TGFβ Drives Metabolic Perturbations during Epithelial Mesenchymal Transition in Pancreatic Cancer: TGFβ Induced EMT in PDAC

TGF beta; Pancreatic cancer; Tumor microenvironmentTGF beta; Cancer de pancreas; Microambiente tumoralTGF beta; Càncer de pàncrees; Microambient tumoralPancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy wherein a majority of patients present metastatic disease at diagnosis. Although the role of epithelial to mesenchymal transition (EMT), mediated by transforming growth factor beta (TGFβ), in imparting an aggressive phenotype to PDAC is well documented, the underlying biochemical pathway perturbations driving this behaviour have not been elucidated. We used high-resolution mass spectrometry (HRMS) based molecular phenotyping approach in order to delineate metabolic changes concomitant to TGFβ-induced EMT in pancreatic cancer cells. Strikingly, we observed robust changes in amino acid and energy metabolism that may contribute to tumor invasion and metastasis. Somewhat unexpectedly, TGFβ treatment resulted in an increase in intracellular levels of retinoic acid (RA) that in turn resulted in increased levels of extracellular matrix (ECM) proteins including fibronectin (FN) and collagen (COL1). These findings were further validated in plasma samples obtained from patients with resectable pancreatic cancer. Taken together, these observations provide novel insights into small molecule dysregulation that triggers a molecular cascade resulting in increased EMT-like changes in pancreatic cancer cells, a paradigm that can be potentially targeted for better clinical outcomes.This study was supported by American Cancer Society (IRG-92-152-17 award number AWD4470404), Georgetown Lombardi Comprehensive Cancer Center Support Grant Developmental Funds and Ruesch Foundation to K.U. and A.K.C

Enabling Metabolomics Based Biomarker Discovery Studies Using Molecular Phenotyping of Exosome-Like Vesicles

Identification of sensitive and specific biomarkers with clinical and translational utility will require smart experimental strategies that would augment expanding the breadth and depth of molecular measurements within the constraints of currently available technologies. Exosomes represent an information rich matrix to discern novel disease mechanisms that are thought to contribute to pathologies such as dementia and cancer. Although proteomics and transcriptomic studies have been reported using Exosomes-Like Vesicles (ELVs) from different sources, exosomal metabolome characterization and its modulation in health and disease remains to be elucidated. Here we describe methodologies for UPLC-ESI-MS based small molecule profiling of ELVs from human plasma and cell culture media. In this study, we present evidence that indeed ELVs carry a rich metabolome that could not only augment the discovery of low abundance biomarkers but may also help explain the molecular basis of disease progression. This approach could be easily translated to other studies seeking to develop predictive biomarkers that can subsequently be used with simplified targeted approaches.This work was supported by the Spanish Ministry of Health (RD12/0036/0035), the Spanish Ministry of Economy and Competitivy (PI14/02043), the AECC (Grupos Estables de Investigacion 2011 - AECC- GCB 110333 REVE), the Fundació La Marató TV3 (2/C/2013), the CIRIT Generalitat de Catalunya (2014 SGR 1330) and the European Commission, 7th Framework Programe, IRSES (PROTBIOFLUID –269285) – Belgium. The authors would like to acknowledge the Proteomics and Metabolomics Shared Resource partially supported by Cancer Center Support Grant NIH/NCI grant P30-CA051008

Enabling Metabolomics Based Biomarker Discovery Studies Using Molecular Phenotyping Of Exosome-like Vesicles

Identification of sensitive and specific biomarkers with clinical and translational utility will require smart experimental strategies that would augment expanding the breadth and depth of molecular measurements within the constraints of currently available technologies. Exosomes represent an information rich matrix to discern novel disease mechanisms that are thought to contribute to pathologies such as dementia and cancer. Although proteomics and transcriptomic studies have been reported using Exosomes-Like Vesicles (ELVs) from different sources, exosomal metabolome characterization and its modulation in health and disease remains to be elucidated. Here we describe methodologies for UPLC-ESI-MS based small molecule profiling of ELVs from human plasma and cell culture media. In this study, we present evidence that indeed ELVs carry a rich metabolome that could not only augment the discovery of low abundance biomarkers but may also help explain the molecular basis of disease progression. This approach could be easily translated to other studies seeking to develop predictive biomarkers that can subsequently be used with simplified targeted approaches

Metabolomic and Lipidomic Profiling Identifies The Role of the RNA Editing Pathway in Endometrial Carcinogenesis

