#METABOLOMICS WORKBENCH Lu_Group_20220325_055333 DATATRACK_ID:3139 STUDY_ID:ST002121 ANALYSIS_ID:AN003473 PROJECT_ID:PR001346 VERSION 1 CREATED_ON March 25, 2022, 11:17 pm #PROJECT PR:PROJECT_TITLE Functional metabolomics analysis of pancreatic cancer tissues with gemcitabine PR:PROJECT_TITLE treatment PR:PROJECT_TYPE Targeted MS quantitative analysis PR:PROJECT_SUMMARY Characteristics of pancreatic cancer tissues metabolomics with gemcitabine PR:PROJECT_SUMMARY treatment in cell derived xenograft models PR:INSTITUTE Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University PR:DEPARTMENT Shanghai Center for Systems Biomedicine PR:LABORATORY Lu Group PR:LAST_NAME Lu PR:FIRST_NAME Haitao PR:ADDRESS 800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China PR:EMAIL haitao_lu@sjtu.edu.cn PR:PHONE 15221478139 #STUDY ST:STUDY_TITLE Functional metabolic molecule were identified as novel therapeutic targets to ST:STUDY_TITLE facilitate gemcitabine treatment against pancreatic cancer ST:STUDY_SUMMARY With the development of frontier technologies in system biology, traditional ST:STUDY_SUMMARY omics-drove phenotypic studies are insufficient to decipher the diseases. ST:STUDY_SUMMARY Therefore, for a thorough understanding of the molecular mechanisms of diseases ST:STUDY_SUMMARY to investigate novel drug targets, traditional phenotypic studies must be broken ST:STUDY_SUMMARY through to the functional exploration of molecules. Meanwhile, the intuitive ST:STUDY_SUMMARY role of small molecule compounds (metabolites) in pathogenesis, precision ST:STUDY_SUMMARY diagnosis and therapy are gradually recognized compared to macromolecules such ST:STUDY_SUMMARY as DNA, RNA and proteins. Therefore, we pioneeringly proposed Spatial Temporal ST:STUDY_SUMMARY Operative Real Metabolomics (STORM) strategy that established a relationship ST:STUDY_SUMMARY between metabolic phenotypes and functions to accurately character abnormal ST:STUDY_SUMMARY metabolisms and further identify operative functional molecules as novel ST:STUDY_SUMMARY therapeutic targets. Here, given the difficulty of pancreatic cancer (PC) ST:STUDY_SUMMARY treatment and the high resistance of clinical drugs, we were committed to ST:STUDY_SUMMARY explore new targets and drugs of pancreatic cancer from a small molecular ST:STUDY_SUMMARY functional perspective via STORM strategy. Fortunately, based on targeted ST:STUDY_SUMMARY metabolomics, we found that gemcitabine, one of the most effective clinical ST:STUDY_SUMMARY anti-PC drugs, served as a dual modulator that promote the accumulation of ST:STUDY_SUMMARY functional metabolic molecules in purine metabolism to activate down-streamed ST:STUDY_SUMMARY kinases. And the quantitative consequences of related enzymes annotated the ST:STUDY_SUMMARY unique molecular mechanisms of purine metabolism regulations by gemcitabine. ST:STUDY_SUMMARY Collectively, we broadened the cognitions of gemcitabine in tumor inhibition, ST:STUDY_SUMMARY providing potential strategies for treating PC with small molecules ST:STUDY_SUMMARY modification. Even more importantly, with the integration of multiple frontier ST:STUDY_SUMMARY technologies, the STORM strategy has proven to be well adapted to the phenotypic ST:STUDY_SUMMARY era of functional