PR1

Analyzing Tumor Microenvironment and Exploiting its Characteristics
for Controlling Malignant Solid Tumors and Distant Metastatic Potential
to decrease the number of lung metastases, and an acutehypoxia-releasing treatment such as the administration of
nicotinamide or bevacizumab may be promising for reducing numbers of lung metastases. Consequently,
BPA-BNCT in combination with nicotinamide and/or
bevacizumab treatment may show a little more potential
to reduce the number of metastases. Now, it has been
elucidated that control of the chronic hypoxia-rich Q cell
population in the primary solid tumor has the potential toimpact the control of local tumors as a whole, and that
control of the acute hypoxia-rich total tumor cell population in the primary solid tumor has the potential to impact
the control of lung metastases.
The aim of this research project is focused on clarifying and analyzing the characteristics of intratumor microenvironment including hypoxia within malignant solid
tumors and optimizing cancer therapeutic modalities,
especially radiotherapy including BNCT in the use of
newly-developed 10B-carriers based on the revealed findings on intratumor microenvironmental characteristics.

S. Masunaga
Research Reactor Institute, Kyoto University
BACKGROUNDS AND PURPOSES: Human solid
tumors contain moderately large fractions of quiescent(Q) tumor cells that are out of the cell cycle and stop cell
division, but are viable compared with established experimental animal tumor cell lines. The presence of Q
cells is probably due, in part, to hypoxia and the depletion of nutrition in the tumor core, which is another consequence of poor vascular supply. As a result, Q cells are
viable and clonogenic, but stop cell division. In general,
radiation and many DNA-damaging chemotherapeutic
agents kill proliferating (P) tumor cells more efficiently
than Q tumor cells, resulting in many clonogenic Q cells
remaining following radiotherapy or chemotherapy.
Therefore, it is harder to control Q tumor cells than tocontrol P tumor cells, and many post-radiotherapy recurrent tumors result partly from the regrowth of Q tumor
cells that could not be killed by radiotherapy. Similarly,
sufficient doses of drugs cannot be distributed into Q
tumor cells mainly due to heterogeneous and poor vascularity within solid tumors. Thus, one of the major causesof post-chemotherapy recurrent tumors is an insufficient
dose distribution into the Q cell fractions.
With regard to boron neutron capture therapy(BNCT), with 10B-compounds, boronophenylalanine-10B(BPA) increased the sensitivity of the total cells to agreater extent than mercaptoundecahydrododecaborate10
B (BSH). However, the sensitivity of Q cells treatedwith BPA was lower than that in BSH-treated Q cells.
The difference in the sensitivity between the total and Q
cells was greater with 10B-compounds, especially with
BPA. These findings concerning the difference in sensitivity, including other recovery and reoxygenation following neutron irradiation after 10B-compound administration were mainly based on the fact that it is difficult
to deliver a therapeutic amount of 10B from 10B-carriers
throughout the target tumors, especially into intratumor
hypoxic cells with low uptake capacities.
Hypoxia is suggested to enhance metastasis by increasing genetic instability. Acute, but not chronic, hypoxia was reported to increase the number of macroscopic metastases in mouse lungs. We recently reported thesignificance of the injection of an acute hypoxia-releasing
agent, nicotinamide, into tumor-bearing mice as a combined treatment with -ray irradiation in terms of repressing lung metastasis. As the delivered total dose increased with irradiation, the number of macroscopic lungmetastases decreased reflecting the decrease in the number of clonogenically viable tumor cells in the primarytumor. The metastasis-repressing effect achieved througha reduction in the number of clonogenic tumor cells by
irradiation is much greater than that achieved by releasing tumor cells from acute hypoxia. On the other hand,more 10B from BPA than from BSH could be distributed
into the acute hypoxia-rich total tumor cell population,
resulting in a greater decrease in the number of highly
clonogenic P tumor cells with BPA-BNCT than with
BSH-BNCT and with neutron beam irradiation only.
BPA-BNCT rather than BSH-BNCT has some potential

