Image-Guided Neutron Capture Therapy Using the Gd-DO3A-BTA Complex as a New Combinatorial Treatment Approach

Gadolinium-neutron capture therapy (Gd-NCT) is based on the nuclear capture reaction that occurs when 157Gd is irradiated with low energy thermal neutrons to primarily produce gamma photons. Herein, we investigated the effect of neutron capture therapy (NCT) using a small molecular gadolinium complex, Gd-DO3A-benzothiazole (Gd-DO3A-BTA), which could be a good candidate for use as an NCT drug due to its ability to enter the intracellular nuclei of tumor cells. Furthermore, MRI images of Gd-DO3A-BTA showed a clear signal enhancement in the tumor, and the images also played a key role in planning NCT by providing accurate information on the in vivo uptake time and duration of Gd-DO3A-BTA. We injected Gd-DO3A-BTA into MDA-MB-231 breast tumor-bearing mice and irradiated the tumors with cyclotron neutrons at the maximum accumulation time (postinjection 6 h); then, we observed the size of the growing tumor for 60 days. Gd-DO3A-BTA showed good therapeutic effects of chemo-Gd-NCT for the in vivo tumor models. Simultaneously, the Gd-DO3A-BTA groups ([Gd-DO3A-BTA(+), NCT(+)]) showed a significant reduction in tumor size (p < 0.05), and the inhibitory effect on tumor growth was exhibited in the following order: [Gd-DO3A-BTA(+), NCT(+)] > [Gd-DO3A-BTA(+), NCT(−)] > [Gd-DO3A-BTA(−), NCT(+)] > [Gd-DO3A-BTA(−), NCT(−)]. On day 60, the [Gd-DO3A-BTA(+), NCT(+)] and [Gd-DO3A-BTA(−), NCT(−)] groups exhibited an approximately 4.5-fold difference in tumor size. Immunohistochemistry studies demonstrated that new combinational therapy with chemo-Gd-NCT could treat breast cancer by both the inhibition of tumor cell proliferation and induction of apoptosis-related proteins, with in vivo tumor monitoring by MRI.


Introduction
Neutron capture therapy (NCT) is a well-known approach to cancer treatment based on the accumulation of neutron capture agents at the tumor site [1], followed by irradiation with thermal neutrons. NCT is a very effective technique for cancer treatment because the thermal neutrons with low energy do not cause damage to normal cells that lack the neutron capture compounds. is technique thus provides a solution to the major problem of radiation therapy, which is the radiation-induced damage to normal tissue. erefore, the strategy for the accumulation of NCT compounds specifically within the tumor is critical to avoid damage to normal tissues [2][3][4][5].
Gadolinium ( 157 Gd)-based NCT (Gd-NCT) has generated recent interest as a cancer treatment due to the following merits. (i) e 157 Gd atom captures neutrons ( 157 Gd + n th (0.025 eV) ⟶ [ 158 Gd] ⟶ 158 Gd + γ + 7.94 MeV) across a very large cross section (σ th � 240,000 b). (ii) e kinetic energy of Gd-NCT, with a mixture of low-and high-energy ionizing particles, is more uniformly distributed throughout tumor tissues, and can be used to solve the shortcomings of heterogeneous in vivo dose distribution. (iii) 157 Gd complexes are widely developed as magnetic resonance imaging (MRI) contrast agents by chelation chemistry and have been routinely used in clinical applications including Gadovist ® (gadobutrol), Dotarem ® (gadoterate meglumine) and Omniscan ® (gadodiamide) [6]. Gadolinium MRI contrast agents could thus be first considered as NCT agents, but it is difficult to specifically target tumor cells in vivo. ese agents do not accumulate well in tumor tissue during neutron irradiation after an intravenous injection for therapy, as shown in Table 1. e data show high uptake of the contrast agents into tumor cells at 5 mins, but they exhibit very low uptake at 2 hrs. To obtain success with 157 Gd-NCT, 157 Gd must be transferred into tumor cells at high concentrations during neutron irradiation. In a previous study, a 50-200 μg 157 Gd/g tumor was reported as an effective cancer treatment.
For the 157 Gd neutron capture reaction, the majority of the energy is released as long-range gamma radiation, while, 0.63% of the time, this emission occurs as Auger and conversion electrons. Auger electrons generated from Gd-NCT have strong in vivo cytotoxicity by high linear energy transfer (LET), which can induce DNA double-strand breaks (DSB) and restrain the proliferation of tumor cells. us, increase in the therapeutic effects is observed when Gd atoms are highly internalized into tumor cells [18,19].
As therapeutic candidates, various benzothiazoles deserve special attention, as they are known to possess diverse biological properties such as anti-inflammatory, antimicrobial, and anticancer effects. Some of the compounds containing the benzothiazole system are in clinical usage for the treatment of various diseases/disorders [20]. In our previous study, the complexes have not only displayed tumor specificity but also enhanced intracellular MR images of the cytosol and nuclei of a series of tumor cells. e antiproliferative activity of Gd-DO3A-BTA (Chart 1), which contains a chelating moiety (DO3A) and a chemoagent region (BTA), was demonstrated by determining the in vitro growth inhibition values (GI 50 and TGI) and monitoring tumor volume regression in vivo. In particular, Gd-DO3A-BTA has been reported to specifically accumulate intracellularly in tumors arising from MDA-MB-231 breast cancer cells [9,21]. On the basis of this result, we make attempts to treat in vivo tumor tissue by neutron beam irradiation with a medical cyclotron, preserving the high tumor uptake of 157 Gd complexes.

