#METABOLOMICS WORKBENCH gpoulogiannis_20251110_151622 DATATRACK_ID:6649 STUDY_ID:ST004372 ANALYSIS_ID:AN007306 PROJECT_ID:PR002770
VERSION             	1
CREATED_ON             	November 20, 2025, 3:36 pm
#PROJECT
PR:PROJECT_TITLE                 	Assessing the effect of Aspirin treatment on Arachidonic acid levels in PIK3CA
PR:PROJECT_TITLE                 	mutant and wild-type isogenic cells
PR:PROJECT_SUMMARY               	Previous work in our lab showed that PIK3CA mutant breast cancer have elevated
PR:PROJECT_SUMMARY               	levels of Arachidonic acid driven by activation of cPLA2. Follow-up work
PR:PROJECT_SUMMARY               	established cPLA2 as a target of Aspirin, thereby reducing Arachidonic acid
PR:PROJECT_SUMMARY               	levels as well as prostaglandin levels. Arachidonic Acid (AA) level was measured
PR:PROJECT_SUMMARY               	to confirm the impact of Aspirin treatment on its abundance downstream of
PR:PROJECT_SUMMARY               	wild-type (WT) or mutant (MUT) PIK3CA activity. Using Liquid Chromatography-Mass
PR:PROJECT_SUMMARY               	Spectrometry (LC-MS), immortalised breast MCF10A PIK3CA isogenic cells, mammary
PR:PROJECT_SUMMARY               	murine 4T1 PIK3CA isogenic cells and the common drug aspirin, we find that
PR:PROJECT_SUMMARY               	Arachidonic acid levels are reduced following treatment in the PIK3CA MUT
PR:PROJECT_SUMMARY               	context with no (MCF10A) or minor (4T1) effect in the WT context.
PR:INSTITUTE                     	The Institute of Cancer Research London
PR:DEPARTMENT                    	Cell and Molecular Biology
PR:LABORATORY                    	Signalling and Cancer Metabolism
PR:LAST_NAME                     	Poulogiannis
PR:FIRST_NAME                    	George
PR:ADDRESS                       	237 Fulham Road SW3 6JB LONDON
PR:EMAIL                         	George.poulogiannis@icr.ac.uk
PR:PHONE                         	+442071535347
#STUDY
ST:STUDY_TITLE                   	Assessing the effect of Aspirin treatment on Arachidonic acid levels in PIK3CA
ST:STUDY_TITLE                   	mutant and wild-type isogenic cells
ST:STUDY_SUMMARY                 	It has been established that PIK3CA-mutant cancers exhibit elevated arachidonic
ST:STUDY_SUMMARY                 	acid levels compared with PIK3CA wild-type tumours, driven in part by activation
ST:STUDY_SUMMARY                 	of cPLA2 downstream of hyperactivated PI3K signalling. Epidemiological and
ST:STUDY_SUMMARY                 	experimental studies have further suggested a strong anti-cancer effect of
ST:STUDY_SUMMARY                 	aspirin in PIK3CA-mutant tumours, although the underlying mechanism and its
ST:STUDY_SUMMARY                 	therapeutic relevance have remained unclear. In the present work, we demonstrate
ST:STUDY_SUMMARY                 	that aspirin suppresses prostaglandin synthesis through a COX-independent
ST:STUDY_SUMMARY                 	mechanism, specifically via acetylation-mediated inhibition of cPLA2, leading to
ST:STUDY_SUMMARY                 	reduced arachidonic acid availability in PIK3CA-mutant cell lines. We validated
ST:STUDY_SUMMARY                 	this mechanism using liquid chromatography–mass spectrometry (LC-MS) in MCF10A
ST:STUDY_SUMMARY                 	PIK3CA-isogenic breast epithelial cells and 4T1 PIK3CA-isogenic murine mammary
ST:STUDY_SUMMARY                 	cells treated with or without aspirin. Consistent with our model, aspirin
ST:STUDY_SUMMARY                 	markedly decreases arachidonic acid levels in PIK3CA-mutant cells, while
ST:STUDY_SUMMARY                 	exerting no detectable effect in wild-type MCF10A cells and only a modest
ST:STUDY_SUMMARY                 	reduction in wild-type 4T1 cells.
