Summary of Study ST003664

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 PR002273. The data can be accessed directly via it's Project DOI: 10.21228/M8TC2W This work is supported by NIH grant, U2C- DK119886.

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Study IDST003664
Study TitleTumour interstitial fluid-enriched phosphoethanolamine suppresses T cell function.
Study SummaryNutrient stress represents a significant barrier for antitumor immunity, and tumor interstitial fluid (TIF) often contains metabolites that hinder immune function. However, it is difficult to isolate the effects of tumor nutrient stress from other suppressive factors. Thus, we employed a chemically-defined cell culture medium based on the metabolomic profile of TIF: Tumor Interstitial Fluid Medium (TIFM). Culture of CD8+ T cells in TIFM limited cell expansion and impaired CD8+ T cell effector functions upon restimulation, suggesting tumor nutrient stress alone is sufficient to drive T cell dysfunction. We identified phosphoethanolamine (pEtn), a phospholipid intermediate, as a driver of T cell dysfunction. pEtn dampened T Cell Receptor (TCR) signaling by depleting T cells of diacylglycerol required for TCR signal transduction. Reduction of pEtn accumulation in tumors improved intratumoral T cell function and tumor control, suggesting pEtn accumulation plays a dominant role in TME immunosuppression.
Institute
University of Chicago
DepartmentComprehensive Cancer Center
LaboratoryMetabolomics Platform
Last NameShah
First NameHardik
Address900 E 57th street, Chicago, IL, 60637, USA
Emailhardikshah@uchicago.edu
Phone7738348830
Submit Date2025-01-14
Num Groups9
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2025-01-20
Release Version1
Hardik Shah Hardik Shah
https://dx.doi.org/10.21228/M8TC2W
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002273
Project DOI:doi: 10.21228/M8TC2W
Project Title:Tumour interstitial fluid-enriched phosphoethanolamine suppresses T cell function
Project Summary:Nutrient stress represents a significant barrier for antitumor immunity, and tumor interstitial fluid (TIF) often contains metabolites that hinder immune function. However, it is difficult to isolate the effects of tumor nutrient stress from other suppressive factors. Thus, we employed a chemically-defined cell culture medium based on the metabolomic profile of TIF: Tumor Interstitial Fluid Medium (TIFM). Culture of CD8+ T cells in TIFM limited cell expansion and impaired CD8+ T cell effector functions upon restimulation, suggesting tumor nutrient stress alone is sufficient to drive T cell dysfunction. We identified phosphoethanolamine (pEtn), a phospholipid intermediate, as a driver of T cell dysfunction. pEtn dampened T Cell Receptor (TCR) signaling by depleting T cells of diacylglycerol required for TCR signal transduction. Reduction of pEtn accumulation in tumors improved intratumoral T cell function and tumor control, suggesting pEtn accumulation plays a dominant role in TME immunosuppression.
Institute:University of Chicago
Department:Comprehensive Cancer Center
Laboratory:UCCC-Metabolomics Platform
Last Name:Shah
First Name:Hardik
Address:900 E 57th street, Chicago, IL, 60637, USA
Email:hardikshah@uchicago.edu
Phone:7738348830

Subject:

Subject ID:SU003796
Subject Type:Cultured cells
Subject Species:Mus musculus
Taxonomy ID:10090

Factors:

Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Sample source Genotype
SA401185Pos_C_D1_35T cells C_D1
SA401186Pos_C_D1_31T cells C_D1
SA401187Pos_C_D1_32T cells C_D1
SA401188Pos_C_D1_34T cells C_D1
SA401189C_D1_31T cells C_D1
SA401190C_D1_32T cells C_D1
SA401191C_D1_34T cells C_D1
SA401192C_D1_35T cells C_D1
SA401193C_D1_36T cells C_D1
SA401194Pos_C_D1_36T cells C_D1
SA401195Pos_C_D3_34T cells C_D3
SA401196Pos_C_D3_32T cells C_D3
SA401197Pos_C_D3_31T cells C_D3
SA401198C_D3_31T cells C_D3
SA401199C_D3_36T cells C_D3
SA401200C_D3_35T cells C_D3
SA401201C_D3_34T cells C_D3
SA401202C_D3_32T cells C_D3
SA401203Pos_C_D3_36T cells C_D3
SA401204Pos_C_D3_35T cells C_D3
SA401205C_D5_35T cells C_D5
SA401206Pos_C_D5_31T cells C_D5
SA401207Pos_C_D5_32T cells C_D5
SA401208C_D5_36T cells C_D5
SA401209C_D5_34T cells C_D5
SA401210C_D5_32T cells C_D5
SA401211C_D5_31T cells C_D5
SA401212Pos_C_D5_34T cells C_D5
SA401213Pos_C_D5_35T cells C_D5
SA401214Pos_C_D5_36T cells C_D5
SA401215Ctrl_32T cells Ctrl
SA401216Pos_Ctrl_36T cells Ctrl
SA401217Pos_Ctrl_35T cells Ctrl
SA401218Pos_Ctrl_34T cells Ctrl
SA401219Pos_Ctrl_32T cells Ctrl
SA401220Ctrl_31T cells Ctrl
SA401221Pos_Ctrl_31T cells Ctrl
SA401222Ctrl_36T cells Ctrl
SA401223Ctrl_35T cells Ctrl
SA401224Ctrl_34T cells Ctrl
SA401225Pos_PC_D1_32T cells PC_D1
SA401226Pos_PC_D1_36T cells PC_D1
SA401227Pos_PC_D1_35T cells PC_D1
SA401228Pos_PC_D1_34T cells PC_D1
SA401229Pos_PC_D1_31T cells PC_D1
SA401230PC_D1_31T cells PC_D1
SA401231PC_D1_32T cells PC_D1
SA401232PC_D1_34T cells PC_D1
SA401233PC_D1_35T cells PC_D1
SA401234PC_D1_36T cells PC_D1
SA401235PC_D3_34T cells PC_D3
SA401236Pos_PC_D3_34T cells PC_D3
SA401237Pos_PC_D3_35T cells PC_D3
SA401238Pos_PC_D3_36T cells PC_D3
SA401239PC_D3_31T cells PC_D3
SA401240PC_D3_32T cells PC_D3
SA401241PC_D3_35T cells PC_D3
SA401242Pos_PC_D3_32T cells PC_D3
SA401243PC_D3_36T cells PC_D3
SA401244Pos_PC_D3_31T cells PC_D3
SA401245PC_D5_32T cells PC_D5
SA401246PC_D5_34T cells PC_D5
SA401247PC_D5_35T cells PC_D5
SA401248PC_D5_36T cells PC_D5
SA401249Pos_PC_D5_32T cells PC_D5
SA401250Pos_PC_D5_34T cells PC_D5
SA401251PC_D5_31T cells PC_D5
SA401252Pos_PC_D5_35T cells PC_D5
SA401253Pos_PC_D5_36T cells PC_D5
SA401254Pos_PC_D5_31T cells PC_D5
SA401255Pos_PE_D1_31T cells PE_D1
SA401256Pos_PE_D1_32T cells PE_D1
SA401257Pos_PE_D1_34T cells PE_D1
SA401258Pos_PE_D1_35T cells PE_D1
SA401259Pos_PE_D1_36T cells PE_D1
SA401260PE_D1_34T cells PE_D1
SA401261PE_D1_35T cells PE_D1
SA401262PE_D1_36T cells PE_D1
SA401263PE_D1_31T cells PE_D1
SA401264PE_D1_32T cells PE_D1
SA401265PE_D3_34T cells PE_D3
SA401266Pos_PE_D3_36T cells PE_D3
SA401267Pos_PE_D3_35T cells PE_D3
SA401268Pos_PE_D3_34T cells PE_D3
SA401269Pos_PE_D3_32T cells PE_D3
SA401270Pos_PE_D3_31T cells PE_D3
SA401271PE_D3_35T cells PE_D3
SA401272PE_D3_31T cells PE_D3
SA401273PE_D3_36T cells PE_D3
SA401274PE_D3_32T cells PE_D3
SA401275PE_D5_31T cells PE_D5
SA401276PE_D5_32T cells PE_D5
SA401277PE_D5_34T cells PE_D5
SA401278PE_D5_35T cells PE_D5
SA401279PE_D5_36T cells PE_D5
SA401280Pos_PE_D5_31T cells PE_D5
SA401281Pos_PE_D5_32T cells PE_D5
SA401282Pos_PE_D5_34T cells PE_D5
SA401283Pos_PE_D5_35T cells PE_D5
SA401284Pos_PE_D5_36T cells PE_D5
Showing results 1 to 100 of 100

Collection:

Collection ID:CO003789
Collection Summary:Single cell suspensions of OT-I splenocytes were cultured with SIINFEKL peptide and 50U/mL of IL-2 in RPMI for 24 hours. After the initial 24 hours of activation, cells were cultured in RPMI with 50U/mL of IL-2 until they were transferred into RPMI.
Sample Type:T-cells

Treatment:

Treatment ID:TR003805
Treatment Summary:RPMI was supplemented with pEtn, choline or pCholine to match metabolite concentrations in TIFM for 1, 3 or 5 days prior to analysis on day 7 after initial activation. After treatment, 2 x 106 cells were pelleted and flash frozen.

