#METABOLOMICS WORKBENCH ariane_busso0903_20210426_103957 DATATRACK_ID:2602 STUDY_ID:ST001755 ANALYSIS_ID:AN002859 PROJECT_ID:PR001125
VERSION             	1
CREATED_ON             	April 29, 2021, 5:15 pm
#PROJECT
PR:PROJECT_SUMMARY               	Metabolomics analysis of cancer-derived extracellular vesicles.
PR:INSTITUTE                     	National Center for Research in Energy and Materials - CNPEM
PR:DEPARTMENT                    	Brazilian Biosciences National Laboratory - LNBio
PR:LABORATORY                    	Mass Spectrometry Laboratory
PR:FIRST_NAME                    	Ariane
PR:ADDRESS                       	R. Giuseppe Máximo Scolfaro, 10000, Bosque das Palmeiras, Campinas, SP,
PR:ADDRESS                       	13083-100, Brazil
PR:EMAIL                         	ariane.lopes@lnbio.cnpem.br
PR:PHONE                         	+55 19 3512-1276
PR:PROJECT_TITLE                 	A reductionist approach using primary and metastatic cell-derived extracellular
PR:PROJECT_TITLE                 	vesicles reveals hub proteins associated with oral cancer prognosis
PR:LAST_NAME                     	Busso Lopes
#STUDY
ST:STUDY_TITLE                   	A reductionist approach using primary and metastatic cell-derived extracellular
ST:STUDY_TITLE                   	vesicles reveals hub proteins associated with oral cancer prognosis
ST:STUDY_SUMMARY                 	Oral squamous cell carcinoma (OSCC) has high mortality rates that are largely
ST:STUDY_SUMMARY                 	associated with lymph node metastasis. However, the molecular mechanisms that
ST:STUDY_SUMMARY                 	drive OSCC metastasis are unknown. Extracellular vesicles (EVs) are
ST:STUDY_SUMMARY                 	membrane-bound particles that play a role in intercellular communication and
ST:STUDY_SUMMARY                 	impact cancer development and progression. Thus, profiling EVs would be of great
ST:STUDY_SUMMARY                 	significance to decipher the role of EV cargo in OSCC metastasis. For that
ST:STUDY_SUMMARY                 	purpose, we used a reductionist approach to map the proteomic, miRNA,
ST:STUDY_SUMMARY                 	metabolomic, and lipidomic profiles of extracellular vesicles (EVs) derived from
ST:STUDY_SUMMARY                 	human primary tumor (SCC-9) cells and matched lymph node metastases (LN1) cells.
ST:STUDY_SUMMARY                 	Distinct omics profiles were associated with the metastatic phenotype, including
ST:STUDY_SUMMARY                 	670 proteins, 217 miRNAs, 26 metabolites, and 64 lipids differentially abundant
ST:STUDY_SUMMARY                 	between LN1- and SCC-9-derived EVs. A multi-omics integration identified 11
ST:STUDY_SUMMARY                 	‘hub proteins’ significantly decreased at the metastatic site compared to
ST:STUDY_SUMMARY                 	primary tumor-derived EVs. We confirmed the validity of these findings with
ST:STUDY_SUMMARY                 	analysis of data from multiple public databases, and found that low abundance of
ST:STUDY_SUMMARY                 	seven hub proteins in metastatic EVs is correlated with reduced survival and
ST:STUDY_SUMMARY                 	tumor aggressiveness in cancer patients. In summary, this multi-omics approach
ST:STUDY_SUMMARY                 	identified proteins transported by EVs that are associated with metastasis, and
ST:STUDY_SUMMARY                 	which may potentially serve as prognostic markers in OSCC.
