{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST000529","ANALYSIS_ID":"AN000807","VERSION":"1","CREATED_ON":"December 30, 2016, 8:07 am"},

"PROJECT":{"PROJECT_TITLE":"Regulation of Metabolism by LSR","PROJECT_TYPE":"Broad spectrum, reverse phase LCMS metabolomics","PROJECT_SUMMARY":"Breast cancer is a global crisis, accounting for nearly one quarter of all cancers in women. In the U.S., African-American (AA) women suffer disproportionately from breast cancer mortality compared to other racial/ethnic groups. Both social (socioeconomic factors, diet, obesity) and biological hypotheses (gene polymorphisms, gene expression profiling) have been advanced to explain these discrepancies, but the differences remain poorly understood. Multiple aspects of tumor aggressiveness have been identified in the AA population, including a high proportion of basal-like tumors. Basal-like tumors are clinically the most aggressive, characterized by enhanced cancer stem cell-like features. Currently, no effective molecular therapies exist for these highly aggressive cancers and patient survival is poor. Escalating this disparity is the disease promoting effects of obesity and metabolic syndrome, which are significantly higher in AA women. Obesity and its associated inflammation have been attributed to poor patient outcomes, resistance to chemotherapeutics, and/or cancer risk. A meta-analysis of 43 studies of obesity and breast cancer revealed that obese patients were 33% more likely than non-obese patients to die of breast cancer. Yet apart from correlative studies, no reports have combined these factors with cancer disparities to ascertain their molecular interactions and physiology on breast tumorigenesis. We tested the integration these factors, with focus on a specific molecule, Lipolysis Stimulated Lipoprotein Receptor (LSR), in the promotion of aggressive cancer behaviors. LSR is a cell surface molecule that regulates post-prandial lipid uptake in the liver, is sensitive to high fat diets, and is regulated by metabolic cues, including leptin. By testing each of these factors as well as their dynamic interactions on breast cancer tumorigenesis, we will gain valuable insight into biological mechanisms that influence cancer risk/disparities, response to therapy, and ultimately patient outcome. We recently reported LSR is overexpressed in breast tumors, directs aggressive breast cancer cell behaviors including proliferation and migration, and enhances cancer stem cell-like and chemotherapeutic resistance features in breast cancer cells.","INSTITUTE":"North Carolina Central Univeristy","DEPARTMENT":"Department of Biology","LAST_NAME":"Fleming","FIRST_NAME":"Jodie","ADDRESS":"1801 Fayetteville Street, Durham, NC 27707","EMAIL":"Jodie.Fleming@nccu.edu","PHONE":"919-530-6216"},

"STUDY":{"STUDY_TITLE":"Regulation of Metabolism by LSR","STUDY_TYPE":"Broad spectrum, reverse phase LCMS metabolomics (Negative ion mode)","STUDY_SUMMARY":"Our aim is to identify the LSR-driven metabolomics profile of breast cancer cells in lean and obesogenic environments. Breast cancer cell models with high or undetectable levels of LSR, including drug resistance models, were cultured in lean and obesogenic environments and comprehensive metabolomics profiling, including lipidomics-focused sub-analyses were performed. The metabolomics analyses using both approaches will help us determine if LSR enhances aggressive breast cancer phenotypes via modulation of cellular bioenergetic metabolism, ultimately contributing to poor patient outcome.","INSTITUTE":"RTI International","DEPARTMENT":"Systems and Translational Sciences","LABORATORY":"NIH Eastern Regional Comphrehensive Metabolomics Resource Core","LAST_NAME":"Sumner","FIRST_NAME":"Susan","ADDRESS":"3040 East Cornwallis Road, RTI International, Research Triangle Park, North Carolina, 27709, USA","EMAIL":"ssumner@rti.org","PHONE":"919-541-7479","NUM_GROUPS":"4","TOTAL_SUBJECTS":"20"},

"SUBJECT":{"SUBJECT_TYPE":"Cells","SUBJECT_SPECIES":"Homo sapiens","SUBJECT_COMMENTS":"Cell pellets stored at -80˚C.","CELL_COUNTS":"1E 7 cells/pellet"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"NEG_RP_EQ _P28_JFtotPool1-6",
"Factors":{"Factor1 Cell Line":"Study Pool"},
"Additional sample data":{"DRCC ID":"P_28"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_P_29_JFtotPool2",
"Factors":{"Factor1 Cell Line":"Study Pool"},
"Additional sample data":{"DRCC ID":"P_29"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_P_30_JFtotPool3",
"Factors":{"Factor1 Cell Line":"Study Pool"},
"Additional sample data":{"DRCC ID":"P_30"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_P_31_JFtotPool4",
"Factors":{"Factor1 Cell Line":"Study Pool"},
"Additional sample data":{"DRCC ID":"P_31"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_28_MCF7-CRISPR",
"Factors":{"Factor1 Cell Line":"MCF7 CRISPR"},
"Additional sample data":{"DRCC ID":"S_28"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_30_MCF7-CRISPR",
"Factors":{"Factor1 Cell Line":"MCF7 CRISPR"},
"Additional sample data":{"DRCC ID":"S_30"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_21_MCF7-1",
"Factors":{"Factor1 Cell Line":"MCF7 parental"},
"Additional sample data":{"DRCC ID":"S_21"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_22_MCF7-2",
"Factors":{"Factor1 Cell Line":"MCF7 parental"},
"Additional sample data":{"DRCC ID":"S_22"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_25_MCF7",
"Factors":{"Factor1 Cell Line":"MCF7 parental"},
"Additional sample data":{"DRCC ID":"S_25"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_31_HS2",
"Factors":{"Factor1 Cell Line":"Hs578t parental"},
"Additional sample data":{"DRCC ID":"S_31"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_32_HS",
"Factors":{"Factor1 Cell Line":"Hs578t parental"},
"Additional sample data":{"DRCC ID":"S_32"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_33_HS#2",
"Factors":{"Factor1 Cell Line":"Hs578t parental"},
"Additional sample data":{"DRCC ID":"S_33"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_34_HS#2",
"Factors":{"Factor1 Cell Line":"Hs578t parental"},
"Additional sample data":{"DRCC ID":"S_34"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_35_HS#2",
"Factors":{"Factor1 Cell Line":"Hs578t parental"},
"Additional sample data":{"DRCC ID":"S_35"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_36_LSRH-L2",
"Factors":{"Factor1 Cell Line":"Hs578t+"},
"Additional sample data":{"DRCC ID":"S_36"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_38_LSRH-L6",
"Factors":{"Factor1 Cell Line":"Hs578t+"},
"Additional sample data":{"DRCC ID":"S_38"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_39_LSRH-L8",
"Factors":{"Factor1 Cell Line":"Hs578t+"},
"Additional sample data":{"DRCC ID":"S_39"}
},
{
"Subject ID":"-",
"Sample ID":"NEG_RP_S_40_LSRH-L10",
"Factors":{"Factor1 Cell Line":"Hs578t+"},
"Additional sample data":{"DRCC ID":"S_40"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"20 breast cancer cell pellets from four cell lines [MCF7 parental (LSR+), MCF7 Crisper (LSR-), Hs578t parental (LSR-) and Hs578t (LSR++)], with five replicate samples for each cell line were stored at -80˚ until sample preparation.","SAMPLE_TYPE":"Cell pellets","STORAGE_CONDITIONS":"-80˚C"},