molecules devoted to innovate molecule mechanism annotation ST:STUDY_SUMMARY and therapeutic discovery. ST:INSTITUTE Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University ST:LAST_NAME Lu ST:FIRST_NAME Haitao ST:ADDRESS 800 Dongchuan RD. Minhang District, Shanghai, ST:EMAIL haitao_lu@sjtu.edu.cn ST:PHONE 15221478139 #SUBJECT SU:SUBJECT_TYPE Human SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - M-1 Treatment:2 times per week with 3 weeks consecutive respectively intraperitoneal injection with 0.2ml mormal saline RAW_FILE_NAME=M-1 SUBJECT_SAMPLE_FACTORS - M-3 Treatment:2 times per week with 3 weeks consecutive respectively intraperitoneal injection with 0.2ml mormal saline RAW_FILE_NAME=M-3 SUBJECT_SAMPLE_FACTORS - M-4 Treatment:2 times per week with 3 weeks consecutive respectively intraperitoneal injection with 0.2ml mormal saline RAW_FILE_NAME=M-4 SUBJECT_SAMPLE_FACTORS - M-5 Treatment:2 times per week with 3 weeks consecutive respectively intraperitoneal injection with 0.2ml mormal saline RAW_FILE_NAME=M-5 SUBJECT_SAMPLE_FACTORS - M-6 Treatment:2 times per week with 3 weeks consecutive respectively intraperitoneal injection with 0.2ml mormal saline RAW_FILE_NAME=M-6 SUBJECT_SAMPLE_FACTORS - M-7 Treatment:2 times per week with 3 weeks consecutive respectively intraperitoneal injection with 0.2ml mormal saline RAW_FILE_NAME=M-7 SUBJECT_SAMPLE_FACTORS - G-1 Treatment:2 times per week with 3 consecutive respectively intraperitoneal injection with 25mg/kg gemcitabine in 0.2ml RAW_FILE_NAME=G-1 SUBJECT_SAMPLE_FACTORS - G-2 Treatment:2 times per week with 3 consecutive respectively intraperitoneal injection with 25mg/kg gemcitabine in 0.2ml RAW_FILE_NAME=G-2 SUBJECT_SAMPLE_FACTORS - G-4 Treatment:2 times per week with 3 consecutive respectively intraperitoneal injection with 25mg/kg gemcitabine in 0.2ml RAW_FILE_NAME=G-4 SUBJECT_SAMPLE_FACTORS - G-5 Treatment:2 times per week with 3 consecutive respectively intraperitoneal injection with 25mg/kg gemcitabine in 0.2ml RAW_FILE_NAME=G-5 SUBJECT_SAMPLE_FACTORS - G-6 Treatment:2 times per week with 3 consecutive respectively intraperitoneal injection with 25mg/kg gemcitabine in 0.2ml RAW_FILE_NAME=G-6 SUBJECT_SAMPLE_FACTORS - G-7 Treatment:2 times per week with 3 consecutive respectively intraperitoneal injection with 25mg/kg gemcitabine in 0.2ml RAW_FILE_NAME=G-7 #COLLECTION CO:COLLECTION_SUMMARY Tumor tissues were stripped from euthanized mice for further assays CO:SAMPLE_TYPE Subcutaneous tumor tissues of pancreatic cancer #TREATMENT TR:TREATMENT_SUMMARY All mice are fed at a constant temperature of 20-26℃ with a free choice of TR:TREATMENT_SUMMARY standard food and water with 12-hour light/dark cycle.After 2 weeks acclimatized TR:TREATMENT_SUMMARY breeding, Aspc-1 cells were injected into right armpit of nude mice when they 8 TR:TREATMENT_SUMMARY weeks old. When solid tumors can be stably observed, treating mice with 25mg/kg TR:TREATMENT_SUMMARY gemcitabine twice a week by intraperitoneal injection of Gem-treated group, TR:TREATMENT_SUMMARY meanwhile, the control group received the same volume of saline. Measuring tumor TR:TREATMENT_SUMMARY volume every two days. 21 days later, tumors were stripped and weighed, parts of TR:TREATMENT_SUMMARY which were embedded in paraffin for immunohistochemistry, and the residues were TR:TREATMENT_SUMMARY kept in -80 degree refrigerator. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 100-130 mg tissue samples tumor tissues have to be broken by oscillation with SP:SAMPLEPREP_SUMMARY 60HZ and 2 minutes in iced 80% methanol solution (mixed with internal standard SP:SAMPLEPREP_SUMMARY 0.001mg/ml 4-chloro-DL-phenylalanine). centrifuging at 20,000 g for 10 min at 4 SP:SAMPLEPREP_SUMMARY °C, and mixing the supernatants with 800 μL ice-cold acetonitrile. Then the SP:SAMPLEPREP_SUMMARY supernatant obtained by centrifugation was filtered through a 0.22 µm organic SP:SAMPLEPREP_SUMMARY phase membrane, blown dry in nitrogen, reconstituted in 100 µL of water and SP:SAMPLEPREP_SUMMARY transferred to vials for metabolomics assay. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters Acquity HSS T3 column (100 mm×2.1, 1.8 μm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6495 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Agilent MassHunter Workstation Data Acquisition Agilent MassHunter #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS 13 MS_METABOLITE_DATA_START Samples M-1 M-3 M-4 M-5 M-6 M-7 G-1 G-2 G-4 G-5 G-6 G-7 Factors Treatment:2 times per week with 3 weeks consecutive respectively intraperitoneal injection with 0.2ml mormal saline Treatment:2 times per week with 3 weeks consecutive respectively intraperitoneal injection with 0.2ml mormal saline Treatment:2 times per week with 3 weeks consecutive respectively intraperitoneal injection with 0.2ml mormal saline Treatment:2 times per week with 3 weeks consecutive respectively intraperitoneal injection with 0.2ml mormal saline Treatment:2 times per week with 3 weeks consecutive respectively intraperitoneal injection with 0.2ml mormal saline Treatment:2 times per week with 3 weeks consecutive respectively intraperitoneal injection with 0.2ml mormal saline Treatment:2 times per week with 3 consecutive respectively intraperitoneal injection with 25mg/kg gemcitabine in 0.2ml Treatment:2 times per week with 3 consecutive respectively intraperitoneal injection with 25mg/kg gemcitabine in 0.2ml Treatment:2 times per week with 3 consecutive respectively intraperitoneal injection with 25mg/kg gemcitabine in 0.2ml Treatment:2 times per week with 3 consecutive respectively intraperitoneal injection with 25mg/kg gemcitabine in 0.2ml Treatment:2 times per week with 3 consecutive respectively intraperitoneal injection with 25mg/kg gemcitabine in 0.2ml Treatment:2 times per week with 3 consecutive respectively intraperitoneal injection with 25mg/kg gemcitabine in 0.2ml Citrate 45172579.98 41062444.66 61394756.16 45003714.71 35228968.89 38090727.16 50774017.92 47699342.89 59684517.03 64543934.42 49528116.85 42501022.41 Taurocholic acid 19645.48376 21730.80117 12886.646 137518.0565 7149.574663 6667.871281 31354.1961 9342.97996 16558.19322 7254.299391 229875.2907 204648.5543 Cinnamoylglycine 30303.20173 27339.15502 62028.42036 38425.03348 16548.90833 26212.46056 23448.09635 39140.95513 26137.42471 42430.85438 15394.30802 57240.26246 Isocitric acid 32665618.55 38601395.89 47171450.63 34620235.97 33590105.33 38254428.26 38649213.77 30106026.86 47333753.32 46826498.89 44592182.89 33906813.05 