Summary of Study ST003154

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 PR001960. The data can be accessed directly via it's Project DOI: 10.21228/M8CF0S 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	ST003154
Study Title	Nucleotide metabolism of tumor interstitial fluid in one murine model of pancreatic cancer
Study Summary	The experiment focus on the nucleotide metabolism of tumor interstitial fluid in one murine model of pancreatic cancer. Briefly, KPC FC1245 cells were genetically engineered using doxycycline inducible CRISPR/Cas9 system platform to target specifically cytidine deaminase (sgCda). sgCda and the control sgNT were then orthotopically injected in the head of the pancreas of 8-10 weeks old C57BL/6J female mice. When the tumor weight reached approximately 0.2-0.4g, mice were sacrificed and tumor interstitial fluid was collected as reported in the Collection section of this study. Standard curves for glutamine, cytidine, uridine, glucose, UTP and UDP were prepared and extracted along with the interstitial fluid samples. The concentrations of glutamine, cytidine, uridine, UDP and UTP were measured by LC-MS, and glucose was measured by GC-MS. A decrease in uridine content, and accordingly in UDP and UTP, and a concomitant accumulation of cytidine was observed in sgCda tumors. On the other hand, no differences were observed in glucose and glutamine abundance.
Institute	VIB-KU Leuven Center for Cancer Biology
Last Name	Mazzone
First Name	Massimiliano
Address	Herestraat 49, box 912, Leuven, Flemish Brabant, 3000, Belgium
Email	massimiliano.mazzone@kuleuven.be
Phone	+32-16-37.32.13
Submit Date	2024-03-12
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	GC/LC-MS
Release Date	2024-04-05
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001960
Project DOI:	doi: 10.21228/M8CF0S
Project Title:	Nucleotide metabolism of tumor interstitial fluid in one murine model of pancreatic cancer
Project Type:	Nucleotide metabolism in murine pancreatic tumors
Project Summary:	The experiment focus on the nucleotide metabolism of tumor interstitial fluid in one murine model of pancreatic cancer. Briefly, KPC FC1245 cells were genetically engineered using doxycycline inducible CRISPR/Cas9 system platform to target specifically cytidine deaminase (sgCda). sgCda and the control sgNT were then orthotopically injected in the head of the pancreas of 8-10 weeks old C57BL/6J female mice. When the tumor weight reached approximately 0.2-0.4g, mice were sacrificed and tumor interstitial fluid was collected as reported in the Collection section of this study. Standard curves for glutamine, cytidine, uridine, glucose, UTP and UDP were prepared and extracted along with the interstitial fluid samples. The concentrations of glutamine, cytidine, uridine, UDP and UTP were measured by LC-MS, and glucose was measured by GC-MS. A decrease in uridine content, and accordingly in UDP and UTP, and a concomitant accumulation of cytidine was observed in sgCda tumors. On the other hand, no differences were observed in glucose and glutamine abundance.
Institute:	VIB Center for Cancer Biology
Last Name:	Mazzone
First Name:	Massimiliano
Address:	Herestraat 49, box 912, Leuven, Flemish Brabant, 3000, Belgium
Email:	massimiliano.mazzone@kuleuven.be
Phone:	+32-16-37.32.13

Subject:

Subject ID:	SU003271
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57BL/6J

