Project Research
Project 9PR9Analyzing Tumor Microenvironment and Exploiting its Characteristics in
Search of Optimizing Cancer Therapy Including Neutron Capture Therapy

S. Masunaga
Particle Radiation Biology, Division of Radiation Life
Science, 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
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.
RESEARCH SUBJECTS:
The collaborators and allotted research subjects (ARS)
were organized as follows;
ARS-1 (27P9-1): 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 (27P9-2): Development of Hypoxic Microenvironment-Oriented 10B-Carriers. (H. Nagasawa, et al.)
ARS-3 (27P9-3): Clarification of Mechanism of Radio-Resistance in Cancer Using Optical Imaging at Tissue Level. (H. Harada, et al.)
ARS-4 (27P9-4)*: Analysis of Radiation-Induced
Cell-Killing Effect in Neutron Capture Reaction. (R.
Hirayama, et al.)
ARS-5 (27P9-5)*: Transdermal Drug Delivery Systemusing Hyaluronan-Conjugated Liposomes as 10B-Carrier
in Boron Neutron Capture Therapy for Melanoma(S. Kasaoka, et al.)
ARS-6 (27P9-6) *: 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 (27P9-7)*: Molecular Design and Synthesis andFunctional Evaluation of Anticancer and Molecular Targeting Agents. (Y. Uto, et al.)
ARS-8 (27P9-8)*: Analyzing Biological Effect of BNCT
from the Viewpoint of the Changes in Oxygenation Level. (H. Yasui, et al.)
ARS-9 (27P9-9): Analyses on the Responsiveness of
Malignant Tumors to BNCT. (M. Masutani, et al.)
ARS-10 (27P9-10) *: 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 (27P9-11)*: Antitumor and MetastasisReppresing Effect of BNCT on Human Breast andPancreatic Cancer Cell Lines. (Y. Matsumoto, et al.)(* Due to the absence of operating our reactor in 2015,
no data were obtained, resulting in no reporting here.)

27P9- 50 -

PR9-1The Effect of p53 Status of Tumor Cells on Radiosensitivity of IrradiatedSolid Tumors with Accelerated Carbon-ions Compared with -rays or Reactor
Neutrons

S. Masunaga, A. Uzawa1, R. Hirayama1, Y. Matsumoto2, Y.
Sakurai, H. Tanaka, K. Tano, Y. Sanada, M. Suzuki, N.
Kondo, T. Watanabe, T. Takata, A. Maruhashi and K. Ono
Research Reactor Institute, Kyoto University
Research Center for Charged Particle Therapy, National
Institute of Radiological Sciences
2
Proton Medical Research Center, Faculty of Medicine,
University of Tsukuba
1

INTRODUCTION: We examined the characteristics ofradio-sensitivity in the total (proliferating (P) plus quiescent) and quiescent (Q) cell populations in solid tumors
irradiated with 290 MeV/u accelerated carbon ion beams
at varying LET values in a 6-cm spread-out Bragg peak
(SOBP) installed at the National Institute of Radiological
Sciences (Chiba, Japan) compared with irradiation with60
C -rays and reactor thermal and epithermal neutron
beams at our institute with our method for selectively
detecting the response of Q cells within solid tumors[1,2], using two different tumor cell lines with identicalgenetic backgrounds except for p53 status.
MATERIALS AND METHODS: Human head andneck squamous cell carcinoma cells transfected with mutant TP53 (SAS/mp53) or with neo vector (SAS/neo)
were injected subcutaneously into hind legs of nude mice.
Tumor-bearing mice received 5-bromo-2’-deoxyuridine(BrdU) continuously to label all intratumor P cells. They
received -rays or accelerated carbon-ion beams at a highor reduced dose–rate. Other tumor-bearing mice received
reactor thermal or epithermal neutrons at a reduced
dose-rate. Immediately or 9 hours after the high dose-rateirradiation (HDRI), or immediately after the reduced
dose-rate irradiation (RDRI), the tumor cells were isolated and incubated with a cytokinesis blocker, and the micronucleus (MN) frequency in cells without BrdU labeling (= Q cells) was determined using immunofluorescence staining for BrdU.
RESULTS: The difference in radio-sensitivity between
the total (= P + Q) and Q cells after -ray irradiation wasmarkedly reduced with reactor neutron beams or carbon
ion beams, especially with a higher linear energy transfer(LET) value. Following -ray irradiation, SAS/neo tumor
cells, especially intratumor Q cells, showed a marked
reduction in sensitivity due to the recovery from radiation-induced damage, compared with the total or Q cells
within SAS/mp53 tumors that showed little repair capacity [3]. In both total and Q cells within both SAS/neo andSAS/mp53 tumors, carbon-ion beam irradiation, especially with a higher LET, showed little recovery capacity
through leaving an interval between HDRI and the assay

