VIII-Ⅱ-1. Project Research
Project 3

PR3

Analyzing Tumor Microenvironment andExploiting its Characteristics for Controlling Malignant Solid Tumors

S. Masunaga
Research Reactor Institute, Kyoto University
BACKGROUNDS AND PURPOSES: Human solid
tumors are thought to contain moderately large fractionsof 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 that havebeen employed for various oncology studies [1]. The
presence of Q cells is probably due, in part, to hypoxia
and the depletion of nutrition in the tumor core, which isanother consequence of poor vascular supply [1]. As aresult, 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 manyclonogenic Q cells remaining following radiotherapy or
chemotherapy [1]. Therefore, it is harder to control Q
tumor cells than to control P tumor cells, and many
post-radiotherapy recurrent tumors result partly from theregrowth of Q tumor cell populations that could not be
sufficiently killed by radiotherapy [1]. Further, sufficient
doses of drugs cannot be distributed within Q tumor cell
populations mainly due to heterogeneous and poor vascular distributions within solid tumors. Thus, one of the
major causes of post-chemotherapy recurrent tumors is aninsufficient dose distribution in 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 a greater extent than mercaptoundecahydrododecaborate- 10B (BSH).
However, the sensitivity of Q cells treated with BPA waslower than that in BSH-treated Q cells. The difference inthe sensitivity between the total and Q cells was greaterwith 10B-compounds, especially with BPA [2]. Q cells
showed greater potentially lethal damage repair (PLDR)
capacities than the total cells. -Ray irradiation and neutron irradiation with BPA induced larger PLDR capacities
in each cell population. In contrast, thermal neutron irradiation without the 10B-compound induced the smallest
PLDR capacity in both cell populations. The use of the10
B-compound, especially BPA, resulted in an increase in
the PLDR capacity in both cell populations, and made the
PLDR patterns of the both cell populations look likethose induced by  -ray irradiation [3]. In both the total
and Q tumor cells, the hypoxic fractions (HFs) immediately after neutron irradiation increased suddenly.
Reoxygenation after each neutron irradiation occurred
more rapidly in the total cells than in the Q cells. In bothcell populations, reoxygenation appeared to be rapidly
induced in the following order: neutron irradiation without 10B-compounds > neutron irradiation following BSH
administration > neutron irradiation following BPA administration >  -ray irradiation [4]. These findings concerning the difference in sensitivity, PLDR andreoxygenation following neutron irradiation after10
B-compound administration were thought to be mainly

based on the fact that it is difficult to deliver a therapeuticamount of 10B from currently used 10B-carriers throughout the target tumors, especially into intratumor hypoxic
cells with low uptake capacities [5,6].
Therefore, 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 radiation therapy including BNCT in theuse of newly-developed 10B-compound based on the revealed findings on intratumor microenvironmental characteristics.
RESEARCH SUBJECTS:
The collaborators and allotted research subjects (ARS)
were organized as follows;
ARS-1 (23P3-1): 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,Y. Sakurai, H. Tanaka, Y. Liu, M.
Takagaki, N. Kondo and Y. Matsumoto)
ARS-2 (23P3-2): Development of Hypoxic Microenvironment-Oriented 10B-Carriers(H. Nagasawa, S. Masunaga, K. Okuda, S. Y. Hirayama
and T. Harada)
ARS-3 (23P3-3): Clarification of Mechanism of Radio-Resistance in Cancer Using Optical Imaging at
Tissue Level(H. Harada, M. Hiraoka, S. Masunaga, S. Itasaka, M.
Ogura and M. Yoshimura)
ARS-4 (23P3-4): Analysis of Radiation-Induced
Cell-Killing Effect in Neutron Capture Reaction(R. Hirayama, S. Masunaga, G. Kashino, Y. Sakurai, H.
Tanaka and Y. Matsumoto)
ARS-5 (23P3-5): Transdermal Drug Delivery Systemusing
Hyaluronan-Conjugated
Liposomes
as10
B-Carrier in Boron Neutron Capture Therapy for
Melanoma(S. Kasaoka, K. Hashimoto and S. Masunaga)
ARS-6 (23P3-6): Evaluation of Inclusion Complex ofCarborane Modified Kojic Acid and Cyclodextrin as
10
B-Carrier in Boron Neutron Capture Therapy(T. Nagasaki, S. Masunaga, M. Kirihata, H. Azuma, K.
Li, U. Shu, Y. Kagoshima, K. Hayashi and R. Kawasaki)(Underline: Representative at each research group)
REFERENCES:[1] P. Vaupel, Semin. Radiat. Oncol. 14 (2004) 197-275.
[2] S. Masunaga et al., Int. J. Radiat. Oncol. Biol. Phys.
41 (1998) 1163-1170.
[3] S. Masunaga et al., J. Cancer Res. Clin. Oncol. 125(1999) 609-614.
[4] S. Masunaga et al., Int. J. Radiat. Oncol. Biol. Phys.
44 (1999) 391-398.
[5] S. Masunaga et al., Radiat. Med. 24(6) (2006) 98-107.
[6] S. Masunaga et al., Clin. Exp. Metastasis 26 (2009)693-700.