採択課題番号 26P1

RESEARCH SUBJECTS:
The collaborators and allotted research subjects (ARS)
were organized as follows;
ARS-1 (26P1-1): Optimization of Radiation Therapy
Including BNCT in terms of the Effect on a Specific
Cell Fraction within a Solid Tumor and the Suppressing
Effect of Distant Metastasis. (S. Masunaga,et al.)ARS-2 (26P1-2): Development of Hypoxic Microenvironment-Oriented 10B-Carriers. (H. Nagasawa, et al.)
ARS-3 (26P1-3): Clarification of Mechanism of Radio-Resistance in Cancer Using Optical Imaging at Tissue Level. (H. Harada, et al.)
ARS-4 (26P1-4)*: Analysis of Radiation-Induced
Cell-Killing Effect in Neutron Capture Reaction. (R.
Hirayama, et al.)
ARS-5 (26P1-5): Transdermal Drug Delivery Systemusing Hyaluronan-Conjugated Liposomes as 10B-Carrier
in Boron Neutron Capture Therapy for Melanoma(S. Kasaoka, et al.)
ARS-6 (26P1-6): Evaluation of Inclusion Complex ofCarborane Modified Kojic Acid and Cyclodextrin as
10
B-Carrier in Boron Neutron Capture Therapy. (T. Nagasaki, et al.)
ARS-7 (26P1-7)*: Molecular Design and Synthesis andFunctional Evaluation of Anticancer and Molecular Targeting Agents. (Y. Uto, et al.)
ARS-8 (26P1-8)*: Analyzing Biological Effect of BNCT
from the Viewpoint of the Changes in Oxygenation Level. (H. Yasui, et al.)
ARS-9 (26P1-9): Analyses on the Responsiveness of
Malignant Tumors to BNCT. (M. Masutani, et al.)
ARS-10 (26P1-10): Assay for Tumor Cell Survival andTumor Growth Delay through Neutron Capture Reaction
according to the Changes in Intracellular Concentrations
within Solid Tumors of Newly- Developed 10B-Carriers.
(K. Nakai, et al.)
ARS-11 (26P1-11)*: Antitumor and MetastasisReppresing Effect of BNCT on Human Breast andPancreatic Cancer Cell Lines. (Y. Matsumoto, et al.)(* Due to the irregular and short operation period of ourreactor in 2014, the data could not be shown here.)

局所腫瘍制御と転移抑制を同時に目指す
治療法開発のためのがん微小環境解析とその応用（京大・原子炉）増永慎一郎
3

プロジェクトPR1-1Significance of Fractionated Thalidomide Administration Combined with γ-Ray
Irradiation from the Viewpoint of Local Tumor Response and Lung Metastasis

S. Masunaga1, Y. Sanada1, T. Moriwaki2, K. Tano1, Y.
Sakurai1, H. Tanaka1, M. Suzuki1, N. Kondo1, M. Narabayashi1, T. Watanabe1, Y. Nakagawa1, A. Maruhashi1
and K. Ono1

