Endocrine Abstracts

Thyroid cancer (TC) is the most common endocrine tumor and its incidence has increased faster than in any other malignancy. Although TCs are usually well differentiated, disease recurrence or persistence is high, because of local and distant metastasis and therapeutic resistance. Among the different genetic alterations, BRAFV600E is the most frequent one. Several studies tried to establish a correlation between BRAFV600E and patients outcome, with controversial results. Nevertheless, the activation of different BRAF downstream pathways influences immune response, matrix remodeling and intra- and extra-cellular pH. All these alterations substantially modify tumor microenvironment and may enhance the survival of cancer-initiating cells and promote therapy resistance. The aim of the study is to investigate the role of BRAF in cancer-associated fibroblast matrix deposition and remodeling in in vitro 2D and 3D systems obtained from immortalized and patients-derived cells. In particular, the use of conditioned media and co--cultures of TC cells with different genetic background and fibroblasts is used to generate different extracellular matrices (ECM). The ECM themselves and their effects on cell growth and survival is then analyzed by different techniques, such as western blot, immunofluorescence, real-time PCR, colony assay, sphere formation assays and proliferation assays. Our results show that TC cells with BRAFV600E mutation can significantly increase the proliferation and activation of fibroblasts in respect with BRAF WT TC cells and normal thyrocytes; fibroblasts that have been conditioned with BRAFV600E TC cells can produce an ECM that is thicker and has a different fiber pattern than the one produced frorm fibroblasts conditioned with BRAF WT TC cells and normal thyrocytes. Moreover, the different matrices differentially influence the survival of BRAF mutated and WT TC cells. As second step, we are currently evaluating the effects of different drugs that acts against BRAF downstream effectors involved in matrix remodeling and metabolism alterations. In conclusion, our in vitro model can partially recapitulate the complex environment of human tumors and can be a useful tool for the screening of different anticancer drugs.