Summary of Study ST003764

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 PR002347. The data can be accessed directly via it's Project DOI: 10.21228/M8854M This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003764
Study Title	Profiling of fatty acid double bond positing ing gemcitabine-resistant pancreatic ductal adenocarcinoma cells
Study Summary	In this work we developed gemcitabine (GEMC) resistant (GEMR) PDAC cell lines from Panc1 and MiaPaCa2 cells. This dataset contains ozone-induced fatty acid discovery (OzFAD) data, which shows differences in carbon-carbon double bond position between Panc1 and MiaPaCa2 cells. Panc1 cells contain significant amounts of 16:1n-10, which is an unusual isomeric fatty acid produced by Δ6 desaturation of palmitic acid by fatty acid desaturase 2 (FADS2). Panc1 GEMR cells show increased stearoyl-CoA desaturase 1 (SCD1)-derived 16:1n-7 alongside decreased 16:1n-10.
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	2025-02-21
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:	PR002347
Project DOI:	doi: 10.21228/M8854M
Project Title:	Profiling of fatty acid double bond positing ing gemcitabine-resistant pancreatic ductal adenocarcinoma 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:	SU003897
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	group	group
SA409244	MIA_1	MiaPaCa2 cells	CON
SA409245	MIA_2	MiaPaCa2 cells	CON
SA409246	MIA_3	MiaPaCa2 cells	CON
SA409247	M64_1	MiaPaCa2 cells	GEMR
SA409248	M64_2	MiaPaCa2 cells	GEMR
SA409249	M64_3	MiaPaCa2 cells	GEMR
SA409250	PANC_1	Panc1 cells	CON
SA409251	PANC_2	Panc1 cells	CON
SA409252	PANC_3	Panc1 cells	CON
SA409253	P150_1	Panc1 cells	GEMR
SA409254	P150_2	Panc1 cells	GEMR
SA409255	P150_3	Panc1 cells	GEMR

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

Collection:

Collection ID:	CO003890
Collection Summary:	Samples were obtained from established immortalised adherent human pancreatic ductal adenocarcinoma cells (Panc1). 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:	TR003906
Treatment Summary:	Panc1 GEMR cells were produced by serial treatment with escalating doses of gemcitabine 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).

Treatment ID:

TR003906

Treatment Summary:

Panc1 GEMR cells were produced by serial treatment with escalating doses of gemcitabine 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).

Sample Preparation:

Sampleprep ID:	SP003903
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:

SP003903

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.

Chromatography:

Chromatography ID:	CH004691
Instrument Name:	Waters Acquity
Column Name:	Waters ACQUITY UPLC CSH C18 (100 x 2.1 mm, 1.7 μm)
Column Temperature:	60℃
Flow Gradient:	0 - 0.5 min at 20% B, then linearly increased to 90% B (16 min), then 100% B (17.35 min), held at 100% B until 20.35 min, reduced to 20% B and held until 21 min.
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

Analysis:

Analysis ID:	AN006179
Analysis Type:	MS
Chromatography ID:	CH004691
Num Factors:	4
Num Metabolites:	22
Units:	normalized peak area