{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST003639","ANALYSIS_ID":"AN005977","PROJECT_ID":"PR002250","VERSION":"1","CREATED_ON":"December 26, 2024, 7:47 am"},
"PROJECT":{"PROJECT_TITLE":"Multilevel Plasticity and Altered Glycosylation Drive Aggressiveness in Hypoxic and Glucose-Deprived Bladder Cancer Cells","PROJECT_TYPE":"Multilevel Plasticity and Altered Glycosylation Drive Aggressiveness in Hypoxic and Glucose-Deprived Bladder Cancer Cells","PROJECT_SUMMARY":"Bladder tumours with aggressive characteristics often present microenvironmental niches marked by low oxygen levels (hypoxia) and limited glucose supply due to inadequate vascularization. The molecular mechanisms facilitating cellular adaptation to these stimuli remain largely elusive. Employing a multi-omics approach, we discovered that hypoxic and glucose-deprived cancer cells enter a quiescent state supported by mitophagy, fatty acid β-oxidation, and amino acid catabolism, concurrently enhancing their invasive capabilities. Reoxygenation and glucose restoration efficiently reversed cell quiescence without affecting cellular viability, highlighting significant molecular plasticity in adapting to microenvironmental challenges. Furthermore, cancer cells exhibited substantial perturbation of protein O-glycosylation, leading to simplified glycophenotypes with shorter glycosidic chains. Exploiting glycoengineered cell models, we established that immature glycosylation contributes to reduced cell proliferation and increased invasion. Our findings collectively indicate that hypoxia and glucose deprivation trigger cancer aggressiveness, reflecting an adaptive escape mechanism underpinned by altered metabolism and protein glycosylation, providing grounds for clinical intervention.","INSTITUTE":"Portuguese Oncology Institute of Porto (IPO-Porto)","DEPARTMENT":"IPO Porto Research Center","LABORATORY":"Lab 2","LAST_NAME":"Peixoto","FIRST_NAME":"Andreia","ADDRESS":"Rua Dr. António Bernardino de Almeida","EMAIL":"andreia.peixoto@ipoporto.min-saude.pt","PHONE":"225084000","FUNDING_SOURCE":"The authors acknowledge FCT/MCTES funding within the projects RESOLVE (PTDC/MED-OUT/2512/2021) and REVERENT (2022.03621.PTDC; DOI 10.54499/2022.03621.PTDC) and funding for the IPO research center (PEst-OE/SAU/UI0776/201, CI-IPOP-29-2016-2022, CI-IPOP-58-2016-2022), for CQUM (UID/QUI/00686/2020), and the LAQV research unit (UIDB/50006/2020 | UIDP/50006/2020).","CONTRIBUTORS":"Andreia Peixoto, Dylan Ferreira, Andreia Miranda, Marta Relvas-Santos, Rui Freitas, Tim S. Veth, Andreia Brandão, Eduardo Ferreira, Paula Paulo, Marta Cardoso, Cristiana Gaiteiro, Sofia Cotton, Janine Soares, Luís Lima, Filipe Teixeira, Rita Ferreira, Carlos Palmeira, Albert J. R. Heck, Maria José Oliveira, André M. N. Silva, Lúcio Lara Santos, José Alexandre Ferreira"},
"STUDY":{"STUDY_TITLE":"Multilevel Plasticity and Altered Glycosylation Drive Aggressiveness in Hypoxic and Glucose-Deprived Bladder Cancer Cells","STUDY_TYPE":"MS quantitative analysis","STUDY_SUMMARY":"Bladder tumours with aggressive characteristics often present microenvironmental niches marked by low oxygen levels (hypoxia) and limited glucose supply due to inadequate vascularization. The molecular mechanisms facilitating cellular adaptation to these stimuli remain largely elusive. Employing a multi-omics approach, we discovered that hypoxic and glucose-deprived cancer cells enter a quiescent state supported by mitophagy, fatty acid β-oxidation, and amino acid catabolism, concurrently enhancing their invasive capabilities. Reoxygenation and glucose restoration efficiently reversed cell quiescence without affecting cellular