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Genotype
SA341014	STD7_b	Calibration Curve	N/A
SA341015	STD6_b	Calibration Curve	N/A
SA341016	STD8_b	Calibration Curve	N/A
SA341017	STD10_b	Calibration Curve	N/A
SA341018	STD11_b	Calibration Curve	N/A
SA341019	STD5_b	Calibration Curve	N/A
SA341020	STD9_b	Calibration Curve	N/A
SA341021	STD4_b	Calibration Curve	N/A
SA341022	STD12_a	Calibration Curve	N/A
SA341023	STD11_a	Calibration Curve	N/A
SA341024	STD13_a	Calibration Curve	N/A
SA341025	STD14_a	Calibration Curve	N/A
SA341026	STD15_a	Calibration Curve	N/A
SA341027	STD12_b	Calibration Curve	N/A
SA341028	STD13_b	Calibration Curve	N/A
SA341029	STD9_c	Calibration Curve	N/A
SA341030	STD8_c	Calibration Curve	N/A
SA341031	STD10_c	Calibration Curve	N/A
SA341032	STD11_c	Calibration Curve	N/A
SA341033	STD12_c	Calibration Curve	N/A
SA341034	STD7_c	Calibration Curve	N/A
SA341035	STD6_c	Calibration Curve	N/A
SA341036	STD2_c	Calibration Curve	N/A
SA341037	STD14_b	Calibration Curve	N/A
SA341038	STD3_c	Calibration Curve	N/A
SA341039	STD4_c	Calibration Curve	N/A
SA341040	STD5_c	Calibration Curve	N/A
SA341041	STD10_a	Calibration Curve	N/A
SA341042	STD3_b	Calibration Curve	N/A
SA341043	STD4_a	Calibration Curve	N/A
SA341044	STD2_a	Calibration Curve	N/A
SA341045	STD9_a	Calibration Curve	N/A
SA341046	STD5_a	Calibration Curve	N/A
SA341047	STD3_a	Calibration Curve	N/A
SA341048	STD7_a	Calibration Curve	N/A
SA341049	STD8_a	Calibration Curve	N/A
SA341050	STD6_a	Calibration Curve	N/A
SA341051	TIF12	Pancreatic tumor interstitial fluid	sgCda
SA341052	TIF7	Pancreatic tumor interstitial fluid	sgCda
SA341053	TIF11_GC	Pancreatic tumor interstitial fluid	sgCda
SA341054	TIF6_GC	Pancreatic tumor interstitial fluid	sgCda
SA341055	TIF6	Pancreatic tumor interstitial fluid	sgCda
SA341056	TIF7_GC	Pancreatic tumor interstitial fluid	sgCda
SA341057	TIF5_GC	Pancreatic tumor interstitial fluid	sgCda
SA341058	TIF19	Pancreatic tumor interstitial fluid	sgCda
SA341059	TIF20	Pancreatic tumor interstitial fluid	sgCda
SA341060	TIF21	Pancreatic tumor interstitial fluid	sgCda
SA341061	TIF13_GC	Pancreatic tumor interstitial fluid	sgCda
SA341062	TIF11	Pancreatic tumor interstitial fluid	sgCda
SA341063	TIF18	Pancreatic tumor interstitial fluid	sgCda
SA341064	TIF13	Pancreatic tumor interstitial fluid	sgCda
SA341065	TIF12_GC	Pancreatic tumor interstitial fluid	sgCda
SA341066	TIF5	Pancreatic tumor interstitial fluid	sgCda
SA341067	TIF4	Pancreatic tumor interstitial fluid	sgNT
SA341068	TIF2	Pancreatic tumor interstitial fluid	sgNT
SA341069	TIF3	Pancreatic tumor interstitial fluid	sgNT
SA341070	TIF8	Pancreatic tumor interstitial fluid	sgNT
SA341071	TIF9	Pancreatic tumor interstitial fluid	sgNT
SA341072	TIF10	Pancreatic tumor interstitial fluid	sgNT
SA341073	TIF4_GC	Pancreatic tumor interstitial fluid	sgNT
SA341074	TIF15	Pancreatic tumor interstitial fluid	sgNT
SA341075	TIF16	Pancreatic tumor interstitial fluid	sgNT
SA341076	TIF17	Pancreatic tumor interstitial fluid	sgNT
SA341077	TIF1	Pancreatic tumor interstitial fluid	sgNT
SA341078	TIF14	Pancreatic tumor interstitial fluid	sgNT
SA341079	TIF1_GC	Pancreatic tumor interstitial fluid	sgNT
SA341080	TIF9_GC	Pancreatic tumor interstitial fluid	sgNT
SA341081	TIF8_GC	Pancreatic tumor interstitial fluid	sgNT
SA341082	TIF3_GC	Pancreatic tumor interstitial fluid	sgNT
SA341083	TIF2_GC	Pancreatic tumor interstitial fluid	sgNT
SA341084	TIF10_GC	Pancreatic tumor interstitial fluid	sgNT

