#METABOLOMICS WORKBENCH nemkovt_20220112_130827 DATATRACK_ID:3034 STUDY_ID:ST002059 ANALYSIS_ID:AN003352 PROJECT_ID:PR001303
VERSION             	1
CREATED_ON             	January 12, 2022, 1:28 pm
#PROJECT
PR:PROJECT_TITLE                 	Unchecked oxidative stress is an insurmountable barrier for tumour cells that
PR:PROJECT_TITLE                 	disseminate to skeletal muscle
PR:PROJECT_SUMMARY               	Skeletal muscle has been recognized as an inhospitable site for disseminated
PR:PROJECT_SUMMARY               	tumour cells (DTCs) for decades, yet its antimetastatic nature has eluded a
PR:PROJECT_SUMMARY               	thorough mechanistic description. Here, we show that DTCs traffic to and persist
PR:PROJECT_SUMMARY               	within skeletal muscle, raising the question as to how this tissue suppresses
PR:PROJECT_SUMMARY               	colonization. We employed mouse and organotypic culture models along with
PR:PROJECT_SUMMARY               	metabolomic profiling and ultimately find that oxidative stress is a principal
PR:PROJECT_SUMMARY               	suppressor of DTC proliferation in skeletal muscle. DTCs bypassed this oxidative
PR:PROJECT_SUMMARY               	constraint upon colonization of more fertile sites, but were unable to in
PR:PROJECT_SUMMARY               	muscle. Functional studies demonstrated that disrupting redox homeostasis via
PR:PROJECT_SUMMARY               	chemogenetic induction of reactive oxygen species slowed proliferation in lung.
PR:PROJECT_SUMMARY               	Conversely, enhancing antioxidant potential of tumour cells via ectopic
PR:PROJECT_SUMMARY               	expression of catalase allowed robust colonization of skeletal muscle. These
PR:PROJECT_SUMMARY               	findings reveal a profound metabolic bottleneck imposed on DTCs and sustained by
PR:PROJECT_SUMMARY               	skeletal muscle. Understanding this biology could reveal novel DTC
PR:PROJECT_SUMMARY               	vulnerabilities.
PR:INSTITUTE                     	University of Colorado Anschutz Medical Campus
PR:LAST_NAME                     	Nemkov
PR:FIRST_NAME                    	Travis
PR:ADDRESS                       	12801 E 17th Avenue, RC-1 South, Rm 9121, Aurora, CO, 80045, USA
PR:EMAIL                         	travis.nemkov@cuanschutz.edu
PR:PHONE                         	303-724-3253
#STUDY
ST:STUDY_TITLE                   	4T1 and SkM cells
ST:STUDY_SUMMARY                 	4T1-parental and 4T1-SkM cells were analyzed by metabolomics
ST:INSTITUTE                     	University of Colorado Anschutz Medical Campus
ST:LAST_NAME                     	Nemkov
ST:FIRST_NAME                    	Travis
ST:ADDRESS                       	12801 E 17th Avenue, RC-1 South, Rm 9403G, Aurora, CO, 80045, USA
ST:EMAIL                         	travis.nemkov@cuanschutz.edu
ST:PHONE                         	303-724-3253
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#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           	-	P1-50K_R14+	Genotype:4T1-Parental	RAW_FILE_NAME=P1-50K_R14+.mzxml