viability, highlighting significant molecular plasticity in adapting to microenvironmental challenges. Furthermore, cancer cells exhibited substantial perturbation of protein O-glycosylation, leading to simplified glycophenotypes with shorter glycosidic chains. Exploiting glycoengineered cell models, we established that immature glycosylation contributes to reduced cell proliferation and increased invasion. Our findings collectively indicate that hypoxia and glucose deprivation trigger cancer aggressiveness, reflecting an adaptive escape mechanism underpinned by altered metabolism and protein glycosylation, providing grounds for clinical intervention.","INSTITUTE":"Portuguese Oncology Institute of Porto (IPO-Porto)","DEPARTMENT":"IPO Porto Research Center","LABORATORY":"Lab 2","LAST_NAME":"Peixoto","FIRST_NAME":"Andreia","ADDRESS":"Rua Dr. António Bernardino de Almeida","EMAIL":"andreia.peixoto@ipoporto.min-saude.pt","PHONE":"225084000 (ext:5111)"},
"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"5637_N1",
"Factors":{"Sample source":"5637 cells","Treatment":"Normoxia"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"5637N1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"5637_N2",
"Factors":{"Sample source":"5637 cells","Treatment":"Normoxia"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"5637N2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"5637_N3",
"Factors":{"Sample source":"5637 cells","Treatment":"Normoxia"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"5637N3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"5637_H1",
"Factors":{"Sample source":"5637 cells","Treatment":"Hypoxia plus glucose restriction"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"5637H1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"5637_H2",
"Factors":{"Sample source":"5637 cells","Treatment":"Hypoxia plus glucose restriction"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"5637H2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"5637_H3",
"Factors":{"Sample source":"5637 cells","Treatment":"Hypoxia plus glucose restriction"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"5637H3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"T24_N1",
"Factors":{"Sample source":"T24 cells","Treatment":"Normoxia"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"T24N1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"T24_N2",
"Factors":{"Sample source":"T24 cells","Treatment":"Normoxia"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"T24N2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"T24_N3",
"Factors":{"Sample source":"T24 cells","Treatment":"Normoxia"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"T24N3.raw"}
},
{
"Subject ID":"-",
"Sample ID":"T24_H1",
"Factors":{"Sample source":"T24 cells","Treatment":"Hypoxia plus glucose restriction"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"T24H1.raw"}
},
{
"Subject ID":"-",
"Sample ID":"T24_H2",
"Factors":{"Sample source":"T24 cells","Treatment":"Hypoxia plus glucose restriction"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"5637H2.raw"}
},
{
"Subject ID":"-",
"Sample ID":"T24_H3",
"Factors":{"Sample source":"T24 cells","Treatment":"Hypoxia plus glucose restriction"},
