#METABOLOMICS WORKBENCH nemkovt_20220112_130827 DATATRACK_ID:3034 STUDY_ID:ST002059 ANALYSIS_ID:AN003353 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 NEGATIVE 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-glutamine 6.74E+04 9.53E+04 6.79E+04 1.72E+05 1.81E+05 2.62E+05 ATP 5.09E+04 4.66E+04 4.29E+04 6.41E+04 3.98E+04 5.38E+04 ADP 8.40E+04 8.27E+04 8.43E+04 1.01E+05 6.71E+04 1.08E+05 AMP 1.81E+05 3.13E+05 3.14E+05 1.74E+05 6.07E+04 2.60E+05 GTP 6.08E+03 7.30E+03 3.80E+03 7.51E+03 3.67E+03 9.47E+03 GDP 2.00E+04 1.92E+04 1.81E+04 2.06E+04 1.06E+04 2.11E+04 GMP 1.84E+04 3.19E+04 3.12E+04 9.83E+03 3.55E+03 1.21E+04 CTP 2.80E+03 3.56E+03 4.58E+03 9.91E+03 8.90E+02 5.56E+03 CDP 1.82E+04 3.13E+04 1.57E+04 4.00E+04 9.03E+03 5.34E+04 CMP 2.84E+04 2.35E+04 3.42E+04 1.59E+04 2.75E+04 9.53E+03 UTP 1.74E+04 1.81E+04 2.28E+04 1.73E+04 1.64E+04 1.64E+04 UDP 3.47E+04 2.81E+04 3.27E+04 3.80E+04 2.01E+04 4.66E+04 UMP 2.12E+04 3.74E+04 2.69E+04 7.52E+03 6.37E+03 8.46E+03 Urate 4.18E+03 2.83E+04 6.27E+03 1.14E+04 4.00E+03 2.20E+04 UDP-glucose 9.86E+04 1.03E+05 8.59E+04 8.48E+04 1.17E+05 8.32E+04 ADP-D-ribose 4.31E+03 1.26E+04 8.61E+03 5.18E+03 1.53E+04 1.03E+04 Flavin mononucleotide 3.06E+03 2.24E+03 1.35E+03 8.02E+03 5.71E+03 7.32E+03 FAD 1.54E+04 1.92E+04 1.51E+04 2.57E+04 3.40E+04 3.00E+04 FADH2 2.07E+03 7.16E+02 1.16E+03 1.91E+03 3.20E+03 2.41E+03 NAD+ 1.06E+05 1.18E+05 1.06E+05 4.81E+04 1.45E+04 3.95E+04 NADH 5.98E+03 6.95E+03 1.21E+03 2.05E+03 4.63E+03 1.21E+03 Diphosphate 3.26E+07 2.58E+07 2.57E+07 2.89E+07 1.99E+07 3.37E+07 D-Glucose 5.03E+04 1.17E+05 8.54E+04 2.69E+05 1.45E+05 4.68E+05 D-Hexose-phosphate 1.67E+05 3.90E+05 2.62E+05 3.89E+05 5.10E+05 5.83E+05 D-Fructose 1-6-bisphosphate 2.20E+04 3.18E+04 1.07E+04 5.89E+04 5.28E+04 1.22E+05 D-Glyceraldehyde 3-phosphate/Glycerone phosphate 2.95E+04 1.80E+04 1.02E+04 1.64E+05 9.30E+04 2.66E+05 1-3-Bisphosphoglycerate 3.54E+04 3.41E+04 3.47E+04 3.46E+04 3.33E+04 5.46E+04 2/3-Phospho-D-glycerate 4.03E+05 5.73E+05 3.31E+05 6.31E+05 1.26E+05 1.07E+06 Phosphoenolpyruvate 8.21E+04 4.16E+04 2.81E+04 1.07E+05 1.48E+04 2.64E+05 Pyruvate 1.35E+05 1.75E+05 1.18E+05 1.48E+05 1.31E+05 1.65E+05 Lactate 3.36E+06 1.21E+07 3.34E+06 8.20E+06 4.55E+06 1.38E+07 Citrate 1.50E+05 1.36E+05 1.43E+05 1.65E+05 1.42E+05 1.76E+05 2-Oxoglutarate 5.24E+03 1.04E+04 5.11E+03 2.73E+04 3.68E+03 3.00E+04 Succinate 4.37E+05 6.60E+05 4.51E+05 6.54E+05 3.29E+05 1.11E+06 Fumarate 7.48E+04 7.87E+04 5.29E+04 1.06E+05 5.58E+04 1.76E+05 Malate 5.23E+05 5.47E+05 3.71E+05 9.16E+05 3.32E+05 1.55E+06 2-Hydroxyglutarate 2.35E+04 