Characterization of epcam in thyroid cancer biology by three-dimensional spheroids in vitro model

Thyroid cancer (TC) is the most common Endocrine malignancy. Nowadays Undifferentiated Thyroid Cancers (UTCs) are still a lethal disease, mostly because of the lack of effective therapeutic options. The insurgence of therapy resistance and disease relapse is believed to be caused by a subpopulation of cancer cells with a stem-like phenotype and specific tumor-initiating abilities, the so called Tumor-Initiating Cells (TICs). Several markers have been identified and described over the years to detect TICs and allow the development of potential therapeutic approaches that could serve to specifically target these cells and promote the eradication of the tumor. In TC, TICs have been identified using precise in vitro and in vivo assays (e.g. sphere-forming assays and tumor grafts), variations of enzymatic activities, expression of well-known stemness markers and expression of membrane markers. In the present work, we provide more insight into the role that Epithelial Cell Adhesion Molecule (EpCAM), a known TICs marker in other epithelial tumors, may have in TC biology. This result was accomplished by the integration of TC tissues examination, through Western Blot and Immunofluorescence, and in vitro characterization of 2D and 3D models from FRO Anaplastic Thyroid Cancer (ATC) cell line. The main methodologies applied to obtain the 3D cultures are the hanging-drop and coating with poly(2-hydroxyethyl methacrylate) non-adhesive substrate. Our data demonstrated that EpCAM is subjected to an intense cleavage process in FRO-derived 3D tumor spheres, and that this model is representative of the variability of EpCAM expression and cleavage that we found in patient-derived tissue samples. We also demonstrated that the expression of EpCAM can be modulated by the regulation of its cleavage and that the integrity of the protein seem to be a crucial factor for the initial phase of the generation of the 3D spheres in FRO, postulating that the cleavage of the protein may occur in a second moment, induced by the ability of cells to adapt to variations in growth conditions and/or to the microenvironment within the spheres. Finally, we observed that EpCAM-expressing cells (EpCAM+) in FRO appeared to be more resistant than EpCAM- cells after treatment with Vemurafenib (PLX-4032), as well as FRO-derived 3D spheres with respect to adherent cells. In conclusions, EpCAM expression and cleavage may play a significant role in putative TC TICs biology and the most promising results should be validated on patient-derived samples.