#METABOLOMICS WORKBENCH Lu_Group_20220325_234038 DATATRACK_ID:3142 STUDY_ID:ST002122 ANALYSIS_ID:AN003474 PROJECT_ID:PR001346 VERSION 1 CREATED_ON March 30, 2022, 9:10 pm #PROJECT PR:PROJECT_TITLE Functional metabolomics analysis of pancreatic cancer cells Aspc-1 with PR:PROJECT_TITLE gemcitabine treatment PR:PROJECT_TYPE Targeted MS quantitative analysis PR:PROJECT_SUMMARY Characteristics of pancreatic cancer cells Aspc-1 metabolomics with gemcitabine PR:PROJECT_SUMMARY and inhibitor IBMX treatment 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 molecules were identified as novel therapeutic targets to ST:STUDY_TITLE facilitate gemcitabine treatment against pancreatic cancer (Cells metabolomics) 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:DEPARTMENT Shanghai Center for Systems Biomedicine ST:LABORATORY Lu Group ST:LAST_NAME Lu ST:FIRST_NAME Haitao ST:ADDRESS 800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China ST:EMAIL haitao_lu@sjtu.edu.cn ST:PHONE 15221478139 #SUBJECT SU:SUBJECT_TYPE Cultured cells 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 - ASPC-C-1 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with 25μl DMSO for 72h RAW_FILE_NAME=ASPC-C-1 SUBJECT_SAMPLE_FACTORS - ASPC-C-2 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with 25μl DMSO for 72h RAW_FILE_NAME=ASPC-C-2 SUBJECT_SAMPLE_FACTORS - ASPC-C-3 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with 25μl DMSO for 72h RAW_FILE_NAME=ASPC-C-3 SUBJECT_SAMPLE_FACTORS - ASPC-C-4 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with 25μl DMSO for 72h RAW_FILE_NAME=ASPC-C-4 SUBJECT_SAMPLE_FACTORS - ASPC-C-5 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with 25μl DMSO for 72h RAW_FILE_NAME=ASPC-C-5 SUBJECT_SAMPLE_FACTORS - ASPC-G-1-r002 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM for 72h RAW_FILE_NAME=ASPC-G-1-r002 SUBJECT_SAMPLE_FACTORS - ASPC-G-2 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM for 72h RAW_FILE_NAME=ASPC-G-2 SUBJECT_SAMPLE_FACTORS - ASPC-G-3 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM for 72h RAW_FILE_NAME=ASPC-G-3 SUBJECT_SAMPLE_FACTORS - ASPC-G-4 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM for 72h RAW_FILE_NAME=ASPC-G-4 SUBJECT_SAMPLE_FACTORS - ASPC-G-5 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM for 72h RAW_FILE_NAME=ASPC-G-5 SUBJECT_SAMPLE_FACTORS - ASPC-GI-1 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM and IBMX with a final concentration of 200μM for 72h RAW_FILE_NAME=ASPC-GI-1 SUBJECT_SAMPLE_FACTORS - ASPC-GI-2 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM and IBMX with a final concentration of 200μM for 72h RAW_FILE_NAME=ASPC-GI-2 SUBJECT_SAMPLE_FACTORS - ASPC-GI-3 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM and IBMX with a final concentration of 200μM for 72h RAW_FILE_NAME=ASPC-GI-3 SUBJECT_SAMPLE_FACTORS - ASPC-GI-4 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM and IBMX with a final concentration of 200μM for 72h RAW_FILE_NAME=ASPC-GI-4 SUBJECT_SAMPLE_FACTORS - ASPC-GI-5 Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM and IBMX with a final concentration of 200μM for 72h RAW_FILE_NAME=ASPC-GI-5 #COLLECTION CO:COLLECTION_SUMMARY After 72 hours of drug treatment, the cells were washed twice with ice PBS and CO:COLLECTION_SUMMARY scraped in 80% ice methanol CO:SAMPLE_TYPE Tumor cells #TREATMENT TR:TREATMENT_SUMMARY Cells were evenly divided into 3 groups of 6 discs each. 24h after cell TR:TREATMENT_SUMMARY inoculation, gemcitabine administration group was treated with gemcitabine with TR:TREATMENT_SUMMARY final concentration of 50μM for 72h, while the GI group was supplemented with TR:TREATMENT_SUMMARY an additional 200μM 3-isobutyl-1-methylxanthine (IBMX) in the culture medium. TR:TREATMENT_SUMMARY Meanwhile, the control group was given the same volume of DMSO. One plate of TR:TREATMENT_SUMMARY cells was taken from each group for counting, and the rest were collected for