Summary of Study ST003680

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 PR002283. The data can be accessed directly via it's Project DOI: 10.21228/M8HZ6G 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	ST003680
Study Title	Ketogenic diet suppresses colorectal cancer through the gut microbiome long chain fatty acid stearate - untargeted LCMS data from CMT experiment
Study Summary	Germ-free mice received cecal content from mice having received a ketogenic diet (KC) or from mice having received a standard diet (SC) and were subjected to AOM/DSS treatment as described in Tsenkova et al. (2025). Mouse fecal pellets were analyzed by a LC-MS approach to characterize the fecal metabolome of mice from different treatment groups. Metabolites were extracted from mouse fecal samples as described in the method file. Resulting samples were used for LC-MS untargeted metabolomics screen using a Vanquish UHPLC (ThermoFisher Scientific), coupled to a Q Exactive HF mass spectrometer (ThermoFisher Scientific).
Institute	University of Luxembourg
Last Name	Letellier
First Name	Elisabeth
Address	6, avenue du Swing, Belval, Esch, 4367, Luxembourg
Email	madita.brauer@uni.lu
Phone	(+352) 46 66 44 6954
Submit Date	2025-01-20
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2025-01-27
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002283
Project DOI:	doi: 10.21228/M8HZ6G
Project Title:	Ketogenic diet suppresses colorectal cancer through the gut microbiome long chain fatty acid stearate
Project Summary:	Our manuscript entitled “Ketogenic diet suppresses colorectal cancer through the gut microbiome long chain fatty acid stearate” describes a reduced colonic tumor burden upon ketogenic diet (KD) consumption in a CRC mouse model with a humanized microbiome. Importantly, we demonstrate a causal relationship through microbiome transplantation into germ-free mice, whereby alterations in the gut microbiota were maintained in the absence of continued selective pressure from the KD. Specifically, we identify a shift toward bacterial species that produce stearic acid in ketogenic conditions, whereas consumers were depleted, resulting in elevated levels of free stearate in the gut lumen. This microbial product demonstrates tumor-suppressing properties by inducing apoptosis in cancer cells and decreasing colonic Th17 immune cell populations. As part of this study, we used different metabolomics workflows to study metabolites in mouse fecal and plasma samples as well as the used rodent diet.
Institute:	University of Luxembourg
Last Name:	Letellier
First Name:	Elisabeth
Address:	6, avenue du Swing, Belval, Esch, 4367, Luxembourg
Email:	madita.brauer@uni.lu
Phone:	(+352) 46 66 44 6954

Subject:

Subject ID:	SU003812
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	Condition
SA402242	KC_42	mouse feces	KC
SA402243	KC_18	mouse feces	KC
SA402244	KC_47	mouse feces	KC
SA402245	KC_16	mouse feces	KC
SA402246	KC_38	mouse feces	KC
SA402247	KC_34	mouse feces	KC
SA402248	KC_28	mouse feces	KC
SA402249	KC_23	mouse feces	KC
SA402250	SC_14	mouse feces	SC
SA402251	SC_20	mouse feces	SC
SA402252	SC_21	mouse feces	SC
SA402253	SC_30	mouse feces	SC
SA402254	SC_32	mouse feces	SC
SA402255	SC_35	mouse feces	SC
SA402256	SC_41	mouse feces	SC
SA402257	SC_11	mouse feces	SC

Showing results 1 to 16 of 16

Showing results 1 to 16 of 16

Collection:

Collection ID:	CO003805
Collection Summary:	Germ-free mice received cecal content from mice having received a ketogenic diet (KC) or from mice having received a standard diet (SC) and were subjected to AOM/DSS treatment as described in Tsenkova et al. (2025). Feces samples were collected and flesh frozen in liquid nitrogen and kept at -80℃ until metabolite extraction.
Sample Type:	Feces
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR003821
Treatment Summary:	Germ-free mice received cecal content from mice having received a ketogenic diet (KC) or from mice having received a standard diet (SC) and were subjected to AOM/DSS treatment as described in Tsenkova et al. (2025). Briefly, mice were administered a single intraperitoneal injection of sterile-filtered AOM (10mg/kg mouse body weight). Microbiomes were replaced with donor microbiomes via oral gavage. Mice were administered a total of three cycles of DSS, with a two-week-long recovery period between each cycle. DSS was refreshed mid-cycle. Half of the mice were randomly allocated to each dietary condition at the end of the last cycle of DSS.

Treatment ID:

TR003821

Treatment Summary:

Germ-free mice received cecal content from mice having received a ketogenic diet (KC) or from mice having received a standard diet (SC) and were subjected to AOM/DSS treatment as described in Tsenkova et al. (2025). Briefly, mice were administered a single intraperitoneal injection of sterile-filtered AOM (10mg/kg mouse body weight). Microbiomes were replaced with donor microbiomes via oral gavage. Mice were administered a total of three cycles of DSS, with a two-week-long recovery period between each cycle. DSS was refreshed mid-cycle. Half of the mice were randomly allocated to each dietary condition at the end of the last cycle of DSS.