SUBJECT_SAMPLE_FACTORS           	-	P2-50K_R6+	Genotype:4T1-Parental	RAW_FILE_NAME=P2-50K_R6+.mzxml
SUBJECT_SAMPLE_FACTORS           	-	P3-50K_R7+	Genotype:4T1-Parental	RAW_FILE_NAME=P3-50K_R7+.mzxml
SUBJECT_SAMPLE_FACTORS           	-	SK1-50K_R15+	Genotype:4T1-SkM	RAW_FILE_NAME=SK1-50K_R15+.mzxml
SUBJECT_SAMPLE_FACTORS           	-	SK2-50K_R10+	Genotype:4T1-SkM	RAW_FILE_NAME=SK2-50K_R10+.mzxml
SUBJECT_SAMPLE_FACTORS           	-	SK3-50K_R5+	Genotype:4T1-SkM	RAW_FILE_NAME=SK3-50K_R5+.mzxml
SUBJECT_SAMPLE_FACTORS           	-	P1-50K_R14-	Genotype:4T1-Parental	RAW_FILE_NAME=P1-50K_R14-.mzxml
SUBJECT_SAMPLE_FACTORS           	-	P2-50K_R6-	Genotype:4T1-Parental	RAW_FILE_NAME=P2-50K_R6-.mzxml
SUBJECT_SAMPLE_FACTORS           	-	P3-50K_R7-	Genotype:4T1-Parental	RAW_FILE_NAME=P3-50K_R7-.mzxml
SUBJECT_SAMPLE_FACTORS           	-	SK1-50K_R15-	Genotype:4T1-SkM	RAW_FILE_NAME=SK1-50K_R15-.mzxml
SUBJECT_SAMPLE_FACTORS           	-	SK2-50K_R10-	Genotype:4T1-SkM	RAW_FILE_NAME=SK2-50K_R10-.mzxml
SUBJECT_SAMPLE_FACTORS           	-	SK3-50K_R5-	Genotype:4T1-SkM	RAW_FILE_NAME=SK3-50K_R5-.mzxml
#COLLECTION
CO:COLLECTION_SUMMARY            	. 4T1-parental and 4T1-SkM cells were plated in 6-well plates at an initial
CO:COLLECTION_SUMMARY            	density of 50,000 cells/well and collected 48h later (final density ~275,000
CO:COLLECTION_SUMMARY            	cells/well). Wells were washed twice with 1x PBS and cells were scraped into 1.5
CO:COLLECTION_SUMMARY            	mL microcentrifuge tubes and spun at 16,000 x g for 3 minutes. Samples were then
CO:COLLECTION_SUMMARY            	flash frozen and stored at -80°C until ready to process.
CO:SAMPLE_TYPE                   	Breast cancer cells
#TREATMENT
TR:TREATMENT_SUMMARY             	A 4T1 subline derived from a rare skeletal muscle metastasis cells was serially
TR:TREATMENT_SUMMARY             	passaged through Balb/c mice to achieve a 4T1 derivative that effectively
TR:TREATMENT_SUMMARY             	colonized skeletal muscle (termed “4T1-SkM”)
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Prior to LC-MS analysis, tissue samples were weighed and resuspended in
SP:SAMPLEPREP_SUMMARY            	pre-chilled (-20°C) methanol:acetonitrile:water (5:3:2, v:v) to a final
SP:SAMPLEPREP_SUMMARY            	concentration of 30 mg/ml, cell samples were placed on ice and re-suspended with
SP:SAMPLEPREP_SUMMARY            	methanol:acetonitrile:water (5:3:2, v:v) at a concentration of 2x106 cells/mL,
SP:SAMPLEPREP_SUMMARY            	and media samples were extracted with the same solution at a dilution of 1:25
SP:SAMPLEPREP_SUMMARY            	(v/v). Suspensions were vortexed continuously for 30 min at 4°C. Insoluble
SP:SAMPLEPREP_SUMMARY            	material was removed by centrifugation at 18,000 g for 10 min at 4°C and
SP:SAMPLEPREP_SUMMARY            	supernatants were isolated for metabolomics analysis by UHPLC-MS.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um)