crosis factor from monocytes, and modulates other cytokine action. Thalidomide is racemic, and contains bothleft and right handed isomers in equal amounts: one enantiomer is effective against morning sickness, and theother is teratogenic. The enantiomers are converted toeach other at physiological conditions (pH = 7.0) in vivo.
That is, if a human is given D-thalidomide ((+)- or R-) or
L-thalidomide ((-)- or S-), both isomers can be found in
the serum. Hence, administering only one enantiomer
will not prevent the teratogenic effect in humans. It is
employed for the acute treatment of the cutaneous manifestations of moderate to severe erythema nodosum leprosum (ENL). Available data from in vitro studies andpreliminary clinical trials suggest that its immunologic
effects can vary substantially under different conditions,but may be related to suppression of excessive tumor
necrosis factor production and down-modulation of selected cell surface adhesion molecules involved in leukocyte migration [3].
As a cancer treatment, thalidomide was shown to inhibit basic fibroblast growth factor (bFGF) as well as
VEGF, two promoters of angiogenesis. It was reported tomeasure the modifications in the tumor environment early after an angiogenic treatment of thalidomide, with a
special focus on possible normalization of the tumor
vasculature that could be beneficial for radiotherapy [4].
Incidentally, angiogenesis also is critical during limb development of the foetus. Thalidomide directly inhibits
angiogenesis induced by bFGF or VEGF in vivo. In 2009,it was confirmed that loss of newly formed blood vessels
is the primary cause of thalidomide teratogenesis, andthat developing limbs are particularly susceptible becauseof their relatively immature, highly angiogenic vessel
network. Thus inhibition of angiogenesis is now thought
to be a main mechanism of its teratogenicity [3,5].
Conclusion: It was elucidated that control of the chronic
hypoxia-rich Q cell population in primary solid tumors
has the potential to impact the control of local tumors as awhole, while control of the acute hypoxia-rich total tumor
cell population has the potential to impact the control oflung metastases. Namely, in conventional radiotherapy,
daily fractionated administration of thalidomide combined with MTH is thought to have a great potential tocontrol both local solid tumors and lung metastases fromthe local tumors [6].
REFERENCES:[1] S. Masunaga, et al., Int J Hyperthermia 13 (1997)
401-411.
[2] S. Masunaga, et al., Clin Exp Metastasis 26 (2009)693-700.
[3] M. E. Franks, et al., Lancet 363 (2004) 1802-1811.
[4] R. Ansiaux, et al.,Clin Cancer Res 11 (2005) 743-750.
[5] C. Therapontos, et al., Proc Natl Acad Sci U S A 106(2009) 8573-8578.
[6] S. Masunaga et al., World J Oncol 5 (2014) 155-165.

1

Research Reactor Institute, Kyoto University
Faculty of Medicine, Kyoto University

2

INTRODUCTION: Thalidomide has been reported toinduce tumor blood vessel normalization in a mousemodel [8, 9]. Today, thalidomide is being mainly applied
as a treatment of certain cancers (multiple myeloma) andof a complication of leprosy. Here, using a readily metastasizing murine melanoma cell line, we tried to analyze the significance of combined treatment with thalidomide in radiotherapy with γ-rays in combination with anacute hypoxia-releasing agent nicotinamide or mild temperature hyperthermia (MTH), already shown to have thepotential to release tumor cells from diffusion-limited
chronic hypoxia [1, 2], in terms of local tumor response
and lung metastatic potential. Concerning the local tumor
response, the effect not only on the total (= proliferating(P) + quiescent (Q)) tumor cell population but also on the
Q cell population was evaluated using our original method for selectively detecting the response of Q cells insolid tumors.
Materials and Methods: B16-BL6 melanoma tumor-bearing C57BL/6 mice were continuously given
5-bromo-2’-deoxyuridine (BrdU) to label all P cells. The
tumor-bearing mice then received γ-ray irradiation after
thalidomide treatment through a single or two consecutive daily intraperitoneal administrations up to a total
dose of 400 mg/kg in combination with nicotinamide or
mild MTH. Immediately after the irradiation, cells fromsome tumors were isolated and incubated with a cytokinesis blocker. The responses of the Q and total (= P + Q)
cell populations were assessed based on the frequency of
micronuclei using immunofluorescence staining for BrdU.
In other tumor-bearing mice, 17 days after irradiation,
macroscopic lung metastases were enumerated.
Results: Thalidomide raised the sensitivity of the total
cell population more remarkably than Q cells in both single and daily administrations. Daily fractionated administration of thalidomide elevated the sensitivity of boththe total and Q cell populations, but especially the total
cell population, compared with single administration.
Daily administration, especially combined with MTH,
decreased the number of lung metastases.
Discussion: Thalidomide was originally introduced as a
non-barbiturate hypnotic, but withdrawn from the market
due to teratogenic effects. However, it has been reintroduced and used for a number of immunological and inflammatory disorders due to its immunosuppressive andanti-angiogenic activity. It inhibits release of tumor ne-4