ST:INSTITUTE                     	The Institute of Cancer Research London
ST:DEPARTMENT                    	Cell and Molecular Biology
ST:LABORATORY                    	Signalling and Cancer Metabolism
ST:LAST_NAME                     	Poulogiannis
ST:FIRST_NAME                    	George
ST:ADDRESS                       	237 Fulham Road SW3 6JB LONDON
ST:EMAIL                         	george.poulogiannis@icr.ac.uk
ST:PHONE                         	+442071535347
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens, Mus musculus
SU:TAXONOMY_ID                   	9606, 10090
SU:GENDER                        	Female
SU:CELL_BIOSOURCE_OR_SUPPLIER    	MCF10A: Horizon discovery / 4T1: EditCo
SU:CELL_STRAIN_DETAILS           	Human: MCF-10A (CVCL_0598) and MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) / Murine:
SU:CELL_STRAIN_DETAILS           	4T1 PIK3CA WT (+/+) (CVCL_0125) and 4T1 PIK3CA (H1047R/+) Clone J6 (Custom made)
SU:SUBJECT_COMMENTS              	MC10A isogenic cells were acquired from Horizon discovery. 4T1 isogenic cells
SU:SUBJECT_COMMENTS              	were custom-made by EditCo.
SU:SPECIES_GROUP                 	Human (MCF10A) and mouse (4T1)
#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           	-	4T1 Parental Control 1	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	RAW_FILE_NAME=4T1 Parental Control 1.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 Parental Control 1.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 Parental Control 2	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	RAW_FILE_NAME=4T1 Parental Control 2.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 Parental Control 2.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 Parental Control 3	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	RAW_FILE_NAME=4T1 Parental Control 3.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 Parental Control 3.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 Parental Control 4	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	RAW_FILE_NAME=4T1 Parental Control 4.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 Parental Control 4.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 Parental Control 5	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	RAW_FILE_NAME=4T1 Parental Control 5.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 Parental Control 5.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 Parental Aspirin 1	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=4T1 Parental Aspirin 1.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 Parental Aspirin 1.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 Parental Aspirin 2	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=4T1 Parental Aspirin 2.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 Parental Aspirin 2.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 Parental Aspirin 3	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=4T1 Parental Aspirin 3.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 Parental Aspirin 3.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 Parental Aspirin 4	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=4T1 Parental Aspirin 4.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 Parental Aspirin 4.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 Parental Aspirin 5	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=4T1 Parental Aspirin 5.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 Parental Aspirin 5.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 PIK3CA Mutant J6 Control 1	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	RAW_FILE_NAME=4T1 PIK3CA Mutant J6 Control 1.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 PIK3CA Mutant J6 Control 1.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 PIK3CA Mutant J6 Control 2	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	RAW_FILE_NAME=4T1 PIK3CA Mutant J6 Control 2.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 PIK3CA Mutant J6 Control 2.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 PIK3CA Mutant J6 Control 3	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	RAW_FILE_NAME=4T1 PIK3CA Mutant J6 Control 3.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 PIK3CA Mutant J6 Control 3.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 PIK3CA Mutant J6 Control 4	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	RAW_FILE_NAME=4T1 PIK3CA Mutant J6 Control 4.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 PIK3CA Mutant J6 Control 4.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 PIK3CA Mutant J6 Control 5	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	RAW_FILE_NAME=4T1 PIK3CA Mutant J6 Control 5.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 PIK3CA Mutant J6 Control 5.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 PIK3CA Mutant J6 Aspirin 1	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=4T1 PIK3CA Mutant J6 Aspirin 1.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 PIK3CA Mutant J6 Aspirin 1.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 PIK3CA Mutant J6 Aspirin 2	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=4T1 PIK3CA Mutant J6 Aspirin 2.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 PIK3CA Mutant J6 Aspirin 2.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 PIK3CA Mutant J6 Aspirin 3	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=4T1 PIK3CA Mutant J6 Aspirin 3.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 PIK3CA Mutant J6 Aspirin 3.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 PIK3CA Mutant J6 Aspirin 4	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=4T1 PIK3CA Mutant J6 Aspirin 4.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 PIK3CA Mutant J6 Aspirin 4.mzML
