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PRECLINICAL STUDY
RANKL-dependent and RANKL-independent mechanismsof macrophage-osteoclast differentiation in breast cancer
Y. S. Lau Æ L. Danks Æ S. G. Sun Æ S. Fox ÆA. Sabokbar Æ A. Harris Æ N. A. Athanasou
Received: 16 July 2006 / Accepted: 24 October 2006 / Published online: 7 December 2006� Springer Science+Business Media B.V. 2006
Abstract The cellular and humoral mechanisms
accounting for tumour osteolysis in metastatic breast
cancer are uncertain. Osteoclasts, the specialised mul-
tinucleated cells responsible for tumour osteolysis, are
derived from monocyte/macrophage precursors. Breast
cancer-derived tumour-associated macrophages
(TAMs) are capable of osteoclast differentiation but
the cellular and humoral mechanisms controlling this
activity are uncertain. In this study, TAMs were iso-
lated from primary breast cancers and cultured in the
presence and absence of cytokines/growth factors
influencing osteoclastogenesis. Extensive TAM-osteo-
clast differentiation occurred only in the presence of
RANKL and M-CSF; this process was inhibited by
OPG and RANK:Fc, decoy receptors for RANKL.
Breast cancer-derived fibroblasts and human bone
stromal cells expressed mRNA for RANKL, OPG and
TRAIL, and co-culture of these fibroblasts with human
monocytes stimulated osteoclast formation by a
RANKL-dependent mechanism. Osteoclast formation
and lacunar resorption also occurred by a RANKL-
independent mechanism when the conditioned med-
ium from breast cancer cells, MDA-MB-231 and MCF-
7, was added (with M-CSF) to monocyte cultures. Our
findings indicate that TAMs in breast cancer are
capable of osteoclast differentiation and that breast
cancer-derived fibroblasts and breast cancer cells con-
tribute to this process by producing soluble factors that
influence osteoclast formation by RANKL-dependent
and RANKL-independent mechanisms respectively.
Keywords Breast cancer � Osteoclast � Bone
resorption � RANKL
Introduction
Skeletal metastasis is a relatively common complica-
tion in patients with cancer of the breast. These met-
astatic lesions are usually osteolytic and may cause
bone pain, pathological fracture and hypercalcaemia
[1]. The cellular and molecular mechanisms whereby
this tumour osteolysis is effected are uncertain. Breast
cancer cells are not capable of lacunar bone resorption
and it is thought that tumour osteolysis is effected by
stimulating the formation and activity of osteoclasts,
multinucleated cells which are specialised to carry out
lacunar bone resorption [2, 3].
Osteoclasts are part of the mononuclear phagocyte
system and are formed by fusion of mononuclear pre-
cursors of haematopoietic origin [4]. In both mouse
and man, mononuclear osteoclast precursors circulate
in the monocyte fraction and express a monocyte/
macrophage antigenic phenotype [5]. Osteoclast
Y. S. Lau � L. Danks � A. Sabokbar � N. A. Athanasou (&)Department of Pathology, Nuffield Department ofOrthopaedic Surgery, University of Oxford, NuffieldOrthopaedic Centre, Oxford OX3 7LD, UKe-mail: [email protected]
S. G. SunDepartment of Orthopaedics, Tangdu Hospital, The FourthMilitary Medical University, Xian 710038, China
S. FoxDepartment of Cellular Pathology, John Radcliffe Hospital,Oxford OX3 7DU, UK
A. HarrisWeatherall Institute of Molecular Medicine, John RadcliffeHospital, Oxford OX3 9DS, UK
123
Breast Cancer Res Treat (2007) 105:7–16
DOI 10.1007/s10549-006-9438-y
differentiation from these mononuclear precursors re-
quires the presence of macrophage-colony stimulating
factor (M-CSF) and involves a receptor-ligand inter-
action with cells of the osteoblast lineage, which ex-
press a membrane-bound osteoclast differentiation
factor termed receptor activator for nuclear factor jB
ligand (RANKL) [6]. RANKL interacts with its
receptor, RANK, which is expressed by mononuclear
osteoclast precursors; this process is inhibited by os-
teoprotegerin (OPG), which is produced by bone
stromal cells and breast cancer cells [7–9]. In addition
to this RANKL-dependent mechanism of osteoclast
formation, it has been shown several cytokines, such as
tumour necrosis factor-a (TNF-a) and interleukin-6
(IL-6), and growth factors, such as transforming growth
factor-b (TGF-b), can induce osteoclast formation
from marrow and circulating osteoclast precursors by a
mechanism independent of RANKL [10–13].
