Summary of Study ST002383

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 PR001533. The data can be accessed directly via it's Project DOI: 10.21228/M8J99C 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.

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Study IDST002383
Study TitleMetabolomics of B-cell Acute Lymphoblastic Leukemia in Response to Adipocyte Conditioned Media
Study TypeUntargeted MS
Study SummaryAdipocyte conditioned media (ACM), stromal cell conditioned media (SCM) and unconditioned media (UCM) were added to B-cell Acute Lymphoblastic Leukemia cells (REH and RCH-AcV) either with or without methotrexate (MTX). The metabolomic profiles of the cells was determined by mass spectrometry.
Institute
Emory University
DepartmentPediatrics
LaboratoryJoshua Chandler, PhD
Last NameChandler
First NameJoshua
Address2015 Uppergate Drive NE, Atlanta, GA 30322
Emailjoshua.chandler@emory.edu
Phone404-727-3536
Submit Date2022-10-07
Num GroupsTwo sets of two sets of three groups (MTX and no-MTX, REH and RCH-AcV, ACM vs SCM vs UCM)
Total Subjects3 replicates of each group (36 total)
Publicationssubmitted to JNCI
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2023-06-07
Release Version1
Joshua Chandler Joshua Chandler
https://dx.doi.org/10.21228/M8J99C
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001533
Project DOI:doi: 10.21228/M8J99C
Project Title:The 'Omics of Obesity in B-cell Acute Lymphoblastic Leukemia
Project Summary:The obesity pandemic currently affects over 70 million Americans and over 650 million individuals worldwide. In addition to increasing susceptibility to pathogenic infections (e.g., SARS-CoV-2 et al.), obesity promotes the development of many cancer subtypes and increases mortality rates in most cases. It has been demonstrated that, in the context of B-cell acute lymphoblastic leukemia (B-ALL), adipocytes promote multi-drug chemoresistance. Furthermore, it has been demonstrated that B-ALL cells exposed to the adipocyte secretome alter their metabolic states to circumvent chemotherapy-mediated cytotoxicity. To better understand how adipocytes impact the function of human B-ALL cells, we used an untargeted metabolomics mass spectroscopy approach to define adipocyte-induced changes in B-cells. These analyses revealed that the adipocyte secretome directly modulates programs in human B-ALL cells which are associated with metabolism. In all, our data increases our understanding of pathways which may promote chemoresistance in human B-ALL.
Institute:Emory University
Department:Pediatrics
Laboratory:Joshua Chandler, PhD
Last Name:Chandler
First Name:Joshua
Address:2015 Uppergate Drive NE, Atlanta, GA 30322
Email:joshua.chandler@emory.edu
Phone:404-727-3536
Funding Source:This study was supported by funding for the CURE Childhood Cancer Foundation (Grant No. 001006916), Swim Across America (Grant No. 00103163), The Mark Foundation for Cancer Research (Grant No. 18-031-ASP), Emory University School of Medicine Bridge Funding (Grant No. 00098174), The American Cancer Society and Emory University Winship Cancer Institute Institutional Research Grant (Grant No. IRG-21-137-07-IRG) and the TREC Training Course (Grant No. R25CA203650) all awarded to Curtis J Henry. In additional, funding for Joshua Chandler included NIH R01 NR018666, R56 HL150658, and support from CF@LANTA, a component of Emory University and Children’s Healthcare of Atlanta.
Publications:submitted to JNCI
Contributors:Delaney K. Geitgey, Miyoung Lee, Kirsten A. Cottrill, Matthew B. Kilgore, Joshua D. Chandler, Curtis J. Henry

Subject:

Subject ID:SU002472
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606
Subject Comments:Human B-ALL cell line (REH and RCH-AcV) were a generous gift from Dr. Christopher Porter at Emory University

