#METABOLOMICS WORKBENCH agoldman_20230620_070147 DATATRACK_ID:4100 STUDY_ID:ST002740 ANALYSIS_ID:AN004443 PROJECT_ID:PR001705 VERSION 1 CREATED_ON June 21, 2023, 3:41 pm #PROJECT PR:PROJECT_TITLE Acetate acts as a metabolic immunomodulator that potentiates anti-tumour PR:PROJECT_TITLE immunity in breast cancer PR:PROJECT_SUMMARY Acetate metabolism is an important metabolic pathway in many cancers and is PR:PROJECT_SUMMARY controlled by acetyl-CoA synthetase 2 (ACSS2), an enzyme that catalyzes the PR:PROJECT_SUMMARY conversion of acetate to acetyl-CoA. While the metabolic role of ACSS2 in cancer PR:PROJECT_SUMMARY is well described, the consequences of blocking tumour acetate metabolism on the PR:PROJECT_SUMMARY tumour microenvironment and anti-tumour immunity are unknown. We demonstrate PR:PROJECT_SUMMARY that blocking ACSS2 switches cancer cells from acetate consumers to producers of PR:PROJECT_SUMMARY acetate thereby freeing acetate for tumour-infiltrating lymphocytes to use as a PR:PROJECT_SUMMARY fuel source. We show that acetate supplementation metabolically bolsters T-cell PR:PROJECT_SUMMARY effector functions and proliferation. Targeting ACSS2 with CRISPR-Cas9 guides or PR:PROJECT_SUMMARY a small molecule inhibitor promotes an anti-tumour immune response and enhances PR:PROJECT_SUMMARY the efficacy of chemotherapy in preclinical breast cancer models. We propose a PR:PROJECT_SUMMARY novel paradigm for targeting acetate metabolism in cancer in which inhibition of PR:PROJECT_SUMMARY ACSS2 dually acts to impair tumour cell metabolism and potentiate anti-tumour PR:PROJECT_SUMMARY immunity. PR:INSTITUTE The Wistar Institute PR:LAST_NAME Schug PR:FIRST_NAME Zachary PR:ADDRESS 3601 Spruce St, Philadelphia PA 19104 PR:EMAIL zschug@wistar.org PR:PHONE 215-898-3705 #STUDY ST:STUDY_TITLE Non-targeted metabolomics screen comparing 13C2-acetate labeling of metabolites ST:STUDY_TITLE in CD8+ T cells and NK cells from mouse spleens. ST:STUDY_SUMMARY Non-targeted metabolomics screen comparing 13C2-acetate labeling of metabolites ST:STUDY_SUMMARY in CD8+ T cells and NK cells from mouse spleens (wild type vs ACSS2 knockout ST:STUDY_SUMMARY C57Bl/6 mice). Metabolites were analyzed using a high-resolution, ST:STUDY_SUMMARY high-performance LC-MS analysis. ST:INSTITUTE The Wistar Institute ST:LAST_NAME Schug ST:FIRST_NAME Zachary ST:ADDRESS 3601 Spruce St, Philadelphia PA 19104 ST:EMAIL zschug@wistar.org ST:PHONE 215-898-3705 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57Bl/6 #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 - Sample_1-100_1 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_1-100_1.raw SUBJECT_SAMPLE_FACTORS - Sample_1-100_2 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_1-100_2.raw SUBJECT_SAMPLE_FACTORS - Sample_1-100_3 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_1-100_3.raw SUBJECT_SAMPLE_FACTORS - Sample_1-500_1 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_1-500_1.raw SUBJECT_SAMPLE_FACTORS - Sample_1-500_2 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_1-500_2.raw SUBJECT_SAMPLE_FACTORS - Sample_1-500_3 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_1-500_3.raw SUBJECT_SAMPLE_FACTORS - Sample_2-100_1 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_2-100_1.raw SUBJECT_SAMPLE_FACTORS - Sample_2-100_2 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_2-100_2.raw SUBJECT_SAMPLE_FACTORS - Sample_2-100_3 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_2-100_3.raw SUBJECT_SAMPLE_FACTORS - Sample_2-500_1 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_2-500_1.raw SUBJECT_SAMPLE_FACTORS - Sample_2-500_2 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_2-500_2.raw SUBJECT_SAMPLE_FACTORS - Sample_2-500_3 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_2-500_3.raw