SUBJECT_SAMPLE_FACTORS           	-	4T1 PIK3CA Mutant J6 Aspirin 5	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=4T1 PIK3CA Mutant J6 Aspirin 5.d; RAW_FILE_NAME(MZML_FILE_NAME)=4T1 PIK3CA Mutant J6 Aspirin 5.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A Parental Control 1	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:DMSO	RAW_FILE_NAME=MCF10A Parental Control 1.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A Parental Control 1.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A Parental Control 2	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:DMSO	RAW_FILE_NAME=MCF10A Parental Control 2.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A Parental Control 2.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A Parental Control 3	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:DMSO	RAW_FILE_NAME=MCF10A Parental Control 3.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A Parental Control 3.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A Parental Control 4	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:DMSO	RAW_FILE_NAME=MCF10A Parental Control 4.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A Parental Control 4.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A Parental Control 5	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:DMSO	RAW_FILE_NAME=MCF10A Parental Control 5.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A Parental Control 5.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A Parental Aspirin 1	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=MCF10A Parental Aspirin 1.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A Parental Aspirin 1.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A Parental Aspirin 2	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=MCF10A Parental Aspirin 2.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A Parental Aspirin 2.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A Parental Aspirin 3	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=MCF10A Parental Aspirin 3.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A Parental Aspirin 3.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A Parental Aspirin 4	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=MCF10A Parental Aspirin 4.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A Parental Aspirin 4.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A Parental Aspirin 5	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=MCF10A Parental Aspirin 5.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A Parental Aspirin 5.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A PIK3CA Mutant H1047R Control 2	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:DMSO	RAW_FILE_NAME=MCF10A PIK3CA Mutant H1047R Control 2.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A PIK3CA Mutant H1047R Control 2.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A PIK3CA Mutant H1047R Control 3	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:DMSO	RAW_FILE_NAME=MCF10A PIK3CA Mutant H1047R Control 3.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A PIK3CA Mutant H1047R Control 3.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A PIK3CA Mutant H1047R Control 4	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:DMSO	RAW_FILE_NAME=MCF10A PIK3CA Mutant H1047R Control 4.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A PIK3CA Mutant H1047R Control 4.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A PIK3CA Mutant H1047R Control 5	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:DMSO	RAW_FILE_NAME=MCF10A PIK3CA Mutant H1047R Control 5.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A PIK3CA Mutant H1047R Control 5.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A PIK3CA Mutant H1047R Aspirin 1	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=MCF10A PIK3CA Mutant H1047R Aspirin 1.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A PIK3CA Mutant H1047R Aspirin 1.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A PIK3CA Mutant H1047R Aspirin 2	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=MCF10A PIK3CA Mutant H1047R Aspirin 2.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A PIK3CA Mutant H1047R Aspirin 2.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A PIK3CA Mutant H1047R Aspirin 3	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=MCF10A PIK3CA Mutant H1047R Aspirin 3.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A PIK3CA Mutant H1047R Aspirin 3.mzML
SUBJECT_SAMPLE_FACTORS           	-	MCF10A PIK3CA Mutant H1047R Aspirin 5	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:Aspirin 1.5mM	RAW_FILE_NAME=MCF10A PIK3CA Mutant H1047R Aspirin 5.d; RAW_FILE_NAME(MZML_FILE_NAME)=MCF10A PIK3CA Mutant H1047R Aspirin 5.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	MCF10A Wild-type and PIK3CA mutant (H1047R/+) isogenic cells were purchased from
CO:COLLECTION_SUMMARY            	Horizon discovery (Cat# HD 101-011). Cells were grown in their recommended
CO:COLLECTION_SUMMARY            	media, as follow: DMEM/F-12 including 2.5 mM L-glutamine and 15 mM HEPES,
CO:COLLECTION_SUMMARY            	supplemented with 5% horse serum, 10 µg/mL insulin, 0.5 µg/mL hydrocortisone,
CO:COLLECTION_SUMMARY            	0.1 µg/mL cholera toxin. 4T1 parental and PIK3CA mutant (H1047R/+) isogenic
CO:COLLECTION_SUMMARY            	cells were custom made by EditCo. Cells were grown in their recommended media as
CO:COLLECTION_SUMMARY            	follow: RPMI 1640 supplemented with 10% foetal bovine serum (FBS). Cells were
CO:COLLECTION_SUMMARY            	cultured at 37°C in a 5% CO2 incubator. The day before treatment, cells were
CO:COLLECTION_SUMMARY            	seeded at a density of 3M (MCF10A) or 4M (4T1) cells per plate in 150mm cell
CO:COLLECTION_SUMMARY            	culture dish.