Endometrial cancer (EC) remains the most common malignancy of the genital tract among women in developed countries. Although much research has been performed at genomic, transcriptomic and proteomic level, there is still a significant gap in the metabolomic studies of EC. In order to gain insights into altered metabolic pathways in the onset and progression of EC carcinogenesis, we used high resolution mass spectrometry to characterize the metabolomic and lipidomic profile of 39 human EC and 17 healthy endometrial tissue samples. Several pathways including lipids, Kynurenine pathway, endocannabinoids signaling pathway and the RNA editing pathway were found to be dysregulated in EC. The dysregulation of the RNA editing pathway was further investigated in an independent set of 183 human EC tissues and matched controls, using orthogonal approaches. We found that ADAR2 is overexpressed in EC and that the increase in expression positively correlates with the aggressiveness of the tumor. Furthermore, silencing of ADAR2 in three EC cell lines resulted in a decreased proliferation rate, increased apoptosis, and reduced migration capabilities in vitro. Taken together, our results suggest that ADAR2 functions as an oncogene in endometrial carcinogenesis and could be a potential target for improving EC treatment strategies

Exosome-like vesicles in uterine aspirates: a comparison of ultracentrifugation-based isolation protocols

Background: Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols for their isolation, characterization, and analysis. Methods: We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases. The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol. Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot. Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples. Results: All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed a higher concentration of vesicles with a mode of 135 +/- 5 nm, and immunoblot showed a higher expression of exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR. Conclusion: We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future comparative studies to foster the field of biomarker research in gynecology

Exosome-like vesicles in uterine aspirates : a comparison of ultracentrifugation-based isolation protocols

Altres ajuts: This work was supported by the "Fondo Europeo de Desarrollo Regional" FEDER (RTC-2014-3110-1), the AECC (Grupos Estables de Investigacion 2011 - AECC- GCB 110333 REVE), the Fundació La Marató TV3 (2/C/2013).Altres ajuts: MSCBS/RD12-0036-0035Uterine aspirates are used in the diagnostic process of endometrial disorders, yet further applications could emerge if its complex milieu was simplified. Exosome-like vesicles isolated from uterine aspirates could become an attractive source of biomarkers, but there is a need to standardize isolation protocols. The objective of the study was to determine whether exosome-like vesicles exist in the fluid fraction of uterine aspirates and to compare protocols for their isolation, characterization, and analysis. We collected uterine aspirates from 39 pre-menopausal women suffering from benign gynecological diseases. The fluid fraction of 27 of those aspirates were pooled and split into equal volumes to evaluate three differential centrifugation-based procedures: (1) a standard protocol, (2) a filtration protocol, and (3) a sucrose cushion protocol. Characterization of isolated vesicles was assessed by electron microscopy, nanoparticle tracking analysis and immunoblot. Specifically for RNA material, we evaluate the effect of sonication and RNase A treatment at different steps of the protocol. We finally confirmed the efficiency of the selected methods in non-pooled samples. All protocols were useful to isolate exosome-like vesicles. However, the Standard procedure was the best performing protocol to isolate exosome-like vesicles from uterine aspirates: nanoparticle tracking analysis revealed a higher concentration of vesicles with a mode of 135 ± 5 nm, and immunoblot showed a higher expression of exosome-related markers (CD9, CD63, and CD81) thus verifying an enrichment in this type of vesicles. RNA contained in exosome-like vesicles was successfully extracted with no sonication treatment and exogenous nucleic acids digestion with RNaseA, allowing the analysis of the specific inner cargo by Real-Time qPCR. We confirmed the existence of exosome-like vesicles in the fluid fraction of uterine aspirates. They were successfully isolated by differential centrifugation giving sufficient proteomic and transcriptomic material for further analyses. The Standard protocol was the best performing procedure since the other two tested protocols did not ameliorate neither yield nor purity of exosome-like vesicles. This study contributes to establishing the basis for future comparative studies to foster the field of biomarker research in gynecology. The online version of this article (doi:10.1186/s12967-016-0935-4) contains supplementary material, which is available to authorized users

Metabolomic and lipidomic profiling identifies the role of the RNA editing pathway in endometrial carcinogenesis