採択課題番号 23P3 腫瘍内微小環境解析とその特性利用による悪性腫瘍制御の試み プロジェクト（京大・原子炉）増永慎一郎

PR3-1

Radiosensitivity and Capacity to Recover from Radiation-Induced Damage
in Oxygenated Intratumor Quiescent Cell Population Depend on p53 Status

S. Masunaga, Y. Sakurai, H. Tanaka, M. Suzuki,
N. Kondo, M. Narabayashi, A. Maruhashi, K. Ono
Research Reactor Institute, Kyoto University
BACKGROUNDS AND PURPOSES: Using our
method for selectively detecting the response of quiescent(Q) cells in solid tumors to DNA-damaging treatment
including conventional irradiation and chemotherapeutic
agents, the following characteristics of Q cells in murine
solid tumors were clarified: Q tumor cells are more radioand chemo-resistant than the total (proliferating (P) + Q)
tumor cell population; Q cells have a greater capacity torecover from potentially lethal damage than the total cell
population; and Q cell populations include a larger hypoxic fraction (HF) than total cell populations. Further, it
was also indicated that the clonogenicity of Q cells is
lower than that of P cells, and that the HF of Q cells islargely comprised of a diffusion-limited chronically HF
with a smaller perfusion-limited acutely HF [1,2].
Meanwhile, the Q cell population in solid tumors has
never been shown to be fully hypoxic. Actually, the sizesof HFs of Q cell populations in SCC VII squamous cell
carcinomas with a diameter of 1 cm implanted in the hindlegs of C3H/He mice were 55.1 ± 6.2 (mean ± SD) %.
The size was significantly less than 100 %. This meansthat the Q cell population undoubtedly includes oxygenated tumor cells [3].
A few years ago, the detection of hypoxic cells in bothtissues and cell cultures universally became possible using pimonidazole, substituted 2-nitroimidazole, and amouse IgG1 monoclonal antibody (MAb1) to stable covalent adducts formed through reductive activation ofpimonidazole in hypoxic cells. Further, the genetic andfunctional status of the p53 gene is thought to be an important factor in guiding therapeutic strategies for cancer
patients. In the present study, the radio-sensitivity of thepimonidazole non-labeled cell fraction of the Q cell populations within p53-wild and –mutated type tumors, following -ray irradiation at both a high dose rate (HDR)
and a reduced dose rate (RDR), was determined.
MATERIALS AND METHODS: Human head andneck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53), or with neo vector as a control(SAS/neo), were inoculated subcutaneously into left hindlegs of Balb/cA nude mice. The tumor-bearing mice received 5-bromo-2’-deoxyuridine (BrdU) continuously tolabel all intratumor P cells. Tumors were irradiated with-rays at a high dose-rate or a reduced dose-rate at 1 h
after the administration of pimonidazole. The responsesof Q and total cell populations were evaluated with thefrequencies of micronucleation and apoptosis using immunofluorescence staining for BrdU. The response ofpimonidazole unlabeled tumor cell fractions was assessedwith apoptosis frequency using immunofluorescencestaining for pimonidazole.

