#METABOLOMICS WORKBENCH juliehaines_20240916_141603 DATATRACK_ID:5196 STUDY_ID:ST003485 ANALYSIS_ID:AN005723 PROJECT_ID:PR002140 VERSION 1 CREATED_ON September 17, 2024, 1:08 pm #PROJECT PR:PROJECT_TITLE Blocking tryptophan catabolism reduces triple-negative breast cancer invasive PR:PROJECT_TITLE capacity PR:PROJECT_SUMMARY Anchorage-independent triple-negative breast cancer (TNBC) cells exhibit PR:PROJECT_SUMMARY elevated levels of the tryptophan (TRP) catabolizing enzyme tryptophan PR:PROJECT_SUMMARY 2,3-dioxygenase 2 (TDO2) compared to the same cells grown in two-dimensional PR:PROJECT_SUMMARY culture. Tracing of 13C11-TRP demonstrated that anchorage-independent culture PR:PROJECT_SUMMARY and/or inflammatory cytokines that activate nuclear factor PR:PROJECT_SUMMARY kappa-light-chain-enhancer of activated B (NFκB) increase TRP catabolism and PR:PROJECT_SUMMARY production of downstream catabolites such as kynurenine (KYN), which activate PR:PROJECT_SUMMARY the aryl hydrocarbon receptor (AhR). TDO2 expression is heterogeneous within PR:PROJECT_SUMMARY TNBC cell lines. To determine the function of TDO2, both pharmacologic PR:PROJECT_SUMMARY inhibition and genetic manipulation were conducted. TDO2 knockdown revealed a PR:PROJECT_SUMMARY compensatory increase in indoleamine 2,3-dioxygenase 1 (IDO1), a non-homologous PR:PROJECT_SUMMARY TRP catabolizing enzyme, indicating that dual inhibition of these two enzymes is PR:PROJECT_SUMMARY necessary to reliably block TRP catabolism. Thus, we tested a newly developed PR:PROJECT_SUMMARY TDO2/IDO1 dual inhibitor, AT-0174, and found that it effectively inhibits TNBC PR:PROJECT_SUMMARY TRP catabolism. Furthermore, AT-0174 treatment or AhR inhibitor significantly PR:PROJECT_SUMMARY decreased TNBC anchorage-independent survival, invasive capacity, and expression PR:PROJECT_SUMMARY of mesenchymal genes and protein, while exogenous KYN increased invasion through PR:PROJECT_SUMMARY AhR-mediated ZEB1 expression. Thus, dual inhibition of TDO2/IDO1 may prove PR:PROJECT_SUMMARY efficacious against TNBC progression. PR:INSTITUTE University of Colorado Anschutz Medical Campus PR:LABORATORY Lab of Angelo D'Alessandro in collaboration with lab of Jennifer Richer PR:LAST_NAME Haines PR:FIRST_NAME Julie PR:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA PR:EMAIL julie.haines@cuanschutz.edu PR:PHONE 3037243339 #STUDY ST:STUDY_TITLE Tryptophan metabolite profiling of cell supernatants (i.e. cell culture media) ST:STUDY_TITLE from TNBC cell lines with either overexpression or stable knockdown of TDO2. ST:STUDY_SUMMARY Indole-focused metabolomics analysis of cell supernatants (i.e. cell culture ST:STUDY_SUMMARY media) from TNBC cell lines with overexpression or knockdown of TDO2. BT549 ST:STUDY_SUMMARY cells were genetically manipulated with stable knockdown of TDO2 (shTDO2). TNBC ST:STUDY_SUMMARY cell line SUM159PT was generated with stable TDO2 overexpression (TDO2 OE) and ST:STUDY_SUMMARY treated with TDO2/IDO1 dual inhibitor AT-0174. ST:INSTITUTE University of Colorado Anschutz Medical Campus ST:LAST_NAME Haines ST:FIRST_NAME Julie ST:ADDRESS 12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA ST:EMAIL julie.haines@cuanschutz.edu ST:PHONE 3037243339 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:GENDER Female #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 - SUM-pEV-D-1M cell line:SUM159PT | TDO2 status:empty vector | Treatment:vehicle | Sample source:conditioned media RAW_FILE_NAME=DS1-52-010+ SUBJECT_SAMPLE_FACTORS - SUM-pEV-D-2M cell line:SUM159PT | TDO2 status:empty vector | Treatment:vehicle | Sample source:conditioned media RAW_FILE_NAME=DS1-52-011+ SUBJECT_SAMPLE_FACTORS - SUM-pEV-D-3M cell line:SUM159PT | TDO2 status:empty vector | Treatment:vehicle | Sample source:conditioned media RAW_FILE_NAME=DS1-52-012+ SUBJECT_SAMPLE_FACTORS - SUM-pTDO2-D-1M cell line:SUM159PT | TDO2 status:overexpression | Treatment:vehicle | Sample source:conditioned media RAW_FILE_NAME=DS1-52-022+ SUBJECT_SAMPLE_FACTORS - SUM-pTDO2-D-2M cell line:SUM159PT | TDO2 status:overexpression | Treatment:vehicle | Sample source:conditioned media RAW_FILE_NAME=DS1-52-023+ SUBJECT_SAMPLE_FACTORS - SUM-pTDO2-D-3M cell line:SUM159PT | TDO2 status:overexpression | Treatment:vehicle | Sample source:conditioned media RAW_FILE_NAME=DS1-52-024+ SUBJECT_SAMPLE_FACTORS - SUM-pTDO2-1A-1M cell line:SUM159PT | TDO2 status:overexpression | Treatment:1 uM AT0174 | Sample source:conditioned media RAW_FILE_NAME=DS1-52-025+ SUBJECT_SAMPLE_FACTORS - SUM-pTDO2-1A-2M cell line:SUM159PT | TDO2 status:overexpression | Treatment:1 uM AT0174 | Sample source:conditioned media RAW_FILE_NAME=DS1-52-026+ SUBJECT_SAMPLE_FACTORS - SUM-pTDO2-1A-3M cell line:SUM159PT | TDO2 status:overexpression | Treatment:1 uM AT0174 | Sample source:conditioned media RAW_FILE_NAME=DS1-52-027+ SUBJECT_SAMPLE_FACTORS - SUM-pTDO2-10A-1M cell line:SUM159PT | TDO2 status:overexpression | Treatment:10 uM AT0174 | Sample source:conditioned media RAW_FILE_NAME=DS1-52-028+ SUBJECT_SAMPLE_FACTORS - SUM-pTDO2-10A-2M cell line:SUM159PT | TDO2 status:overexpression | Treatment:10 uM AT0174 | Sample source:conditioned media RAW_FILE_NAME=DS1-52-029+ SUBJECT_SAMPLE_FACTORS - SUM-pTDO2-10A-3M cell line:SUM159PT | TDO2 status:overexpression | Treatment:10 uM AT0174 | Sample source:conditioned media RAW_FILE_NAME=DS1-52-030+ SUBJECT_SAMPLE_FACTORS - BT-shNEG-1M cell line:BT549 | TDO2 status:sh negative control | Treatment:none | Sample source:conditioned media RAW_FILE_NAME=DS1-52-034+ SUBJECT_SAMPLE_FACTORS - BT-shNEG-2M cell line:BT549 | TDO2 status:sh negative control | Treatment:none | Sample source:conditioned media RAW_FILE_NAME=DS1-52-035+ SUBJECT_SAMPLE_FACTORS - BT-shNEG-3M cell line:BT549 | TDO2 status:sh negative control | Treatment:none | Sample source:conditioned media RAW_FILE_NAME=DS1-52-036+ SUBJECT_SAMPLE_FACTORS - BT-shTDO2-1-1M cell line:BT549 | TDO2 status:shTDO2-82 | Treatment:none | Sample source:conditioned media RAW_FILE_NAME=DS1-52-037+ SUBJECT_SAMPLE_FACTORS - BT-shTDO2-1-2M cell line:BT549 | TDO2 status:shTDO2-82 | Treatment:none | Sample source:conditioned media RAW_FILE_NAME=DS1-52-038+ SUBJECT_SAMPLE_FACTORS - BT-shTDO2-1-3M cell line:BT549 | TDO2 status:shTDO2-82 | Treatment:none | Sample source:conditioned media RAW_FILE_NAME=DS1-52-039+ SUBJECT_SAMPLE_FACTORS - BT-shTDO2-2-1M cell line:BT549 | TDO2 status:shTDO2-98 | Treatment:none | Sample source:conditioned media RAW_FILE_NAME=DS1-52-040+ SUBJECT_SAMPLE_FACTORS - BT-shTDO2-2-2M cell line:BT549 | TDO2 status:shTDO2-98 | Treatment:none | Sample source:conditioned media RAW_FILE_NAME=DS1-52-041+ SUBJECT_SAMPLE_FACTORS - BT-shTDO2-2-3M cell line:BT549 | TDO2 status:shTDO2-98 | Treatment:none | Sample source:conditioned media RAW_FILE_NAME=DS1-52-042+ #COLLECTION CO:COLLECTION_SUMMARY At the end of the cell culturing timecourse, the media were harvested and CO:COLLECTION_SUMMARY centrifuged at 1500 rpm, 4℃. The resulting supernatants were collected and CO:COLLECTION_SUMMARY frozen at -80 ℃. CO:SAMPLE_TYPE Breast cancer cells #TREATMENT TR:TREATMENT_SUMMARY TNBC cell line BT549 with stable TDO2 knockdown (shTDO2) or scramble control TR:TREATMENT_SUMMARY (shSCR) was cultured for 48 hours. TNBC cell line SUM159PT with stable TDO2 TR:TREATMENT_SUMMARY overexpression (TDO2 OE) was cultured for 24 hours then treated with DMSO TR:TREATMENT_SUMMARY (control), 1 µM AT-0174, or 10 µM AT-0174 for 48 hours. Culture media was not TR:TREATMENT_SUMMARY changed during the duration of the experiments. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Supernatant aliquots were thawed on ice then 20 uL was treated with 480 uL of SP:SAMPLEPREP_SUMMARY ice cold 5:3:2 methanol:acetonitrile:water (v/v/v). Samples were vortexed 30 min SP:SAMPLEPREP_SUMMARY at 4 degrees C followed by centrifugation for 10 min at 18,000 g. Aliquots of SP:SAMPLEPREP_SUMMARY supernatant (50 uL) were dried using a speedvac then reconstituted in an SP:SAMPLEPREP_SUMMARY equivalent volume of 0.1% formic acid. Samples were maintained at 4°C until SP:SAMPLEPREP_SUMMARY analysis that same day. SP:PROCESSING_STORAGE_CONDITIONS 4℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Positive C18 CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (150 x 2.1mm,1.7um) CH:SOLVENT_A 100% water; 0.1% formic acid CH:SOLVENT_B 100% acetonitrile; 0.1% formic acid CH: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 CH:FLOW_GRADIENT 95-5% B, 3-5 min hold at 5% B CH:FLOW_RATE 450 uL/min CH:COLUMN_TEMPERATURE 45 CH:SAMPLE_INJECTION 6 uL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms, MS:MS_COMMENTS microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV, MS:MS_COMMENTS capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas MS:MS_COMMENTS 0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were MS:MS_COMMENTS annotated and integrated using Maven in conjunction with the KEGG database. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples SUM-pEV-D-1M SUM-pEV-D-2M SUM-pEV-D-3M SUM-pTDO2-D-1M SUM-pTDO2-D-2M SUM-pTDO2-D-3M SUM-pTDO2-1A-1M SUM-pTDO2-1A-2M SUM-pTDO2-1A-3M SUM-pTDO2-10A-1M SUM-pTDO2-10A-2M SUM-pTDO2-10A-3M BT-shNEG-1M BT-shNEG-2M BT-shNEG-3M BT-shTDO2-1-1M BT-shTDO2-1-2M BT-shTDO2-1-3M BT-shTDO2-2-1M BT-shTDO2-2-2M BT-shTDO2-2-3M Factors cell line:SUM159PT | TDO2 status:empty vector | Treatment:vehicle | Sample source:conditioned media cell line:SUM159PT | TDO2 status:empty vector | Treatment:vehicle | Sample source:conditioned media cell line:SUM159PT | TDO2 status:empty vector | Treatment:vehicle | Sample source:conditioned media cell line:SUM159PT | TDO2 status:overexpression | Treatment:vehicle | Sample source:conditioned media cell line:SUM159PT | TDO2 status:overexpression | Treatment:vehicle | Sample source:conditioned media cell line:SUM159PT | TDO2 status:overexpression | Treatment:vehicle | Sample source:conditioned media cell line:SUM159PT | TDO2 status:overexpression | Treatment:1 uM AT0174 | Sample source:conditioned media cell line:SUM159PT | TDO2 status:overexpression | Treatment:1 uM AT0174 | Sample source:conditioned media cell line:SUM159PT | TDO2 status:overexpression | Treatment:1 uM AT0174 | Sample source:conditioned media cell line:SUM159PT | TDO2 status:overexpression | Treatment:10 uM AT0174 | Sample source:conditioned media cell line:SUM159PT | TDO2 status:overexpression | Treatment:10 uM AT0174 | Sample source:conditioned media cell line:SUM159PT | TDO2 status:overexpression | Treatment:10 uM AT0174 | Sample source:conditioned media cell line:BT549 | TDO2 status:sh negative control | Treatment:none | Sample source:conditioned media cell line:BT549 | TDO2 status:sh negative control | Treatment:none | Sample source:conditioned media cell line:BT549 | TDO2 status:sh negative control | Treatment:none | Sample source:conditioned media cell line:BT549 | TDO2 status:shTDO2-82 | Treatment:none | Sample source:conditioned media cell line:BT549 | TDO2 status:shTDO2-82 | Treatment:none | Sample source:conditioned media cell line:BT549 | TDO2 