Summary of Study ST004464

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 PR002390. The data can be accessed directly via it's Project DOI: 10.21228/M8Q250 This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

Study ID	ST004464
Study Title	Polar metabolite profiling of SEM-luciferase cells purified from the skulls and spleens of mice on copper replete diet (CuR+vehicle) or copper depleted diet with zinc acetate drinking water (CuD+ZnAc) and pulsed with 3-13C serine tracer.
Study Summary	This study aimed to measure differences in purine synthesis between leukemia cells from the central nervous system (CNS) and spleens of mice, and also to determine the effect of copper depletion on purine synthesis in these two sites. Mice were irradiated, engrafted with leukemia cells and randomized to either CuR+vehicle or CuD+ZnAc treatments. 16 hours prior to harvest, mice were dosed with 800mg/kg 3-13C serine by intraperitoneal injection. Leukemia cells were harvested from the CNS and spleens of mice using magnet-activated cell sorting (MACS). This experiment revealed accumulation of purine synthesis intermediates in CNS leukemia cells compared to splenic leukemia cells, and that copper depletion leads to reduced label incorporation into purines.
Institute	Boston Childrens Hospital
Last Name	Wong
First Name	Alan
Address	300 Longwood Avenue
Email	alan.wong@childrens.harvard.edu
Phone	(617) 355-7433
Submit Date	2025-12-04
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-12-22
Release Version	1

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Project:

Project ID:	PR002390
Project DOI:	doi: 10.21228/M8Q250
Project Title:	In vivo CRISPR screen identifies copper metabolism as a vulnerability in acute lymphoblastic leukemia
Project Summary:	The nutrient-sparse cerebrospinal fluid (CSF) poses a significant challenge to spreading cancer cells. Despite this challenge, leukemia often spreads to the CSF and represents a significant clinical complication. To uncover nutritional dependencies of leukemia cells in the CSF that could be targeted therapeutically, we conducted an in vivo targeted CRISPR screen in a xenograft model of leukemia. We found that SLC31A1, the primary cell surface copper importer, is a genetic dependency of leukemia in both the central nervous system as well as in the hematopoietic organs. Perturbation of copper metabolism leads to complex IV deficiency, perturbed nucleotide metabolism and slowed leukemia cell proliferation. Furthermore, nutritional copper depletion reduced cancer progression in cell line based and patient-derived xenograft models of leukemia. Copper thus appears to be an actionable micronutrient in leukemia.
Institute:	Boston Children's Hospital
Department:	Pathology
Laboratory:	Naama Kanarek
Last Name:	Wong
First Name:	Alan
Address:	300 Longwood Avenue, Boston, MA, 02115, USA
Email:	alan.wong@childrens.harvard.edu
Phone:	(617) 355-7433
Funding Source:	NCI 1R01CA282477-01A1

Subject:

Subject ID:	SU004640
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	treatment
SA530612	20251030_QE1_HILIC_SEMserineMACS_AYW1371	CNS	CuD+ZnAc
SA530613	20251030_QE1_HILIC_SEMserineMACS_AYW1379	CNS	CuD+ZnAc
SA530614	20251030_QE1_HILIC_SEMserineMACS_AYW1377	CNS	CuD+ZnAc
SA530615	20251030_QE1_HILIC_SEMserineMACS_AYW1375	CNS	CuD+ZnAc
SA530616	20251030_QE1_HILIC_SEMserineMACS_AYW1373	CNS	CuD+ZnAc
SA530617	20251030_QE1_HILIC_SEMserineMACS_AYW1369	CNS	CuD+ZnAc
SA530618	20251030_QE1_HILIC_SEMserineMACS_AYW1357	CNS	CuR+Vehicle
SA530619	20251030_QE1_HILIC_SEMserineMACS_AYW1367	CNS	CuR+Vehicle
SA530620	20251030_QE1_HILIC_SEMserineMACS_AYW1359	CNS	CuR+Vehicle
SA530621	20251030_QE1_HILIC_SEMserineMACS_AYW1363	CNS	CuR+Vehicle
SA530622	20251030_QE1_HILIC_SEMserineMACS_AYW1365	CNS	CuR+Vehicle
SA530623	20251030_QE1_HILIC_SEMserineMACS_AYW1361	CNS	CuR+Vehicle
SA530624	20251030_QE1_HILIC_SEMserineMACS_AYW1376	Spleen	CuD+ZnAc
SA530625	20251030_QE1_HILIC_SEMserineMACS_AYW1380	Spleen	CuD+ZnAc
SA530626	20251030_QE1_HILIC_SEMserineMACS_AYW1378	Spleen	CuD+ZnAc
SA530627	20251030_QE1_HILIC_SEMserineMACS_AYW1370	Spleen	CuD+ZnAc
SA530628	20251030_QE1_HILIC_SEMserineMACS_AYW1372	Spleen	CuD+ZnAc
SA530629	20251030_QE1_HILIC_SEMserineMACS_AYW1374	Spleen	CuD+ZnAc
SA530630	20251030_QE1_HILIC_SEMserineMACS_AYW1364	Spleen	CuR+Vehicle
SA530631	20251030_QE1_HILIC_SEMserineMACS_AYW1360	Spleen	CuR+Vehicle
SA530632	20251030_QE1_HILIC_SEMserineMACS_AYW1358	Spleen	CuR+Vehicle
SA530633	20251030_QE1_HILIC_SEMserineMACS_AYW1368	Spleen	CuR+Vehicle
SA530634	20251030_QE1_HILIC_SEMserineMACS_AYW1366	Spleen	CuR+Vehicle
SA530635	20251030_QE1_HILIC_SEMserineMACS_AYW1362	Spleen	CuR+Vehicle

