Summary of Study ST003762

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002345. The data can be accessed directly via it's Project DOI: 10.21228/M8HN9H This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST003762
Study Title	Stable-label palmitate tracing of gemcitabine-based chemotherapy in PDAC cells
Study Summary	In this work we developed both gemcitabine resistant (GEMR) Panc1 and MiaPaCa2 cell lines and combination gemcitabine/paclitaxel attenuated (CombAT) Panc1 and MiaPaCa2 cells. We found significant upregulation of de novo lipogenesis in both cell lines following GEMR or CombAT. In this study, we traced uniformly 13C labelled palmitate into all cell lines and compared it with controls (CON) to derived relative activity of stearoyl-CoA desaturase 1 (SCD1) and fatty acid desaturase 2 (FADS2). We found upregulation of SCD1 in Panc1 GEMR through increased ratio 16:1n-7/16:0, with no change in SCD1 activity with CombAT in either cell line. FADS2 activity was substantially suppressed in Panc1 CombAT cells through decreased 16:1n-10/16:0. This study contains the following groups: source - cell lines Panc1 or MiaPaCa2, blank or fatty acid methyl ester reference (FAMEs) group - control (CON), gemcitabine resistant (GEMR), or combination gemcitabine/paclitaxel attenuated (CombAT). matched_CON - GEMR or CombAT each had their own matched control cells plated at the same time label - cells cultured in either unlabelled (UL) or labelled (13C) palmitate (12.5 uM) conjugated to bovine serum albumin (2% w/v).
Institute	Victor Chang Cardiac Research Institute
Last Name	Hancock
First Name	Sarah
Address	Level 7 Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW 2010, Australia
Email	s.hancock@victorchang.edu.au
Phone	+61414537526
Submit Date	2025-02-20
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2025-03-21
Release Version	1

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Project:

Project ID:	PR002345
Project DOI:	doi: 10.21228/M8HN9H
Project Title:	Stable-label palmitate tracing of gemcitabine-based chemotherapy in PDAC cells
Project Summary:	Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease with few treatment options and poor survivability. In this work we sought to characterise metabolic adaptations to gemcitabine (GEMC)-based chemotherapy exposure to discover new therapeutic targets for improving treatment efficacy. We show that GEMC resistance (GEMR) upregulates de novo lipogenesis in Panc1 and MiaPaCa2 cells.
Institute:	Victor Chang Cardiac Research Institute
Last Name:	Hancock
First Name:	Sarah
Address:	Level 7 Lowy Packer Building, 405 Liverpool Street, Darlinghurst, NSW 2010, Australia
Email:	s.hancock@victorchang.edu.au
Phone:	+61414537526

Subject:

Subject ID:	SU003895
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	group	group	matched_CON	label
SA409146	Blank	-	Blank	Blank	-	-
SA409147	FAMEs-mix_02	-	FAMEs	FAMEs	-	-
SA409148	FAMEs-mix_03	-	FAMEs	FAMEs	-	-
SA409149	FAMEs-mix_01	-	FAMEs	FAMEs	-	-
SA409162	M8C_2_13C_p1	Pancreas	MiaPaCa2 cells	CombAT	2	13C
SA409163	M8C_2_13C_p3	Pancreas	MiaPaCa2 cells	CombAT	2	13C
SA409164	M8C_2_13C_p2	Pancreas	MiaPaCa2 cells	CombAT	2	13C
SA409165	M8C_2_UL_p2	Pancreas	MiaPaCa2 cells	CombAT	2	UL
SA409166	M8C_2_UL_p3	Pancreas	MiaPaCa2 cells	CombAT	2	UL
SA409167	M8C_2_UL_p1	Pancreas	MiaPaCa2 cells	CombAT	2	UL
SA409150	MiaPaCa2_1_13C_p1	Pancreas	MiaPaCa2 cells	CON	1	13C
SA409151	MiaPaCa2_1_13C_p3	Pancreas	MiaPaCa2 cells	CON	1	13C
SA409152	MiaPaCa2_1_13C_p2	Pancreas	MiaPaCa2 cells	CON	1	13C
SA409153	MiaPaCa2_1_UL_p3	Pancreas	MiaPaCa2 cells	CON	1	UL
SA409154	MiaPaCa2_1_UL_p2	Pancreas	MiaPaCa2 cells	CON	1	UL
SA409155	MiaPaCa2_1_UL_p1	Pancreas	MiaPaCa2 cells	CON	1	UL
SA409156	MiaPaCa2_2_13C_p3	Pancreas	MiaPaCa2 cells	CON	2	13C
SA409157	MiaPaCa2_2_13C_p2	Pancreas	MiaPaCa2 cells	CON	2	13C
SA409158	MiaPaCa2_2_13C_p1	Pancreas	MiaPaCa2 cells	CON	2	13C
SA409159	MiaPaCa2_2_UL_p1	Pancreas	MiaPaCa2 cells	CON	2	UL
SA409160	MiaPaCa2_2_UL_p3	Pancreas	MiaPaCa2 cells	CON	2	UL
SA409161	MiaPaCa2_2_UL_p2	Pancreas	MiaPaCa2 cells	CON	2	UL
SA409168	M64_1_13C_p3	Pancreas	MiaPaCa2 cells	GEMR	1	13C
SA409169	M64_1_13C_p2	Pancreas	MiaPaCa2 cells	GEMR	1	13C
SA409170	M64_1_13C_p1	Pancreas	MiaPaCa2 cells	GEMR	1	13C
SA409171	M64_1_UL_p3	Pancreas	MiaPaCa2 cells	GEMR	1	UL
SA409172	M64_1_UL_p1	Pancreas	MiaPaCa2 cells	GEMR	1	UL
SA409173	M64_1_UL_p2	Pancreas	MiaPaCa2 cells	GEMR	1	UL
SA409186	P28C_2_13C_p1	Pancreas	Panc1 cells	CombAT	2	13C
SA409187	P28C_2_13C_p3	Pancreas	Panc1 cells	CombAT	2	13C
SA409188	P28C_2_13C_p2	Pancreas	Panc1 cells	CombAT	2	13C
SA409189	P28C_2_UL_p3	Pancreas	Panc1 cells	CombAT	2	UL
SA409190	P28C_2_UL_p2	Pancreas	Panc1 cells	CombAT	2	UL
SA409191	P28C_2_UL_p1	Pancreas	Panc1 cells	CombAT	2	UL
SA409174	Panc1_1_13C_p3	Pancreas	Panc1 cells	CON	1	13C
SA409175	Panc1_1_13C_p1	Pancreas	Panc1 cells	CON	1	13C
SA409176	Panc1_1_13C_p2	Pancreas	Panc1 cells	CON	1	13C
SA409177	Panc1_1_UL_p3	Pancreas	Panc1 cells	CON	1	UL
SA409178	Panc1_1_UL_p2	Pancreas	Panc1 cells	CON	1	UL
SA409179	Panc1_1_UL_p1	Pancreas	Panc1 cells	CON	1	UL
SA409180	Panc1_2_13C_p2	Pancreas	Panc1 cells	CON	2	13C
SA409181	Panc1_2_13C_p1	Pancreas	Panc1 cells	CON	2	13C
SA409182	Panc1_2_13C_p3	Pancreas	Panc1 cells	CON	2	13C
SA409183	Panc1_2_UL_p1	Pancreas	Panc1 cells	CON	2	UL
SA409184	Panc1_2_UL_p2	Pancreas	Panc1 cells	CON	2	UL
SA409185	Panc1_2_UL_p3	Pancreas	Panc1 cells	CON	2	UL
SA409192	P150_1_13C_p3	Pancreas	Panc1 cells	GEMR	1	13C
SA409193	P150_1_13C_p2	Pancreas	Panc1 cells	GEMR	1	13C
SA409194	P150_1_13C_p1	Pancreas	Panc1 cells	GEMR	1	13C
SA409195	P150_1_UL_p3	Pancreas	Panc1 cells	GEMR	1	UL
SA409196	P150_1_UL_p2	Pancreas	Panc1 cells	GEMR	1	UL
SA409197	P150_1_UL_p1	Pancreas	Panc1 cells	GEMR	1	UL