"TREATMENT":{"TREATMENT_SUMMARY":"Cultured in an obesogenic media."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Cell pellets were resuspended in volume of ice cold Lipid Extractions Solvent (50 µg/mL) based on biomass. Contents were transferred to new, pre-labeled 2.0 mL LoBind tube with 10-15 ceramic beads, and homogenized using 2 pulses at 2,000 rpm for 30 sec on a MagNA Lyser. Volume of HPLC-grade water (with 0.02 mg/mL Tryptophan-d5 in water) was added based on biomass. Samples were allowed to sit at room temperature for 10 minutes, then centrifuged at 16,000 rcf for 10 min at 10˚C. For reverse phase, transferred the maximum clean volume of the upper aqueous layer to a new, pre-labeled LoBind tube then a 150 µL aliquot was transferred to a new 1.5 mL, LoBind tube for analysis. An additional 50 µL aliquot of each study sample was combined in a 2 mL LoBind tube to make a QC pool, which was vortexed for 30 sec and aliquoted into 5 Total Pool samples (150 µL each), and the remainder used for 1 Equilibrium sample (for column conditioning). Samples were placed at -80˚C for 60 mins and lyophilized to dryness. Samples were reconstituted in H2O/Methanol (95:5 v/v), vortexed at 5,000 rpm for 2 mins and centrifuged at 16,000 rcf for 4 min. 100 µL of each supernatant was transferred to pre-labeled autosampler vials and 5 µL was injected Synapt G2Si ESI-Q-TOF for data acquisition."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Reversed-Phase Gradient Seperation","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Waters Acquity","COLUMN_NAME":"Waters Acquity HSS T3 (100 x 2.1mm, 1.8um)","FLOW_GRADIENT":"Time(min) Flow Rate(mL/min) %A %B Curve 1. Initial 0.400 99.0 1.0 2. 1.00 0.400 99.0 1.0 3. 16.00 0.400 1.0 99.0 4. 20.00 0.400 1.0 99.0 5. 20.50 0.400 99.0 1.0 6. 22.00 0.400 99.0 1.0","FLOW_RATE":"0.4 mL/min","COLUMN_TEMPERATURE":"50 °C","METHODS_FILENAME":"RTI-RCMRC-RP","SOLVENT_A":"Water with 0.1% formic acid","SOLVENT_B":"Methanol with 0.1% formic acid","METHODS_ID":"RTI-RCMRC-RP","INTERNAL_STANDARD":"L-Tryptophan-d5","ANALYTICAL_TIME":"22 min","WEAK_WASH_SOLVENT_NAME":"10% MeOH","WEAK_WASH_VOLUME":"1000 µL","STRONG_WASH_SOLVENT_NAME":"80% MeOH","STRONG_WASH_VOLUME":"1000 µL","TARGET_SAMPLE_TEMPERATURE":"8 °C","SAMPLE_LOOP_SIZE":"10 µL","SAMPLE_SYRINGE_SIZE":"100 µL","RANDOMIZATION_ORDER":"Yes"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"MS_COMMENTS":"-","INSTRUMENT_NAME":"Waters Synapt G2 Si QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","CAPILLARY_TEMPERATURE":"110 °C","CAPILLARY_VOLTAGE":"1.0 kV","COLLISION_ENERGY":"4","FRAGMENTATION_METHOD":"CID","HELIUM_FLOW":"180","IONIZATION":"ES-","IONIZATION_POTENTIAL":"5 ppm","SOURCE_TEMPERATURE":"100 °C","DATAFORMAT":"Continuum","DESOLVATION_GAS_FLOW":"400 L/Hr","DESOLVATION_TEMPERATURE":"400 °C","RESOLUTION_SETTING":"18000","SCAN_RANGE_MOVERZ":"50-1000 m/z","SCANNING_CYCLE":"1.0 s","TUBE_LENS_VOLTAGE":"74"},

"MS_METABOLITE_DATA":{
"Units":"m/z",

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