#METABOLOMICS WORKBENCH yefeifei0418_20250815_014618 DATATRACK_ID:6293 STUDY_ID:ST004136 ANALYSIS_ID:AN006857 PROJECT_ID:PR002601 VERSION 1 CREATED_ON August 20, 2025, 6:48 pm #PROJECT PR:PROJECT_TITLE Bifidobacterium animalis subsp. lactis secretes acetate to delay the progression PR:PROJECT_TITLE of epithelial ovarian cancer via regulating the NRF2-HO-1-ferroptosis pathway PR:PROJECT_SUMMARY Recent advances highlight the anti-tumor effect exerted by gut microbiota and PR:PROJECT_SUMMARY derived metabolites via modulating ferroptosis, while their role in epithelial PR:PROJECT_SUMMARY ovarian cancer (EOC) has not been fully explored. Based on the data of qPCR and PR:PROJECT_SUMMARY previous 16S rRNA sequencing, we firstly identified Bifidobacterium to be PR:PROJECT_SUMMARY markedly depleted in gut microbiota of EOC females. And acetic acid, an PR:PROJECT_SUMMARY essential bacterial metabolite, was also found to be significantly differed in PR:PROJECT_SUMMARY dual-species of both human and mouse by GC-MS technique. By oral gavage of the PR:PROJECT_SUMMARY Bifidobacterium animalis subsp. lactis NCU-01 and sodium acetate to EOC mice PR:PROJECT_SUMMARY model, their impact in inhibiting tumor growth, rehabilitating intestinal PR:PROJECT_SUMMARY barrier function, and alleviating systematic inflammation was validated. H&E PR:PROJECT_SUMMARY staining and TEM scan visualized the distinctive features of ferroptosis in PR:PROJECT_SUMMARY tumor cells such as cytoplasmic staining and mitochondrial cristae degeneration PR:PROJECT_SUMMARY from histopathological perspective. Western blot and ELISA assays further PR:PROJECT_SUMMARY quantified the disruption of iron homeostasis regulators TFR1 and FTH1, the PR:PROJECT_SUMMARY alteration of lipid peroxidation and cellular stress molecules including MDA, PR:PROJECT_SUMMARY GSH, and SOD, together with the down-regulation of NRF2/HO-1/GPX4 signaling PR:PROJECT_SUMMARY cascade that opposes ferroptosis in the EOC tissues. This study unveiled a novel PR:PROJECT_SUMMARY microbiota-metabolite axis anchoring the NRF2/HO-1/GPX4 to balance ferroptosis PR:PROJECT_SUMMARY and antioxidant defense, offering a therapeutic tactic for EOC intervention in PR:PROJECT_SUMMARY clinic, and underscoring the translational potential of probiotics in precision PR:PROJECT_SUMMARY oncology PR:INSTITUTE Nanchang university PR:LAST_NAME ye PR:FIRST_NAME feifei PR:ADDRESS hongjiaozhou, nanchang, jiangxi, 330006, China PR:EMAIL huihuizi0418@126.com PR:PHONE 13762306123 #STUDY ST:STUDY_TITLE Bifidobacterium animalis subsp. lactis secretes acetate to delay the progression ST:STUDY_TITLE of epithelial ovarian cancer via regulating the NRF2-HO-1-ferroptosis pathway ST:STUDY_SUMMARY Recent advances highlight the anti-tumor effect exerted by gut microbiota and ST:STUDY_SUMMARY derived metabolites via modulating ferroptosis, while their role in epithelial ST:STUDY_SUMMARY ovarian cancer (EOC) has not been fully explored. Based on the data of qPCR and ST:STUDY_SUMMARY previous 16S rRNA sequencing, we firstly identified Bifidobacterium to be ST:STUDY_SUMMARY markedly depleted in gut microbiota of EOC females. And acetic acid, an ST:STUDY_SUMMARY essential bacterial metabolite, was also found to be significantly differed in ST:STUDY_SUMMARY dual-species of both human and mouse by GC-MS technique. By oral gavage of the ST:STUDY_SUMMARY Bifidobacterium animalis subsp. lactis NCU-01 and sodium acetate to EOC mice ST:STUDY_SUMMARY model, their impact in inhibiting tumor growth, rehabilitating intestinal ST:STUDY_SUMMARY barrier function, and alleviating systematic inflammation was validated. H&E ST:STUDY_SUMMARY staining and TEM scan visualized the distinctive features of ferroptosis in ST:STUDY_SUMMARY tumor cells such as cytoplasmic staining and mitochondrial cristae degeneration ST:STUDY_SUMMARY from histopathological perspective. Western blot and ELISA assays further ST:STUDY_SUMMARY quantified the