#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