#METABOLOMICS WORKBENCH lgafeira_20241119_065522 DATATRACK_ID:5383 STUDY_ID:ST003585 ANALYSIS_ID:AN005888 PROJECT_ID:PR002215
VERSION             	1
CREATED_ON             	November 21, 2024, 6:33 pm
#PROJECT
PR:PROJECT_TITLE                 	Glutaminase 1 (GLS1)-based therapy targets glutamine reliance in glioblastoma
PR:PROJECT_TITLE                 	(GBM)
PR:PROJECT_SUMMARY               	Glioblastomas (GBMs) are the most lethal central nervous system (CNS) neoplasms,
PR:PROJECT_SUMMARY               	accounting for this the inexistence of specific therapies. Glutamine plays a key
PR:PROJECT_SUMMARY               	role in CNS physiology, and metabolic adjustments have been reported in GBM,
PR:PROJECT_SUMMARY               	which are considered glutamine-addicted. Therefore, our goal was to affect GBM
PR:PROJECT_SUMMARY               	cells by decreasing the systemic bioavailability of glutamine with a glutaminase
PR:PROJECT_SUMMARY               	(GLS1)-based therapy. The in vitro results showed that the GBM cell lines were
PR:PROJECT_SUMMARY               	indeed glutamine-dependent, triggering a metabolic remodeling upon the
PR:PROJECT_SUMMARY               	presence/absence of glutamine. GLS1 treatment increased cell death, decreased
PR:PROJECT_SUMMARY               	cell proliferation, and induced U-87MG invasion, due to glutamine scarcity. This
PR:PROJECT_SUMMARY               	intervention triggered a metabolic shift, evident in altered gene expression and
PR:PROJECT_SUMMARY               	metabolite profiles in cell extracts and conditioned media. Results from the in
PR:PROJECT_SUMMARY               	vivo orthotopic xenograft mouse model of GBM showed an increase in overall
PR:PROJECT_SUMMARY               	survival (OS) with the GLS1 treatment, along with lower cachexia. Moreover,
PR:PROJECT_SUMMARY               	metabolomics’ multivariate analysis of serum collected during tumor induction
PR:PROJECT_SUMMARY               	and the therapeutic course showed statistically different metabolic profiles of
PR:PROJECT_SUMMARY               	GLS1-treated mice midst treatment, which could help monitor the therapy
PR:PROJECT_SUMMARY               	response.
PR:INSTITUTE                     	ITQB NOVA
PR:LAST_NAME                     	Gonçalves
PR:FIRST_NAME                    	Luís
PR:ADDRESS                       	Avenida Republica, Oeiras, Not USCanada, 2780-157 Oeiras, Portugal
PR:EMAIL                         	lgafeira@itqb.unl.pt
PR:PHONE                         	214469464
PR:CONTRIBUTORS                  	Filipa Martins, Céline S. Gonçalves, Fernanda Silva2, David van der Kellen,
PR:CONTRIBUTORS                  	Eduarda P. Martins, Renata Arada, Saudade André, Lúcia Roque, Marta Pojo,
PR:CONTRIBUTORS                  	Luís G. Gonçalves, Bruno M. Costa, Jacinta Serpa
#STUDY
ST:STUDY_TITLE                   	Glutaminase 1 (GLS1)-based therapy targets glutamine reliance in glioblastoma
ST:STUDY_TITLE                   	(GBM)- cell line assays supernants
ST:STUDY_SUMMARY                 	Glioblastomas (GBMs) are the most lethal central nervous system (CNS) neoplasms,
ST:STUDY_SUMMARY                 	accounting for this the inexistence of specific therapies. Glutamine plays a key