disruption of iron homeostasis regulators TFR1 and FTH1, the ST:STUDY_SUMMARY alteration of lipid peroxidation and cellular stress molecules including MDA, ST:STUDY_SUMMARY GSH, and SOD, together with the down-regulation of NRF2/HO-1/GPX4 signaling ST:STUDY_SUMMARY cascade that opposes ferroptosis in the EOC tissues. This study unveiled a novel ST:STUDY_SUMMARY microbiota-metabolite axis anchoring the NRF2/HO-1/GPX4 to balance ferroptosis ST:STUDY_SUMMARY and antioxidant defense, offering a therapeutic tactic for EOC intervention in ST:STUDY_SUMMARY clinic, and underscoring the translational potential of probiotics in precision ST:STUDY_SUMMARY oncology ST:INSTITUTE Nanchang university ST:LAST_NAME ye ST:FIRST_NAME feifei ST:ADDRESS hongjiaozhou, nanchang, jiangxi, 330006, China ST:EMAIL huihuizi0418@126.com ST:PHONE 13762306123 #SUBJECT SU:SUBJECT_TYPE SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Female #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 H H1 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H1.mzML SUBJECT_SAMPLE_FACTORS H H2 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H2.mzML SUBJECT_SAMPLE_FACTORS H H3 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H3.mzML SUBJECT_SAMPLE_FACTORS H H4 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H4.mzML SUBJECT_SAMPLE_FACTORS H H5 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H5.mzML SUBJECT_SAMPLE_FACTORS H H6 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H6.mzML SUBJECT_SAMPLE_FACTORS H H7 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H7.mzML SUBJECT_SAMPLE_FACTORS H H8 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H8.mzML SUBJECT_SAMPLE_FACTORS H H9 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H9.mzML SUBJECT_SAMPLE_FACTORS H H10 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H10.mzML SUBJECT_SAMPLE_FACTORS H H11 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H11.mzML SUBJECT_SAMPLE_FACTORS H H12 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H12.mzML SUBJECT_SAMPLE_FACTORS H H13 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H13.mzML SUBJECT_SAMPLE_FACTORS H H14 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H14.mzML SUBJECT_SAMPLE_FACTORS H H15 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H15.mzML SUBJECT_SAMPLE_FACTORS H H16 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H16.mzML SUBJECT_SAMPLE_FACTORS H H17 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H17.mzML SUBJECT_SAMPLE_FACTORS H H18 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H18.mzML SUBJECT_SAMPLE_FACTORS H H19 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H19.mzML SUBJECT_SAMPLE_FACTORS H H20 Sample source:serum | factor:healty females RAW_FILE_NAME(mzML flie name)=H20.mzML SUBJECT_SAMPLE_FACTORS OC OC1 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC1.mzML SUBJECT_SAMPLE_FACTORS OC OC2 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC2.mzML SUBJECT_SAMPLE_FACTORS OC OC3 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC3.mzML SUBJECT_SAMPLE_FACTORS OC OC4 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC4.mzML SUBJECT_SAMPLE_FACTORS OC OC5 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC5.mzML SUBJECT_SAMPLE_FACTORS OC OC6 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC6.mzML SUBJECT_SAMPLE_FACTORS OC OC7 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC7.mzML SUBJECT_SAMPLE_FACTORS OC OC8 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC8.mzML SUBJECT_SAMPLE_FACTORS OC OC9 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC9.mzML SUBJECT_SAMPLE_FACTORS OC OC10 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC10.mzML SUBJECT_SAMPLE_FACTORS OC OC11 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC11.mzML SUBJECT_SAMPLE_FACTORS OC OC12 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC12.mzML SUBJECT_SAMPLE_FACTORS OC OC13 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC13.mzML