SUBJECT_SAMPLE_FACTORS - Sample_KO1-100_1 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_KO1-100_1.raw SUBJECT_SAMPLE_FACTORS - Sample_KO1-100_2 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_KO1-100_2.raw SUBJECT_SAMPLE_FACTORS - Sample_KO1-100_3 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_KO1-100_3.raw SUBJECT_SAMPLE_FACTORS - Sample_KO1-500_1 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_KO1-500_1.raw SUBJECT_SAMPLE_FACTORS - Sample_KO1-500_2 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_KO1-500_2.raw SUBJECT_SAMPLE_FACTORS - Sample_KO1-500_3 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_KO1-500_3.raw SUBJECT_SAMPLE_FACTORS - Sample_KO2-100_1 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_KO2-100_1.raw SUBJECT_SAMPLE_FACTORS - Sample_KO2-100_2 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_KO2-100_2.raw SUBJECT_SAMPLE_FACTORS - Sample_KO2-100_3 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_KO2-100_3.raw SUBJECT_SAMPLE_FACTORS - Sample_KO2-500_1 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_KO2-500_1.raw SUBJECT_SAMPLE_FACTORS - Sample_KO2-500_2 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_KO2-500_2.raw SUBJECT_SAMPLE_FACTORS - Sample_KO2-500_3 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_KO2-500_3.raw SUBJECT_SAMPLE_FACTORS - Sample_KO2-500_IN_1 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):Y RAW_FILE_NAME=Schug_m22-L258_Sample_KO2-500_IN_1.raw SUBJECT_SAMPLE_FACTORS - Sample_KO2-500_IN_2 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):Y RAW_FILE_NAME=Schug_m22-L258_Sample_KO2-500_IN_2.raw SUBJECT_SAMPLE_FACTORS - Sample_KO2-500_IN_3 Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):Y RAW_FILE_NAME=Schug_m22-L258_Sample_KO2-500_IN_3.raw SUBJECT_SAMPLE_FACTORS - Sample_N3-100_1 Mouse model:WT C57Bl/6 | Cell Type:NK cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_N3-100_1.raw SUBJECT_SAMPLE_FACTORS - Sample_N3-100_2 Mouse model:WT C57Bl/6 | Cell Type:NK cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_N3-100_2.raw SUBJECT_SAMPLE_FACTORS - Sample_N3-100_3 Mouse model:WT C57Bl/6 | Cell Type:NK cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_N3-100_3.raw SUBJECT_SAMPLE_FACTORS - Sample_N3-500_1 Mouse model:WT C57Bl/6 | Cell Type:NK cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_N3-500_1.raw SUBJECT_SAMPLE_FACTORS - Sample_N3-500_2 Mouse model:WT C57Bl/6 | Cell Type:NK cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_N3-500_2.raw SUBJECT_SAMPLE_FACTORS - Sample_N3-500_3 Mouse model:WT C57Bl/6 | Cell Type:NK cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_N3-500_3.raw SUBJECT_SAMPLE_FACTORS - Sample_WT2-100_1 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_WT2-100_1.raw SUBJECT_SAMPLE_FACTORS - Sample_WT2-100_2 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_WT2-100_2.raw SUBJECT_SAMPLE_FACTORS - Sample_WT2-100_3 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_WT2-100_3.raw SUBJECT_SAMPLE_FACTORS - Sample_WT2-500_1 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_WT2-500_1.raw SUBJECT_SAMPLE_FACTORS - Sample_WT2-500_2 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_WT2-500_2.raw SUBJECT_SAMPLE_FACTORS - Sample_WT2-500_3 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N RAW_FILE_NAME=Schug_m22-L258_Sample_WT2-500_3.raw SUBJECT_SAMPLE_FACTORS - Sample_WT2-500-IN_1 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):Y RAW_FILE_NAME=Schug_m22-L258_Sample_WT2-500-IN_1.raw SUBJECT_SAMPLE_FACTORS - Sample_WT2-500-IN_2 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):Y RAW_FILE_NAME=Schug_m22-L258_Sample_WT2-500-IN_2.raw SUBJECT_SAMPLE_FACTORS - Sample_WT2-500-IN_3 Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):Y RAW_FILE_NAME=Schug_m22-L258_Sample_WT2-500-IN_3.raw #COLLECTION CO:COLLECTION_SUMMARY CD8+ T cells and NK cells were purified by negative selection from freshly CO:COLLECTION_SUMMARY prepared mouse splenocytes (wild type vs ACSS2 knockout C57Bl/6 mice). CO:SAMPLE_TYPE Splenocytes #TREATMENT TR:TREATMENT_SUMMARY Cells were incubated up to 3 hours in the in 100 or 500 µM 13C2-acetate and TR:TREATMENT_SUMMARY treated with vehicle (DMSO) or an ACSS2 inhibitor (VY-3-135). #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells were pelleted and washed with ice-cold PBS before extraction of SP:SAMPLEPREP_SUMMARY metabolites in 80% methanol. Samples were centrifuged at 4 °C two times (18,000 SP:SAMPLEPREP_SUMMARY x g). Deproteinated supernatants were stored at -80 °C prior to analysis. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Hydrophilic interaction liquid chromatography (HILIC) was performed at 0.2 CH:CHROMATOGRAPHY_SUMMARY ml/min on a ZIC-pHILIC column (150 x 2.1 mm, 5 µM particle size, EMD Millipore) CH:CHROMATOGRAPHY_SUMMARY with a ZIC-pHILIC guard column (20 x 2.1 mm, EMD Millipore) at 30 °C. Solvent A CH:CHROMATOGRAPHY_SUMMARY was 20 mM ammonium carbonate, 0.1% ammonium hydroxide, pH 9.2, 5 µM medronic CH:CHROMATOGRAPHY_SUMMARY acid, and solvent B was acetonitrile. The gradient was 85% B for 2 min, 85% B to CH:CHROMATOGRAPHY_SUMMARY 20% B over 15 min, 20% B to 85% B over 0.1 min, and 85% B for 8.9 min. The CH:CHROMATOGRAPHY_SUMMARY autosampler was held at 4 °C. For each analysis, 5 µl of sample was injected. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME SeQuant ZIC-pHILIC (150 x 2.1mm, 5um) CH:SOLVENT_A 20 mM ammonium carbonate, 0.1% ammonium hydroxide, pH 9.2, 5 µM medronic acid CH:SOLVENT_B acetonitrile CH:FLOW_GRADIENT 85% B for 2 min, 85% B to 20% B over 15 min, 20% B to 85% B over 0.1 min, and CH:FLOW_GRADIENT 85% B for 8.9 min CH:FLOW_RATE 0.2 ml/min CH:COLUMN_TEMPERATURE 30 #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 The following parameters were used for the MS analysis: sheath gas flow rate, MS:MS_COMMENTS 30; auxiliary gas flow rate, 5; sweep gas flow rate, 0; auxiliary gas heater MS:MS_COMMENTS temperature, 200 °C; spray voltage, 3.6 kV for positive and negative modes; MS:MS_COMMENTS capillary temperature, 325 °C; and funnel RF level, 65. Samples were analyzed MS:MS_COMMENTS by full MS with polarity switching. Full MS scans were acquired at 60,000 MS:MS_COMMENTS resolution with 2 microscans and a scan range of 72-1080 m/z, automatic gain MS:MS_COMMENTS control (AGC) target of 5e6, and maximum injection time (IT) of 200 ms. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples Sample_1-100_1 Sample_1-100_2 Sample_1-100_3 Sample_1-500_1 Sample_1-500_2 Sample_1-500_3 Sample_2-100_1 Sample_2-100_2 Sample_2-100_3 Sample_2-500_1 Sample_2-500_2 Sample_2-500_3 Sample_KO1-100_1 Sample_KO1-100_2 Sample_KO1-100_3 Sample_KO1-500_1 Sample_KO1-500_2 Sample_KO1-500_3 Sample_KO2-100_1 Sample_KO2-100_2 Sample_KO2-100_3 Sample_KO2-500_1 Sample_KO2-500_2 Sample_KO2-500_3 Sample_KO2-500_IN_1 Sample_KO2-500_IN_2 Sample_KO2-500_IN_3 Sample_N3-100_1 Sample_N3-100_2 Sample_N3-100_3 Sample_N3-500_1 Sample_N3-500_2 Sample_N3-500_3 Sample_WT2-100_1 Sample_WT2-100_2 Sample_WT2-100_3 Sample_WT2-500_1 Sample_WT2-500_2 Sample_WT2-500_3 Sample_WT2-500-IN_1 Sample_WT2-500-IN_2 Sample_WT2-500-IN_3 Factors Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):Y Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):Y Mouse model:Acss2-/- C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):Y Mouse model:WT C57Bl/6 | Cell Type:NK cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:NK cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:NK cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:NK cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:NK cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:NK cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:100 | Time (hours):1 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):N Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):Y Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):Y Mouse model:WT C57Bl/6 | Cell Type:CD8+ T cells | [13C2-acetate] in mM:500 | Time (hours):2 | ACSS2 inhibitor (Y/N):Y alpha-Ketoglutaric acid 3543113 3463554 3380395 1635549 1336395 1447294 10603245 10148443 11102770 7394011 6965021 7738371 5046568 4461495 4475825 4952437 4751793 5342288 3273977 3679792 3814855 2154854 2186893 2219570 2022788 1792595 1857396 7593082 7651623 7561177 4976112 5623879 5525904 5170933 4901837 4719258 2571844 2217707 2537994 3156367 3660426 3420260 alpha-Ketoglutaric acid +1 388356 416414 431622 198354 150990 191159 1549945 1466776 1624480 966050 968229 1062093 389147 356487 343955 665975 652499 721825 418069 485641 509233 388290 336737 397988 359596 290407 371441 914882 894871 859933 862912 1012521 974252 738516 739019 684569 379025 349838 392478 579168 672009 635747 alpha-Ketoglutaric acid +2 888435 855326 918602 1266328 1043645 1159956 2301375 2154035 2381049 4365700 4287511 4546739 861749 823710 795607 3520798 3416320 3754869 887874 1057000 1040599 1660643 1608780 1676110 1460451 1246630 1398342 1146534 1110882 1027539 1933393 2188014 2042067 1392512 1333337 1189251 2042517 1885047 1843113 2496338 2771282 2524961 alpha-Ketoglutaric acid +3 150725 115735 157431 542495 434535 473057 510221 460027 506847 2363170 2247425 2449246 93427 70737 87406 1145812 1045604 1181428 161372 193986 199924 913184 794977 893305 817508 689919 849471 167033 148431 149868 980764 1182938 1105672 282092 228197 258634 1169018 1018573 1080114 1313047 1489090 1427643 alpha-Ketoglutaric acid +4 73331 90088 89277 697583 538814 629965 198111 207168 284301 2904274 3044734 3333867 51448 0 19797 1215909 1166229 1390234 74545 121623 137501 1088496 1101060 1151782 979553 896176 887842 0 0 45423 946032 1021134 1022704 121027 107473 104828 1802942 1412769 1562678 1605076 2076981 1881161 alpha-Ketoglutaric acid +5 0 0 0 236811 185488 233910 0 24650 57060 1612579 1491070 1655055 0 32633 0 266494 195687 317211 24993 0 40026 506557 463706 483198 479209 422345 494910 36451 37585 30512 492979 527775 526241 0 36683 26984 986267 802793 996798 733429 913515 834444 Citric acid 127072208 112042739 128108646 83090462 71009077 90796731 155079417 157638603 173870940 88984217 109532620 121170780 176934435 171998837 166423811 117225998 112819647 128164907 157390311 178658640 180730302 112036398 127319587 121559584 106893039 110359765 113877662 196014435 242383169 248219185 137999260 137434213 149385872 183723076 193299273 202858826 113178608 106626398 123850030 118884168 126387735 133705190 Citric acid +1 15424230 14685391 16582454 9628921 8763468 9751632 26810656 28255609 29573867 15740179 17827555 19283728 18970319 18797701 16948654 17269860 16155972 19448023 25243008 27708495 28032258 26215586 29637231 27518417 24749417 25537495 26020566 28914620 34684695 33058657 33738335 33996188 34497047 29687230 30215116 30065735 26944274 24507267 23495556 28083812 33013658 31325744 Citric acid +2 28939502 26455043 29660553 47203259 42122697 44255311 50219529 48572643 55716364 80034103 92151000 98542303 43993467 42844920 38809262 106354375 102952794 109571409 36679198 40612562 40700215 81214788 84777306 80673695 68423561 72279518 72791703 43499844 49006441 48243644 83716783 83401966 84099508 44583786 41077627 42513973 87162158 80385140 80706127 84734695 103438650 98064173 Citric acid +3 5399300 8668010 9307013 16633060 15513320 18465279 13790726 13481252 13585022 35495358 39735887 43799045 5363846 5758183 5239261 30343876 29486602 33239868 10807900 11666992 12750370 54149802 57756766 56794818 51543050 