"Additional sample data":{"RAW_FILE_NAME(Raw file name)":"5637H3.raw"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Human BLCA cell lines were purchased from American Type Culture Collection (ATCC). Cells were maintained with complete RPMI 1640 GlutaMAX™ medium (Gibco) supplemented with 10% FBS (Gibco, 10500-064). Cells were kept at 37 °C in a 5% CO2 humidified atmosphere (Normoxia). Cells were also grown for 24h under hypoxia and nutrient deprivation (Hypoxia -Glc) at 37 °C in a 5% CO2, 99.9% N2 and 0.1% O2 atmosphere, using a BINDER C-150 incubator (BINDER GmbH), and complete RPMI 1640 without glucose (Gibco). For re-oxygenation experiments, cells under oxygen and glucose deprivation were restored to standard culture conditions 24 h prior to analysis. Cells were detached with Trypsin-EDTA (Gibco) and dispersed in 80% methanol (Merck, 34860-1L-R), sonicated for 30 min at 4 °C and kept at -20 °C for 1 h. Samples were then centrifuged, and the supernatant was analysed by UHPLC-ESI-MS/MS in positive and negative mode.","SAMPLE_TYPE":"Cultured cells","STORAGE_CONDITIONS":"-20℃"},
"TREATMENT":{"TREATMENT_SUMMARY":"Human BLCA cell lines 5637 (white male patient, HTB-9™) and T24 (white female patient, HTB-4™) were were maintained with complete RPMI 1640 GlutaMAX™ medium (Gibco) supplemented with 10% FBS (Gibco, 10500-064). Cells were kept at 37 °C in a 5% CO2 humidified atmosphere (Normoxia). Cells were also grown for 24h under hypoxia and nutrient deprivation (Hypoxia -Glc) at 37 °C in a 5% CO2, 99.9% N2 and 0.1% O2 atmosphere, using a BINDER C-150 incubator (BINDER GmbH), and complete RPMI 1640 (Gibco)."},
"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Cells were dispersed in 80% methanol (Merck, 34860-1L-R), sonicated for 30 min at 4 °C and kept at -20 °C for 1 h. Samples were then centrifuged, and the supernatant was analysed by UHPLC-ESI-MS/MS in positive and negative mode.","PROCESSING_STORAGE_CONDITIONS":"On ice","EXTRACT_STORAGE":"-20℃"},
"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000","COLUMN_NAME":"Waters Acquity UPLC HSS T3 (150 x 2.1mm,1.8um)","SOLVENT_A":"100% water; 0.1% formic acid","SOLVENT_B":"100% acetonitrile","FLOW_GRADIENT":"0-1.5 min, 95-70% A; 1.5-9.5 min, 70-5% A; 9.5-14.5 min, 5% A; 14.5-14.6 min, 5-95% A; 14.6-18.0 min, 95% A","FLOW_RATE":"0.3 mL/min","COLUMN_TEMPERATURE":"40 °C","METHODS_FILENAME":"Chromatography Methods.txt","CHROMATOGRAPHY_COMMENTS":"column Acquity UPLC HSS T3; 100 Å, 1.8 μm, 2.1 mm × 150 mm"},
"ANALYSIS":{"ANALYSIS_TYPE":"MS"},
"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"Metabolites were identified by retention time and corresponding MS/MS spectra. For metabolomics data pre-processing and analysis, raw data matrices were blank subtracted (a mean blank value was calculated per metabolite) and normalised to the number of cells for each condition. The resulting matrices were then imported to Metaboanalyst 4.0 and log-transformed to reduce heteroscedasticity and Pareto-scaled to adjust for differences in fold changes between metabolites."},
"MS_METABOLITE_DATA":{
"Units":"AUC",
"Data":[{"Metabolite":"Oxalic acid","5637_H1":"7.91936252272529","5637_H2":"8.3570877082561","5637_H3":"7.94676440754994","5637_N1":"9.38223235420137","5637_N2":"6.35887318296166","5637_N3":"8.15442835632007","T24_H1":"0.381138434657924","T24_H2":"0.286382200177148","T24_H3":"7.14777872412389","T24_N1":"8.98455483394742","T24_N2":"4.88377276162899","T24_N3":"6.17340671834574","":""},{"Metabolite":"L-Lactic acid","5637_H1":"1785.90283865064","5637_H2":"1740.98866915753","5637_H3":"1091.11886395125","5637_N1":"5493.87567051567","5637_N2":"1203.12472346314","5637_N3":"7146.6805086066","T24_H1":"2638.96344123422","T24_H2":"891.277083330252","T24_H3":"3309.84877033688","T24_N1":"3817.45916849547","T24_N2":"4355.20753213383","T24_N3":"4667.00924744152","":""},{"Metabolite":"Sulfate","5637_H1":"2001.17332610813","5637_H2":"2463.75148280613","5637_H3":"2984.2406937313","5637_N1":"2361.4816272967","5637_N2":"1842.12032033938","5637_N3":"1971.94935866374","T24_H1":"2795.20818650307","T24_H2":"199478.567385672","T24_H3":"2641.14599515866","T24_N1":"1520.65275470877","T24_N2":"1767.74152400163","T24_N3":"1755.10442569559","":""},{"Metabolite":"Phosphate","5637_H1":"19348.8801370213","5637_H2":"16547.7829853434","5637_H3":"13238.4944491106","5637_N1":"22518.6355666463","5637_N2":"21546.1079106735","5637_N3":"21729.1676308939","T24_H1":"9599.19049706771","T24_H2":"1941.17316462508","T24_H3":"17411.1427442559","T24_N1":"22029.6932676014","T24_N2":"21243.7037976296","T24_N3":"20111.6018987068","":""},{"Metabolite":"Valeric