In Vitro Evaluation System of Pharmacokinetics and Irradiation Effect in Boron Neutron Capture Therapy (BNCT) Using Three-Dimensional Artificial Human Tumor Tissue Model

Boron neutron capture therapy (BNCT) is based on the incorporation of boron-containing drugs to cancer cells and the nuclear reaction of 10 B atoms by thermal neutron irradiation results in tumor degeneration. For the development of this therapy, currently, long time and high cost consuming experiments using many animals are required. In this study, we constructed a new in vitro evaluation system for BNCT by combination of an artificial tumor tissue model, comprised of normal human dermal-derived fibroblast (NHDF) and human pancreatic cancer cell line BxPC3, and the optical plastic material CR-39 as a solid state nuclear track detector. Administration of boronophenylalanine ( 10 BPA) as a boron-containing drug and neutron irradiation up to 2.52 × 10 12 n/cm 2 to BxPC3 cells and NHDF was evaluated as 5.46 and 1.71, respectively. The tumor and normal tissue ratio (T/N ratio) was 3.19, which was corresponded with those of BPA as 2 - 4 that reported in the previous studies. This new in vitro evaluation system may provide a useful tool for a low cost, labor-saving, and non-animal method for the development of new boron-containing drugs or improvement of BNCT conditions.


Introduction
Boron neutron capture therapy (BNCT) is based on the nuclear reaction of nonradioactive isotope 10 B atoms that absorb low-energy (<0.5 eV) neutrons (thermal neutrons) disintegrate into an alpha ( 4 He) particle and a recoiled lithium nucleus ( 7 Li) that deposit high energy along their very short path (<10 μm) and result in destroying of only malignant cells with 10 B following thermal neutron irradiation [1]- [7]. Recently, ground-breaking results of BNCT for refractory cancer have been reported by clinical use of two boron-containing drugs, borocaptate sodium ( 10 BSH) and Boronophenylalanine ( 10 BPA). In regard to this new therapy, the improvement of the techniques and the development of new boron-containing drugs for higher performance are vigorously promoted [8] [9].
However, currently, long time and high cost consuming experiments using many animals are required for these studies. Moreover, the analysis and evaluation of pharmacokinetics and irradiation effects in human tissue at microscopic level are still difficult. Therefore, the establishment of more efficient in vitro experimental models is demanded for effective development of BNCT.
For this purpose, we focused on the usage of the artificially engineered tumor tissue models constructed by an extracellular matrix (ECM)-based three-dimensional tissue-constructing method, known as cell-accumulation technique [10] [11] [12] [13]. In previous studies, the in vitro human artificial tumor tissue models were prepared by seeding human cancer cells on the three-dimensional culture of normal human dermal-derived fibroblast (NHDF) involving vascular networks to apply for drug discovery, pathological model and diagnostic tool [10] [11] [12] [13].
In this study, we constructed new evaluation system for BNCT by com-  [7]. Using this in vitro system and 10 BPA as a boron-containing drug, we evaluated the irradiation damage to the cancer cells on the tissue by analyzing the alpha-ray/recoiled Li particle tracks on CR-39.

Preparation of in Vitro Human Three-Dimensional Tumor Tissue Model
Human three-dimensional tumor tissue was prepared by cell accumulation method [10] [11] [12] [13]. First, connective tissue-like structures were fabricated by three-dimensional lamination of NHDFs. As shown in Figure 1 [13]. The cells are seeded on the transwell inserts at a density of 27.2 × 10 5 cells/insert (8 layers) and cultured under the conditions of 5% carbon dioxide at 37˚C for 12 to 24 hours. We regarded this connective tissue-like structure as an artificial human normal tissue model, termed as NHDF3D.
Next, RFP-labeled BxPC3 cells which have been cultured and proliferated were collected by trypsin treatment, washed, and uniformly seeded on the upper surface of NHDF3D at a density of 300 cells/mm 2 , then further cultured for 24 hours under the above culture conditions. As shown in Figure 2(e) and Figure   2(f), BxPC3 cells proliferated on the surface of NHDF3D forming the groups with flat shape. We regarded this cancer-loaded NHDF3D as an artificial human tumor tissue model, termed as NHDF3D/BxPC3. In the present study, one case of NHDF3D and two cases of NHDF3D/BxPC3 were prepared by the methods mentioned above and were used for the experiments.