27P9-1- 51-

or decreasing the dose-rate. The recovery from radiation-induced damage after -ray irradiation was a
p53-dependent event, but little recovery was found after
carbon-ion beam irradiation. With RDRI, the radiosensitivity to reactor thermal and epithermal neutron beams
was slightly higher than that to carbon ion beams.
DISCUSSION: Two major pathways for the repair ofpotentially lethal DNA dsbs exist in mammalian cells.
The non-homologous end-joining (NHEJ) pathway isimprecise, error-prone, and mutagenic, and mutant celllines lacking key components of this pathway all exhibit
impaired kinetics of DNA dsb repair and exquisite radio-sensitivity. Homologous recombination (HR) is amore precise (error-free) repair mechanism and is more
important for the repair of dsbs in late-S and G2 when asister chromatid is available for the recombination reaction. Cell lines with defects in HR also exhibit increased
radio-sensitivity and decreased fidelity of repair.
A cellular safeguard against genetic destabilization is
activation of the p53 tumor suppressor protein, which
commonly responds to DNA damage signals by inducing
apoptosis or regulating the cell cycle including DNA
damage repair. As also shown in our previous report, thenet MN frequencies in SAS/neo tumor cells were lower
than those in SAS/mp53 tumor cells under all conditions(P < 0.05), probably resulting from exclusion of a higher
number of radiation-induced apoptotic SAS/neo cells
than SAS/mp53 cells.
Loss-of-function of wild-type TP53 can result in lossof the G1/S cell-cycle checkpoint and an increase in HR.
As p53 seems to interact with RAD51, the absence of
normal p53 function is thought to enhance
RAD51-mediated DNA pairing activity and HR, due tooverexpression of RAD51 out of regulation by normal
p53. Thus, HR is thought to be a principal mechanism of
DNA dsb repair in SAS/mp53 cells. The very small repair
capacity of SAS/mp53 cells in vivo may show that the
repair in solid tumors with a mutant p53 is mainly due to,
if anything, the NHEJ rather than HR.
CONCLUSION: For tumor control, including intratumor Q-cell control, accelerated carbon-ion beams, especially with a higher LET, and reactor thermal and epithermal neutron beams were very useful for suppressing
the recovery from radiation-induced damage irrespectiveof p53 status of tumor cells [4].
REFERENCES:[1] S. Masunaga, et al., Acta Oncol 47 (2008) 1087-1093.
[2] S. Masunaga, et al., Int J Radiat Oncol Biol Phys
70(1) (2008) 221-228.
[3] S. Masunaga, et al., Oncol Rep 18 (2007) 1589-1596.
[4] S. Masunaga, et al., World J Oncol 6(4) (2015)398-409.

PR9-2Development of New Membrane Penetrating Boron Carriers Comprised of
BSH and Pepducins

A. Isono, T. Hirayama, K. Okuda1, S. Masunaga2 and H.
Nagasawa
Laboratory of Medicinal & Pharmaceutical Chemistry,
Gifu Pharmaceutical University
1
Laboratory of Organic Chemistry, Kobe Pharmaceutical
University
2

Research Reactor Institute, Kyoto University

INTRODUCTION: Selective delivery of sufficientquantity of 10B to tumor cells is essential for the successof boron neutron capture therapy (BNCT). Although sodium borocaptate (BSH) has been used clinically as aboron carrier for BNCT, it is impermeable to plasma
membrane due to its high hydrophilicity and anioniccharges. Recently, we found that pepducin, which are
artificial lipopeptides derived from an inner loop domain
of G protein-coupled receptors (GPCRs), 1 enabled anionic molecule such as fluorescein to penetrate membrane
directly.2 These findings let us to envisage that an anionicboron cluster can be delivered into cytosol using the
pepducin delivery unit.
In this study, we designed and synthesized novel
pepducin-boron cluster hybrid compounds as boron carriers for BNCT and evaluated the intracellular delivery ofthem.
MOLECULAR DESIGN: As shown in Fig. 1, we designed new hybrid molecules comprising pepducin (Pep)
as a vehicle, and boron cluster (BS) as a cargo through anappropriate linker. When this molecule is internalized
into the cytosol, intracellular glutathione can cleave thedisulfide bond (SS) to release the boron cluster cargo into
the cytosol. 10B atoms are accumulated in the cells due
to hydrophilicity of the anionic property.

