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Tumor Biology |
B Ligand (RANKL) Is a Key Molecule of Osteoclast Formation for Bone Metastasis in a Newly Developed Model of Human Neuroblastoma1
Department of Environmental Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, Osaka 594-1101, Japan
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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B
ligand (RANKL) in the coculture compared with the culture of bone
marrow cells alone. Interleukin-1
and cyclooxygenase-2 expression in
the murine marrow cells was also increased in the presence of NB-19
cells. To further study the role of RANKL in the OC-like cell formation
in the coculture of NB-19 cells and murine marrow cells, an expression
vector encoding the active portion of the murine osteoprotegerin, which
is the native inhibitor of RANKL action, was constructed and introduced
into COS-7 cells. The conditioned media of the COS-7 cells transfected
with the osteoprotegerin expression vector effectively blocked OC-like
cell formation in the coculture of the bone marrow cells and NB-19
cells. These results suggested that in the bone microenvironment of
NB-19-bearing mice, the stimulated expression of RANKL plays an
important role in OC formation, leading to osteolytic bone metastasis. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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7%
(9)
. In contrast, the patients with stage IV-S disease,
where the tumor does not cross the midline despite the presence of
metastasis to liver, skin, or bone marrow, show a rather favorable
prognosis, and the survival rate has been reported to be 75%
(10
, 11)
. These data suggest the urgent need to develop an
effective bone-targeting therapy in neuroblastoma. In some cancers such as breast cancer and myeloma as well, bone metastasis causes clinical complications including pain, pathological fractures, and nerve compression (12) . In the case of breast cancer, the role of osteoclastic bone resorption enhanced by the secretion of PTHrP3 has been demonstrated in the development of bone metastasis (13) , and adjuvant therapy with inhibitors of the osteoclastic bone resorption including bisphosphonates was proven to be beneficial (14, 15, 16, 17) . In neuroblastoma, however, the mechanisms involved in the bone metastasis have not been elucidated. In addition, because treatment with bisphosphonates may have adverse effects on bone growth in infants and children, other therapies may be required to treat childhood patients with neuroblastoma, although bisphosphonates have proven to be effective in treating bone complications in breast cancer patients and myeloma patients (14 , 18) .
The interaction between cancer cells and the bone marrow microenvironment plays an important role in bone metastasis. In the case of breast cancer, it is reported that estrogen-independent MDA-MB-231 cells respond to produce more PTHrP in the bone microenvironment when exposed to transforming growth factor ß, which is stored in bone matrix and released by osteoclastic bone resorption (19) . The increased production of PTHrP may accelerate the osteoclastic destruction of bone, creating the space for cancer proliferation in bone. Another line of studies using the murine mammary tumor cell line MMT060562 has demonstrated that PGE2 plays a role in in vitro osteoclastogenesis in a coculture system of tumor cells and bone marrow cells, although this tumor cell line does not possess metastatic potential in vivo (20) .
Very recently, RANKL, a novel cytokine also known as OPG ligand, OC differentiation factor, and TNF-related activation-induced cytokine, has been identified as being critical to OC recruitment (21, 22, 23, 24) . RANKL, expressed in bone marrow stromal cells that support OC differentiation, encodes a 316-aa type II transmembrane protein and belongs to the TNF ligand family. RANKL exerts its effects through its cognate receptor, RANK, which is expressed on the OC precursors (25) . OPG is a soluble OC-inhibitory molecule that is a member of the TNF receptor family, along with RANK (23) . OPG binds to RANKL as a decoy receptor and inhibits OC recruitment by interrupting the interaction between RANKL and RANK. There is no doubt that RANKL is a key regulator of osteoclastogenesis because RANKL-knock out mice show severe osteopetrosis and a defect of tooth eruption as a result of an inability of osteoblasts to support osteoclastogenesis (26) . It has also been revealed that a variety of extracellular factors including 1,25-dihydroxyvitamin D3, parathyroid hormone, PGE2, IL-1, and IL-6 induce osteoclastogenesis through the stimulation of RANKL expression in bone marrow stromal cells. It is likely that increased cytokine production may cause the stimulated RANKL expression and accelerate osteoclastic bone destruction in the bone marrow microenvironment where cancer cells are colonized.
To study the mechanisms of metastasis and develop a new strategy for treating patients with cancer, appropriate animal models are required. However, there are few good models of bone metastasis; one example is the breast cancer model by intracardiac inoculation of MDA-MB-231 cells (15 , 27) . The number of human cancer cell lines that are heterotransplantable to animals is limited, and only a few of them metastasize to distant organs (28 , 29) .