#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                   	The Q Exactive mass spectrometer (ThermoFisher) was operated independently in
MS:MS_COMMENTS                   	positive or negative ion mode, scanning in Full MS mode (2 scans) from 60 to
MS:MS_COMMENTS                   	900 m/z at 70,000 resolution, with 4 kV spray voltage, 45 sheath gas, 15
MS:MS_COMMENTS                   	auxiliary gas. Calibration was performed prior to analysis using the PierceTM
MS:MS_COMMENTS                   	Positive and Negative Ion Calibration Solutions (ThermoFisher). Acquired data
MS:MS_COMMENTS                   	was then converted from raw to mzXML file format using Mass Matrix (Cleveland,
MS:MS_COMMENTS                   	OH). Samples were analyzed in randomized order with a technical mixture injected
MS:MS_COMMENTS                   	periodically through analysis to qualify instrument performance. Metabolite
MS:MS_COMMENTS                   	assignments, isotopologue distributions, and correction for expected natural
MS:MS_COMMENTS                   	abundances of deuterium, 13C, and 15N isotopes were performed using MAVEN
MS:MS_COMMENTS                   	(Princeton, NJ)
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	AU
MS_METABOLITE_DATA_START
Samples	P1-50K_R14+	P2-50K_R6+	P3-50K_R7+	SK1-50K_R15+	SK2-50K_R10+	SK3-50K_R5+
Factors	Genotype:4T1-Parental	Genotype:4T1-Parental	Genotype:4T1-Parental	Genotype:4T1-SkM	Genotype:4T1-SkM	Genotype:4T1-SkM
L-alanine	1.67E+05	3.52E+05	1.70E+05	3.52E+05	3.78E+05	5.19E+05
L-arginine	1.02E+05	2.12E+05	1.95E+05	4.38E+05	1.53E+06	6.12E+05
L-asparagine	9.76E+03	6.88E+03	1.46E+04	7.10E+03	2.73E+04	8.59E+03
L-aspartate	8.88E+04	1.43E+05	9.42E+04	8.46E+04	1.09E+05	1.05E+05
L-cysteine	1.03E+04	2.16E+04	1.49E+04	2.66E+04	3.49E+04	3.98E+04
L-glutamate	9.38E+05	1.17E+06	8.68E+05	1.60E+06	1.06E+06	2.35E+06
glycine	2.09E+06	1.92E+06	2.15E+06	1.83E+06	2.06E+06	2.07E+06
L-histidine	1.10E+05	1.57E+05	1.08E+05	2.86E+05	3.16E+05	4.61E+05
L-leucine	2.06E+07	3.26E+07	1.94E+07	3.74E+07	4.69E+07	7.71E+07
L-isoleucine	1.24E+07	2.04E+07	1.23E+07	2.42E+07	1.34E+07	4.35E+07
L-lysine	1.56E+05	3.87E+05	2.00E+05	3.37E+05	6.55E+05	5.21E+05
L-methionine	3.56E+06	6.77E+06	3.98E+06	9.64E+06	1.19E+07	1.95E+07
L-phenylalanine	9.71E+06	1.99E+07	8.89E+06	1.98E+07	1.51E+07	2.99E+07
L-proline	7.63E+05	1.55E+06	8.87E+05	8.22E+05	8.00E+05	1.41E+06
L-serine	1.91E+04	1.04E+05	2.49E+04	2.66E+04	5.06E+04	5.86E+04
L-threonine	1.01E+05	2.03E+05	1.08E+05	1.98E+05	2.04E+05	2.99E+05
L-tryptophan	2.23E+06	5.60E+06	2.29E+06	4.33E+06	2.80E+06	5.61E+06
L-tyrosine	6.48E+06	1.08E+07	6.05E+06	1.13E+07	8.79E+06	2.11E+07
L-valine	5.73E+06	1.16E+07	6.67E+06	1.11E+07	7.03E+06	2.08E+07
Adenosine	1.91E+06	1.67E+06	1.48E+06	6.09E+06	3.09E+06	4.96E+06
Adenine	2.18E+05	2.54E+05	2.64E+05	1.33E+06	9.97E+05	1.15E+06
Guanosine	2.04E+06	2.90E+06	1.65E+06	1.21E+06	2.67E+06	2.05E+06
