Summary of Study ST002058

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 PR001303. The data can be accessed directly via it's Project DOI: 10.21228/M89135 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	ST002058
Study Title	Muscle/Lung/Tumor metabolomics
Study Summary	Skeletal muscle (SkM, tibialis anterior and upper arm) and lung samples analyzed by LC-MS metabolomics. These tissue types are analyzed as healthy control, Mcherry+ tumor, or adjacent tissue.
Institute	University of Colorado Anschutz Medical Campus
Last Name	Nemkov
First Name	Travis
Address	12801 East 17th Ave. Research 1 South Rm 9121 Aurora CO 80045
Email	travis.nemkov@cuanschutz.edu
Phone	303-724-3253
Submit Date	2022-01-12
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2022-02-14
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001303
Project DOI:	doi: 10.21228/M89135
Project Title:	Unchecked oxidative stress is an insurmountable barrier for tumour cells that disseminate to skeletal muscle
Project Summary:	Skeletal muscle has been recognized as an inhospitable site for disseminated tumour cells (DTCs) for decades, yet its antimetastatic nature has eluded a thorough mechanistic description. Here, we show that DTCs traffic to and persist within skeletal muscle, raising the question as to how this tissue suppresses colonization. We employed mouse and organotypic culture models along with metabolomic profiling and ultimately find that oxidative stress is a principal suppressor of DTC proliferation in skeletal muscle. DTCs bypassed this oxidative constraint upon colonization of more fertile sites, but were unable to in muscle. Functional studies demonstrated that disrupting redox homeostasis via chemogenetic induction of reactive oxygen species slowed proliferation in lung. Conversely, enhancing antioxidant potential of tumour cells via ectopic expression of catalase allowed robust colonization of skeletal muscle. These findings reveal a profound metabolic bottleneck imposed on DTCs and sustained by skeletal muscle. Understanding this biology could reveal novel DTC vulnerabilities.
Institute:	University of Colorado Anschutz Medical Campus
Last Name:	Nemkov
First Name:	Travis
Address:	12801 E 17th Avenue, RC-1 South, Rm 9403G, Aurora, CO, 80045, USA
Email:	travis.nemkov@cuanschutz.edu
Phone:	303-724-3253

Subject:

Subject ID:	SU002140
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	Tissue	Treatment
SA193901	CTL-8_R13-	Lung	Control
SA193902	CTL-4_R20+	Lung	Control
SA193903	CTL-12_R14-	Lung	Control
SA193904	CTL-8_R13+	Lung	Control
SA193905	CTL-12_R14+	Lung	Control
SA193906	CTL-4_R20-	Lung	Control
SA193907	4T1-25_R16-	Lung	Tumor
SA193908	4T1-17_R4+	Lung	Tumor
SA193909	4T1-21_R22-	Lung	Tumor
SA193910	4T1-21_R22+	Lung	Tumor
SA193911	4T1-17_R4-	Lung	Tumor
SA193912	4T1-25_R16+	Lung	Tumor
SA193913	CTL-9_R1+	Tibialis_anterior	Control
SA193914	CTL-5_R2+	Tibialis_anterior	Control
SA193915	CTL-1_R23-	Tibialis_anterior	Control
SA193916	CTL-9_R1-	Tibialis_anterior	Control
SA193917	CTL-1_R23+	Tibialis_anterior	Control
SA193918	CTL-5_R2-	Tibialis_anterior	Control
SA193919	4T1-1_R7+	Tibialis_anterior	Tumor
SA193920	4T1-15_R11-	Tibialis_anterior	Tumor
SA193921	4T1-15_R11+	Tibialis_anterior	Tumor
SA193922	4T1-1_R7-	Tibialis_anterior	Tumor
SA193923	CTL-7_R3-	Upper_Arm_SkM	Control
SA193924	CTL-3_R6-	Upper_Arm_SkM	Control
SA193925	CTL-3_R6+	Upper_Arm_SkM	Control
SA193926	CTL-11_R21-	Upper_Arm_SkM	Control
SA193927	CTL-11_R21+	Upper_Arm_SkM	Control
SA193928	CTL-7_R3+	Upper_Arm_SkM	Control
SA193929	4T1-19_R15+	Upper_Arm_SkM	Tumor
SA193930	4T1-19_R15-	Upper_Arm_SkM	Tumor
SA193931	4T1-27_R18-	Upper_Arm_SkM	Tumor
SA193932	4T1-27_R18+	Upper_Arm_SkM	Tumor

Showing results 1 to 32 of 32

Showing results 1 to 32 of 32

Collection:

