#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