#METABOLOMICS WORKBENCH mdm_20250120_021247 DATATRACK_ID:5544 STUDY_ID:ST003681 ANALYSIS_ID:AN006042 PROJECT_ID:PR002283 VERSION 1 CREATED_ON January 21, 2025, 1:09 pm #PROJECT PR:PROJECT_TITLE Ketogenic diet suppresses colorectal cancer through the gut microbiome long PR:PROJECT_TITLE chain fatty acid stearate PR:PROJECT_SUMMARY Our manuscript entitled “Ketogenic diet suppresses colorectal cancer through PR:PROJECT_SUMMARY the gut microbiome long chain fatty acid stearate” describes a reduced colonic PR:PROJECT_SUMMARY tumor burden upon ketogenic diet (KD) consumption in a CRC mouse model with a PR:PROJECT_SUMMARY humanized microbiome. Importantly, we demonstrate a causal relationship through PR:PROJECT_SUMMARY microbiome transplantation into germ-free mice, whereby alterations in the gut PR:PROJECT_SUMMARY microbiota were maintained in the absence of continued selective pressure from PR:PROJECT_SUMMARY the KD. Specifically, we identify a shift toward bacterial species that produce PR:PROJECT_SUMMARY stearic acid in ketogenic conditions, whereas consumers were depleted, resulting PR:PROJECT_SUMMARY in elevated levels of free stearate in the gut lumen. This microbial product PR:PROJECT_SUMMARY demonstrates tumor-suppressing properties by inducing apoptosis in cancer cells PR:PROJECT_SUMMARY and decreasing colonic Th17 immune cell populations. As part of this study, we PR:PROJECT_SUMMARY used different metabolomics workflows to study metabolites in mouse fecal and PR:PROJECT_SUMMARY plasma samples as well as the used rodent diet and human feces samples. PR:INSTITUTE University of Luxembourg PR:LAST_NAME Letellier PR:FIRST_NAME Elisabeth PR:ADDRESS 6, avenue du Swing, Belval, Esch, 4367, Luxembourg PR:EMAIL madita.brauer@uni.lu PR:PHONE (+352) 46 66 44 6954 #STUDY ST:STUDY_TITLE Ketogenic diet suppresses colorectal cancer through the gut microbiome long ST:STUDY_TITLE chain fatty acid stearate - untargeted LCMS data from human fecal samples ST:STUDY_SUMMARY Stool samples from healthy human donors were analyzed by a LC-MS. Metabolites ST:STUDY_SUMMARY were extracted from human stool samples as described in the method file. ST:STUDY_SUMMARY Resulting samples were used for LC-MS untargeted metabolomics screen using a ST:STUDY_SUMMARY Vanquish UHPLC (ThermoFisher Scientific), coupled to a Q Exactive HF mass ST:STUDY_SUMMARY spectrometer (ThermoFisher Scientific). ST:INSTITUTE University of Luxembourg ST:LAST_NAME Letellier ST:FIRST_NAME Elisabeth ST:ADDRESS 6, avenue du Swing, Belval, Esch, 4367, Luxembourg ST:EMAIL madita.brauer@uni.lu ST:PHONE (+352) 46 66 44 6954 #SUBJECT SU:SUBJECT_TYPE Mammal 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 HD_2 HD_2 Sample source:human feces | Condition:healthy donor RAW_FILE_NAME(raw_file_name)=MT002_LC-EMunt_Feces_HD_2.mzML SUBJECT_SAMPLE_FACTORS HD_3 HD_3 Sample source:human feces | Condition:healthy donor RAW_FILE_NAME(raw_file_name)=MT002_LC-EMunt_Feces_HD_3.mzML SUBJECT_SAMPLE_FACTORS HD_4 HD_4 Sample source:human feces | Condition:healthy donor RAW_FILE_NAME(raw_file_name)=MT002_LC-EMunt_Feces_HD_4.mzML SUBJECT_SAMPLE_FACTORS HD_1 HD_1 Sample source:human feces | Condition:healthy donor RAW_FILE_NAME(raw_file_name)=MT002_LC-EMunt_Feces_HD_1.mzML SUBJECT_SAMPLE_FACTORS HD_5 HD_5 Sample source:human feces | Condition:healthy donor RAW_FILE_NAME(raw_file_name)=MT002_LC-EMunt_Feces_HD_5.mzML #COLLECTION CO:COLLECTION_SUMMARY Human samples were obtained and handled in accordance with institutional CO:COLLECTION_SUMMARY guidelines. All human samples used in this project were donated freely, upon CO:COLLECTION_SUMMARY informed consent. Samples were collected at the donors’ homes in feces CO:COLLECTION_SUMMARY collection tubes, then aliquoted and stored at -80°C until further use. CO:SAMPLE_TYPE Feces CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY A humanized gut microbiome mouse model of CRC was established through the TR:TREATMENT_SUMMARY transplantation of stool samples from five healthy human donors into germ-free TR:TREATMENT_SUMMARY and antibiotic-treated mice. Prior to this, the stool samples of the five TR:TREATMENT_SUMMARY healthy donors used where characterized by metagenomics sequencing and an TR:TREATMENT_SUMMARY untargeted metabolomics approach. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Circa 1g of frozen human fecal material was placed in a microvial pre-cooled in SP:SAMPLEPREP_SUMMARY liquid nitrogen. The samples were milled in a cryogenic grinder (6875D SP:SAMPLEPREP_SUMMARY Freezer/Mill®, SPEX® SamplePrep, Instrument Solutions; four minutes of SP:SAMPLEPREP_SUMMARY precooling, followed by three cycles of two-minute-long runtime at a rate of 12 SP:SAMPLEPREP_SUMMARY cycles per second, spaced by one minute of cooling), reducing them to a fine SP:SAMPLEPREP_SUMMARY powder. Upon removal from the grinder, the samples were maintained in liquid SP:SAMPLEPREP_SUMMARY nitrogen, aliquoted and then stored at -80°C until processing. The samples were SP:SAMPLEPREP_SUMMARY not thawed at any time during homogenization. 50mg of homogenized fecal powder SP:SAMPLEPREP_SUMMARY was resuspended in 500μL of MilliQ® water and further homogenized at 6000rpm SP:SAMPLEPREP_SUMMARY for 30 seconds at 4°C in a ThermoMixer. Samples were centrifuged at maximum SP:SAMPLEPREP_SUMMARY speed for five minutes at 4°C. An internal standard mix (ISM) was prepared SP:SAMPLEPREP_SUMMARY (1mg/mL of 6-chloropurine riboside (Sigma-Aldrich), SP:SAMPLEPREP_SUMMARY 2-chloroquinoline-3-carboxylic acid (Sigma-Aldrich), 4-chloro-DL-phenylalanine SP:SAMPLEPREP_SUMMARY (Sigma-Aldrich), Nε-trifluoroacetyl-L-lysine(Sigma-Aldrich), sucralose SP:SAMPLEPREP_SUMMARY (Sigma-Aldrich), caffeine-trimethyl (Sigma-Aldrich) in MilliQ® water). SP:SAMPLEPREP_SUMMARY Supernatants were collected and filled up to 75μL with MilliQ water, then 75μL SP:SAMPLEPREP_SUMMARY of the ISM were added to each sample and they were vortexed thoroughly. Proteins SP:SAMPLEPREP_SUMMARY were precipitated by the addition of 600μL of ACN (Carl ROTH) +1% formic acid SP:SAMPLEPREP_SUMMARY (v/v, Sigma-Aldrich) at 4°C to 120μL of sample and then vortexed thoroughly. SP:SAMPLEPREP_SUMMARY Samples were incubated for five minutes at 4°C in a ThermoMixer Comfort SP:SAMPLEPREP_SUMMARY (Eppendorf), then centrifuged for five minutes at maximum speed at 4°C. SP:SAMPLEPREP_SUMMARY Phospholipids were removed by transferring the supernatants to a phospholipid SP:SAMPLEPREP_SUMMARY removal plate (Phree) and vacuum for 2-7 inches Hg was applied until the SP:SAMPLEPREP_SUMMARY filtrate collected in the deep well plate. 500μL were transferred into 1.5mL SP:SAMPLEPREP_SUMMARY Eppendorf tubes and the solvents were evaporated in a rotary vacuum evaporator SP:SAMPLEPREP_SUMMARY at 4°C overnight. Then, the temperature of the rotary vacuum evaporator was SP:SAMPLEPREP_SUMMARY increased to room temperature for 25 minutes to avoid water condensation on the SP:SAMPLEPREP_SUMMARY surface of the tube. Samples were stored at -80°C until LC-MS analysis was SP:SAMPLEPREP_SUMMARY performed. An eluent was prepared (20mM ammonium acetate (AmAc, Sigma-Aldrich) SP:SAMPLEPREP_SUMMARY in 90% ACN+ 0.1% formic acid). Samples were reconstituted in 50μL of a 5% SP:SAMPLEPREP_SUMMARY solution of the eluent in MilliQ® water, then filtered through PHENEX-RC 4mm SP:SAMPLEPREP_SUMMARY syringe filters (Phenomenex) into glass LC vials (Chromatographie Zubehör SP:SAMPLEPREP_SUMMARY Trott) with microinserts (Chromatographie Zubehör Trott) and screwcaps SP:SAMPLEPREP_SUMMARY (Chromatographie Zubehör Trott). 5μL per sample were pooled in a separate SP:SAMPLEPREP_SUMMARY vial. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME SeQuant ZIC- pHILIC (150 x 2.1mm,5um) CH:SOLVENT_A 100% water, 0.01% formic acid CH:SOLVENT_B 90% acetonitrile, 0.1% formic acid, 20 mM ammonium acetate CH:FLOW_GRADIENT 90 % B for 1.5 min, followed by a decrease to 20% B within 15 min, then 20% B CH:FLOW_GRADIENT for 2 min, increase to 90% B within 2 min, then 90% B for 13 min CH:FLOW_RATE 200 µl/ml CH:COLUMN_TEMPERATURE 50°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF-X Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE UNSPECIFIED MS:MS_COMMENTS Resolution - 120,000, m/z range - 60-900, AGC target - 1e6, maximum injection MS:MS_COMMENTS time – 70 ms. ddMS2 was applied using the following settings: Resolution - MS:MS_COMMENTS 30,000, AGC target - 5e5, maximum injection time – 70, topN - 5, Normalized MS:MS_COMMENTS collision energy – 20. Samples were acquired in positive and negative MS:MS_COMMENTS ionization mode simultaneously (polarity switching). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS area MS_METABOLITE_DATA_START Samples HD_2 HD_3 HD_4 HD_1 HD_5 Factors Sample source:human feces | Condition:healthy donor Sample source:human feces | Condition:healthy donor Sample source:human feces | Condition:healthy donor Sample source:human feces | Condition:healthy donor Sample source:human feces | Condition:healthy donor Succinic acid 250644684.2 234072612.9 194441518.9 161453373.6 333557973.5 Niacin 31772705.18 34457258.62 21264211.07 63086232.2 51872939.88 Histidine 109108110.6 55425159.57 43496024.41 55782641.92 61451309.44 D-PANTOTHENIC ACID 38027485.25 27125322.89 15293937.33 58405550.83 32868482.99 2-Oxobutyric acid 20176097.79 14160832.78 13334638.14 13026890.34 19682372.78 Betaine 1544239181 346268111.6 2821040079 554118591.9 1517214654 alpha-Ketoglutaric acid 47678280.13 36494037.13 31530973.57 35288305.59 47820470.53 Serine 38222080.79 21783494.18 18647803.8 21623959.63 26129841.95 Choline 526154295.4 2615274643 1879798849 443703984 883451517.3 Pyridoxine 32578012.75 14477550.82 17591721.97 18669677.9 32282926.59 N-Acetylaspartic acid 54815935.35 36027540.94 42855009.5 104610323.7 127061503.8 Arginine 287684986.5 16298496.87 140465424.9 323533529.6 101743256.9 Tryptophan 33817458.27 18196507.66 14889011.19 17091182.07 18303488.04 Indole-3-acrylic acid 30378714.75 16392118.54 12886529.62 15776155.23 15628441.95 Lactic Acid 82122332.23 275343900.2 96692288.69 156197807.4 254227139.3 Phenylalanine 565283244.3 238304200.8 240743627.5 304613308.5 342980349.1 Proline 556229449.2 290180763.8 259169211.7 401963931.4 394688528.1 Malic Acid 151124456.6 114877165.4 80281530.67 41201080.81 141993047.3 Carnitine 55631972.59 124504595.8 61088980.29 114038892.5 199248557.3 Adenine 13381682.09 27150212.98 157628023.6 26982835.22 95304879.92 Fumaric acid 35374219.43 23839740.14 14158654.76 9443107.979 23188512.21 Cytosine 10438934.97 25133215.67 6519110.713 46735145.11 21727625.45 Citrulline 206308048.4 267388787.5 118268014 164453898.8 253066716.6 Isoleucine 1443113237 685642896.4 637959149.9 776503707 998371657.6 Valine 343903580.8 277533499.5 145203345.9 185431590.5 315096708.6 Guanine 6708766.733 8642506.945 5276317.649 26072361.75 10029448.81 Creatine 17718033.45 348186564.2 18124352.18 151259220.2 10223975.8 2-Hydroxybutyric acid 8031263.188 36674980.54 3678151.944 20243290.86 15185578.41 Tyrosine 142851340.3 69176249.2 62640520.25 95677957.28 85914255.09 Lysine 122422680 23331328.35 27913882.33 86762327.45 83031646.38 Uracil 119407286.7 219109878.8 62710300.73 184312214.8 151006958.6 Asparagine 6016529.62 1482731.459 6064423.941 5598972.73 8241294.118 Methionine 220013184.1 78763367.89 65709234.45 151114755.8 146364993.2 Glutamic acid 402223370.8 219526869.3 234711148.2 501075541.2 844545455.6 Kynurenic acid 8461358.869 7387386.259 6704173.764 6746880.416 2649376.662 Threonine 82409505.89 38437427.24 35809262.16 38163850.76 56584406.19 Creatinine 43460620.72 459222225.5 232822793.2 2362667948 22831693.12 2-Hydroxyglutaric acid 63328446.56 38355224.71 33377508.13 87506342.64 132602805.6 Taurine 784906.2891 51391912.18 1018603.497 58802204.46 1737929.7 Glutamine 318041656.4 188565349.4 181209450.1 128757067.4 107650634.5 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Succinic acid Niacin Histidine D-PANTOTHENIC ACID 2-Oxobutyric acid Betaine alpha-Ketoglutaric acid Serine Choline Pyridoxine N-Acetylaspartic acid Arginine Tryptophan Indole-3-acrylic acid Lactic Acid Phenylalanine Proline Malic Acid Carnitine Adenine Fumaric acid Cytosine Citrulline Isoleucine Valine Guanine Creatine 2-Hydroxybutyric acid Tyrosine Lysine Uracil Asparagine Methionine Glutamic acid Kynurenic acid Threonine Creatinine 2-Hydroxyglutaric acid Taurine Glutamine METABOLITES_END #END