A prominent macrophage infiltrate is commonly
found in both primary and secondary breast cancers
[14, 15]. We have previously shown that TAMs isolated
from primary human and mouse mammary carcinomas
are capable of osteoclast differentiation when these
cells are co-cultured with bone-derived stromal cells in
the presence of 1,25 dihydroxyvitamin D3 and M-CSF
[16, 17]. We have also shown that breast cancer cells
secrete factors that dose-dependently influence human
osteoclast formation [18]. The precise cellular and
molecular mechanisms whereby TAMs in breast can-
cer differentiate into osteoclasts are not known. In this
study, we have analysed the role of RANKL-depen-
dent and RANKL-independent mechanisms in TAM-
osteoclast differentiation in breast cancer. We have
also examined whether the other major cellular com-
ponents found in a breast cancer metastasis (i.e. breast
cancer cells, tumour fibroblasts and bone stromal cells)
influence osteoclast formation from TAMs. As in
previous studies, we isolated TAMs and tumour fi-
broblasts from primary breast cancers rather than
skeletal metastases of breast cancer as the latter would
contain mature bone-resorbing osteoclasts and thus
make it impossible to assess osteoclast formation in
culture.
Materials and methods
This study was approved by the Oxford Clinical Re-
search Ethics Committee. Alpha minimum essential
medium (MEM) and fetal bovine serum (FBS) were
purchased from Gibco Laboratories (Paisley, UK);
MEM containing 10% FBS, 100 U/ml penicillin, and
10 lg/ml streptomycin (MEM/FBS) was used for all
cell culture experiments unless otherwise specified.
Recombinant human M-CSF, OPG, RANK:Fc, and
anti-human TNF-a antibody were obtained from R&D
Systems Europe (Abingdon, UK). Soluble RANKL
was obtained from Peprotech (London, UK). All re-
agents used in reverse transcription and DNA ampli-
fication were obtained from Invitrogen (Paisley, UK).
All cultures were incubated at 37�C in a humidified
atmosphere of 5% CO2 and 95% air, and carried out in
triplicate.
TAMs (and tumour fibroblasts) were isolated from
primary invasive ductal breast carcinomas were ob-
tained from eight female patients (age range 51–76).
TAM isolation and culture
The tumour tissue was washed in sterile phosphate
buffered saline. Fragments of the tumour were then
placed in 1 mg/ml of collagenase Type 1 (Sigma-Al-
drich, Dorset, UK) and incubated for 1 h. The digested
tissue suspension was passed through a Falcon� 70 lm
pore size cell strainer (Becton Dickinson, Oxford,
UK). The filtrate was centrifuged at 1800g for 10 min
and the cell pellet resuspended in 2 ml of MEM/FBS.
The cell yield was determined using a haemocytometer
after lysis of red blood cells with 5% (v/v) acetic acid.
1 · 105 cells per well were added to 96-well tissue
culture plates containing glass coverslips and dentine
slices prepared as previously described [19]. After 2 h
incubation, dentine slices and coverslips were removed
from the wells, washed vigorously in MEM/FBS to
remove non-adherent cells and then placed in a 24-well
tissue culture plate containing 1 ml of MEM/FBS
supplemented with M-CSF (25 ng/ml) and/or RANKL
(30 ng/ml). Negative controls contained no added fac-
tors. All cultures were maintained for 24 h and up to
21 days. Culture medium containing these factors was
replenished every 3–4 days. To determine macrophage
purity in the isolated TAM cell population, 24-h cell
cultures were stained immunohistochemically by an
indirect immunoperoxidase technique with monoclonal
antibody GSR1 (Dakopatts, Glostrup, Denmark) di-
rected against CD14 (a monocyte/macrophage marker)
[20], breast cancer cell markers E29 and MNF116
(Dakopatts, Glostrup, Denmark), directed against
epithelial membrane antigen (EMA) and cytokeratin
respectively.
