Multicellular spheroids: a new model to screen novel drugs for incurable forms of thyroid cancer

Thyroid cancer (TC) is the most common endocrine malignancy and its incidence is increasing worldwide even if the mortality rate remains low. There is accumulating evidence that solid tumors contain a distinct subpopulation of Cancer Stem-like Cells (CSCs), or tumor-initiating cells (TICs), that play important roles in cancer initiation, progression, recurrence and metastasis. Whether thyroid cancer cells are derived from mutated adult stem cells or whether they display a newly acquired stem-like phenotype due to cancer mutations remains controversial, but they are believed to be one main actor in therapy resistance and disease progression. The aim of the project is to understand the biology of thyroid TICs, by in vitro characterization of thyrosphere-forming cells, and use this 3D model to investigate thyroid TICs sensibility to different anticancer treatments. To overcome the intrinsic limitations of primary cultures and fresh sample availability, we are currently developing a standardized thyrosphere model based on immortalized cell lines with different genetic background. Hanging drop cell cultures and coating with poly(2-hydroxyethyl methacrylate) non-adhesive substrate are the main methods to obtain thyrosphere-forming cells. In appropriate growth condition, all the cell lines tested are able to generate thyrospheres when seeded at clonal density, with an efficiency significantly higher that what has been previously reported. In particular, we’ve obtained a sphere-forming efficiency of 61.22% for B-CPAP, 52.22% for HTC/C3, 61.20% for SW579, 70.24% for FRO, 51.09% for SW1736 and 50.09% for HTH-74. Furthermore, we selected different anticancer drugs and analysed their effects on both 3D and 2D cultures. In this regard, we’ve tested two inhibitors so far: PLX-4720, a potent and selective inhibitor of ERK phosphorylation in BRAFV600E cells, and Cariporide, a selective inhibitor of NHE-1, a Na+/H+ exchanger. We evaluated the effects of these compounds by MTT colorimetric assay and observed that cell lines with different genetic background respond differently when seeded as adherent cells or as 3D thyrospheres. In conclusion, this 3D model can partially mimic the tumor complexity in vitro, and the most promising results shall be validated on patients-derived samples.