ST:INSTITUTE                     	National Center for Research in Energy and Materials
ST:DEPARTMENT                    	Brazilian Biosciences National Laboratory - LNBio
ST:LABORATORY                    	Mass Spectrometry Laboratory
ST:LAST_NAME                     	Busso Lopes
ST:FIRST_NAME                    	Ariane
ST:ADDRESS                       	R. Giuseppe Máximo Scolfaro, 10000
ST:EMAIL                         	ariane.lopes@lnbio.cnpem.br
ST:NUM_GROUPS                    	2
ST:TOTAL_SUBJECTS                	10
ST:PHONE                         	+55 19 3512-1276
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:GENOTYPE_STRAIN               	SCC-9 and LN1
SU:CELL_STRAIN_DETAILS           	Primary tumor (SCC-9) and metastatic (LN1) oral cancer cell lines
#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           	SCC-9-EV	SCC-9_1-EV intensity	Factor:Primary tumor EV	RAW_FILE_NAME=EV_SCC9_09fev2017.cdf
SUBJECT_SAMPLE_FACTORS           	SCC-9-EV	SCC-9_2-EV intensity	Factor:Primary tumor EV	RAW_FILE_NAME=EV_SCC9_24abril2017.cdf
SUBJECT_SAMPLE_FACTORS           	SCC-9-EV	SCC-9_3-EV intensity	Factor:Primary tumor EV	RAW_FILE_NAME=EV_SCC9_25abril2017.cdf
SUBJECT_SAMPLE_FACTORS           	SCC-9-EV	SCC-9_4-EV intensity	Factor:Primary tumor EV	RAW_FILE_NAME=SCC9_B.cdf
SUBJECT_SAMPLE_FACTORS           	SCC-9-EV	SCC-9_5-EV intensity	Factor:Primary tumor EV	RAW_FILE_NAME=SCC9_C.cdf
SUBJECT_SAMPLE_FACTORS           	LN1-EV	LN1_1-EV intensity	Factor:Metastatic EV	RAW_FILE_NAME=EV_LN1_02maio2017.cdf
SUBJECT_SAMPLE_FACTORS           	LN1-EV	LN1_2-EV intensity	Factor:Metastatic EV	RAW_FILE_NAME=EV_LN1_03maio2017.cdf
SUBJECT_SAMPLE_FACTORS           	LN1-EV	LN1_3-EV intensity	Factor:Metastatic EV	RAW_FILE_NAME=EV_LN1_07fev2017.cdf
SUBJECT_SAMPLE_FACTORS           	LN1-EV	LN1_4-EV intensity	Factor:Metastatic EV	RAW_FILE_NAME=LN1_A.cdf
SUBJECT_SAMPLE_FACTORS           	LN1-EV	LN1_5-EV intensity	Factor:Metastatic EV	RAW_FILE_NAME=LN1_B.cdf
#COLLECTION
CO:COLLECTION_SUMMARY            	EVs were isolated from SCC-9 and LN1 cell cultures through differential
CO:COLLECTION_SUMMARY            	centrifugation. Cells were cultured until 80% cell confluence in 150 mm diameter
CO:COLLECTION_SUMMARY            	plates, washed three times with phosphate buffered saline (PBS) and further
CO:COLLECTION_SUMMARY            	cultivated for 48 h in media without FBS, at 37°C and 5% CO2. After serum
CO:COLLECTION_SUMMARY            	deprivation treatment, the conditioned media (200 mL) was collected and
CO:COLLECTION_SUMMARY            	centrifuged at 200 x g for 5 min, 2,000 x g for 15 min, 3,500 x g for 30 min and
CO:COLLECTION_SUMMARY            	10,000 x g for 90 min. The cleared supernatant was further ultracentrifuged at
CO:COLLECTION_SUMMARY            	100,000 x g for 90 min at 4°C and vesicle-containing pellets were washed with
CO:COLLECTION_SUMMARY            	PBS by ultracentrifugation for 1.5 h at the same speed. The samples were stored
CO:COLLECTION_SUMMARY            	at -80°C until further use.
