Three-dimensional multicellular spheroids: an alternative model to screen new drugs for incurable forms of thyroid cancer

Thyroid cancer (TC) is the most prevalent endocrine malignancy, and its incidence has been increasing in the last decades even if the mortality rate remains low. This is a consequence of a favourable prognosis for the more frequent well-differentiated forms. In contrast, the rare poorly differentiated and anaplastic TCs (PDTC and ATC) have a very poor prognosis because of their invasiveness and metastatic behaviour, as well as their insensitivity to radioactive-iodine treatment. There is accumulating evidence that solid tumors contain a distinct subpopulation of Cancer Stem-like Cells (CSCs), or tumor-initiating cells (TICs), that exerts important roles in cancer initiation, progression, recurrence and metastasis. Thyroid tumors have been traditionally hypothesized to follow a classic multistep carcinogenesis model. Nevertheless, an alternative stem cell model has been proposed. According to this model, only a subset of cancer cells with self-renew abilities and tumorigenic potential gives rise to progenitor cells that drive tumor growth. Whether thyroid cancer cells are derived from mutated adult stem cells or whether they show a newly acquired stem-like phenotype due to cancer mutations is still not clear, but they are believed to be essential contributors for therapy resistance and disease progression. The aim of the study is to investigate the biology of thyroid TICs, by in vitro characterization of thyrosphere-forming cells, and apply this 3D model to evaluate thyroid TICs sensibility to different anticancer treatments. We are currently developing a standardized thyrosphere model, based on immortalized cell lines displaying different genetic background, to overcome the limitations of primary cultures and the availability of fresh samples. In particular, the main methodologies applied to obtain thyrosphere-forming cells cultures are the hanging drop and coating with poly(2-hydroxyethyl methacrylate) non-adhesive substrate. In appropriate growth condition, all the cell lines tested are able to generate thyrospheres when seeded at clonal density. We have obtained a sphere-forming efficiency of 61.22% for B-CPAP (PDTC), 52.22% for HTC/C3 (PDTC), 61.20% for SW579 (PDTC), 70.24% for FRO (ATC), 51.09% for SW1736 (ATC) and 50.09% for HTH-74 (ATC). Moreover, we selected different anticancer drugs and examined 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 mutation-bearing cells, and Cariporide, a selective inhibitor of NHE-1, a Na+/H+ exchanger. Using the MTT colorimetric assay we assessed the effects of these compounds and observed that cell lines respond differently when seeded as adherent cells or as 3D thyrospheres. In conclusion, although the study of therapy resistance in isolated cancer cells is somewhat a simplistic model, as tumor microenvironment is much more complex, this 3D model allows the identification and maintenance of thyroid TICs and therefore permits the study of drug response in a more accurate way than in other in vitro models. Moreover, the most promising results shall be validated on patients-derived samples.