採択課題番号 23P3-1

RESULTS: The pimonidazole-unlabeled tumor cell fraction showed significantly enhanced radio-sensitivity
compared with the whole tumor cell fraction more remarkably in Q cells and p53-mutated tumors than total
cells and p53-wild type tumors, respectively. However, asignificantly greater decrease in radio-sensitivity in thepimonidazole-unlabeled than the whole cell fraction,
evaluated using a delayed assay or a decrease in radiationdose rate, was more clearly observed in Q cells andp53-wild type tumors than total cells and p53-mutatedtype tumors, respectively. Concerning the whole tumor
cell fraction, the Q cells showed significantly greater radio-resistance and recovery capacity from radiation-induced damage than the total cells both in p53-wildand p53-mutated type tumors.
DISSCUSSION: In recent years, the concept of cancer
stem cells (CSCs) has ignited a great deal of interest because of the potential clinical implications associatedwith these cells. In part, these cells are thought to exist ina patho-physiological microenvironment where hypoxia,
low pH, and nutrient deprivation occur. In addition,
within the tumor microenvironment, significant heterogeneity, both spatial and temporal, also occurs. The tumor
microenvironment has very similar conditions to theintratumor area where dividing tumor cells become quiescent. Further, a subset of CSCs is thought to be
non-dividing quiescent cells. Thus, we tried to clarify theradio-biological characteristics of this sub-population of
the intratumor Q cell population, hoping to contribute to
CSC research.
One mechanism of CSC resistance to cytotoxic treatment
was reported to result from an enhanced DNA repair capacity. Here, the pimonidazole unlabeled cell fraction
among Q cells showed a much greater repair capacity
than the whole Q cell population even if the repair capacity was significantly larger in the whole Q cell population
than the total tumor cell population as a whole. In otherwords, from the viewpoint of not only quiescent status
but also enhanced DNA repair capacity, the characteristics of the pimonidazole unlabeled cell fraction in the Q
cell population was found to be very similar to those of
CSCs.
Although there was similarity between the pimonidazole
unlabeled Q cell fraction and the CSCs in terms of quiescent status and enhanced repair capacity, CSCs arethought to exist under rather hypoxic conditions. Therefore, we would like to further analyze the characteristicsof the intratumor Q cell population in connection withthose of CSCs, including the use of human tumor celllines, in future [4,5].
REFERENCES:[1] S. Masunaga et al., J. Radiat. Res. 43 (2002) 11-25.
[2] N. Hamada et al., J. Radiat. Res. 51 (2010) 365-383.
[3] S. Masunaga et al., Int. J. Radiat. Oncol. Biol Phys.
49 (2001) 1361-1368.
[4] S. Masunaga et al. Radiat. Res. 174 (2010) 459-466.
[5] S. Masunaga et al. World J. Oncol. 2 (2011), 1-9.

腫瘍内各特定細胞集団の制御及び転移抑制効果をも加味した プロジェクト
BNCT を含む放射線治療の最適化（京大・原子炉）増永慎一郎、櫻井良憲、田中裕基、劉 勇、近藤夏子（藍野学院短期大）高垣政雄、（放医研）松本孔貴

PR3-2 Evaluating the Usefulness of a Novel Tumor Targeting 10B-carrier, GPU-201 in Boron Neutron
Capture Therapy
T. Harada1, S. Masunaga2, S. Kimura1, K. Okuda1, T.
Hirayama1, and H. Nagasawa1
1
Laboratory of Medicinal & Pharmaceutical Chemistry,
Gifu Pharmaceutical University
2
Research Reactor Institute, Kyoto University
INTRODUCTION: Boron-containing agents play a keyrole in successful boron neutron capture therapy (BNCT).
We developed new 10B carriers containing the
Arg-Gly-Asp (RGD) motif which is the minimum
recognition element for the v3 integrin. Integrin v3 is
an attractive target for antitumor drug delivery because of
its specific expression in proliferating endothelial andtumor cells of various origins. Therefore, icosahedralboron cluster-Arg-Gly-Asp (RGD) peptide conjugate,
GPU-201(Fig. 1) was developed as a tumor-selective
boron carrier. 1 We evaluated the usefulness of GPU-201
in BNCT.2

an inclusion complex with -CD by NMR study. The
stoichiometry and association constant of the complex
were estimated by using 1H-NMR techniques. These
analysis showed that the stoichiometric ratio was 1 : 1
and the Ka value was 229.8 M-1. The complex of BSH
with HP--CD is effective for sustainable localizationin tumor. Based on the findings concerning these 10B
biodistribution patterns, irradiation was started from 60
min and 30 min after the intraperitoneal administration of
BSH-CD or GPU-201 at a dose of 0.75 mole･g-1 and
BSH at a dose of 1.2 mole･g-1, respectively.