SUBJECT_SAMPLE_FACTORS OC OC14 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC14.mzML SUBJECT_SAMPLE_FACTORS OC OC15 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC15.mzML SUBJECT_SAMPLE_FACTORS OC OC16 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC16.mzML SUBJECT_SAMPLE_FACTORS OC OC17 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC17.mzML SUBJECT_SAMPLE_FACTORS OC OC18 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC18.mzML SUBJECT_SAMPLE_FACTORS OC OC19 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC19.mzML SUBJECT_SAMPLE_FACTORS OC OC20 Sample source:serum | factor:EOC patients RAW_FILE_NAME(mzML flie name)=OC20.mzML #COLLECTION CO:COLLECTION_SUMMARY The blood was collected in centrifuge tubes and placed at 37 °C for 1 h to CO:COLLECTION_SUMMARY solidify and separate into layers. Then, centrifuge the sample at 3,000 rpm for CO:COLLECTION_SUMMARY 10 min at room temperature to attain the supernatant, followed by centrifuging CO:COLLECTION_SUMMARY the supernatant at 12,000 rpm for 10 min (4 °C) and aliquot the newly-collected CO:COLLECTION_SUMMARY supernatants into centrifuge tubes (1.5 mL), with 0.2 mL in each. Store the CO:COLLECTION_SUMMARY samples at -80 °C for further detections. CO:SAMPLE_TYPE Blood (serum) #TREATMENT TR:TREATMENT_SUMMARY A 100 mg/mL mixed standard stock solutions of 6 SCFAs (acetic acid, propionic TR:TREATMENT_SUMMARY acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid) and 100 TR:TREATMENT_SUMMARY mg/mL caproic acid stock solution were prepared by water and ether respectively. TR:TREATMENT_SUMMARY 6 SCFAs and caproic acid working solution series were both prepared by TR:TREATMENT_SUMMARY appropriate dilutions of standard stock solution. 75 μg/mL of internal standard TR:TREATMENT_SUMMARY (IS) solution containing 4-methylvaleric acid was similarly prepared with ether. TR:TREATMENT_SUMMARY Ten points calibration curve was made by adding 220 μL the working solutions TR:TREATMENT_SUMMARY which containing 200 μL six acids working solition series and 20 μL caproic TR:TREATMENT_SUMMARY acid working solution series, 100 μL of 15% phosphoric acid, 20 μL of 75 TR:TREATMENT_SUMMARY μg/mL IS solution and 260 μL of ether covering a range from 0.02 to 100 μg/mL TR:TREATMENT_SUMMARY (0.02, 0.1, 0.5, 1, 2, 5, 10, 25, 50, 100 μg/mL). Stock solutions were stored TR:TREATMENT_SUMMARY at -20℃ before use and working solutions were prepared when using. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY A 100 mg/mL mixed standard stock solutions of 6 SCFAs (acetic acid, propionic SP:SAMPLEPREP_SUMMARY acid, isobutyric acid, butyric acid, isovaleric acid and valeric acid) and 100 SP:SAMPLEPREP_SUMMARY mg/mL caproic acid stock solution were prepared by water and ether respectively. SP:SAMPLEPREP_SUMMARY 6 SCFAs and caproic acid working solution series were both prepared by SP:SAMPLEPREP_SUMMARY appropriate dilutions of standard stock solution. 75 μg/mL of internal standard SP:SAMPLEPREP_SUMMARY (IS) solution containing 4-methylvaleric acid was similarly prepared with ether. SP:SAMPLEPREP_SUMMARY Ten points calibration curve was made by adding 220 μL the working solutions SP:SAMPLEPREP_SUMMARY which containing 200 μL six acids working solition series and 20 μL caproic SP:SAMPLEPREP_SUMMARY acid working solution series, 100 μL of 15% phosphoric acid, 20 μL of 75 SP:SAMPLEPREP_SUMMARY μg/mL IS solution and 260 μL of ether covering a range from 0.02 to 100 μg/mL SP:SAMPLEPREP_SUMMARY (0.02, 0.1, 0.5, 1, 2, 5, 10, 25, 50, 100 μg/mL). Stock solutions were stored SP:SAMPLEPREP_SUMMARY at -20℃ before use and working solutions were prepared when using #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY GC column: trace 1310 (30 m × 0.25 mm ID × 0.25 μm Thermo Fisher Scientific, CH:CHROMATOGRAPHY_SUMMARY USA) CH:CHROMATOGRAPHY_TYPE GC CH:INSTRUMENT_NAME Thermo Trace 1310 CH:COLUMN_NAME Agilent HP5-MS (30m x 0.25mm, 0.25 um) CH:SOLVENT_A none (GCMS) CH:SOLVENT_B none (GCMS) CH:FLOW_GRADIENT none (GCMS) CH:FLOW_RATE The carrier flow was set to 10 mL/min with helium. CH:COLUMN_TEMPERATURE Injection was made in split mode at 10:1 with an injection volume of 1 μL and CH:COLUMN_TEMPERATURE an injector temperature of 250℃. The temperature of the ion source and MS CH:COLUMN_TEMPERATURE transfer line were 300℃ and 250℃, respectively. The column temperature was CH:COLUMN_TEMPERATURE programmed to increase from an initial temperature of 90℃, followed by an CH:COLUMN_TEMPERATURE increase to 120℃ at 10℃/min, and to 150℃ at 5℃/min, and finally to CH:COLUMN_TEMPERATURE 250℃ at 25℃/min which was maintained for 2 min. #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo ISQ LT MS:INSTRUMENT_TYPE Single quadrupole MS:MS_TYPE EI MS:ION_MODE POSITIVE MS:MS_COMMENTS Mass spectrometric detection of metabolites was performed on ISQ LT (Thermo MS:MS_COMMENTS Fisher Scientific, USA) with electron impact ionization mode. Single ion MS:MS_COMMENTS monitoring (SIM) mode was used with the electron energy of 70 eV. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS μg/mL MS_METABOLITE_DATA_START Samples H1 H2 H3 H4 H5 H6 H7 H8 H9 H10 H11 H12 H13 H14 H15 H16 H17 H18 H19 H20 OC1 OC2 OC3 OC4 OC5 OC6 OC7 OC8 OC9 OC10 OC11 OC12 OC13 OC14 OC15 OC16 OC17 OC18 OC19 OC20 Factors Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:healty females Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Sample source:serum | factor:EOC patients Acetic acid 1.6923 2.1915 0.9976 1.2183 1.4843 1.8126 2.6487 2.2803 2.346 1.5156 2.9123 2.5175 2.8009 1.1429 2.0944 2.8084 1.7963 1.0584 3.0142 3.3462 2.4241 1.7561 1.4001 1.4819 1.7317 2.1383 1.3162 1.2669 1.4147 1.4033 2.3555 2.1779 1.4463 0.7961 1.0686 1.2769 0.9543 2.2856 1.8405 0.8428 Propionic acid 0.1424 0.07095 0.1199 0.09103 0.1768 0.5212 0.1904 0.1799 0.282 0.09786 0.219 0.1602 0.1726 0.07618 0.1342 0.2511 0.08555 0.08464 0.2524 0.1979 0.0711 0.1052 0.1063 0.05588 0.1187 0.1544 0.05388 0.1043 0.1151 0.09113 0.1373 0.08326 0.05828 0.0262 0.1053 0.09762 0.07362 0.1683 0.1444 0.07553 Isobutyric acid 0.03946 0.02695 0.04178 0.01587 0.01124 0.02451 0.02476 0.06144 0.02922 0.02928 0.03463 0.02752 0.02466 0.01515 0.01737 0.02756 0.01192 0.0354 0.03844 0.04935 0.03563 0.04195 0.03637 0.02533 0.01614 0.03977 0.01976 0.02259 0.04421 0.01451 0.05053 0.01238 0.02436 0.01468 0.01176 0.02352 0.02772 0.03965 0.02963 0.02054 Butyric acid 0.04637 0.02649 0.008774 0.02378 0.04208 0.3241 0.09178 0.283 0.294 0.3046 0.4875 0.4206 0.4075 0.03172 0.2712 0.403 0.03121 0.0004526 0.3504 0.3347 0.166 0.0255 0.04076 0.01173 0.1397 0.02795 0.003601 0.021 0.04563 0.02438 0.1294 0.01409 0.03159 0.006176 0.01212 0.04001 0.007114 0.2195 0.0158 0.009264 Isovaleric acid 0.06962 0.05233 0.03234 0.03457 0.0184 0.0563 0.03726 0.01949 0.01998 0.01386 0.02314 0.01962 0.01777 0.01578 0.01163 0.02398 0.03571 0.01536 0.04408 0.02413 0.02251 0.03434 0.03485 0.03524 0.02767 0.1274 0.01658 0.02729 0.01827 0.03544 0.02226 0.01352 0.01808 0.001599 0.02536 0.0432 0.04473 0.02404 0.0387 0.03815 Valeric acid 0.01673 0.01277 0.005901 0.008551 0.01503 0.009801 0.009258 0.009735 0.009938 0.007718 0.006691 0.006537 0.004299 0.006076 0.004374 0.02339 0.01274 0.004503 0.005708 0.006572 0.006563 0.008033 0.006818 0.006549 0.007893 0.002087 0.004527 0.005092 0.008365 0.004716 0.00293 0.01506 0.004556 0.002213 0.003531 0.00729 0.003853 0.005041 0.004307 0.004607 Caproic acid 0.02572 0.01343 0.006982 0.03427 0.00941 0.01781 0.008147 0.0112 0.01133 0.0186 0.007024 0.006563 0.01462 0.00974 0.01365 0.01655 0.008681 0.002907 0.01045 0.01121 0.01919 0.01266 0.01311 0.005765 0.003517 0.01367 0.01192 0.001628 0.005235 0.008492 0.002559 0.01368 0.003931 0.005129 0.007716 0.005801 0.001059 0.001836 0.005251 0.006138 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Acetic acid Propionic acid Isobutyric acid Butyric acid Isovaleric acid Valeric acid Caproic acid METABOLITES_END #END