In the present study, we screened several human neuroblastoma cell
lines for tumorigenicity and metastatic capacity in immunocompromised
nude mice and developed a new bone metastasis model of human
neuroblastoma. We then examined whether the bone-metastasizing cell
line NB-19 exhibited OC-inducing activity, and we found that the
presence of NB-19 cells in murine bone marrow cells induced OC-like
cell formation in culture through increased expression of IL-1,
COX-2, and RANKL.
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MATERIALS AND METHODS |
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-MEM (Nikken) with 10% FCS and antibiotics under a 5%
CO2 atmosphere.
Tumor Cell Injection into Nude Mice.
Animal protocols were approved by the Institutional Animal Care and Use
Committee at Osaka Medical Center and Research Institute for Maternal
and Child Health. Four-week-old female nude mice (BALB/cA Jcl-nu,
nu/nu) were supplied by Clea Japan (Tokyo, Japan) and
maintained under pathogen-free conditions. For heterotransplantation,
NB-16 and NB-1 cells were mechanically detached from the culture dish,
and NB-19 and NH-6 cells were harvested by treatment with 0.25%
trypsin-0.5 mM EDTA solution (Life Technologies,
Inc.) following the manufacturer’s instructions. Then the cells were
washed once with PBS and suspended in PBS at a concentration of
1 x 108 cells/ml. One hundred
µl of the cell suspension were mixed with the same volume of Matrigel
(Becton Dickinson Collaborative Biomedical Products, Bedford, MA) and
injected s.c. into the right back of each mouse. Tumorigenicity of each
cell line in mice was evaluated as the mass size 8 weeks after tumor
cell injection.
X-ray of Mice.
Animals in a prone position against the film were X-rayed using SOFTEX
CMB-2 (SOFTEX Co. Ltd., Tokyo, Japan) while under anesthesia.
Histological Examination.
Bones of tumor-bearing mice and control mice were fixed in 10%
buffered formalin, followed by decalcification in 14% EDTA solution
for 2 weeks at room temperature with gentle stirring. Sections were
made by the conventional method and stained with H&E.
Micrometastasis Assay Based on Genomic PCR for Alu Sequence and
RT-PCR for Tyrosine Hydroxylase.
To detect the micrometastases of human neuroblastoma cells in the bone
marrow of the host mice, we performed a PCR-based metastasis assay
using the amplification of Alu sequences that are specific to the human
genome (30)
. Genomic DNA was isolated from the bone marrow
of tumor-bearing mice and control mice using Trizol reagent (Life
Technologies, Inc.) following the manufacturer’s instructions.
Specific primers for human Alu sequences were Alu-sense
(5'-ACGCCTGTAATCCCAGCACTT-3') and Alu-antisense
(5'-TCGCCCAGGCTGGAGTGCA-3'), which produced a band of 224 bp
(30)
. PCR was carried out using 100 ng of genomic DNA as
template and recombinant Taq DNA polymerase (Takara Syuzo, Kyoto,
Japan) under the following thermocycling conditions: an initial
denaturation step of 94°C for 4 min; 25 cycles comprised of 94°C
for 30 s, 60°C for 45 s, and 72°C for 45 s; and a
final extension step of 72°C for 7 min.
We also performed another micrometastasis assay using RT-PCR that amplified cDNA of tyrosine hydroxylase, a specific marker for neuroblastoma (31) . Total RNA was extracted from the bone marrow of the mice bearing each cell line using Trizol reagent at the same time as the genomic DNA was obtained. Two µg each of total RNA were then reverse transcribed using random hexamers (Promega, Madison, WI) and SuperScript II reverse transcriptase (Life Technologies, Inc.). For PCR, the following primers were used: (a) hTH340F (sense), 5'-AGCCAAAATCCACCATCTAG-3'; (b) hTH528F (sense), 5'-TGTCAGAGCTGGACAAGTGT-3'; (c) hTH826R (antisense), 5'-GATATTGTCTTCCCGGTAGC-3'; and (d) hTH866R (antisense), 5'-TGCGCTCCTTCAGGAAGCGG-3'. The first-round PCR was performed using the primer set of hTH340F and hTH866R, which was expected to amplify a 526-bp fragment in the presence of tyrosine hydroxylase mRNA. Thirty cycles of amplification were carried out with 94°C for 30 s, 55°C for 1 min, and 72°C for 1 min. For the second-round PCR, the primer set of hTH528F and hTH826R was used with 2-µl aliquots of the first-round PCR products (20 µl) as template, amplifying a 298-bp fragment. The thermocycling program for the second-round PCR was the same as that used for the first-round PCR.
