{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003484","ANALYSIS_ID":"AN005721","PROJECT_ID":"PR002140","VERSION":"1","CREATED_ON":"September 18, 2024, 7:26 pm"},

"PROJECT":{"PROJECT_TITLE":"Blocking tryptophan catabolism reduces triple-negative breast cancer invasive capacity","PROJECT_SUMMARY":"Anchorage-independent triple-negative breast cancer (TNBC) cells exhibit elevated levels of the tryptophan (TRP) catabolizing enzyme tryptophan 2,3-dioxygenase 2 (TDO2) compared to the same cells grown in two-dimensional culture. Tracing of 13C11-TRP demonstrated that anchorage-independent culture and/or inflammatory cytokines that activate nuclear factor kappa-light-chain-enhancer of activated B (NFκB) increase TRP catabolism and production of downstream catabolites such as kynurenine (KYN), which activate the aryl hydrocarbon receptor (AhR). TDO2 expression is heterogeneous within TNBC cell lines. To determine the function of TDO2, both pharmacologic inhibition and genetic manipulation were conducted. TDO2 knockdown revealed a compensatory increase in indoleamine 2,3-dioxygenase 1 (IDO1), a non-homologous TRP catabolizing enzyme, indicating that dual inhibition of these two enzymes is necessary to reliably block TRP catabolism. Thus, we tested a newly developed TDO2/IDO1 dual inhibitor, AT-0174, and found that it effectively inhibits TNBC TRP catabolism. Furthermore, AT-0174 treatment or AhR inhibitor significantly decreased TNBC anchorage-independent survival, invasive capacity, and expression of mesenchymal genes and protein, while exogenous KYN increased invasion through AhR-mediated ZEB1 expression. Thus, dual inhibition of TDO2/IDO1 may prove efficacious against TNBC progression.","INSTITUTE":"University of Colorado Anschutz Medical Campus","LABORATORY":"Lab of Angelo D''Alessandro in collaboration with lab of Jennifer Richer","LAST_NAME":"Haines","FIRST_NAME":"Julie","ADDRESS":"12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA","EMAIL":"julie.haines@cuanschutz.edu","PHONE":"3037243339"},