2.1.
In Vivo MR Imaging. Figure 1(a) shows the in vivo T1weighted MR images of mice that were injected with Gd-DO3A-BTA (0.1 mmol Gd/kg) via the tail vein. T1-weighted MR images were used because the T1 shortening effect is more dominant at the relatively low Gd concentrations used (∼0.1 mmol Gd/kg). e MR image reveals clear tumor enhancement, which increased for 6 h before gradually decreasing (Figure 1(b)). is result is consistent with the biodistribution data reported in our previous study [9], demonstrating that we can effectively define the starting point of NCT with in vivo MR imaging.

Gd-NCT Inhibited Tumor Growth with Gd-DO3A-BTA.
A small animal study was performed in which mice were irradiated with a neutron beam at 6 h p.i. to determine the optimal time at which the highest uptake of Gd-DO3A-BTA into the tumor tissue occurred. All other tissues, except the tumor tissues, were covered with a plastic box of Teflon to protect them from irregular showering with neutron beams.  11.99 ± 5.05, which was much smaller than that in the other groups (p < 0.05) on day 60. Due to the tumor suppressive effects of BTA, the antitumor activity of Gd-DO3A-BTA alone (no neutron beam irradiation) was higher than that of neutron irradiation alone. Consistent with earlier reports [10,22,23], the neutron irradiation [Gd-DO3A-BTA(−), NCT(+)] alone group also showed some inhibition of tumor growth than the group that did not receive either treatment [Gd-DO3A-BTA(−), NCT(−)]. After irradiation by neutron beams, changes in the body weight of the mice were measured as well as tumor volume. As shown in Figure 2(b), no significant weight loss was observed, suggesting the safety of Gd-DO3A-BTA for NCT. e suppression of tumor growth could be clearly seen from the MR images and morphological findings shown in Figure 3.

