Patient-Derived in vitro models for unraveling medullary thyroid cancer microenvironment and therapy resistance

Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor arising from parafollicular C-cells. Nowadays different targeted therapies are available, directed mostly against the main genetic driver of MTC, RET, and against the common pro-oncogenic pathways of VEGF, EGFR and c-MYC. Despite this, an escape phenomenon has been observed with sudden disease progression during treatment, leaving surgery as the only curative treatment. The evaluation of pre and post treatment genetic landscape of RET-mutated cancers recently demonstrated that Selpercatinib resistance is acquired either by appearance of secondary RET solvent front mutations or by the positive selection of a minor RET-wild type tumor subpopulation. So far, practically no attempts have been made to explain the intrinsic biological mechanisms responsible for medical therapy failure in MTC, mainly because of the lack of in vitro models closely representing cancer complexity. We hypothesized that, similarly than in other cancers, therapy resistance may be the result of a shift in between different cellular subpopulations and of biological changes induced in cancer cells by specific microenvironmental factors. The main aim of this study was the establishment of patient-derived MTC primary cell lines that can be grown in tridimensional models and conserve the ability to shift toward a more mesenchymal stem-like phenotype. We successfully established primary cell lines from 4 patients. We characterized their phenotype and response to targeted therapies through Western Blot, Confocal Microscopy, ELISAs, ELDAs, RT-qPCR and proliferation assays. Our data demonstrated that these cells secrete high levels of the MTC markers calcitonin and CEA when grown in differentiation media. When cultivated in a tridimensional setting they all show a significant increase in the stem and neuroendocrine precursor markers such as SOX2, OCT4, EPAS1, TUJI and FOXA1. Anyway, among the cell lines we observed significant differences in the markers expression, which correlate with sphere forming abilities, probably reflecting the differences in the composition of the original tumors. Our studies show that these cells are generally more resistant to TKIs treatment than the widely used immortalized MTC cell line (TT), as observed in the MTC patients in vivo. Moreover, changes of the growth conditions, observed during induction of a pseudo-hypoxic state or growth in adhesion-free environment, have a great impact on the cell lines phenotypes and their response to the anticancer drugs currently used for MTC treatment.In conclusion, we report here the generation of an unprecedented valid patient-derived in vitro model that can provide further insight in MTC biology complexity and will allow the future study of therapy resistance mechanisms evolution.