#METABOLOMICS WORKBENCH Lu_Group_20220325_234038 DATATRACK_ID:3142 STUDY_ID:ST002122 ANALYSIS_ID:AN003475 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 NEGATIVE 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 2'-Deoxyinosine 159.3811336 147.1643998 174.0027585 155.0079996 207.3309451 299.3246171 314.052782 326.5100276 277.9930606 304.4480807 233.3072243 178.5450285 172.0457137 192.7079941 267.2831479 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z RT 2'-Deoxyinosine 251.1 3.79 METABOLITES_END #END