Summary of Study ST001903

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 PR001198. The data can be accessed directly via it's Project DOI: http://dx.doi.org/10.21228/M8VD7F 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	ST001903
Study Title	Effect of ketogenic diet on the plasma and tumor metabolome of melanoma-bearing mice
Study Type	Metabolomics analysis
Study Summary	Growing evidence supports the use of low-carbohydrate/high-fat ketogenic diets (KDs) together with standard therapies to improve cancer treatment outcomes. However, conflicting data exist regarding the efficacy of KDs in melanoma, the deadliest skin cancer. Here, we show that two different KD formulations effectively reduced tumor growth in immunocompromised mice bearing genetically and metabolically heterogeneous human melanoma xenografts. Furthermore, the KDs exerted a metastasis-reducing effect in an immunocompetent syngeneic melanoma mouse model. Ketone bodies did not directly influence melanoma cell proliferation; therefore, we performed an in-depth metabolomics analysis using the MxP® Quant 500 kit combined with a acylcarnitine assay (biocrates life sciences ag)to elucidate potential anti-tumor mechanisms in vivo. Targeted analysis of plasma and tumor metabolomes revealed distinct changes in amino acid metabolism induced by the KDs. Moreover, the KDs increased sphingomyelin synthesis and hydroxylation of certain lipids. Thus, KDs simultaneously affect multiple metabolic pathways to create an unfavorable environment for melanoma cell proliferation, supporting their potential as a complementary nutritional approach to melanoma therapy.
Institute	University Hospital of the Paracelsus Medical University Salzburg
Last Name	Weber
First Name	Daniela
Address	Müllner Hauptstraße 48, 5020 Salzburg, Austria
Email	d.weber@salk.at
Phone	0043 57255 26274
Submit Date	2021-08-10
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS/MS(Dir. Inf.)
Release Date	2022-07-11
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001198
Project DOI:	http://dx.doi.org/10.21228/M8VD7F
Project Title:	Effect of ketogenic diet on the plasma and tumor metabolome of melanoma-bearing mice
Project Type:	Metabolomics analysis
Project Summary:	Growing evidence supports the use of low-carbohydrate/high-fat ketogenic diets (KDs) together with standard therapies to improve cancer treatment outcomes. However, conflicting data exist regarding the efficacy of KDs in melanoma, the deadliest skin cancer. Here, we show that two different KD formulations effectively reduced tumor growth in immunocompromised mice bearing genetically and metabolically heterogeneous human melanoma xenografts. Furthermore, the KDs exerted a metastasis-reducing effect in an immunocompetent syngeneic melanoma mouse model. Ketone bodies did not directly influence melanoma cell proliferation; therefore, we performed an in-depth metabolomics analysis using the MxP® Quant 500 kit combined with a acylcarnitine assay (biocrates life sciences ag)to elucidate potential antitumor mechanisms in vivo. Targeted analysis of plasma and tumor metabolomes revealed distinct changes in amino acid metabolism induced by the KDs. Moreover, the KDs increased sphingomyelin synthesis and hydroxylation of certain lipids. Thus, KDs simultaneously affect multiple metabolic pathways to create an unfavorable environment for melanoma cell proliferation, supporting their potential as a complementary nutritional approach to melanoma therapy.
Institute:	University Hospital of the Paracelsus Medical University Salzburg
Last Name:	Weber
First Name:	Daniela
Address:	Müllner Hauptstraße 48, Salzburg, Salzburg, 5020, Austria
Email:	d.weber@salk.at
Phone:	0043 57255 26274

Subject:

Subject ID:	SU002424
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 description	Tissue
SA232171	plasma # 237	A375 CTRL	plasma
SA232172	plasma # 235	A375 CTRL	plasma
SA232173	plasma # 238	A375 CTRL	plasma
SA232174	plasma # 239	A375 CTRL	plasma
SA232175	plasma # 227	A375 CTRL	plasma
SA232176	plasma # 234	A375 CTRL	plasma
SA232177	plasma # 236	A375 CTRL	plasma
SA232178	plasma # 233	A375 CTRL	plasma
SA232179	plasma # 228	A375 CTRL	plasma
SA232180	plasma # 230	A375 CTRL	plasma
SA232181	plasma # 229	A375 CTRL	plasma
SA232182	plasma # 231	A375 CTRL	plasma
SA232183	plasma # 232	A375 CTRL	plasma
SA232184	tumor # 236	A375 CTRL	tumor
SA232185	tumor # 234	A375 CTRL	tumor
SA232186	tumor # 233	A375 CTRL	tumor
SA232187	tumor # 238	A375 CTRL	tumor
SA232188	tumor # 239	A375 CTRL	tumor
SA232189	tumor # 237	A375 CTRL	tumor
SA232190	tumor # 235	A375 CTRL	tumor
SA232191	tumor # 230	A375 CTRL	tumor
SA232192	tumor # 232	A375 CTRL	tumor
SA232193	tumor # 228	A375 CTRL	tumor
SA232194	tumor # 229	A375 CTRL	tumor
SA232195	tumor # 231	A375 CTRL	tumor
SA232196	tumor # 227	A375 CTRL	tumor
SA232197	plasma # 261	A375 LCT-MCT	plasma
SA232198	plasma # 262	A375 LCT-MCT	plasma
SA232199	plasma # 259	A375 LCT-MCT	plasma
SA232200	plasma # 263	A375 LCT-MCT	plasma
SA232201	plasma # 253	A375 LCT-MCT	plasma
SA232202	plasma # 258	A375 LCT-MCT	plasma
SA232203	plasma # 255	A375 LCT-MCT	plasma
SA232204	plasma # 254	A375 LCT-MCT	plasma
SA232205	plasma # 257	A375 LCT-MCT	plasma
SA232206	plasma # 256	A375 LCT-MCT	plasma
SA232207	tumor # 261	A375 LCT-MCT	tumor
SA232208	tumor # 259	A375 LCT-MCT	tumor
SA232209	tumor # 262	A375 LCT-MCT	tumor
SA232210	tumor # 258	A375 LCT-MCT	tumor
SA232211	tumor # 253	A375 LCT-MCT	tumor
SA232212	tumor # 263	A375 LCT-MCT	tumor
SA232213	tumor # 254	A375 LCT-MCT	tumor
SA232214	tumor # 255	A375 LCT-MCT	tumor
SA232215	tumor # 256	A375 LCT-MCT	tumor
SA232216	tumor # 257	A375 LCT-MCT	tumor
SA232217	plasma # 246	A375 LCT	plasma
SA232218	plasma # 247	A375 LCT	plasma
SA232219	plasma # 249	A375 LCT	plasma
SA232220	plasma # 251	A375 LCT	plasma
SA232221	plasma # 245	A375 LCT	plasma
SA232222	plasma # 244	A375 LCT	plasma
SA232223	plasma # 240	A375 LCT	plasma
SA232224	plasma # 241	A375 LCT	plasma
SA232225	plasma # 243	A375 LCT	plasma
SA232226	plasma # 252	A375 LCT	plasma
SA232227	plasma # 248	A375 LCT	plasma
SA232228	tumor # 241	A375 LCT	tumor
SA232229	tumor # 244	A375 LCT	tumor
SA232230	tumor # 240	A375 LCT	tumor
SA232231	tumor # 251	A375 LCT	tumor
SA232232	tumor # 252	A375 LCT	tumor
SA232233	tumor # 245	A375 LCT	tumor
SA232234	tumor # 243	A375 LCT	tumor
SA232235	tumor # 246	A375 LCT	tumor
SA232236	tumor # 250	A375 LCT	tumor
SA232237	tumor # 248	A375 LCT	tumor
SA232238	tumor # 249	A375 LCT	tumor
SA232239	tumor # 247	A375 LCT	tumor
SA232240	plasma # 281	WM3000 CTRL	plasma
SA232241	plasma # 280	WM3000 CTRL	plasma
SA232242	plasma # 279	WM3000 CTRL	plasma
SA232243	plasma # 278	WM3000 CTRL	plasma
SA232244	plasma # 282	WM3000 CTRL	plasma
SA232245	plasma # 288	WM3000 CTRL	plasma
SA232246	plasma # 287	WM3000 CTRL	plasma
SA232247	plasma # 277	WM3000 CTRL	plasma
SA232248	plasma # 286	WM3000 CTRL	plasma
SA232249	plasma # 285	WM3000 CTRL	plasma
SA232250	plasma # 284	WM3000 CTRL	plasma
SA232251	plasma # 283	WM3000 CTRL	plasma
SA232252	tumor # 279	WM3000 CTRL	tumor
SA232253	tumor # 284	WM3000 CTRL	tumor
SA232254	tumor # 277	WM3000 CTRL	tumor
SA232255	tumor # 283	WM3000 CTRL	tumor
SA232256	tumor # 285	WM3000 CTRL	tumor
SA232257	tumor # 288	WM3000 CTRL	tumor
SA232258	tumor # 286	WM3000 CTRL	tumor
SA232259	tumor # 287	WM3000 CTRL	tumor
SA232260	tumor # 281	WM3000 CTRL	tumor
SA232261	tumor # 282	WM3000 CTRL	tumor
SA232262	tumor # 280	WM3000 CTRL	tumor
SA232263	tumor # 278	WM3000 CTRL	tumor
SA232264	plasma # 305	WM3000 LCT-MCT	plasma
SA232265	plasma # 304	WM3000 LCT-MCT	plasma
SA232266	plasma # 303	WM3000 LCT-MCT	plasma
SA232267	plasma # 310	WM3000 LCT-MCT	plasma
SA232268	plasma # 302	WM3000 LCT-MCT	plasma
SA232269	plasma # 308	WM3000 LCT-MCT	plasma
SA232270	plasma # 311	WM3000 LCT-MCT	plasma