PR1-2Development of Nucler-targeting Boron Carriers Based on the Chemistry of Direct
Cell-penetrating Pepducins

A. Isono1, K. Okuda1, T. Hirayama1, S. Masunaga2, and
H. Nagasawa1

was likely to be anchored on the plasma membrane but
could not be translocated into the cytosol. To evaluate thestructure activity relationship of the peptide sequence forcontrolling cellular distribution, we synthesized pepducins containing various peptides to conjugate with thecargo moiety.

1

Laboratory of Medicinal & Pharmaceutical Chemistry,
Gifu Pharmaceutical University
2
Research Reactor Institute, Kyoto University
INTRODUCTION: For the success of boron neutron
capture therapy (BNCT), it is essential to selectively deliver sufficient amount of 10B atoms to tumor cells. To
achieve intracellular molecular targeting of drug delivery,
we focused on the direct cell-penetrating lipopeptides,
pepducins which were developed as allosteric modulators
of GPCRs[1]. By the study of fluorescence resonanceenergy transfer (FRET)-based imaging of pepducins, wedemonstrated that they penetrate directly into cells bytrans-membrane lipid translocation [2]. Accordingly, we
envisaged that the pepducin moiety may be exploitablefor intracellular deliver of membrane impermeable
molecules such as anionic boron claster, sodium borocaptate (BSH).
MOLECULAR DESIGN: As shown in Fig. 1, we designed new hybrid molecules comprising pepducin (Pep)
as a vehicle, and boron cluster (BS) attached with fluorescent dyes such as 7-hydroxycoumarin (Cou) andHoechst 33342 dye (Hoe) as a cargo; Pep-SS-BS-Cou
and Pep-SS-BS-Hoe. The cargo is supposed to be released into cytosol after reductive cleavage of the disulfide bond by intracellular glutathione when it is carried
into the cytosol.

We also synthesized Pep-SS-BSs by connecting of various pepducins to the boron cluster without any fluorophores. To quantify their intracellular boron concentration, ICP-MS analyses of nuclear and cytoplasmic extracts will be conducted.

RESULTS AND DISCUSSION: The lipidated peptides
were prepared by solid-phase synthesis and then combined with BSH and the corresponding fluorophorethrough an appropriate liker to afford the boron carriers(Fig. 2 and 3). We performed live cell imaging experiments using MCF-7 cells with the probes and confocal
microscopy. By the treatment with Pep-SS-BS-Cou, thefluorescence signal was localized on cell-membrane formore than 30 min, which suggested that the boron carrier

REFERENCES:[1] L. Covic et al., Proc. Natl. Acad. Sci. USA., 99(2002) 643–648.
[2] M. Tsuji et al., Org. Biomol. Chem., 11(2013),
3030-3037/.?E6! 261 A<,(4" 08  
C7

F*D>$>'G9B =%5+) :12;&F$ &3G#1-@

5PR1-3

Genetic Screening of Upstream Activators of Hypoxia-inducible Factor 1 for the
Devrlopment of Novel Anti-cancer Strategies