Sample Preparation:

Sampleprep ID:	SP003819
Sampleprep Summary:	Mouse fecal pellets were placed in 0.5mL Precellys® tubes (VWR) containing five ceramic beads each and MilliQ® water was added to each sample at a 1:16 dry weight to water ratio. The samples were homogenized at 6000rpm, for two 30-second-long cycles at 4°C, in a Precellys®24 Homogenizer (Bertin Corp.). Samples were then incubated at 4°C for ten minutes, then centrifuged at maximum speed for 10 minutes at 4°C. Samples were maintained on ice and in the dark. The supernatant was used for further downstream processing. An ISM was prepared (2μg/mL of ribitol, pentanedioic-d6 acid and d-mannose and 10μg/mL tridecanoic-d25 acid, 6-chloropurine riboside, 4-chloro-DL-phenylalanine, Nε-trifluoroacetyl-L-lysine and thionicotinamide adenine dinucleotide (Sigma-Aldrich) in MilliQ® water). 40μL of the ISM was added to 100μL of the fecal supernatant fluid. 80 μL of this mixture was added to 320μL of methanol, vortexed thoroughly, incubated for five minutes at 4°C at maximum speed in an Eppendorf ThermoMixer, and then centrifugated for five minutes at 4°C at maximum speed. 350 μL of supernatant were added to 280μL of chloroform. 180μL of MilliQ® water were added, the samples were vortexed thoroughly, incubated for ten minutes at 4°C at maximum speed in an ThermoMixer (Eppendorf), then centrifugated for five minutes at 4°C at maximum speed. The extract was then split – 200 μL of the upper (polar) phase were aliquoted into a GC vial with a micro-insert and the rest of the upper (polar) phase was filtered through a PHENEX-RC syringe filter (Phenomenex), and 200 μL were retained in Eppendorf tubes for further processing. Then, the temperature of the SpeedVac® was increased to 25°C for 25 minutes to avoid water condensation on the surface of the glass vial. Samples were protected from light exposure and stored at -80°C. Samples for LC-MS analysis (polar) were reconstituted in 80μL 50% ACN in water, transferred into LC vials for LC-MS analysis and analyzed on a Vanquish UHPLC (ThermoFisher Scientific), coupled to a Q Exactive HF mass spectrometer (ThermoFisher Scientific).

Sampleprep ID:

SP003819

Sampleprep Summary:

Mouse fecal pellets were placed in 0.5mL Precellys® tubes (VWR) containing five ceramic beads each and MilliQ® water was added to each sample at a 1:16 dry weight to water ratio. The samples were homogenized at 6000rpm, for two 30-second-long cycles at 4°C, in a Precellys®24 Homogenizer (Bertin Corp.). Samples were then incubated at 4°C for ten minutes, then centrifuged at maximum speed for 10 minutes at 4°C. Samples were maintained on ice and in the dark. The supernatant was used for further downstream processing. An ISM was prepared (2μg/mL of ribitol, pentanedioic-d6 acid and d-mannose and 10μg/mL tridecanoic-d25 acid, 6-chloropurine riboside, 4-chloro-DL-phenylalanine, Nε-trifluoroacetyl-L-lysine and thionicotinamide adenine dinucleotide (Sigma-Aldrich) in MilliQ® water). 40μL of the ISM was added to 100μL of the fecal supernatant fluid. 80 μL of this mixture was added to 320μL of methanol, vortexed thoroughly, incubated for five minutes at 4°C at maximum speed in an Eppendorf ThermoMixer, and then centrifugated for five minutes at 4°C at maximum speed. 350 μL of supernatant were added to 280μL of chloroform. 180μL of MilliQ® water were added, the samples were vortexed thoroughly, incubated for ten minutes at 4°C at maximum speed in an ThermoMixer (Eppendorf), then centrifugated for five minutes at 4°C at maximum speed. The extract was then split – 200 μL of the upper (polar) phase were aliquoted into a GC vial with a micro-insert and the rest of the upper (polar) phase was filtered through a PHENEX-RC syringe filter (Phenomenex), and 200 μL were retained in Eppendorf tubes for further processing. Then, the temperature of the SpeedVac® was increased to 25°C for 25 minutes to avoid water condensation on the surface of the glass vial. Samples were protected from light exposure and stored at -80°C. Samples for LC-MS analysis (polar) were reconstituted in 80μL 50% ACN in water, transferred into LC vials for LC-MS analysis and analyzed on a Vanquish UHPLC (ThermoFisher Scientific), coupled to a Q Exactive HF mass spectrometer (ThermoFisher Scientific).

Combined analysis:

Analysis ID	AN006041
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish
Column	SeQuant ZIC- pHILIC (150 x 2.1 mm, 5 μm)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF-X Orbitrap
Ion Mode	UNSPECIFIED
Units	normalized area

Chromatography:

Chromatography ID:	CH004591
Instrument Name:	Thermo Vanquish
Column Name:	SeQuant ZIC- pHILIC (150 x 2.1 mm, 5 μm)
Column Temperature:	50°C
Flow Gradient:	90 % B for 1.5 min, followed by a decrease to 20% B within 15 min, then 20% B for 2 min, increase to 90% B within 2 min, then 90% B for 13 min
Flow Rate:	200 µL/ml
Solvent A:	100% water; 0.01% formic acid
Solvent B:	90% acetonitrile/10% water; 0.1% formic acid; 20 mM ammonium acetate
Chromatography Type:	HILIC

MS:

MS ID:	MS005750
Analysis ID:	AN006041
Instrument Name:	Thermo Q Exactive HF-X Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Resolution - 120,000, m/z range - 60-900, AGC target - 1e6, maximum injection time – 70 ms. ddMS2 was applied using the following settings: Resolution - 30,000, AGC target - 5e5, maximum injection time – 70, topN - 5, Normalized collision energy – 20. Samples were acquired in positive and negative ionization mode simultaneously (polarity switching).
Ion Mode:	UNSPECIFIED