Summary of Study ST004457

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 PR002810. The data can be accessed directly via it's Project DOI: 10.21228/M8FC3V This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST004457
Study TitleCompare lipid metabolism in lung tumors from mice harboring Kras G12D/+ and either STK1-/- (KL) or Tp53 -/- mutations (KP).
Study SummaryLung from mice with KL or KP tumors were inflated using a solution of 0.3% low melt agarose. The lobes were then sliced using a vibratome instrument to a thickness of 300µM and allowed to recover overnight in RPM1640 media containing 2% FBS. After recovery, slices were washed with 0.9% w/V saline twice and tracing media was added. The tracing media was DMEM containing 1either 10mM 13C glucose or 0.4mM 13C serine and 2% FBS. At the end of the 24 hours of tracing, wells were washed three times with 0.9% w/V saline, then placed on a metal block chilled in a slurry of liquid nitrogen and ethanol to flash freeze the slices. A 3mM dermal punch was used to isolate portions of the slice with tumors and transferred to eppendorfs and kept frozen until extracted. At the time of extracting, 3 ceramic beads were added to the punches along with 500µL of methanol and internal standards. Punches were homogenized using a Retsch instrument for 10 minutes at a setting of 30Hz. 50µL of the homogenate was taken for protien measurement, while the rest was spun down at 21000gs for 15minutes. The supernatant was transferred to a new tube and dried under nitrogen. The dried metabolites were kept in a -80C freezer until they were resuspended in 60uL of methanol and analyzed on a C18 column. MS data was analyzed using EL-Maven v0.12.1 with a mass error of 5ppm and maximum retention time shift between parent ions and isotopomer ions of 5 seconds. An in-house matlab script was used for natural abundance correction.
Institute
Salk Institute for Biological Studies
Last NameWessendorf-Rodriguez
First NameKarl
Address10010 N Torrey Pines Rd
Emailkwessendorf@salk.edu
Phone7874490440
Submit Date2025-12-08
Raw Data AvailableYes
Raw Data File Type(s)mzXML
Analysis Type DetailLC-MS
Release Date2025-12-22
Release Version1
Karl Wessendorf-Rodriguez Karl Wessendorf-Rodriguez
https://dx.doi.org/10.21228/M8FC3V
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002810
Project DOI:doi: 10.21228/M8FC3V
Project Title:Modeling Lipid Homeostasis Using Stable Isotope Tracing and Flux Analysis
Project Summary:Lipids represent the most diverse pool of metabolites found in cells, facilitating compartmentation, signaling, and other functions. Dysregulation of lipid metabolism is linked to disease states ranging from cancer to neurodegeneration. However, limited tools are available for quantifying metabolic fluxes across the lipidome. To directly measure reaction fluxes encompassing membrane lipid homeostasis, we apply stable isotope tracing, liquid chromatography-high-resolution mass spectrometry, and network-based mass isotopomer modeling to non-small cell lung cancer (NSCLC) models. Lipid metabolic flux analysis (MFA) enables the concurrent quantitation of fatty acid synthesis, elongation, headgroup assembly, and salvage reactions within virtually any biological system. Lipid-MFA highlights distinct changes in fatty acid synthase and very long-chain fatty acid (VLCFA) elongation fluxes in typical culture conditions. Using this approach, we resolve differences in sphingolipid recycling in p53-deficient versus liver kinase B1 (LKB1)-deficient NSCLC tumors using precision-cut lung slice culture. Finally, Lipid-MFA demonstrates the unique trafficking of long-chain versus very long-chain ceramide fluxes as well as the isozyme specificity of a classical ceramide synthase inhibitor. These results illustrate the ability of Lipid-MFA to quantify lipid homeostasis and elucidate molecular mechanisms in membrane lipid metabolism.
Institute:Salk Insititute for Biological Studies
Last Name:Wessendorf-Rodriguez
First Name:Karl
Address:10010N Torrey Pines Rd, La Jolla, California, 92037, USA
Email:kwessendorf@salk.edu
Phone:7874490440

Subject:

Subject ID:SU004624
Subject Type:Mammal
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

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

mb_sample_id local_sample_id Sample source Genotype Treatment Injection order
SA527197KL_Ser_3Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C3]serine B4
SA527198KL_Ser_12Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C3]serine B4
SA527199KL_Ser_11Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C3]serine B4
SA527200KL_Ser_10Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C3]serine B4
SA527201KL_Ser_9Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C3]serine B4
SA527202KL_Ser_8Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C3]serine B4
SA527203KL_Ser_7Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C3]serine B4
SA527204KL_Ser_6Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C3]serine B4
SA527205KL_Ser_5Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C3]serine B4
SA527206KL_Ser_4Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C3]serine B4
SA527207KL_Ser_1Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C3]serine B4
SA527208KL_Ser_2Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C3]serine B4
SA527209KL_Gluc_2Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C6]glucose B4
SA527210KL_Gluc_1Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C6]glucose B4
SA527211KL_Gluc_12Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C6]glucose B4
SA527212KL_Gluc_10Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C6]glucose B4
SA527213KL_Gluc_9Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C6]glucose B4
SA527214KL_Gluc_8Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C6]glucose B4
SA527215KL_Gluc_7Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C6]glucose B4
SA527216KL_Gluc_6Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C6]glucose B4
SA527217KL_Gluc_5Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C6]glucose B4
SA527218KL_Gluc_4Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C6]glucose B4
SA527219KL_Gluc_3Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C6]glucose B4
SA527220KL_Gluc_11Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Stk11-/- 2% FBS and [U-13C6]glucose B4
SA527221KP_Ser_6Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C3]serine B4
SA527222KP_Ser_3Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C3]serine B4
SA527223KP_Ser_4Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C3]serine B4
SA527224KP_Ser_5Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C3]serine B4
SA527225KP_Ser_11Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C3]serine B4
SA527226KP_Ser_7Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C3]serine B4
SA527227KP_Ser_8Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C3]serine B4
SA527228KP_Ser_9Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C3]serine B4
SA527229KP_Ser_10Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C3]serine B4
SA527230KP_Ser_2Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C3]serine B4
SA527231KP_Ser_12Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C3]serine B4
SA527232KP_Ser_1Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C3]serine B4
SA527233KP_Gluc_9Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C6]glucose B4
SA527234KP_Gluc_12Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C6]glucose B4
SA527235KP_Gluc_11Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C6]glucose B4
SA527236KP_Gluc_10Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C6]glucose B4
SA527237KP_Gluc_8Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C6]glucose B4
SA527238KP_Gluc_7Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C6]glucose B4
SA527239KP_Gluc_6Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C6]glucose B4
SA527240KP_Gluc_5Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C6]glucose B4
SA527241KP_Gluc_4Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C6]glucose B4
SA527242KP_Gluc_3Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C6]glucose B4
SA527243KP_Gluc_2Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C6]glucose B4
SA527244KP_Gluc_1Precision Cut Lung Slices (Mus musculus) Kras G12D/+ and Tp53-/- 2% FBS and [U-13C6]glucose B4
Showing results 1 to 48 of 48

