Effect to Therapy of Sodium-Iodine Symporter Expression by Alpha-Ray Therapeutic Agent via Sodium/Iodine Symporter

This study confirmed the effect of sodium/iodine symporter (NIS) expression on existing drugs by in vitro and in vivo tests using cultured cell lines. The tumor growth inhibitory effect of sodium astatide ([211At]NaAt) was evaluated by in vitro and in vivo tests using human thyroid cancer cells (K1, K1/NIS and K1/NIS-DOX). NIS expression in cancer cells was controlled using the Tet-On system. [131I]NaI was used as control existing drug. From the results of the in vitro studies, the mechanism of [211At]NaAt uptake into thyroid cancer cells is mediated by NIS, analogous to [131I]NaI, and the cellular uptake rate correlates with the expression level of NIS. [211At]NaAt’s ability to inhibit colony formation was more than 10 times that of [131I]NaI per becquerel (Bq), and [211At]NaAt’s DNA double-strand breaking (DSB) induction was more than ten times that of [131I]NaI per Bq, and [211At]NaAt was more than three times more cytotoxic than [131I]NaI (at 1000 kBq each). In vivo studies also showed that the tumor growth inhibitory effect of [211At]NaAt depended on NIS expression and was more than six times that of [131I]NaI per Bq.


Introduction
The large uptake of iodine by the thyroid gland has provided opportunities for the use of [ 131 I]NaI in radioactive iodine therapy for patients with differentiated thyroid cancer [1][2][3][4]. However, a number of patients do not show sufficient therapeutic effects with [ 131 I]NaI therapy, even with sufficient uptake of [ 131 I]NaI [4,5]. For these patients, we considered an alpha-ray therapy targeting the sodium/iodide symporter (NIS) as a more effective therapy. Short-lived alpha rays have a short range and strong cytotoxicity, which makes them suitable for treatment. We are focused on astatine-211 ( 211 At, half-time: 7.2 h), an alpha-emitting nuclide that can be produced in an accelerator.
We have already started clinical trials of [ 211 At]NaAt to solve the problems associated with [ 131 I]NaI in radioiodine therapy at Osaka University Hospital. However, although it has been reported that [ 211 At]NaAt uptake is via NIS, there are no clear data on whether there is a correlation between the expression level of NIS and therapeutic effects. The purpose of this study was to evaluate the influence of NIS expression on the therapeutic effects of [ 131 I]NaI and [ 211 At]NaAt, even in a model with low expression of NIS.
One of the reasons for the failure of RAI treatment is the decreased expression of NIS. Various mechanisms have been proposed as the cause of NIS decrease [6], but in any case, it is speculated that when the expression of NIS decreases, the amount of nuclide uptake Int. J. Mol. Sci. 2022, 23, 15509 2 of 8 decreases and the therapeutic effect decreases. In the case of reduced NIS expression, it may be necessary to use nuclides with high biological efficacy [7]. Investigation of the correlation between NIS expression and therapeutic efficacy is also of great importance in confirmation the usefulness of astatine.

Examination of Uptake, Colony-Forming Ability and DSB Induction of K1-NIS/DOX Cells Whose NIS Expression Level Was Controlled by Tet-On System
It had already confirmed that the expression of NIS in K1-NIS/DOX cells increases according to the amount of doxycycline treated. The uptake of [ 211 At]NaAt by K1-NIS/DOX cells was nearly double that of [ 131 I]NaI (Figure 1a,b). For both nuclides, colony formation inhibition increased with increasing DOX concentration (i.e., increasing NIS expression level) (Figure 1c,d). [ 211 At]NaAt was more than ten times more effective than [ 131 I] NaI in inhibiting colony formation. The uptake of both nuclides is considered to be dependent on the expression levels of NIS. The frequency of DSB increased with increasing DOX concentration (NIS expression level) for both nuclides, and was compared using % of cells with >5 γH2AX foci/cell. Despite the [ 211 At]NaAt treatment of 1/10, the radioactivity of [ 131 I]NaI strongly caused damage to the cells (Figure 1e,f). effects of [ 131 I]NaI and [ 211 At]NaAt, even in a model with low expression of NIS.
One of the reasons for the failure of RAI treatment is the decreased expressio Various mechanisms have been proposed as the cause of NIS decrease [6], but in a it is speculated that when the expression of NIS decreases, the amount of nuclid decreases and the therapeutic effect decreases. In the case of reduced NIS expre may be necessary to use nuclides with high biological efficacy [7]. Investigatio correlation between NIS expression and therapeutic efficacy is also of great impo confirmation the usefulness of astatine.

