Summary of Study ST003830

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 PR002357. The data can be accessed directly via it's Project DOI: 10.21228/M8ZN7S 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	ST003830
Study Title	Overcoming resistance to immunotherapy by targeting CD38 in human tumor explants - Extracellular metabolomics of B7.H3 human CAR-T cells
Study Type	Extracellular metabolomics
Study Summary	CD38 is an ectoenzyme on the surface of T cells and controlling both intracellular and extracellular NAD+ levels, which are important for T cell function. In this study we profiled the extracellular metabolomics of B7.H3 human CAR-T cells that were chronically stimulated using TCR activation for 10 days and were treated with CD38 inhibitor (78c).
Institute	Massachusetts General Hospital
Department	KF-CCR
Laboratory	Jenkins lab
Last Name	Revach
First Name	Or-Yam
Address	55 Fruit street, Boston, MA, 02114, USA
Email	oryamush@gmail.com
Phone	(617) 726-5130
Submit Date	2025-02-10
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-05-14
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR002357
Project DOI:	doi: 10.21228/M8ZN7S
Project Title:	Overcoming resistance to immunotherapy by targeting CD38 in human tumor explants
Project Summary:	T cell exhaustion is a major driver of immune checkpoint blockade (ICB) resistance and clinically effective strategies to prevent or reverse itT cell exhaustion to restore ICB sensitivity are lacking. CD38, an ecto-enzyme involved in NAD+ catabolism, is highly expressed in exhausted CD8+ T cells in human melanoma, yet its role in T cell exhaustion remains to be elucidated. Here we show that CD38+CD8+ T cells are enriched during tumor progression and are strongly associated with ICB resistance in melanoma. Chronic TCR activation and type I interferon stimulation upregulate CD38 in human T cells, leading to mitochondrial dysfunction and reduced anti-tumor activity. Disrupting CD38 restored cellular NAD+ pools and T cell bioenergetics, leading to increased TCF7 expression, improved T cell proliferation, and enhanced effector function. Importantly, targeting CD38 in a cohort of patient-derived organotypic tumor spheroid (PDOTS), human living tumor explants, demonstrates that CD38-directed therapies can overcome ICB resistance in clinically resistant human melanoma, an effect that is furthered induced by supplementation with NAD+. These results emphasize the need for further preclinical and clinical evaluation of CD38 directed therapies in melanoma and underscore the importance of NAD+ as a vital metabolite to enhance those therapies.
Institute:	Massachusetts General Hospital
Department:	KF-CCR
Laboratory:	Jenkins lab
Last Name:	Revach
First Name:	Or-Yam
Address:	55 Fruit street, Boston, MA, 02114, USA
Email:	oryamush@gmail.com
Phone:	(617) 726-5130

Subject:

Subject ID:	SU003964
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

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

mb_sample_id	local_sample_id	Sample source	Stimulation
SA419061	OYR103	human CAR-T cell media	CART acute
SA419062	OYR105	human CAR-T cell media	CART acute
SA419063	OYR104	human CAR-T cell media	CART acute
SA419064	OYR114	human CAR-T cell media	CART chronic
SA419065	OYR113	human CAR-T cell media	CART chronic
SA419066	OYR112	human CAR-T cell media	CART chronic
SA419067	OYR108	human CAR-T cell media	CART chronic
SA419068	OYR107	human CAR-T cell media	CART chronic
SA419069	OYR106	human CAR-T cell media	CART chronic
SA419070	OYR116	human CAR-T cell media	CART chronic +78c
SA419071	OYR115	human CAR-T cell media	CART chronic +78c
SA419072	OYR111	human CAR-T cell media	CART chronic +78c
SA419073	OYR110	human CAR-T cell media	CART chronic +78c
SA419074	OYR109	human CAR-T cell media	CART chronic +78c
SA419075	OYR117	human CAR-T cell media	CART chronic +78c
SA419049	OYR118	RPMI control media	RPMI
SA419050	OYR119	RPMI control media	RPMI
SA419051	OYR123	RPMI control media	RPMI+78c
SA419052	OYR122	RPMI control media	RPMI+78c
SA419059	OYR127	RPMI control media	RPMI+aFAS
SA419060	OYR126	RPMI control media	RPMI+aFAS
SA419053	OYR129	RPMI control media	RPMI+CD3/CD38
SA419054	OYR128	RPMI control media	RPMI+CD3/CD38
SA419055	OYR121	RPMI control media	RPMI+DMSO
SA419056	OYR120	RPMI control media	RPMI+DMSO
SA419057	OYR125	RPMI control media	RPMI+IL-2
SA419058	OYR124	RPMI control media	RPMI+IL-2

Showing results 1 to 27 of 27

Showing results 1 to 27 of 27

Collection:

Collection ID:	CO003957
Collection Summary:	100 μL of 3 days culture conditioned media of CAR-T cells cultures from two different donors (d1 and d2) (chronic/ chronic+CD38i). Media was collected and centrifuged at 1500 RPM for 5 min. Supernatant was collected and 30 μL of media was resuspended in 300 μL of lysis buffer, followed by sample prep processing
Sample Type:	Media

Treatment:

Treatment ID:	TR003973
Treatment Summary:	Acute or chronically stimulated CAR-T cells or chronically stimulated +3 days treatment of CD38 inhibitor 1uM 78c (6391, Toris) from two different donors (d1 and d2) were analyzed. For prepare the T cells, B7-H3.CAR-T cells were activated with washed αCD3/αCD28 Dynabeads ® (ThermoFisher Scientific, 11161D), according to the manufacturer protocols, in a 1:1 ratio of beads to cells, or 3 μg anti-human CD3 and CD28 antibodies (Biolegend, 300438, 377604), 20 ng/mL of IL-2 (PeproTech, 200-02), 0.6 μg/mL aFAS neutralizing antibody (EMD Millipore, 05-338) and RPMI (Corning, 10-040-CV), supplemented with 10% FBS and 1% penicillin/streptomycin. After 3 days, the beads were removed using a magnet/antibodies were washed and cells were divided into acute and chronic conditions. Acute and chronic were cultured in the media above, and only the chronic got restimulated with 1:1 Dynabeads ® to T cells ratio. Media and beads were replaced every 2-4 days for a total of 10-14 days. Donor 1 conditions: chronic stimulation, chronic+CD38i(78c) Donor 2 conditions: chronic stimulation, chronic+CD38i(78c)

Treatment ID:

TR003973

Treatment Summary:

Acute or chronically stimulated CAR-T cells or chronically stimulated +3 days treatment of CD38 inhibitor 1uM 78c (6391, Toris) from two different donors (d1 and d2) were analyzed. For prepare the T cells, B7-H3.CAR-T cells were activated with washed αCD3/αCD28 Dynabeads ® (ThermoFisher Scientific, 11161D), according to the manufacturer protocols, in a 1:1 ratio of beads to cells, or 3 μg anti-human CD3 and CD28 antibodies (Biolegend, 300438, 377604), 20 ng/mL of IL-2 (PeproTech, 200-02), 0.6 μg/mL aFAS neutralizing antibody (EMD Millipore, 05-338) and RPMI (Corning, 10-040-CV), supplemented with 10% FBS and 1% penicillin/streptomycin. After 3 days, the beads were removed using a magnet/antibodies were washed and cells were divided into acute and chronic conditions. Acute and chronic were cultured in the media above, and only the chronic got restimulated with 1:1 Dynabeads ® to T cells ratio. Media and beads were replaced every 2-4 days for a total of 10-14 days. Donor 1 conditions: chronic stimulation, chronic+CD38i(78c) Donor 2 conditions: chronic stimulation, chronic+CD38i(78c)

Sample Preparation:

Sampleprep ID:	SP003970
Sampleprep Summary:	At the day of the metabolomic analysis cells were cultured in fresh full RPMI media in equal concentrations for 2h. Four million cultured CAR-T cells (donor 1) or two million cultured CAR-T cells (donor 2), in three technical replicates were collected, centrifuged, washed with 0.9% NaCl, and resuspended in 300 μL lysis buffer (80% Methanol, 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water, supplemented with isotopically-labeled amino acid standards [Cambridge Isotope Laboratories, MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope Laboratories, CNLM-6245-10]). Samples were vortexed for 10 sec, then centrifuged for 10 minutes at 18,000g to pellet cell debris. The supernatant was dried on ice using a liquid nitrogen dryer. Dried samples were resuspended in 25 μL water and 2 μL was injected into a ZIC-pHILIC 150 x 2.1 mm (5 um particle size) column (EMD Millipore). Operated on Vanquish™ Flex UHPLC Systems (Thermo Fisher Scientific, San Jose, CA, USA).

Sampleprep ID:

SP003970

Sampleprep Summary:

At the day of the metabolomic analysis cells were cultured in fresh full RPMI media in equal concentrations for 2h. Four million cultured CAR-T cells (donor 1) or two million cultured CAR-T cells (donor 2), in three technical replicates were collected, centrifuged, washed with 0.9% NaCl, and resuspended in 300 μL lysis buffer (80% Methanol, 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water, supplemented with isotopically-labeled amino acid standards [Cambridge Isotope Laboratories, MSK-A2-1.2], aminopterin, and reduced glutathione standard [Cambridge Isotope Laboratories, CNLM-6245-10]). Samples were vortexed for 10 sec, then centrifuged for 10 minutes at 18,000g to pellet cell debris. The supernatant was dried on ice using a liquid nitrogen dryer. Dried samples were resuspended in 25 μL water and 2 μL was injected into a ZIC-pHILIC 150 x 2.1 mm (5 um particle size) column (EMD Millipore). Operated on Vanquish™ Flex UHPLC Systems (Thermo Fisher Scientific, San Jose, CA, USA).

Chromatography:

Chromatography ID:	CH004774
Instrument Name:	Thermo Vanquish
Column Name:	Merck SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
Column Temperature:	25 °C
Flow Gradient:	0–20 min: linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B
Flow Rate:	150 uL/min
Solvent A:	100% acetonitrile
Solvent B:	100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide
Chromatography Type:	HILIC

Analysis:

Analysis ID:	AN006291
Analysis Type:	MS
Chromatography ID:	CH004774
Num Factors:	9
Num Metabolites:	141
Rt Units:	Minutes
Units:	a.u.