2.81E+04 1.78E+04 2.51E+04 1.44E+04 4.72E+04 6-Phospho-D-gluconate 1.07E+04 1.07E+04 4.63E+03 4.07E+04 2.29E+04 4.64E+04 Sedoheptulose 7-phosphate 6.33E+04 1.19E+05 4.78E+04 1.98E+04 6.14E+04 8.54E+04 Pentose phosphates 9.73E+05 1.04E+06 6.70E+05 5.02E+05 9.41E+05 8.38E+05 Glutathione 2.34E+06 2.84E+06 1.97E+06 1.53E+06 1.03E+06 1.72E+06 Glutathione disulfide 5.42E+04 4.79E+04 3.73E+04 4.23E+04 4.61E+04 5.63E+04 5-Oxoproline 8.30E+05 3.70E+06 8.12E+05 2.13E+06 9.38E+05 3.47E+06 S-Glutathionyl-L-cysteine 1.96E+03 1.77E+03 0.00E+00 4.27E+03 6.59E+03 7.60E+03 Phosphoserine 7.70E+03 6.90E+03 4.57E+03 1.96E+04 1.49E+04 2.56E+04 alpha-D-Glucosamine 1-phosphate 6.49E+03 3.56E+02 0.00E+00 0.00E+00 1.83E+04 5.30E+03 Glucoronate/Galacturonate/Fructuronate/Iduronate/Mannuronate 7.04E+02 4.14E+02 1.04E+03 5.44E+03 1.94E+04 1.04E+04 UDP-D-glucuronate 3.53E+04 4.29E+04 3.14E+04 2.08E+04 2.28E+04 3.57E+04 Arabinose/Xylulose/Xylose 8.96E+03 1.57E+04 9.19E+03 1.66E+04 2.54E+04 2.25E+04 UDP-N-acetyl-D-glucosamine 7.64E+04 8.84E+04 6.38E+04 7.51E+04 1.27E+05 7.08E+04 N-acetyl-D-glucosamine 1.91E+04 1.54E+04 1.22E+04 8.94E+03 2.30E+04 9.16E+03 Pantothenate 1.45E+05 1.85E+05 1.23E+05 3.24E+05 1.45E+05 4.36E+05 Pantetheine 4--phosphate 3.91E+04 4.68E+04 2.35E+04 1.15E+05 2.18E+05 2.11E+05 Pantetheine 1.29E+03 2.63E+03 2.80E+03 2.62E+03 8.79E+03 4.96E+03 sn-glycero-3-Phosphoethanolamine 3.68E+03 7.26E+03 3.07E+03 1.01E+04 9.52E+03 1.45E+04 Glycerol 3-phosphate 2.60E+04 3.46E+04 2.00E+04 8.54E+03 8.10E+03 1.98E+04 Phosphoethanolamine 5.19E+04 7.55E+04 5.01E+04 0.00E+00 0.00E+00 0.00E+00 Dodecanoic acid 2.07E+05 1.90E+05 1.65E+05 1.67E+05 1.80E+05 1.86E+05 Tetradecanoic acid 4.10E+05 4.60E+05 4.90E+05 4.65E+05 8.70E+05 5.48E+05 Hexadecanoic acid 5.42E+06 5.80E+06 7.62E+06 5.65E+06 8.27E+06 7.56E+06 Octadecanoic acid 2.76E+06 3.35E+06 4.69E+06 3.39E+06 5.33E+06 4.01E+06 Tetradecenoic acid 1.92E+04 1.41E+04 1.60E+04 1.18E+04 1.92E+04 1.66E+04 Octadecenoic acid 4.50E+05 5.44E+05 5.64E+05 9.94E+05 2.92E+06 1.52E+06 Octadecadienoic acid 7.67E+04 8.24E+04 7.29E+04 6.81E+04 1.92E+05 1.16E+05 Octadecatrienoic acid 4.44E+03 4.84E+03 4.20E+03 3.98E+03 1.27E+04 6.48E+03 Eicosatetraenoic acid 9.76E+04 1.10E+05 9.36E+04 1.24E+05 4.26E+05 2.39E+05 Eicosapentaenoic acid 1.08E+04 9.72E+03 9.07E+03 1.03E+04 3.57E+04 1.45E+04 Docosahexaenoic acid 4.30E+04 4.48E+04 3.70E+04 4.06E+04 1.56E+05 5.89E+04 3-Phospho-D-glyceroyl phosphate 3.54E+04 3.41E+04 3.47E+04 3.46E+04 3.33E+04 5.46E+04 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name CmpdID parent medRt L-glutamine