Showing results 1 to 52 of 52

Showing results 1 to 52 of 52

Collection:

Collection ID:	CO003888
Collection Summary:	Samples were obtained from established immortalised adherent human pancreatic ductal adenocarcinoma cells (Panc1 & MiaPaCa2). Cells were cultured in high glucose DMEM containing 4.5 g/L glucose and 4 mM glutamine and were supplemented with 10% FCS. Cells were cultured under 5% CO2 at 37ºC and routinely passaged every 2-3 days (when ~80-90% confluent). Cell lines were regularly screened for mycoplasma infection.
Sample Type:	Pancreas

Treatment:

Treatment ID:	TR003904
Treatment Summary:	Gemcitabine (GEMC) resistant (GEMR) cells were produced by serial treatment with escalating doses of GEMC for approximately 12 weeks. The final concentration of GEMC for Panc1 cells was 150 nM, resulting in a half-maximal inhibitory concentration of 720.5 nM (approximately 2.5 times increase over control (CON) cells, 273.3 nM). The final concentration of GEMC for MiaPaCa2 cells was 64 nM, resulting in a half-maximal inhibitory concentration of 92.2 nM (approximately 3x increase over control (CON) cells, 30.6 nM) Combination gemcitabine/paclitaxel attenuated (CombAT) cells were produced by serial treatment with escalating doses of gemcitabine/paclitaxel (10:1) for approximately 12 weeks. The final concentration of gemcitabine for Panc1 cells was 28 nM (2.8 nM paclitaxel), and 8 nM (0.8 nM paclitaxel) for MiaPaCa2 cells. For labelling, cells were seeded in 6 well plates at a density of 3x10^5 cells/well and allowed to adhere overnight. The following day, cell culture media was changed to FCS-free high glucose DMEM containing 12.5 µM 13C16-labelled or unlabelled palmitate conjugated to 2% fatty acid-free BSA. Palmitate was prepared in cell culture media from 100 mM ethanolic stocks of palmitic acid and conjugated to fatty acid-free BSA by incubating at 55 °C in media for 2 h. Lipids were extracted and derivatised as described under Sample prep after 72 h of incubation with labelled or unlabelled palmitate.

Treatment ID:

TR003904

Treatment Summary:

Gemcitabine (GEMC) resistant (GEMR) cells were produced by serial treatment with escalating doses of GEMC for approximately 12 weeks. The final concentration of GEMC for Panc1 cells was 150 nM, resulting in a half-maximal inhibitory concentration of 720.5 nM (approximately 2.5 times increase over control (CON) cells, 273.3 nM). The final concentration of GEMC for MiaPaCa2 cells was 64 nM, resulting in a half-maximal inhibitory concentration of 92.2 nM (approximately 3x increase over control (CON) cells, 30.6 nM) Combination gemcitabine/paclitaxel attenuated (CombAT) cells were produced by serial treatment with escalating doses of gemcitabine/paclitaxel (10:1) for approximately 12 weeks. The final concentration of gemcitabine for Panc1 cells was 28 nM (2.8 nM paclitaxel), and 8 nM (0.8 nM paclitaxel) for MiaPaCa2 cells. For labelling, cells were seeded in 6 well plates at a density of 3x10^5 cells/well and allowed to adhere overnight. The following day, cell culture media was changed to FCS-free high glucose DMEM containing 12.5 µM 13C16-labelled or unlabelled palmitate conjugated to 2% fatty acid-free BSA. Palmitate was prepared in cell culture media from 100 mM ethanolic stocks of palmitic acid and conjugated to fatty acid-free BSA by incubating at 55 °C in media for 2 h. Lipids were extracted and derivatised as described under Sample prep after 72 h of incubation with labelled or unlabelled palmitate.

