#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