status:shTDO2-82 | Treatment:none | Sample source:conditioned media cell line:BT549 | TDO2 status:shTDO2-98 | Treatment:none | Sample source:conditioned media cell line:BT549 | TDO2 status:shTDO2-98 | Treatment:none | Sample source:conditioned media cell line:BT549 | TDO2 status:shTDO2-98 | Treatment:none | Sample source:conditioned media 5-Hydroxyindoleacetate 97040.94 61665.84 64606.62 220599.05 192755.02 93882.06 175313.55 39992.5 46730.77 98202.47 61634.82 92952.09 122104.65 116636.34 97928.38 83004.51 88905.66 120847.63 112782.56 182384.81 162376.92 3-Methyldioxyindole 5445.99 4625.6 10318.68 4795.65 16064.66 4142.26 9765.93 4227.87 9481.48 4281.25 3710.83 5668.26 31634.76 22894.63 22050.68 27966.52 17424.04 27030.1 41172.05 37953.55 51765 3-Methyleneoxindole 4262386 2189287.5 2795398.25 3827281.25 3160821.75 1432195.62 4758756.5 1454660.12 1569580.38 4623616.5 2393056 3597460.75 6055972 5548808.5 5020800 4607612.5 5208801.5 6347501.5 9555396 14740551 11002784 Indole 2942188 1519605 1892677.12 2730257 2246463 1078610.88 3412519.75 1049833.62 1090769.88 3325653.25 1802033.12 2558821.75 4394192 3790066.25 3449896.25 3330080.25 3660150 4379832 6638200.5 10008459 7763002.5 Indole-3-acetaldehyde 210293.42 103581.06 135647.09 186660.8 152464.55 57023.25 221226.52 66604.19 78908.11 245412.45 104300.06 164762.12 326164.28 287814 281286.25 227082.94 242102.05 299770.88 443814 730582.5 564736.5 Indole-3-acetate 7268.77 48888.16 28050.85 17747.12 7951.88 6087.02 14327.17 6591.04 7683.9 9816.31 4311.39 13851.49 68180.44 57230.78 62103.36 13290 12743 15347.71 15889.16 37898.1 27720.41 Indoxyl 476157.25 254273.86 265978.47 472885.5 356026.53 236160.58 506290.66 235362.42 215132.56 620073.62 341737.28 388793.78 8213328 6992764.5 8378161.5 7747488.5 9202295 10468844 9869198 13524637 12897464 kynurenine 150448.2 82410.34 66247.65 302290.38 272983.88 127082.54 203133.61 58715.51 58997.61 138733.5 80877.3 126429.09 175015.64 166476.56 145334.44 118222.21 124993.66 183421.11 184139.08 271336.09 211980.02 N-formyl kynurenine 43496.55 50545.75 22413.92 66009.7 48046.52 33255.83 52263.28 19403.62 22881.01 32878.31 17452.34 24885.93 79870.88 80732.04 63185.82 58205.75 52187.37 76448.19 81958.66 120567.12 103446.04 Anthranilate 100002.59 52779.75 39537.24 60153.18 121446.59 75840.12 107120.45 101083.59 129002.46 61247.36 87972.28 33304.5 767819.5 640035.06 571133.06 177802.23 203086.14 154590.69 1491458.38 2039308.62 1808475 Picolinic acid 63600.11 41734.81 41254.91 58243.86 53437.19 31620.24 87877.34 42091.39 45857.92 79330.34 63487.96 74697.21 2092694.88 1732777.38 1689892.12 198303.5 211727.25 262108.89 91415.53 111394.37 102314.76 5-hydroxytryptophan 58890.65 55103.84 50215.75 84298.43 68580.53 33500.11 97820.88 25512.21 33445.44 97496.94 60465.98 68091.6 179878.05 155756.78 149018.3 62099.36 63245.49 74991.59 101777.09 106724.15 98390.13 L-tryptophan 7051529.5 3301004.75 4397090.5 6000423.5 5153119.5 2459488.25 7778225.5 2282205.25 2318613.25 7363408 3971832.75 5892922.5 9563539 9044752 7374281.5 7522712.5 8940104 10870347 15447547 24667318 18716400 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID m/z RT 5-Hydroxyindoleacetate C05635 192.06517 1.68 3-Methyldioxyindole C05834 164.070328 1.22 3-Methyleneoxindole C02796 146.060043 1.756 Indole C00463 118.064972 1.751 Indole-3-acetaldehyde C00637 160.075684 1.751 Indole-3-acetate C00954 176.070419 1.891 Indoxyl C05658 134.059952 1.222 kynurenine C00328 209.091782 1.681 N-formyl kynurenine C02700 237.086655 1.724 Anthranilate C00108 138.054947 1.791 Picolinic acid C10164 124.039146 1.16 5-hydroxytryptophan C00643 221.09169 1.384 L-tryptophan C00078 205.096909 1.75 METABOLITES_END #END