Examination of Uptake, Colony-Forming Ability and DSB Induction of K1-NIS/DO whose NIS Expression Level Was Controlled by Tet-On System
It had already confirmed that the expression of NIS in K1-NIS/DOX cells i according to the amount of doxycycline treated. The uptake of [ 211 At]NaAt NIS/DOX cells was nearly double that of [ 131 I]NaI (Figure 1a,b). For both nuclides formation inhibition increased with increasing DOX concentration (i.e., increasing pression level) (Figure 1c,d). [ 211 At]NaAt was more than ten times more effective t NaI in inhibiting colony formation. The uptake of both nuclides is considered t pendent on the expression levels of NIS. The frequency of DSB increased with in DOX concentration (NIS expression level) for both nuclides, and was compared of cells with >5 γH2AX foci/cell. Despite the [ 211 At]NaAt treatment of 1/10, the rad ity of [ 131 I]NaI strongly caused damage to the cells (Figure 1e,f).

Comparison of Cytotoxically Effects in K1 Cells with and without NIS
Using K1 and K1-NIS cells, we confirmed the cytotoxically effects of NIS. NIS-independent cytotoxicity was lower for [ 211 At]NaAt. In contrast, NIS-dependent cytotoxicity was higher for [ 211 At]NaAt (Figure 2a,

Comparison of Cytotoxically Effects in K1 Cells with and without NIS
Using K1 and K1-NIS cells, we confirmed the cytotoxically effects of NIS. NIS-independent cytotoxicity was lower for [ 211 At]NaAt. In contrast, NIS-dependent cytotoxicity was higher for [ 211 At]NaAt (Figure 2a, (Figure 2c,d).

Comparison of Cellular Uptake in K1 Cells with and without NIS
In both the [ 131 I]NaI and [ 211 At]NaAt groups, there was a positive correlation between cell uptake and radioactivity added per well, indicating that both nuclides were almost

Comparison of Cellular Uptake in K1 Cells with and without NIS
In both the [ 131 I]NaI and [ 211 At]NaAt groups, there was a positive correlation between cell uptake and radioactivity added per well, indicating that both nuclides were almost identical. K1-NIS cells showed a radioactivity uptake more than three times that of K1 cells (Figure 3a,b). These results indicate that the cellular uptake of [ 131 I]NaI and [ 211 At]NaAt is due to a specific NIS-mediated mechanism and that the affinities of both nuclides for NIS are almost identical. identical. K1-NIS cells showed a radioactivity uptake more than three times that of K1 cells (Figure 3a,b). These results indicate that the cellular uptake of [ 131 I]NaI and [ 211 At]NaAt is due to a specific NIS-mediated mechanism and that the affinities of both nuclides for NIS are almost identical.

Colony-Forming Ability
In the case of [ 131 I]NaI, almost no decrease in the colony-forming ability was observed in any well (Figure 4c,d). This difference is clear when compared with the decrease in the [ 211 At]NaAt wells. [ 211 At]NaAt-treated K1-NIS cells showed a marked decrease in colonyforming ability in all wells, except the control wells (0 kBq). [ 211 At]NaAt-treated K1 cells inhibited colony formation only in wells with radioactivity ≥ 300 kBq (Figure 4a). These results showed that remarkably, [ 211 At]NaAt inhibited colony-forming ability and was affected by the presence or absence of NIS expression. However, since the colony-forming ability of K1 cells is also suppressed by high radioactivity (300 kBq or more), a small amount of NIS may be expressed in K1 cells; that is, even with a small amount of NIS expression the non-specific effects of [ 211 At]NaAt in the high radioactivity group cannot be completely denied (Figure 4b).

