Heterogeneity of sodium iodide symporter (NIS) expression in thyroid cancer impairs therapeutic efficacy: An in vitro study

Introduction: Differentiated thyroid carcinoma (DTC) is one of the most prevalent malignancies in endocrinological organs, with DCT alone accounting for approximately 90% of all thyroid cancers. DTC is typically treated with surgery and radioactive iodine (RAI) therapy based on the risk of recurrence and generally has a favorable prognosis. However, patients with RAI-refractory metastases have a poor prognosis with high rates of recurrence and mortality. Most studies have concluded that low ¹³¹I uptake associated with low sodium iodide symporter (NIS) expression is the primary cause of RAI-refractoriness. However, we have observed that even lesions with significant ¹³¹I uptake can show disease progression. Multiple reports have suggested a correlation between heterogeneous intratumoral distribution of radioactivity and poor therapeutic efficacy in radionuclide therapy with beta-emitting radionuclides. To our knowledge, no studies have investigated the relationship between intratumoral heterogeneity of ¹³¹I distribution in DTC and the efficacy of RAI treatment. This study aimed to determine if heterogeneity of NIS-expressing cancer cell distribution affects ¹³¹I therapeutic efficacy in vitro.

Methods: We used K1 cells which are human thyroid papillary carcinoma with low NIS expression and K1-NIS cells, NIS-expressing K1 cells, for this study. These cells were plated at a density of 1 x 10⁵ cells per well in 6‑well plates and incubated for 24 h prior to undergoing Cell surviving assay or Uptake assay. For Cell surviving assay, the culture medium was replaced with fresh culture medium containing approximately 1 MBq/ml ¹³¹I or no radioactive iodine, and incubated for an additional 48 h. The number of viable cells was counted and the ratio of viable cells in the ¹³¹I-containing culture medium (VR) was calculated by dividing the number of viable cells in the ¹³¹I-containing culture medium by the number of viable cells in the control culture medium. For Uptake assay, the culture medium was replaced with fresh culture medium containing 3 KBq/ml ¹³¹I and incubated for an additional 48 h. The cells were then dissolved in 1 M NaOH and radioactivity was measured using a gamma-ray counter. Evaluation of Cross-fire effect (Fig. 1): K1 and K1-NIS cells were suspended in culture medium in various proportions and plated into 6‑well plates. The VR was determined by Cell surviving assay with 0.9 MBq/ml ¹³¹I, and the results were compared to the estimated VR based on the proportions of each cell type, without taking into account the cross-fire effect. Comparison between homogenous and heterogenous conditions (Fig. 2): K1 and K1-NIS cells were also co-cultured in 6-well plates under conditions of homogenous (condition A) and heterogeneous (condition B) distribution of K1-NIS cells. In condition A, K1 cells and 1/9th of the number of K1-NIS cells were suspended in culture medium and were plated into the wells. In condition B, K1-NIS cells were plated in 37 equally-spaced locations in the wells, and 1 h later, suspension containing 9-fold more K1 cells was gently added. The VR was determined by Cell surviving assay with 0.8 MBq/ml ¹³¹I and radioactivity was measured by Uptake assay.

Results: VR was significantly lower than the estimated VR when only 1 % of the total cells were K1-NIS cells (0.42 vs 0.69, P < 0.01, Fig. 1). The VR was also significantly lower than the estimated VR in 10, 25, and 50 % of K1-NIS cells. When 10 % of the total cells were K1-NIS cells, the VR in condition A was significantly lower than in condition B (0.58 vs 0.76, P = 0.02, Fig. 2). There was no significant difference in radioactivity between conditions A and B.

Conclusions: The cytotoxic effect of ¹³¹I was impaired in the condition where K1-NIS cells were heterogeneously distributed compared to the homogenous condition. This suggested that heterogeneous distribution of NIS-positive cells within the tumor may decrease the therapeutic effect, even if the overall tumor accumulation of radioactive iodine is the same.

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