C00064 145.0608 0.67 ATP C00002 505.9882 3.81 ADP C00008 426.0224 3.81 AMP C00020 346.0557 0.90 GTP C00044 521.9832 3.88 GDP C00035 442.0170 3.82 GMP C00144 362.0509 1.26 CTP C00063 481.9771 1.83 CDP C00112 402.0108 0.79 CMP C00055 322.0443 0.61 UTP C00075 482.9610 1.92 UDP C00015 402.9948 3.14 UMP C00105 323.0287 1.38 Urate C00366 167.0201 0.86 UDP-glucose C00029 565.0479 0.76 ADP-D-ribose C01882 558.0645 0.75 Flavin mononucleotide C00061 455.0978 1.70 FAD C00016 784.1509 1.69 FADH2 C01352 786.1562 1.67 NAD+ C00003 662.1023 0.87 NADH C00004 664.1122 0.86 Diphosphate C00013 176.9350 0.60 D-Glucose C00031 179.0556 0.67 D-Hexose-phosphate C02965 259.0223 0.60 D-Fructose 1-6-bisphosphate C00354 338.9886 2.74 D-Glyceraldehyde 3-phosphate/Glycerone phosphate C00118 168.9893 0.59 1-3-Bisphosphoglycerate C00236 264.9520 3.12 2/3-Phospho-D-glycerate C00631 184.9849 0.95 Phosphoenolpyruvate C00074 166.9742 1.32 Pyruvate C00022 87.0073 0.76 Lactate C01432 89.0230 0.77 Citrate C00158 191.0190 3.89 2-Oxoglutarate C00026 145.0142 0.72 Succinate C00042 117.0179 0.77 Fumarate C00122 115.0023 0.80 Malate C00149 133.0130 0.54 2-Hydroxyglutarate C02630 147.0291 0.76 6-Phospho-D-gluconate C00345 275.0173 0.88 Sedoheptulose 7-phosphate C05382 289.0330 0.61 Pentose phosphates C00199 229.0114 0.61 Glutathione C00051 306.0771 0.76 Glutathione disulfide C00127 611.1448 0.78 5-Oxoproline C01879 128.0341 0.77 S-Glutathionyl-L-cysteine C05526 425.0773 1.06 Phosphoserine C02532 184.0006 0.59 alpha-D-Glucosamine 1-phosphate C06156 258.0380 0.65 Glucoronate/Galacturonate/Fructuronate/Iduronate/Mannuronate C00191 193.0345 0.56 UDP-D-glucuronate C00167 579.0271 0.79 Arabinose/Xylulose/Xylose C00259 149.0459 0.89 UDP-N-acetyl-D-glucosamine C00043 606.0746 0.77 N-acetyl-D-glucosamine C00140 220.0825 0.77 Pantothenate C00864 218.1031 1.00 Pantetheine 4--phosphate C01134 357.0892 1.06 Pantetheine C00831 277.1224 1.72 sn-glycero-3-Phosphoethanolamine C01233 214.0479 0.91 Glycerol 3-phosphate C00093 171.0070 0.89 Phosphoethanolamine C00346 140.0106 0.61 Dodecanoic acid C02679 199.1697 2.33 Tetradecanoic acid C06424 227.2014 2.56 Hexadecanoic acid C00249 255.2329 2.91 Octadecanoic acid C01530 283.2643 3.40 Tetradecenoic acid C08322 225.1857 2.37 Octadecenoic acid C00712 281.2487 2.94 Octadecadienoic acid C01595 279.2331 2.64 Octadecatrienoic acid C06427 277.2174 2.47 Eicosatetraenoic acid C00219 303.2329 2.55 Eicosapentaenoic acid C06428 301.2174 2.40 Docosahexaenoic acid C06429 327.2332 2.46 3-Phospho-D-glyceroyl phosphate C00236 264.9520 3.12 METABOLITES_END #END