Guanine	8.16E+06	1.20E+07	7.08E+06	3.89E+06	4.82E+06	7.37E+06
Cytidine	2.51E+06	3.47E+06	1.65E+06	1.50E+06	2.58E+06	2.08E+06
Cytosine	1.01E+06	1.37E+06	6.73E+05	5.69E+05	1.00E+06	8.80E+05
Thymine	6.07E+04	9.17E+04	6.16E+04	5.89E+04	8.31E+04	8.83E+04
Uracil	1.38E+06	1.95E+06	1.23E+06	8.78E+05	2.32E+06	1.76E+06
Inosine	4.90E+06	6.40E+06	4.88E+06	4.31E+06	6.47E+06	5.64E+06
Hypoxanthine	4.51E+07	6.04E+07	4.04E+07	1.81E+07	2.76E+07	4.15E+07
Xanthosine	9.99E+03	6.64E+03	3.00E+03	8.93E+03	9.79E+03	9.21E+03
Xanthine	6.05E+04	1.15E+05	5.42E+04	8.31E+04	6.25E+04	1.50E+05
3',5'-Cyclic AMP	2.66E+04	6.10E+04	3.84E+04	1.83E+03	6.13E+03	0.00E+00
5-6-Dihydrothymine	4.62E+04	4.78E+04	4.85E+04	1.37E+05	5.97E+04	1.52E+05
Pyridoxal	2.59E+04	2.69E+04	3.01E+04	4.99E+04	6.05E+04	5.94E+04
Nicotinamide	1.06E+07	1.74E+07	1.28E+07	2.15E+07	2.75E+07	3.81E+07
Phosphate	4.42E+07	4.96E+07	4.79E+07	4.84E+07	4.86E+07	4.67E+07
Cys-Gly	2.20E+05	3.07E+05	2.02E+05	1.81E+05	1.00E+05	2.77E+05
gamma-L-Glutamyl-L-cysteine	1.23E+05	2.34E+05	1.37E+05	1.04E+05	1.06E+05	1.30E+05
gamma-L-Glutamyl-D-alanine	1.92E+04	2.38E+04	1.06E+04	3.74E+04	2.30E+04	5.16E+04
gamma-Glutamyl-gamma-aminobutyrate	2.22E+04	4.46E+04	3.92E+04	5.95E+04	8.76E+04	8.89E+04
gamma-L-Glutamylputrescine	0.00E+00	0.00E+00	0.00E+00	8.05E+03	5.78E+04	1.68E+04
L-Homocysteine	1.65E+03	4.37E+03	1.54E+03	1.54E+03	3.41E+03	9.37E+03
Cystathionine	0.00E+00	0.00E+00	1.34E+03	7.46E+03	5.93E+03	2.46E+04
Dimethylglycine	5.47E+04	5.85E+04	5.82E+04	5.86E+04	6.41E+04	1.13E+05
Betaine	7.33E+06	8.46E+06	6.67E+06	9.44E+06	6.67E+06	1.00E+07
S-Adenosyl-L-homocysteine	1.84E+05	2.83E+05	1.65E+05	1.72E+05	2.08E+05	3.46E+05
S-Adenosyl-L-methionine	1.22E+04	2.31E+04	2.51E+04	1.63E+04	1.78E+04	2.81E+04
Folate	1.22E+04	1.24E+04	1.10E+04	2.66E+04	3.24E+03	6.02E+04
Ornithine	6.27E+03	2.39E+05	1.82E+04	1.22E+04	9.58E+03	1.37E+04
5-Methylthioadenosine	1.85E+05	1.54E+05	1.36E+05	1.95E+05	9.41E+04	1.92E+05
Putrescine	1.76E+05	2.60E+05	1.94E+05	1.99E+05	9.28E+04	2.21E+05
Spermidine	2.83E+07	1.68E+07	1.08E+07	2.24E+07	4.39E+05	2.25E+07
Spermine	2.72E+06	1.20E+06	7.27E+05	2.48E+06	5.30E+04	2.10E+06
N1-Acetylspermidine	1.22E+05	1.98E+05	1.48E+05	7.15E+04	1.76E+05	7.00E+04
N-Acetylneuraminate	1.56E+04	3.67E+04	3.88E+04	3.23E+04	1.12E+05	4.71E+04
D-Glucosamine	5.48E+03	2.35E+04	1.02E+04	7.08E+03	1.00E+04	2.48E+04
Asymmetric dimethyl arginine	6.02E+03	1.24E+04	6.11E+03	1.33E+04	7.99E+04	4.88E+04
Creatine	6.76E+06	6.03E+06	4.86E+06	1.71E+07	5.16E+06	1.94E+07
Taurine	1.35E+05	1.81E+05	1.28E+05	2.56E+05	2.01E+05	2.64E+05
Hypotaurine	1.08E+04	1.10E+04	0.00E+00	3.91E+04	0.00E+00	4.99E+04
L-Methionine S-oxide	4.37E+04	5.55E+04	3.91E+04	6.87E+04	7.50E+04	9.86E+04
Kynurenine	7.34E+03	1.05E+04	5.62E+03	1.48E+04	6.36E+03	2.66E+04
Diethanolamine	1.37E+06	1.31E+06	1.30E+06	9.86E+05	1.99E+06	1.14E+06
Triethanolamine	1.07E+07	9.73E+06	8.99E+06	8.19E+06	5.58E+06	1.04E+07
N-Methylethanolamine phosphate	1.30E+04	1.04E+04	2.54E+04	2.35E+04	4.35E+04	2.52E+04