BPA Immersion Treatment and Fixation
The boronophenylalanine (BPA) solution (3% w/v) was diluted to a concentration Journal of Cancer Therapy of 40 ppm with DMEM containing 10% FBS. This is referred to as a BPA treatment solution. After removing the culture solution of NHDF3D or NHDF3D/ BxPC3, 750 μL of BPA treatment solution was added, and incubated for 2 hours (BPA exposure) under conditions of 5% carbon dioxide at 37˚C (Figure 1(b)).
After that, the BPA treatment solution was removed and the tissues were washed 3 times with 0.01 M phosphate-buffered saline (PBS, pH 7.3). Subsequently, the tissues were fixed by 4% paraformaldehyde/0.1M phosphate buffer (pH 7.3) for 30 minutes at room temperature shading the light. After the fixation, the cellular nucleus was stained by 4',6-diamidino-2-phenylindole (DAPI).

Observation of BxPC3 Cell Distribution on the Artificial Tissues
After the fixation, three holes were provided on the tissue using an 18 G injec-

Neutron Irradiation Experiment
The above-mentioned NHDF3D or NHDF3D/BxPC3 in the transwell inserts were cut out with a knife together with the polyester base, mounted on the solid track detector CR-39 [6] [7] with close contact, and used as a sample for track image acquisition (Figure 1(d)). Irradiation experiments using these samples were conducted at the Heavy Water Neutron Irradiation Facility of Kyoto University Reactor (KUR), and irradiation was performed for 30 minutes under an irradiation flux of 1.4 × 10 9 n/cm 2 /s (total flux = 2.25 × 10 12 n/cm 2 ) ( Figure   1(d)). After the neutron irradiation, the above sample was etched (6 N NaOH, 70˚C × 2 hours) to visualize the α-ray/recoiled Li particle tracks generated on the CR-39 surface.

Analysis of α-Ray/Recoiled Li Particle Track Images
The α-ray/recoiled Li particle track image of etched CR-39 was taken using the bright field function of the fluorescence microscope BZ-X700 (Figure 1(e)). A low magnification image including the entire tissue mount and 10 random high magnification images were obtained respectively, and the following analysis was performed.
1) The whole tissue images of fluorescent BxPC3 cell distribution and the α-ray/recoiled Li particle track distribution were compared referring to the position of three-hole markers, and the relationship of these distributions was observed.
2) Alpha-ray/recoiled Li particle tracks in the high magnification images were regarded as the particles, and quantitatively analyzed by using software FIJI (https://fiji.sc). Briefly, after binarizing the image, the tracks with more than 4 μm diameter were detected, and their number per unit area (0.01 mm 2 ) and the track size were measured. Significant difference between the data from the artificial normal tissue model (NHDF3D) and artificial tumor tissue model (NHD-F3D/BxCP3) was confirmed using student t-test.

