#METABOLOMICS WORKBENCH VincentCarbonnier_20250505_074309 DATATRACK_ID:5882 STUDY_ID:ST003957 ANALYSIS_ID:AN006504 PROJECT_ID:PR002480 VERSION 1 CREATED_ON June 10, 2025, 8:50 am #PROJECT PR:PROJECT_TITLE Spatial transcriptomic profiling of gene expression alterations following PR:PROJECT_TITLE ACBP/DBI inhibition in a MASH-Driven HCC mouse model PR:PROJECT_SUMMARY We identified ACBP/DBI as a critical regulator in the pathogenesis of PR:PROJECT_SUMMARY hepatocellular carcinoma (HCC), with its inhibition significantly impairing PR:PROJECT_SUMMARY hepatocarcinogenesis in MASH-driven HCC models. To further elucidate the PR:PROJECT_SUMMARY underlying transcriptional mechanisms, we performed spatial transcriptomic PR:PROJECT_SUMMARY analyses using formalin-fixed, paraffin-embedded (FFPE) liver tissues from the PR:PROJECT_SUMMARY following two experimental models: (i) Western diet (WD) and Carbon PR:PROJECT_SUMMARY tetrachloride treated (CCl₄-treated) mice with tamoxifen-inducible ACBP/DBI PR:PROJECT_SUMMARY knockout (Dbi⁻/⁻ mice), with Dbi⁺/⁺ littermates serving as controls, PR:PROJECT_SUMMARY after a total of 27 weeks; (ii) WD and CCl₄-treated mice immunized with either PR:PROJECT_SUMMARY keyhole limpet hemocyanin (KLH) alone or KLH-ACBP conjugate, after a total of 34 PR:PROJECT_SUMMARY weeks. PR:INSTITUTE INSERM PR:DEPARTMENT UMRS1138 PR:LABORATORY Kroemer Laboratory PR:LAST_NAME Carbonnier PR:FIRST_NAME Vincent PR:ADDRESS 15 rue de l'École de Médecine PR:EMAIL vincent.carbonnier@inserm.fr PR:PHONE +33 1 44 27 76 61 PR:CONTRIBUTORS Sijing, Li; Sylvère, Durand; Alexandra, Cerone; Guido, Kroemer #STUDY ST:STUDY_TITLE Spatial transcriptomic profiling of gene expression alterations following ST:STUDY_TITLE ACBP/DBI inhibition in a MASH-Driven HCC mouse model ST:STUDY_SUMMARY We identified ACBP/DBI as a critical regulator in the pathogenesis of ST:STUDY_SUMMARY hepatocellular carcinoma (HCC), with its inhibition significantly impairing ST:STUDY_SUMMARY hepatocarcinogenesis in MASH-driven HCC models. To further elucidate the ST:STUDY_SUMMARY underlying transcriptional mechanisms, we performed spatial transcriptomic ST:STUDY_SUMMARY analyses using formalin-fixed, paraffin-embedded (FFPE) liver tissues from the ST:STUDY_SUMMARY following two experimental models: (i) Western diet (WD) and CCl₄-treated mice ST:STUDY_SUMMARY with tamoxifen-inducible ACBP/DBI knockout (Dbi⁻/⁻ mice), with Dbi⁺/⁺ ST:STUDY_SUMMARY littermates serving as controls, after a total of 27 weeks; (ii) WD and ST:STUDY_SUMMARY CCl₄-treated mice immunized with either keyhole limpet hemocyanin (KLH) alone ST:STUDY_SUMMARY or KLH-ACBP conjugate, after a total of 34 weeks. Spatially resolved mass ST:STUDY_SUMMARY spectrometric imaging (MSI)-based metabolomics enabled precise differentiation ST:STUDY_SUMMARY between tumor (T) and non-tumor (NT) regions in mice liver tissues across ST:STUDY_SUMMARY distinct dietary and genetic backgrounds. Hierarchical clustering of metabolomic ST:STUDY_SUMMARY profiles revealed clear segregation of malignant and non-malignant areas, ST:STUDY_SUMMARY particularly in livers from WD+CCl₄-treated mice, with distinct metabolic ST:STUDY_SUMMARY signatures observed between Dbi⁺/⁺ and Dbi⁻/⁻ genotypes. MSI-defined ST:STUDY_SUMMARY regions of interest (ROIs) showed strong concordance with histologically ST:STUDY_SUMMARY validated areas, where Dbi⁺/⁺ livers displayed both tumor and non-tumor ST:STUDY_SUMMARY regions under stress, while Dbi⁻/⁻ livers exhibited minimal or absent tumor ST:STUDY_SUMMARY development. This integrated approach offers high spatial resolution for ST:STUDY_SUMMARY metabolic tissue profiling and underscores the tumor-suppressive effect of Dbi ST:STUDY_SUMMARY deficiency in a pathological liver environment. ST:INSTITUTE INSERM ST:DEPARTMENT UMRS1138 ST:LABORATORY Kroemer Laboratory ST:LAST_NAME Carbonnier ST:FIRST_NAME Vincent ST:ADDRESS 15 rue de l'École de Médecine ST:EMAIL vincent.carbonnier@inserm.fr ST:PHONE +33 1 44 27 76 61 