51755174 53031780 7845557 10447664 10008360 48199668 50005484 50653524 11527614 12775892 12044478 64286344 60480551 61622595 62250432 69925190 69776627 Citric acid +4 2529424 4743281 5097414 27075593 25332290 26979182 8191610 8505135 9336337 49378039 54107372 60391242 3406445 3422257 3309303 47449091 45816625 50917007 3997610 4213098 4631639 45656110 48023138 46128500 41061778 41944218 42083041 3237882 4375695 4330231 40519745 40670404 39658277 3021811 3783686 3734726 61658145 58083940 61704544 50027607 57444260 60307189 Citric acid +5 381920 1406309 1703780 19270992 16426950 19189202 2216710 2529341 2754402 34068873 38026969 41342721 355707 476923 385773 24608022 21768721 25647766 822619 840717 989059 24932450 26004722 26046357 22277827 23314304 23590567 422770 1082161 931690 21250558 21411383 21003755 524622 700778 858916 34550608 31351857 31884848 24797212 30625010 29379794 Citric acid +6 0 181530 276927 5261371 4803200 5093050 140641 178459 82508 16755031 18902454 20509395 0 0 0 3994530 3604117 3906865 0 0 0 9806887 10539306 10584977 8828281 9241615 9546732 0 0 0 10280975 10073373 10675735 0 0 0 18085435 16341520 17145539 10644216 12658575 12384441 Glutamic acid 510064773 506689201 496785551 290322016 243766993 243345781 947956479 936865362 960066216 657071066 674371834 682560362 804788110 751346748 744455453 547774678 552925485 583707259 716053096 722404323 692731825 507489838 496564658 479126365 490677338 445387367 455795926 959948534 910645109 883610547 557425188 573439517 548205939 842848912 774522424 786993232 521559925 496577361 506168992 556707863 573985027 537606678 Glutamic acid +1 64731065 64033748 62963865 40840901 34560033 34453834 142515892 140551558 143829559 99150907 102575980 104250948 75069440 69931271 68945497 76728760 77506541 81996006 113182291 114256911 109109033 105120960 102515904 98824393 103297411 94260050 95971253 136259323 128817527 125846386 116889981 119625083 114881151 134239275 123769364 124333864 93785471 89393240 91389490 117220512 121170659 112838392 Glutamic acid +2 146119761 145026551 141697707 241705677 202769601 202165141 229194743 224700117 228881377 428100001 441749984 446347242 162858637 152544635 153722433 425237749 430215487 453421981 229157096 230172659 220780715 418368374 408749868 395104217 395891495 357052369 365876720 182728492 170007120 166708742 281672768 291581376 277371038 264565017 242956312 248577781 432668918 410542950 419680385 476549803 490745226 460883400 Glutamic acid +3 28775717 28547057 27919693 108794249 90592850 91211917 49836428 49218234 50632230 232442788 240208289 240655733 18978655 17941076 17586699 139025485 139735941 149372474 51094524 51831614 49813698 245922937 240742165 232627128 242582188 220605896 225426187 30791358 29132358 28458650 166866212 172406910 164995021 57958707 53526549 54670307 272158609 259170516 264848072 287674751 294749998 275421047 Glutamic acid +4 17713748 17389439 17125746 141116435 118651391 118177306 24639221 24403863 25138721 296076626 305059386 310941408 9961391 9691770 8910523 153681867 156004007 165811991 29272291 30098371 28512795 307023458 299479255 288755721 300374317 271478750 277589323 12484306 11228802 11394814 156748615 161197023 154300165 33953585 31068070 31401558 384674647 366190144 371472798 354508599 364856765 341677903 Glutamic acid +5 2107561 2079499 2118887 47447668 40215542 39536657 3679007 3651905 3702859 161834954 166055488 168097979 639826 672645 639378 29994419 30424944 32225285 3930565 3929399 3861359 149213206 146006067 140941926 156654249 142052518 145302140 1472620 1417444 1275593 87997979 89457891 85996125 5202572 4768249 5087316 235155502 224761347 228073404 181291678 187332059 174243428 Malic Acid 21210911 22661716 21639114 11475994 10456354 10386552 48969219 45012731 49546364 32644112 36057921 34812926 34067699 30826455 