Summary of Study ST003485

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002140. The data can be accessed directly via it's Project DOI: 10.21228/M80R74 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST003485
Study TitleTryptophan metabolite profiling of cell supernatants (culture media) from TNBC cell lines with either overexpression or stable knockdown of TDO2.
Study SummaryIndole-focused metabolomics analysis of cell supernatants (i.e. cell culture media) from TNBC cell lines with overexpression or knockdown of TDO2. BT549 cells were genetically manipulated with stable knockdown of TDO2 (shTDO2). TNBC cell line SUM159PT was generated with stable TDO2 overexpression (TDO2 OE) and treated with TDO2/IDO1 dual inhibitor AT-0174.
Institute
University of Colorado Anschutz Medical Campus
Last NameHaines
First NameJulie
Address12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
Emailjulie.haines@cuanschutz.edu
Phone3037243339
Submit Date2024-09-16
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2024-10-03
Release Version1
Julie Haines Julie Haines
https://dx.doi.org/10.21228/M80R74
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002140
Project DOI:doi: 10.21228/M80R74
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

Subject:

Subject ID:SU003613
Subject Type:Cultured cells
Subject Species:Homo sapiens
Gender:Female

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id cell line TDO2 status Treatment Sample source
SA383964BT-shNEG-1MBT549 sh negative control none culture media
SA383965BT-shNEG-3MBT549 sh negative control none culture media
SA383966BT-shNEG-2MBT549 sh negative control none culture media
SA383967BT-shTDO2-1-3MBT549 shTDO2-82 none culture media
SA383968BT-shTDO2-1-2MBT549 shTDO2-82 none culture media
SA383969BT-shTDO2-1-1MBT549 shTDO2-82 none culture media
SA383970BT-shTDO2-2-3MBT549 shTDO2-98 none culture media
SA383971BT-shTDO2-2-2MBT549 shTDO2-98 none culture media
SA383972BT-shTDO2-2-1MBT549 shTDO2-98 none culture media
SA383973SUM-pEV-D-1MSUM159PT empty vector vehicle culture media
SA383974SUM-pEV-D-2MSUM159PT empty vector vehicle culture media
SA383975SUM-pEV-D-3MSUM159PT empty vector vehicle culture media
SA383979SUM-pTDO2-10A-2MSUM159PT overexpression 10 uM AT0174 culture media
SA383980SUM-pTDO2-10A-1MSUM159PT overexpression 10 uM AT0174 culture media
SA383981SUM-pTDO2-10A-3MSUM159PT overexpression 10 uM AT0174 culture media
SA383976SUM-pTDO2-1A-3MSUM159PT overexpression 1 uM AT0174 culture media
SA383977SUM-pTDO2-1A-2MSUM159PT overexpression 1 uM AT0174 culture media
SA383978SUM-pTDO2-1A-1MSUM159PT overexpression 1 uM AT0174 culture media
SA383982SUM-pTDO2-D-3MSUM159PT overexpression vehicle culture media
SA383983SUM-pTDO2-D-2MSUM159PT overexpression vehicle culture media
SA383984SUM-pTDO2-D-1MSUM159PT overexpression vehicle culture media
Showing results 1 to 21 of 21

Collection:

Collection ID:CO003606
Collection Summary:At the end of the cell culturing timecourse, the media were harvested and centrifuged at 1500 rpm, 4℃. The resulting supernatants were collected and frozen at -80 ℃.
Sample Type:Culture Media

Treatment:

Treatment ID:TR003622
Treatment Summary:TNBC cell line BT549 with stable TDO2 knockdown (shTDO2) or scramble control (shSCR) was cultured for 48 hours. TNBC cell line SUM159PT with stable TDO2 overexpression (TDO2 OE) was cultured for 24 hours then treated with DMSO (control), 1 µM AT-0174, or 10 µM AT-0174 for 48 hours. Culture media was not changed during the duration of the experiments.

Sample Preparation:

Sampleprep ID:SP003620
Sampleprep Summary:Culture media aliquots were thawed on ice then 20 uL was treated with 480 uL of ice cold 5:3:2 methanol:acetonitrile:water (v/v/v). Samples were vortexed 30 min at 4 degrees C followed by centrifugation 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℃

Combined analysis:

Analysis ID AN005723
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Vanquish
Column Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode POSITIVE
Units peak area

Chromatography:

Chromatography ID:CH004342
Chromatography Summary:Positive C18
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
Column Temperature:45
Flow Gradient:0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min 95-5% B, 3-5 min hold at 5% B
Flow Rate:450 uL/min
Sample Injection:6 uL
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS005446
Analysis ID:AN005723
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
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.
Ion Mode:POSITIVE
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