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Collection:

Collection ID:	CO002417
Collection Summary:	Human melanoma xenografts were established in mice using A375 and WM47 (BRAF mutant), WM3311 (BRAF/NRAS wild-type) and WM3000 (NRAS mutant) cells. Blood was taken by cardiac puncture, collected in heparin-coated tubes and centrifuged for 3 min at 2000 g. Plasma samples were stored at -80 °C until analysis. Tumors were harvested and snap frozen in liquid nitrogen and stored at -80°C for metabolomics.
Sample Type:	Blood (plasma) and tumor tissue

Treatment:

Treatment ID:	TR002436
Treatment Summary:	Human melanoma xenografts were established in mice using A375 and WM47 (BRAF mutant), WM3311 (BRAF/NRAS wild-type) and WM3000 (NRAS mutant) cells. Once the tumor volume reached 100 mm3, mice were equally assigned to control diet (CTRL) and two ketogenic diet (KDs) differing in their fat composition (LCT: long-chain triglyceride-based KD and LCT-MCT: long-chain triglyceride-based KD supplemented with C8 and C10 medium-chain triglycerides, ratio of fat to the sum of carbohydrate and protein of 8:1). Both KDs were administered ad libitum as monotherapy.

Treatment ID:

TR002436

Treatment Summary:

Human melanoma xenografts were established in mice using A375 and WM47 (BRAF mutant), WM3311 (BRAF/NRAS wild-type) and WM3000 (NRAS mutant) cells. Once the tumor volume reached 100 mm3, mice were equally assigned to control diet (CTRL) and two ketogenic diet (KDs) differing in their fat composition (LCT: long-chain triglyceride-based KD and LCT-MCT: long-chain triglyceride-based KD supplemented with C8 and C10 medium-chain triglycerides, ratio of fat to the sum of carbohydrate and protein of 8:1). Both KDs were administered ad libitum as monotherapy.