Taurochenodeoxycholic acid 5265.52459 4895.72457 11960.95575 19372.04797 1831.421914 8263.859124 8128.820797 5741.647108 8938.733105 7067.714455 26865.27291 33887.97652 Citraconic acid 347822.5884 235341.5354 292062.8603 601573.0478 240792.7042 406274.0196 252203.1673 340120.3446 411193.1294 334125.1724 372738.385 303879.6155 Methylmalonic acid 10262175.02 12304771.87 10566728.61 9527978.565 15479423.84 13072131.55 10343217.98 11027647.65 16523961.64 12976911 16094005.42 11128933.26 3-Hydroxybenzoic acid 27636.71874 27186.96957 41239.55769 47380.66736 17564.42741 32382.61177 35769.81874 29705.29206 37677.80839 37293.44251 24847.47792 29987.27688 Uridine 3647511.293 5151176.633 2747837.169 3137497.862 3606412.076 3550243.332 3274481.58 3029662.833 5396667.477 3026953.215 3616212.682 3887594.113 L-Lactate 13430319.25 13291584.69 12080827.05 10593406.31 13897443.59 12285680.49 12416600.85 11639375.42 13513366.36 13773044.12 13373707.42 12831753.11 Oxalic acid 1929631.662 2075042.938 1855046.851 1663249.743 1962335.447 1887535.116 1800374.462 1774855.57 2153950.58 1902151.709 2023540.496 1979743.143 Malonic acid 2990925.077 2196115.487 819300.9076 2989902.914 2616162.087 3009578.096 554604.1869 1451534.782 4165497.884 2370677.724 4642656.133 1586875.397 4-Hydroxybenzoic acid 32302.09226 34064.60709 46397.07714 49653.05696 19114.42894 33753.58971 37793.73164 34588.27019 42174.69539 48219.9026 26488.98887 34958.54169 Azelaic acid 928120.875 599379.5366 897255.7432 1026170.948 486079.9367 723083.1296 755238.8513 542680.1834 500950.0973 866609.7098 822168.9801 283769.555 Maleic acid 843369.8122 725568.8775 730540.7571 476044.8251 752089.7314 894694.1559 857859.8184 1162977.578 923850.7099 1247035.129 847698.8558 1578256.603 N-Acetyl-L-tryptophan 12637.37194 10846.5588 26151.9833 8261.903403 13328.69448 8764.747042 12343.48711 13822.34458 37318.86708 33347.71032 21339.28871 18697.59019 Sebacic acid 115903.8727 75391.9655 97200.24903 143956.0855 53048.29695 82262.02195 103775.048 86282.41491 84105.29679 115567.7102 93229.97832 118330.0225 N-Acetylglutamic acid 157552.7928 103400.2267 269395.5845 62630.43931 116565.9989 104370.3221 139923.9752 147399.3324 174411.7635 341631.9781 274490.0694 132865.0132 Allantoin 26173.45168 24442.00645 41401.2416 50587.23924 21493.25741 30947.11775 22660.25814 19177.23681 28816.07689 22901.85985 17760.74241 22996.13844 dUMP 36602.89418 41296.2468 43677.29564 32824.35072 19377.01624 34322.47741 47211.32536 70281.91161 46626.90529 74559.40476 50399.00819 73438.81532 Indoxyl sulfate 216711.5429 386174.652 912610.4466 342709.1821 199493.803 416573.9966 1437799.549 1040026.035 889238.3142 970177.399 594867.4892 954022.7126 2'-Deoxyinosine 1706577.201 1610218.805 1002763.952 910480.1672 2028078.448 1776796.79 1470159.873 1489556.31 1898027.912 1255986.28 1760496.379 1285836.913 Cis-Aconitic acid 406025.2483 366621.2472 390788.0905 699300.7983 381030.848 354044.8371 491166.5654 335073.4401 623720.2539 409939.4765 409816.016 404354.693 D-Glucose 6-phosphate 876994.6114 920505.7641 587570.1181 515015.9433 1155988.626 1364810.188 1073200.778 1174495.731 1321224.188 862294.9038 1045095.14 1359042.976 Flavin adenine dinucleotide 2511513.233 2331275.424 2202276.345 1748250.814 2052786.907 2066503.748 2445232.545 