CO:COLLECTION_PROTOCOL_FILENAME  	Eicosanoids_aspirin.pdf
CO:SAMPLE_TYPE                   	Cultured cells
CO:STORAGE_CONDITIONS            	Described in summary
#TREATMENT
TR:TREATMENT_SUMMARY             	Cells were treated the day after seeding with 1.5mM Aspirin or DMSO as negative
TR:TREATMENT_SUMMARY             	control for 24 hours.
TR:TREATMENT_COMPOUND            	Aspirin
TR:TREATMENT_DOSE                	1.5mM
TR:CELL_PCT_CONFLUENCE           	40-50%
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	On the day of the extraction, cells were placed on ice and washed twice with
SP:SAMPLEPREP_SUMMARY            	cold PBS. Then cells were scraped using 1mL of extraction buffer (MeOH + 0.05%
SP:SAMPLEPREP_SUMMARY            	butylated-hydroxytoluene (BHT) + AA-D11 (100ng/mL) + PGE2-D4 (100ng/ml)) and
SP:SAMPLEPREP_SUMMARY            	transferred into Eppendorf tubes. Samples were then placed on shaker for 15min
SP:SAMPLEPREP_SUMMARY            	at 4OC and then centrifugated for 15 min at 4OC, 3000g. Supernatant was
SP:SAMPLEPREP_SUMMARY            	transferred to a 15mL Falcon tube and LC-MS grade water added to reach a final
SP:SAMPLEPREP_SUMMARY            	volume of 11mL to decrease methanol concentration below 10% for efficient
SP:SAMPLEPREP_SUMMARY            	extraction using solid phase extraction (SPE) cartridges. SPE was achieved using
SP:SAMPLEPREP_SUMMARY            	Oasis HLB 6cc Vac Cartridge (SKU: WAT106202). Cartridges were washed with 3mL of
SP:SAMPLEPREP_SUMMARY            	Methanol and equilibrated with 3mL of water. Samples were then loaded and passed
SP:SAMPLEPREP_SUMMARY            	through the column at an approximate rate of 1 drop per second. After loading,
SP:SAMPLEPREP_SUMMARY            	the cartridges were flushed with 6mL of 1:9 v:v Methanol:Water + 0.05% BHT for
SP:SAMPLEPREP_SUMMARY            	sample cleaning and analytes elution was performed with 1mL Methanol + 0.05%
SP:SAMPLEPREP_SUMMARY            	BHT. Samples were then dried under nitrogen stream at RT in a fume hood and
SP:SAMPLEPREP_SUMMARY            	resuspended in 80l of 4:3:3 v:v:v Methanol:Acetonitrile:Water before
SP:SAMPLEPREP_SUMMARY            	transferring into glass LC/MS vials.