OC-like Cell Formation Assay.
OC-like cell formation assay was carried out following the methods
originally described by Takahashi et al. (32)
,
with some modifications. Murine bone marrow cells were collected
aseptically from C57BL/6N mice (Clea Japan). The collected cells were
washed and resuspended in -MEM supplemented with 10% FCS (Hyclone,
Logan, UT), and the cell suspension was incubated in 10-cm culture
dishes (Becton Dickinson Labware, Franklin Lakes, NJ) at 37°C for
2 h. After the incubation, the nonadherent cells were collected
and plated onto 48-well plates at a density of 1 x 106 cells/well with various numbers of NB-19
cells (0–1 x 104 cells/well) and
cultured for 6 days in -MEM supplemented with 10% FCS in the
absence of 1,25-dihydroxyvitamin D or any other OC-inducing reagent.
When the cultures were terminated, the cells were fixed and stained for
TRAP activity using a commercial kit (Sigma, St. Louis, MO). The
TRAP(+)MNCs containing two or more nuclei were counted as OC-like
cells.
RT-PCR Analysis for Cytokines, COX-2, RANKL, and OPG.
To examine what kind of cytokines were involved in the TRAP(+) MNC
formation in the coculture of murine bone marrow cells and NB-19
neuroblastoma cells, RT-PCR analysis was performed. Murine bone marrow
cells (1 x 107 cells/well) and
NB-19 cells (1 x 105 cells/well)
were plated together or separately to each well of 6-well plates and
cultured for 6 days in -MEM supplemented with 10% FCS in the
absence of 1,25-dihydroxyvitamin D or other osteoclastogenic
stimulators. When the cultures were terminated, total RNA was extracted
from each culture using Trizol reagent and treated with DNase (Takara
Syuzo) to remove the contaminated genomic DNA. Two µg each of
DNase-treated RNA were then reverse transcribed using SuperScript II.
The cDNA samples were then subjected to PCR using the primers listed in
Table 1
. The amplification of the expected fragments was confirmed by
sequencing of the PCR products using an automated sequencer (a 377
model; PE Applied Biosystems, Tokyo, Japan).
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Statistical Analysis.
The data were analyzed by ANOVA. All values were represented as the
mean ± SE.
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RESULTS |
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summarizes the tumorigenicity of the four cell lines of human
neuroblastoma examined as described in "Materials and Methods."
NB-19 and NB-16, originating from the involved bone marrow of patients
with advanced neuroblastoma, exhibited stronger tumorigenicity
than NB-1 and NH-6, which were established from the primary sites of
the disease. Interestingly, NB-19-bearing mice showed splenomegaly.
Radiological examination of NB-19-bearing mice revealed the development
of osteolytic lesions 8 weeks after tumor cell injection (Fig. 1)
. Of the 10 mice that had been inoculated with NB-19 cells, all of them
had a mass at the inoculation site, and 7 of them exhibited osteolytic
lesions detectable by X-ray. The total number of these regions was 14
(5 lesions in right tibiae, 6 lesions in left tibiae, and 3 lesions in
left femurs), and the diameter of these lesions was 1.71 ± 0.67 mm (mean ± SD). In the histological
analysis, although the decrease in trabecular volume was obvious in
NB-19-bearing mice, massive metastasis was not apparent (Fig. 2)
. To confirm the presence of NB-19 cells in the bone marrow of the host
animals, we performed genomic PCR for Alu sequences using the genomic
DNA extracted from the tumor-bearing mice. Specific signals were
detected when DNA from the bone marrow of NB-19-bearing mice was used
as template, but not when DNA obtained from the bone marrow of nude
mice bearing other cell lines was used (Fig. 3)
. In the RT-PCR assay for tyrosine hydroxylase as well, specific
signals were detected only when the RNA from the NB-19-bearing mice was
used as template (Fig. 3)
, confirming the presence of human
neuroblastoma cells in the bone marrow of NB-19-bearing mice.
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TRAP(+)MNC Formation in the Coculture of Murine Bone Marrow Cells
and NB-19 Human Neuroblastoma Cells.
Murine bone marrow cells were cultured for 6 days in the presence or
absence of NB-19 cells, and the number of TRAP(+)MNCs was determined.