acid","5637_H1":"6.88761326380408","5637_H2":"8.70905585246088","5637_H3":"14.2168180844795","5637_N1":"27.042822659478","5637_N2":"7.68966213211453","5637_N3":"5.93649204335091","T24_H1":"2.60868010453371","T24_H2":"3.85881263707814","T24_H3":"16.471235153598","T24_N1":"50.5447063643972","T24_N2":"30.7388770443288","T24_N3":"58.6325062487876","":""},{"Metabolite":"Piperidic acid","5637_H1":"330.268327023066","5637_H2":"415.952327000657","5637_H3":"245.07273843979","5637_N1":"86.677649703054","5637_N2":"73.4229770816222","5637_N3":"60.3975126993004","T24_H1":"251.223074993506","T24_H2":"20.1472162264088","T24_H3":"703.342128863436","T24_N1":"107.95996776339","T24_N2":"115.456738575777","T24_N3":"152.154391102231","":""},{"Metabolite":"Uracil","5637_H1":"118.621827289966","5637_H2":"78.5054763153336","5637_H3":"66.5958282979539","5637_N1":"478.73705512091","5637_N2":"569.000458061474","5637_N3":"421.679881147704","T24_H1":"22.8035777270368","T24_H2":"4.92546976223166","T24_H3":"74.3530998574672","T24_N1":"187.477846047035","T24_N2":"205.696693420523","T24_N3":"107.547934778186","":""},{"Metabolite":"Cadmium","5637_H1":"213.985778779739","5637_H2":"225.071639710269","5637_H3":"102.330606065021","5637_N1":"171.279590108434","5637_N2":"414.134183114475","5637_N3":"387.485980798319","T24_H1":"259.990802008805","T24_H2":"25.707859038722","T24_H3":"327.815131231334","T24_N1":"377.188121159672","T24_N2":"403.309187446463","T24_N3":"405.822721137824","":""},{"Metabolite":"Thiosulfate","5637_H1":"347.101417877097","5637_H2":"392.394297734059","5637_H3":"458.78911007614","5637_N1":"241.306025819663","5637_N2":"450.247913704271","5637_N3":"443.494725885667","T24_H1":"337.842988375556","T24_H2":"437.629913896689","T24_H3":"409.547029449811","T24_N1":"516.016276219444","T24_N2":"304.475268398734","T24_N3":"283.585237464561","":""},{"Metabolite":"Trifluoroacetic acid","5637_H1":"7994.59827957979","5637_H2":"9248.78174438152","5637_H3":"8401.76829155183","5637_N1":"7187.43420511254","5637_N2":"6469.81164033898","5637_N3":"7275.05445175625","T24_H1":"7800.10792379227","T24_H2":"2544.08316566198","T24_H3":"7638.20821842281","T24_N1":"7164.13437840512","T24_N2":"6367.86141679093","T24_N3":"6220.9108796267","":""},{"Metabolite":"L-Proline","5637_H1":"31.9015095142475","5637_H2":"25.5110268212953","5637_H3":"12.8914136046961","5637_N1":"51.7218418026478","5637_N2":"61.0940069365276","5637_N3":"61.1458635681062","T24_H1":"13.1356862385661","T24_H2":"11.4471821734507","T24_H3":"36.2562927841874","T24_N1":"48.1932540133295","T24_N2":"52.8138948439386","T24_N3":"52.9760861343026","":""},{"Metabolite":"Fumaric acid","5637_H1":"111.538485866178","5637_H2":"71.2530580754563","5637_H3":"81.949557323279","5637_N1":"445.879463463394","5637_N2":"231.167347845172","5637_N3":"181.672318125562","T24_H1":"84.2989510722595","T24_H2":"20.9611507762166","T24_H3":"243.484550534885","T24_N1":"167.691362960865","T24_N2":"260.523230090218","T24_N3":"350.761101428378","":""},{"Metabolite":"Indole","5637_H1":"67.0271747849637","5637_H2":"87.6479386668347","5637_H3":"78.154383426027","5637_N1":"70.9751653737971","5637_N2":"55.4093401427262","5637_N3":"52.5018471867265","T24_H1":"64.8826777380111","T24_H2":"29.6419748858837","T24_H3":"120.203497358907","T24_N1":"70.6273566462076","T24_N2":"55.7308261980702","T24_N3":"64.8922809320685","":""},{"Metabolite":"Methylmalonic