Summary of Study ST003884

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 PR002436. The data can be accessed directly via it's Project DOI: 10.21228/M8RN89 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	ST003884
Study Title	Respiration defects limit serine synthesis required for lung cancer growth and survival - Effect of Polg mutation in NSCLC Em tumor derived cell lines (TDCLs)
Study Summary	This study investigates the impact of mtDNA mutation burden induced by the PolGD256A mutation in NSCLC tumor derived cell lines (TDCLs). We characterized the metabolic profile of TDCLs harboring this mutation compared to controls. KP: NSCLC TDCLs generated from conditional animals PGKP: NSCLC TDCLs generated from conditional animals bearing PolG mutation. Here, we show that PolG mutation causes mitochondrial dysfunction, impacting serine/glycine metabolism as well as energy metabolism through glycolysis due intensive usage of glucose over time. Here, we show that the consumption of glucose, serine, glycine, and glycolytic intermediates varied over time (at 24, 48, and 72 hours) in a manner dependent on mitochondrial function, which is linked to the genotype of the TDCLs—either KP or PGKP cells.
Institute	Rutgers Cancer Institute
Last Name	Cararo Lopes
First Name	Eduardo
Address	195 Little Albany Street
Email	edu.llopes@gmail.com
Phone	732-235-5795
Submit Date	2025-03-20
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2025-05-06
Release Version	1

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Project:

Project ID:	PR002436
Project DOI:	doi: 10.21228/M8RN89
Project Title:	Respiration defects limit serine synthesis required for lung cancer growth and survival
Project Type:	Pool Size Metabolomic in vivo
Project Summary:	Mitochondrial function supports energy and anabolic metabolism. Pathogenic mitochondrial DNA (mtDNA) mutations impair these processes, causing mitochondrial diseases. Their role in human cancers is less clear; while some cancers harbor high mtDNA mutation burden, others do not. Here we show that a proofreading mutant of DNA polymerase gamma (PolGD256A) increases the mtDNA mutation burden in non-small-cell lung cancer (NSCLC). This mutation promotes the accumulation of defective mitochondria, reduces tumor cell proliferation and viability, and improves cancer survival. In NSCLC, pathogenic mtDNA mutations enhance glycolysis and create a glucose dependency to support mitochondrial energy, but at the expense of a lower NAD⁺/NADH ratio that hinders de novo serine synthesis. Thus, mitochondrial function in NSCLC is essential for maintaining adequate serine synthesis, which in turn supports the anabolic metabolism and redox homeostasis required for tumor growth, explaining why these cancers preserve functional mtDNA.
Institute:	Rutgers University
Department:	Rutgers Cancer Institute
Laboratory:	Eileen White
Last Name:	Cararo Lopes
First Name:	Eduardo
Address:	195 Little Albany Street
Email:	edu.llopes@gmail.com
Phone:	732-235-5795
Funding Source:	NIH
Publications:	Respiration defects limit serine synthesis required for lung cancer growth and survival

Subject:

Subject ID:	SU004019
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male and female

Factors:

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

mb_sample_id	local_sample_id	Genotype	Time (H)
SA427082	24h_31-3_1_KP_24	KP	24
SA427083	24h_31-1_1_KP_24	KP	24
SA427084	24h_31-1_2_KP_24	KP	24
SA427085	24h_31-1_3_KP_24	KP	24
SA427086	24h_31-2_1_KP_24	KP	24
SA427087	24h_31-2_2_KP_24	KP	24
SA427088	24h_31-2_3_KP_24	KP	24
SA427089	24h_31-3_2_KP_24	KP	24
SA427090	24h_31-3_3_KP_24	KP	24
SA427091	24h_31-4_1_KP_24	KP	24
SA427092	24h_31-4_2_KP_24	KP	24
SA427093	24h_31-4_3_KP_24	KP	24
SA427094	48h_31-2_3_KP_48	KP	48
SA427095	48h_31-1_2_KP_48	KP	48
SA427096	48h_31-1_3_KP_48	KP	48
SA427097	48h_31-2_1_KP_48	KP	48
SA427098	48h_31-2_2_KP_48	KP	48
SA427099	48h_31-3_1_KP_48	KP	48
SA427100	48h_31-1_1_KP_48	KP	48
SA427101	48h_31-3_3_KP_48	KP	48
SA427102	48h_31-4_1_KP_48	KP	48
SA427103	48h_31-4_2_KP_48	KP	48
SA427104	48h_31-4_3_KP_48	KP	48
SA427105	48h_31-3_2_KP_48	KP	48
SA427106	72h_31-3_2_KP_72	KP	72
SA427107	72h_31-4_3_KP_72	KP	72
SA427108	72h_31-4_2_KP_72	KP	72
SA427109	72h_31-4_1_KP_72	KP	72
SA427110	72h_31-3_3_KP_72	KP	72
SA427111	72h_31-2_1_KP_72	KP	72
SA427112	72h_31-3_1_KP_72	KP	72
SA427113	72h_31-2_3_KP_72	KP	72
SA427114	72h_31-2_2_KP_72	KP	72
SA427115	72h_31-1_3_KP_72	KP	72
SA427116	72h_31-1_2_KP_72	KP	72
SA427117	72h_31-1_1_KP_72	KP	72
SA427118	24h_04-1_2_PGKP_24	PGKP	24
SA427119	24h_04-1_1_PGKP_24	PGKP	24
SA427120	24h_26-4_3_PGKP_24	PGKP	24
SA427121	24h_26-4_2_PGKP_24	PGKP	24
SA427122	24h_26-4_1_PGKP_24	PGKP	24
SA427123	24h_26-3_3_PGKP_24	PGKP	24
SA427124	24h_26-3_2_PGKP_24	PGKP	24
SA427125	24h_26-3_1_PGKP_24	PGKP	24
SA427126	24h_26-2_3_PGKP_24	PGKP	24
SA427127	24h_26-2_2_PGKP_24	PGKP	24
SA427128	24h_26-2_1_PGKP_24	PGKP	24
SA427129	24h_26-1_3_PGKP_24	PGKP	24
SA427130	24h_26-1_2_PGKP_24	PGKP	24
SA427131	24h_26-1_1_PGKP_24	PGKP	24
SA427132	24h_04-4_3_PGKP_24	PGKP	24
SA427133	24h_04-4_2_PGKP_24	PGKP	24
SA427134	24h_04-4_1_PGKP_24	PGKP	24
SA427135	24h_04-3_3_PGKP_24	PGKP	24
SA427136	24h_04-3_2_PGKP_24	PGKP	24
SA427137	24h_04-3_1_PGKP_24	PGKP	24
SA427138	24h_04-2_3_PGKP_24	PGKP	24
SA427139	24h_04-2_2_PGKP_24	PGKP	24
SA427140	24h_04-2_1_PGKP_24	PGKP	24
SA427141	24h_04-1_3_PGKP_24	PGKP	24
SA427142	48h_04-2_2_PGKP_48	PGKP	48
SA427143	48h_04-2_1_PGKP_48	PGKP	48
SA427144	48h_26-3_1_PGKP_48	PGKP	48
SA427145	48h_04-1_1_PGKP_48	PGKP	48
SA427146	48h_04-3_3_PGKP_48	PGKP	48
SA427147	48h_04-1_2_PGKP_48	PGKP	48
SA427148	48h_26-2_2_PGKP_48	PGKP	48
SA427149	48h_26-2_1_PGKP_48	PGKP	48
SA427150	48h_26-1_3_PGKP_48	PGKP	48
SA427151	48h_26-1_2_PGKP_48	PGKP	48
SA427152	48h_26-1_1_PGKP_48	PGKP	48
SA427153	48h_04-4_3_PGKP_48	PGKP	48
SA427154	48h_04-4_2_PGKP_48	PGKP	48
SA427155	48h_04-4_1_PGKP_48	PGKP	48
SA427156	48h_04-3_2_PGKP_48	PGKP	48
SA427157	48h_26-2_3_PGKP_48	PGKP	48
SA427158	48h_04-3_1_PGKP_48	PGKP	48
SA427159	48h_04-2_3_PGKP_48	PGKP	48
SA427160	48h_04-1_3_PGKP_48	PGKP	48
SA427161	48h_26-3_2_PGKP_48	PGKP	48
SA427162	48h_26-3_3_PGKP_48	PGKP	48
SA427163	48h_26-4_1_PGKP_48	PGKP	48
SA427164	48h_26-4_2_PGKP_48	PGKP	48
SA427165	48h_26-4_3_PGKP_48	PGKP	48
SA427166	72h_26-3_2_PGKP_72	PGKP	72
SA427167	72h_26-4_3_PGKP_72	PGKP	72
SA427168	72h_26-4_2_PGKP_72	PGKP	72
SA427169	72h_26-4_1_PGKP_72	PGKP	72
SA427170	72h_26-3_3_PGKP_72	PGKP	72
SA427171	72h_26-1_3_PGKP_72	PGKP	72
SA427172	72h_26-3_1_PGKP_72	PGKP	72
SA427173	72h_04-3_2_PGKP_72	PGKP	72
SA427174	72h_04-1_1_PGKP_72	PGKP	72
SA427175	72h_04-1_2_PGKP_72	PGKP	72
SA427176	72h_04-2_1_PGKP_72	PGKP	72
SA427177	72h_04-2_2_PGKP_72	PGKP	72
SA427178	72h_04-2_3_PGKP_72	PGKP	72
SA427179	72h_04-3_1_PGKP_72	PGKP	72
SA427180	72h_04-3_3_PGKP_72	PGKP	72
SA427181	72h_26-2_3_PGKP_72	PGKP	72

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Collection:

Collection ID:	CO004012
Collection Summary:	TDCLs were plated in 6 multiwell plates (Corning) and incubated (37° C in 5% CO2) for 24h hours. After this period, the medium was replaced and cells were further incubated and extracted at the indicated time points. Three wells were used for the metabolite extraction, and the other three were harvested using trypsin 0.25% to measure the wet cell volume using a PCV-packed cell volume tube (TPP). One aliquot of the medium (15µL) of each well was collected and immediately introduced in extraction buffer 40:40:20 (Methanol: Acetonitrile: Water) with 0.05% formic acid allowed to rest on ice for 5 minutes. For the TDCLs the metabolites extraction was performed by washing the wells twice with cold PBS, and 400 µL of extraction buffer 40:40:20 with 0.05% formic acid was added to each well. The plate was allowed to rest on ice for 5 minutes, and then the cells and buffer were scraped. The samples, extraction from medium and cells, were placed in a 1.5 mL microtube with 22 µL of 15% NH4HCO3 vortexed and centrifuged for 10 min at 15,000g at 4° C. 380 µL of the supernatant were collected and stored in -80° C freezer until analysis by LC-MS.
Sample Type:	Tumor cells
Volumeoramount Collected:	400uL of organic phase of metabolites extraction
Storage Conditions:	-80℃
Collection Vials:	1.5 mL Eppendorf tubes
Storage Vials:	1.5 mL Eppendorf tubes

Treatment:

Treatment ID:	TR004028
Treatment Summary:	TDCLs derived from NSCLC tumors of PGKP and KP animals were plated, and their culture medium was replaced after 24 hours. Following this initial period, metabolites were extracted every 24 hours, resulting in a kinetic analysis at three time points: 24, 48, and 72 hours of cell growth without further medium changes.

Sample Preparation:

Sampleprep ID:	SP004025
Sampleprep Summary:	TDCLs were plated in 6 multiwell plates (Corning) and incubated (37° C in 5% CO2) for 24h hours. After this period, the medium was replaced and cells were further incubated and extracted at the indicated time points (24, 48, and 72h). Three wells were used for the metabolite extraction, and the other three were harvested using trypsin 0.25% to measure the wet cell volume using a PCV-packed cell volume tube (TPP). One aliquot of the medium (15µL) of each well was collected and immediately introduced in extraction buffer 40:40:20 (Methanol: Acetonitrile: Water) with 0.05% formic acid allowed to rest on ice for 5 minutes. For the TDCLs the metabolites extraction was performed by washing the wells twice with cold PBS, and 400 µL of extraction buffer 40:40:20 with 0.05% formic acid was added to each well. The plate was allowed to rest on ice for 5 minutes, and then the cells and buffer were scraped. The samples, extraction from medium and cells, were placed in a 1.5 mL microtube with 22 µL of 15% NH4HCO3 vortexed and centrifuged for 10 min at 15,000g at 4° C. 380 µL of the supernatant were collected and stored in -80° C freezer until analysis by LC-MS.

Sampleprep ID:

SP004025

Sampleprep Summary:

TDCLs were plated in 6 multiwell plates (Corning) and incubated (37° C in 5% CO2) for 24h hours. After this period, the medium was replaced and cells were further incubated and extracted at the indicated time points (24, 48, and 72h). Three wells were used for the metabolite extraction, and the other three were harvested using trypsin 0.25% to measure the wet cell volume using a PCV-packed cell volume tube (TPP). One aliquot of the medium (15µL) of each well was collected and immediately introduced in extraction buffer 40:40:20 (Methanol: Acetonitrile: Water) with 0.05% formic acid allowed to rest on ice for 5 minutes. For the TDCLs the metabolites extraction was performed by washing the wells twice with cold PBS, and 400 µL of extraction buffer 40:40:20 with 0.05% formic acid was added to each well. The plate was allowed to rest on ice for 5 minutes, and then the cells and buffer were scraped. The samples, extraction from medium and cells, were placed in a 1.5 mL microtube with 22 µL of 15% NH4HCO3 vortexed and centrifuged for 10 min at 15,000g at 4° C. 380 µL of the supernatant were collected and stored in -80° C freezer until analysis by LC-MS.

Chromatography:

Chromatography ID:	CH004839
Chromatography Summary:	Tumor derived cell Lines (TDCLs) from NSCLC.
Methods Filename:	Chromatography_method.pdf
Instrument Name:	Thermo Vanquish
Column Name:	Waters XBridge BEH Amide (150 × 2.1mm, 2.5um)
Column Temperature:	25 °C
Flow Gradient:	0 min, 100% B; 3 min, 100% B; 3.2 min, 90% B; 6.2 min, 90% B; 6.5 min, 80% B; 10.5 min, 80% B; 10.7 min, 70% B; 13.5 min, 70% B; 13.7 min, 45% B; 16 min, 45% B; 16.5 min, 100% B; and 22 min, 100% B
Flow Rate:	300 μL/min
Solvent A:	95% water/5% acetonitrile; 20mM acetic acid; 40mM ammonium hydroxide (pH 9.4)
Solvent B:	20% water/80% acetonitrile; 20mM acetic acid, 40mM ammonium hydroxide (pH 9.4)
Chromatography Type:	HILIC

Analysis:

Analysis ID:	AN006379
Analysis Type:	MS
Chromatography ID:	CH004839
Num Factors:	6
Num Metabolites:	158
Units:	ion count

Analysis ID:	AN006380
Analysis Type:	MS
Chromatography ID:	CH004839
Num Factors:	6
Num Metabolites:	163
Units:	ion count