#METABOLOMICS WORKBENCH shancock_20250220_142626 DATATRACK_ID:5660 STUDY_ID:ST003762 ANALYSIS_ID:AN006177 PROJECT_ID:PR002345 VERSION 1 CREATED_ON February 21, 2025, 5:09 pm #PROJECT PR:PROJECT_TITLE Stable-label palmitate tracing of gemcitabine-based chemotherapy in PDAC cells PR:PROJECT_SUMMARY Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with few PR:PROJECT_SUMMARY treatment options and poor survivability. In this work we sought to characterise PR:PROJECT_SUMMARY metabolic adaptations to gemcitabine (GEMC)-based chemotherapy exposure to PR:PROJECT_SUMMARY discover new therapeutic targets for improving treatment efficacy. We show that PR:PROJECT_SUMMARY GEMC resistance (GEMR) upregulates de novo lipogenesis in Panc1 and MiaPaCa2 PR:PROJECT_SUMMARY cells. PR:INSTITUTE Victor Chang Cardiac Research Institute PR:LAST_NAME Hancock PR:FIRST_NAME Sarah PR:ADDRESS Level 7 Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW 2010, PR:ADDRESS Australia PR:EMAIL s.hancock@victorchang.edu.au PR:PHONE +61414537526 #STUDY ST:STUDY_TITLE Stable-label palmitate tracing of gemcitabine-based chemotherapy in PDAC cells ST:STUDY_SUMMARY In this work we developed both gemcitabine resistant (GEMR) Panc1 and MiaPaCa2 ST:STUDY_SUMMARY cell lines and combination gemcitabine/paclitaxel attenuated (CombAT) Panc1 and ST:STUDY_SUMMARY MiaPaCa2 cells. We found significant upregulation of de novo lipogenesis in both ST:STUDY_SUMMARY cell lines following GEMR or CombAT. In this study, we traced uniformly 13C ST:STUDY_SUMMARY labelled palmitate into all cell lines and compared it with controls (CON) to ST:STUDY_SUMMARY derived relative activity of stearoyl-CoA desaturase 1 (SCD1) and fatty acid ST:STUDY_SUMMARY desaturase 2 (FADS2). We found upregulation of SCD1 in Panc1 GEMR through ST:STUDY_SUMMARY increased ratio 16:1n-7/16:0, with no change in SCD1 activity with CombAT in ST:STUDY_SUMMARY either cell line. FADS2 activity was substantially suppressed in Panc1 CombAT ST:STUDY_SUMMARY cells through decreased 16:1n-10/16:0. This study contains the following groups: ST:STUDY_SUMMARY source - cell lines Panc1 or MiaPaCa2, blank or fatty acid methyl ester ST:STUDY_SUMMARY reference (FAMEs) group - control (CON), gemcitabine resistant (GEMR), or ST:STUDY_SUMMARY combination gemcitabine/paclitaxel attenuated (CombAT). matched_CON - GEMR or ST:STUDY_SUMMARY CombAT each had their own matched control cells plated at the same time label - ST:STUDY_SUMMARY cells cultured in either unlabelled (UL) or labelled (13C) palmitate (12.5 uM) ST:STUDY_SUMMARY conjugated to bovine serum albumin (2% w/v). ST:INSTITUTE Victor Chang Cardiac Research Institute ST:LAST_NAME Hancock ST:FIRST_NAME Sarah ST:ADDRESS Level 7 Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW 2010, ST:ADDRESS Australia ST:EMAIL s.hancock@victorchang.edu.au ST:PHONE +61414537526 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - Blank Sample source:Blank | group1:Blank | matched_CON:- | label:- RAW_FILE_NAME(Raw file name)=Blank.mzML; protein amount=- SUBJECT_SAMPLE_FACTORS - FAMEs-mix_01 Sample source:FAMEs | group1:FAMEs | matched_CON:- | label:- RAW_FILE_NAME(Raw file name)=FAMEs-mix_01.mzML; protein amount=- SUBJECT_SAMPLE_FACTORS - FAMEs-mix_02 Sample source:FAMEs | group1:FAMEs | matched_CON:- | label:- RAW_FILE_NAME(Raw file name)=FAMEs-mix_02.mzML; protein amount=- SUBJECT_SAMPLE_FACTORS - FAMEs-mix_03 Sample source:FAMEs | group1:FAMEs | matched_CON:- | label:- RAW_FILE_NAME(Raw file name)=FAMEs-mix_03.mzML; protein amount=- SUBJECT_SAMPLE_FACTORS - M64_1_13C_p1 Sample source:MiaPaCa2 cells | group1:GEMR | matched_CON:1 | label:13C RAW_FILE_NAME(Raw file name)=M64_1_13C_p1.mzML; protein amount=234.4564935 