Collection ID:	CO002133
Collection Summary:	Lung and skeletal muscle (TA and bulk muscles from the upper arm) were collected upon necropsy and imaged for mCherry+ 4T1-SkM lesions (Zeiss AxioZoom). Once a mCherry+ lesion was identified, the region was excised, leaving as little tissue margins as possible. The sample was weighed and then flash frozen using LN2. Healthy lung and skeletal muscle samples were collected through the same process, without the mCherry imaging step. 4T1-parental and 4T1-SkM cells were plated in 6-well plates at an initial density of 50,000 cells/well and collected 48h later (final density ~275,000 cells/well). Wells were washed twice with 1x PBS and cells were scraped into 1.5 mL microcentrifuge tubes and spun at 16,000 x g for 3 minutes. Samples were then flash frozen and stored at -80°C until ready to process.
Sample Type:	Lung

Treatment:

Treatment ID:	TR002152
Treatment Summary:	Lung and skeletal muscle (TA and bulk muscles from the upper arm) were collected upon necropsy and imaged for mCherry+ 4T1-SkM lesions (Zeiss AxioZoom). Once a mCherry+ lesion was identified, the region was excised, leaving as little tissue margins as possible. The sample was weighed and then flash frozen using LN2. Healthy lung and skeletal muscle samples were collected through the same process, without the mCherry imaging step. 4T1-parental and 4T1-SkM cells were plated in 6-well plates at an initial density of 50,000 cells/well and collected 48h later (final density ~275,000 cells/well). Wells were washed twice with 1x PBS and cells were scraped into 1.5 mL microcentrifuge tubes and spun at 16,000 x g for 3 minutes. Samples were then flash frozen and stored at -80°C until ready to process.

Treatment ID:

TR002152

Treatment Summary:

Lung and skeletal muscle (TA and bulk muscles from the upper arm) were collected upon necropsy and imaged for mCherry+ 4T1-SkM lesions (Zeiss AxioZoom). Once a mCherry+ lesion was identified, the region was excised, leaving as little tissue margins as possible. The sample was weighed and then flash frozen using LN2. Healthy lung and skeletal muscle samples were collected through the same process, without the mCherry imaging step. 4T1-parental and 4T1-SkM cells were plated in 6-well plates at an initial density of 50,000 cells/well and collected 48h later (final density ~275,000 cells/well). Wells were washed twice with 1x PBS and cells were scraped into 1.5 mL microcentrifuge tubes and spun at 16,000 x g for 3 minutes. Samples were then flash frozen and stored at -80°C until ready to process.

Sample Preparation:

Sampleprep ID:	SP002146
Sampleprep Summary:	Prior to LC-MS analysis, tissue samples were weighed and resuspended in pre-chilled (-20°C) methanol:acetonitrile:water (5:3:2, v:v) to a final concentration of 30 mg/ml, cell samples were placed on ice and re-suspended with methanol:acetonitrile:water (5:3:2, v:v) at a concentration of 2x106 cells/mL, and media samples were extracted with the same solution at a dilution of 1:25 (v/v). Suspensions were vortexed continuously for 30 min at 4°C. Insoluble material was removed by centrifugation at 18,000 g for 10 min at 4°C and supernatants were isolated for metabolomics analysis by UHPLC-MS. Analyses were performed as previously published85,86. Injection volumes for tissue, cell, and media extracts were 2 L, 10 L, and 10L, respectively. The analytical platform employs a Vanquish UHPLC system (ThermoFisher) coupled online to a Q Exactive mass spectrometer (ThermoFisher). Samples were resolved over a Kinetex C18 column, 2.1 x 150 mm, 1.7 m particle size (Phenomenex, Torrance, CA) equipped with a guard column (SecurityGuardTM Ultracartridge – UHPLC C18 for 2.1 mm ID Columns – AJO-8782 – Phenomenex, Torrance, CA) using an aqueous phase (A) of water and 0.1% formic acid and a mobile phase (B) of acetonitrile and 0.1% formic acid for positive ion polarity mode, and an aqueous phase (A) of water:acetonitrile (95:5) with 1 mM ammonium acetate, and a mobile phase (B) of acetonitrile:water (95:5) with 1 mM ammonium acetate for negative ion polarity mode. Samples were eluted from the column using either an isocratic elution of 5% B flowed at 250 L/min and 25°C or a gradient from 5% to 95% B over 1 min, followed by an isocratic hold at 95% B for 2 min, flowed at 400 L/min and 45°C. The Q Exactive mass spectrometer (ThermoFisher) was operated independently in positive or negative ion mode, scanning in Full MS mode (2 scans) from 60 to 900 m/z at 70,000 resolution, with 4 kV spray voltage, 45 sheath gas, 15 auxiliary gas. Calibration was performed prior to analysis using the PierceTM Positive and Negative Ion Calibration Solutions (ThermoFisher). Acquired data was then converted from raw to mzXML file format using Mass Matrix (Cleveland, OH). Samples were analyzed in randomized order with a technical mixture injected periodically through analysis to qualify instrument performance. Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of deuterium, 13C, and 15N isotopes were performed using MAVEN (Princeton, NJ)92.