Sample Preparation:

Sampleprep ID:	SP002430
Sampleprep Summary:	To capture a broad spectrum of metabolites, we used the MxP® Quant 500 kit combined with the in-house acylcarnitine (AC) assay (biocrates life sciences ag). Samples were prepared the same way for both assays. Therefore, 50-80 mg of tumor tissues were transferred to 0.5 ml Precellys® vials followed by the addition of 85:15 ethanol:0.01 M phosphate as a lysis buffer with a volume [µl] of 3 times the tissue weight [mg]. Tissue homogenization was carried out at 4 °C and 5800 rpm using a Precellys® 24 homogenizer coupled to a Cryolys® cooling unit. The homogenates were centrifuged at 10,000 g for 2 min at 2-4 °C. Supernatants were collected and stored at -80 °C until analysis. Plasma samples as well as tissue homogenate supernatant were thawed on ice, then vortexed before use.

Sampleprep ID:

SP002430

Sampleprep Summary:

To capture a broad spectrum of metabolites, we used the MxP® Quant 500 kit combined with the in-house acylcarnitine (AC) assay (biocrates life sciences ag). Samples were prepared the same way for both assays. Therefore, 50-80 mg of tumor tissues were transferred to 0.5 ml Precellys® vials followed by the addition of 85:15 ethanol:0.01 M phosphate as a lysis buffer with a volume [µl] of 3 times the tissue weight [mg]. Tissue homogenization was carried out at 4 °C and 5800 rpm using a Precellys® 24 homogenizer coupled to a Cryolys® cooling unit. The homogenates were centrifuged at 10,000 g for 2 min at 2-4 °C. Supernatants were collected and stored at -80 °C until analysis. Plasma samples as well as tissue homogenate supernatant were thawed on ice, then vortexed before use.

Combined analysis:

Analysis ID	AN003815	AN003816	AN003817
Analysis type	MS	MS	MS
Chromatography type	Reversed phase	None (Direct infusion)	Reversed phase
Chromatography system	Waters Acquity	Waters Acquity	Thermo Dionex Ultimate 3000
Column	MxP Quant 500 Kit System	MxP Quant 500 Kit System	Biocrates Acylcarnitine
MS Type	ESI	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole	Triple quadrupole
MS instrument name	Waters TQ-S MS	Waters TQ-S MS	Thermo TQS Vantage
Ion Mode	POSITIVE	UNSPECIFIED	POSITIVE
Units	uM	uM	uM

Chromatography:

Chromatography ID:	CH002822
Instrument Name:	Waters Acquity
Column Name:	MxP Quant 500 Kit System
Chromatography Type:	Reversed phase

Chromatography ID:	CH002823
Instrument Name:	Waters Acquity
Column Name:	MxP Quant 500 Kit System
Chromatography Type:	None (Direct infusion)

Chromatography ID:	CH002824
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Biocrates Acylcarnitine
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003557
Analysis ID:	AN003815
Instrument Name:	Waters TQ-S MS
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	MS acquisition data were obtained using MRM transition (Q1 and Q3 pair) in positive ion mode. Raw data of the MxP® Quant 500 assay were exported and quantified using MetIDQTM software (Biocrates Life Sciences). Downstream analysis was performed using MetaboAnalyst 5.0.
Ion Mode:	POSITIVE

MS ID:	MS003558
Analysis ID:	AN003816
Instrument Name:	Waters TQ-S MS
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	MS acquisition data were obtained using MRM transition (Q1 and Q3 pair) in positive and negative ion mode. Raw data of the MxP® Quant 500 assay were exported and quantified using MetIDQTM software (Biocrates Life Sciences). Downstream analysis was performed using MetaboAnalyst 5.0.
Ion Mode:	UNSPECIFIED

MS ID:	MS003559
Analysis ID:	AN003817
Instrument Name:	Thermo TQS Vantage
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	MS acquisition data were obtained using MRM transition (Q1 and Q3 pair) in positive ion mode. Raw data files of the AC assay were generated by using XcaliburTM software (Thermo Fisher) and exported to MetIDQTM for further analysis, including peak integration, retention time correction, area calculation, calibration curve preparation and concentration calculation. Downstream analysis was performed using MetaboAnalyst 5.0.
Ion Mode:	POSITIVE