Endocrine Abstracts

Introduction: Due to their remarkable self-organizing structures and functional properties, organoid has become a powerful tool to model diseases ‘in a dish’. The use of organoids in cancer research emerged to better understand tumor behavior. Originally, adult stem cells (aSC)-derived organoids were derived from primary tumors while recent studies have reported the generation of cancer models arising from healthy cells by controlling oncogene expression. SC-derived cancer organoids can access the effects of oncogenes and early events driving tumorigenesis, role of cancer stem cells in tumor induction, genomic stability, effect of treatments and screening of new therapeutics.

Objectives: To generate a PTC organoid model by inducing the Braf^V637E mutation in mouse ESC-derived functional thyroid follicles to better comprehend the oncogenic events driven by Braf-oncogene and develop a drug screening tool.

Methods: TRE-Nkx2-1-Pax8_bTg-NES-Braf^V637E-ERT² mESCs were differentiated into thyroid follicles with doxycycline and hrTSH/cAMP. After follicle enrichment Braf^V637E activation was induced with 4-Hydroxytamoxifen (4OHT). Organoids were then treated with agents previously described to inhibit Braf oncogenesis (MEK, PI3K and histone deacetylase (VPA) inhibitors).

Results: TRE-Nkx2-1-Pax8_bTg-NES-Braf ^V637E-ERT² mESCs were able to differentiate into functional thyroid follicles with iodinate-Tg (Tg-I) luminal accumulation. Starting six hours after 4OHT addition time-dependent dedifferentiation was observed; as evidenced by a decrease in mRNA expression of TSHR, Tg, Nis and Tpo. It was associated with an increase in ERK phosphorylation and proliferation. Activation of Braf ^V637E disrupted follicular organization and decreased Tg-I accumulation, ¹²⁵I uptake and organification. Transcriptomic analysis revealed hyperactivation of PI3K-AKT-mTOR, TNF, cytokine signaling and promotion of Epithelial Mesenchymal Transition. Isolated inhibition of MEK and PI3K resulted in partial increase of Slc5a5/Nis levels, whereas treatment with VPA resulted in complete recovery. The combination of MEK and PI3K inhibitors resulted in complete re-expression of Nis, Tg, TSHR, Tpo and reorganization into functional follicles confirmed by the detection of Tg-I.

Conclusions: We demonstrate the generation of an mESC-derived organoid model that recapitulates the transcriptomic and 3D-histological features of PTC. The combination of MEK and PI3K inhibitors promoted Nis reexpression and thyrocyte redifferentiation with recovery of follicular functionality. This mESC-derived PTC in vitro model opens new opportunities to study early mechanisms of carcinogenesis while providing a simple and efficient tool for screening new treatments for thyroid cancer.