Summary of Study ST003761

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 PR002344. The data can be accessed directly via it's Project DOI: 10.21228/M8NC1Z 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	ST003761
Study Title	Fatty acids profiling of gemcitabine based chemotherapy resistant PDAC cells
Study Summary	In this work produced combination gemcitabine/paclitaxel attenuated (CombAT) Panc1 and MiaPaCa2 cells. We profiled AMP-derivatised fatty acids from both cell lines and compared them to control (CON) cells. Also included are a reference standard produced from AMP-derivatised fatty acid methyl esters (FAMEs), internal quality controls (iQC) made from pooled samples, and a blank sample for background subtraction. This study found increased 16:1n-7 and decreased 16:1n-10 in Panc1 CombAT cells, with no change in MiaPaCa2 CombAT cells. Also apparent were changes in polyunsaturated fatty acids in Panc1 CombAT cells consistent with a decrease in fatty acid desaturase 2 (FADS2) activity. Data were analysed using Agilent Profinder 10.0 with fatty acids identified using a Personal Compound Databases and Libraries (PCDL) developed from the FAMEs reference standard.
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:	PR002344
Project DOI:	doi: 10.21228/M8NC1Z
Project Title:	Fatty acids profiling of gemcitabine based chemotherapy resistant 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 gemcitabine-based chemotherapy 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:	SU003894
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
SA409129	Blank	-	Blank	Blank
SA409130	FAMEs-mix_02	-	FAMEs	FAMEs
SA409131	FAMEs-mix_01	-	FAMEs	FAMEs
SA409132	iQC_01	-	pool	iQC
SA409133	iQC_02	-	pool	iQC
SA409137	MiaPaCa2_COMBR_p1	Pancreas	MiaPaCa2 cells	CombAT
SA409138	MiaPaCa2_COMBR_p2	Pancreas	MiaPaCa2 cells	CombAT
SA409139	MiaPaCa2_COMBR_p3	Pancreas	MiaPaCa2 cells	CombAT
SA409134	MiaPaCa2_CON_p1	Pancreas	MiaPaCa2 cells	CON
SA409135	MiaPaCa2_CON_p3	Pancreas	MiaPaCa2 cells	CON
SA409136	MiaPaCa2_CON_p2	Pancreas	MiaPaCa2 cells	CON
SA409143	Panc1_COMBR_p1	Pancreas	Panc1 cells	CombAT
SA409144	Panc1_COMBR_p2	Pancreas	Panc1 cells	CombAT
SA409145	Panc1_COMBR_p3	Pancreas	Panc1 cells	CombAT
SA409140	Panc1_CON_p1	Pancreas	Panc1 cells	CON
SA409141	Panc1_CON_p2	Pancreas	Panc1 cells	CON
SA409142	Panc1_CON_p3	Pancreas	Panc1 cells	CON

Showing results 1 to 17 of 17

Showing results 1 to 17 of 17

Collection:

Collection ID:	CO003887
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:	TR003903
Treatment Summary:	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.

Sample Preparation:

Sampleprep ID:	SP003900
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:

SP003900

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:	CH004688
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

Analysis:

Analysis ID:	AN006176
Analysis Type:	MS
Chromatography ID:	CH004688
Num Factors:	7
Num Metabolites:	37
Rt Units:	Minutes
Units:	nmol/mg protein