Showing results 1 to 71 of 71

Showing results 1 to 71 of 71

Collection:

Collection ID:	CO003264
Collection Summary:	8-10 weeks old C57BL/6J female mice were euthanized with 75 µl of a 60 mg ml-1 Dolethal solution (pentobarbital sodium, Vetoquinol). Subsequently, sgNT and sgCda primary tumors were collected by surgical resection, washed with blood bank saline and dried from liquid excess. Tissues were then placed in a home-made filtered centrifugation tube supplemented with a 20 µm nylon mesh filter (Repligen) and centrifuged at 400 x g at 4 °C for 10 minutes. Between 1-14 µl of tumor interstitial fluid were collected and stored on dry ice. Interstitial fluid volume was used to determine the metabolites concentration measured by mass spectrometry.
Sample Type:	Interstitial fluid

Treatment:

Treatment ID:	TR003280
Treatment Summary:	KPC FC1245 cells were genetically engineered using doxycycline inducible CRISPR/Cas9 system platform to target specifically cytidine deaminase (sgCda). sgCda and the control sgNT were then orthotopically injected in the head of the pancreas of 8-10 weeks old C57BL/6J female mice. When the tumor weight reached approximately 0.2-0.4g, mice were sacrificed and tumor interstitial fluid was collected

Sample Preparation:

Sampleprep ID:	SP003278
Sampleprep Summary:	Standard curves for Glucose (Sigma-Aldrich, G7021), UDP (Sigma-Aldrich, 94330), UTP (Jena Bioscience, NU-1024S), cytidine (Sigma-Aldrich, C4654), uridine (Sigma-Aldrich, U3003) and glutamine (Gibco, 25030-34) were used to calculate the concentration of these metabolites in the samples. The standard curve was prepared with a dilution series starting from 5 mM Glucose and Glutamine, 1 mM Cytidine, Uridine, UDP and UTP. Specific dilutions are indicated in the Study Design table. Metabolites and calibration curves were extracted simultaneously by addition of 800 μl of MS-grade methanol-water buffer (MeOH:H2O, 5:3, v/v) containing the internal standards glutaric acid (5 μg ml-1, Sigma-Aldrich, G3407) and 13C6-Glucose (30 μg ml-1, Cambridge Isotope Laboratories, Inc., CLM-1396), followed by 500 μl of chloroform. Samples were then vortexed and centrifuged (4 °C for 10 min each). The polar (upper) phases were collected, divided into two equal parts for gas chromatography (GC) and liquid chromatography (LC) mass spectrometry analysis, and dried using a vacuum concentrator. The dried metabolite extracts were stored at -80 °C until analysis.

Sampleprep ID:

SP003278

Sampleprep Summary:

Standard curves for Glucose (Sigma-Aldrich, G7021), UDP (Sigma-Aldrich, 94330), UTP (Jena Bioscience, NU-1024S), cytidine (Sigma-Aldrich, C4654), uridine (Sigma-Aldrich, U3003) and glutamine (Gibco, 25030-34) were used to calculate the concentration of these metabolites in the samples. The standard curve was prepared with a dilution series starting from 5 mM Glucose and Glutamine, 1 mM Cytidine, Uridine, UDP and UTP. Specific dilutions are indicated in the Study Design table. Metabolites and calibration curves were extracted simultaneously by addition of 800 μl of MS-grade methanol-water buffer (MeOH:H2O, 5:3, v/v) containing the internal standards glutaric acid (5 μg ml-1, Sigma-Aldrich, G3407) and 13C6-Glucose (30 μg ml-1, Cambridge Isotope Laboratories, Inc., CLM-1396), followed by 500 μl of chloroform. Samples were then vortexed and centrifuged (4 °C for 10 min each). The polar (upper) phases were collected, divided into two equal parts for gas chromatography (GC) and liquid chromatography (LC) mass spectrometry analysis, and dried using a vacuum concentrator. The dried metabolite extracts were stored at -80 °C until analysis.

