Analysis of extracellular vesicles in cancer immunomodulation and liquid biopsy

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