Radiation Sensitivity of in Vitro Evaluation System of Pharmacokinetics in Boron Neutron Capture Therapy (BNCT) Using Three-Dimensional Artificial Human Tumor Tissue Model

One of the important matters that must be determined in advance when per-forming BNCT treatment is the optimization of neutron irradiation time and dose. In this article, following the previous article (2.52 × 10 12 n/cm 2 ) (Case 1), double irradiation (5.04 × 10 12 n/cm 2 ) was further performed (Case 2) by verifying the radiation sensitivity performance of the artificial tumor tissue NHDF3D/BxPC3 and the possibility of evaluating the optimum neutron dose required for treatment was examined. As a result, although the radiation damage rate in the normal tissue NHDF3D and the tumor tissue BxPC3 in-creased in proportion to the irradiation dose due to heavy irradiation in Case 1 or more, the increase in the damage rate in the normal tissue exceeded the tumor tissue. Furthermore, the tumor/normal tissue damage ratio T/N ratio showed the maximum value in Case 1, and the dose ratio in Case 2 with a higher dose showed a tendency to decrease. From the above experimental facts, it was shown that irradiation dose optimization is possible to some extent by an evaluation method using an artificial tumor tissue.


Introduction
In the previous paper [1], the authors made a bilayer 3D artificial tumor tissue (BxPC3/NHDF3D) using human pancreatic cancer cell line BxPC3 and normal human dermal-derived fibroblast NHDF3D, and pharmacokinetic study for 10BPA-BNCT treatment [2]- [10] by neutron irradiation from reactor Went. As a result, optical observation of the irradiated tissue gave a T/N ratio of 3.19 at a neutron dose of 2.52 × 10 12 n/cm 2 , indicating the effectiveness of the BNCT pharmacokinetics test using 3D artificial tissue [11]- [16].
On the other hand, what is important in BNCT treatment of tumor patients is to secure the accumulation in the tumor affected area after the boron drug administration and to optimize the irradiation dose during the treatment. Therefore, in this paper, we examined the applicability of the 3D artificial tumor tissue used in the previous paper [1] as a testing technique for optimizing the radiation dose at the time of BNCT treatment while verifying the sensitivity performance to radiation dose.

Cells, Reagents and Instruments
Normal human dermal-derived fibroblast (NHDFs) and red fluorescent protein (RFP) -labeled human pancreatic cancer cell line BxPC3 used in the experiment were purchased from LONZA (Walkersville, MD) and Anti-Cancer Japan (Ibaraki, Japan), respectively. Dulbecco's modified Eagle's medium (DMEM) (Wako, Osaka, Japan) containing 10% fetal bovine serum (FBS) (Nichirei, Tokyo, Japan) was used to proliferate cells prior to construction of the tumor tissue model. The cells were cultivated at 37˚C, 5% carbon dioxide. Bovine plasma-derived fibronectin (FN) and porcine skin gelatin (G) were purchased from Sigma-Aldrich

Preparation of in Vitro Human Three-Dimensional Tumor Tissue Model
Human three-dimensional tumor tissue was prepared by cell accumulation method [11] [12] [13] [14]. First, connective tissue-like structures were fabricated by three-dimensional lamination of NHDFs. As shown in Figure 1 In present paper, we adopted combination of an artificial tumor tissue model, comprised of normal human dermal-derived fibroblast (NHDF) and human pancreatic cancer cell line BxPC3.
[11] [12] [13] [14]. 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 [1].
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. BxPC3 cells proliferated on the surface of NHDF3D forming the groups with a 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 three 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 concen-

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

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 [7] [8] 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 60 minutes under an irradiation flux of 1.4 × 10 9 n/cm 2 /s (total flux = 4.5 × 10 12 n/cm 2 ). After the neutron irradiation, the above sample was etched (6N NaOH, 70˚C × 2 hours) to visualize the α-ray/recoiled Li particle tracks generated on the CR-39 surface [1].

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. 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. A significant difference between the data from the artificial normal tissue model (NHDF3D) and artificial tumor tissue model (NHDF3D/BxCP3) was confirmed using student t test [1].

Distribution of the BxPC3 Cells and Alpha-Ray/Recoiled Li Particle Tracks on the Tissue Models
Low-magnification images of NHDF3D and NHDF3D/BxPC3 (three cases) detecting RFP fluorescence were shown in Figure 2  In these figures, the α-ray/recoiled Li particle tracks were observed as small dots at #1 -#12, reflecting the intensity of the emission. These tracks distribution and density at different observation points in each tissue are approximated for each group. This means that the α-ray/recoiled Li particle damage occurs almost uniformly in each tissue.

Radiation Sensitivity of NHDF3D and NHDF3D/BxCP3
Here, we verified the suitability as a tool to determine the dose optimization during BNCT treatment by using NHDF3D/BxPC3.
There is a clear difference in track density between the two, and it can be seen that the higher the neutron irradiation amount, the higher the track density. Similarly, when the track density of NHDF3D/BxPC3 was compared between Figure 5(B) and Figure 5(b'), it was found that the track density was high at the high dose NHDF3D/BxPC3.    were prepared from the photo images and particle analysis was performed to count the number of the tracks and measure the of their size and the number of the particle tracks in 0.01 mm 2 (track density). 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 in Case 1 and Case 2.
The track density increases as the irradiation dose increases in this way, meaning that 10 B absorbed in the sample remains in the tissue in an unreacted state even after neutron irradiation for a short time of about 30 minutes [1]. Figure 6 shows the relationship between neutron irradiation dose (n/cm 2 ) and track density (counts/0.01 mm 2 ). According to this, the increase rate of NHDF3D and BxPC3 is different with the increase of the irradiation amount, and the increase rate of the track density of NHDF3D is remarkable. On the other hand, it can be seen that the increase rate of BxPC3 is almost saturated, however, in Case 2, the rate of increase is similar to that of normal cells.
This means that when neutrons dose reached up to 4.25 × 10 12 n/cm 2 or more, the effect of radiation damage on normal cells must be considered in addition to the original BNCT treatment effect. Figure 7 shows the relationship between the neutron irradiation dose and the T/N ratio, where the definition of T/N ratio here is defined as the track density ratio of NHDF3D and BxPC3 in Table 1. According to this, although the T/N ratio increases as the neutron irradiation dose increases, it turns to decrease when the irradiation dose in Case 1 is exceeded. In other words, in Case 2 from the viewpoint of BNCT treatment, radiation damage to the normal tissue becomes significant and there is a possibility of over-irradiation.
S. Ishiyama, M. Suzuki Figure 6. The relationship between irradiation dose and track density. The increase rate of NHDF3D and BxPC3 is different with the increase of the irradiation amount, and the increase rate of the track density of NHDF3D is remarkable. On the other hand, it can be seen that the increase rate of BxPC3 is almost saturated, however, in Case 2, the rate of increase is similar to that of normal cells. 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 [1]. Furthermore, in this paper, following the previous paper, heavy irradiation was performed using the same artificial tumor tissue. As a result, it became clear that selection of the optimal dose, which is one of the most important treatment conditions for BNCT treatment, is possible to some extent by an evaluation method using an artificial tumor tissue.