CHARACTERIZATION OF EPCAM IN THYROID CANCER BIOLOGY BY THREE-DIMENSIONAL SPHEROIDS IN VITRO MODEL.

Thyroid cancer (TC) is the most common Endocrine malignancy. Currently, the mechanisms responsible for insensitivity to therapeutic treatments, tumor aggressiveness and tumor relapse described in undifferentiated thyroid cancers (UTCs) haven’t been fully elucidated yet. The insurgence of therapy resistance and disease relapse is believed to be due to a subpopulation of cancer cells within the tumor bulk that shows stem-like phenotype and specific tumor-initiating abilities. These cells are called tumor-initiating cells (TICs), and a comprehensive understanding of how to specifically isolate and target them is necessary. In thyroid cancer, TICs have been identified using specific in vitro and in vivo assays, variations of enzymatic activities, expression of well-known stemness markers and expression of membrane markers. Among the possible membrane markers described in literature to identify TICs, Epithelial Cell Adhesion Molecule (EpCAM) has already been defined as a marker for cancer cells with tumor-initiating properties in many solid tumors, where its possible role in tumor progression and aggressiveness has been described. However, this protein is poorly studied in TC and its role in TC pathology, especially in the undifferentiated forms, needs further comprehension. Hence, the aim of my PhD project was to: 1) characterize EpCAM as a putative thyroid TICs marker on poorly differentiated and anaplastic thyroid cancer cell lines, applying both classical monolayer cultures and 3D spheres cultures; 2) establish if the 3D model is a valid in vitro approach to study the expression and role of EpCAM in the context of TC; 3) observe whether TC cell lines respond differently when treated as adherent cells or as 3D spheres. Our data demonstrated that EpCAM is subjected to an intense cleavage process in FRO-derived 3D tumor spheres and that the 3D model is representative of the variability of EpCAM expression and cleavage that we observed in patient-derived tissue samples, with the different gradient of EpCAM cleavages corresponding to different areas of the tumor sections. We also observed that the expression of EpCAM can be modulated by the regulation of its cleavages and that the integrity of the protein seem to be a crucial factor for the initial phase of the generation of 3D spheres in FRO, while the cleavage of the protein may occur in a subsequent phase, induced by the ability of cells to adapt to variations in growth conditions and/or to the microenvironment within the spheres. Moreover, we demonstrated that EpCAM+ and EpCAM- subpopulations respond differently to treatment with a well-known drug commonly used in clinical practice, Vemurafenib (PLX-4032), both in FRO and HTCC3 adherent cells, and EpCAM+ cells appeared to be more resistant. Finally, the 3D sphere model is also a valid in vitro approach to assess cell response to PLX-4032 and we observed that FRO-derived 3D spheres seemed to be more resistant than HTCC3-derived spheres upon PLX-4032 treatment. In conclusion, we believe that EpCAM expression and its cleavage in 3D spheres could play a significant role in putative TC TICs biology and may partially explain the therapeutic failure observed in the more undifferentiated TCs, concomitantly to other mechanisms of resistance and/or escape that may occur.