Endometrial cancer (EC) remains the most common malignancy of the genital tract among women in developed countries. Although much research has been performed at genomic, transcriptomic and proteomic level, there is still a significant gap in the metabolomic studies of EC. In order to gain insights into altered metabolic pathways in the onset and progression of EC carcinogenesis, we used high resolution mass spectrometry to characterize the metabolomic and lipidomic profile of 39 human EC and 17 healthy endometrial tissue samples. Several pathways including lipids, Kynurenine pathway, endocannabinoids signaling pathway and the RNA editing pathway were found to be dysregulated in EC. The dysregulation of the RNA editing pathway was further investigated in an independent set of 183 human EC tissues and matched controls, using orthogonal approaches. We found that ADAR2 is overexpressed in EC and that the increase in expression positively correlates with the aggressiveness of the tumor. Furthermore, silencing of ADAR2 in three EC cell lines resulted in a decreased proliferation rate, increased apoptosis, and reduced migration capabilities in vitro. Taken together, our results suggest that ADAR2 functions as an oncogene in endometrial carcinogenesis and could be a potential target for improving EC treatment strategies.This work was supported by the Spanish Ministry of Health (RD12/0036/0035), the Spanish Ministry of Economy and Competitivy (PI14/02043), the AECC (Grupos Estables de Investigacion 2011 - AECC- GCB 110333 REVE), the Fundació La Marató TV3 (2/C/2013), the CIRIT Generalitat de Catalunya (2014 SGR 1330) and the European Commission, 7th Framework Program, IRSES (PROTBIOFLUID –269285) – Belgium. Te Spanish Ministry of Economy and Competitiveness (IJCI-2015-25000) granted Dr. Colás and and the AGAUR Generalitat de Catalunya (2015FI_B00703) granted Tatiana Altadill. Te authors would like to acknowledge the Proteomics and Metabolomics Shared Resource partially supported by Cancer Center Support Grant NIH/NCI grant P30-CA051008. Te Institut de Salud Carlos III (FIS (PI13/01701)) also supported this project. Tissue samples were obtained with the support of “Xarxa Catalana de Bancs de Tumors” and “Plataforma de Biobancos” ISCIII (PT13/0010/0014)

Metabolomic study for the identification of diagnostic markers and the characterization of the dissemination process of endometrial cancer