Sampleprep ID:

SP002146

Sampleprep Summary:

Prior to LC-MS analysis, tissue samples were weighed and resuspended in pre-chilled (-20°C) methanol:acetonitrile:water (5:3:2, v:v) to a final concentration of 30 mg/ml, cell samples were placed on ice and re-suspended with methanol:acetonitrile:water (5:3:2, v:v) at a concentration of 2x106 cells/mL, and media samples were extracted with the same solution at a dilution of 1:25 (v/v). Suspensions were vortexed continuously for 30 min at 4°C. Insoluble material was removed by centrifugation at 18,000 g for 10 min at 4°C and supernatants were isolated for metabolomics analysis by UHPLC-MS. Analyses were performed as previously published85,86. Injection volumes for tissue, cell, and media extracts were 2 L, 10 L, and 10L, respectively. The analytical platform employs a Vanquish UHPLC system (ThermoFisher) coupled online to a Q Exactive mass spectrometer (ThermoFisher). Samples were resolved over a Kinetex C18 column, 2.1 x 150 mm, 1.7 m particle size (Phenomenex, Torrance, CA) equipped with a guard column (SecurityGuardTM Ultracartridge – UHPLC C18 for 2.1 mm ID Columns – AJO-8782 – Phenomenex, Torrance, CA) using an aqueous phase (A) of water and 0.1% formic acid and a mobile phase (B) of acetonitrile and 0.1% formic acid for positive ion polarity mode, and an aqueous phase (A) of water:acetonitrile (95:5) with 1 mM ammonium acetate, and a mobile phase (B) of acetonitrile:water (95:5) with 1 mM ammonium acetate for negative ion polarity mode. Samples were eluted from the column using either an isocratic elution of 5% B flowed at 250 L/min and 25°C or a gradient from 5% to 95% B over 1 min, followed by an isocratic hold at 95% B for 2 min, flowed at 400 L/min and 45°C. The Q Exactive mass spectrometer (ThermoFisher) was operated independently in positive or negative ion mode, scanning in Full MS mode (2 scans) from 60 to 900 m/z at 70,000 resolution, with 4 kV spray voltage, 45 sheath gas, 15 auxiliary gas. Calibration was performed prior to analysis using the PierceTM Positive and Negative Ion Calibration Solutions (ThermoFisher). Acquired data was then converted from raw to mzXML file format using Mass Matrix (Cleveland, OH). Samples were analyzed in randomized order with a technical mixture injected periodically through analysis to qualify instrument performance. Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of deuterium, 13C, and 15N isotopes were performed using MAVEN (Princeton, NJ)92.

Combined analysis:

Analysis ID	AN003350	AN003351
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	QExactive	QExactive
Column	Kinetix C18 (100 x 2.1mm)	Kinetix C18 (100 x 2.1mm)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	AU	AU

Chromatography:

Chromatography ID:	CH002481
Instrument Name:	QExactive
Column Name:	Kinetix C18 (100 x 2.1mm)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003119
Analysis ID:	AN003350
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The Q Exactive mass spectrometer (ThermoFisher) was operated independently in positive or negative ion mode, scanning in Full MS mode (2 scans) from 60 to 900 m/z at 70,000 resolution, with 4 kV spray voltage, 45 sheath gas, 15 auxiliary gas. Calibration was performed prior to analysis using the PierceTM Positive and Negative Ion Calibration Solutions (ThermoFisher). Acquired data was then converted from raw to mzXML file format using Mass Matrix (Cleveland, OH). Samples were analyzed in randomized order with a technical mixture injected periodically through analysis to qualify instrument performance. Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of deuterium, 13C, and 15N isotopes were performed using MAVEN (Princeton, NJ)
Ion Mode:	POSITIVE

MS ID:	MS003120
Analysis ID:	AN003351
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	The Q Exactive mass spectrometer (ThermoFisher) was operated independently in positive or negative ion mode, scanning in Full MS mode (2 scans) from 60 to 900 m/z at 70,000 resolution, with 4 kV spray voltage, 45 sheath gas, 15 auxiliary gas. Calibration was performed prior to analysis using the PierceTM Positive and Negative Ion Calibration Solutions (ThermoFisher). Acquired data was then converted from raw to mzXML file format using Mass Matrix (Cleveland, OH). Samples were analyzed in randomized order with a technical mixture injected periodically through analysis to qualify instrument performance. Metabolite assignments, isotopologue distributions, and correction for expected natural abundances of deuterium, 13C, and 15N isotopes were performed using MAVEN (Princeton, NJ)
Ion Mode:	NEGATIVE