Three-dimensional spheroids: a new approach for the identification and characterization of novel markers for tumor-initiating cells subpopulations in thyroid cancer

Thyroid cancer (TC) is the most common endocrine malignancy, with an increasing number of diagnosis in the last decades. Of all TC histotypes, the poorly differentiated and anaplastic TCs (PDTC and ATC) have a very poor prognosis because of their invasiveness and metastatic behavior, as well as their insensitivity to radioactive-iodine treatment. In the last years, the stem-cell model has been proposed to further explain TC carcinogenesis. According to this model, only a subset of cancer cells, identified as Cancer Stem-like Cells (CSCs) or Tumor Initiating Cells (TICs), give rise to progenitor cells that may drive tumor growth. These cells exert stem-like properties, tumorigenic potential and the ability to grow as non-adherent spheres. An intriguing candidate for the identification of putative TICs in TC, especially for the most dedifferentiated forms, is EpCAM (epithelial cell adhesion molecule), a transmembrane glycoprotein that is highly expressed in TICs of other tumors of epithelial origin, playing a role in balancing cell proliferation and differentiation. We are aiming to investigate the biology of putative thyroid TICs, by in vitro characterization of thyrosphere-forming cells. We’ve developed a standardized thyrosphere model, based on PDTC and ATC cell lines displaying different genetic background. The main methodologies applied to obtain the 3D cultures are the hanging-drop and coating with poly(2-hydroxyethyl methacrylate) non-adhesive substrate. In appropriate growth condition, all the cell lines tested were able to generate thyrospheres when seeded at clonal density. We applied the ELDA web-tool to estimate the frequency of TICs with potential stem-like properties able to generate the 3D spheres, and we observed the highest stem cell frequency on spheres derived from FRO (ATC cell line). The screening of eight cell lines by Western Blot revealed that only FRO express EpCAM. Immunofluorescence on FRO-derived 3D spheres have shown an increase in EpCAM cleavage according to a radial gradient, and a variable expression of E-cadherin, a typical epithelial marker, in EpCAM-expressing cells. Moreover, immunofluorescence on cryosections of healthy and pathological patient-derived tissue samples have shown homogeneous expression and cleavage of EpCAM in the healthy epithelium, while a highly heterogeneous distribution is seen in tumor sections. To conclude, EpCAM displays a distinct expression among TC cell lines and human tissues, and appears to correlate with the ability to generate 3D spheres in vitro. This thyrosphere model is a promising approach to better investigate the biology of aggressive TCs, including the sensitivity of thyroid TICs to different anticancer drugs.