Measurement of Nuclear Size and Cell Size of BxPC3
For

Distribution of the BxPC3 Cells and Alpha-Ray/Recoiled Li Particle Tracks on the Tissue Models
Low-magnification images of NHDF3D and NHDF3D/BxPC3 (two cases) detecting RFP fluorescence were shown in Figures 3(a)-(c). Although the BxPC3 cells were seeded over the artificial tissue in regular cell concentration, slight heterogeneity of cell distribution was observed after the cultivation for 24 hours. The parts with intense fluorescence denoting high density of BxPC3 cells were indicated by yellow circles in the figures. The α-ray/recoiled Li particle tracks in corresponded parts of Figures 3(a)-(c) were shown in Figures 3(a')-(c'). Asterisks indicate the marking holes to match the position of the tissue images and the track images. The concentration of α-ray/recoiled Li particle tracks in NHDF3D/BxCP3 is higher than those of NHDF3D. Moreover, relatively high concentration of the α-ray/recoiled Li particle tracks was observed at the yellow circles in Figure 3(b) and Figure 3(c). These results suggested that the preferential incorporation of 10 B into BxPC3 cells and their distribution on the tumor tissue model were reflected by the α-ray/recoiled Li particle track images.   Figure 4 in high magnification. (a')-(c'): α-ray/recoiled Li particle track distribution of (a)-(c). The tracks are observed as the gray colors with several concentration. The relatively high concentration parts in (b') and (c') indicated by hatched yellow circles correspond to those of (b) and (c). By observing the fluorescence optical tissue, we were able to grasp the nuclei, cellular tissue structure, shape, and distribution state of the manufactured artificial tumor tissue before BNCT treatment.  Figures 4(a')-(c'), the α-ray/recoiled Li particle tracks were observed as small dots, reflecting the intensity of the emission. Since the contrast between NHDF3D (a') and NHDF3D/BxPC3 (b' or c') was obvious, the BxPC3 cells-dependent incorporation of 10 B resulting the α-ray/recoiled Li particle emission was clearly detected in this system. Furthermore, as shown by the area surrounded with yellow and red hatched lines, the high density of BxPC3 cells on the tissue in Figure 4(b) and Figure 4(c) was comparative with the high intensity of the α-ray/recoiled Li particle emission in (b') and (c'). From these results, it was suggested that 10 BPA was abundantly taken in proportion to the tumor concentration locally at a high tumor concentration site.

Quantitative Analysis of α-Ray/Recoiled Li Particle Tracks in NHDF3D and NHDF3D/BxCP3 Samples
To investigate detailed α-ray/recoiled  Figure 4(d) and Figure   4(e). The number of α-ray/recoiled Li particle tracks of NHDF3D/BxCP3 was about 1.6 times higher than that of NHDF3D ( Figure 5(d)).
Next, we evaluated the hit number of α-ray/recoiled Li particle tracks per single cell on the top layer of NHDF3D and NHDF3D/BxCP3 as shown in Table 1.
In the control tissue NHDF3D, the number of α-ray and recoiled Li particles tracks (H B ) was 51.52/0.01 mm 2 as the value of Figure 5 (1) and (2) according to the seeded cell number and duplication at 24 hours after seeding that was confirmed in previous study (data not shown  (1) and NHDF3D/BxPC3 (2). The binary data were prepared from the photo images and particle analysis was performed to count the number of the tracks and measure the of their size. (d): the number of the particle tracks in 0.01 mm 2 . By further expanding the tissue change sites in the normal tissue and tumor tissue in the artificial tumor tissue after BNCT treatment, it was possible to grasp in detail the α track shape and distribution that occurred after BNCT treatment. experiments [16].
From these results, it was suggested that our in vitro method can reproduce the in vivo T/N ratio of BPA and available for evaluation system of pharmacokinetics.
In conclusion, our in vitro model of tumor tissue for BNCT demonstrated the pharmacokinetics of BPA and the efficacy of neutron irradiation by direct observation of α-ray/recoiled Li particle tracks that are corresponding to the distribution of BxPC3 cells. Moreover, the evaluated number of α-ray/recoiled Li particle tracks per single BxPC3 cell or NHDF provided the comparable value with T/N ratio of BPA in the previous studies. These results suggested that our in vitro model can be applied for a reproducible and high throughput method which enables to develop new boron-containing reagents from many drug can-Journal of Cancer Therapy didates for BNCT without any animal experiments. Our model also can be used to evaluate the optimum conditions of BNCT such as dose of boron-containing drugs and the irradiated intensity (power and time) of neutron to the patients.
Particularly, the examination using cancer cells obtained from each individual patient may enable to provide the safety and effectiveness for personalized BNCT.
As a limitation of this system, the detectable α-ray/recoiled Li particle tracks by CR-39 is restricted only those from the top layer of the artificial tissue, so that the other cancer cells such that rapidly infiltrate into the tissue cannot be applied. Three-dimensional detection of boron-containing drugs or irradiation damages distribution may extremely increase the value of this evaluation system as a tool for the development of BNCT and related medical studies.