Analysis of extracellular vesicles in cancer immunomodulation and liquid biopsy
Título (trad.)
Análisis de vesículas extracelulares en inmunomodulación y biopsia líquida del cáncerAutor (es)
Campos Silva, CarmenDirector (es)
Valés Gómez, María del MarEntidad
UAM. Departamento de Biología Molecular; CSIC. Centro Nacional de Biotecnología (CNB)Fecha de edición
2022-10-18Materias
Biomarcadores; Melanoma; Inmunoterapia; Biología y Biomedicina / BiologíaNota
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de Lectura: 18-10-2022Esta Tesis tiene embargado el acceso al texto completo hasta el 18-04-2024
Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Resumen
Extracellular Vesicles (EVs), that are released by most cell types to the extracellular milieu or to body
fluids, carry multiple molecules which can provide information on the state or characteristics of their
cell of origin. The definition of EV-associated molecules with clinical value, released to body fluids,
opens new possibilities for the identification of biomarkers in liquid biopsy. However, even though numerous candidate EV biomarkers have been suggested for different pathologies, few of these have been validated in studies of large cohorts of patients due to the lack of widespread, sensitive methodologies for high-throughput EV analyses. This thesis describes the optimization of highthroughput immunoassays for the characterization of EV-resident proteins directly in body fluids, that
can be used with technologies available in any standard or clinical laboratory. The critical parameters to increase sensitivity for the detection of EV-proteins were analysed, and time, temperature, bead
number, antibody combinations and geometry of the assay were shown to be important variables for best assay performance. The results confirmed that EVs in solution show physico-chemical properties
typical of colloidal suspensions, and that these features could be exploited for detection of tumourderived EVs in immunocapture experiments. The optimised methods were validated by detecting general EV and tumour-associated markers, such as EpCAM or the NKG2D-L, MICA, in EVs present in
body fluids (urine, plasma, serum and ascites) from patients suffering different types of cancer. Antigen detection was semi-quantitative and showed considerably higher sensitivity than analyses
performed in parallel by Western Blot.
The optimised immunocapture methods were then applied to the study of metastatic melanoma, a
cancer with very low overall survival rates, when treated with chemotherapy or targeted therapies,
mainly due to the emergence of drug-resistant tumours. A commonly used targeted therapy for
BRAFV600E metastatic melanoma, the BRAF kinase inhibitor vemurafenib, has been shown to decrease cell surface expression of immune activating NKG2D-L by metastatic melanoma in vitro, and this
immune evasion mechanism could contribute to treatment failure. Currently, therapy combining BRAF inhibitors with an inhibitor of the downstream MAPK, MEK, is one strategy used to delay drug resistance development. However, this thesis demonstrates that in vitro treatment of BRAFV600E
melanoma cells with a MEK inhibitor or its combination with vemurafenib, also induces a decrease in
surface NKG2D-L expression, that in general, was paralleled by decreased levels of soluble and EVNKG2D-
L. Using the immunoassays optimised here, circulating soluble and EV-associated NKG2D-L
levels were analysed ex vivo in sera from advanced melanoma patients receiving targeted therapy
treatment and were compared to the data obtained in vitro. Based on these preliminary observations, possible clinical associations are discussed and a rationale for combination strategies involving
immunotherapies is provided
Lista de ficheros
Google Scholar:Campos Silva, Carmen
Lista de colecciones del ítem
Registros relacionados
Mostrando ítems relacionados por título, autor, creador y materia.