OC48 - Generation of patient-derived in vitro models to unravel medullary thyroid cancer microenvironment and therapy resistance

Background/Introduction Medullary thyroid carcinoma (MTC) is a rare neuroendocrine tumor arising from parafollicular C-cells in the thyroid. Unlike other neuroendocrine tumors, knowledge about the MTC tumor microenvironment and its role in therapy resistance remains limited, mainly due to the lack of reliable in vitro models. We hypothesized that, as observed in other cancer types, MTC cells may exhibit a degree of phenotypic plasticity that could be modulated by various microenvironmental factors. Purpose The primary aim of this study was to establish patient-derived MTC in vitro models capable of growing under different three-dimensional (3D) culture conditions while retaining the ability to shift toward a more mesenchymal, stem-like phenotype. Methods We successfully established eight patient-derived primary MTC cell lines from tumors with different genetic backgrounds and variable degrees of aggressiveness. These lines were characterized in terms of phenotype and responses to different microenvironmental stimuli and targeted therapies through Western Blot, confocal microscopy, ELISA assays, ELDA, RT-qPCR, and proliferation assays. Results Notably, the cells maintained their plasticity over extended passaging when cultured under stem-promoting conditions. In differentiation media, they preserved the ability to secrete high levels of classic MTC markers, such as calcitonin and chromogranin A. Under 3D culture conditions, all cell lines showed a significant increase in stem and neuroendocrine precursor markers, including SOX2, OCT4, TBB3 and FOXA1, along with spatially dependent shifts in Epithelial-to- Mesenchymal Transition (EMT) markers E-cadherin and Vimentin. These phenotypic variations partially recapitulated the molecular alterations observed in MTC tissue samples. Moreover, changes in culture conditions—such as inducing a pseudo- hypoxic state or culturing in non-adherent environments—profoundly affected both cell phenotype and sensitivity to therapeutic agents currently available for MTC treatment, including Pralsetinib, Selpercatinib, Cabozantinib, Lenvatinib, and Vandetanib. Conclusion(s) In conclusion, we present here the generation of a new valid patient- derived in vitro model that can provide new opportunities to explore MTC biology complexity and will allow future translational studies, including those involved in uncovering the mechanisms underlying the insurgence of therapy resistance.