H. Harada1, S. Masunaga2, M.

Fig. 1

1

Department of Radiation Oncology and Image-applied
Therapy, Graduate School of Medicine, Kyoto University.
2
Research Reactor Institute, Kyoto University
INTRODUCTION:
Accumulating evidence has
shown that hypoxia-inducible factor 1 (HIF-1) plays critical roles in distant tumor metastases at multiple steps [1]and eventually causes death among cancer patients. Clinical studies have demonstrated consistent data that HIF-1
could be used as an adverse prognostic factor for not only
local tumor recurrence but also distant tumor metastasis
in cancer patients. These findings justify targeting HIF-1
for cancer therapies.
HIF-1, a heterodimeric transcription factor composed ofan -subunit (HIF-1) and a -subunit (HIF-1), is
known to become activate mainly through the stabilization of HIF-1 protein. Under normoxic conditions,prolyl-4-hydroxylases (PHDs) hydroxylate proline residues, P402 and P564, in the oxygen-dependent degradation domain (ODD domain) of HIF-1 in an oxygen-dependent manner. The hydroxylations trigger
polyubiquitilation and subsequent proteasomal degradation of HIF-1. On the contrary, HIF-1 becomes active
under hypoxic conditions because of the inactivation of
the hydroxylases, and then, interacts with its binding
partner, HIF-1. Resultant heterodimer, HIF-1, binds to
its cognate enhancer sequence, the hypoxia-responsive
element (HRE), and induces transcriptions of various
genes related to the escape from hypoxia (invasion andmetastasis of cancer cells) as well as the improvement ofoxygen-availability (angiogenesis) and adaptation of cellular metabolism to hypoxia (metabolic reprogramming).
In order to explore novel genes which are responsible
for the HIF-1-mediated tumor metastasis, we recently
established a new genetic screening method [2] andfound that overexpression of ubiquitin C-terminal hydrolase L1 (UCHL1) is responsible for the activation of
HIF-1. In the present study, we analyzed both the molecular mechanisms underlying the UCHL1-mediated activation of HIF-1 and the involvement of UCHL1-HIF-1
axis in distant tumor metastases.

A murine model of pulmonary metastasis demonstrated
that the forced expression of UCH-L1 facilitated tumor
metastases in a HIF-1-dependent manner (Fig. 2) [3].

Fig. 2On the other hand, silencing of the aberrantly expressed
UCH-L1 suppressed the metastatic tumor formation byinactivating HIF-1[3].
Moreover, we revealed that UCH-L1 expression levels
were associated with poor prognosis of patients withbreast and lung cancers (Fig. 3) [3].

Fig. 3DISCUSSIONS: Together, these results demonstratethat UCH-L1 functions in distant tumor metastasis as a
deubiquitinating enzyme for HIF-1, and thus, justify
exploiting it as a prognostic marker and therapeutic target
for individualization of cancer therapy.

EXPERIMENTS & RESULTS: We established stabletransfectants of HeLa cells with the UCHL1 expression
vector (HeLa/UCHL1) or its empty vector (HeLa/EV) [3].
We found that aberrant expression of UCH-L1 in cancer
cells abrogated von Hippel-Lindau-mediated ubiquitination of HIF-1, leading to the stabilization of HIF-1 andsubsequent activation of HIF-1 (Fig. 1) [3].

採択課題番号26 P1-3

REFERENCES (papers published from our lab):[1] Zhao T et al. Sci Rep. 4:3793. 2014.
[2] Zeng L et al. Oncogene. in press.
[3] Goto Y et al. Nature Communications. 6:6153. 2015.

低酸素誘導性因子1（HIF-1）を活性化する新規遺伝子の探索と機能解析、および局所腫瘍制御への展開（京大・院医）原田浩（京大・原子炉）増永慎一郎
6

プロジェクトPR1-4

Quantitative Detection of Metastasized B16BL6 Cells in Lung by Using
TaqMan RT-PCR Assay

T. Nagasaki, J. Kawaguchi, R. Kawasaki, M. Sakuramoto,
M. Kirihata1, Y. Hattori1, S. Masunaga2 and Y. Sakurai2

tion (Q-PCR) was performed according to the manufac(Roche) and Q-PCR was run in a STRAGENE Mx3000P
using the following program:. Primers and probe were
AGC ACC TGG AAC CACGAG GGC GTTATC TATGT GTA GT 3probe: 5'Hex-CAC TAC AAA
AGT TGT GGG TAC TAC ACC TG-BHQ-1-3'.