30312891 24624483 22499364 26044301 20397324 20101199 21440448 14218958 13145230 12879555 12595569 12470751 13264909 67201871 68123310 65228783 40202353 39405223 39129868 25190009 25248945 22440808 13626943 13269445 12885269 17540425 16922417 16540587 Malic Acid +1 2546059 2657854 2240746 2086676 1822722 1701177 7352626 6742489 7396704 6496733 7249159 7380622 2439425 2244082 1941838 3066921 2996627 3244652 2697092 2643792 2777105 2911794 2725209 2624240 2674956 2504026 2703439 8302219 8123769 8153223 10695781 10009819 9850016 3293892 3214989 2745069 2968412 2554839 2564959 3913490 3819812 3665406 Malic Acid +2 4283542 4212206 3822815 6623949 5636034 5735135 8628152 7876801 8366209 17512771 19997842 19012970 3903524 3288566 3278450 9461077 9128353 9868579 3793041 3935313 3645372 6633427 5956091 5939458 5727753 5495102 5546311 7643570 7884370 7459413 15818546 15443399 15442997 4603750 4239304 3618398 7475568 6799094 7004058 9347983 8607559 8029345 Malic Acid +3 1026433 856385 1013252 4991552 4413956 4068851 2177583 2094990 2244228 15172827 16751704 16363805 387014 281534 244099 4554275 4250832 4644165 887703 898102 859024 5329790 5211141 4902814 4716276 4813546 5053966 1314796 1316489 1218596 12099856 12117893 11363154 1035406 1169555 884354 6960220 6514674 6567372 7626086 7604532 6907872 Malic Acid +4 0 0 0 1606516 1279600 1201508 195166 133006 185813 7240285 8395875 8188979 0 0 0 710800 816463 924355 0 0 0 2023915 1875018 1982871 2327213 2011710 2048920 0 0 0 5331941 4977635 5305274 0 0 0 3628734 3280978 3277500 3084047 3201285 2931704 Palmitic acid 1284155540 1299963847 1364778616 1366130660 1405327113 1428422321 1207383134 1129856642 1275765194 1237203955 1254987428 1225049781 888740161 946520594 916821287 707426293 746991177 730293505 803866168 780790931 793302553 848075142 866202214 950716110 856601586 890835996 898770799 1163527158 1244400860 1247677309 1129445100 1142573278 1156909267 762713560 851112925 882342149 853287319 934408941 993227464 945618969 1040527748 1184905358 Palmitic acid +2 17473993 18188077 19141124 18940198 19422104 19099628 16193761 15979848 16157605 16592753 16117721 16928892 12119943 12738883 11936257 10132483 8249164 10274200 10116156 10152358 10110018 11726297 11714822 12540615 11624682 11486342 12570296 15658725 16645433 16639281 15365631 16264704 16815773 10626361 10804510 11011420 11871808 13070401 13149591 13955418 12006497 15572796 Palmitic acid +4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Formula m/z RT (min) Adduct alpha-Ketoglutaric acid C5H6O5 145.0143 9.85 M-H alpha-Ketoglutaric acid +1 [C13]C4H6O5 146.0176 9.85 M-H alpha-Ketoglutaric acid +2 [C13]2C3H6O5 147.021 9.85 M-H alpha-Ketoglutaric acid +3 [C13]3C2H6O5 148.0243 9.85 M-H alpha-Ketoglutaric acid +4 [C13]4CH6O5 149.0277 9.85 M-H alpha-Ketoglutaric acid +5 [C13]5H6O5 150.031 9.85 M-H Citric acid C6H8O7 191.0197 11.21 M-H Citric acid +1 [C13]C5H8O7 192.0231 11.21 M-H Citric acid +2 [C13]2C4H8O7 193.0264 11.21 M-H Citric acid +3 [C13]3C3H8O7 194.0298 11.21 M-H Citric acid +4 [C13]4C2H8O7 195.0332 11.21 M-H Citric acid +5 [C13]5C1H8O7 196.0365 11.21 M-H Citric acid +6 [C13]6H8O7 197.0399 11.21 M-H Glutamic acid C5H9NO4 148.0604 9.37 M+H Glutamic acid +1 [C13]C4H9NO4 149.0638 9.37 M+H Glutamic acid +2 [C13]2C3H9NO4 150.0671 9.37 M+H Glutamic acid +3 [C13]3C2H9NO4 151.0705 9.37 M+H Glutamic acid +4 [C13]4CH9NO4 152.0739 9.37 M+H Glutamic acid +5 [C13]5H9NO4 153.0772 9.37 M+H Malic Acid C4H6O5 133.0143 10.16 M-H Malic Acid +1 [C13]C3H6O5 134.0176 10.16 M-H Malic Acid +2 [C13]2C2H6O5 135.021 10.16 M-H Malic Acid +3 [C13]3CH6O5 136.0243 10.16 M-H Malic Acid +4 [C13]4H6O5 137.0277 10.16 M-H Palmitic acid C16H32O2 255.233 1.31 M-H Palmitic acid +2 [C13]2C14H32O2 257.2397 1.31 M-H Palmitic acid +4 [C13]4C12H32O2 259.2464 1.31 M-H METABOLITES_END #END