In the absence of NB-19 cells, the murine bone marrow cells did not
form TRAP(+)MNCs without the addition of OC-inducing reagents such as
1,25-dihydroxyvitamin D (Fig. 4B)
. In the presence of NB-19 cells, a number of TRAP(+)MNCs
were formed even in the absence of an osteoclastogenesis stimulator
(Fig. 4A)
.
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expression was stimulated in the coculture of bone
marrow cells and NB-19 cells compared with the marrow cells cultured in
the absence of NB-19 cells (Fig. 5)
. These PCR products were revealed to be identical to murine IL-1
cDNA by sequencing analysis (data not shown). No signals were detected
when the primer set for human IL-1 was used (data not shown). Murine
TNF- and TNF-ß were detected in the culture of murine bone marrow
cells alone and in the coculture of murine bone marrow cells and NB-19
cells at similar levels but were not detected in the culture of NB-19
alone (Fig. 5)
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has been reported to increase PG production, we also
examined the expression of COX-2 in this coculture system. In the
RT-PCR analysis, the expression of COX-2 appeared to be increased in
the coculture of NB-19 cells and bone marrow cells compared with the
culture of bone marrow cells alone (Fig. 5)
. The PCR product amplified
from the cDNA of the coculture was subjected to cloning into
pT7-Blue vector (Novagen), followed by sequencing. All of the eight
clones examined were identical to the cDNA for murine COX-2 (data not
shown).
RANKL expression was up-regulated in the coculture of NB-19 and marrow
cells compared with the culture of marrow cells alone, whereas the
expression level of OPG was unchanged (Fig. 5)
. Sequencing analysis
revealed that these PCR products were identical to murine sequences
(data not shown).
Biological Activities of Recombinant RANKL and OPG.
The biological activity of recombinant RANKL and OPG was examined in
murine bone marrow culture. In the presence of recombinant human M-CSF
(10 ng/ml; Genzyme/Techne, Minneapolis, MN), the conditioned media from
the RANKL-transfected COS-7 cells stimulated TRAP(+)MNC formation in a
dose-dependent manner (Fig. 6, A and C)
. Crude conditioned media harvested from
the COS-7 cells transfected with the OPG expression vector or empty
vector pFLAG-CMV-1 were added to the murine bone marrow culture in the
presence of 10 ng/ml M-CSF and the conditioned media from the
RANKL-transfected cells (20% concentration of the culture media). The
conditioned media from OPG-transfected cells inhibited RANKL-induced
TRAP(+)MNC formation in a dose-dependent manner, whereas the
conditioned media from empty vector-transfected cells did not (Fig. 6, B and D)
. These data suggested that recombinant
RANKL and recombinant OPG prepared in the study were biologically
active.
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DISCUSSION |
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In addition to the osteolytic lesions revealed in rentgenography, two different PCR-based assays detected micrometastasis of NB-19 cells in the host bone marrow. One of the assays was based on the genomic PCR amplification of Alu sequences specific to the human genome, which had been previously used in the chick embryo metastasis model by Kim et al. (30) . The other assay, reverse transcription-nested PCR for tyrosine hydroxylase, has been used to detect occult neuroblastoma cells in the bone marrow and peripheral blood of patients on therapy (31) . In both assays, the signals were detected in the bone marrow of NB-19-bearing mice but not in those of the control animals, confirming the presence of human neuroblastoma cells in the bone marrow of NB-19-bearing mice. Neither of the assays detected signals when bone marrow obtained from mice bearing one of the other three cell lines (NB-1, NB-16, and NH-6) was used as the source of the template.
A number of reports have demonstrated that osteoclastic bone resorption
is important to the development of bone metastasis in some cancer types
including breast cancer, lung cancer, and prostate cancer
(35)
. These cancers induce OC formation via secretion of
PTHrP, IL-1, or PGE2 by themselves (13
, 20
, 36)
. Consistent with the role of bone resorption in cancer
metastasis, antiresorption therapy such as administration of
bisphosphonates or anti-PTHrP neutralizing antibody was reported to be
effective in an animal model of breast cancer in which direct
intracardiac injection of MDA-MB-231 cells was performed on nude mice
(13
, 37) .
