#METABOLOMICS WORKBENCH juliehaines_20240917_091515 DATATRACK_ID:5200 STUDY_ID:ST003486 ANALYSIS_ID:AN005724 PROJECT_ID:PR002140
VERSION             	1
CREATED_ON             	September 18, 2024, 8:45 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 (culture media) from TNBC
ST:STUDY_TITLE                   	cell line BT549 grown in suspension with TDO inhibition.
ST:STUDY_SUMMARY                 	Indole-focused metabolomics analysis of cell supernatants (i.e. cell culture
ST:STUDY_SUMMARY                 	media) from TNBC cell lines with pharmacological inhibition of TDO2 by AT-0174
ST:STUDY_SUMMARY                 	or 680c91.
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                  	Human
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
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           	-	BTS-68048-1M	treatment:10 uM 680c91 | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=DS1-30-13+
SUBJECT_SAMPLE_FACTORS           	-	BTS-68048-2M	treatment:10 uM 680c91 | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=DS1-30-14+
SUBJECT_SAMPLE_FACTORS           	-	BTS-68048-3M	treatment:10 uM 680c91 | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=DS1-30-15+
SUBJECT_SAMPLE_FACTORS           	-	BTS-10A48-1M	treatment:10 uM AT0174 | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=DS1-30-16+
SUBJECT_SAMPLE_FACTORS           	-	BTS-10A48-2M	treatment:10 uM AT0174 | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=DS1-30-17+
SUBJECT_SAMPLE_FACTORS           	-	BTS-10A48-3M	treatment:10 uM AT0174 | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=DS1-30-18+
SUBJECT_SAMPLE_FACTORS           	-	BTS-1A48-1M	treatment:1 uM AT0174 | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=DS1-30-19+
SUBJECT_SAMPLE_FACTORS           	-	BTS-1A48-2M	treatment:1 uM AT0174 | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=DS1-30-20+
SUBJECT_SAMPLE_FACTORS           	-	BTS-1A48-3M	treatment:1 uM AT0174 | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=DS1-30-21+
SUBJECT_SAMPLE_FACTORS           	-	BTS-D48-1M	treatment:DMSO vehicle | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=DS1-30-22+
SUBJECT_SAMPLE_FACTORS           	-	BTS-D48-2M	treatment:DMSO vehicle | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=DS1-30-23+
SUBJECT_SAMPLE_FACTORS           	-	BTS-D48-3M	treatment:DMSO vehicle | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=DS1-30-24+
#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                   	Culture Media
#TREATMENT
TR:TREATMENT_SUMMARY             	TNBC cell line BT549 was cultured for 24 hours then treated with DMSO (control),
TR:TREATMENT_SUMMARY             	1 µM AT-0174, 10 µM AT-0174, or 10 uM 680c91 for 48 hours. Culture media was
TR:TREATMENT_SUMMARY             	not 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	BTS-68048-1M	BTS-68048-2M	BTS-68048-3M	BTS-10A48-1M	BTS-10A48-2M	BTS-10A48-3M	BTS-1A48-1M	BTS-1A48-2M	BTS-1A48-3M	BTS-D48-1M	BTS-D48-2M	BTS-D48-3M
Factors	treatment:10 uM 680c91 | Sample source:conditioned media	treatment:10 uM 680c91 | Sample source:conditioned media	treatment:10 uM 680c91 | Sample source:conditioned media	treatment:10 uM AT0174 | Sample source:conditioned media	treatment:10 uM AT0174 | Sample source:conditioned media	treatment:10 uM AT0174 | Sample source:conditioned media	treatment:1 uM AT0174 | Sample source:conditioned media	treatment:1 uM AT0174 | Sample source:conditioned media	treatment:1 uM AT0174 | Sample source:conditioned media	treatment:DMSO vehicle | Sample source:conditioned media	treatment:DMSO vehicle | Sample source:conditioned media	treatment:DMSO vehicle | Sample source:conditioned media