Colony-Forming Ability
In the case of [ 131 I]NaI, almost no decrease in the colony-forming ability was observed in any well (Figure 4c,d). This difference is clear when compared with the decrease in the [ 211 At]NaAt wells. [ 211 At]NaAt-treated K1-NIS cells showed a marked decrease in colony-forming ability in all wells, except the control wells (0 kBq). [ 211 At]NaAt-treated K1 cells inhibited colony formation only in wells with radioactivity ≥ 300 kBq (Figure 4a). These results showed that remarkably, [ 211 At]NaAt inhibited colony-forming ability and was affected by the presence or absence of NIS expression. However, since the colonyforming ability of K1 cells is also suppressed by high radioactivity (300 kBq or more), a small amount of NIS may be expressed in K1 cells; that is, even with a small amount of NIS expression the non-specific effects of [ 211 At]NaAt in the high radioactivity group cannot be completely denied (Figure 4b). identical. K1-NIS cells showed a radioactivity uptake more than three times that of K1 cells (Figure 3a,b). These results indicate that the cellular uptake of [ 131 I]NaI and [ 211 At]NaAt is due to a specific NIS-mediated mechanism and that the affinities of both nuclides for NIS are almost identical.

Colony-Forming Ability
In the case of [ 131 I]NaI, almost no decrease in the colony-forming ability was observed in any well (Figure 4c,d). This difference is clear when compared with the decrease in the [ 211 At]NaAt wells. [ 211 At]NaAt-treated K1-NIS cells showed a marked decrease in colonyforming ability in all wells, except the control wells (0 kBq). [ 211 At]NaAt-treated K1 cells inhibited colony formation only in wells with radioactivity ≥ 300 kBq (Figure 4a). These results showed that remarkably, [ 211 At]NaAt inhibited colony-forming ability and was affected by the presence or absence of NIS expression. However, since the colony-forming ability of K1 cells is also suppressed by high radioactivity (300 kBq or more), a small amount of NIS may be expressed in K1 cells; that is, even with a small amount of NIS expression the non-specific effects of [ 211 At]NaAt in the high radioactivity group cannot be completely denied (Figure 4b).

Contribution of NIS in Therapeutic Efficacy
The therapeutic effects of NIS on tumors with and without NIS were compared. Tumors with a higher expression of NIS (K1-NIS) were more efficacious. In addition, [ 211 At]NaAt showed a stronger inhibitory effect on tumor growth (Figure 5a,c). Compared to [ 131 I]NaI (Figure 5b,d), [ 211 At]NaAt also inhibited the growth of K1 tumors.

Contribution of NIS in Therapeutic Efficacy
The therapeutic effects of NIS on tumors with and without NIS were compared. Tumors with a higher expression of NIS (K1-NIS) were more efficacious. In addition, [ 211 At]NaAt showed a stronger inhibitory effect on tumor growth (Figure 5a,c). Compared to [ 131 I]NaI (Figure 5b,d), [ 211 At]NaAt also inhibited the growth of K1 tumors.