ST:STUDY_SUMMARY                 	role in CNS physiology, and metabolic adjustments have been reported in GBM,
ST:STUDY_SUMMARY                 	which are considered glutamine-addicted. Therefore, our goal was to affect GBM
ST:STUDY_SUMMARY                 	cells by decreasing the systemic bioavailability of glutamine with a glutaminase
ST:STUDY_SUMMARY                 	(GLS1)-based therapy. In order to understand how Glutaminase treatment affects
ST:STUDY_SUMMARY                 	the metabolism of GBM cell lines (U251 and U-87MG), we exposed these cell lines
ST:STUDY_SUMMARY                 	to GLS1 for 24 h and the conditioned media (supernatants) and cell extracts
ST:STUDY_SUMMARY                 	(aqueous and organic phase) were further analyzed by 1H-NMR. The in vitro
ST:STUDY_SUMMARY                 	results showed that the GBM cell lines were indeed glutamine-dependent,
ST:STUDY_SUMMARY                 	triggering a metabolic remodeling upon the presence/absence of glutamine. GLS1
ST:STUDY_SUMMARY                 	treatment increased cell death, decreased cell proliferation, and induced U-87MG
ST:STUDY_SUMMARY                 	invasion, due to glutamine scarcity. This intervention triggered a metabolic
ST:STUDY_SUMMARY                 	shift, evident in altered gene expression and metabolite profiles in cell
ST:STUDY_SUMMARY                 	extracts and conditioned media. Results from the in vivo orthotopic xenograft
ST:STUDY_SUMMARY                 	mouse model of GBM showed an increase in overall survival (OS) with the GLS1
ST:STUDY_SUMMARY                 	treatment, along with lower cachexia. Moreover, metabolomics’ multivariate
ST:STUDY_SUMMARY                 	analysis of serum collected during tumor induction and the therapeutic course
ST:STUDY_SUMMARY                 	showed statistically different metabolic profiles of GLS1-treated mice midst
ST:STUDY_SUMMARY                 	treatment, which could help monitor the therapy response.
ST:INSTITUTE                     	ITQB NOVA
ST:LAST_NAME                     	Gonçalves
ST:FIRST_NAME                    	Luís
ST:ADDRESS                       	Avenida Republica, Oeiras, Not USCanada, 2780-157 Oeiras, Portugal
ST:EMAIL                         	lgafeira@itqb.unl.pt
ST:PHONE                         	214469464
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	C_U_ 1	Condition:Control | Sample source:U251 brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super1_210113
SUBJECT_SAMPLE_FACTORS           	-	C_U_ 2	Condition:Control | Sample source:U251 brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super2_210113
SUBJECT_SAMPLE_FACTORS           	-	C_U_ 3	Condition:Control | Sample source:U251 brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super201_210519
SUBJECT_SAMPLE_FACTORS           	-	C_U_ 4	Condition:Control | Sample source:U251 brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super202_210519a
SUBJECT_SAMPLE_FACTORS           	-	Gln_U_1	Condition:Glutamine | Sample source:U251 brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super5_210113
SUBJECT_SAMPLE_FACTORS           	-	Gln_U_2	Condition:Glutamine | Sample source:U251 brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super6_210113