El càncer d'endometri (CE) és la malaltia ginecològica més habitual dels països desenvolupats i la quarta causa de càncer en dones. Presenta una incidència que es troba en continu creixement en pacients joves. Donada aquesta problemàtica, en les darreres dècades diverses línies de recerca s'han focalitzat en entendre quins són els canvis moleculars i metabòlics associats al CE. El seu diagnòstic recau en l'avaluació d'una biòpsia endometrial, mètode considerat mínimament invasiu. A més a més, el maneig dels pacients es basa en l'avaluació de les característiques clínico-patològiques del tumor; aproximació que no és del tot acurada, ja que alguns casos recauen impredictiblement. Per tal de solucionar els reptes clínics existents, la meta principal d'aquesta tesi va ser la identificació d'un panell novell de marcadors diagnòstics pel CE així com indagar en les vies metabòliques alterades que es donen de manera paral·lela a l'establiment i el procés de disseminació del CE. Hem aconseguit aquesta meta mitjançant l'ús d'aproximacions basades en la metabolòmica per finalment poder acabar millorant la cura del pacient i reduir la taxa de mortalitat d'aquesta malaltia. Per aconseguir aquests objectius, hem dividit aquesta tesi en tres capítols, cada un d'ells dirigit a un objectiu específic: 1) Optimitzar els mètodes d'extracció per a l'anàlisi metabolòmic de les vesícules extracel·lulars (VEs) contingudes en biofluids mitjançant l'ús de l'espectrometria de masses associada a cromatografia líquida; 2) Identificació, verificació i validació de marcadors diagnòstics del CE a partir de biofluids humans (plasma, aspirats uterins i VEs aïllats d'ambdós biofluids); 3) Estudi metabolòmic en mostres de teixit de CE per tal de detectar les alteracions moleculars que es donen en la iniciació i en el transcurs de la malaltia. Com a descobriments importants de la tesi, descrivim aproximacions estandarditzades per a tècniques basades en espectrometria de masses per estudiar els perfils metabolòmics de les VEs que es podran traslladar a altres línies de recerca de cerca de biomarcadors i que podran tenir impacte en clínica translacional així com en la millora del seguiment dels pacients amb CE. També presentem evidències que el cargo enriquit contingut en les VEs ofereixen noves oportunitats per al descobriment de metabòlits poc abundants com a biomarcadors de malalties. Cal destacar que les dades exposades en aquest treball representen un avanç en la comprensió de les alteracions moleculars que es donen en en CE. Els nostres resultats així com els nostres models in vitro posen en evidència el rol oncogènic d' ADAR2 així com el rol que té la via de RNA editing en el CE. Així doncs, aquests estudis suposen un avanç en el camp de recerca del CE demostrant l'ús i la optimització de noves metodologies (metabolòmics en VEs) i guanyant idees cap a les pertorbacions bioquímiques específiques que poden tenir un paper clau en el procés carcinogen del CE.Endometrial cancer (EC) is the most common gynaecological malignancy in developed countries and the fourth most common cancer in women. It shows a continuous increasing incidence among younger patients. Concerning this problematic, many new research lines focused on the better understanding of the molecular and metabolic changes associated to EC have appeared the last decades. Its diagnosis relies on the evaluation of an endometrial biopsy, which is a minimally-invasive method. Moreover, the management of patients is based on the assessment of the clinic-pathological features of the tumor, which is not completely accurate since some cases still recur unpredictably. In order to overcome the existing clinical challenges, the major goal of this thesis work was to identify a novel panel of EC diagnostic markers and to gain insights into altered metabolic pathways that are concomitant with the establishment and dissemination process of EC. We achieved our goal by using metabolomic-based approaches to finally improve patient care and overcome the mortality rate of this malignancy. To reach these objectives, we divided the thesis work into three chapters each one of them addressing a specific objective: 1) Optimization of the extraction methods for the metabolomic analysis of extracellular vesicles (EVs) contained in biofluids using liquid chromatography mass spectrometry (LC-MS) techniques; 2) Identification, verification and validation of EC diagnostic markers from human biofluids (use of plasma, uterine aspirates, and EVs isolated from both biofluids); 3) Metabolomic profiling of EC tissues to elucidate the molecular alterations that take place in the initiation and progression of the disease. As main findings of the dissertation, we describe standardized approaches for MS based metabolomics profiling of extracellular vesicles that can be translated to other biomarker research lines and may have big impact in translational clinics and improving the outcome of EC patients. We also present evidence that the enriched cargo contained in EVs offers new opportunities for the discovery of low abundance metabolites as disease biomarkers. Importantly, we highlight the relevance of the use of proximal biofluids, specifically UA, and EVs in the biomarker research and we opened a new avenue for identification of more specific EC biomarkers. Moreover, the data presented in this dissertation depicts a significant advance in the understanding of the metabolic alterations that take place in EC tumorogenesis. Our results and in vitro models evidenced the oncogenic role of ADAR2 and thus the role of RNA editing pathway in EC. Taken together, these studies help further the field of EC research by demonstrated use and optimization of new methodologies (EVs metabolomics) and gaining insights into specific biochemical perturbations that may have a critical role in endometrial carcinogenesis

Metabolomic study for the identification of diagnostic markers and the characterization of the dissemination process of endometrial cancer