SUBJECT_SAMPLE_FACTORS - M64_1_13C_p2 Sample source:MiaPaCa2 cells | group1:GEMR | matched_CON:1 | label:13C RAW_FILE_NAME(Raw file name)=M64_1_13C_p2.mzML; protein amount=215.0601274 SUBJECT_SAMPLE_FACTORS - M64_1_13C_p3 Sample source:MiaPaCa2 cells | group1:GEMR | matched_CON:1 | label:13C RAW_FILE_NAME(Raw file name)=M64_1_13C_p3.mzML; protein amount=328.7749983 SUBJECT_SAMPLE_FACTORS - M64_1_UL_p1 Sample source:MiaPaCa2 cells | group1:GEMR | matched_CON:1 | label:UL RAW_FILE_NAME(Raw file name)=M64_1_UL_p1.mzML; protein amount=229.5226983 SUBJECT_SAMPLE_FACTORS - M64_1_UL_p2 Sample source:MiaPaCa2 cells | group1:GEMR | matched_CON:1 | label:UL RAW_FILE_NAME(Raw file name)=M64_1_UL_p2.mzML; protein amount=218.3641978 SUBJECT_SAMPLE_FACTORS - M64_1_UL_p3 Sample source:MiaPaCa2 cells | group1:GEMR | matched_CON:1 | label:UL RAW_FILE_NAME(Raw file name)=M64_1_UL_p3.mzML; protein amount=333.8193232 SUBJECT_SAMPLE_FACTORS - M8C_2_13C_p1 Sample source:MiaPaCa2 cells | group1:CombAT | matched_CON:2 | label:13C RAW_FILE_NAME(Raw file name)=M8C_2_13C_p1.mzML; protein amount=225.5124255 SUBJECT_SAMPLE_FACTORS - M8C_2_13C_p2 Sample source:MiaPaCa2 cells | group1:CombAT | matched_CON:2 | label:13C RAW_FILE_NAME(Raw file name)=M8C_2_13C_p2.mzML; protein amount=245.1506245 SUBJECT_SAMPLE_FACTORS - M8C_2_13C_p3 Sample source:MiaPaCa2 cells | group1:CombAT | matched_CON:2 | label:13C RAW_FILE_NAME(Raw file name)=M8C_2_13C_p3.mzML; protein amount=304.3879635 SUBJECT_SAMPLE_FACTORS - M8C_2_UL_p1 Sample source:MiaPaCa2 cells | group1:CombAT | matched_CON:2 | label:UL RAW_FILE_NAME(Raw file name)=M8C_2_UL_p1.mzML; protein amount=289.878918 SUBJECT_SAMPLE_FACTORS - M8C_2_UL_p2 Sample source:MiaPaCa2 cells | group1:CombAT | matched_CON:2 | label:UL RAW_FILE_NAME(Raw file name)=M8C_2_UL_p2.mzML; protein amount=248.0786945 SUBJECT_SAMPLE_FACTORS - M8C_2_UL_p3 Sample source:MiaPaCa2 cells | group1:CombAT | matched_CON:2 | label:UL RAW_FILE_NAME(Raw file name)=M8C_2_UL_p3.mzML; protein amount=299.4749735 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_1_13C_p1 Sample source:MiaPaCa2 cells | group1:CON | matched_CON:1 | label:13C RAW_FILE_NAME(Raw file name)=MiaPaCa2_1_13C_p1.mzML; protein amount=306.4188303 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_1_13C_p2 Sample source:MiaPaCa2 cells | group1:CON | matched_CON:1 | label:13C RAW_FILE_NAME(Raw file name)=MiaPaCa2_1_13C_p2.mzML; protein amount=275.3659849 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_1_13C_p3 Sample source:MiaPaCa2 cells | group1:CON | matched_CON:1 | label:13C RAW_FILE_NAME(Raw file name)=MiaPaCa2_1_13C_p3.mzML; protein amount=300.357848 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_1_UL_p1 Sample source:MiaPaCa2 cells | group1:CON | matched_CON:1 | label:UL RAW_FILE_NAME(Raw file name)=MiaPaCa2_1_UL_p1.mzML; protein amount=352.7149027 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_1_UL_p2 Sample source:MiaPaCa2 cells | group1:CON | matched_CON:1 | label:UL RAW_FILE_NAME(Raw file name)=MiaPaCa2_1_UL_p2.mzML; protein amount=298.7694553 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_1_UL_p3 Sample source:MiaPaCa2 cells | group1:CON | matched_CON:1 | label:UL RAW_FILE_NAME(Raw file name)=MiaPaCa2_1_UL_p3.mzML; protein amount=316.9341875 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_2_13C_p1 Sample source:MiaPaCa2 cells | group1:CON | matched_CON:2 | label:13C RAW_FILE_NAME(Raw file name)=MiaPaCa2_2_13C_p1.mzML; protein amount=269.5479532 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_2_13C_p2 Sample source:MiaPaCa2 cells | group1:CON | matched_CON:2 | label:13C RAW_FILE_NAME(Raw file name)=MiaPaCa2_2_13C_p2.mzML; protein amount=230.5828978 