Graduate School of Engineering, Osaka City University
1
BNCT Research Center, Osaka Prefecture University,
2
Research Reactor Institute, Kyoto University
INTRODUCTION:
Although melanoma boron neutron capture therapy (BNCT) with L-10B-p-boronophenyl
alanine has been treated for melanoma patient, the enhancement of 10B accumulation in melanoma cells would
lead to improved efficacy of malignant melanoma BNCT.
Recently, we have evaluated the inclusion complex of
kojic acid-appended carborane (CKA) with hydroxypropyl- -cyclodextrin (HP- -CD) as a novel boron agent for
BNCT toward melanoma. CKA/HP- -CD complex
showed melanoma cells selectivity, unique nuclear localization, and high tumor-suppression effect on BNCT towardmelanoma-bearing
mice.
Furthermore,
CKA/HP- -CD complex was found to be the potent inhibitor of hypoxia-induced HIF-1 and to have a strong
effect on tumor metastasis [1]. Herein, in order to evaluate quantitatively metastasis-suppression, TaqMan
RT-PCR assay of melanoma gene GP100 was carried out
toward melanoma B16BL6 cells metastasis from thighsubcutaneous to lung in melanoma-bearing mice [2].

Fig.1 Relative mRNA expression of melanoma gene
GP100 in lungs.

RESULTS AND DISCUSSION: GP100 is highly
expressed in melanoma B16BL6 cells, but not to any
substantial degree in normal tissues. GP100 expression
exceeded background levels when at least 5 103 B16BL6
cells are contained in metastasized tissue. In order toquntify the increase of metastasized B16BL6 cells, lungs
were collected 1, 3, 7, or 14 days post tumor implantation.
On day 7 increase of GP100 expression reached a levelof about 100 times the lowest point (Fig. 1).
TaqMan RT-PCR Assay toward GP100 is potent toestimate quantitatively the metastasis of melanoma cells
to lung. The quantitative evaluation of metastasis-suppression effect of CKA/HP- -CD complex is proceeding in our laboratory.

Structure of CKA/HP- -CD

EXPERIMENTS: B16BL6 cells (2.5 1012 cells) were
implanted into thigh subcutaneous. Mice were euthanized
and lung and spleen were isolated and snap frozen in liquid nitrogen before being stored at Total RNA wasextracted using NucleoSpinRNA Kit (Takara). Total
RNA concentration and purity were measured using
NanoDrop 2000c Spectrometer (Thermo Scientific).
Subsequently, cDNA synthesis was performed by using
PrimeScriptTM RT reagent Kit (Perfect Real Time)(Takara). Quantitative real time polymerase chain reac-

REFERENCES:[1] K. Shimizu et al., Bioorg. Med. Chem. Lett., 20(2010) 1453-1456.
[2] M. R. Sorensen et al., PLoS One., 9 (2001)
e87831.

メラノーマ中性子捕捉療法への適応を目指した薬物送達システムに関する研究7

PR1-5

Response of Tumor Cells to BNCTS. Imamichi1, T. Itoh1,2, S. Kikuhara1,2, H. Fujimori1, A.
Sato3, T. Hirai1,4, S. Masunaga5 and M. Masutani1,6

minescence dosimeter. These physical radiation doses
were measured with the kind help of Drs. Yoshinori Sakurai and Hiroki Tanaka of KUR.

1

Division of Chemotherapy and Clinical Research, National Cancer Center Research Institute, 2Faculty of Industrial Science and Technology, Tokyo University of
Science, 3Faculty of Pharmaceutical Sciences, Tokyo
University of Science, 4Department of Radiation Oncology, Juntendo University Faculty of Medicine, 5 Research
Reactor Institute, Kyoto University, 6Department ofFrontier Life Sciences, Nagasaki University Graduate
School of Biomedical Sciences