Discussion
In vitro studies revealed that the uptake mechanism of [ 211 At]NaAt in thyroid cancer cells, such as [ 131 I]NaI, was mediated by NIS, and cytotoxicity was correlated with the expression level of NIS. The colony formation inhibition of [ 211 At]NaAt was more than ten times that of [ 131 I]NaI, and [ 211 At]NaAt DSB was also more than ten times that of [ 131 I]NaI; [ 211 At]NaAt was more than three times more cytotoxic than [ 131 I]NaI (1000 kBq). In vivo studies have shown that the tumor growth-inhibitory effect of [ 211 At]NaAt depends on the amount of NIS expressed in human thyroid carcinomas and is more than six times (per Bq) that of [ 131 I]NaI.
Previous reports suggest that the expression of NIS often decreases with differentiation, thus reducing the effects of radioactive iodine ([ 131 I]NaI) therapy. In this experiment, it was confirmed that the expression of NIS contributed significantly to treatment with [ 131 I]NaI or [ 211 At]NaAt. However, while NIS expression was strongly important in the effect of RAI therapy, notable therapeutic effects were observed in [ 211 At]NaAt therapy, even in cells with low NIS expression (K1). In other words, it was suggested that [ 211 At]NaAt is effective even in patients with a low response to [ 131 I]NaI. However, the transport mechanism of [ 211 At]NaAt still remains unclear. Initially, it was thought that astatine might also be transported to tumors by symporters other than NIS, however, the

Discussion
In vitro studies revealed that the uptake mechanism of [ 211 At]NaAt in thyroid cancer cells, such as [ 131 I]NaI, was mediated by NIS, and cytotoxicity was correlated with the expression level of NIS. The colony formation inhibition of [ 211 At]NaAt was more than ten times that of [ 131 I]NaI, and [ 211 At]NaAt DSB was also more than ten times that of [ 131 I]NaI; [ 211 At]NaAt was more than three times more cytotoxic than [ 131 I]NaI (1000 kBq). In vivo studies have shown that the tumor growth-inhibitory effect of [ 211 At]NaAt depends on the amount of NIS expressed in human thyroid carcinomas and is more than six times (per Bq) that of [ 131 I]NaI.
Previous reports suggest that the expression of NIS often decreases with differentiation, thus reducing the effects of radioactive iodine ([ 131 I]NaI) therapy. In this experiment, it was confirmed that the expression of NIS contributed significantly to treatment with [ 131 I]NaI or [ 211 At]NaAt. However, while NIS expression was strongly important in the effect of RAI therapy, notable therapeutic effects were observed in [ 211 At]NaAt therapy, even in cells with low NIS expression (K1). In other words, it was suggested that [ 211 At]NaAt is effective even in patients with a low response to [ 131 I]NaI. However, the transport mechanism of [ 211 At]NaAt still remains unclear. Initially, it was thought that astatine might also be transported to tumors by symporters other than NIS, however, the results of uptake experiments indicated that the main reason for the high therapeutic effect of astatine was the high cellular effect of alpha rays.
To prevent non-specific accumulation of [ 211 At]NaAt, elemental blockade is considered effective, as is treatment with iodine [8]. In addition, [ 211 At]NaAt is highly accumulated not only in the thyroid but also in the stomach [9][10][11]. This is believed to be due to NIS expression. Of course, it is possible that the chlorine transporter for producing gastric juice is active, but this has not been verified. Since the authors previously confirmed that [ 211 At]NaAt accumulation was suppressed using H2 blockers [data not shown], it is necessary to investigate the involvement of transporters other than NIS in [ 211 At]NaAt transport.
Although NIS expression in K1-NIS/DOX was easily controlled with doxycycline in vitro, it was more difficult to control the expression levels of NIS in K1-NIS//DOX in vivo. This is because K1 and K1-NIS have distinctly different proliferation rates, and drug-treated cells consist of a heterogeneous population. However, it is possible that the heterogeneous population may mimic clinical patient tissue. Our model was preliminary; thus, we attempted to establish an animal model.

