The sodium/iodide symporter (NIS) mediates iodide uptake in the thyroid. Since decades, NIS-mediated iodide uptake is a very useful tool for radioactive ablation of thyroid cancer cells. NIS-based gene therapy is a promising tool for the treatment of tumor cells of extrathyroidal origin. Some preclinical studies on this approach produced very promising results, but other studies concluded that the required radioactive iodine administrations were too high or resistance to treatment occurs. The reason of this variability was not well-defined. This work is based on the observations made by the TIRO group from the University of Nice in France. They inoculated subcutaneously HT29NIS cells, colon adenocarcinoma cells (HT29 line) stably expressing exogenous mNIS to induce tumors in nude mice. Using SPECT imaging and immunohistochemistry, they found an unexpected heterogeneous distribution of iodide uptake capacity and NIS expression localized at the border of the xenografts. In this study, the aim was to analyze factors of the tumor microenvironment such as hypoxia and cellular heterogeneity (this term refers to the presence of cells in a proliferative or quiescent state) on the activity of the NIS protein. We studied HT29NIS cells in vitro in conditions that induce quiescent and/or hypoxic states. In this in vitro model, the expression, function and localization of NIS protein were analyzed. Changes in the proteome and metabolome of HT29NIS induced by quiescence and / or hypoxia were also analyzed. Also, change at the cellular redox state level and its effects on glycolytic and oxidative metabolism under quiescence and hypoxia conditions and its effect on the expression of the NIS protein were analyzed. Finally, possible changes in cellular behavior such as viability, redox status, and glycolytic and oxidative metabolism of wild type HT29 cells when transfected with the NIS gene were analyzed. We found that the iodide uptake, NIS-expression level and NIS localization at the plasma membrane were reduced by quiescence and hypoxia. Our proteomics results indicate that quiescent and hypoxic states are associated to a 16 decrease in the expression of proteins involved in protein localization to membrane and changes in protein linked to energy metabolism. In addition, transfection of the NIS protein in HT29WT cells was found to reduce glycolytic metabolism and increase oxidative metabolism. In conclusion, the results showed that hypoxia and quiescence impair NIS expression and NIS cellular localization, and consequently iodide uptake. These findings also indicate that the use of cell lines for preclinical evaluation of NIS-based gene therapy could lead to underestimate the efficiency of the approach. It is also important to consider possible changes in cellular behavior when an exogenous gene is introduced into cell lines that are subjected to conditions such as hypoxia and quiescence. More generally, our study show that tumor microenvironment is an important parameter in the use of NIS in the treatment of cancer cells. Resolving some limitations of the use of NIS in gene therapy, given by factors of the tumor microenvironment such as cellular heterogeneity, variations in the availability of oxygen and nutrients, pH and ROS production will allow in the future to delimit areas of the tumor, dosage of radioisotopes, decrease of side effects in healthy tissues, increasing the radiosensitivity of the tumor and improving the prognosis of the cancer patient.