L-Carnitine	1.31E+06	1.54E+06	1.19E+06	2.10E+06	9.66E+05	2.55E+06
Acetylcarnitine	8.79E+05	1.06E+06	8.69E+05	1.86E+06	9.91E+05	2.76E+06
Butyrylcarnitine	5.80E+05	4.59E+05	4.74E+05	1.75E+06	5.74E+05	2.26E+06
Valerylcarnitine	2.98E+05	2.77E+05	2.00E+05	1.77E+05	1.68E+05	2.44E+05
Malonylcarnitine	1.18E+04	8.27E+03	8.31E+03	8.71E+03	9.13E+03	1.23E+04
Hexanoylcarnitine	5.74E+04	7.90E+04	7.66E+04	3.93E+04	6.59E+04	8.59E+04
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	CmpdID	parent	medRt
L-alanine	C00041	90.0550	0.67
L-arginine	C00062	175.1190	0.74
L-asparagine	C00152	133.0608	0.89
L-aspartate	C00049	134.0448	0.66
L-cysteine	C00097	122.0270	0.69
L-glutamate	C00025	148.0604	0.67
glycine	C00037	76.0393	0.85
L-histidine	C00135	156.0768	0.86
L-leucine	C00123	132.1019	1.03
L-isoleucine	C00407	132.1019	0.97
L-lysine	C00047	147.1128	0.62
L-methionine	C00073	150.0584	0.88
L-phenylalanine	C00079	166.0863	1.39
L-proline	C00148	116.0706	0.70
L-serine	C00065	106.0499	0.66
L-threonine	C00188	120.0655	0.67
L-tryptophan	C00078	205.0972	1.72
L-tyrosine	C00082	182.0812	0.91
L-valine	C00183	118.0862	0.83
Adenosine	C00212	268.1035	0.91
Adenine	C00147	136.0619	0.83
Guanosine	C00387	284.0985	0.94
Guanine	C00242	152.0567	0.82
Cytidine	C00475	244.0925	0.81
Cytosine	C00380	112.0505	0.84
Thymine	C00178	127.0502	1.44
Uracil	C00106	113.0350	0.90
Inosine	C00294	269.0876	0.95
Hypoxanthine	C00262	137.0459	0.90
Xanthosine	C01762	285.0826	1.06
Xanthine	C00385	153.0407	0.89
3',5'-Cyclic AMP	C00575	330.0601	0.91
5-6-Dihydrothymine	C00906	129.0658	0.73
Pyridoxal	C00250	168.0657	0.90
Nicotinamide	C00153	123.0552	0.89
Phosphate	C00009	98.9841	0.69
Cys-Gly	C01419	179.0484	0.89
gamma-L-Glutamyl-L-cysteine	C00669	251.0695	0.90
gamma-L-Glutamyl-D-alanine	C03738	219.0975	0.86
gamma-Glutamyl-gamma-aminobutyrate	C15767	233.1132	0.87
gamma-L-Glutamylputrescine	C15699	218.1499	0.71
L-Homocysteine	C00155	136.0429	0.75
Cystathionine	C00542	223.0744	1.00
Dimethylglycine	C01026	104.0706	0.77
Betaine	C00719	118.0861	0.68
S-Adenosyl-L-homocysteine	C00021	385.1282	0.89
S-Adenosyl-L-methionine	C00019	399.1441	0.66
Folate	C00504	442.1470	1.71
Ornithine	C01602	133.0972	0.60
5-Methylthioadenosine	C00170	298.0970	1.70
Putrescine	C00134	89.1073	0.61
Spermidine	C00315	146.1652	0.59
Spermine	C00750	203.2229	0.60
N1-Acetylspermidine	C00612	188.1756	0.65
N-Acetylneuraminate	C00270	310.1127	0.71
D-Glucosamine	C00329	180.0874	0.79
Asymmetric dimethyl arginine	C03626	203.1503	0.76
Creatine	C00300	132.0768	0.69
Taurine	C00245	126.0219	0.68
Hypotaurine	C00519	110.0269	0.67
L-Methionine S-oxide	C02989	166.0533	0.87
Kynurenine	C00328	209.0918	1.36
Diethanolamine	C06772	106.0862	0.67
Triethanolamine	C06771	150.1125	0.70
N-Methylethanolamine phosphate	C01210	156.0421	0.64
L-Carnitine	C00318	162.1124	0.68
Acetylcarnitine	HMDB0000201	204.1230	0.91
Butyrylcarnitine	HMDB0002013	232.1542	1.73
Valerylcarnitine	HMDB0013128	246.1697	1.81
Malonylcarnitine	HMDB0062592	248.1125	1.11
Hexanoylcarnitine	HMDB0000705	260.1853	1.85
METABOLITES_END
#END