boron carrier. T98G cells were treated with the boron
carriers (10 μM) at 37 °C for various time, then, washedwith PBS three times, and dissolved in 200 μL HNO3 for
1 h. The boron concentrations of the extracts were measured by inductively coupled plasma-atomic emission
spectrometry (ICP-AES: HORIBA JOVIN YBON ULTIMA2) detected at 249.773 nm.
RESULTS AND DISCUSSION: Various lipopeptide-BSH hybrids were synthesized (Table 1). Compound
13Pep showed the highest boron concentration in cells

among the test compounds examined by ICP-AES. The
boron concentration increased over time from 2h to 12 h.
(Fig. 2). These data suggested that the palmitoyl tail andCargo
reductively cleavable linker
linker

S

10B
10BH

S S
linker

C15H31

peptide2

NH2

O
cell-penetrating pepducin

Vehicle.
Fig 1 Design of membrane penetrating boron carriers

METHODS: To explore the structural requirements ofmembrane penetrating boron carrier for intracellular uptake, we evaluated the sequence and length of peptide
and structure of lipid moiety. Various lipidated peptides
were prepared by solid-phase synthesis and then combined with BSH through an appropriate liker to afford thehydrophobic residues at C-terminal end are necessary forthe intracellular penetration.
We are performing the further optimization of peptides
and linker structures to promote the intracellular accumulation.
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.

27P9-2- 52 -

PR9-3

LY6E; a Conductor of Malignant Tumor Growth through Modulation of the
PTEN/PI3K/Akt/HIF-1 Axis

H. Harada and S. Masunaga1
Department of Radiation Oncology and Image-applied
Therapy, Graduate School of Medicine, Kyoto University
1
Research Reactor Institute, Kyoto University
INTRODUCTION:
Accumulating evidence has
shown that hypoxia-inducible factor 1 (HIF-1) plays critical roles in radioresistance of cancer cells and tumor
recurrence after radiation therapy and eventually causes
death among cancer patients. Clinical studies havedemonstrated 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 [1,2].
HIF-1, a heterodimeric transcription factor com-posed ofan α-subunit (HIF-1α) and a β-subunit (HIF-1α), its activity is known to depend on the expression levels of
HIF-1α protein. Under normoxic conditions, HIF-1α
protein is actively degraded through the hydroxylationand subsequent ubiquitination-mediated proteolysis reactions. On the contrary, HIF-1α becomes active under hypoxic conditions because of the inactivation of the hydroxylases, and then, interacts with its binding partner,
HIF-1β. 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 and metastasis ofcancer cells) as well as the improvement of
oxy-gen-availability (angiogenesis) and adaptation ofcellular metabolism to hypoxia (metabolic reprogramming).
In order to explore novel genes which are responsible for
the HIF-1-mediated tumor malignant progression, we
recently established a new genetic screening method andfound that overexpression of lymphocyte antigen 6
complex, locus E (LY6E) is responsible for the activation of HIF-1. In the present study, we analyzed both themolecular mechanisms underlying the LY6E-mediatedactivation of HIF-1 and the involvement of LY6E-HIF-1
axis in malignant progression of cancers.
Fig. 1

EXPERIMENTS & RESULTS: Forced expression of
LY6E using a plasmid-based expression vector for LY6E
increased HIF-1α gene expression principally at the transcription level (Fig. 1).
This, in turn, led to the expression of the pro-angiogenic
factors, VEGFA and PDGFB, through decreases in theexpression levels of PTEN mRNA and subsequent activation of the PI3K/Akt pathway. The LY6E-HIF-1 axis
functioned to increase tumor blood vessel density andpromoted tumor growth in immune-deficient mice (Fig.
2).

Fig. 2LY6E expression levels were significantly higher in human breast cancers than in normal breast tissues, andwere strongly associated with the poor prognoses of various cancer patients (Fig. 3). Moreover we found that
LY6E induced radioresistance of cancer cells via the activation of HIF-1.

Fig. 3

Our results characterized LY6E as a novel conductor oftumor growth and tumor radioresistance through its modulation of the PTEN/PI3K/Akt/HIF-1 axis and demonstrated the validity of targeting this pathway for cancer
therapy.
REFERENCES:[1] Inoue M, Yoshimura M, Kobayashi M, Morinibu A,
Itasaka S, Hiraoka M, *Harada H. Sci Rep. 5:15666.
2015.
[2] *Harada H. J Radiat Res. in press.

27P9-3- 53 -

PR-

Response of Squamous Cell Carcinoma SAS Cells after BNCR

S. Imamichi1, T. Itoh1, 2, S. Kikuhara1, 2, A. Sato3, H. Fujimori1,4, T. Hirai1, 5, S. Masunaga6 and M. Masutani1,4

Yoshinori Sakurai and Hiroki Tanaka of KUR. 10B concentration was measured by prompt-gamma ray analysis(PGA) as described elsewhere.