RESULTS:
Fig. 1. The physical dose measurement.
Themeasuredresults of total
physicaldose,
neutronandgamma ray dosewere shown in
Fig. 1 and confirmed that thedose estimation
was in the expected
range.
Twenty-four hrs
after 24 Gy-eq
irradiation, SAScells showed thecleavageof
PARP1, caspase-9 and caspase-3, suggesting that apoptosis started to occur. Meanwhile, the low levels of necroticcleavage pattern of PARP1 were also observed, implyingthat necrotic cell death is also involved after BNCR. The
isolated RNA 6 and 24 hrs after irradiation was evaluatedby electrophoresis and RT-PCR. As shown in Fig. 2, theexpression levels of PARP1 and GUSB were not different
among the irradiation conditions, confirming the qualityof RNA. In the microarray analysis, we found that theexpression of the genes related to cell death, transcription,and inflammatory and immune responses was augmented
after BNCR and the further evaluation is ongoing.
By proteome analysis, proteins involved in the vesicle
regulation, mRNA processing, transcription were observedwithchanges
after
BNCR and being
studiedfurther.
Theobservationsuggests that dynamic changes in cellular responses could
be induced after BNCR reaction at an early phase. SAS
xenograft model in nude mice was also set up, and theboron concentration in the blood and tissues was measured. Using the experimental system, the genes and proteins associated with BNCR response will be further elucidated.

INTRODUCTION: Tumors are consisted of heterogenous populations of cells, including hypoxic cells,
cancer stem cells and differentiating cells. These populations affect the consequence of the boron neutron capture
therapy (BNCT) [1]. Boron neutron capture reaction(BNCR) causes extensive DNA double strand breaks andmicronuclei formation has been used to estimate the levelof DNA damage induced by BNCR [2]. In rat xenograftmodel, BNCR caused the up-regulation of H2AX and
HMGB1, then induction of poly(ADP-ribose) [3]. The
cell death process and the factors affecting tumor cellkilling, side effects and effectiveness of BNCT are notfully understood. To elucidate the cell death process after
BNCR, we have taken approaches of comprehensive
analysis of the tumor cells by using transcriptome andproteome analysis.
EXPERIMENTS: Human oral squamous cancer SAS
cells [1] was used for the comprehensive analysis withandproteome
analysis.
transcriptome
10
B-boronophenylalanine (10B-BPA)-fructose solution
was prepared as described previously [4]. SAS cell was
suspended and incubated 2 h with or without 25 ppm of
10
B-BPA as previously reported [3]. After neutron-beamirradiation operated at 1 MW, cells were inoculated forcolony formation, RNA and protein preparation. To analyze the early response of cancer cells, RNA and protein
were prepared 6 and 24 hrs after irradiation at the supposal doses of 4 Gy-eq and 24 Gy-eq conditions.
For mouse xenograft model, male BALB/c-nu/nu
mice were used and SAS cells were injected subcutaneously into left legs of the mice. 10B-BPA at the dose of
250 mg/kg bodyweight was intraperitoneally injected 1 h
before irradiation operated at 1 MW. Tumors, tissues, andblood were sampled for the analysis of early responses
after irradiation. The animal studies were performed inaccordance with the relevant laws and institutional guidelines of KUR and NCC. 10B concentration was measured by prompt-gamma ray analysis (PGA). Thermal
neutron fluence was measured with gold foils activation
analysis. Gamma-ray dose was measured with thermalu-

採択課題番号 26P1-9

REFERENCES:[1] Masunaga et al, Jpn. J. Cancer Res., 93(12) (2002)
1366-77.
[2] Kinashi et al, Radiat. Oncol., 5;6 (2011) 106.
[3] M. Masutani et al, Appl. Radiat Isot.88 (2014) 104-8.
[4] Yoshino et al, Strahlen. Onkol., 165: (1998) 127-12ホウ素中性子捕捉療法に対する腫瘍応答機構の分析と奏効バイオマーカーの探索