El càncer d'endometri (CE) és la malaltia ginecològica més habitual dels països desenvolupats i la quarta causa de càncer en dones. Presenta una incidència que es troba en continu creixement en pacients joves. Donada aquesta problemàtica, en les darreres dècades diverses línies de recerca s'han focalitzat en entendre quins són els canvis moleculars i metabòlics associats al CE. El seu diagnòstic recau en l'avaluació d'una biòpsia endometrial, mètode considerat mínimament invasiu. A més a més, el maneig dels pacients es basa en l'avaluació de les característiques clínico-patològiques del tumor; aproximació que no és del tot acurada, ja que alguns casos recauen impredictiblement. Per tal de solucionar els reptes clínics existents, la meta principal d'aquesta tesi va ser la identificació d'un panell novell de marcadors diagnòstics pel CE així com indagar en les vies metabòliques alterades que es donen de manera paral·lela a l'establiment i el procés de disseminació del CE. Hem aconseguit aquesta meta mitjançant l'ús d'aproximacions basades en la metabolòmica per finalment poder acabar millorant la cura del pacient i reduir la taxa de mortalitat d'aquesta malaltia. Per aconseguir aquests objectius, hem dividit aquesta tesi en tres capítols, cada un d'ells dirigit a un objectiu específic: 1) Optimitzar els mètodes d'extracció per a l'anàlisi metabolòmic de les vesícules extracel·lulars (VEs) contingudes en biofluids mitjançant l'ús de l'espectrometria de masses associada a cromatografia líquida; 2) Identificació, verificació i validació de marcadors diagnòstics del CE a partir de biofluids humans (plasma, aspirats uterins i VEs aïllats d'ambdós biofluids); 3) Estudi metabolòmic en mostres de teixit de CE per tal de detectar les alteracions moleculars que es donen en la iniciació i en el transcurs de la malaltia. Com a descobriments importants de la tesi, descrivim aproximacions estandarditzades per a tècniques basades en espectrometria de masses per estudiar els perfils metabolòmics de les VEs que es podran traslladar a altres línies de recerca de cerca de biomarcadors i que podran tenir impacte en clínica translacional així com en la millora del seguiment dels pacients amb CE. També presentem evidències que el cargo enriquit contingut en les VEs ofereixen noves oportunitats per al descobriment de metabòlits poc abundants com a biomarcadors de malalties. Cal destacar que les dades exposades en aquest treball representen un avanç en la comprensió de les alteracions moleculars que es donen en en CE. Els nostres resultats així com els nostres models in vitro posen en evidència el rol oncogènic d' ADAR2 així com el rol que té la via de RNA editing en el CE. Així doncs, aquests estudis suposen un avanç en el camp de recerca del CE demostrant l'ús i la optimització de noves metodologies (metabolòmics en VEs) i guanyant idees cap a les pertorbacions bioquímiques específiques que poden tenir un paper clau en el procés carcinogen del CE.Endometrial cancer (EC) is the most common gynaecological malignancy in developed countries and the fourth most common cancer in women. It shows a continuous increasing incidence among younger patients. Concerning this problematic, many new research lines focused on the better understanding of the molecular and metabolic changes associated to EC have appeared the last decades. Its diagnosis relies on the evaluation of an endometrial biopsy, which is a minimally-invasive method. Moreover, the management of patients is based on the assessment of the clinic-pathological features of the tumor, which is not completely accurate since some cases still recur unpredictably. In order to overcome the existing clinical challenges, the major goal of this thesis work was to identify a novel panel of EC diagnostic markers and to gain insights into altered metabolic pathways that are concomitant with the establishment and dissemination process of EC. We achieved our goal by using metabolomic-based approaches to finally improve patient care and overcome the mortality rate of this malignancy. To reach these objectives, we divided the thesis work into three chapters each one of them addressing a specific objective: 1) Optimization of the extraction methods for the metabolomic analysis of extracellular vesicles (EVs) contained in biofluids using liquid chromatography mass spectrometry (LC-MS) techniques; 2) Identification, verification and validation of EC diagnostic markers from human biofluids (use of plasma, uterine aspirates, and EVs isolated from both biofluids); 3) Metabolomic profiling of EC tissues to elucidate the molecular alterations that take place in the initiation and progression of the disease. As main findings of the dissertation, we describe standardized approaches for MS based metabolomics profiling of extracellular vesicles that can be translated to other biomarker research lines and may have big impact in translational clinics and improving the outcome of EC patients. We also present evidence that the enriched cargo contained in EVs offers new opportunities for the discovery of low abundance metabolites as disease biomarkers. Importantly, we highlight the relevance of the use of proximal biofluids, specifically UA, and EVs in the biomarker research and we opened a new avenue for identification of more specific EC biomarkers. Moreover, the data presented in this dissertation depicts a significant advance in the understanding of the metabolic alterations that take place in EC tumorogenesis. Our results and in vitro models evidenced the oncogenic role of ADAR2 and thus the role of RNA editing pathway in EC. Taken together, these studies help further the field of EC research by demonstrated use and optimization of new methodologies (EVs metabolomics) and gaining insights into specific biochemical perturbations that may have a critical role in endometrial carcinogenesis

Metabolomic study for the identification of diagnostic markers and the characterization of the dissemination process of endometrial cancer

