Using TGF-β1 biology in radioiodine refractory differentiated thyroid cancer to re-establish sodium iodide symporter (NIS) expression using engineered mesenchymal stem cells as therapy vehicles

Based on its role in mediating iodide uptake from the blood into the thyroid, the sodium iodide symporter (NIS) allows radioiodine imaging and therapy of differentiated thyroid cancer. This biology is largely responsible for the favorable prognosis of most thyroid cancers. However, due to the loss of functional NIS expression, a number of thyroid cancers remain refractory to radioiodine (RAI) including RAI-refractory differentiated thyroid cancer (DTC) and anaplastic thyroid cancer (ATC). The delivery of NIS as a transgene into refractory tumors represents a therapeutic strategy for RAI-refractory thyroid cancer and metastasis. Previous studies have extensively investigated the transfer of the NIS gene into experimental tumor models using viruses, polymers or mesenchymal stem cells (MSCs) as delivery vehicles. MSCs are under development for NIS gene transfer based on their excellent tumor homing abilities. In this regard, various strategies have been applied to enhance the tumor-specificity and effectiveness of MSC-mediated NIS gene transfer. Based on the critical role of Transforming growth factor beta (TGF-β) in thyroid cancer including driving tumor-directed migration of MSCs, and the identification of an autocrine TGF-β loop as a mechanism through which BRAFV600E fosters NIS repression, in the present thesis we proposed to re-induce NIS-mediated radioiodine uptake in tumor mouse models using a synthetic TGF-β1-inducible/SMAD-responsive promoter in engineered MSCs (SMAD-NIS-MSCs) to drive NIS expression. As the first step, TGF-β levels were evaluated in different thyroid cancer cell lines by qRT-PCR and ELISA. Tumor cell-conditioned medium was then used to evaluate stimulation of NIS expression in SMAD-NIS-MSCs. In parallel experiments validation of TGF-β induced NIS expression in SMAD-NIS-MSCs was studied through co-culture of the thyroid cancer cells with the engineered MSCs. SMAD-NIS-MSCs demonstrated different levels of radioiodine uptake after stimulation with either tumor conditioned medium or by co-culture with thyroid cancer cells. A 3D live chemotaxis assay confirmed the directed migration of MSCs towards supernatants derived from K1 and BCPAP papillary thyroid cancer (PTC) cells as compared to control culture medium. This tumor directed migration of MSCs was further validated in vivo using K1 and BCPAP xenograft mouse models. Systemic injection of SMAD-NIS-MSCs showed effective tumor homing of the cells with functional NIS gene transfer resulting in a high level of tumoral radioiodine uptake in both subcutaneous tumor models as compared to WT-MSCs analyzed by ${}^{123}$I-scintigraphy. Tumor-specific NIS expression in tumor bearing mice treated with SMADNIS-MSCs was further confirmed ex vivo by NIS immunohistochemistry. A therapy study of the experimental tumors using systemically administered SMAD-NIS-MSCs followed by ${}^{131}$I treatment resulted in a significantly prolonged survival with reduced tumor growth in the therapy groups in both animal models. Immunofluorescence analysis of Ki67 and CD31 expression in the resected tumors demonstrated a decrease in tumor proliferation index and blood vessel density in therapy mice. Taken together, the work presented in this thesis supports the exciting prospect of using NIS-mediated radioiodine therapy for the treatment of RAI refractory thyroid tumors by taking advantage of the tumor-selective homing of MSCs and targeting the TGF-β biology in RAI refractory thyroid cancer. We were able to hit the tumor with its own weapons by targeting the TGF-β/SMAD signaling to re-induce radioiodine accumulation in the tumor stroma and make use of the crossfire effect of ${}^{131}$I to achieve effective therapy.