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 3 ST:NUM_FEMALES 3 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN WT; ACBP-KO SU:AGE_OR_AGE_RANGE 34 weeks 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 1 LiverCtrl-NEG-1 Sample source:Liver tissue | Genotype:Wild-type | Diet:Normal diet RAW_FILE_NAME(Raw file name)=LiverCtrl-NEG-1_1.imzml; RAW_FILE_NAME(Raw file name 2)=LiverCtrl-NEG-1_1.ibd SUBJECT_SAMPLE_FACTORS 1 LiverCtrl-NEG-2 Sample source:Liver tissue | Genotype:Wild-type | Diet:Normal diet RAW_FILE_NAME(Raw file name)=LiverCtrl-NEG-2_1.imzml; RAW_FILE_NAME(Raw file name 2)=LiverCtrl-NEG-2_1.ibd SUBJECT_SAMPLE_FACTORS 1 LiverCtrl-NEG-3 Sample source:Liver tissue | Genotype:Wild-type | Diet:Normal diet RAW_FILE_NAME(Raw file name)=LiverCtrl-NEG-3_1.imzml; RAW_FILE_NAME(Raw file name 2)=LiverCtrl-NEG-3_1.ibd SUBJECT_SAMPLE_FACTORS 2 Liver29-Ctrl-NEG-2 Sample source:Liver tissue | Genotype:Wild-type | Diet:Western diet RAW_FILE_NAME(Raw file name)=Liver29-Ctrl-NEG-2_1.imzml; RAW_FILE_NAME(Raw file name 2)=Liver29-Ctrl-NEG-2_1.ibd SUBJECT_SAMPLE_FACTORS 2 Liver29-Ctrl-NEG-3 Sample source:Liver tissue | Genotype:Wild-type | Diet:Western diet RAW_FILE_NAME(Raw file name)=Liver29-Ctrl-NEG-3_1.imzml; RAW_FILE_NAME(Raw file name 2)=Liver29-Ctrl-NEG-3_1.ibd SUBJECT_SAMPLE_FACTORS 3 Liver6-ACBP-KO-NEG-1 Sample source:Liver tissue | Genotype:ACBP-KO | Diet:Western diet RAW_FILE_NAME(Raw file name)=Liver6-ACBP-KO-NEG-1_1.imzml; RAW_FILE_NAME(Raw file name 2)=Liver6-ACBP-KO-NEG-1_1.ibd SUBJECT_SAMPLE_FACTORS 3 Liver6-ACBP-KO-NEG-2 Sample source:Liver tissue | Genotype:ACBP-KO | Diet:Western diet RAW_FILE_NAME(Raw file name)=Liver6-ACBP-KO-NEG-2_1.imzml; RAW_FILE_NAME(Raw file name 2)=Liver6-ACBP-KO-NEG-2_1.ibd SUBJECT_SAMPLE_FACTORS 3 Liver6-ACBP-KO-NEG-3 Sample source:Liver tissue | Genotype:ACBP-KO | Diet:Western diet RAW_FILE_NAME(Raw file name)=Liver6-ACBP-KO-NEG-3_1.imzml; RAW_FILE_NAME(Raw file name 2)=Liver6-ACBP-KO-NEG-3_1.ibd #COLLECTION CO:COLLECTION_SUMMARY Mice tissues were fresh frozen. Liver tissues were harvested from mice, rinsed CO:COLLECTION_SUMMARY with PBS and placed in embedding cassette with one piece of foam pads then CO:COLLECTION_SUMMARY gently frozen in steam bath of liquid nitrogen. Cassettes were stored at -80°C. CO:SAMPLE_TYPE Liver CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Normal diet and western diet on wild type mice or ACBP-KO mice. Western diet is TR:TREATMENT_SUMMARY a high-fat, high-fructose and high-cholesterol diet, TD.120528 from Envigo. TR:TREATMENT_VEHICLE Food #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For sectioning, tissues were equilibrated from –80°C to –15°C over 20 SP:SAMPLEPREP_SUMMARY minutes, mounted with Milli-Q water on a peltier platform, and fast-frozen for SP:SAMPLEPREP_SUMMARY 10 minutes. Sections (20 µm thick) were cut using a cryotome (HM 500 O, Micro) SP:SAMPLEPREP_SUMMARY and placed on labeled slides. Slides were alternately assigned for positive-mode SP:SAMPLEPREP_SUMMARY MSI, negative-mode MSI, or Hematoxylin and Eosin (H&E) staining. MSI slides were SP:SAMPLEPREP_SUMMARY stored at –80°C; H&E staining was performed immediately after cutting. For SP:SAMPLEPREP_SUMMARY H&E staining, slides were sequentially immersed in 100%, 70%, and 50% ethanol (2 SP:SAMPLEPREP_SUMMARY min each), rinsed with tap water (2 min), stained with hematoxylin (4.5 min), SP:SAMPLEPREP_SUMMARY rinsed (4 min), stained with eosin (2.2 min), rinsed (1.5 min), dehydrated SP:SAMPLEPREP_SUMMARY through graded ethanol (50%, 70%, 100%; 1 min each), rinsed with xylene, and SP:SAMPLEPREP_SUMMARY mounted with Entellan. For MSI acquisition, full-scan mass spectra (50–1200 SP:SAMPLEPREP_SUMMARY m/z) were acquired on a SYNAPT XS (Waters) with a DESI XS source. Positive- and SP:SAMPLEPREP_SUMMARY negative-polarity acquisitions were performed on separate sections. SP:SAMPLEPREP_SUMMARY Negative-mode tissues were sprayed with 2 µL/min methanol/water (96:4) SP:SAMPLEPREP_SUMMARY containing 