyglutarate_M+3 48831.36 10229.25 25287.14 40946.81 21633.34 20089.14 30299.75 41190.26 36235.1 22929.09 44587.86 85753.06 21175.85 30727.04 33232.93 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG parent medRt Glucose_M+0 C00031 179.0548 0.6841307 Glucose_M+6 C00031 185.075 0.677248 Hexose-6-phosphate_M+0 C02965 259.0218 0.6097583 Hexose-6-phosphate_M+6 C02965 265.042 0.6053211 D-Fructose 1-6-bisphosphate_M+0 C00354 338.993 0.6730736 D-Fructose 1-6-bisphosphate_M+6 C00354 345.0137 0.758325 Pyruvate_M+0 C00022 87.00704 0.6787521 Pyruvate_M+3 C00022 90.01715 0.685328 lactate_M+0 C01432 89.02271 0.6687 lactate_M+3 C01432 92.0328 0.6844542 Citrate_M+0 C00158 191.0184 0.6155801 Citrate_M+2 C00158 193.0227 0.6190151 Citrate_M+3 C00158 194.0263 0.60818 2-Oxoglutarate_M+0 C00026 145.0127 0.607075 Itaconate_M+0 C00490 129.0177 0.612636 Succinate_M+0 C00042 117.0177 0.608344 Succinate_M+4 C00042 121.0313 0.6825637 Fumarate_M+0 C00122 115.0021 0.6087667 Fumarate_M+4 C00122 119.0154 0.6766375 (S)-Malate_M+0 C00149 133.0126 0.601675 L-Aspartate_M+0 C00049 132.0286 0.6369441 Aspartate_M+2 C00049 134.0346 0.673596 L-Glutamate_M+0 C00025 146.0444 0.6356361 Glutamate_M+2 C00025 148.0503 0.671004 Glutamate_M+3 C00025 149.0537 0.6787166 2-Hydroxyglutarate_M+0 C02630 147.0284 0.6045336 2-Hydroxyglutarate_M+3 C02630 150.0382 0.7093033 METABOLITES_END #END