#METABOLOMICS WORKBENCH juliehaines_20240918_063241 DATATRACK_ID:5204 STUDY_ID:ST003489 ANALYSIS_ID:AN005728 PROJECT_ID:PR002140
VERSION             	1
CREATED_ON             	September 18, 2024, 7:13 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                    	Core director Angelo D'Alessandro, study PI 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 pathway profiling of conditioned media from TNBC cell line MDA-MB-231
ST:STUDY_TITLE                   	+/- inhibitors of the TDO/IDO pathway.
ST:STUDY_SUMMARY                 	Conditioned media harvested from TNBC cell line MDA-MB-231 with and without
ST:STUDY_SUMMARY                 	treatment with TDO2/IDO1 dual inhibitor AT-0174 and IDO1 inhibitor epacadostat.
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           	-	AA90-M231-1	treatment:Vehicle | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=AA90-M231-1
SUBJECT_SAMPLE_FACTORS           	-	AA90-M231-2	treatment:Vehicle | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=AA90-M231-2
SUBJECT_SAMPLE_FACTORS           	-	AA90-M231-3	treatment:Vehicle | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=AA90-M231-3
SUBJECT_SAMPLE_FACTORS           	-	AA90-M231-4	treatment:10uM AT0174 | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=AA90-M231-4
SUBJECT_SAMPLE_FACTORS           	-	AA90-M231-5	treatment:10uM AT0174 | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=AA90-M231-5
SUBJECT_SAMPLE_FACTORS           	-	AA90-M231-6	treatment:10uM AT0174 | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=AA90-M231-6
SUBJECT_SAMPLE_FACTORS           	-	AA90-M231-7	treatment:10uM Epacadostat | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=AA90-M231-7
SUBJECT_SAMPLE_FACTORS           	-	AA90-M231-8	treatment:10uM Epacadostat | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=AA90-M231-8
SUBJECT_SAMPLE_FACTORS           	-	AA90-M231-9	treatment:10uM Epacadostat | Sample source:conditioned media	RAW_FILE_NAME(raw file name)=AA90-M231-9
#COLLECTION
CO:COLLECTION_SUMMARY            	At the end of the experimental timecourse, media were harvested and centrifuged
CO:COLLECTION_SUMMARY            	at 1500 rpm, 4℃. The supernatants were collected and frozen at -80℃ prior to
CO:COLLECTION_SUMMARY            	metabolomics analysis.
CO:SAMPLE_TYPE                   	Culture Media
#TREATMENT
TR:TREATMENT_SUMMARY             	The TNBC cell line MDA-MB-231 was cultured for 24 hours then treated with DMSO
TR:TREATMENT_SUMMARY             	(control), 10 µM AT-0174 or 10 µM epacadostat for 48 hours. Culture media was
TR:TREATMENT_SUMMARY             	not changed during the duration of the experiments.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Media aliquots were thawed on ice then metabolites extracted from a 10 uL
SP:SAMPLEPREP_SUMMARY            	aliquot using 240 uL of cold 5:3:2 MeOH:acetonitrile:water. Samples were
SP:SAMPLEPREP_SUMMARY            	vortexed 30 min at 4 degrees C then supernatants clarified by centrifugation (10
SP:SAMPLEPREP_SUMMARY            	min, 10,000 g, 4 degrees C). A 150 uL of supernatant was dried using a speedvac
SP:SAMPLEPREP_SUMMARY            	then resuspended in 150 uL of 0.1% formic acid and transferred to autosampler
SP:SAMPLEPREP_SUMMARY            	vials.
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              	10 uL
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Orbitrap Exploris 120
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	We use a Thermo Orbitrap Exploris 120. Resolution 120,000, scan range 65-975
MS:MS_COMMENTS                   	m/z, maximum injection time 100 ms, microscans 1, automatic gain control (AGC)
MS:MS_COMMENTS                   	detection duration 20 msec, source voltage 3.5 kV, capillary temperature 320 C,
MS:MS_COMMENTS                   	vaporizer temp 200 C, and sheath gas 50, auxiliary gas 10, and sweep gas 1 (all
MS:MS_COMMENTS                   	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	AA90-M231-1	AA90-M231-2	AA90-M231-3	AA90-M231-4	AA90-M231-5	AA90-M231-6	AA90-M231-7	AA90-M231-8	AA90-M231-9
Factors	treatment:Vehicle | Sample source:conditioned media	treatment:Vehicle | Sample source:conditioned media	treatment:Vehicle | Sample source:conditioned media	treatment:10uM AT0174 | Sample source:conditioned media	treatment:10uM AT0174 | Sample source:conditioned media	treatment:10uM AT0174 | Sample source:conditioned media	treatment:10uM Epacadostat | Sample source:conditioned media	treatment:10uM Epacadostat | Sample source:conditioned media	treatment:10uM Epacadostat | Sample source:conditioned media
L-tryptophan	31292354	31103488	33853740	58413684	67764552	66651572	30845518	25539704	22852898
5-Hydroxyindoleacetate	8356312	9219125	9943541	6359090	7236370	7932280	10445584	8945125	9311665
3-Methyleneoxindole	308098	320594	326180	624078	688217	598747	287896	268434	239343
Indole-3-acetaldehyde	927350	894774	1011945	860051	946350	934462	976407	833005	764581
Indole-3-acetate	11844325	10881296	12814083	10128045	13371160	12938152	12242081	9549732	8347080
Indoxyl	712127	748455	706803	714810	758057	882641	790029	653086	661689
5-hydroxytryptophan	809606	778747	926784	692409	856075	830430	920578	685033	677585
kynurenine	36824784	38962892	41959956	28586208	31733554	30490298	43555296	38784884	36420048
N-formyl kynurenine	3868778	3879298	4466581	2688372	3035013	2997742	4172619	3416164	3093764
Anthranilate	21055676	24237514	25260006	18662134	19611090	19940924	26898814	22919814	20083426
2-Aminomuconate	123326	73409	89276	54844	152109	50335	94379	79649	60280
Kynurenate	250586	248224	288567	223798	230473	212341	278782	229880	213129
8-Methoxykynurenate	103483	162124	98213	81539	75535	76464	91254	194082	91536
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	KEGG ID	m/z	RT
L-tryptophan	C00078	205.0971	1.7170
5-Hydroxyindoleacetate	C05635	192.06546	1.355
3-Methyleneoxindole	C02796	146.060074	1.713
Indole-3-acetaldehyde	C00637	160.075684	1.211
Indole-3-acetate	C00954	176.070587	1.916
Indoxyl	C05658	134.060104	0.824
5-hydroxytryptophan	C00643	221.092102	1.022
kynurenine	C00328	209.092056	1.352
N-formyl kynurenine	C02700	237.086975	1.41
Anthranilate	C00108	138.055023	1.855
2-Aminomuconate	C02220	158.044754	0.823
Kynurenate	C01717	190.049835	1.735
8-Methoxykynurenate	C05830	220.06041	1.853
METABOLITES_END
#END