Sample Preparation:

Sampleprep ID:	SP003901
Sampleprep Summary:	Lipids were extracted from cells grown to 80-90% confluency in 6 well plates using a modified methyl tert-butyl ether (MTBE) method. Adherent cells were washed with ice-cold PBS and scraped into methanol containing 0.01% butylated hydroxytoluene and 100 pmol each of internal standards (phosphatidylcholine 19:0/19:0, phosphatidylethanolamine 17:0/17:0, phosphatidylserine 17:0/17:0, lysophosphatidylcholine 17:0, lysophosphatidylethanolamine 14:0, phosphatidylglycerol 17:0/17:0, phosphatidic acid 17:0/17:0, ceramide d18:1/17:0, dihydrosphingomyelin 12:0, cholesteryl ester 22:1, diacylglycerol 17:0/17:0, D5-triacylglycerol 16:0/16:0/16:0 and cardiolipin 14:0/14:0/14:0/14:0, and 20 nmol of methyl nonadecanoate). An empty well was also scraped for use in background subtraction. To this, MTBE was added at a ratio of 3:1 v/v, and extracts were rotated overnight at 4ºC. Following overnight rotation, 1 part of ice-cold 150 mM ammonium acetate was added, samples were vortexed vigorously and centrifuged at 2000 x g for 5 min. The upper organic phase containing lipids was transferred into a 1.5 mL autosampler vial and dried under N2 at 37ºC Dried lipids were reconstituted in chloroform:methanol:water (60:30:4.5 v/v/v), transferred to a sleeved vial, and stored at -20ºC until analysis. The aqueous phase containing the protein pellet was dried under N2 and digested with 1M NaOH overnight at 4ºC. Digested protein was diluted 1:2 v/v with water and protein concentration was determined using a Pierce BCA assay kit (ThermoFisher Scientific) Following MTBE extraction of lipids, an aliquot was taken and hydrolysed in 0.6 M KOH in 75% v/v methanol at 60ºC for 30 min. Samples were then cooled to room temperature and neutralised with 25% v/v acetic acid. Water and n-hexane (3:2 v/v) were added separately and samples were vortexed vigorously before being centrifuged at 2000 x g for 5 min. The upper phase containing hydrolysed fatty acids was removed, and the aqueous phase was washed with a second volume of n-hexane and combined. The combined organic phase was then dried under N2 at 37°C. Dried hydrolysed fatty acids were derivatised with an AMP+ MaxSpec kit (Cayman Chemical, Ann Arbor, MI, USA) as per manufacturer instructions. Following this, AMP-derivatised fatty acids were re-extracted using MTBE:water (1:1 v/v), and the upper MTBE phase was dried under N2 before being resuspended in methanol. Samples were stored at -20°C until analysis. A 37-component fatty acid methyl ester standard (Merck Life Sciences) containing an additional 3.184 nmol of methyl nonadecanoate was concurrently subjected to the same derivatisation procedure and used as an external quality control for fatty acid identification by LC-MS.

Sampleprep ID:

SP003901

Sampleprep Summary:

Lipids were extracted from cells grown to 80-90% confluency in 6 well plates using a modified methyl tert-butyl ether (MTBE) method. Adherent cells were washed with ice-cold PBS and scraped into methanol containing 0.01% butylated hydroxytoluene and 100 pmol each of internal standards (phosphatidylcholine 19:0/19:0, phosphatidylethanolamine 17:0/17:0, phosphatidylserine 17:0/17:0, lysophosphatidylcholine 17:0, lysophosphatidylethanolamine 14:0, phosphatidylglycerol 17:0/17:0, phosphatidic acid 17:0/17:0, ceramide d18:1/17:0, dihydrosphingomyelin 12:0, cholesteryl ester 22:1, diacylglycerol 17:0/17:0, D5-triacylglycerol 16:0/16:0/16:0 and cardiolipin 14:0/14:0/14:0/14:0, and 20 nmol of methyl nonadecanoate). An empty well was also scraped for use in background subtraction. To this, MTBE was added at a ratio of 3:1 v/v, and extracts were rotated overnight at 4ºC. Following overnight rotation, 1 part of ice-cold 150 mM ammonium acetate was added, samples were vortexed vigorously and centrifuged at 2000 x g for 5 min. The upper organic phase containing lipids was transferred into a 1.5 mL autosampler vial and dried under N2 at 37ºC Dried lipids were reconstituted in chloroform:methanol:water (60:30:4.5 v/v/v), transferred to a sleeved vial, and stored at -20ºC until analysis. The aqueous phase containing the protein pellet was dried under N2 and digested with 1M NaOH overnight at 4ºC. Digested protein was diluted 1:2 v/v with water and protein concentration was determined using a Pierce BCA assay kit (ThermoFisher Scientific) Following MTBE extraction of lipids, an aliquot was taken and hydrolysed in 0.6 M KOH in 75% v/v methanol at 60ºC for 30 min. Samples were then cooled to room temperature and neutralised with 25% v/v acetic acid. Water and n-hexane (3:2 v/v) were added separately and samples were vortexed vigorously before being centrifuged at 2000 x g for 5 min. The upper phase containing hydrolysed fatty acids was removed, and the aqueous phase was washed with a second volume of n-hexane and combined. The combined organic phase was then dried under N2 at 37°C. Dried hydrolysed fatty acids were derivatised with an AMP+ MaxSpec kit (Cayman Chemical, Ann Arbor, MI, USA) as per manufacturer instructions. Following this, AMP-derivatised fatty acids were re-extracted using MTBE:water (1:1 v/v), and the upper MTBE phase was dried under N2 before being resuspended in methanol. Samples were stored at -20°C until analysis. A 37-component fatty acid methyl ester standard (Merck Life Sciences) containing an additional 3.184 nmol of methyl nonadecanoate was concurrently subjected to the same derivatisation procedure and used as an external quality control for fatty acid identification by LC-MS.

Combined analysis:

Analysis ID	AN006177
Chromatography ID	CH004689
MS ID	MS005882
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 1290 Infinity II
Column	Thermo Accucore C30 (150 x 2.1 mm, 2.6 μm)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Agilent 6560 Ion Mobility
Ion Mode	POSITIVE
Units	peak area (cps)

Chromatography:

Chromatography ID:	CH004689
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Thermo Accucore C30 (150 x 2.1 mm, 2.6 μm)
Column Temperature:	30°C
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 min increase to 80% B, 7.5-9.5 min increase to 100% B, hold at 100% B for 1.5 mins, re-equilibrated at 50% B for 4.5 mins.
Flow Rate:	0.4 mL/min
Solvent A:	100% Water; 0.1% Formic acid
Solvent B:	100% Acetonitrile; 0.1% Formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS005882
Analysis ID:	AN006177
Instrument Name:	Agilent 6560 Ion Mobility
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	AMP-derivatised fatty acids were detected as positive ions in QTOF-only mode on an Agilent 6560 IM-MS QTOF mass spectrometer (Agilent Technologies). Source conditions were as follows: capillary voltage of 3.5 kV, nozzle voltage of 1 kV and fragmentor at 360 V. Nitrogen was used as the sheath and collision gas, with drying gas set at 5 L/min and sheath gas at 12 L/min (both gases heated to 300°C) and the nebuliser pressure set at 20 psi. Ions were acquired in Auto MS/MS mode at an MS1 mass range of m/z 100-1000 and MS2 mass range of m/z 50-600. Spectra were acquired in MS1 mode at 5 spectra/s and 200 ms/spectrum, and in MS2 mode at 10 spectra/s and 100 ms/spectrum. Precursor ions were isolated using a window of 1.3 Th, acquisition time of 50 ms/spectra, and collision energy of 45 eV. Calibration mix A was infused throughout and masses were automatically detected and corrected using a 100 ppm window and minimum height of 1x10^3 counts to ensure 5 ppm mass accuracy, with m/z 121.050873 and 922.009798 used as reference ions. AMP-derivatised fatty acids as [M+H]+ were aligned using Mass Profinder v10.0 (Agilent Technologies) with an RT tolerance of 0.5 min and MS1 tolerance of 10 ppm. Lipids were identified using a custom personal compound database and library (PCDL) generated from the RT of external quality control samples (attached) and MS/MS spectra were manually inspected to ensure correct MUFA isomer identification. Using this method, FA 18:1 isomers were only partially separated and so these data were not analysed further. Data were exported, blank subtracted and quantified from internal standards before being normalised to total protein.
Ion Mode:	POSITIVE