Project 5 The effect of BNCT on normal tissues (R4P5)

概要

I-1.

PROJECT RESEARCHES
Project 5

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PR5

The effect of BNCT on normal tissues

M. Suzuki
Institute for Integrated Radiation and Nuclear Science, Kyoto
University

In 2020, accelerator-based boron neutron capture
therapy (BNCT) for head and neck cancer was approved by Japanese Ministry of Health, Labor, and Welfare. At two medical institutes, BNCT for head and neck
cancer has been started under the insurance coverage.
At Institute for Integrated Radiation and Nuclear Science, Kyoto University (KURNS), BNCT for body
trunk tumors such as malignant pleural mesothelioma,
liver tumors, and breast cancers had been carried out
using Kyoto University Research Reactor (KUR) until
2019. These malignancies will be expected to be treated
with accelerator-based BNCT in the medical institutes.
For planning clinical trials of BNCT, the preclinical
studies are very important. Especially the scientific
findings on safety of BNCT are indispensable for firstin-men clinical trials. Therefore, I have started this project investigating the effect of BNCT on normal tissues
or organs. Unfortunately, due to shortening of operation
of KUR in 2022, three of six researches were not carried
out. Details of three projects are referred to each progress report. I hope that this project will arouse much
interest in BNCT researchers and shed new light on radiation biology.

Boron neutron capture irradiation (BNCR) using boronophenylalanine (BPA) to whole thorax of mice induced severe weight loss and death within one week when irradiation time was over 20 minutes. We speculated the cause
of the severe acute adverse events was ascribed to the damage of esophagus. According to the report investigating
the effect on esophagus by X-ray irradiation, we examined
the change of numbers of basal cells in esophageal membrane. The number of basal cells was lowest at 7 days after
BNCR to the whole thorax. In further experiments, the
number of the basal cells at 7 days after various treatment
will be compared to investigate the effect of BNCT on
esophagus.

P5-6: The Influence of Boron Neutron Capture
Therapy on Bone Grow in Young Mice.
According to the experiment performed in this project last year, the tibial growth in the young mice was
slightly suppressed in the higher dose of boron neutron
capture irradiation (BNCR) using BPA. In this year,
the pathological analysis was carried out to elucidate
the morphological change of bones in young mice at 3
months after BNCR.
The tibias were collected from the mice of the following the four cohorts, Control (no irradiation), X-ray irradiation, Neutron irradiation, and BNCR. Histological changes in the tibias in the young mice after BNCR
were less than dose in the X-ray group.

P5-3: Phenotypic Change of Macrophage/Micro-

glia in the Brain after BNCT.
Glioblastoma (GBM) had been treated with BNCT
using Kyoto University Research Reactor (KUR) until
2019. With BNCT, better control and survival benefit
was achieved. However, adverse effects such as brain
edema or brain necrosis may sometimes occur after
BNCT. It is known that microglia and macrophages
change phenotype to control the inflammation in brain.
Typically, M1type is known to induce inflammation and
M2 type is known to reduce inflammation in tissue repair.
In this study, the phenotypic change of microglia/macrophage after BNCT in the surrounding normal brain in
glioma baring mice. At 2 days after BNCT, M1 marker
tended to increase compared to non-irradiated control.
At 8 days after BNCT, M2 marker tended to increase
compared to non-irradiated control.
P5-4: The effect of boron neutron capture therapy
(BNCT) on esophagus in mice

R4P5

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PR5-1

Phenotypic Change of Macrophage/Microglia in the Brain after BNCT

N. Kondo1, Y. Sakurai1 T. Takata1 and M. Suzuki1

Next, we showed the tissue of non-irradiated control and
8 days after BNCT stained with M2 marker in fig. 2A
and B respectively. After 8 days, M2 marker tended to
increase compared to non-irradiated control.

Institute for Integrated Radiation and Nuclear Science,
Kyoto University (KURNS)
1

(A)

INTRODUCTION: Boron Neutron Capture Therapy
(BNCT) have been applied to recurrent malignant glioma
and even after standard therapy (surgery, chemo-radiation
therapy) because of the selective damage to the tumor.
Especially, glioblastoma (GBM) is the most miserable
cancer, whose patient survival is 14.6 months and remarkably resistant to chemo-radiation and immunotherapy. With BNCT, we achieved better local control and
survival benefit in malignant glioma using thermal neutrons produced by the reactor in Kyoto University. However, adverse effects (brain edema or brain necrosis, etc)
may sometimes occur after BNCT. Microglia and macrophages change phenotype to control the inflammation
in brain. Typically, M1 type is known to induce inflammation and M2 type is known to reduce inflammation in
tissue repair. In this study, we examined the phenotypic
change of microglia/ macrophage after BNCT in the surrounding normal brain in glioma bearing mice.

Fig. 2. Typical immunostaining of brain tissue with
M2 marker, CD206 in non-irradiated control (A) and 8
days after BNCT (B) .
We will continue to immune-staining of these samples till
we obtain enough numbers of samples.