El càncer d'endometri (CE) és la malaltia ginecològica més habitual dels països desenvolupats i la quarta causa de càncer en dones. Presenta una incidència que es troba en continu creixement en pacients joves. Donada aquesta problemàtica, en les darreres dècades diverses línies de recerca s'han focalitzat en entendre quins són els canvis moleculars i metabòlics associats al CE. El seu diagnòstic recau en l'avaluació d'una biòpsia endometrial, mètode considerat mínimament invasiu. A més a més, el maneig dels pacients es basa en l'avaluació de les característiques clínico-patològiques del tumor; aproximació que no és del tot acurada, ja que alguns casos recauen impredictiblement. Per tal de solucionar els reptes clínics existents, la meta principal d'aquesta tesi va ser la identificació d'un panell novell de marcadors diagnòstics pel CE així com indagar en les vies metabòliques alterades que es donen de manera paral·lela a l'establiment i el procés de disseminació del CE. Hem aconseguit aquesta meta mitjançant l'ús d'aproximacions basades en la metabolòmica per finalment poder acabar millorant la cura del pacient i reduir la taxa de mortalitat d'aquesta malaltia. Per aconseguir aquests objectius, hem dividit aquesta tesi en tres capítols, cada un d'ells dirigit a un objectiu específic: 1) Optimitzar els mètodes d'extracció per a l'anàlisi metabolòmic de les vesícules extracel·lulars (VEs) contingudes en biofluids mitjançant l'ús de l'espectrometria de masses associada a cromatografia líquida; 2) Identificació, verificació i validació de marcadors diagnòstics del CE a partir de biofluids humans (plasma, aspirats uterins i VEs aïllats d'ambdós biofluids); 3) Estudi metabolòmic en mostres de teixit de CE per tal de detectar les alteracions moleculars que es donen en la iniciació i en el transcurs de la malaltia. Com a descobriments importants de la tesi, descrivim aproximacions estandarditzades per a tècniques basades en espectrometria de masses per estudiar els perfils metabolòmics de les VEs que es podran traslladar a altres línies de recerca de cerca de biomarcadors i que podran tenir impacte en clínica translacional així com en la millora del seguiment dels pacients amb CE. També presentem evidències que el cargo enriquit contingut en les VEs ofereixen noves oportunitats per al descobriment de metabòlits poc abundants com a biomarcadors de malalties. Cal destacar que les dades exposades en aquest treball representen un avanç en la comprensió de les alteracions moleculars que es donen en en CE. Els nostres resultats així com els nostres models in vitro posen en evidència el rol oncogènic d' ADAR2 així com el rol que té la via de RNA editing en el CE. Així doncs, aquests estudis suposen un avanç en el camp de recerca del CE demostrant l'ús i la optimització de noves metodologies (metabolòmics en VEs) i guanyant idees cap a les pertorbacions bioquímiques específiques que poden tenir un paper clau en el procés carcinogen del CE.Endometrial cancer (EC) is the most common gynaecological malignancy in developed countries and the fourth most common cancer in women. It shows a continuous increasing incidence among younger patients. Concerning this problematic, many new research lines focused on the better understanding of the molecular and metabolic changes associated to EC have appeared the last decades. Its diagnosis relies on the evaluation of an endometrial biopsy, which is a minimally-invasive method. Moreover, the management of patients is based on the assessment of the clinic-pathological features of the tumor, which is not completely accurate since some cases still recur unpredictably. In order to overcome the existing clinical challenges, the major goal of this thesis work was to identify a novel panel of EC diagnostic markers and to gain insights into altered metabolic pathways that are concomitant with the establishment and dissemination process of EC. We achieved our goal by using metabolomic-based approaches to finally improve patient care and overcome the mortality rate of this malignancy. To reach these objectives, we divided the thesis work into three chapters each one of them addressing a specific objective: 1) Optimization of the extraction methods for the metabolomic analysis of extracellular vesicles (EVs) contained in biofluids using liquid chromatography mass spectrometry (LC-MS) techniques; 2) Identification, verification and validation of EC diagnostic markers from human biofluids (use of plasma, uterine aspirates, and EVs isolated from both biofluids); 3) Metabolomic profiling of EC tissues to elucidate the molecular alterations that take place in the initiation and progression of the disease. As main findings of the dissertation, we describe standardized approaches for MS based metabolomics profiling of extracellular vesicles that can be translated to other biomarker research lines and may have big impact in translational clinics and improving the outcome of EC patients. We also present evidence that the enriched cargo contained in EVs offers new opportunities for the discovery of low abundance metabolites as disease biomarkers. Importantly, we highlight the relevance of the use of proximal biofluids, specifically UA, and EVs in the biomarker research and we opened a new avenue for identification of more specific EC biomarkers. Moreover, the data presented in this dissertation depicts a significant advance in the understanding of the metabolic alterations that take place in EC tumorogenesis. Our results and in vitro models evidenced the oncogenic role of ADAR2 and thus the role of RNA editing pathway in EC. Taken together, these studies help further the field of EC research by demonstrated use and optimization of new methodologies (EVs metabolomics) and gaining insights into specific biochemical perturbations that may have a critical role in endometrial carcinogenesis