Under neutron beam irradiation, the sensitivity oftumor cells was increased with any 10B-carrier, however GPU-201 and BSH- CD showed a greaterrdiosensitizing effect than BSH. In contrast, no reliable
radio- sensitizing effect was obtained with any
10
B-carrier under -ray irradiation. (Fig.1) There wasno significant difference of tumor growth 14 days after

Figure 1

EXPERIMENTS: Chemistry: Synthesis of GPU-201:
The cyclo(-Arg-Gly-Asp-D-Phe-Lys-) {c[RGDfK]} wassynthesized by Fmoc solid-phase method and conjugated10toB cluster 1,2-dicarba-closo-dodecaborane-10B
through alkyl amide linker chain.
Enhancement of BNCT and -ray effect by GPU-201:
Mercaptododecaborate-10B(BSH)
dissolved
inphysiological saline and BSH and GPU-201 dissolved
with 10% HP--cyclodextrin (HP--CD) (pH 7) as asolubilizing and dispersing agent were intraperitoneallyadministered to SCC VII tumor-bearing mice. Then, the10
B concentrations in the tumors and normal tissues were
measured by -ray spectrometry. Subsequently,
tumor-bearing
mice
were
continuously
given
5-bromo-2’-deoxyuridine (BrdU) to label all proliferating(P) cells in the tumors, then treated with GPU-201,
BSH-CD, or BSH. Immediately after reactor neutron
beam or -ray irradiation, during which intratumor 10B
concentrations were kept at levels similar to each other,cells from some tumors were isolated and incubated witha cytokinesis blocker. The responses of the Q and total (=P + Q) cell populations were assessed based on thefrequency of micronuclei using immunofluorescencestaining for BrdU.
RESULTS: In the evaluation of time course of the 10B
concentration in tumor and organs, the retention of 10B
from BSH-CD and GPU-201 was similar except in blood,
where the concentration of 10B from GPU-201 was higher
for longer. We also demonstrated that the BSH formed

Figure 1 Clonogenic cell survival curves after invivo irradiation

Figure 2 Tumor growth curves after neutron beamirradiation with/without 10B-carriers.

the tumor cell inoculation without irradiation, with orwithout a 10B-carrier. With irradiation at a physicallyabsorbed dose of 1.825 Gy, the period required wassignificantly prolonged compared with no irradiation(p < 0.05), except for neutrons only without a
10
B-carrier, and the treatments ranked in the following
order; without a 10B-carrier < with BSH < with
BSH-CD < GPU-201(Fig. 2).
Conclusion: A novel 10B-carrier conjugated with an integrin-binding RGD peptide (GPU-201) that sensitizedtumor cells more markedly than conventional 10B-carriers
may be a promising candidate for use in BNCT. However,its toxicity needs to be tested further.
REFERENCES:[1]S. Kimura et al., Bioorg. Med. Chem., 19 (2011)1721-1728.
[2]S. Masunaga et al., World J. Oncol., in press (2012)

採択課題番号 23P3-2 低酸素微小環境を標的とするボロンキャリアの開発（岐阜薬大・薬化学）原田友宏、木村禎亮、奥田健介、平山 祐、永澤秀子（京大・原子炉）増永慎一郎

