#METABOLOMICS WORKBENCH jamoslandgraf_20181010_120926 DATATRACK_ID:1539 STUDY_ID:ST001075 ANALYSIS_ID:AN001757 PROJECT_ID:PR000720 VERSION 1 CREATED_ON October 17, 2018, 11:38 am #PROJECT PR:PROJECT_TITLE Integrated metabolome and transcriptome analyses provide novel insight into PR:PROJECT_TITLE colon cancer modulation by the gut microbiota PR:PROJECT_SUMMARY Colon cancer onset and progression is strongly associated with the presence, PR:PROJECT_SUMMARY absence, or relative abundances of certain microbial taxa in the PR:PROJECT_SUMMARY gastrointestinal tract. However, specific mechanisms affecting disease PR:PROJECT_SUMMARY susceptibility related to complex commensal bacterial mixtures are poorly PR:PROJECT_SUMMARY understood. We used a multi-omics approach to determine how differences in the PR:PROJECT_SUMMARY complex gut microbiome (GM) influence the metabolome and host transcriptome and PR:PROJECT_SUMMARY ultimately affect susceptibility to adenoma development. Fecal samples collected PR:PROJECT_SUMMARY from a preclinical rat model of colon cancer harboring distinct complex GMs were PR:PROJECT_SUMMARY analyzed using ultra-high performance liquid chromatography mass spectrometry PR:PROJECT_SUMMARY (UHPLC-MS). We collected samples prior to observable disease onset and PR:PROJECT_SUMMARY identified putative metabolite profiles that predicted future disease severity, PR:PROJECT_SUMMARY independent of GM status. Transcriptome analyses performed after disease onset PR:PROJECT_SUMMARY from normal epithelium and tumor tissues between the high and low tumor GMs PR:PROJECT_SUMMARY suggests that the GM is also correlated with altered host gene expression. PR:PROJECT_SUMMARY Integrated pathway (IP) analyses of the metabolome and transcriptome based on PR:PROJECT_SUMMARY putatively identified metabolic features indicate that bile acid biosynthesis PR:PROJECT_SUMMARY was enriched in rats with high tumors (GM:F344) along with increased fatty acid PR:PROJECT_SUMMARY metabolism and mucin biosynthesis. These data emphasize the utility of using PR:PROJECT_SUMMARY untargeted metabolomics to reveal signatures of susceptibility and resistance PR:PROJECT_SUMMARY and integrated analysis reveals common pathways that are likely to be universal PR:PROJECT_SUMMARY targets for intervention. PR:INSTITUTE University of Missouri PR:DEPARTMENT Veterinary Pathobiology PR:LABORATORY Amos-Landgraf PR:LAST_NAME Busi PR:FIRST_NAME Susheel Bhanu PR:ADDRESS 4011 Discovery Drive, N121 PR:EMAIL sb6f4@mail.missouri.edu PR:PHONE 2404094390 PR:CONTRIBUTORS Zhentian Lei, James Amos-Landgraf, Lloyd W. Sumner, #STUDY ST:STUDY_TITLE Integrated metabolome and transcriptome analyses provide novel insight into ST:STUDY_TITLE colon cancer modulation by the gut microbiota ST:STUDY_SUMMARY Colon cancer onset and progression is strongly associated with the presence, ST:STUDY_SUMMARY absence, or relative abundances of certain microbial taxa in the ST:STUDY_SUMMARY gastrointestinal tract. However, specific mechanisms affecting disease ST:STUDY_SUMMARY susceptibility related to complex commensal bacterial mixtures are poorly ST:STUDY_SUMMARY understood. We used a multi-omics approach to determine how differences in the ST:STUDY_SUMMARY complex gut microbiome (GM) influence the metabolome and host transcriptome and ST:STUDY_SUMMARY ultimately affect susceptibility to adenoma development. Fecal samples collected ST:STUDY_SUMMARY from a preclinical rat model of colon cancer harboring distinct complex GMs were ST:STUDY_SUMMARY analyzed using ultra-high performance liquid chromatography mass spectrometry ST:STUDY_SUMMARY (UHPLC-MS). We collected samples prior to observable disease onset and ST:STUDY_SUMMARY identified putative metabolite profiles that predicted future disease severity, ST:STUDY_SUMMARY independent of GM status. Transcriptome analyses performed after disease onset ST:STUDY_SUMMARY from normal epithelium and tumor tissues between the high and low tumor GMs ST:STUDY_SUMMARY suggests that the GM is also correlated with altered host gene expression. ST:STUDY_SUMMARY Integrated pathway (IP) analyses of the metabolome and transcriptome based on ST:STUDY_SUMMARY putatively identified metabolic features indicate that bile acid biosynthesis ST:STUDY_SUMMARY was enriched in rats with high tumors (GM:F344) along with increased fatty acid ST:STUDY_SUMMARY