1 mM ammonium acetate and leucine-enkephalin (250 ng/mL); capillary SP:SAMPLEPREP_SUMMARY voltage was 0.70 V, sampling cone 40 V, source temperature 150°C. Positive-mode SP:SAMPLEPREP_SUMMARY tissues used methanol/water (82:18) with 0.1% formic acid and SP:SAMPLEPREP_SUMMARY leucine-enkephalin; capillary voltage 0.40 V. DESI scanning was performed at 50 SP:SAMPLEPREP_SUMMARY µm² spatial resolution with a 0.153 s scan time. SP:PROCESSING_STORAGE_CONDITIONS -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE None (Direct infusion) CH:INSTRUMENT_NAME none CH:COLUMN_NAME none CH:SOLVENT_A NA CH:SOLVENT_B NA CH:FLOW_GRADIENT NA CH:FLOW_RATE NA CH:COLUMN_TEMPERATURE NA #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Waters Synapt-XS MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE DESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS DESI MSI (mass spectrometry imaging) MSI acquisition Full scan (50 m/z to 1200 MS:MS_COMMENTS m/z) acquisitions were performed in sensitive mode with a Synapt XS Waters MS:MS_COMMENTS equipped with a DESI XS source. Three slices were acquired in positive polarity MS:MS_COMMENTS acquisition mode, and three others were used for negative polarity acquisition MS:MS_COMMENTS mode. Tissue sections acquired in negative polarity were sprayed with 2 µl/min MS:MS_COMMENTS of methanol/water buffer (96/4, with 1 mmol of ammonium acetate) with lockspray MS:MS_COMMENTS leucine-enkephaline (250 ng/ml). Capillary voltage was set at 0.70V, sampling MS:MS_COMMENTS cone at 40V, source temperature at 150°C. Regarding the negative polarity MS:MS_COMMENTS acquisition mode, the 2 µl/min of buffer was composed of methanol/water (82/18) MS:MS_COMMENTS with 0.1% of formic acid and 250 ng/ml of leucine-enkephaline. Capillary voltage MS:MS_COMMENTS was set at 0.40V, sampling cone at 40V, source temperature at 150°C. DESI MS:MS_COMMENTS source was scanning the tissue spatial resolution with a spatial resolution of MS:MS_COMMENTS 50 µm² per pixel, and a scan time of 0.153s. Acquisition parameters were set MS:MS_COMMENTS with HDI v1.6 and acquired with MassLynx v4.2. Data treatment Raw data were MS:MS_COMMENTS processed by HDI v1.6. A target list was built from one replicate, based on the MS:MS_COMMENTS thousand most intense ion signals. Then we added to this data-driven built list MS:MS_COMMENTS our own targeted list of special interest. It is resulting to an identical list MS:MS_COMMENTS of m/z searched throughout pixels (corresponding to mass spectra) of the whole MS:MS_COMMENTS set of MSI files. HDI raw outputs were exported in text files and import in R MS:MS_COMMENTS software to further data treatments. Data handling with R included raw data MS:MS_COMMENTS cleaning (image cropping, mis-acquired replicate exclusion) and normalization by MS:MS_COMMENTS total ion count (TIC). A smooth function was applied on the MSI files, in order MS:MS_COMMENTS to limit the artefactual signal variations between pixels. Then, artefactual MS:MS_COMMENTS metabolites were removed: if the mean of the tissue region of interest (tissue MS:MS_COMMENTS ROI, based on Kmeans pixels clutering) was less than the mean of the glass ROI MS:MS_COMMENTS for more than the half of the MSI files, then metabolites were excluded from MS:MS_COMMENTS ulterior analysis. All pixel values corresponding to the tissue replicates were MS:MS_COMMENTS then gathered and analyzed by a centered and non-scaled principal component MS:MS_COMMENTS analysis (PCA). PCA values were then clusterized by a Kmeans method, and each MS:MS_COMMENTS pixel was assigned by a color code depending on its cluster. Images of the MS:MS_COMMENTS replicated tissue sections were then reconstructed from this color code MS:MS_COMMENTS assignation, with common ROI to the whole set of files. Finally, each ions MS:MS_COMMENTS signal was meaned by ROI and value were computed in a heatmap. MS:MS_RESULTS_FILE ST003957_AN006504_Results.txt UNITS:intensity Has m/z:Yes Has RT:No RT units:No RT data #END