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_2_13C_p3 Sample source:MiaPaCa2 cells | group1:CON | matched_CON:2 | label:13C RAW_FILE_NAME(Raw file name)=MiaPaCa2_2_13C_p3.mzML; protein amount=218.8342804 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_2_UL_p1 Sample source:MiaPaCa2 cells | group1:CON | matched_CON:2 | label:UL RAW_FILE_NAME(Raw file name)=MiaPaCa2_2_UL_p1.mzML; protein amount=247.8955469 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_2_UL_p2 Sample source:MiaPaCa2 cells | group1:CON | matched_CON:2 | label:UL RAW_FILE_NAME(Raw file name)=MiaPaCa2_2_UL_p2.mzML; protein amount=239.6736103 SUBJECT_SAMPLE_FACTORS - MiaPaCa2_2_UL_p3 Sample source:MiaPaCa2 cells | group1:CON | matched_CON:2 | label:UL RAW_FILE_NAME(Raw file name)=MiaPaCa2_2_UL_p3.mzML; protein amount=269.9188485 SUBJECT_SAMPLE_FACTORS - P150_1_13C_p1 Sample source:Panc1 cells | group1:GEMR | matched_CON:1 | label:13C RAW_FILE_NAME(Raw file name)=P150_1_13C_p1.mzML; protein amount=396.8667546 SUBJECT_SAMPLE_FACTORS - P150_1_13C_p2 Sample source:Panc1 cells | group1:GEMR | matched_CON:1 | label:13C RAW_FILE_NAME(Raw file name)=P150_1_13C_p2.mzML; protein amount=275.4691808 SUBJECT_SAMPLE_FACTORS - P150_1_13C_p3 Sample source:Panc1 cells | group1:GEMR | matched_CON:1 | label:13C RAW_FILE_NAME(Raw file name)=P150_1_13C_p3.mzML; protein amount=310.1604171 SUBJECT_SAMPLE_FACTORS - P150_1_UL_p1 Sample source:Panc1 cells | group1:GEMR | matched_CON:1 | label:UL RAW_FILE_NAME(Raw file name)=P150_1_UL_p1.mzML; protein amount=428.1118929 SUBJECT_SAMPLE_FACTORS - P150_1_UL_p2 Sample source:Panc1 cells | group1:GEMR | matched_CON:1 | label:UL RAW_FILE_NAME(Raw file name)=P150_1_UL_p2.mzML; protein amount=288.249109 SUBJECT_SAMPLE_FACTORS - P150_1_UL_p3 Sample source:Panc1 cells | group1:GEMR | matched_CON:1 | label:UL RAW_FILE_NAME(Raw file name)=P150_1_UL_p3.mzML; protein amount=313.5947605 SUBJECT_SAMPLE_FACTORS - P28C_2_13C_p1 Sample source:Panc1 cells | group1:CombAT | matched_CON:2 | label:13C RAW_FILE_NAME(Raw file name)=P28C_2_13C_p1.mzML; protein amount=245.3334858 SUBJECT_SAMPLE_FACTORS - P28C_2_13C_p2 Sample source:Panc1 cells | group1:CombAT | matched_CON:2 | label:13C RAW_FILE_NAME(Raw file name)=P28C_2_13C_p2.mzML; protein amount=211.5529831 SUBJECT_SAMPLE_FACTORS - P28C_2_13C_p3 Sample source:Panc1 cells | group1:CombAT | matched_CON:2 | label:13C RAW_FILE_NAME(Raw file name)=P28C_2_13C_p3.mzML; protein amount=237.4875664 SUBJECT_SAMPLE_FACTORS - P28C_2_UL_p1 Sample source:Panc1 cells | group1:CombAT | matched_CON:2 | label:UL RAW_FILE_NAME(Raw file name)=P28C_2_UL_p1.mzML; protein amount=239.5824709 SUBJECT_SAMPLE_FACTORS - P28C_2_UL_p2 Sample source:Panc1 cells | group1:CombAT | matched_CON:2 | label:UL RAW_FILE_NAME(Raw file name)=P28C_2_UL_p2.mzML; protein amount=210.0285672 SUBJECT_SAMPLE_FACTORS - P28C_2_UL_p3 Sample source:Panc1 cells | group1:CombAT | matched_CON:2 | label:UL RAW_FILE_NAME(Raw file name)=P28C_2_UL_p3.mzML; protein amount=195.0329264 SUBJECT_SAMPLE_FACTORS - Panc1_1_13C_p1 Sample source:Panc1 cells | group1:CON | matched_CON:1 | label:13C RAW_FILE_NAME(Raw file name)=Panc1_1_13C_p1.mzML; protein amount=342.3277071 SUBJECT_SAMPLE_FACTORS - Panc1_1_13C_p2 Sample source:Panc1 cells | group1:CON | matched_CON:1 | label:13C RAW_FILE_NAME(Raw file name)=Panc1_1_13C_p2.mzML; protein amount=306.3121521 SUBJECT_SAMPLE_FACTORS - Panc1_1_13C_p3 Sample source:Panc1 cells | group1:CON | matched_CON:1 | label:13C RAW_FILE_NAME(Raw file name)=Panc1_1_13C_p3.mzML; protein amount=261.0277605 SUBJECT_SAMPLE_FACTORS - Panc1_1_UL_p1 Sample source:Panc1 cells | group1:CON | matched_CON:1 | label:UL RAW_FILE_NAME(Raw file name)=Panc1_1_UL_p1.mzML; protein amount=186.4569944 SUBJECT_SAMPLE_FACTORS - Panc1_1_UL_p2 Sample source:Panc1 cells | group1:CON | matched_CON:1 | label:UL RAW_FILE_NAME(Raw file name)=Panc1_1_UL_p2.mzML; protein amount=351.7042812 SUBJECT_SAMPLE_FACTORS - Panc1_1_UL_p3 Sample source:Panc1 cells | group1:CON | matched_CON:1 | label:UL RAW_FILE_NAME(Raw file name)=Panc1_1_UL_p3.mzML; protein amount=258.0843237 SUBJECT_SAMPLE_FACTORS - Panc1_2_13C_p1 Sample source:Panc1 cells | group1:CON | matched_CON:2 | label:13C RAW_FILE_NAME(Raw file name)=Panc1_2_13C_p1.mzML; protein amount=353.0568568 SUBJECT_SAMPLE_FACTORS - Panc1_2_13C_p2 Sample source:Panc1 cells | group1:CON | matched_CON:2 | label:13C RAW_FILE_NAME(Raw file name)=Panc1_2_13C_p2.mzML; protein amount=296.3644573 SUBJECT_SAMPLE_FACTORS - Panc1_2_13C_p3 Sample source:Panc1 cells | group1:CON | matched_CON:2 | label:13C RAW_FILE_NAME(Raw file name)=Panc1_2_13C_p3.mzML; protein amount=274.5617606 SUBJECT_SAMPLE_FACTORS - Panc1_2_UL_p1 Sample source:Panc1 cells | group1:CON | matched_CON:2 | label:UL RAW_FILE_NAME(Raw file name)=Panc1_2_UL_p1.mzML; protein amount=390.2989558 SUBJECT_SAMPLE_FACTORS - Panc1_2_UL_p2 Sample source:Panc1 cells | group1:CON | matched_CON:2 | label:UL RAW_FILE_NAME(Raw file name)=Panc1_2_UL_p2.mzML; protein amount=300.7018904 SUBJECT_SAMPLE_FACTORS - Panc1_2_UL_p3 Sample source:Panc1 cells | group1:CON | matched_CON:2 | label:UL RAW_FILE_NAME(Raw file name)=Panc1_2_UL_p3.mzML; protein amount=313.1152001 #COLLECTION CO:COLLECTION_SUMMARY Samples were obtained from established immortalised adherent human pancreatic CO:COLLECTION_SUMMARY ductal adenocarcinoma cells (Panc1 & MiaPaCa2). Cells were cultured in high CO:COLLECTION_SUMMARY glucose DMEM containing 4.5 g/L glucose and 4 mM glutamine and were supplemented CO:COLLECTION_SUMMARY with 10% FCS. Cells were cultured under 5% CO2 at 37ºC and routinely passaged CO:COLLECTION_SUMMARY every 2-3 days (when ~80-90% confluent). Cell lines were regularly screened for CO:COLLECTION_SUMMARY mycoplasma infection. CO:SAMPLE_TYPE Pancreas #TREATMENT TR:TREATMENT_SUMMARY Gemcitabine (GEMC) resistant (GEMR) cells were produced by serial treatment with TR:TREATMENT_SUMMARY escalating doses of GEMC for approximately 12 weeks. The final concentration of TR:TREATMENT_SUMMARY GEMC for Panc1 cells was 150 nM, resulting in a half-maximal inhibitory TR:TREATMENT_SUMMARY concentration of 720.5 nM (approximately 2.5 times increase over control (CON) TR:TREATMENT_SUMMARY cells, 273.3 nM). The final concentration of GEMC for MiaPaCa2 cells was 64 nM, TR:TREATMENT_SUMMARY resulting in a half-maximal inhibitory concentration of 92.2 nM (approximately TR:TREATMENT_SUMMARY 3x increase over control (CON) cells, 30.6 nM) Combination TR:TREATMENT_SUMMARY gemcitabine/paclitaxel attenuated (CombAT) cells were produced by serial TR:TREATMENT_SUMMARY treatment with escalating doses of gemcitabine/paclitaxel (10:1) for TR:TREATMENT_SUMMARY approximately 12 weeks. The final concentration of gemcitabine for Panc1 cells TR:TREATMENT_SUMMARY was 28 nM (2.8 nM paclitaxel), and 8 nM (0.8 nM paclitaxel) for MiaPaCa2 cells. TR:TREATMENT_SUMMARY For labelling, cells were seeded in 6 well plates at a density of 3x10^5 TR:TREATMENT_SUMMARY cells/well and allowed to adhere overnight. The following day, cell culture TR:TREATMENT_SUMMARY media was changed to FCS-free high glucose DMEM containing 12.5 µM TR:TREATMENT_SUMMARY 13C16-labelled or unlabelled palmitate conjugated to 2% fatty acid-free BSA. TR:TREATMENT_SUMMARY Palmitate was prepared in cell culture media from 100 mM ethanolic stocks of TR:TREATMENT_SUMMARY palmitic acid and conjugated to fatty acid-free BSA by incubating at 55 °C in TR:TREATMENT_SUMMARY media for 2 h. Lipids were extracted and derivatised as described under Sample TR:TREATMENT_SUMMARY prep after 72 h of incubation with labelled or unlabelled palmitate. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Lipids were extracted from cells grown to 80-90% confluency in 6 well plates SP:SAMPLEPREP_SUMMARY using a modified methyl tert-butyl ether (MTBE) method. Adherent cells were SP:SAMPLEPREP_SUMMARY washed with ice-cold PBS and scraped into methanol containing 0.01% butylated SP:SAMPLEPREP_SUMMARY hydroxytoluene and 100 pmol each of internal standards (phosphatidylcholine SP:SAMPLEPREP_SUMMARY 19:0/19:0, phosphatidylethanolamine 17:0/17:0, phosphatidylserine 17:0/17:0, SP:SAMPLEPREP_SUMMARY lysophosphatidylcholine 17:0, lysophosphatidylethanolamine 14:0, SP:SAMPLEPREP_SUMMARY phosphatidylglycerol 17:0/17:0, phosphatidic acid 17:0/17:0, ceramide SP:SAMPLEPREP_SUMMARY d18:1/17:0, dihydrosphingomyelin 12:0, cholesteryl ester 22:1, diacylglycerol SP:SAMPLEPREP_SUMMARY 17:0/17:0, D5-triacylglycerol 16:0/16:0/16:0 and cardiolipin SP:SAMPLEPREP_SUMMARY 14:0/14:0/14:0/14:0, and 20 nmol of methyl nonadecanoate). An empty well was SP:SAMPLEPREP_SUMMARY also scraped for use in background subtraction. To this, MTBE was added at a SP:SAMPLEPREP_SUMMARY ratio of 3:1 v/v, and extracts were rotated overnight at 4ºC. Following SP:SAMPLEPREP_SUMMARY overnight rotation, 1 part of ice-cold 150 mM ammonium acetate was added, SP:SAMPLEPREP_SUMMARY samples were vortexed vigorously and centrifuged at 2000 x g for 5 min. The SP:SAMPLEPREP_SUMMARY upper organic phase containing lipids was transferred into a 1.5 mL autosampler SP:SAMPLEPREP_SUMMARY vial and dried under N2 at 37ºC Dried lipids were reconstituted in SP:SAMPLEPREP_SUMMARY chloroform:methanol:water (60:30:4.5 v/v/v), transferred to a sleeved vial, and SP:SAMPLEPREP_SUMMARY stored at -20ºC until analysis. The aqueous phase containing the protein pellet SP:SAMPLEPREP_SUMMARY was dried under N2 and digested with 1M NaOH overnight at 4ºC. Digested protein SP:SAMPLEPREP_SUMMARY was diluted 1:2 v/v with water and protein concentration was determined using a SP:SAMPLEPREP_SUMMARY Pierce BCA assay kit (ThermoFisher Scientific) Following MTBE extraction of SP:SAMPLEPREP_SUMMARY lipids, an aliquot was taken and hydrolysed in 0.6 M KOH in 75% v/v methanol at SP:SAMPLEPREP_SUMMARY 60ºC for 30 min. Samples were then cooled to room temperature and neutralised SP:SAMPLEPREP_SUMMARY with 25% v/v acetic acid. Water and n-hexane (3:2 v/v) were added separately and SP:SAMPLEPREP_SUMMARY samples were vortexed vigorously before being centrifuged at 2000 x g for 5 min. SP:SAMPLEPREP_SUMMARY The upper phase containing hydrolysed fatty acids was removed, and the aqueous SP:SAMPLEPREP_SUMMARY phase was washed with a second volume of n-hexane and combined. The combined SP:SAMPLEPREP_SUMMARY organic phase was then dried under N2 at 37°C. Dried hydrolysed fatty acids SP:SAMPLEPREP_SUMMARY were derivatised with an AMP+ MaxSpec kit (Cayman Chemical, Ann Arbor, MI, USA) SP:SAMPLEPREP_SUMMARY as per manufacturer instructions. Following this, AMP-derivatised fatty acids SP:SAMPLEPREP_SUMMARY were re-extracted using MTBE:water (1:1 v/v), and the upper MTBE phase was dried SP:SAMPLEPREP_SUMMARY under N2 before being resuspended in methanol. Samples were stored at -20°C SP:SAMPLEPREP_SUMMARY until analysis. A 37-component fatty acid methyl ester standard (Merck Life SP:SAMPLEPREP_SUMMARY Sciences) containing an additional 3.184 nmol of methyl nonadecanoate was SP:SAMPLEPREP_SUMMARY concurrently subjected to the same derivatisation procedure and used as an SP:SAMPLEPREP_SUMMARY external quality control for fatty acid identification by LC-MS. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Thermo Accucore C30 (150 x 2.1mm,2.6um) CH:SOLVENT_A Water (0.1% formic acid) CH:SOLVENT_B Acetonitrile (0.1% formic acid) CH:FLOW_GRADIENT 0-3.25 min increase from 50% B to 56% B, 3.25-6.5 min increase to 58% B, 6.5-7.5 CH:FLOW_GRADIENT min increase to 80% B, 7.5-9.5 min increase to 100% B, hold at 100% B for 1.5 CH:FLOW_GRADIENT mins, re-equilibrated at 50% B for 4.5 mins. CH:FLOW_RATE 0.4 mL/min CH:COLUMN_TEMPERATURE 30 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6560 Ion Mobility MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS AMP-derivatised fatty acids were detected as positive ions in QTOF-only mode on MS:MS_COMMENTS an Agilent 6560 IM-MS QTOF mass spectrometer (Agilent Technologies). Source MS:MS_COMMENTS conditions were as follows: capillary voltage of 3.5 kV, nozzle voltage of 1 kV MS:MS_COMMENTS and fragmentor at 360 V. Nitrogen was used as the sheath and collision gas, with MS:MS_COMMENTS drying gas set at 5 L/min and sheath gas at 12 L/min (both gases heated to MS:MS_COMMENTS 300°C) and the nebuliser pressure set at 20 psi. Ions were acquired in Auto MS:MS_COMMENTS MS/MS mode at an MS1 mass range of m/z 100-1000 and MS2 mass range of m/z MS:MS_COMMENTS 50-600. Spectra were acquired in MS1 mode at 5 spectra/s and 200 ms/spectrum, MS:MS_COMMENTS and in MS2 mode at 10 spectra/s and 100 ms/spectrum. Precursor ions were MS:MS_COMMENTS isolated using a window of 1.3 Th, acquisition time of 50 ms/spectra, and MS:MS_COMMENTS collision energy of 45 eV. Calibration mix A was infused throughout and masses MS:MS_COMMENTS were automatically detected and corrected using a 100 ppm window and minimum MS:MS_COMMENTS height of 1x10^3 counts to ensure 5 ppm mass accuracy, with m/z 121.050873 and MS:MS_COMMENTS 922.009798 used as reference ions. AMP-derivatised fatty acids as [M+H]+ were MS:MS_COMMENTS aligned using Mass Profinder v10.0 (Agilent Technologies) with an RT tolerance MS:MS_COMMENTS of 0.5 min and MS1 tolerance of 10 ppm. Lipids were identified using a custom MS:MS_COMMENTS personal compound database and library (PCDL) generated from the RT of external MS:MS_COMMENTS quality control samples (attached) and MS/MS spectra were manually inspected to MS:MS_COMMENTS ensure correct MUFA isomer identification. Using this method, FA 18:1 isomers MS:MS_COMMENTS were only partially separated and so these data were not analysed further. Data MS:MS_COMMENTS were exported, blank subtracted and quantified from internal standards before MS:MS_COMMENTS being normalised to total protein. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area (cps) MS_METABOLITE_DATA_START Samples M64_1_13C_p1 M64_1_13C_p2 M64_1_13C_p3 M64_1_UL_p1 M64_1_UL_p2 M64_1_UL_p3 M8C_2_13C_p1 M8C_2_13C_p2 M8C_2_13C_p3 M8C_2_UL_p1 M8C_2_UL_p2 M8C_2_UL_p3 MiaPaCa2_1_13C_p1 MiaPaCa2_1_13C_p2 MiaPaCa2_1_13C_p3 MiaPaCa2_1_UL_p1 MiaPaCa2_1_UL_p2 MiaPaCa2_1_UL_p3 MiaPaCa2_2_13C_p1 MiaPaCa2_2_13C_p2 MiaPaCa2_2_13C_p3 MiaPaCa2_2_UL_p1 MiaPaCa2_2_UL_p2 MiaPaCa2_2_UL_p3 P150_1_13C_p1 P150_1_13C_p2 P150_1_13C_p3 P150_1_UL_p1 P150_1_UL_p2 P150_1_UL_p3 P28C_2_13C_p1 P28C_2_13C_p2 P28C_2_13C_p3 P28C_2_UL_p1 P28C_2_UL_p2 P28C_2_UL_p3 Panc1_1_13C_p1 Panc1_1_13C_p2 Panc1_1_13C_p3 Panc1_1_UL_p1 Panc1_1_UL_p2 Panc1_1_UL_p3 Panc1_2_13C_p1 Panc1_2_13C_p2 Panc1_2_13C_p3 Panc1_2_UL_p1 Panc1_2_UL_p2 Panc1_2_UL_p3 Factors Sample source:MiaPaCa2 cells | group1:GEMR | matched_CON:1 | label:13C Sample source:MiaPaCa2 cells | group1:GEMR | matched_CON:1 | label:13C Sample source:MiaPaCa2 cells | group1:GEMR | matched_CON:1 | label:13C Sample source:MiaPaCa2 cells | group1:GEMR | matched_CON:1 | label:UL Sample source:MiaPaCa2 cells | group1:GEMR | matched_CON:1 | label:UL Sample source:MiaPaCa2 cells | group1:GEMR | matched_CON:1 | label:UL Sample source:MiaPaCa2 cells | group1:CombAT | matched_CON:2 | label:13C Sample source:MiaPaCa2 cells | group1:CombAT | matched_CON:2 | label:13C Sample source:MiaPaCa2 cells | group1:CombAT | matched_CON:2 | label:13C Sample source:MiaPaCa2 cells | group1:CombAT | matched_CON:2 | label:UL Sample source:MiaPaCa2 cells | group1:CombAT | matched_CON:2 | label:UL Sample source:MiaPaCa2 cells | group1:CombAT | matched_CON:2 | label:UL Sample source:MiaPaCa2 cells | group1:CON | matched_CON:1 | label:13C Sample source:MiaPaCa2 cells | group1:CON | matched_CON:1 | label:13C Sample source:MiaPaCa2 cells | group1:CON | matched_CON:1 | label:13C Sample source:MiaPaCa2 cells | group1:CON | matched_CON:1 | label:UL Sample source:MiaPaCa2 cells | group1:CON | matched_CON:1 | label:UL Sample source:MiaPaCa2 cells | group1:CON | matched_CON:1 | label:UL Sample source:MiaPaCa2 cells | group1:CON | matched_CON:2 | label:13C Sample source:MiaPaCa2 cells | group1:CON | matched_CON:2 | label:13C Sample source:MiaPaCa2 cells | group1:CON | matched_CON:2 | label:13C Sample source:MiaPaCa2 cells | group1:CON | matched_CON:2 | label:UL Sample source:MiaPaCa2 cells | group1:CON | matched_CON:2 | label:UL Sample source:MiaPaCa2 cells | group1:CON | matched_CON:2 | label:UL Sample source:Panc1 cells | group1:GEMR | matched_CON:1 | label:13C Sample source:Panc1 cells | group1:GEMR | matched_CON:1 | label:13C Sample source:Panc1 cells | group1:GEMR | matched_CON:1 | label:13C Sample source:Panc1 cells | group1:GEMR | matched_CON:1 | label:UL Sample source:Panc1 cells | group1:GEMR | matched_CON:1 | label:UL Sample source:Panc1 cells | group1:GEMR | matched_CON:1 | label:UL Sample source:Panc1 cells | group1:CombAT | matched_CON:2 | label:13C Sample source:Panc1 cells | group1:CombAT | matched_CON:2 | label:13C Sample source:Panc1 cells | group1:CombAT | matched_CON:2 | label:13C Sample source:Panc1 cells | group1:CombAT | matched_CON:2 | label:UL Sample source:Panc1 cells | group1:CombAT | matched_CON:2 | label:UL Sample source:Panc1 cells | group1:CombAT | matched_CON:2 | label:UL Sample source:Panc1 cells | group1:CON | matched_CON:1 | label:13C Sample source:Panc1 cells | group1:CON | matched_CON:1 | label:13C Sample source:Panc1 cells | group1:CON | matched_CON:1 | label:13C Sample source:Panc1 cells | group1:CON | matched_CON:1 | label:UL Sample source:Panc1 cells | group1:CON | matched_CON:1 | label:UL Sample source:Panc1 cells | group1:CON | matched_CON:1 | label:UL Sample source:Panc1 cells | group1:CON | matched_CON:2 | label:13C Sample source:Panc1 cells | group1:CON | matched_CON:2 | label:13C Sample source:Panc1 cells | group1:CON | matched_CON:2 | label:13C Sample source:Panc1 cells | group1:CON | matched_CON:2 | label:UL Sample source:Panc1 cells | group1:CON | matched_CON:2 | label:UL Sample source:Panc1 cells | group1:CON | matched_CON:2 | label:UL 13C16-16:1n7 713216.9 718528.03 580628.42 0 0 0 741697.26 649089.26 1012427.81 0 0 0 750943.72 1057333.8 851008.28 0 0 0 903182.89 856892.24 1065543.13 0 0 0 422083.29 614305.49 712641.16 0 0 0 630574.79 678470.93 634219.26 0 0 0 440629.7 475578.38 331074.45 0 0 0 386093.41 375159.39 578682.2 0 0 0 13C16-16:1n10 30953.34 24490.85 33586.21 0 0 0 111270.74 60955.61 83342.88 0 0 0 51660.74 66643.9 62189.2 0 0 0 70354.67 60942.34 75011.87 0 0 0 236166.37 321125.77 339981.64 0 0 0 26251.9 19832.68 31012.87 0 0 0 362215.62 398519.59 256084.59 0 0 0 349931.4 287360.7 503593.46 0 0 0 16:1n-7 6359363.38 4990908.84 6116137.54 7128708.11 5004497.7 7832196.1 11021669.47 10603777.59 13595672.57 13574624.92 11031992.64 10396131.62 8298445.87 11942158.92 9445163.19 9099727.35 9566557.48 8901096.04 10198565.09 