Exosomes in uterine aspirates: characterization and identification of diagnostic biomarkers in endometrial cancer /
- Autores: Irene Campoy Moncayo
- Directores de la Tesis: Jaume Reventós Puigjaner (dir. tes.), Eva Colas Ortega (dir. tes.), Antonio Gil Moreno (dir. tes.), Immaculada Ponsa Arjona (tut. tes.)
- Lectura: En la Universitat Autònoma de Barcelona ( España ) en 2017
- Idioma: español
- Tribunal Calificador de la Tesis: Juan Manuel Falcón Pérez (presid.), María Asunción Pérez Benavente (secret.), Gisela María Hirschfeld (voc.)
- Programa de doctorado: Programa Oficial de Doctorado en Biología Celular
- Materias:
- Enlaces
- Tesis en acceso abierto en: DDD
- Resumen
Endometrial cancer (EC) is the most common cancer of the female genital tract and the fourth most frequent cancer in women in the United States. Discouraging, EC deaths are increasing rapidly and rates grew about a 2% per year from 2010 to 2014. Even though early-stage diagnosed EC restricted to the uterus represent 67% of the cases with a 5-year survival rate of 95%, it still remains around a 30% of patients diagnosed at advanced stages of the disease, when the 5-year survival rate decreases dramatically to 69% and 17% in case of regional and distant spreading, respectively. EC is divided into the more common estrogen dependent type 1 and the less common but more aggressive estrogen independent type 2. There is an urgent need to develop non-invasive tests that can provide early detection of EC and that can discriminate EC subtypes.
The current diagnostic process is laborious and includes a pelvic examination and a transvaginal ultrasonography followed by a confirmatory histopathological evaluation of an endometrial biopsy, which can either be obtained by aspiration or by hysteroscopy. To improve EC diagnosis, the use of uterine aspirates (UAs) meet some features that makes this biofluid a promising source of biomarkers for screening, diagnosis and monitoring of pathologies related to the female genital tract: (i) sampling of UAs is minimally invasive and it is feasible to retrieve between 50-1000 µl of sample from a patient, which is sufficient to extract material for molecular studies; (ii) UAs are in direct contact with the uterine cavity, representing faithfully its complex environment. UA’s composition derives from secretions and cells flaking from a variety of surrounding tissues, from the luminal epithelium and glands, from proteins selectively transudated from blood, and likely contributors from tubal fluid; and (iii) UAs exhibit molecular alterations present in EC and are useful to study the EC tumor heterogeneity. UAs highly express abundant proteins coming from plasma; and those mask the detection and analysis of less abundant proteins, which are generally the most promising candidates for biomarkers discovery. To solve this dynamic-range problem, and to expand research in the field of biomarkers discovery for EC, exosome-like vesicles (ELVs), nanosized-membranous vesicles released by multivesicular bodies fusing with the cell membrane, arise as a promising source of EC biomarkers. This study focuses on the identification of protein markers in ELVs isolated from uterine aspirates.
Protein extracts from purified ELVs were obtained following ultracentrifugation of UAs from age-matched groups of control, type1 and type2 EC patients (10 patients/group). The quality of isolated ELVs was monitored by Nanoparticle Tracking Analysis, and immunoblots. To profile protein abundance across different groups, we develop a super-SILAC approach where ELV proteins from 3 different EC cell lines grown in heavy Lys and Arg amino acids were combined with ELV protein extracts of each patient. Proteins were separated by SDS-PAGE and gel-isolated bands were digested with trypsin and analyzed by Mass Spectrometry. We generated a list of 54 protein candidates that was further validated by LC-SRM in an independent cohort of 107 patients including 3 age-matched groups: type 1 EC (n=45) EC, type 2 EC (n=21) and healthy individuals (n=41).
Our targeted mass spectrometry approach confirmed that ELVs from UAs contain proteins that can discriminate between cancer and healthy patients. More importantly, a 2-protein signature improves this capacity to discriminate healthy from EC patients (ROC AUC=0.94). This protein signature can detect endometrial cancer independently of the cancer type. In addition, we also report a new protein signature that can differentiate type1 versus type2 EC (ROC AUC=0.93). This study has important implications in early detection of EC and in patient stratification.