acid","5637_H1":"367.284357441757","5637_H2":"289.256350534712","5637_H3":"236.35875590164","5637_N1":"752.615109792191","5637_N2":"808.780339171418","5637_N3":"834.662165427316","T24_H1":"472.058080989124","T24_H2":"230.107754382463","T24_H3":"522.91652264587","T24_N1":"1193.07776293651","T24_N2":"826.18698876513","T24_N3":"1029.47703241928","":""},{"Metabolite":"2-Hydroxyvaleric acid","5637_H1":"2.82516642335184","5637_H2":"15.3389773170531","5637_H3":"20.6518142341038","5637_N1":"11.2438275878057","5637_N2":"12.087076056702","5637_N3":"4.56455507298278","T24_H1":"13.4428825324685","T24_H2":"1.61257144072584","T24_H3":"5.78960040937593","T24_N1":"47.1662076379728","T24_N2":"42.8245194814388","T24_N3":"98.1260165369715","":""},{"Metabolite":"Bovinocidin","5637_H1":"9818.760429376","5637_H2":"11143.0959888895","5637_H3":"20297.1183653171","5637_N1":"9660.51936049334","5637_N2":"8687.38841307668","5637_N3":"9988.05421404027","T24_H1":"8828.30001371075","T24_H2":"6155.97059468717","T24_H3":"9367.58121904864","T24_N1":"10277.7143102434","T24_N2":"8223.58415835063","T24_N3":"10357.0234783123","":""},{"Metabolite":"L-Threonine","5637_H1":"129.545013355263","5637_H2":"134.245144538424","5637_H3":"88.6836549045776","5637_N1":"165.602562992562","5637_N2":"81.468439867596","5637_N3":"111.740672506524","T24_H1":"64.6087671816001","T24_H2":"14.1975789024298","T24_H3":"69.9314200398449","T24_N1":"163.31848063148","T24_N2":"126.424687790497","T24_N3":"145.430943812044","":""},{"Metabolite":"Benzoic acid","5637_H1":"19.1687654210739","5637_H2":"25.6810242553175","5637_H3":"73.4353382082044","5637_N1":"6.35905378303585","5637_N2":"12.9500638183028","5637_N3":"17.2224588392313","T24_H1":"106.102786508087","T24_H2":"62.7874327232071","T24_H3":"24.6689738097353","T24_N1":"27.4296161949507","T24_N2":"6.65686457439913","T24_N3":"24.559592509568","":""},{"Metabolite":"Taurine","5637_H1":"1584.97793978941","5637_H2":"1943.10707409916","5637_H3":"1038.15874772894","5637_N1":"3123.52855102404","5637_N2":"2482.63805725029","5637_N3":"2120.21370592446","T24_H1":"96.7367172990519","T24_H2":"14.0879512821603","T24_H3":"349.51872314767","T24_N1":"1708.41779533199","T24_N2":"1516.49485347736","T24_N3":"1910.54041465166","":""},{"Metabolite":"2-Hydroxyethanesulfonate","5637_H1":"8.64685557517935","5637_H2":"10.2923990420393","5637_H3":"9.96142221637597","5637_N1":"8.31773139776231","5637_N2":"6.91780616599556","5637_N3":"8.39344590845706","T24_H1":"8.08824318662219","T24_H2":"1157.66634862983","T24_H3":"8.50032601936951","T24_N1":"7.94049672223681","T24_N2":"6.81884955707924","T24_N3":"9.01450484059101","":""},{"Metabolite":"Ethylphosphate","5637_H1":"41.7211122710869","5637_H2":"35.7449131191535","5637_H3":"42.0703449043214","5637_N1":"135.184889635537","5637_N2":"59.4443787952502","5637_N3":"101.464614125295","T24_H1":"27.499441186629","T24_H2":"6.18728496740747","T24_H3":"47.4272680914272","T24_N1":"119.115966793769","T24_N2":"97.9282913545731","T24_N3":"201.202594183725","":""},{"Metabolite":"Thymine","5637_H1":"3.01326665042765","5637_H2":"4.88117680570439","5637_H3":"1.82673736080862","5637_N1":"10.8477884883953","5637_N2":"16.3015784402442","5637_N3":"12.3793666626171","T24_H1":"4.02942917573279","T24_H2":"2.12919764848201","T24_H3":"7.65728046131395","T24_N1":"9.32116846511273","T24_N2":"20.8196033114131","T24_N3":"7.78372387427591","":""},{"Metabolite":"Methylselenic 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}
}