9093170.19 10866223.71 9542946.84 12061793.95 9634079.75 9920193.81 10212482.77 123871.98.49 15434675.67 11162100.75 11508874.01 8317410.92 7355083.34 6300165.9 8815655.78 7488430.49 10008807.15 9913868.01 9913868.01 6086353.87 10295226.31 10175295.74 5966412.12 9748759.92 7500337.54 9670210.78 9746935.31 7709405.16 8489115.93 16:1n-10 301445.99 221864.51 301343.99 284602.19 267806.81 222147.72 1468183.82 1103560.64 1096977.94 1915415.17 1122875.14 1118547.74 680941.02 906816.82 685807.12 443170.71 687109.87 550930.96 754834.85 580129.7 973940.69 624578.9 1050782.01 892969.37 5625444.78 5494340.29 5641937.28 8292518.66 5899475.22 4971568.83 294695.93 351493.37 249001.49 317986.26 238868.69 351329.95 7593430.08 7593430.08 4257636.86 7614066.32 6915711.41 4013218.66 7353211.51 4805250.22 7033651.94 7039599.64 5706392.17 6273832.33 13C16-16:0 1443993.47 1217216.47 1189656.78 0 0 0 733088.91 774207.96 949263.18 0 0 0 1121219.46 1305737.68 1300967.87 0 0 0 1141078.92 1130625.92 1246579.16 0 0 0 597117.02 720897.33 777393.37 0 0 0 886954.25 1311770.02 1214873.89 0 0 0 825585.94 831668.35 665772.23 0 0 0 647325.98 650852.04 1099165.34 0 0 0 16:0 25358945.37 20338294.78 22841659.25 27814141.36 23349044.09 24296243.48 19773588.05 21419993.2 22140697.84 20425791.45 20199964.88 26807309.75 25317057.31 26660456.83 28076035.69 22851502.41 24785028.86 23198326 25726643.09 23886850 24499247.24 22733602.24 29057752.06 22106481.84 19138172.79 20525803.88 20542278.14 24070699.16 18550962.49 19362058.88 20617016.86 23787046.23 21987396.39 21004333.2 23010870.12 28910845.54 25909945.97 27020609.17 18929578.85 25653073.51 27327645.87 16785122.37 23984877.09 20171987.81 26810628.79 26263176.49 22516185.43 23897983.09 13C16 16:0/16:0 0.056942174 0.059848502 0.052082765 0 0 0 0.037074147 0.036144174 0.042874131 0 0 0 0.044287116 0.048976568 0.046337306 0 0 0 0.044353976 0.047332567 0.050882345 0 0 0 0.031200315 0.035121515 0.037843581 0 0 0 0.043020494 0.055146402 0.055253195 0 0 0 0.031863669 0.030779038 0.035171001 0 0 0 0.026988922 0.032265141 0.040997373 0 0 0 13C16-16:1n7/13C16 16:0 0.493919754 0.590304229 0.488063809 0 0 0 1.011742573 0.838391354 1.066540693 0 0 0 0.669756231 0.809759737 0.65413474 0 0 0 0.791516585 0.757891912 0.854773739 0 0 0 0.70686863 0.852140054 0.916705991 0 0 0 0.710943986 0.517217896 0.522045346 0 0 0 0.533717544 0.571836574 0.497278852 0 0 0 0.596443557 0.576412713 0.526474206 0 0 0 13C16-16:1 n-10/13C16 16:0 0.021435928 0.020120373 0.028231849 0 0 0 0.151783417 0.078732864 0.087797443 0 0 0 0.046075494 0.051039272 0.047802257 0 0 0 0.06165627 0.053901418 0.060174173 0 0 0 0.395511034 0.44545285 0.437335399 0 0 0 0.029597806 0.015119022 0.025527646 0 0 0 0.438737632 0.479180902 0.384642943 0 0 0 0.540579879 0.441514634 0.45815988 0 0 0 16:0 norm to protein 108160.5589 94570.27215 69475.04941 121182.5304 106927.071 72782.61559 87682.92039 87374.82617 72738.41445 70463.18369 81425.63359 89514.35719 82622.39396 96818.26475 93475.28582 64787.45933 82957.03734 73196.03538 95443.65959 103593.329 111953.4252 91706.37603 121238.8466 81900.47477 48223.16953 74512.16074 66231.14044 56225.25223 64357.3975 61742.29076 84036.70127 112440.1362 92583.35804 87670.57589 109560.668 148235.7162 75687.55153 88212.65819 72519.40872 137581.7174 77700.63468 65037.35728 67934.88535 68064.801 97648.80854 67289.89689 74878.76248 76323.29278 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention time molecular weight 13C16-16:1n7 2.572 437.3755 13C16-16:1n10 2.801 437.3755 16:1n-7 2.6 421.3213 16:1n-10 2.8 421.3213 13C16-16:0 3.9 439.3912 16:0 3.9 423.337 13C16 16:0/16:0 - - 13C16-16:1n7/13C16 16:0 - - 13C16-16:1 n-10/13C16 16:0 - - 16:0 norm to protein - - METABOLITES_END #END