1

Division of Chemotherapy and Clinical Research, National Cancer Center Research Institute2
Faculty of Industrial Science and Technology, Tokyo
University of Science
3
Faculty of Pharmaceutical Sciences, Tokyo University of
Science4Department of Frontier Life Sciences, Nagasaki University Graduate School of Biomedical Sciences
5
Department of Radiation Oncology, Juntendo University
Faculty of Medicine
6
Department of Radiation Oncology, National Cancer
Center Hospital
8
Research Reactor Institute, Kyoto University
INTRODUCTION: Boron neutron capture reaction(BNCR) efficiently introduces DNA double strand breaks
in the cells [2], however, tumor cell killing is affected byvarious factors [3] including the uptake of boron compounds. Heterogeneiety of cancer cells in the tumor tissues, such as the presence of cancer stem-like cells andhypoxic cells, may potentially cause resistant populations
to the boron neutron capture therapy (BNCT) [1]. It istherefore important to analyze the responses of the cells
to boron neutron capture reaction (BNCR) in various
conditions. We have performed comprehensive analysis
of mRNA expressions and proteome of human squamous
carcinoma SAS cells after BNCR [4]. Changes in theprotein levels involved in the various functions, such as
endoplasmic reticulum, DNA repair, and RNA processing were observed within 24 hrs after neutron-beamirradiation. We also showed that BNCR induced generation of fragments from endoplasmic reticulum-localized
lymphoid-restricted protein (LRMP). The elucidation of
biological significance of fragmentation of LRMP isfurther necessary.
EXPERIMENTS: Because the neutron-beam irradiation
with KUR nuclear reactor was not carried out during
FY2015, we analyzed the irradiated cell samples prepared before. We used the cell extract of human oral
squamous cancer SAS cells [1] to perform the mRNA
expression and proteome analysis. Previously,
10
B-boronophenylalanine (10B-BPA)-fructose solution
was prepared as described [5]. SAS cells were suspended
in the polypropyrene vials and incubated 2 hrs with orwithout 25 ppm of 10B-BPA [5]. After 6 and 24 hrs irradiation operated at 1 MW in the KUR facility, cells wereseparated to supernatant and cell pellet. Proteome analysis was performed using two dimensional polyacrylamide
gel electrophoresis and mass spectrometry. Thermal neutron fluence and gamma-ray dose was measured withthermaluminescence dosimeter. These physical radiation
doses were measured with the kind help of Drs.

RESULTS: For the analysis of RNA and protein dynamics, we used therapeutic dose conditions. The irradiated total dose of BNCR-treated SAS cells was about
17.5 Gy and that of neutron beam-irradiated SAS cells
was about 4.0 Gy. Cell lysates of SAS cells with BPA(BNCR-sample) or without BPA (control) harvested 6
and 24 hrs post-irradiation were analyzed. Twenty-fourhours after irradiation,	  apoptosis was observed as amajor cell death and cleavage of caspase-9, caspase-3 and
PARP-1 was observed. The sign of necrosis was also
observed at this time point. The expression analysis ofmRNA demonstrated 	  dynamic changes of various
genes related to inflammation and immune responses,
and transcription. In the proteomic analysis [4], the peptide sequences from twenty-two spots have been determined by MALDI-TOF/MS from the twenty-nine spotsthat showed changes in the intensities between
BNCR-sample and control.
We observed that proteins involved in the vesicle regulation, mRNA processing, transcription, rRNA processing, GTPase activity, ribosome biogenesis, DNA
replication, and respiratory electron transport chain
showed dynamic changes. Notably, lymphoid-restricted
membrane protein (LRMP) and steroid hormone receptor(ERR1) were detected in multiple spots of different molecular mass. LRMP/ Jaw1 is known to be present at thecytoplasmic face of the ER, but its function has not been
well understood [6]. We observed fragmentation of
LRMP in the grafted lymphosarcoma of the rat 20 hrs
after BNCR [4].
We further analyzed the LRMP in BNCR-sample andcontrol by western blotting. Truncated LRMP of around
22 kDa was induced 24 hrs after BNCR-irradiation. The
study for the biological significance of truncated LRMPin BNCR-induced cell death is ongoing.
REFERENCES:

27P9-9- 54 -[1] D. Spanjaard, Ph.D. Thesis, Oxford Univ. (1969).
[2] S.G. Hussein et al., Proc. Montreal Int. Conf. Eds
Harvey, Cusson, Geiger, Pearson (U. Mont Press)(1969) 91.
[3] K. Okano et al., Nucl. Instr. and Meth, 186 (1981)
115-120.
[4] A. Sato et al., Appl. Rad. Iso., 106 (2015) 213-219[5] R. B. Firestone, in Table of Isotopes, 8th ed., edited
by V. S. Shirley (Wiley, New York, 1996), Vol. 1.
[6] T.W. Behrens et al., J. Immunol., 153 (1994)
682-690.