プロジェクトPR3-3

A Biological Mechanism behind the Radioresistance of Hypoxic Tumor Cells

H. Harada1,2, S. Itasaka2, M. Yoshimura2, M. Ogura2, S.
Masunaga3 and M. Hiraoka2
1

Group of Radiation and Tumor Biology, Career-Path
Promotion Unit, Kyoto University.
2
Department of Radiation Oncology and Image-applied
Therapy, Kyoto University Graduate School of Medicine.
3
Research Reactor Institute, Kyoto University.
INTRODUCTION: Because of the limited distancemolecular oxygen can diffuse, most malignant tumors
grow individually as a conglomerate of so-called ‘micro
tumor cords’, in each of which a blood vessel is surrounded by well-oxygenated (normoxic), then oxygen-insufficient (chronic hypoxic), and finally oxygen-depleted (anoxic/necrotic) cancer cells [1-3].
Immunohistochemical analysis with antibodies against awell-known hypoxia marker, pimonidazole, and against atranscriptional factor HIF-1 recently revealed thatHIF-1-positive cells are not necessarily stained withpimonidazole. Namely, in some types of malignant tumors, chronic hypoxia can be histologically categorized
into 2 sectors; HIF-1+/pimonidazole- (herein: HIF-1+
regions)andpimonidazole+/HIF-1(herein:+pimonidazole regions). Tumor hypoxia and HIF-1 havelong been associated with tumor radioresistance and tumor recurrence after radiation therapy; however detailed
molecular mechanisms behind them are largely unknown.
To address these questions, we performed following two
basic researches.
EXPERIMENTS:
1. A Molecular Mechanism behind Tumor Recurrence
after Radiation Therapy: 【Purpose & Methods】To
directly examine the involvement of hypoxia in tumor
recurrence, we developed a novel cell-tagging strategy
mediated by a hypoxia-inducible Cre-loxP system. We
tracked the post-irradiation fate of the cells which existed
in perinecrotic regions at the time of radiation [4].
【Results】Although the perinecrotic/Pimonidazole+ cells
were originally HIF-1-negative, they acquired HIF-1
activity after surviving radiation, which triggered theirtranslocation toward tumor blood vessels [4]. HIF-1
inhibitors suppressed the translocation and decreased theincidence of post-irradiation tumor recurrence [4].
【Conclusions】For the first time, our data unveil the
HIF-1-dependent cellular dynamics during postirradiation
tumor recurrence and provide a rational basis fortargeting HIF-1 after radiation therapy [4].
2. A Biological Mechanism behind Radioresistance of
Perinecrotic Hypoxic Tumor Cells: 【 Purpose &採択課題番号 23P3-3

Methods 】 Accumulated evidence has suggested thatsome intracellular and extracellular factors, such as oxygen-availability, transcriptional activity of HIF-1 and cell
cycle status, have been suggested to affect radioresistanceof cancer cells. We next analyzed mechanistic andspatio-temporal relationships among them in highly heterogeneous tumor microenvironments in order to elucidate a mechanism behind the predominant survival of
perinecrotic tumor cells after radiation. 【 Results 】
Experiments in vitro demonstrated that a decrease in the
glucose concentration reduced the transcriptional activityof HIF-1 and expression of a downstream gene for thecell cycle regulator p27Kip1 even under hypoxic conditions. Then, the proportion of cells in radioresistantS-phase increased, whereas those in radiosensitive
G1-phase decreased, significantly. Immunohistochemical
analyses showed that cancer cells in perinecrotic hypoxic
regions, which should be under low glucose conditions,expressed little HIF-1; and therefore, were mainly in
S-phase and less damaged by radiation treatment. Continuous administration of glucagon, which increased
blood glucose concentration and resultantly improved
glucose-availability in perinecrotic hypoxic regions, induced HIF-1 expression and increased radiation-induced DNA damage. 【Conclusions】Cancer cells
in perinecrotic regions, which would be under low glucose and hypoxic conditions, acquire radioresistance bydecreasing the level of both HIF-1 activity and p27Kip1
expression and adjusting their cell cycle to theradioresistant S-phase [5].
REFERENCES:[1] *Harada H. Gene Therapy Strategy for Tumor Hypoxia. Targets in Gene Therapy. 11: 185-200. 2011.
[2] Yeom CJ, Zeng L, Zhu Y, Hiraoka M, *Harada H.
Strategies to assess hypoxic/HIF-1-active cancer cells
for the development of innovative radiation therapy.
Cancers. 3: 3610-3631. 2011.
[3] *Harada H. How can we overcome tumor hypoxia inradiation therapy? J Radiat Res. 52: 545-556. 2011.
[4] *Harada H, Inoue M, Itasaka S, Hirota K, Morinibu A,
Shinomiya K, Zeng L, Ou G, Zhu Y, Yoshimura M,
McKenna WG, Muschel RJ, Hiraoka M. Cancer cells
that survive radiation therapy acquire HIF-1 activity
and translocate toward tumor blood vessels. Nat
Commun. in press.
[5] Zhu Y, Zhao T, Itasaka S, Zeng L, Yeom CJ, Hirota K,
Suzuki K, Morinibu A, Shinomiya K, Ou G,
Toshimura M, Hiraoka M, *Harada H. Involvement of
Decreased Hypoxia-Inducible Factor 1 Activity and
Resultant G1-S Cell Cycle Transition inRadioresistance of Perinecrotic Tumor Cells. Oncogene. in pres