"STUDY":{"STUDY_TITLE":"Metabolism of TNBC cell line MDA-MB-453 is altered by cytokines, TDO2 inhibition, or suspension growth conditions - cellular steady state metabolome and in vitro tracing with 13C11 tryptophan","STUDY_SUMMARY":"TNBC cell line MDA-MB-453 was cultured in 13C11 tryptophan and experimental treatments were as follows: a) cytokine treatment (TNFα+IL1β); b) treated with TDO2/IDO1 dual inhibitor AT-0174 for 24 or 48 hours with an without labeled tryptophan; c) cultured in 13C11 tryptophan and suspension condition.","INSTITUTE":"University of Colorado Anschutz Medical Campus","LAST_NAME":"Haines","FIRST_NAME":"Julie","ADDRESS":"12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA","EMAIL":"julie.haines@cuanschutz.edu","PHONE":"3037243339"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","GENDER":"Female"},
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"Factors":{"Growth condition":"Attached","time (h)":"24","treatment":"1µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Attached","time (h)":"24","treatment":"1µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Attached","time (h)":"24","treatment":"DMSO","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Attached","time (h)":"24","treatment":"DMSO","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Attached","time (h)":"24","treatment":"DMSO","Sample source":"Breast cancer cells"},
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{
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"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"10µM 68c091","Sample source":"Breast cancer cells"},
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{
"Subject ID":"-",
"Sample ID":"MS610-24-2",
"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"10µM 68c091","Sample source":"Breast cancer cells"},
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},
{
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"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"10µM 68c091","Sample source":"Breast cancer cells"},
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},
{
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"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"10µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"10µM AT0174","Sample source":"Breast cancer cells"},
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"Subject ID":"-",
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"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"10µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"1µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"1µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"1µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"DMSO","Sample source":"Breast cancer cells"},
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"Subject ID":"-",
"Sample ID":"MSD-24-2",
"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"DMSO","Sample source":"Breast cancer cells"},
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{
"Subject ID":"-",
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"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"DMSO","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"10µM 68c091","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"10µM 68c091","Sample source":"Breast cancer cells"},
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"Sample ID":"MA610-48-3",
"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"10µM 68c091","Sample source":"Breast cancer cells"},
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{
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"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"10µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"10µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"10µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"1µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"1µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"1µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"DMSO","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"DMSO","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"10µM 68c091","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"10µM 68c091","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"10µM 68c091","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"10µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"10µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"10µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"1µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"1µM AT0174","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"1µM AT0174","Sample source":"Breast cancer cells"},
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},
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"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"DMSO","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"DMSO","Sample source":"Breast cancer cells"},
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"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"DMSO","Sample source":"Breast cancer cells"},
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},
{
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"Factors":{"Growth condition":"Attached","time (h)":"24","treatment":"NFkB cocktail  10 ng/ml IL1b + 10 ng/ml TNFa","Sample source":"Breast cancer cells"},
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},
{
"Subject ID":"-",
"Sample ID":"MAT-24-2",
"Factors":{"Growth condition":"Attached","time (h)":"24","treatment":"NFkB cocktail  10 ng/ml IL1b + 10 ng/ml TNFa","Sample source":"Breast cancer cells"},
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},
{
"Subject ID":"-",
"Sample ID":"MAT-24-3",
"Factors":{"Growth condition":"Attached","time (h)":"24","treatment":"NFkB cocktail  10 ng/ml IL1b + 10 ng/ml TNFa","Sample source":"Breast cancer cells"},
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},
{
"Subject ID":"-",
"Sample ID":"MAV-24-1",
"Factors":{"Growth condition":"Attached","time (h)":"24","treatment":"Vehicle","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-121"}
},
{
"Subject ID":"-",
"Sample ID":"MAV-24-2",
"Factors":{"Growth condition":"Attached","time (h)":"24","treatment":"Vehicle","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-122"}
},
{
"Subject ID":"-",
"Sample ID":"MAV-24-3",
"Factors":{"Growth condition":"Attached","time (h)":"24","treatment":"Vehicle","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-123"}
},
{
"Subject ID":"-",
"Sample ID":"MAT-48-1",
"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"NFkB cocktail  10 ng/ml IL1b + 10 ng/ml TNFa","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-118"}
},
{
"Subject ID":"-",
"Sample ID":"MAT-48-2",
"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"NFkB cocktail  10 ng/ml IL1b + 10 ng/ml TNFa","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-119"}
},
{
"Subject ID":"-",
"Sample ID":"MAT-48-3",
"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"NFkB cocktail  10 ng/ml IL1b + 10 ng/ml TNFa","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-120"}
},
{
"Subject ID":"-",
"Sample ID":"MAV-48-1",
"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"Vehicle","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-124"}
},
{
"Subject ID":"-",
"Sample ID":"MAV-48-2",
"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"Vehicle","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-125"}
},
{
"Subject ID":"-",
"Sample ID":"MAV-48-3",
"Factors":{"Growth condition":"Attached","time (h)":"48","treatment":"Vehicle","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-126"}
},
{
"Subject ID":"-",
"Sample ID":"MST-24-1",
"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"NFkB cocktail  10 ng/ml IL1b + 10 ng/ml TNFa","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-127"}
},
{
"Subject ID":"-",
"Sample ID":"MST-24-2",
"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"NFkB cocktail  10 ng/ml IL1b + 10 ng/ml TNFa","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-128"}
},
{
"Subject ID":"-",
"Sample ID":"MST-24-3",
"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"NFkB cocktail  10 ng/ml IL1b + 10 ng/ml TNFa","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-129"}
},
{
"Subject ID":"-",
"Sample ID":"MSV-24-1",
"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"Vehicle","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-133"}
},
{
"Subject ID":"-",
"Sample ID":"MSV-24-2",
"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"Vehicle","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-134"}
},
{
"Subject ID":"-",
"Sample ID":"MSV-24-3",
"Factors":{"Growth condition":"Suspension","time (h)":"24","treatment":"Vehicle","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-135"}
},
{
"Subject ID":"-",
"Sample ID":"MST-48-1",
"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"NFkB cocktail  10 ng/ml IL1b + 10 ng/ml TNFa","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-130"}
},
{
"Subject ID":"-",
"Sample ID":"MST-48-2",
"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"NFkB cocktail  10 ng/ml IL1b + 10 ng/ml TNFa","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-131"}
},
{
"Subject ID":"-",
"Sample ID":"MST-48-3",
"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"NFkB cocktail  10 ng/ml IL1b + 10 ng/ml TNFa","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-132"}
},
{
"Subject ID":"-",
"Sample ID":"MSV-48-1",
"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"Vehicle","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-136"}
},
{
"Subject ID":"-",
"Sample ID":"MSV-48-2",
"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"Vehicle","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-137"}
},
{
"Subject ID":"-",
"Sample ID":"MSV-48-3",
"Factors":{"Growth condition":"Suspension","time (h)":"48","treatment":"Vehicle","Sample source":"Breast cancer cells"},
"Additional sample data":{"RAW_FILE_NAME":"MR09-138"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"The cells were detached using 0.5% trypsin/EDTA and then centrifuged at 1500rpm for 5 mins at 4℃, and the supernatants were discarded. Next, PBS was used to resuspend the cell pellets and centrifuged again at 13000rpm for 15 mins at 4℃. The supernatant then discarded. The cell pellets were frozen at -80℃.","SAMPLE_TYPE":"Breast cancer cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"TNBC cell line MDA-MB-453 was first cultured in 13C11 tryptophan for 24 hours and treated with cytokines (10ng/ml TNFα+ 10ng/ml IL1β) for 24 hours and 48 hours. Culture media was not changed during the duration of the experiments."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Metabolites from frozen pellets were extracted at 2e6 cells per mL using ice cold 5:3:2 methanol:acetonitrile:water (v/v/v) with vigorous vortexing at 4 degrees C followed by centrifugation as described for 10 min at 18,000 g. Aliquots of supernatant (50 uL) were dried using a speedvac then reconstituted in an equivalent volume of 0.1% formic acid. Samples were maintained at 4°C until analysis that same day.","PROCESSING_STORAGE_CONDITIONS":"4℃","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Negative C18","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Vanquish","COLUMN_NAME":"Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)","SOLVENT_A":"95% water/5% acetonitrile; 1 mM ammonium acetate","SOLVENT_B":"95% acetonitrile/5% water; 1 mM ammonium acetate","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 100-0% B, 3-5 min hold at 0% B","FLOW_RATE":"450 uL/min","COLUMN_TEMPERATURE":"45","SAMPLE_INJECTION":"6 uL"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were annotated and integrated using Maven in conjunction with the KEGG database."},

"MS_METABOLITE_DATA":{
"Units":"peak area",

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