SP:SAMPLEPREP_PROTOCOL_FILENAME  	Eicosanoids_aspirin.pdf
SP:PROCESSING_STORAGE_CONDITIONS 	Described in summary
SP:EXTRACT_ENRICHMENT            	Oasis HLB 6cc Vac Cartridge (SKU: WAT106202)
SP:SAMPLE_RESUSPENSION           	80ul of 4:3:3 v:v:v Methanol:Acetonitrile:Water
SP:SAMPLE_SPIKING                	AA-D11 (100ng/mL) + PGE2-D4 (100ng/ml)
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Eicosanoid analysis was performed using a UHPLC-Q/TOF system. Chromatographic
CH:CHROMATOGRAPHY_SUMMARY        	separation was carried out on an Agilent 1290 Infinity II Binary Pump using a
CH:CHROMATOGRAPHY_SUMMARY        	Agilent InfinityLab Poroshell 120 Phenyl-Hexyl column (2.7um, 3.0cm*150mm)
CH:CHROMATOGRAPHY_SUMMARY        	(Agilent – Cat.: 693675-312) maintained at 35°C. Samples were maintained at
CH:CHROMATOGRAPHY_SUMMARY        	20°C in an autosampler and injection volume was set to 20μL. The elution of
CH:CHROMATOGRAPHY_SUMMARY        	metabolites through the column was achieved at a flow rate of 0.600mL/min using
CH:CHROMATOGRAPHY_SUMMARY        	as solvent A, 0.05% acetic acid in Water and solvent B, 1:1 v:v
CH:CHROMATOGRAPHY_SUMMARY        	Acetonitrile:Methanol. The following gradient was used for elution: 0min, 45%B;
CH:CHROMATOGRAPHY_SUMMARY        	12min, 45%B; 12.5min, 65%B; 21min, 65%B; 21.1min, 98%B; 23min, 98%B; 23.1min,
CH:CHROMATOGRAPHY_SUMMARY        	100%B; 25min, 100%B. Post time of 3 min was added between samples to achieve
CH:CHROMATOGRAPHY_SUMMARY        	re-equilibration.
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Agilent 1290 Infinity II
CH:COLUMN_NAME                   	Agilent InfinityLab Poroshell 120 Phenyl-Hexyl column (150 x 30mm, 2.7um)
CH:SOLVENT_A                     	100% Water; 0.05% acetic acid
CH:SOLVENT_B                     	50% Acetonitrile/50% Methanol
CH:FLOW_GRADIENT                 	0min, 45%B; 12min, 45%B; 12.5min, 65%B; 21min, 65%B; 21.1min, 98%B; 23min, 98%B;
CH:FLOW_GRADIENT                 	23.1min, 100%B; 25min, 100%B
CH:FLOW_RATE                     	0.600 mL/min
CH:COLUMN_TEMPERATURE            	35℃
CH:INJECTION_TEMPERATURE         	20℃
CH:SAMPLE_INJECTION              	20 µL
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:LABORATORY_NAME               	Signalling and Cancer Metabolism
AN:ACQUISITION_DATE              	08/2025 - 10/2025
AN:DATA_FORMAT                   	.d
#MS
MS:INSTRUMENT_NAME               	Agilent 6546 QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Mass spectrometry analysis was performed on an Agilent 6546 LC/Q-TOF mass
MS:MS_COMMENTS                   	spectrometer in MS negative polarity mode. Settings for MS mode were as follows:
MS:MS_COMMENTS                   	Gas Temperature: 200°C; Gas flow: 12L/min; Nebulizer pressure: 45psig; Sheath
MS:MS_COMMENTS                   	gas temperature: 350°C; Sheath gas flow: 12L/min; Capillary Voltage: 3500V;
MS:MS_COMMENTS                   	Fragmentor: 175V; Skimmer 65V; Octopole RF Vpp 750V. Data was acquired in
MS:MS_COMMENTS                   	centroid mode at a rate of 1 spectra/s between m/z value 100 to 170. Agilent
MS:MS_COMMENTS                   	Masshunter Workstation Data Acquisition software was used for data acquisition.
MS:MS_COMMENTS                   	For data analysis, Agilent Profinder B.8.0.00 was used with retention time
MS:MS_COMMENTS                   	determined using MS/MS and standards for AA and PGE2.