腫瘍組織レベルの光イメージングで迫る

プロジェクト

癌の放射線抵抗性機構の解明（京大・キャリアパス）原田 浩（京大院・医）板坂 聡、小倉 昌和、吉村 通央、平岡 真寛（京大・原子炉）増永 慎一郎

PR3-4

An Influence of Boron Neutron Capture Reaction on HSG Cell Inactivation

R. Hirayama, Y. Matsumoto, S. Masunaga1, Y. Sakurai1,
H. Tanaka1 and Y. Furusawa
Research Center for Charged Particle Therapy, National
Institute of Radiological Sciences
1
Research Reactor Institute, Kyoto University

stained with 1 % methylene blue in water. Colonies
consisting of more than 50 surviving cells were scored.
The survival curves were fitted by the single-hit model:
SF = exp (-α∙D). SF and D are the surviving fraction
and the dose, respectively.

INTRODUCTION:
Excellent dose distribution ofneutron capture reaction of boron atom induces high relative biological effectiveness (RBE) and the low oxygen
enhancement ratio (OER). These phenomena are commonly assumed to be an interaction between cells andlow energy heavy particles (α and Li) resulting from theboron atom fissions in the cells. However, there has
been little study done concerning the action of the particles on living cells. We have investigated contributionsof indirect actions of radiation in cell killing by heavy
ions with radical scavenger that selectively reduces theindirect action [1-3].
Therefore, it is important that how these mechanisms
can be made to clear through a thorough basic research inboron neutron capture therapy is urgently discussed.
The main object of this year is to disclose an influence ofboron neutron capture reaction on HSG cell inactivation.

RESULTS: The survival curves for neutrons is shownin Fig. 1. The D10 value for with and without BPA were1.62 and 2.79, respectively. The D10 value of X-rays
was 4.70 Gy. The RBE values for with and without
BPA were 1.68 and 2.90, respectively. The survival
curves of the coefficient of determination (R2) wereabove 0.9. We will retest and add the dose points.
These results suggest that he large RBE was induced bylow energy heavy particles (α and Li) from boron neutron
capture reaction.

BPABPA+(3hr)
0.1Surviving Fraction

EXPERIMENTS: Human salivary gland tumor (HSG)
cells were grown in Eagle’s minimum essential medium
supplemented with 10% fetal bovine serum and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin)
under humidified air with 5% CO2 at 37˚C. The HSG
cells
were
incubated
with
25
µg/ml
10
B-para-boronophenylalanine (BPA) for 3 h prior to irradiations. The cells were suspended at a density of
about 7 × 105 cells/ml. The cells in Polypropylene tubes(NUNC) were irradiated at the remodeled heavy water
facility at the KURRI.
Total fluencies of thermal neutron, epithermal neutronand fast neutron were measured by means of gold foil
activation analysis. The gamma ray dose including
secondary gamma rays was measured with a thermo luminescence dosimeter. Boron concentrations in thecells were taken to be equivalent to those in the medium
as reported previously [4]. The total absorbed dose resulting from thermal neutron irradiation was calculatedby the sum of the absorbed doses mainly from the1
H(n,g)2D, 14N(n,r)14C and 10B(n,a)7Li reactions according to Kobayashi’s model [5].
After irradiation, cells were seeded in triplicate onto 60
mm (Φ) culture dishes at densities to give approximately
100 colonies per dish. After 14 days of incubation, thecolonies were fixed with 10 % formalin solution and

1

0.01

0.001

0.0001
0

2

4

6

8

Dose (Gy)Fig. 1. Survival curves for HSG cells exposed to neutrons in the absence (circle) and presence (square) of 25µg/ml BPA. The survival parameters were calculated
from the data by a curve fitting using: SF = exp (-α∙D).
REFERENCES:[1] A. Ito et al., Radiat. Res., 165 (2006) 703-712.
[2] R. Hirayama et al., Radiat. Res., 171 (2009) 212-218.
[3] R. Hirayama et al., Radiat. Phys. Chem., 78 (2009)1175-1178.
[4] H. Fukuda et al., Int. J. Radiat. Biol., 51 (1987)
167-175.
[5] T. Kobayashi et al., Radiat. Res., 91 (1982) 77-94.