EXPERIMENTS: We implanted 2x105 GL261 murine
glioma cells in the C57BL/6 mouse brain stereotactically
after anesthsia. About two weeks later, we administrated
500mg/kg of boronophenylalanine (BPA) subcutaneously
and irradiated thermal neutrons to mouse brain in the
Heavy Water Neutron Irradiation Facility of the KUR.
We collected brains with time course (2, 8, and 14 days)
after BNCT and fixed them in 10 % formalin, replaced
with 10 to 20 % sucrose and kept samples in deep freezer.
We immune-stained the thin-sliced tissue with M1 (iNOS: inducible nitric oxide synthase) and M2 (CD206,
Macrophage mannose receptor 1) markers.
RESULTS: We showed the tissue of non-irradiated
control and 2days after BNCT stained with M1 marker in
fig. 1A and B respectively. After 2 days, M1 marker
tended to increase compared to non-irradiated control.
(A)

(B)

(B)

Fig. 1

Fig.1. Typical immunostaining of brain tissue with
M1 marker, iNOS in non-irradiated control (A) and 2
days after BNCT (B) .

R4P5-3
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The effect of boron neutron capture therapy (BNCT) on esophagus in mice
Fig.1. HE staining of mouse esophagus.

M. Suzuki, H. Tanaka, Y. Sakurai, T. Takata

Institute for Integrated Radiation and Nuclear Science
Kyoto University
INTRODUCTION: In 2020, accelerator-based boron
neutron capture therapy for head and neck cancer was
approved by Japanese Ministry of Health, Labor, and
Welfare. At two medical institutes, BNCT for head and
neck cancer has started under the insurance coverage.
For BNCT to be applied to other malignancies such as
lung cancer or liver cancers, the effect of BNCT on normal
tissues should be investigated. To investigate the effect of
BNCT on lung tissues, we performed boron neutron
capture irradiation (BNCR) to whole thorax of mice using
boronophenylalanine (BPA) as a boron compound. In the
cohort of mice irradiated for 30-m BNCR, all the mice
were dead within 7 days after the irradiation. No apparent
abnormality was observed in the extracted lungs from the
dead mice.
We speculated that cause of death would be acute side
effect of the esophagus since esophagitis is known to acute
side effect in radiotherapy for lung cancer or esophageal
cancer. Ando et al. reported the effect of X-ray irradiation
on cell kinetics of esophageal membrane cells in mice.
According to this report, numbers of basal cell decreased
up to 4 days after the irradiation, then rapidly increased.
With reference to this report, we investigated the effect of
BNCT on esophagus using mice.
EXPERIMENTS:
Mice
Ten- to twelve-week-old female C3H/He mice were used.
The mice were purchased from Japan SLC, Inc.
BNCR and measurement of thermal neutron and -ray
In this study, BPA was administered subcutaneously at the
dose of 500 mg/kg before the whole thorax irradiation. At
the each BNCR, three mice were held within a specially
designed acrylic box. LiF plates (5-mm thick) were used to
shield the whole body except for chest.
Neutron fluences were measured by radio activation of gold
foils (3mm diameter; 0.05 mm thick) on the anterior and
dorsal surface of the mice. Thermoluminesent dosimeters
were used for -ray dosimetry.
Assessment of numbers of basal sells in mouse esophageal
membrane
The extracted esophagus was fixed in the 10% neutral
formaldehyde. The fixed samples were processed for
preparation of the section and hematoxylin-eosin (HE)
staining. Using a software, basal cell numbers per 90-120 m
length were counted at 10 parts in each sample as shown in
Fig.1.

RESULTS
Dosimetry
The anterior and posterior surface of mice were irradiated
with thermal neutron beam at the thermal neutron flux of
5.3 and 1.3 n/cm2/s, respectively. The -ray was irradiated
at the dose rate of 0.045 Gy/min.
Change in numbers of esophageal basal cells after BNCR
To investigate the change of numbers of esophageal basal
cell, BNCR with fixed irradiation time (20 min) was
performed. Irradiated mice were sacrificed at 2, 4, 7, and
10 days after the BNCR. Three mice were irradiated at
each timepoint. Fig.2 shows the numbers of esophageal
basal cells at each time interval after BNCR.

Fig.2. Change in number of esophageal basal cells after BNCR.
At 7 days after BNCT, the number of basal cells in
esophageal membrane was lowest.
In the further
experiments to compare the effect of BNCR on esophagus
at different fluence of thermal neutrons or X-ray irradiation,
7-day interval after BNCR will be selected for the
assessment of the numbers of basal cells.

R4P5-4

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The Influence of Boron Neutron Capture Therapy on Bone Growth in Young Mice

R. Iwasaki, R. Yoshikawa, R. Kido, T. Mori, K. Ono1, Y.
Sakurai2, and M. ...