MS:CAPILLARY_VOLTAGE             	3500
MS:DRY_GAS_FLOW                  	12l/min
MS:DRY_GAS_TEMP                  	200
MS:FRAGMENT_VOLTAGE              	175
MS:NEBULIZER                     	45 psi
MS:OCTPOLE_VOLTAGE               	750
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Arbitrary unit/ug protein
MS_METABOLITE_DATA_START
Samples	4T1 Parental Control 1	4T1 Parental Control 2	4T1 Parental Control 3	4T1 Parental Control 4	4T1 Parental Control 5	4T1 Parental Aspirin 1	4T1 Parental Aspirin 2	4T1 Parental Aspirin 3	4T1 Parental Aspirin 4	4T1 Parental Aspirin 5	4T1 PIK3CA Mutant J6 Control 1	4T1 PIK3CA Mutant J6 Control 2	4T1 PIK3CA Mutant J6 Control 3	4T1 PIK3CA Mutant J6 Control 4	4T1 PIK3CA Mutant J6 Control 5	4T1 PIK3CA Mutant J6 Aspirin 1	4T1 PIK3CA Mutant J6 Aspirin 2	4T1 PIK3CA Mutant J6 Aspirin 3	4T1 PIK3CA Mutant J6 Aspirin 4	4T1 PIK3CA Mutant J6 Aspirin 5	MCF10A Parental Control 1	MCF10A Parental Control 2	MCF10A Parental Control 3	MCF10A Parental Control 4	MCF10A Parental Control 5	MCF10A Parental Aspirin 1	MCF10A Parental Aspirin 2	MCF10A Parental Aspirin 3	MCF10A Parental Aspirin 4	MCF10A Parental Aspirin 5	MCF10A PIK3CA Mutant H1047R Control 2	MCF10A PIK3CA Mutant H1047R Control 3	MCF10A PIK3CA Mutant H1047R Control 4	MCF10A PIK3CA Mutant H1047R Control 5	MCF10A PIK3CA Mutant H1047R Aspirin 1	MCF10A PIK3CA Mutant H1047R Aspirin 2	MCF10A PIK3CA Mutant H1047R Aspirin 3	MCF10A PIK3CA Mutant H1047R Aspirin 5
Factors	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:DMSO	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	Sample source:4T1 (CVCL_0125) | Species:Murine | Drug treatment:Aspirin 1.5mM	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:DMSO	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:DMSO	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:DMSO	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:DMSO	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:DMSO	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:Aspirin 1.5mM	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:Aspirin 1.5mM	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:Aspirin 1.5mM	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:Aspirin 1.5mM	Sample source:MCF-10A (CVCL_0598) | Species:Human | Drug treatment:Aspirin 1.5mM	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:DMSO	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:DMSO	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:DMSO	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:DMSO	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:Aspirin 1.5mM	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:Aspirin 1.5mM	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:Aspirin 1.5mM	Sample source:MCF-10A PIK3CA (H1047R/+)(CVCL_LD55) | Species:Human | Drug treatment:Aspirin 1.5mM
Arachidonic Acid	317889.6124	292176.1844	248516.9752	169597.2801	120309.8258	202697.3348	155568.6584	92697.14429	107457.3861	169593.6998	283358.0326	469763.0513	452683.0096	388194.4405	291300.7882	152160.7356	212222.5041	138755.0566	160574.9704	158990.444	621569.7991	616407.3582	695165.1374	727373.2559	757793.4181	574469.4545	825108.5313	599149.4428	691641.5992	735690.3518	1089208.378	804773.9747	1033115.455	868426.0676	723387.3624	425508.2561	477353.2774	451585.1762
Prostaglandin E2	26209.37123	30272.26748	30665.85693	20187.58444	34401.00001	13541.3547	10356.59178	3093.23839	10232.06949	11591.74829	46703.06417	38139.67414	40013.2106	43178.53943	22667.00651	9056.258258	10479.8876	8301.306868	10466.44569	9746.147272																		
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Formula	Mass	RT	CAS ID	KEGG	ChEBI
Arachidonic Acid	C20 H32 O2	304.2397	22.49	506-32-1	C00219	CHEBI:15843
Prostaglandin E2	C20 H32 O5	352.2242	6.23	363-24-6	C00584	CHEBI:15551
METABOLITES_END
#END