採択課題番号 23P3-4 中性子捕捉反応における細胞致死機構の放射線作用解析（放医研・重粒子医科学センター）平山亮一、松本孔貴、古澤佳也（京大・原子炉）増永慎一郎、櫻井良憲、田中浩基

プロジェクトPR3-5

Boron-conjugated hyaluronan nanoparticles for Tumor Targetingin Boron Neutron Capture Therapy

S. Kasaoka, K. Masuda, K. Hashimoto, S. Masunaga1, Y.
Sakurai1, H. Tanaka1 and K. Ono1
Department of Pharmaceutical Science, Hiroshima International University
1
Research Reactor Institute, Kyoto University

INTRODUCTION: Hyaluronan is a candidate foractive targeting ligand to melanoma, many of which
overexpress the hyaluronan receptors CD44 andRHAMM [1]. In this study, we have developed a novel
boron delivery system for BNCT using hyaluronan conjugated with borocaptate (BSH). This BSH-bearing
hyaluronan nanoparticles (B-HA-NPs) are expected todeliver boron atoms into melanoma cells by receptor-mediated endocytosis for antitumor application in
BNCT. Transdermal drug delivery system has been accepted as potential non-invasive route of drug administration, with advantages of sustained therapeutic action
and better patient compliance, though, its prevalent use isrestricted due to excellent impervious nature of skin.
Thus, many approaches have been attempted to perturb
skin barrier and enhance the transdermal delivery of drug[2]. The major approaches for enhancing transdermaldeliveryarephysicalenhancers(ultrasound,iontophoresis and electroporation), vesicles, particulate
systems(liposome,niosome,transfersome,microemulsion, solid lipid nanoparticle) and chemicalenhancers. Electroporation (EP) is a physical method thatenhances delivery of molecules to tissues in vivo. In thisstudy, a unique nanoparticle-EP-mediated approach has
been developed for dermal and intracellular delivery of10
B into melanocytic tumors present in skin to retard
melanoma.
EXPERIMENTS: BSH was dissolved in acetone andadded to N-Succinimidyl 6-Maleimidobenzoate with triethylamine catalysis. The mixture was incubated at room
temperature for 2 h under argon. Sodium hyaluronate wasdissolved in deionized water. To this solution, the highly
acidic ion exchange resin, Dowex-100, was added, andthe slurry was stirred for eight hours. The acidic solution
was neutralized with 0.2M tetrabutylammonium hydroxide, forming a quaternary ammonium salt of hyaluronate
and the tetrabutylammonium group (HA-TBA). The solution was frozen and lyophilized to yield the dry product.
The HA-TBA was dissolved in DMSO and added to BSH
derivative was of conjugation with diethylamine catalysis.

The mixture was incubated at room temperature for 48 hunder argon. The solution was dialyzed and lyophilized
to yield the dry product.
B16F10 murine melanoma cells were pre-incubated with
25 ppm of B-HA-NPs, B-Folate-conjugates and BSH
solution for 6 hours at 37℃ before neutron irradiation.
The cells were rinsed twice in PBS and suspended in
fresh medium. After irradiation the cells were plated into
plastic Petri dishes 60 mm in diameter at 200 cells per
dish. They were incubated for an additional 7 days toallow colony formation.
RESULTS: Novel BSH-bearing hyaluronan nanoparticles were obtained by the self-assembly in water. The
critical micellar concentration of B-HA-NPs (diameter:
90-180 nm) was in the range of 300-800 mg/L determined by the fluorescence probe technique using pyrene.
B-HA-NPs had high stability (90-97%) in the retention of10
B during storage at 4-37 ℃. All boborocaptate-loaded
formulations had no cytotoxic effects. B-HA-NPs werereadily bound to melanoma cells, and were internalized
by receptor-mediated endocytosis. As shown in Fig. 1,
survival of the washed cells pre-incubated with
B-HA-NPs was lowest compared to survival of control
cells that were pre-incubated with B-PEG-liposomes,
B-HA-liposomes, B-FA-conjugates and BSH solution.
This result suggested cytotoxicity depended on internalization of 10B.
We are now ongoing to investigate the therapeutic potential of EP-mediated approach for transcutaneous BSH
delivery in a three-dimensional skin reconstruction modelof melanoma.