Combined analysis:

Analysis ID	AN005174	AN005175
Analysis type	MS	MS
Chromatography type	Reversed phase	GC
Chromatography system	Thermo Dionex Ultimate 3000	Agilent 8860
Column	Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um)	Agilent J&W DB-35ms (30 m x 0.25 mm, 0.25 µm)
MS Type	ESI	EI
MS instrument type	Orbitrap	Single quadrupole
MS instrument name	Thermo Q Exactive Orbitrap	Agilent 5977C
Ion Mode	NEGATIVE	POSITIVE
Units	Ion Counts (AUC)	Ion Counts (AUC)

Chromatography:

Chromatography ID:	CH003914
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Waters ACQUITY UPLC HSS T3 (100 x 2.1mm,1.8um)
Column Temperature:	40
Flow Gradient:	The gradient started with 5% of solvent B and 95% solvent A and remained at 5% B until 2 min post injection. A linear gradient to 37% B was carried out until 7 min and increased to 41% until 14 min. Between 14 and 26 minutes the gradient increased to 95% of B and remained at 95% B for 4 minutes. At 30 min the gradient returned to 5% B. The chromatography was stopped at 40 min.
Flow Rate:	0.25 mL/min
Solvent A:	100% water; 10 mM tributyl-amine; 15 mM acetic acid
Solvent B:	100% methanol
Chromatography Type:	Reversed phase

Chromatography ID:	CH003915
Chromatography Summary:	Inlet Temperature: 270; Helium flow rate: 0.88 mL/min; The GC oven was kept at 100°C and for 1 min, increased with a gradient of 2.5°C min-1 to 105°C for 2 min, then ramped with a gradient of 15°C min-1 to 260°, and after that ramped with a gradient of 20°C min-1 up to 300°C for 5 min.
Instrument Name:	Agilent 8860
Column Name:	Agilent J&W DB-35ms (30 m x 0.25 mm, 0.25 µm)
Column Temperature:	100-300
Flow Gradient:	N/A
Flow Rate:	N/A
Solvent A:	N/A
Solvent B:	N/A
Chromatography Type:	GC

MS:

MS ID:	MS004909
Analysis ID:	AN005174
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The MS was operated in negative full scan mode (m/z range: 70–900 and 400-500) using a spray voltage of 4.0 kV, capillary temperature of 350 °C, sheath gas at 50.0, auxiliary gas at 10.0. The AGC target was set at 3.0E6 using a resolution if 140000, with a maximum IT fill time of 512 ms. Data was collected and analyzed using the Xcalibur software (Thermo Scientific) considering a 5 ppm error. The chromatographic peaks for UDP, UTP, cytidine, uridine and glutamine were normalized to the internal standard glutaric acid. Tumor interstitial fluid samples were interpolated to the standard curve and normalized for tumor interstitial fluid volume in order to quantify metabolite concentration.
Ion Mode:	NEGATIVE

MS ID:	MS004910
Analysis ID:	AN005175
Instrument Name:	Agilent 5977C
Instrument Type:	Single quadrupole
MS Type:	EI
MS Comments:	5977C quipped with EI inert source. Mass spectrometry was performed at 70 eV and a mass range of 150-650 atomic mass units was measured. The chromatographic peaks were extracted from raw chromatograms with a custom MATLAB Script. The chromatographic peaks for 12C-Glucose were normalized to the internal standard 13C6-glucose. Tumor interstitial fluid samples were interpolated to the standard curve and normalized for tumor interstitial fluid volume to quantify metabolite concentration.
Ion Mode:	POSITIVE