To study the OC-forming activity induced by the presence of NB-19 cells in the host bone marrow, we performed an in vitro osteoclastogenesis assay using a murine bone marrow culture system. In this assay, OC-like TRAP(+)MNCs were formed in the coculture of NB-19 neuroblastoma cells and murine bone marrow cells without the requirement for any osteoclastogenic stimulator, including 1,25-dihydroxyvitamin D and PTHrP. These results suggested that production of some OC-inducing activity is involved in osteolysis in NB-19-bearing mice, which was triggered by the presence of neuroblastoma cells in the host bone marrow.
We further examined which factors were involved in OC-like cell
formation induced by the presence of NB-19 cells. We found that the
expression of IL-1 was stimulated in the coculture of NB-19 cells
and murine bone marrow cells compared with the cultures of NB-19 alone
or marrow cells alone. IL-1 has been reported to induce COX-2, which
is the key enzyme for PGE2 synthesis.
PGE2 is known as a potent inducer of OC formation
(38)
, and recent studies have revealed that
PGE2 is one of the inducers for RANKL
(22)
. Consistent with these findings, the expressions of
both COX-2 and RANKL as well as IL-1 were up-regulated in the
coculture of NB-19 cells and murine bone marrow cells compared with
cultures of NB-19 or marrow cells alone. The data suggested that the
presence of NB-19 cells stimulated the expression of IL-1, COX-2,
and RANKL in the murine marrow cells.
In the present study, the significant role of RANKL in the osteolysis found in our model was enhanced by the blockage of OC-like cell formation by recombinant OPG in the coculture of NB-19 cells and murine bone marrow cells. These results may lead to a new therapeutic approach to bone metastasis in neuroblastoma such as the administration of recombinant OPG or gene therapy using OPG expression vector. Recombinant RANKL constructed in the study required M-CSF to induce osteoclastogenesis in vitro, which was consistent with the previous reports (39) .
Cell-cell interaction via adhesion molecules between tumor cells and
bone marrow stromal cells also appears to play a role in bone
metastasis. The overexpression of integrin
4ß1 promoted the bone
metastasis of Chinese hamster ovary cells, suggesting that the
interaction between integrin
4ß1 and vascular cell
adhesion molecule 1 expressed on the bone marrow stromal cells is
important in bone metastasis (40)
. The Chinese hamster
ovary cells overexpressing integrin
4ß1 were also reported
to stimulate OC-like cell formation in vitro
(41)
. However, NB-19 cells did not express integrin
4ß1 (data not shown).
Some surface molecule other than integrin
4ß1 might be
responsible for osteoclastogenesis and metastatic osteolysis in
neuroblastoma.
In conclusion, we have developed a unique model of osteolytic bone
metastasis where the presence of human neuroblastoma NB-19 cells in
bone marrow was proven by two kinds of PCR-based assays 8 weeks after
the s.c. injection of tumor cells into nude mice. Tumor-host
cell interaction between NB-19 cells and murine bone marrow cells
resulted in the increased expressions of IL-1, COX-2, and RANKL,
leading to OC formation. Recombinant OPG was capable of blocking this
OC formation, demonstrating the critical role of RANKL in osteolysis
induced by neuroblastoma cells.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by grants from Japan Space
Forum (to T. M. and K. O.) and Ministry of Education, Japan (to
T.M.). 
2 To whom requests for reprints should be
addressed, at Department of Environmental Medicine, Osaka Medical
Center and Research Institute for Maternal and Child Health, 840
Murodo-cho, Izumi, Osaka 594-1101, Japan. Phone: 81-725-56-1220; Fax:
81-725-57-3021; E-mail: j61642{at}center.osaka-u.ac.jp
3 The abbreviations used are: PTHrP, parathyroid
hormone-related protein; OC, osteoclast; IL, interleukin; RT-PCR,
reverse transcription-PCR; COX, cyclooxygenase; RANKL, receptor
activator of nuclear factor B ligand; TRAP, tartrate-resistant acid
phosphatase; TRAP(+) MNC, TRAP-positive multinucleated cell; OPG,
osteoprotegerin; PG, prostaglandin; TNF, tumor necrosis factor; aa,
amino acid(s); CMV, cytomegalovirus; M-CSF, macrophage
colony-stimulating factor.
Received 1/31/00. Accepted 12/13/00.
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4ß1 integrin stimulate osteoclast formation in vitro.. J. Bone Miner. Res., 13: 1251-1259, 1998.[Medline]
4ß1 into tumor cells.. Am. J. Pathol., 148: 55-61, 1996.[Abstract]
4ß1 integrin stimulate osteoclast formation in vitro.. J. Bone Miner. Res., 13: 1251-1259, 1998.
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