Discussion
For the success of Gd-NCT as a therapeutic modality for cancer, it is important to effectively deliver and sufficiently accumulate Gd into tumors. For an effective in vivo Gd-NCT, an optimal dose of 50-200 µg·Gd/g tumor tissue has been reported [11,25]. As shown in Table 1, 0.1 mmol/kg of Gd-DO3A-BTA administered as an intravenous injection resulted in approximately 221 µg of Gd/g tumor tissue [9].
Various nano-based formulations have been developed to deliver sufficient amounts of Gd into tumor cells for Gd-NCT, and these include nanoparticles [15][16][17][26][27][28], liposomes [10,11,12], emulsions [13,29], and microcapsules [7,30]. Most of these particles were designed to be approximately 100 nm in size to avoid high uptake by the reticuloendothelial system (RES). However, these particles have limited specific tumor targeting ability, since their delivery completely depends on the enhanced permeability and retention (EPR) effect around tumor sites [11,14,31]. Furthermore, these nanoparticles often show high in vivo instability by releasing Gd from the nano-based formulations due to the high solubility of Gd-DTPA in an aqueous solution [11,13,15]. erefore, some nano-based formulations for Gd-NCT use a lipophilic complex such as Gd-DPTA/pLL or Gd-acetylacetonate (Gd-acac) to prevent the possible release of Gd [11,29]. However, as shown in Table 1, these nano-based formulations can be injected only by intraperitoneal or intratumoral methods and cannot be administered intravenously.
Gd-DO3A-BTA, however, is readily administered intravenously. Furthermore, Gd-DO3A-BTA has a relatively  e MR imaging data demonstrated that Gd-DO3A-BTA preferentially accumulated in tumor tissue in vivo. Gd-DO3A-BTA delivers more Gd to tumor tissue than conventional Gd-based MRI contrast agents, which may result in a more efficient uptake by the tumor. e effective tumor ablation by Gd-DO3A-BTA could therefore be associated with the high accumulation of Gd in tumor tissue, which in turn captures sufficient thermal neutrons to kill the tumor. Although enriched 157 Gd compound would be preferable for Gd-NCT, in this experiment, we utilized the natural Gd compound. e natural abundance of 157 Gd is only 15.65%, while enriched compounds may contain up to 90% 157 Gd [20]. erefore, in the near future, it would be important to perform Gd-NCT experiment with enriched 157 Gd compounds to confirm the current results using the natural Gd compound. e tumors in mice that were irradiated and injected with Gd-DO3A-BTA were much smaller than those observed in other groups, demonstrating that the Gd-DO3A-BTA could significantly enhance the therapeutic effect of Gd-NCT. In addition, the tumor-therapeutic effects of Gd-DO3A-BTA in combination with NCT were confirmed by MR imaging and histological evaluation. Furthermore, the tumor suppressive effects were evident even after 60 days, suggesting that early treatment of the tumor with a Gd-DO3A-BTA injection, and NCT has the potential to suppress further tumor progression. However, the limitation of the current study should be mentioned. In this study, the neutron beam was generated by irradiating a proton beam upon a beryllium target using an MC-50 cyclotron, and the thermal neutron flux was low. Because the high flux of thermal neutrons is usually used for   Contrast Media & Molecular Imaging NCT, it is necessary to perform NCT experiment with low energy neutron at high flux to be sure that the effect shown in the current study is due to NCT.

Conclusions
e present study presented the potential for Gd-NCT using low molecular weight Gd chelate as a new combinatorial chemo-NCT approach in cancer treatment. Despite the simple chemical structure of the metal complex, Gd-DO3A-BTA shows a high therapeutic effect when used in Gd-NCT against solid tumors. e high accumulation of Gd-DO3A-BTA in tumor tissues effectively damaged the tumor cells. Furthermore, as an MR contrast agent, Gd-DO3A-BTA can guide NCT and monitor tumor growth via MR imaging.

General.
e Gd-DO3A-BTA (nonenriched Gd) was prepared as described previously [10,21]. All animal experiments were conducted in compliance with the Guidelines for the Care and Use of Research Animals under protocols approved by the Korea Institute of Radiological and Medical Sciences (KIRAMS) Animal Studies Committee. e neutron beam irradiations including low content thermal neutrons were performed using an MC-50 cyclotron (Scanditronix, Sweden, 1985). When the neutron beam was generated by irradiating a proton beam (20 μA, 35 MeV) upon a beryllium target of 15 mm thickness, the following results were previously reported: the thermal neutron (0.24% of total neutrons) flux was approximately 1.94 × 10 4 n/cm 2 ·sec and the cross section was 13.79 ± 0.45 barn (a). At that time, the absorbed dose of neutron beams was 9.36-8.69 cGy/min (including the gamma ray of 1.42-1.57 cGy/min) at the depth of 15-30 mm with the field size of 26 × 26 cm 2 (b). e irradiation time of neutron beams in our studies was typically performed as the standard point of approximately 1 Gy/12 min [32,33].