SUBJECT_SAMPLE_FACTORS           	-	Gln_U_3	Condition:Glutamine | Sample source:U251 brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super237_210519
SUBJECT_SAMPLE_FACTORS           	-	Gln_U_4	Condition:Glutamine | Sample source:U251 brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super238_210519
SUBJECT_SAMPLE_FACTORS           	-	C_MG_1	Condition:Control | Sample source:U-87MG brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super9_210113
SUBJECT_SAMPLE_FACTORS           	-	C_MG_2	Condition:Control | Sample source:U-87MG brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super10_210113
SUBJECT_SAMPLE_FACTORS           	-	C_MG_3	Condition:Control | Sample source:U-87MG brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super215_210519
SUBJECT_SAMPLE_FACTORS           	-	C_MG_4	Condition:Control | Sample source:U-87MG brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super216_210519
SUBJECT_SAMPLE_FACTORS           	-	Gln_MG_1	Condition:Glutamine | Sample source:U-87MG brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super13_210113
SUBJECT_SAMPLE_FACTORS           	-	Gln_MG_2	Condition:Glutamine | Sample source:U-87MG brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super14_210113
SUBJECT_SAMPLE_FACTORS           	-	Gln_MG_3	Condition:Glutamine | Sample source:U-87MG brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super229_210519
SUBJECT_SAMPLE_FACTORS           	-	Gln_MG_4	Condition:Glutamine | Sample source:U-87MG brain cancer cells	RAW_FILE_NAME(Spectra)=FM_Super230_210519
#COLLECTION
CO:COLLECTION_SUMMARY            	Two commercial GBM cell lines, U-87 MG (HTB-14, American Type Culture Collection
CO:COLLECTION_SUMMARY            	– ATCC) and U251 (09063001, European Collection of Authenticated Cell Cultures
CO:COLLECTION_SUMMARY            	– ECACC) were cultured in Dulbecco’s modified Eagle medium F-12 (DMEM/F-12;
CO:COLLECTION_SUMMARY            	11330-032, Gibco, Life Technologies), supplemented with 10% fetal bovine serum
CO:COLLECTION_SUMMARY            	(FBS; P40-37500, PAN Biotech), 1% Antibiotic-Antimycotic (AA; P06-07300, PAN
CO:COLLECTION_SUMMARY            	Biotech) and 50 µg/mL gentamicin (15750-060, Gibco, Life Technologies). Cells
CO:COLLECTION_SUMMARY            	(2.6 x 10^7 cells/flask) were seeded in 175 cm2 culture flasks. After exposure
CO:COLLECTION_SUMMARY            	to the experimental conditions, supernatants were collected and stored at
CO:COLLECTION_SUMMARY            	-80ºC.
CO:COLLECTION_PROTOCOL_FILENAME  	Glutamine_Protocols.pdf
CO:SAMPLE_TYPE                   	Culture Media
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Cells were grown in presence of 6 mM L-glutamine (25030-024, Gibco) or in
TR:TREATMENT_SUMMARY             	absence
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	60 µL solution of 0.35 mM KPi (pH 7.4) and 0.16 mM in D2O was added to 540 µL
SP:SAMPLEPREP_SUMMARY            	of supernatants
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
#ANALYSIS
AN:ANALYSIS_PROTOCOL_FILE        	Glutamine_Protocols.pdf
#NMR
NM:INSTRUMENT_NAME               	Bruker Avance II+ 800 MHz
NM:INSTRUMENT_TYPE               	FT-NMR
NM:NMR_EXPERIMENT_TYPE           	1D-1H
NM:SPECTROMETER_FREQUENCY        	800