5-Hydroxyindoleacetate	4782790	5522946	4656318	5238430	4643834	3895843	4909326	4539077	4807516	5368992	3861478	5009230
3-methyldioxyindole	35894.18	79072.48	44826.86	134354.3	46453.96	23426.96	128968.9	53579.77	137227.4	33605.43	33889.6	30887.63
3-Methyleneoxindole	1171177	1458937	1625569	1818440	1382399	1877524	1767991	1344006	1462200	1036507	1290743	669262.5
Indole	595884.5	808628.4	848601.8	939298.8	774896.8	985219.2	991921.5	741315.7	719764.8	602300.9	746742.1	349614.6
Indole-3-acetaldehyde	60346.61	109900.9	106981	95176.19	64155.45	112182.6	97425.27	107252.5	68961.32	76529.27	110541.2	27602.64
Indole-3-acetate	88076.13	8585.562	13814.44	170058.1	7822.836	4427.31	151384.2	109205.9	136063.5	167108.5	16548.61	119299.8
Indolepyruvate	138920.7	354823.6	245524.2	117093.4	155083.2	195729.6	140696.1	192602.4	189127	212597.1	137944.5	190921
Indoxyl	187043.6	314643.3	301213.5	292693.5	196120.3	278353.9	303171.1	200493.2	246457.2	258711.1	248473.2	142128.8
kynurenine	4303696	1624258	2353268	5552108	4612482	5666338	6346780	5389814	6029606	10600000	13200000	7945914
N-formyl kynurenine	199256.7	64711.69	101129	213784.1	306485.2	278787.7	444084.4	356343.3	489090.4	1230629	1643594	866418.2
Anthranilate	1872647	8827103	10300000	2512118	1754224	2810072	2743607	2339228	2317618	3074926	2670855	1349049
Picolinic acid	5697850	5468235	5132160	5651640	6059556	4786610	5196138	5919378	5397002	5070434	4947866	5474274
g-Oxalo-crotonate	17400000	12300000	23000000	23100000	16700000	24300000	31200000	23900000	22200000	20800000	25900000	11300000
Formyl-5-hydroxy-kynurenamine	4303696	1624258	2353268	5552108	4612482	5666338	6346780	5389814	6029606	10600000	13200000	7945914
Hydroxyindole-acetylglycine	29460.27	17027.29	27203.62	32826.7	46286.27	41925.05	51849.01	39880.16	28087.94	36204.3	42188.82	15222.45
Kynurenate	247151.8	331872.8	376158.8	217843.2	395637.8	313797.4	250834.6	279259.8	253730.4	265516.3	219812	282174.9
5-hydroxytryptophan	357986.8	408908.6	445912.7	408035.8	368746	436405.8	408396.4	395606.2	348659.9	362454.8	410447.4	202044
L-tryptophan	69214660	82505580	101126700	105818000	75851700	105405200	119800400	101846100	87619690	61683320	77534970	36570020
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	KEGG ID	m/z	RT
5-Hydroxyindoleacetate	C05635	192.0618	0.5667723
3-methyldioxyindole	C05834	164.0704	0.8904966
3-Methyleneoxindole	C02796	146.0603	1.742382
Indole	C00463	118.0656	1.748629
Indole-3-acetaldehyde	C00637	160.0761	1.733175
Indole-3-acetate	C00954	176.0708	1.934664
Indolepyruvate	C00331	204.0636	0.877594
Indoxyl	C05658	134.0603	0.8851032
kynurenine	C00328	209.0922	1.337228
N-formyl kynurenine	C02700	237.0872	1.390947
Anthranilate	C00108	138.0552	1.757466
Picolinic acid	C10164	124.0402	0.5641276
g-Oxalo-crotonate	C03453	159.03	0.8567833
Formyl-5-hydroxy-kynurenamine	C05647	209.0922	1.325983
Hydroxyindole-acetylglycine	C05832	249.085	1.591549
Kynurenate	C01717	190.0499	0.9183065
5-hydroxytryptophan	C00643	221.0923	1.048222
L-tryptophan	C00078	205.0975	1.736209
METABOLITES_END
#END