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Cell Type Media Treatment
SA237879QC-10n/a n/a n/a
SA237880Standard-1n/a n/a n/a
SA237881QC-4n/a n/a n/a
SA237882QC-2n/a n/a n/a
SA237883Blank-2n/a n/a n/a
SA237884QC-11n/a n/a n/a
SA237885QC-5n/a n/a n/a
SA237886QC-3n/a n/a n/a
SA237887SRM1950-1n/a n/a n/a
SA237888QC-1n/a n/a n/a
SA237889QC-7n/a n/a n/a
SA237890QC-6n/a n/a n/a
SA237891Blank-1n/a n/a n/a
SA237892QC-8n/a n/a n/a
SA237893Blank-3n/a n/a n/a
SA237894QC-ddMS2-1n/a n/a n/a
SA237895Blank-5n/a n/a n/a
SA237896Blank-4n/a n/a n/a
SA237897QC-9n/a n/a n/a
SA237843RCH-AcV-ACM-MTX-2RCH-AcV ACM MTX
SA237844RCH-AcV-ACM-MTX-3RCH-AcV ACM MTX
SA237845RCH-AcV-ACM-MTX-1RCH-AcV ACM MTX
SA237846RCH-AcV-ACM-1RCH-AcV ACM n/a
SA237847RCH-AcV-ACM-3RCH-AcV ACM n/a
SA237848RCH-AcV-ACM-2RCH-AcV ACM n/a
SA237849RCH-AcV-RPMI-MTX-1RCH-AcV RPMI MTX
SA237850RCH-AcV-RPMI-MTX-2RCH-AcV RPMI MTX
SA237851RCH-AcV-RPMI-MTX-3RCH-AcV RPMI MTX
SA237852RCH-AcV-RPMI-3RCH-AcV RPMI n/a
SA237853RCH-AcV-RPMI-1RCH-AcV RPMI n/a
SA237854RCH-AcV-RPMI-2RCH-AcV RPMI n/a
SA237855RCH-AcV-SCM-MTX-3RCH-AcV SCM MTX
SA237856RCH-AcV-SCM-MTX-2RCH-AcV SCM MTX
SA237857RCH-AcV-SCM-MTX-1RCH-AcV SCM MTX
SA237858RCH-AcV-SCM-3RCH-AcV SCM n/a
SA237859RCH-AcV-SCM-1RCH-AcV SCM n/a
SA237860RCH-AcV-SCM-2RCH-AcV SCM n/a
SA237861REH-ACM-MTX-3REH ACM MTX
SA237862REH-ACM-MTX-2REH ACM MTX
SA237863REH-ACM-MTX-1REH ACM MTX
SA237864REH-ACM-1REH ACM n/a
SA237865REH-ACM-3REH ACM n/a
SA237866REH-ACM-2REH ACM n/a
SA237867REH-RPMI-MTX-3REH RPMI MTX
SA237868REH-RPMI-MTX-2REH RPMI MTX
SA237869REH-RPMI-MTX-1REH RPMI MTX
SA237870REH-RPMI-3REH RPMI n/a
SA237871REH-RPMI-2REH RPMI n/a
SA237872REH-RPMI-1REH RPMI n/a
SA237873REH-SCM-MTX-1REH SCM MTX
SA237874REH-SCM-MTX-2REH SCM MTX
SA237875REH-SCM-MTX-3REH SCM MTX
SA237876REH-SCM-1REH SCM n/a
SA237877REH-SCM-2REH SCM n/a
SA237878REH-SCM-3REH SCM n/a
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Collection:

Collection ID:CO002465
Collection Summary:Human B-ALL cell lines (REH and RCH-AcV) were cultured for 24 hours in UCM, SCM, or ACM. Additionally, MTX-treated human B-ALL cells were cultured for an additional 12 hours of treatment with MTX (50nM for REH and 70 nM for RCH-AcV). This timepoint was chosen due to our prior work in this system to avoid submitting mostly apoptotic cells for analysis [18]. After the prescribed time, leukemia cells were harvested for analysis. Notably, cell viability at the time of harvest exceeded 85% in each culture (n=3 biological replicates). Cell viability was measured using cell a Bio-Rad cell counter (cat#TC20, Bio-Rad, Hercules, CA) and over 1 million viable cells were collected and washed with 1X cold PBS (pH7.4). Cell pellets were snap-frozen in dry ice and stored at -80oC until samples were submitted to our core facility.
Sample Type:B cells
Storage Conditions:-80℃

Treatment:

Treatment ID:TR002484
Treatment Summary:REH and RCH-AcV were maintained in RPMI1640 (cat#10-041-CV, Corning, Glendale, AZ), supplemented with 20% FBS (cat#S11550, BioTechne, Minneapolis, MN). OP-9 bone marrow stromal cells were maintained in α-MEM (cat#15-012-CV, Corning, Glendale, AZ) supplemented with 20% FBS. For adipocyte differentiation, 105 OP-9 cells were plated in 6-well plates in DMEM (cat#10-017-CV, Corning, Glendale, AZ) supplemented with 10% FBS as previously described [18, 26]. After 24 hours of culture, the media was removed and switched to differentiation media which is composed of α-MEM supplemented with 1.8mM oleate (cat#O7501, Sigma, St. Louis, MO) bound to BSA (cat#A6003, Sigma, St. Louis, MO) with molar ratio 5.5:1 along with 175nM insulin (cat#I6634, Sigma, St. Louis, MO) and 0.2% FBS. ACM was collected after 3 days of differentiation and used in the experiments describe in this study. For SCM, OP-9 cells were plated in DMEM supplemented with 10% FBS and conditioned media were collected on day 3 of culture. Human B-ALL cell lines (REH and RCH-AcV) were cultured for 24 hours in UCM, SCM, or ACM. Additionally, methotrexate (MTX)-treated human B-ALL cells were cultured for an additional 12 hours of treatment with MTX (50nM for REH and 70 nM for RCH-AcV). This timepoint was chosen due to our prior work in this system to avoid submitting mostly apoptotic cells for analysis.
Treatment Compound:adipocyte- , stromal cell-, and un- conditioned media; methotrexate or not
Treatment Dosevolume:methotrexate: 50-70 nM
Treatment Doseduration:conditioned medias: 24 hr; methotrexate: additional 12 hr
Treatment Vehicle:methotrexate: DMSO

Sample Preparation:

Sampleprep ID:SP002478
Sampleprep Summary:Cell samples (1x106) were mixed with 300 µl of ice-cold 2:1 acetonitrile:water and vortexed. All samples were incubated 30 min on ice and centrifuged at 20,627 g for 10 min at 4 ˚C. The supernatant was transferred to autosampler vials (ThermoFisher) held at 4 ˚C for LC-MS acquisition.
Processing Storage Conditions:On ice

Combined analysis:

Analysis ID AN003882
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Vanquish
Column SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE
Units normalized peak area

Chromatography:

Chromatography ID:CH002876
Chromatography Summary:A VanquishTM Horizon Binary ultrahigh performance liquid chromatography system coupled to a Q Exactive High Field Hybrid Orbitrap mass spectrometer (ThermoFisher) was used for data collection. A SeQuant® ZIC-HILIC column (Millipore Sigma, 3.5 µm, 100 Å, 150 x 2.1 mm, PEEK) was pumped with a mixture of mobile phases of 0.1% formic acid in water (mobile phase A) or 0.1% formic acid in acetonitrile (mobile phase B) (all reagents from Millipore Sigma). The column was equilibrated with 90% B for 8.3 min at 0.25 mL/min, after which 2.5 mL of extracted sample was injected. After this, a gradient ran from 90% to 20% B over 17.5, then was held at 20% B at 0.35 mL/min for 7.5 min.
Instrument Name:Thermo Vanquish
Column Name:SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
Flow Gradient:he column was equilibrated with 90% B for 8.3 min at 0.25 mL/min, after which 2.5 mL of extracted sample was injected. After this, a gradient ran from 90% to 20% B over 17.5 min, then was held at 20% B at 0.35 mL/min for 7.5 min.
Flow Rate:0.25 mL/min-0.35 mL/min
Solvent A:100% water; 0.1% formic acid
Solvent B:100% acetonitrile; 0.1% formic acid
Chromatography Type:HILIC

MS:

MS ID:MS003622
Analysis ID:AN003882
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Compound Discoverer 3.3 (Thermo Scientific) was used for data analysis of metabolomic experiments, including retention time alignment across runs, cubic spline normalization of areas for each metabolite in quality control pooled samples, and gap-filling based on real peak detection. The minimum peak intensity was 5x105, and metabolites were excluded if they had >50% relative standard deviation after correction or a max sample area <5-fold that of the blank. Furthermore, to be considered a high-confidence metabolite for analysis, metabolites required a) an accurate mass (5 ppm) match and either (i) retention time (±5%) matching of reference standards, and/or (ii) matching to MS2 spectra in mzCloud (mzcloud.org) (including at least one matching product ion); b) an accurate mass and retention time match to a lipid identity determined by LipidSearch (ThermoFisher, all default parameters used) based on MS2 data; and/or c) an MS2 fragment ion matching to a particular class of compounds (184.1 for phosphatidylcholine). When a chromatographic peak matched to two standards of similar retention time that were indistinguishable by MS2, we prioritized the metabolite with higher abundance in human or retained both annotations. The naming convention for the metabolites reported herein is as follows – a) if the metabolite was an accurate mass and retention time match to our curated library or to our LipidSearch library, it is listed only as the metabolite name; b) if the metabolite had an MS2 library match but no match in the aforementioned libraries, it is listed as the MS2 match and the retention time; c) if the metabolite had an MS2 compound class match, it is listed as the compound class, the chemical formula (or the molecular weight if formula was not available), and the retention time.
Ion Mode:POSITIVE
Capillary Temperature:275
Ionization Potential:+3.5 kV
Automatic Gain Control:1E6
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