metabolism and mucin biosynthesis. These data emphasize the utility of using ST:STUDY_SUMMARY untargeted metabolomics to reveal signatures of susceptibility and resistance ST:STUDY_SUMMARY and integrated analysis reveals common pathways that are likely to be universal ST:STUDY_SUMMARY targets for intervention. ST:INSTITUTE UNIVERSITY OF MISSOURI - COLUMBIA ST:LAST_NAME Busi ST:FIRST_NAME Susheel Bhanu ST:ADDRESS 4011 Discovery Drive N121 ST:EMAIL SB6F4@MAIL.MISSOURI.EDU ST:PHONE 2404094390 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 13 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Rattus norvegicus SU:TAXONOMY_ID 10116 SU:GENOTYPE_STRAIN F344/Ntac-Apc-+/Pirc SU:AGE_OR_AGE_RANGE 30 SU:ANIMAL_ANIMAL_SUPPLIER Taconic and Envigo SU:ANIMAL_HOUSING Conventional, SU:ANIMAL_FEED Labdiet 5058 SU:ANIMAL_WATER Autoclaved, purified with sulfuric acid #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS - SB1 Group:GM;F344 ColonicTumors=11 SUBJECT_SAMPLE_FACTORS - SB2 Group:GM;F344 ColonicTumors=8 SUBJECT_SAMPLE_FACTORS - SB3 Group:GM;F344 ColonicTumors=34 SUBJECT_SAMPLE_FACTORS - SB4 Group:GM;F344 ColonicTumors=19 SUBJECT_SAMPLE_FACTORS - SB5 Group:GM;LEW ColonicTumors=11 SUBJECT_SAMPLE_FACTORS - SB6 Group:GM;LEW ColonicTumors=10 SUBJECT_SAMPLE_FACTORS - SB7 Group:GM;LEW ColonicTumors=6 SUBJECT_SAMPLE_FACTORS - SB8 Group:GM;LEW ColonicTumors=10 SUBJECT_SAMPLE_FACTORS - SB9 Group:GM;SD ColonicTumors=10 SUBJECT_SAMPLE_FACTORS - SB10 Group:GM;SD ColonicTumors=15 SUBJECT_SAMPLE_FACTORS - SB11 Group:GM;SD ColonicTumors=20 SUBJECT_SAMPLE_FACTORS - SB12 Group:GM;SD ColonicTumors=10 SUBJECT_SAMPLE_FACTORS - SB13 Group:GM;SD ColonicTumors=6 #COLLECTION CO:COLLECTION_SUMMARY Fecal samples collected at 1 month of age, prior to any observable disease CO:COLLECTION_SUMMARY onset. F344 refers to Fisher (F344) rats, whereas as GM:SD refers to the GM from CO:COLLECTION_SUMMARY Sprague-Dawley rats, while GM:LEW refers to the gut microbiota (GM) from Lewis CO:COLLECTION_SUMMARY rats CO:COLLECTION_PROTOCOL_ID 8732 CO:SAMPLE_TYPE Feces CO:COLLECTION_METHOD Sterile, asceptic method. Animals were placed in clean, sterile cages and feces CO:COLLECTION_METHOD were speared with sterile, autoclaved toothpicks. CO:COLLECTION_LOCATION Discovery Ridge, Columbia MO CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_VIALS Glass CO:STORAGE_VIALS Glass #TREATMENT TR:TREATMENT_SUMMARY Pirc rats were rederived using 3 surrogate dams, each with distinct gut TR:TREATMENT_SUMMARY microbiota profiles TR:TREATMENT_PROTOCOL_ID 8732 #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Fecal samples were lyophilized at -20 ˚C using 0.1 millibar of vacuum pressure, SP:SAMPLEPREP_SUMMARY following which dried samples (30 mg) were extracted sequentially for both SP:SAMPLEPREP_SUMMARY UHPLC-MS and GC-MS. The dried samples were first treated with 1.0 mL of 80% MeOH SP:SAMPLEPREP_SUMMARY containing 18 µg/mL umbelliferone, sonicated for 5 minutes and centrifuged for SP:SAMPLEPREP_SUMMARY 40 minutes at 3000 g at 10 ºC. 0.5 mL of supernatant was used for UHPLC-MS SP:SAMPLEPREP_SUMMARY analysis after a subsequent spin at 5000 g at 10 ºC for 20 minutes and SP:SAMPLEPREP_SUMMARY transferring 250 µL of the sample into glass autosampler vials with inserts. SP:SAMPLEPREP_SUMMARY For GC-MS (Gas Chromatography-Mass Spectrometry) analyses of primary polar SP:SAMPLEPREP_SUMMARY metabolites, 0.5 mL water was added the remaining extract used above for the SP:SAMPLEPREP_SUMMARY UHPLC preparation, sonicated for 5 min, extracted for 30min, and centrifuged at SP:SAMPLEPREP_SUMMARY 3000 g. 0.5 mL of the polar extract was subsequently dried under nitrogen and SP:SAMPLEPREP_SUMMARY derivatized using previously established protocols (84). Briefly, SP:SAMPLEPREP_SUMMARY N-Methyl-N-(trimethylsilyl) trifluoroacetamide (MSTFA) with 1 % TMCS SP:SAMPLEPREP_SUMMARY (2,2,2-Trifluoro-N-methyl-N-(trimethylsilyl)-acetamide, Chlorotrimethylsilane) SP:SAMPLEPREP_SUMMARY was used to derivatize the polar metabolites, after treatment with SP:SAMPLEPREP_SUMMARY methoxyamine-HCl-pyridine. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 CH:COLUMN_NAME Waters Acquity BEH C18 (150 x 2.1mm, 1.7um) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Bruker maXis Impact qTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_RESULTS_FILE ST001075_AN001757_Results.txt UNITS:Peak Area #END