2145927.788 2676720.165 2380210.519 2363701.728 2364256.717 Fumarate 843369.8122 748994.081 730540.7571 476044.8251 420448.1679 894694.1559 857859.8184 1162977.578 923850.7099 1229747.049 830777.4665 1578256.603 Malic acid 22108980.08 19328863.01 20726060.72 18614506.36 18904180.7 20960258.57 21685047.71 22488028.68 23320485.41 23345238 21663062.83 23191530.74 D-Fructose 2445575.712 3342083.218 2596066.752 1863970.718 2895747.837 2480991.012 2898635.726 2306808.227 2842074.383 2917088.777 3161895.67 2189390.102 Succinic acid 10293883.96 11039360.88 10661151.27 9108569.868 14007734.3 12099899.93 10322404.97 11043405.73 15637607.51 11794805.2 16152471.17 9709185.817 Gluconic acid 67730.10465 83469.80968 113102.9895 84095.15218 59598.30233 101128.632 86274.71298 73545.11188 131775.9376 175161.0839 94043.46465 109513.6684 Glucose 3453200.26 4056576.207 3407530.455 3101665.953 4123298.188 3841600.466 4072175.34 3374661.022 4166156.066 3757564.823 4611976.391 3531337.118 2-hydroxy-2-phenylpropanoic acid 633396.2675 476599.989 804634.4719 1105647.527 470868.6053 634929.7779 709441.2235 814818.6217 655515.834 983600.8236 564194.111 570318.7426 p-Hydroxyphenylpropionic acid 288898.4919 253652.8129 516426.0674 431988.739 120742.2804 411942.7118 228315.8946 232263.049 429908.8878 277792.666 330104.1834 364564.8206 3-Hydroxyphenylpropionic acid 633396.2675 476599.989 804634.4719 1105647.527 470868.6053 634929.7779 709441.2235 814818.6217 655515.834 983600.8236 564194.111 531550.4489 Docosahexaenoic acid 186139.8519 167942.3595 173444.2973 185820.9929 151750.9329 176675.4474 155481.8168 159615.7004 198064.4012 148035.7328 157308.8714 181711.4826 Phenyllactic acid 151466.7912 126050.0094 262114.0609 207357.7433 122072.967 96149.93788 184208.979 101854.256 86986.27215 95715.52155 105778.6531 142711.665 Citramalic acid 59105.66189 59254.12699 70884.84967 75344.01181 46744.3431 47075.89934 44151.20247 41096.20078 66919.44919 80960.68998 45835.07173 69753.51536 Glutaric acid 63142.17921 75629.6069 86429.68119 68563.98679 55387.93047 65062.23398 76791.60217 76355.69319 102432.5639 96176.66158 77918.89656 90312.66878 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z RT Citrate 191.11 1.63 Taurocholic acid 514.28 10.94 Cinnamoylglycine 204.06 9.53 Isocitric acid 191.02 1.64 Taurochenodeoxycholic acid 498.29 12.39 Citraconic acid 129.09 1.73 Methylmalonic acid 117.02 2.11 3-Hydroxybenzoic acid 137.02 6.46 Uridine 243.1 1.88 L-Lactate 89.02 1.51 Oxalic acid 89 1.51 Malonic acid 103 2.29 4-Hydroxybenzoic acid 137.02 6.46 Azelaic acid 187.1 9.8 Maleic acid 115 1.88 N-Acetyl-L-tryptophan 245.09 9.06 Sebacic acid 201.1 11.11 N-Acetylglutamic acid 188.05 1.83 Allantoin 157.03 1.01 dUMP 307.03 1.47 Indoxyl sulfate 212 6.59 2'-Deoxyinosine 251.1 3.88 Cis-Aconitic acid 173 1.73 D-Glucose 6-phosphate 259.02 1.02 Flavin adenine dinucleotide 784.15 6.23 Fumarate 115 1.84 Malic acid 133.01 1.25 D-Fructose 179.05 1.51 Succinic acid 117.02 2.09 Gluconic acid 195.05 1 Glucose 179.05 1.51 2-hydroxy-2-phenylpropanoic acid 165.05 6.91 p-Hydroxyphenylpropionic acid 165.05 6.91 3-Hydroxyphenylpropionic acid 165.05 7.08 Docosahexaenoic acid 327.23 14.98 Phenyllactic acid 165.05 8.52 Citramalic acid 147.03 2.15 Glutaric acid 131.03 3.74 METABOLITES_END #END