CO:SAMPLE_TYPE                   	Keratinocytes
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	EVs were not submitted to any treatment prior to metabolites extraction.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	SCC-9 and LN1-derived EVs (5x10e10 particles; 5 biological replicates for each
SP:SAMPLEPREP_SUMMARY            	group) were submitted to metabolite extraction using the MTBE method.
SP:SAMPLEPREP_SUMMARY            	Derivatization of metabolites was performed as outlined previously (Lisec et
SP:SAMPLEPREP_SUMMARY            	al., Nat Protoc. 2015;10(9):1457).
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	GC
CH:INSTRUMENT_NAME               	Agilent 7890A
CH:COLUMN_NAME                   	Agilent DB-35MS (30m x 0,32mm x 0,25um)
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Leco Pegasus HT TOF
MS:INSTRUMENT_TYPE               	GC-TOF
MS:MS_TYPE                       	EI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	GC-TOF-MS data were obtained using a PAL-Combi XT autosampler (PAL System,
MS:MS_COMMENTS                   	Switzerland, http://www.palsystem.com/), coupled to an Agilent 7890 A gas
MS:MS_COMMENTS                   	chromatograph - Leco Pegasus HT time-of-flight mass spectrometer (LECO, USA) in
MS:MS_COMMENTS                   	both split (1:15 and 1:50) and splitless modes (Weckwerth et al., 2004;
MS:MS_COMMENTS                   	101:7809–14). Chromatograms were exported from Leco ChromaTOF software
MS:MS_COMMENTS                   	(version 3.25) to R. Peak detection, retention time alignment, and library
MS:MS_COMMENTS                   	matching were obtained using the TargetSearch package from Bioconductor
MS:MS_COMMENTS                   	(Cuadros-Inostroza et al., BMC Bioinformatics. 2009;10:428). Metabolites were
MS:MS_COMMENTS                   	quantified by peak intensity of a selective mass, and metabolite intensities
MS:MS_COMMENTS                   	were normalized dividing by the sum of the total ion count followed by log2
MS:MS_COMMENTS                   	transformation.
MS:COLLISION_ENERGY              	70 eV
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	intensity log2
MS_METABOLITE_DATA_START
Samples	SCC-9_1-EV intensity	SCC-9_2-EV intensity	SCC-9_3-EV intensity	SCC-9_4-EV intensity	SCC-9_5-EV intensity	LN1_1-EV intensity	LN1_2-EV intensity	LN1_3-EV intensity	LN1_4-EV intensity	LN1_5-EV intensity
Factors	Factor:Primary tumor EV	Factor:Primary tumor EV	Factor:Primary tumor EV	Factor:Primary tumor EV	Factor:Primary tumor EV	Factor:Metastatic EV	Factor:Metastatic EV	Factor:Metastatic EV	Factor:Metastatic EV	Factor:Metastatic EV
Lactic Acid	16.0921	15.0396	15.2940	15.8178	15.2035	16.4507	16.1413	16.1772	16.4353	16.4956
Alanine	13.1668	12.8646	13.0186	12.8674	13.2408	14.4325	13.8098	14.2609	13.4742	13.8846
Pyruvate	NA	NA	NA	NA	NA	10.2393	9.6254	9.8553	9.6397	9.6110