NM:NMR_PROBE                     	5 mm TCI cryoprobe
NM:NMR_SOLVENT                   	10 % D2O/ 90 % H20
NM:NMR_TUBE_SIZE                 	5 mm
NM:PULSE_SEQUENCE                	noesygppr1d
#NMR_METABOLITE_DATA
NMR_METABOLITE_DATA:UNITS	mM
NMR_METABOLITE_DATA_START
Samples	C_U_ 1	C_U_ 2	C_U_ 3	C_U_ 4	Gln_U_1	Gln_U_2	Gln_U_3	Gln_U_4	C_MG_1	C_MG_2	C_MG_3	C_MG_4	Gln_MG_1	Gln_MG_2	Gln_MG_3	Gln_MG_4
Factors	Condition:Control | Sample source:U251 brain cancer cells	Condition:Control | Sample source:U251 brain cancer cells	Condition:Control | Sample source:U251 brain cancer cells	Condition:Control | Sample source:U251 brain cancer cells	Condition:Glutamine | Sample source:U251 brain cancer cells	Condition:Glutamine | Sample source:U251 brain cancer cells	Condition:Glutamine | Sample source:U251 brain cancer cells	Condition:Glutamine | Sample source:U251 brain cancer cells	Condition:Control | Sample source:U-87MG brain cancer cells	Condition:Control | Sample source:U-87MG brain cancer cells	Condition:Control | Sample source:U-87MG brain cancer cells	Condition:Control | Sample source:U-87MG brain cancer cells	Condition:Glutamine | Sample source:U-87MG brain cancer cells	Condition:Glutamine | Sample source:U-87MG brain cancer cells	Condition:Glutamine | Sample source:U-87MG brain cancer cells	Condition:Glutamine | Sample source:U-87MG brain cancer cells
1-Methylnicotinamide	2.7	3.4	13	11.7	3.6	3.3	9.4	9.5								
2-Hydroxybutyrate	90.7	111.4	91.3	72	25.4	32.3	28.7	30.1	37	29.5	38.5	27.2	26.7	23.1	23.9	24.3
2-Oxoisocaproate	53.5	68.4	66	65.2	8.6	10.8	12.8		11.6	11.8	17.9	9.7	10	7.8	12.3	13.4
3-Hydroxyisobutyrate			55.2	49.5			18	51.4								
Acetate	86.1	42	78.7	35.8		60.2	63.2	30.1	121	170.4	152.3	203.5	162.3	99.2	147.2	157.1
Alanine	26.5	26.4	9.6	9.6	234.2	349.3	313.5	298.8	72.2	69.2	88.7	76.4	123.3	100.7	83.3	88.1
Arginine	250.9	180.8	240.9	285.6	244.9	209.3	294.6	195.2	479.7	516.3	362.7	408.7	455	507.2	480	623.9
Asparagine					152.4	312.6										
Aspartate					267.9	91.9	165.8	220.4	49	84.7	51.3	57.6	84.6	79.6	45	66.4
Choline	46.8	28.9	28.2	22.5	42	36.7	78.7	44	61	64.3	61.8	68.2	62.6	57.4	65.5	68.1
Citrate	10.6	31.8	36.5	29.4			50.9						17.2	14.9	22.5	17.1
Ethanol	104.5	132.2	230.2	185.3	72.7	140	176.7	205.2	199		213.7	410.6	217.9	170.9	346.5	351.8
Formate	197.9	227.9	579.9	406.6	195.4	189.9	316.4	303	126.2	90.2	125.2	80.7	92.9	76	53.6	55.6
Fumarate			2.9	2.3			4.9	5.3								
Glucose									121.8	273.8	968.3	337.7	123.5	151.6	322.1	299.6
Glutamate	75.8				495.2	314.7	423.4	419.3					229.6			118.9
Glutamine					1438.5	1965.3	1694.1	1664.3					3677.3	3244.7	3168.2	3212.2
Glycine	221.2	220.7	553	392.2	169.3	185.2	296	281.6	292	298.3	295.6	282.8	267.6	241.6	236.8	245.4
Histidine	81.8	92	26.5	60.1	44	82	56.6	56.1	35.9	80.3	61.8	104.7	29.2	49.4	37.9	36.2
Hypoxanthine	16.6	5.6	12.3	7.9	7.8	4.5	3.1	3.7	9.9	12.2	11.1	15.2	8.9	8	11.4	12.1
Isobutyrate	31.2	46.9	39.7	38.9	6.7	9.8	8.2	9.5	12.4	7.7	13.5	7.2	6.3	5.6	4.1	222.7
Isoleucine	211.5	86	146.6	125.7	216.7	212.9	212.2	295.9	309.2	351.8	291.5	346.8	332.3	280.4	327.6	280.7
Lactate	336.4	444.4	164.8	279.1	319.8	816.3	484.1	401.9	2223.9	2012.5	3005.1	1427	2051	2223	1234.3	1545
Leucine	204.8	130.3	131.5	124.2	246.9	219.7	210.8	205.2	304.3	342	298.8	339.2	329.4	301	337.4	353.3