Sample Preparation:

Sampleprep ID:SP003803
Sampleprep Summary:For lipidomic analysis, 20uL of Avanti SPLASH deuterated internal standard mix (1:100 dilution) was added to frozen cell pellets, followed by extraction with a modified 44 method. In brief, ice-cold methanol containing 0.1mg/mL butylated hydroxytoluene was added to the cell pellet followed by sonication for 3 minutes and shaking for 5 mins at 15°C and 1000 rpm using Thermomixer. Then 900 uL of methyl-tert-butyl ether (MTBE) was added to the cell pellets tubes to extract the lipid species. The tubes were vortexed for 15 mins at 4°C and 1000 rpm on a Thermomixer. The phase separation was induced by adding 300 µL of ice-cold water followed by sonication for 3 mins, vortex for 15 mins at 15°C and 1000 rpm, and centrifuge at 21,000g and 4°C for 15 mins. The upper organic layer (400µL) was dried using the Genevac EZ-2.4 elite evaporator and stored at -80°C. One aliquot was reserved for the positive ionization mode and the second aliquot for the negative ionization mode. On the day of analysis, the dried lipid extract was re-suspended in 40µL of 3/2/1 isopropanol/acetonitrile/water at room temperature, sonicated for 3 minutes, vortex for 15 mins at 15°C and 2000 rpm, and 25µL of supernatant was transferred to LC-MS vial. A pooled QC sample was generated using the remaining samples.

Combined analysis:

Analysis ID AN006020 AN006021
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Scientific Vanquish Horizon UHPLC Thermo Scientific Vanquish Horizon UHPLC
Column Thermo Scientific Accucore C30 (150 x 2.1 mm, 2.6 µm) Thermo Scientific Accucore C30 (150 x 2.1 mm, 2.6 µm)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Orbitrap IQ-X Tribrid Thermo Orbitrap IQ-X Tribrid
Ion Mode POSITIVE NEGATIVE
Units A.U. A.U.

Chromatography:

Chromatography ID:CH004574
Chromatography Summary:Lipids were separated on Thermo Scientific Accucore C30 (2.1x150 mm, 2.6µm) column connected to a Vanquish Horizon UHPLC system and IQ-X tribrid mass spectrometers. The column temperature, injection volume, and flow rate were 45°C, 4 µL, and 0.26 mL/min, respectively. The mobile phase A (MPA) was 60/40 acetonitrile/water, 10mM ammonium formate + 0.1% formic acid and MPB was 89.1/9.9/0.99 isopropanol/acetonitrile/water, 10 mM ammonium formate + 0.1% formic acid. The chromatographic gradient was 0 minute: 30%B, 2.00 minutes (mins): 43% B, 2.1 mins: 55%B, 12.00 mins: 65%B, 18.00 mins: 85%B, 20.00 mins: 100%B, 25.00 mins:100%B, 25.1 mins:30%B and 30.00 mins:30%B.
Instrument Name:Thermo Scientific Vanquish Horizon UHPLC
Column Name:Thermo Scientific Accucore C30 (150 x 2.1 mm, 2.6 µm)
Column Temperature:45℃
Flow Gradient:0 minute: 30%B, 2.00 minutes (mins): 43% B, 2.1 mins: 55%B, 12.00 mins: 65%B, 18.00 mins: 85%B, 20.00 mins: 100%B, 25.00 mins:100%B, 25.1 mins:30%B and 30.00 mins:30%B.
Flow Rate:0.260 mL/min
Injection Temperature:15℃
Solvent A:60% Acetonitrile/40% Water; 10mM ammonium formate; 0.1% formic acid
Solvent B:89.1% isopropanol/9.9% acetonitrile/0.99% water; 10 mM ammonium formate; 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS005731
Analysis ID:AN006020
Instrument Name:Thermo Orbitrap IQ-X Tribrid
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:MS1 parameters were as follows: spray voltage: 3500 V for positive ionization and 2500 V for negative ionization modes, sheath gas: 40, auxiliary gas: 10, sweep gas: 1, ion transfer tube temperature: 300°C, vaporizer temperature: 350°C, orbitrap resolution: 120K, scan range(m/z): 250-2000 for pos, 200-2000 for neg,, RF lens(%): 60, automatic gain control (AGC) target: 50%, and a maxIT of 100 milliseconds (ms). Quadrupole isolation and Internal calibration using Easy IC were enabled. Lipids were identified by performing the MS2 and MS3 experiments in the orbitrap mass analyzer. A comprehensive data-dependent HCD MS2 experiment with conditional CID MS2 and MS3 data-acquisition strategy was applied for the in-depth characterization of lipid species. Lipid fragmentation was obtained by first MS1 data acquisition in full scan mass range (200-2000) followed by the data-dependent (dd) MS2 with the normalized stepped HCD collision energy (%) at 25, 30, 35, OT-30K resolution, maxIT-54 ms. If the HCD fragmentation had the fragment ion- 184.0733 m/z (phosphocholine head group) then the same ions were subjected either to ddMS2 CID (fixed collision energy-32% , activation time-10 ms & Q-0.25, 30K resolution, 100 % normalized AGC target and maxIT-54 ms) or CID MS3 scans (fixed collision energy-35%, activation Q-0.25) triggered on the top 3 most intense ions that lost neutral fatty acids plus ammonia (only for triacylglycerols). A total of six injections were made to generate fragmentation data using the AcquireX workflow on the pooled QC samples. The Thermo Scientific LipidSearchTM software version 5.0 was used to generate the list of identified lipid (Precursor tolerance ± 3 ppm, production tolerance ±5.0 ppm, product threshold- 1.0). [M+H] adduct was used to identify and quantify HexCer,SM, SPH,MePC, CoQ, AcylCarnitine,LPC,LPE,PC and PE species while [M+NH] was for the TG, DG, cholesteryl ester species. These identified lipid species were quantify using the Compound Discoverer 3.3 and Skyline45 (v24.1) software.
Ion Mode:POSITIVE
  
MS ID:MS005732
Analysis ID:AN006021
Instrument Name:Thermo Orbitrap IQ-X Tribrid
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:MS1 parameters were as follows: spray voltage: 3500 V for positive ionization and 2500 V for negative ionization modes, sheath gas: 40, auxiliary gas: 10, sweep gas: 1, ion transfer tube temperature: 300°C, vaporizer temperature: 350°C, orbitrap resolution: 120K, scan range(m/z): 250-2000 for pos, 200-2000 for neg,, RF lens(%): 60, automatic gain control (AGC) target: 50%, and a maxIT of 100 milliseconds (ms). Quadrupole isolation and Internal calibration using Easy IC were enabled. Lipids were identified by performing the MS2 and MS3 experiments in the orbitrap mass analyzer. A comprehensive data-dependent HCD MS2 experiment with conditional CID MS2 and MS3 data-acquisition strategy was applied for the in-depth characterization of lipid species. Lipid fragmentation was obtained by first MS1 data acquisition in full scan mass range (200-2000) followed by the data-dependent (dd) MS2 with the normalized stepped HCD collision energy (%) at 25, 30, 35, OT-30K resolution, maxIT-54 ms. If the HCD fragmentation had the fragment ion- 184.0733 m/z (phosphocholine head group) then the same ions were subjected either to ddMS2 CID (fixed collision energy-32% , activation time-10 ms & Q-0.25, 30K resolution, 100 % normalized AGC target and maxIT-54 ms) or CID MS3 scans (fixed collision energy-35%, activation Q-0.25) triggered on the top 3 most intense ions that lost neutral fatty acids plus ammonia (only for triacylglycerols). A total of six injections were made to generate fragmentation data using the AcquireX workflow on the pooled QC samples. The Thermo Scientific LipidSearchTM software version 5.0 was used to generate the list of identified lipid (Precursor tolerance ± 3 ppm, production tolerance ±5.0 ppm, product threshold- 1.0). [M+H] adduct was used to identify and quantify HexCer,SM, SPH,MePC, CoQ, AcylCarnitine,LPC,LPE,PC and PE species while [M+NH] was for the TG, DG, cholesteryl ester species. These identified lipid species were quantify using the Compound Discoverer 3.3 and Skyline45 (v24.1) software.
Ion Mode:NEGATIVE
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