Tumor Model.
e human breast adenocarcinoma cancer cell line MDA-MB-231 (ATCC CRM-HTB-26) was purchased from the American Type Culture Collection (ATCC). MDA-MB-231 is a highly aggressive, invasive, and poorly differentiated triple-negative breast cancer (TNBC; estrogen receptor (ER), progesterone receptor (PR), and HER2 (human epidermal growth factor receptor 2)) cell line. e cells were maintained in RPMI-1640 containing 10% fetal bovine serum (FBS) and 1% antibiotics and were grown in a humidified incubator at 37°C and 5% CO 2 . MDA-MB-231 tumor cells (1×10 8 cells·mL −1 ) suspended in RPMI-1640 medium without FBS, and antibiotics were injected into the subcutaneous tissue (sc) of female BALB/c nude mice (aged 6 weeks, 18-25 g of body weight) in both legs. One week after tumor cell implantation, the mice were divided into two groups (n � 5/group). To compare the effect of chemotherapy and NCT on solid tumor, the mice were administered (A) none or (B) Gd-DO3A-BTA (0.1 mmol/kg) intravenously through the tail vein. Only the right-sided tumors were irradiated by neutrons.

In Vivo MR Imaging.
e mice were anesthetized with 1.5% isoflurane in oxygen. Tumor measurements were made before and after injection of 0.1 mmol·Gd/kg via the tail vein. MR images were taken with a 3 T MR unit (Magnetom Tim Trio, Siemens Medical solution, Erlangen, Germany) using an animal coil. e T1-weighted fast spin-echo imaging was performed under the following conditions: repetition time � 9.9 ms; echo time � 3.2 ms; 10 mm field of view; 256 × 256 matrix size; 1 mm slice thickness; and average � 3. e contrast to noise ratio (CNR) was defined as the difference in signal-to-noise ratio (SNR) between adjacent anatomic structures.

In Vivo Gd-NCT.
Gd-DO3A-BTA was administered intravenously as a bolus (0.1 mmol/kg) into the tail vein of female mice with MDA-MB-231 tumors. After 6 h, the mice were locally irradiated on one side with a 0.3 Gy neutron beam, while the other side was shielded using Teflon as shown. Figure 5 shows the overall experimental scheme for Gd-NCT. e tumor size was measured before and after irradiation, and the volume (V) was calculated using the following equation: where a and b are the major and minor axes of the tumor measured by a caliper.

Tissue Preparation and Histological
Evaluation. e animals were sacrificed after 60 days, and tumor tissues were obtained. e samples were fixed in 10% neutral buffered formalin, processed, and embedded in paraffin, according to standard procedures. e sections were then stained with hematoxylin and eosin (H&E) using an automated processor (Tissue-Tek, PrismaE2, Sakura, Japan). e images were acquired using a digital scanner (Panoramic MIDI, 3D HISTECH Ltd, Hungary).

Immunohistochemical Analysis.
e tumor samples were serially sectioned (4 μm sections), deparaffinized, and rehydrated. e sections were incubated in 0.3% hydrogen peroxide to quench the endogenous peroxidase after antigen retrieval by heating in 10 mM citrate buffer (pH 6.0). e immunohistochemistry was performed according to the avidin-biotinylated-HRP complex (ABC) method using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA, USA). e primary antibodies used were Ki-67 and caspase 8 (both 1 : 100, Abcam, Cambridge, MA, USA) and caspase 3 (1 : 100, Santa Cruz, Santa Cruz, CA, USA). Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL; Roche Diagnostics, Indianapolis, IN, USA) according to the manufacturer's instructions. All immunohistochemistry reactions and TUNEL staining were visualized by Vector SG (Vector Laboratories) and counterstained with fast nuclear Contrast Media & Molecular Imaging solutions (Vector Laboratories). Staining was quantified using ImageJ image analysis software (NIH, Bethesda, MD, USA).

Statistical
Analysis. Data in the manuscript are expressed as the mean and standard deviation (SD), and the significance of the results was analyzed by Student's t-test. p < 0.05 was considered statistically significant.

Data Availability
e data used to support the findings of this study are included within the article.

Conflicts of Interest
e authors declare that they have no conflicts of interest.