Preparation of [ 211 At]NaAt and [ 131 I]NaI Solution
211 At was acquired from the National Institute for Quantum Science and Technology (QST) and RIKEN through a supply platform of short-lived radioisotopes. 211 At was produced according to the 209 Bi(α, 2n) 211 At reaction and separated from the Bi target using the dry distillation method. The separated 211 At was then dissolved in pure water. Ascorbic acid (used as a reducing agent) and sodium bicarbonate (used as a pH adjuster) were added to the crude 211 At solution to a final concentration of 1% (w/v) at pH 8.0, and the solution was allowed to stand for 1 h at 23 ± 2 • C. The concentration of At was 10 MBq/mL. Solutions of [ 131 I]NaI were purchased from the Japan Radioisotope Association (JRIA). Ascorbic acid (used as a reducing agent) and sodium bicarbonate (used as a pH adjuster) were added to the 131 I solution to a final concentration of 1% (w/v) at pH 8.0, and the solution was allowed to stand for 1 h at 23 ± 2 • C. The concentration of I was 50 MBq/mL. To measure DSBs, K1-NIS cells were seeded in an eight-well chamber slide at a density of 1 × 10 5 cells/mL. After two days of incubation, the cells were treated with 10 µL medium/well as the control group; 10, 30, 100, 300, and 1000 kBq [ 211 At]NaAt solution/well as 211 At groups; and 10, 30, 100, 300, and 1000 kBq [ 131 I]NaI solution/well as 131 I groups for 20 min. The volume of the solution was approximately 325 µL/well during treatment. After washing with phosphate-buffered saline (PBS), the cells were stained using the HCS DNA Damage Kit (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The fluorescence signals were observed using a fluorescence microscope (BZX-810; Keyence Corporation, Osaka, Japan). The ability to induce DSBs was calculated as the percentage of cells with >5 γH2AX foci/cells treated with both solutions. Colony formation was calculated using ImageJ software and compared between the groups. Cells of interest were selected, and areas of nuclear morphology (Hoechst 33342) and DNA damage (pH2AX antibody) were observed. K1, K1-NIS, and K1-NIS/DOX cells were seeded in 24-well plates to 70-80% confluence and detached for the colony formation assay. The cells in each well were treated with 0, 10, 30, 100, 300 and 1000 kBq [ 211 At]NaAt solution and 0, 10, 30, 100, 300 and 1000 kBq [ 131 I]NaI solution. The volume of the solution was 0.5 mL. After 1 h of treatment at 37 • C in a humidified atmosphere of 5% CO 2 , cells were counted and seeded in fresh medium in 24-well plates at a density of 500 cells/well. After 14 days of incubation, the cells were fixed and stained with a crystal violet solution. The cells were viewed and counted under a microscope (Primo Star, Carl Zeiss AG, Oberkochen, Germany).

Preparation of Animals
Male nude mice were purchased from Japan SLC, Inc. (Hamamatsu, Shizuoka, Japan), housed under a 12-h light/12-h dark cycle, and allowed free access to food and water.

In Vitro and In Vivo NIS Control Model
For For NIS control experiments, K1-NIS/DOX cells were cultured in special serum to eliminate the effects of tetracycline analogs in normal serum. One day before nuclide treatment, the K1-NIS/DOX cells were treated with doxycycline at the determined concentrations. In the K1-NIS/DOX tumor-bearing model, doxycycline was intraperitoneal administrated two days before the injection (2 mg/mouse). Doxycycline was administered every two days, while [ 131 I]NaI was expected to remain in the body.

Statistical Analysis
Results are expressed as the mean ± standard deviation. Comparisons between groups were performed using unpaired t-tests in Microsoft Excel (version 2016, Microsoft Corp., Redmond, WA, USA). For multiple comparisons among the three groups, Bonferroni correction was performed. Differences were considered statistically significant at p < 0.05.

Conclusions
In this study, 211 At showed effective DSBs induction with higher cellular toxicity, and the administration of [ 211 At]NaAt was more effective in an NIS-expressing thyroid cancer model than the administration of [ 131 I]NaI. These results suggest that [ 211 At]NaAt therapy is a more promising option than [ 131 I]NaI treatment for patients with iodine-avid thyroid cancer refractory. It was also confirmed that the amount of uptake was proportional to the expression level of NIS and that the therapeutic effect was also proportional to the expression level of NIS. However, with [ 211 At]NaAt, a certain degree of therapeutic effect was observed even when the expression level of NIS was low and no side effects were observed, indicating the usefulness of [ 211 At]NaAt.