Valine	13.0952	12.1752	13.5783	13.1598	13.0098	14.0988	13.7853	14.2176	13.9546	13.8121
Glycerol	14.0373	14.4563	13.2972	14.1792	NA	11.1201	13.1929	12.0184	13.0211	13.0238
L-Leucine	12.8235	12.0704	13.2021	12.9284	12.7326	13.7552	13.5457	13.9583	13.6585	13.5401
Isoleucine	12.4242	11.6299	12.7996	12.5180	12.3602	13.4868	13.3125	13.7442	13.4141	13.3334
Glycine	12.8895	11.8366	12.3384	13.4657	13.2691	13.0704	12.9783	13.0551	12.2988	12.3568
Phosphate	17.2896	17.3816	17.1890	17.0924	17.0970	14.9993	16.1555	15.7408	15.9901	16.0882
Urea	9.7215	NA	9.4539	11.7530	11.9517	8.1289	NA	8.5547	10.8239	10.6132
L-Serine	10.0675	11.3485	10.6641	10.9856	NA	11.5676	11.4755	11.6152	11.3652	11.2261
(-)-Riboflavin	13.7394	13.3399	13.4336	12.8848	13.1643	11.3187	NA	12.1027	11.3587	11.5578
L-Threonine	11.5923	10.9651	12.0999	11.5168	11.6979	13.2146	12.7762	13.1432	12.5101	12.4616
Uracil	NA	NA	NA	11.3522	10.8951	9.9931	NA	NA	9.7885	9.5520
Malic Acid	7.3770	7.0828	7.5357	NA	NA	6.7566	6.3742	6.7673	7.0322	6.4291
4-Aminobutyric Acid	8.8249	9.1759	8.9203	9.0028	NA	7.9740	7.4800	NA	7.2634	7.3775
L-Aspartic Acid	9.7459	10.2826	9.9403	NA	NA	10.0228	9.7130	10.3724	9.9191	9.8316
L-Methionine	10.1815	9.3633	NA	10.0725	9.4452	11.3230	11.1869	11.4892	11.1116	10.9769
Arginine	10.2629	10.0791	NA	10.8022	10.2666	12.7151	12.3776	12.5485	11.8576	11.5650
Pyroglutamic Acid	13.5743	13.0675	14.4204	14.4433	14.5286	14.6990	14.2551	14.9469	13.9998	13.7931
L-Glutamic Acid	NA	9.2882	NA	11.4346	11.6617	NA	10.5948	NA	12.9167	12.8924
Phenilalanine	10.7977	9.9155	11.3620	10.8972	10.6564	12.0021	11.6883	12.0355	11.9314	11.7805
D-Mannitol	11.2935	NA	13.2577	13.5748	13.8864	14.1459	14.0782	12.8636	14.0171	12.5683
Glucose	11.7863	10.6781	12.4153	11.9711	11.4171	13.6445	12.8252	13.3604	12.8735	12.7412
Citric Acid	9.3271	8.7663	9.6340	9.4327	9.2548	NA	8.5483	NA	8.0222	7.9275
Glutamine	9.9959	11.8413	8.6882	12.2040	11.4274	13.6464	NA	12.7043	13.6671	13.5215
L-Lysine	11.3068	11.0430	12.0281	12.0086	11.3451	13.4363	13.1488	13.3027	13.1493	12.9833
Myo-inositol	10.3035	9.2828	10.8221	10.3499	10.2312	11.4017	11.1261	11.3900	11.0508	11.0331
Tyrosine	12.1325	11.4047	12.8047	12.1618	11.9136	13.9590	13.4521	13.8175	13.3924	13.2678
Adenine	NA	NA	NA	9.6688	9.0265	8.4177	NA	NA	8.1710	7.7007
L-Histidine	NA	NA	NA	NA	NA	NA	8.7549	9.1321	9.1387	8.2306
Tryptophan	9.1659	8.8963	9.0523	8.8958	NA	9.8611	8.5923	9.2947	9.2565	9.1102
Sucrose	11.1732	11.0491	8.6898	10.0799	11.5495	7.5461	NA	8.1274	8.7090	8.4451
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
Lactic Acid
Alanine
Pyruvate
Valine
Glycerol
L-Leucine
Isoleucine
Glycine
Phosphate
Urea
L-Serine
(-)-Riboflavin
L-Threonine
Uracil
Malic Acid
4-Aminobutyric Acid
L-Aspartic Acid
L-Methionine
Arginine
Pyroglutamic Acid
L-Glutamic Acid
Phenilalanine
D-Mannitol
Glucose
Citric Acid
Glutamine
L-Lysine
Myo-inositol
Tyrosine
Adenine
L-Histidine
Tryptophan
Sucrose
METABOLITES_END
#END