Fig. 1. Suppression of the colony formation of B16F10
cells after in vitro BNCT. Values are means±S.D.
REFERENCES:[1] D. Peer and R. Magalit, Neoplasia, 6 (2004)343-353[2] M. Nino et al., Dermatol. Online J. 16(2010)

採択課題番号 23P3-5 腫瘍内微小環境の解析とその利用による悪性腫瘍制御の試み プロジェクト（広国大・薬）笠岡 敏、橋本佳奈（京大・原子炉）増永慎一郎

PR3-6

Kojic Acid-Appended Carorane/Hydroxypropyl--Cyclodextrin Complex:
A Novel Boron Carrier for Melanoma Targeting

R. Kawasaki, T. Nagasaki, J. Li, Y. Zhu, H. Azuma, S.
Masunaga2, M. Kirihata1, Y. Hattori1 and N. Kadono2
Graduate School of Engineering, Osaka City University
1
Graduate School of Life and Environmental Sciences,
Osaka Prefecture University
2
Research Reactor Institute, Kyoto University
INTRODUCTION: Metastatic melanoma remains s
highly lethal cancer. Boron neutron capture therapy(BNCT) has been attracted great deal of attention as apotentially useful modality for this disease [1]. The delivery of 10B compounds deeply inside the tumor cells
and, possibly, close the nucleus is also an important requirement in order to improve the efficacy of BNCT.
Kojic acid is well known to work as an excellent whitening agent for melanocytes by a strong tyrosinase inhibition [2]. This fact suggests that kojic acid possess a specific affinity for melanocytes. In this paper, we evaluate
kojic acid-appended o-carborane (CKA) as BNCT boron
agent for melanoma.
EXPERIMENTS: Since CKA has some disadvantages
water-insoluble compound, water-soluble complex of
CKA with hydroxypropyl--cyclodextrin (CKA/HP-CD,
Fig. 1) was prepared. B16BL6 murine melanoma cells
and colon 26 murine colon carcinoma cells were grownin RPMI and DMM mediums, respectively, containing
10% FBS, 100 U/ml penicillin, 100 g/ml streptomycin
at 37°C with CO2 atmosphere. In order to evaluate theinfluence of kojic acid (KA) on internalization of CKA
complex, cells were treated in the presence of 10 mM
kojic acid. Boron concentration was measured by
ICP-AES (Vista-MPX, Seiko instruments Inc.). Intracellular Trafficking of CKA/HP-CD was estimated byimmuno-fluorescent method with anti-BSH antibody and
Zenon® immunolabeling technology (Invitrogen).

RESULTS AND DISCUSSION: Inclusion rate of obtained water-soluble complex was calculated as 88% by
1
H-NMR and UV spectroscopy. Evaluation of in vitro
uptake of boron containing CKA/HP-CD complex was
carried out with B16BL6 and colon 26 cells (Fig. 2A).
The 50 times higher uptake of boron by B16BL6 cells
was observed than that of colon 26 cells. Inhibition ofboron uptake by free kojic acid was confirmed with
B16BL6 cells (Fig. 2B). The boron uptake was suppressed one-fourth in the presence of excess kojic acid. It
seems that CKA has melanoma affinity based on possessing of kojic acid moiety although detail mechanism isunclear yet. In B16BL6 cells, CKA was interestingly localized in the nucleus. Morrison et al. reported that nuclear localization of boron drug increased tumor-killing
effect by BNCT [3]. Kojic acid-appended carborane(CKA) is promising to be an efficient boron agent towardmelanoma BNCT.

Fig. 2. In vitro cellular uptake of CKA by melanoma andcolon carcinoma cell lines (A). Inhibition experiment of
CKA uptake by kojic acid (B).

A

B

C

Fig. 3. Intracellular distribution of CKA (transmission, [A]; nucleus stained with DAPI [B]; CKA [C]).
REFERENCES:[1] H. Fukuda et al., Radiat. Res., 138 (1994) 435-442.
[2] Y. Mishima, Pigment Cell Res., 14 (2001) 47-70.
[3] G.H. Morrison et al., Biol. Cell, 74 (1992) 105-108.
Fig. 1. Water-soluble CKA/HPβ-CD complex.

採択課題番号 23P3-6

メラノーマ中性子捕捉療法への適応を目指した薬剤送達システムに関する研究

プロジェクト

（阪市大院・工）長﨑 健、東 秀紀、李 家暐、朱 宇翔、鹿子嶋祐太、林高一郎、河崎 陸（阪府大院・生命環境科）切畑光統、服部能英、門野尚之（京大・原子炉）増永慎一郎