Showing results 1 to 24 of 24

Showing results 1 to 24 of 24

Collection:

Collection ID:	CO004633
Collection Summary:	Mice were anesthetized with ketamine/xylazine (100-120mg/kg; and 10mg/kg by intraperitoneal injection), and cells were harvested from the skulls and spleens of mice by blunt dissection into 1xPBS with 2% FBS and 2mM EDTA on ice (PBE). Cells were pelleted, then resuspended in 160uL of PBE, and 50uL of Mouse Cell Depletion Cocktail (Miltenyi Biotec cat #130-104-694) was added per sample and samples incubated on ice for 5 minutes. While incubating, one LS column (Miltenyi Biotec 130-042-401) per sample was affixed to a QuadroMACS magnet (Miltenyi Biotec 130-091-051) and primed using 3mL of ice-cold PBE. The cell suspension was then applied to each column and the flow-through containing unlabelled, human leukemia cell-enriched fraction was collected. The column was washed with an additional 2x1mL of PBE and the flow-through collected as well. The columns were then discarded. The flow-through was spun at 4000g for 1 minute at 4C, and the cell pellet resuspended in 1mL of 0.9% NaCl. After an additional 20 second spin at 4C and 21,000rcf, pelleted cells were resuspended in 100% LC-MS grade methanol for metabolite detection.
Sample Type:	Leukemia cells

Treatment:

Treatment ID:	TR004649
Treatment Summary:	NOD-SCID mice (5 weeks or older) were randomized by cage to either a copper replete diet (6ppm, Teklad TD.240097) with 2% sucrose in the drinking water (SigmaAldrich S0389-1kg) or copper deficient diet (0.3-0.7ppm, Teklad TD.240098) with 2% sucrose and zinc acetate (SigmaAldrich 383317-25g) in the drinking water. The zinc acetate dose was 2g/L for 4 weeks (2 weeks prior to injection and 2 weeks after); after this treatment period, all mice were switched to autoclaved water. Mice were allowed to consume food and water ad libitum, and the food and water was replaced every week. Two weeks after starting these treatments, mice were irradiated (250 rads) and then engrafted with 3 million SEM-luciferase cells in 200uL sterile 0.9% NaCl by tail vein injection. On the 25th day after injection, 16 hours prior to euthanasia, all mice were dosed with 800mg/kg 3-13C labelled serine in sterile 0.9% NaCl by intraperitoneal injection.

Treatment ID:

TR004649

Treatment Summary:

NOD-SCID mice (5 weeks or older) were randomized by cage to either a copper replete diet (6ppm, Teklad TD.240097) with 2% sucrose in the drinking water (SigmaAldrich S0389-1kg) or copper deficient diet (0.3-0.7ppm, Teklad TD.240098) with 2% sucrose and zinc acetate (SigmaAldrich 383317-25g) in the drinking water. The zinc acetate dose was 2g/L for 4 weeks (2 weeks prior to injection and 2 weeks after); after this treatment period, all mice were switched to autoclaved water. Mice were allowed to consume food and water ad libitum, and the food and water was replaced every week. Two weeks after starting these treatments, mice were irradiated (250 rads) and then engrafted with 3 million SEM-luciferase cells in 200uL sterile 0.9% NaCl by tail vein injection. On the 25th day after injection, 16 hours prior to euthanasia, all mice were dosed with 800mg/kg 3-13C labelled serine in sterile 0.9% NaCl by intraperitoneal injection.

Sample Preparation:

Sampleprep ID:	SP004646
Sampleprep Summary:	Cell pellet was resuspended in 400uL of 100% LC-MS grade methanol supplemented with isotopically-labelled amino acid standards [Cambridge Isotope Laboratories, MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope Laboratories, CNLM-6245-10]) with repeated pipetting up and down and vortexing for 10 seconds. Then, 100uL of LCMS-grade water containing 125 mM Ammonium Acetate, 10 mM Na-Ascorbate, and 7.9 mg/mL 5,5-dithio-bis-(2-nitrobenzoic acid (Ellman's reagent) was added, and the sample vortex for another 10 seconds. Samples were then centrifuged for 10 minutes at 18,000 g to pellet cell debris. The supernatant was transferred to a new tube and dried on ice using a liquid nitrogen dryer. The dried metabolites were then resuspended in 30uL and 2uL was injected.