プロジェクト(京大・原子炉）増永慎一郎（国立がん研究センター）益谷美都子、今道祥二、佐藤聡、伊藤祐
菊原颯太、平井崇久、藤森浩彰

8PR1-6

Additive effect of Gadolinium to cell survival analysis
for neutron capture therapy

K. Nakai1, F. Yoshida1, T. Kurita1, Y. Yamamoto1, M.
Shirakawa2, T. Yamamoto1, H. Tanaka3, Y. Sakurai3, S.
Masunaga3

RESULTS: As shown in Fig. 1, Radiation-related
damage significantly increased with the increase of gadolinium concentration. When 5 ppm of Gd-DTPA wasadded to 40 ppm of BPA in C6 cells, the additive effectwas obtaine. In radiosensitive V79 cells the survival fraction dramatically decreased after irradiation and was
lower than 10% at boron concentration of 40ppm.

1

Department of Nueurosurgery ,Faculty of Medicine,
University of Tsukuba
2
Department of Pharmaceutical Sciences,International
University of Health and Welfare
3
Research Reactor Institute, Kyoto University
INTRODUCTION: In neutron capture therapy; thetherapeutic effect of a boron compound is based on alphaparticle production in a 10B(n, α) 7Li reaction, while agadolinium compound effect is based on producing
gamma rays derived from a 157Gd(n, γ) reaction[1,2].
Gadolinium containing agents has been used for contrast
enhance of MRI scan, and are commercially available as
pharmaceutical products.
If gadolinium is administered simultaneously with boron and irradiated with neutrons, it might become asource of additional local gamma rays, which correspondwith recent BNCT clinical trial protocol for glioblastoma.
We analyzed the additive effect of gadolinium on boron
based neutron capture reaction.
Materials and Methods:
Boron and Gadolinium compounds
p-boronophenylalanine (BPA) was obtained from Interpharma Praha (Praha, Czech Republic). Gd-DTPA(Magnevist) was purchased from Shering (Berlin, Germany). BPA and fructose ware dissolved and prepared aspreviously described. Briefly, BPA was converted to
BPA-fructose complex by mixing BPA and fructose in
NaOH. The pH was adjusted to 7.4 with HCl. Dissolution
solvent for Gd-DTPA was cell culture medium.
Cell lines; C6 rat glioma cell lines and V79 Chinese
hamster cell lines were maintained in Eagle’s Minimum
Essential Medium (MEM; Sigma-Aldrich), supplemented
with 10% fetal bovine serum.
Colony Formation Assay ;V79 cells and C6 cells after
exposition with the compounds were irradiated with neutron mixed beam for 90 min at the Kyoto University Research Reactor (KUR). We use the combination of BPA(0, 10, 20, 40 Bu µµg/mL) and Gd-DTPA (0, 0.5, 5, 10
Gdµg/mL) with 104 of cells of each line. The number ofcells was sufficient to produce 20-100 colonies at eachboron concentration. After 7days of incubation, the plates
were stained with 0.25 % methylene blue in 90 % ethanol.
Colonies of more than 50 cells were counted.

Fig. 1. Influence of Gd on BNCT in C6 (upper), V79(lower) cell lines. The symbols ◆ show the control(BPA only); ■ show the result of additional 0.5 ppm of
Gd; ▲ show the result of additional 5 ppm of Gd and× show the result of additional 10 ppm of Gd.

REFERENCES:[1] A. Matsumura et al.,J Exp Clin Cancer Res. (2005)24.
[2] Shikata F., et al. Eur. J. Pharm. Biopharm(2002)53.

	
 採択課題番号26P1-10	
 新規ホウ素化合物等を用いた中性子捕捉反応による細胞生存試験および腫瘍増殖抑制効果	 	 	  	  プロジェクト	
  （筑波大・医）中井	
  啓、吉田文代、山本陽平、高	
  振宇、
栗田	
  正（国際医療大・薬）白川	  真（京大・原子炉）田中浩基、櫻井良憲、増永慎一郎	
 9