Lysine	384.6	344	335.4	303	263.1	358.4	346.2	298.6	396.8	393.1	420.5	358.4	608.4	319.5	421.4	530.1
Methionine	76.7	64.2	71.2	72.4	62.2	56.2	57.9	63.7	92.7	95.8	75.6	79.4	96.5	87.7	95.5	96.2
Nicotinate	11.3	9.8	4.9	5.1	10	9.5	3.6	4.6	16.9	15.4	17.3	16.2	15.9	12.6	16.2	14.7
Ornithine	157.4	201.5	99.4	80.4	255.1	272.7	175.6	237.6	21.7	38.6	102.9	85	61.3	46.6		
Phenylalanine	157.1	206.6	160.9	162.1	134.3	177.9	130	151.2	189.7	176.3	174.7	154.4	133	141.1	156.7	177.1
Proline	118.8	139.9	169.7	132.4	235.8	205.9	220.3	243	142.1	146.5	179.4	142.3	149.4	175.2	230.7	134.7
Pyroglutamate	104.7	201.7	195.4	189.7	466.5	500.9	464.9	416.9	104.2	94.4	127	108.8	407.7	456.7	424.6	441.6
Pyruvate	8.8	12.9	3.7	15.4		12.5	38.5	25	179.4	243.8	296.6	411.7	162.3	158	387	416.8
Serine	107.4	90.3	87.1	83.5	250.9	144.7	202.9	181.9	148.9	178.8	193.8	169.2	170.8	157	193.4	188.3
Threonine	324.3	274.9	338.4	297.3	268.2	272.8	302.1	266.9	325.3	362.3	351.8	371.6	341.2	310.9	246.7	328.4
Tryptophan	31.1	22.8	22.6	17.5	25.8	23.3	22.2	24.6	28.5	27.3	11.6	28.4	27.9	27.8	12.2	26.7
Tyrosine	161.2	130.5	143.2	134.8	149.9	136.7	137.3	161.4	154.7	134	162	162	172.8	157.2	170.4	179.4
Uracil	19.8	10.3	28.8	18	11.8	6.8	19.6	20.7								
Valine	232.2	180.6	203.4	189.7	250.6	226.7	252.1	244.4	305.9	366	342.4	354.9	305	339	325.2	341
myo-Inositol	53.4	51.1	69.6	62	56.2	48.7	61.8	62.6	62.3	58.8	74.6	69.1	62.9	59	74.7	91
NMR_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	HMDB	PubChem	ChEBI	KEGG	METLIN
1-Methylnicotinamide	HMDB0000699	457	16797	C02918	5667
2-Hydroxybutyrate	HMDB0000008	440864	50613	C05984	3783
2-Oxoisocaproate	HMDB0000695	70	48430	C00233	5663
3-Hydroxyisobutyrate	HMDB0000023	440873	37373	C06001	483
Acetate	HMDB0000042	176	15366	C00033	3206
Alanine	HMDB0000161	5950	16977	C00041	NA
Arginine	HMDB0000517	6322	16467	C00062	5502
Asparagine	HMDB0000168	6267	17196	C00152	14
Aspartate	HMDB0000191	5960	17053	C00049	5206
Choline	HMDB0000097	305	15354	C00114	56
Citrate	HMDB0000094	311	30769	C00158	124
Ethanol	HMDB0000108	702	16236	C00469	3203
Formate	HMDB0304356	283	15740	NA	NA
Fumarate	HMDB0000134	444972	18012	C00122	3242
Glucose	HMDB0000122	5793	15903	C00031	133
Glutamate	HMDB0000148	33032	16015	C00025	5174
Glutamine	HMDB0000641	5961	18050	C00064	5614
Glycine	HMDB0000123	750	15428	C00037	20
Histidine	HMDB0000177	6274	15971	C00135	21
Hypoxanthine	HMDB0000157	790	17368	C00262	83
Isobutyrate	HMDB0001873	6590	16135	C02632	106
Isoleucine	HMDB0000172	6306	17191	C00407	5193
Lactate	HMDB0000190	107689	422	C00186	5205
Leucine	HMDB0000687	6106	15603	C00123	24
Lysine	HMDB0000182	5962	18019	C00047	5200
Methionine	HMDB0000696	6137	16643	C00073	5664
Nicotinate	HMDB0001488	938	15940	C00253	6272
Ornithine	HMDB0000214	6262	15729	C00077	27
Phenylalanine	HMDB0000159	6140	17295	C00079	28
Proline	HMDB0000162	145742	17203	C00148	29
Pyroglutamate	HMDB0000267	7405	18183	C01879	3251
Pyruvate	HMDB0000243	1060	32816	C00022	117
Serine	HMDB0000187	5951	17115	C00065	5203
Threonine	HMDB0000167	6288	16857	C00188	32
Tryptophan	HMDB0000929	6305	16828	C00078	5879
Tyrosine	HMDB0000158	6057	17895	C00082	34
Uracil	HMDB0000300	1174	17568	C00106	258
Valine	HMDB0000883	6287	16414	C00183	5842
myo-Inositol	HMDB0000211	NA	17268	C00137	5221
METABOLITES_END
#END