Sampleprep ID:

SP004646

Sampleprep Summary:

Cell pellet was resuspended in 400uL of 100% LC-MS grade methanol supplemented with isotopically-labelled amino acid standards [Cambridge Isotope Laboratories, MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope Laboratories, CNLM-6245-10]) with repeated pipetting up and down and vortexing for 10 seconds. Then, 100uL of LCMS-grade water containing 125 mM Ammonium Acetate, 10 mM Na-Ascorbate, and 7.9 mg/mL 5,5-dithio-bis-(2-nitrobenzoic acid (Ellman's reagent) was added, and the sample vortex for another 10 seconds. Samples were then centrifuged for 10 minutes at 18,000 g to pellet cell debris. The supernatant was transferred to a new tube and dried on ice using a liquid nitrogen dryer. The dried metabolites were then resuspended in 30uL and 2uL was injected.

Combined analysis:

Analysis ID	AN007481	AN007482
Chromatography ID	CH005673	CH005673
MS ID	MS007177	MS007178
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	SeQuant ZIC-HILIC (150 x 2.1mm,5um)	SeQuant ZIC-HILIC (150 x 2.1mm,5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	UNSPECIFIED	UNSPECIFIED
Units	Peak area	Normalized peak area

Chromatography:

Chromatography ID:	CH005673
Chromatography Summary:	2 μL of each sample was injected into a ZIC-pHILIC 150 x 2.1 mm (5 μm particle size) column (EMD Millipore) operated on a Vanquish™ Flex UHPLC system (Thermo Fisher Scientific). Chromatographic separation was achieved using the following conditions: buffer A was 95% acetonitrile and 5% 20 mM ammonium carbonate, 0.1% ammonium hydroxide in water; buffer B was 95% 20 mM ammonium carbonate, 0.1% ammonium hydroxide in water and 5% acetonitrile. Gradient conditions used were: 0-17.5 min: linear gradient from 16.6% to 75.0% B; 17.5-18.0 min: linear gradient from 75.0% to 85.0% B; 18-20.0 min: hold at 85.0% B; 20.0-20.5 min: from 85.0% to 16.6% B; 20.5-24 min: hold at 16.6% B at 0.150 mL/min flow rate. The column oven and autosampler tray were held at 25 °C and 4 °C, respectively.
Instrument Name:	Thermo Vanquish
Column Name:	SeQuant ZIC-HILIC (150 x 2.1mm,5um)
Column Temperature:	25
Flow Gradient:	0-17.5 min: linear gradient from 16.6% to 75.0% B; 17.5-18.0 min: linear gradient from 75.0% to 85.0% B; 18-20.0 min: hold at 85.0% B; 20.0-20.5 min: from 85.0% to 16.6% B; 20.5-24 min: hold at 16.6% B
Flow Rate:	0.15 mL/min
Solvent A:	95% acetonitrile/5% water; 20mM ammonium carbonate; 0.1% ammonium hydroxide
Solvent B:	95% water/5% acetonitrile; 20mM ammonium carbonate; 0.1% ammonium hydroxide
Chromatography Type:	HILIC

MS:

MS ID:	MS007177
Analysis ID:	AN007481
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	MS data acquisition was performed using a QExactive benchtop orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe (Thermo Fisher Scientific) and polarity switching was used. Two scans were used: full scans in both positive and negative ionization mode in a range of m/z = 70–1000, with the resolution set at 70,000, the AGC target at 1 × 10e6, and the maximum injection time (Max IT) at 20 msec from 0-20 minutes. Tune file parameters were: spray voltage = 3.5kV, capillary temperature = 320C, S-lens RF = 50, auxillary gas temperature = 350C. Targeted TraceFinder analysis for unlabelled metabolites, normalized to internal standards and total well-detected polar metabolites.
Ion Mode:	UNSPECIFIED

MS ID:	MS007178
Analysis ID:	AN007482
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	MS data acquisition was performed using a QExactive benchtop orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe (Thermo Fisher Scientific) and polarity switching was used. Two scans were used: full scans in both positive and negative ionization mode in a range of m/z = 70–1000, with the resolution set at 70,000, the AGC target at 1 × 10e6, and the maximum injection time (Max IT) at 20 msec from 0-20 minutes. Tune file parameters were: spray voltage = 3.5kV, capillary temperature = 320C, S-lens RF = 50, auxillary gas temperature = 350C. Targeted TraceFinder analysis for 13C-labelled metabolites.
Ion Mode:	UNSPECIFIED