TR:TREATMENT_SUMMARY metabolite extraction #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The cells were cultured as described above and fixed with 1 ml of 80% ice-cold SP:SAMPLEPREP_SUMMARY menthol after being washed twice with ice-cold PBS. The cells were scraped from SP:SAMPLEPREP_SUMMARY the plates, and 0.5-mm beads were added to process the cells by grinding and SP:SAMPLEPREP_SUMMARY shaking. The supernatants were collected after centrifugation and deproteinized SP:SAMPLEPREP_SUMMARY by mixing with 800μL acetonitrile on ice. Then, the supernatants collected and SP:SAMPLEPREP_SUMMARY spun down under nitrogen at room temperature. The samples were resuspended in SP:SAMPLEPREP_SUMMARY 100 μL of distilled H2O, and 5 μL was used for LC-TQ-MS-based metabolome SP:SAMPLEPREP_SUMMARY 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 POSITIVE MS:MS_COMMENTS Agilent MassHunter Workstation Data Acquisition Agilent MassHunter #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS counts MS_METABOLITE_DATA_START Samples ASPC-C-1 ASPC-C-2 ASPC-C-3 ASPC-C-4 ASPC-C-5 ASPC-G-1-r002 ASPC-G-2 ASPC-G-3 ASPC-G-4 ASPC-G-5 ASPC-GI-1 ASPC-GI-2 ASPC-GI-3 ASPC-GI-4 ASPC-GI-5 Factors Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with 25μl DMSO for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with 25μl DMSO for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with 25μl DMSO for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with 25μl DMSO for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with 25μl DMSO for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM and IBMX with a final concentration of 200μM for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM and IBMX with a final concentration of 200μM for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM and IBMX with a final concentration of 200μM for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM and IBMX with a final concentration of 200μM for 72h Treatment:After inoculating cells for 24h in 10cm dishes, treating cells with gemcitabine with a final concentration of 50μM and IBMX with a final concentration of 200μM for 72h ATP 434250.62 532911.1446 732486.0849 808356.5781 732246.1247 1015876.132 887428.432 1183220.652 984235.0784 1101187.344 512948.1636 595094.365 520935.9288 580656.959 776794.4286 2-Deoxyadenosine 145437.3938 169201.3123 229617.277 242758.5989 407183.5824 42753.73781 48477.56265 48142.98339 43080.46573 45986.01672 30676.76336 39035.97638 35478.03218 42307.79709 36866.22579 Adenosine 19087600.76 24806280.95 26892679 27345577.11 35756956.32 44918786.15 33957890.42 46181003.71 44650736.47 43876717.79 30121152.48 38235586.8 39487687.38 46218488.82 38356066.45 Inosine-5'-monophosphate 27795.78179 19612.13356 36237.27931 26397.07759 38044.14573 64821.40863 172997.0716 103011.6829 119583.6483 94267.21935 73926.92734 81257.60205 59902.42506 51103.60011 55609.26732 Adenosine 5'-monophosphate 803614.0528 566254.7464 1082537.32 811667.4394 1138038.398 1328055.516 3019003.309 1970622.656 2342649.518 1950667.963 1611003.677 1602068.921 1431925.071 1237743.025 1175629.464 3-Isobutyl-1-methylxanthine 6007.267394 3623.849061 1983.157593 1493.337642 1514.176238 130590.6731 2419559.715 9495.494113 13400.81694 10904537.66 28728742.99 25251453.52 26224885.33 23519451.31 28944874.09 cAMP 34259.0578 32323.87926 30644.01852 30385.77372 26978.38562 50691.97382 54543.89698 53023.34764 49548.39595 53173.87645 33155.10858 29973.76835 32542.06815 31658.97033 32597.64737 Inosine 1113371.124 1552869.981 1760874.816 1801967.859 2724419.13 2573613.509 1950981.21 2720401.945 2529655.388 2544227.103 1771833.954 2350199.824 2440117.107 2904926.202 2290984.068 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z RT ATP 508.01 1.94 2-Deoxyadenosine 252.1 3.03 Adenosine 268.25 2.7 Inosine-5'-monophosphate 349.06 1.32 Adenosine 5'-monophosphate 348.07 1.32 3-Isobutyl-1-methylxanthine 223.25 9.08 cAMP 330.22 3.13 Inosine 269.09 2.7 METABOLITES_END #END