Collection:

Collection ID:CO004617
Collection Summary:Lung from mice with KL or KP tumors were inflated using a solution of 0.3% low melt agarose. The lobes were then sliced using a vibratome instrument to a thickness of 300µM and allowed to recover overnight in RPM1640 media containing 2% FBS. After recovery, slices were washed with 0.9% w/V saline twice and tracing media was added. The tracing media was DMEM containing 1either 10mM 13C glucose or 0.4mM 13C serine and 2% FBS. At the end of the 24 hours of tracing, wells were washed three times with 0.9% w/V saline, then placed on a metal block chilled in a slurry of liquid nitrogen and ethanol to flash freeze the slices. A 3mM dermal punch was used to isolate portions of the slice with tumors and transferred to eppendorfs and kept frozen until extracted. At the time of extracting, 3 ceramic beads were added to the punches along with 500µL of methanol and internal standards. Punches were homogenized using a Retsch instrument for 10 minutes at a setting of 30Hz. 50µL of the homogenate was taken for protein measurement, while the rest was spun down at 21000gs for 15minutes. The supernatant was transferred to a new tube and dried under nitrogen. The dried metabolites were kept in a -80C freezer until they were resuspended in 60uL of methanol and analyzed on a C18 column10 nanomoles of norvaline was added to each sample along with a mixture of Equisplash (Avanti Polar Lipids, Cat# 330731), and SPB 18:0;O2 [D7] (Avanti Polar Lipids, Cat# 860658), SPB 18:1;O2 [D7] (Avanti Polar Lipids, Cat# 860657), glucosylceramide 18:1;O2[D7]-15:0 (Avanti Polar Lipids, Cat# 330729), lactosylceramide 18:1;O2[D7] (Avanti Polar Lipids, Cat# 330727), and GM3-d3 18:1;O2/18:0 [D3] (Cayman Chemicals, item No. 39226). The Eppendorfs with 100% methanol, used for polar lipid analysis on C18, were centrifuged at 21,000 g for 15 min at 4°C. 300 μL of methanol were collected and evaporated under nitrogen and stored at -80°C until resuspended for analysis.
Sample Type:Lung

Treatment:

Treatment ID:TR004633
Treatment Summary:After overnight recovery in RPMI 1640, slices were washed twice with PBS and then cultured in DMEM containing 10mM glucose and 2% FBS for 24 hours. Media used contained either 13C glucose or 13C serine to enable stable isotope tracing.

Sample Preparation:

Sampleprep ID:SP004630
Sampleprep Summary:Lipids were extracted from cell pellets using 100% methanol for polar lipids on a C18 column. Lipid containing phases were dried under nitrogen and stored at -80C until they were resuspended for the appropriate analysis.

Combined analysis:

Analysis ID AN007463
Chromatography ID CH005656
MS ID MS007159
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Vanquish
Column Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode POSITIVE
Units Relative Abundance

Chromatography:

Chromatography ID:CH005656
Chromatography Summary:All metabolites abundances were calculated from C18 analysis.
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm,2.6um)
Column Temperature:35
Flow Gradient:0 min, 30% B; 1 min, 30% B; 2 min, 70% B; 11 min, 95%B; 17 min, 30%B; 21.5 min, 30%B; 27 min, 30% B
Flow Rate:0.2 mL/min
Solvent A:98:2 v/v water: methanol with 5 mM ammonium acetate
Solvent B:50:50 v/v methanol: isopropanol with 5 mM ammonium acetate
Chromatography Type:Reversed phase

MS:

MS ID:MS007159
Analysis ID:AN007463
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Lipids were analyzed in positive mode using spray voltage 3.5 kV. Sweep gas flow was 1 arbitrary units, auxiliary gas flow 10 arbitrary units and sheath gas flow 50 arbitrary units, with a capillary temperature of 325 °C. Full mass spectrometry (scan range 220–2,500 m/z) was used at 140,000 resolution with 10E6 automatic gain control and a maximum injection time of 100 ms. Data were analyzed using EI-Maven. Mass error was set to 5 ppm for metabolite identification. Isotopologues were filtered to elute within 5 seconds of the unlabeled parent ion to ensure all labeled species came from the same metabolite. Mass isotopologue distributions were analyzed with an in-house MATLAB script which integrated the metabolite fragment ions and corrected for natural isotope abundances.
Ion Mode:POSITIVE
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