Isolation and culture of human peripheral blood
mononuclear cells (PBMCs)
Human PBMCs were obtained by density gradient
centrifugation of 50 ml of buffy coat cell preparation
8 Breast Cancer Res Treat (2007) 105:7–16
123
provided by the National Blood Transfusion Service
(Bristol, UK). The buffy coat preparation was mixed
with an equal volume of MEM and purified over
Histopaque (Sigma-Aldrich, Dorset, UK). After cen-
trifugation at 2250 rpm for 25 min, the cell layer above
the Histopaque was collected, suspended in MEM, and
centrifuged at 1800 rpm for 10 min. The cell pellet was
resuspended in MEM and centrifuged again. 5 ml of
MEM/FBS was then added and the number of cells
counted in a haematocytometer following lysis of red
blood cells with 5% (v/v) acetic acid. 5 · 105 cells per
well in 100 ll of MEM/FBS were plated immediately
onto dentine slices and glass coverslips in a 96-well
tissue culture plate. After 3 h incubation, the dentine
slices and glass coverslips were washed in MEM/FBS
to remove any non-adherent cells, and then transferred
to 24-well tissue culture plates containing MEM/FBS
and M-CSF (25 ng/ml). Positive controls were set up in
the presence of M-CSF (25 ng/ml) and RANKL
(30 ng/ml).
Cytochemical and functional assessment
of osteoclast differentiation
Histochemistry for the expression of the osteoclast-
associated enzyme, tartrate-resistant acid phosphatase
(TRAP) was carried out on 14-day cell cultures on
glass coverslips using a commercially available kit
(Sigma-Aldrich, Dorset, UK) [21]. These cell cultures
were also stained immunohistochemically with mono-
clonal antibody 23C6 (Serotec, Oxford, UK) directed
against the vitronectin receptor (VNR) (an osteoclast-
associated antigen) [22].
Functional evidence of osteoclast differentiation was
determined by a lacunar resorption assay system using
cell cultures on dentine slices as previously described
[19]. After 21-day incubation, the cells were removed
from the dentine slices by treatment with 1 M ammo-
nium hydroxide. The dentine slices were washed in
distilled water, ultrasonicated to remove adherent
cells, then stained with 0.5% (w/v) toluidine blue to
reveal areas of lacunar resorption and examined by
light microscopy.
Generation of breast cancer-derived fibroblasts
and human bone stromal cells
Following collagenase digestion of the tumour tissue,
isolated cells were suspended in MEM/FBS and placed
in 25 cm2 tissue culture flasks and incubated for up to
3 weeks. The medium was changed after 24-h incuba-
tion and then at 5–7 day intervals until the cell
cultures were confluent. These cultures, containing
spindle-shaped fibroblast-like cells, were passaged by
treatment with trypsin (0.25%)/EDTA (1 mM) at least
3 times before removal in preparation for RNA
extraction and co-culture experiments.
Cultures of bone stromal cells were also derived
from explants of femoral cancellous bone derived from
patients undergoing hip arthroplasty for osteoarthritis
as previously described [23]. The bone pieces were cut
into small fragments, washed vigorously in sterile PBS
to remove blood and fat, then suspended in MEM/FBS
and placed in 25 cm2 tissue culture flasks. The medium
was changed after 24-h incubation and subsequently at
5–7 day intervals. These cultures, containing spindle-
shaped cells, were passaged twice before being re-
moved and used for RNA extraction.
Both tumour-derived fibroblasts and bone stromal
cell cultures were stained for alkaline phosphatase, an
osteoblast-associated marker, and immunohistochemi-
cally with antibodies directed against prolyl-4-hydrox-
ylase and vimentin (both from Dakopatts); these
antigenic markers are expressed by both fibroblasts
and osteoblasts. The fibroblast and osteoblast cultures
were also stained immunohistochemically for leucocyte
common antigen, using monoclonal antibodies PD7/26
(Dakopatts), as well as for TRAP, CD14 and VNR as
described above.
Breast cancer-derived fibroblast total RNA
extraction and RT-PCR
Total RNA extraction was carried out using the
RNeasy� mini kit (QIAGEN, Hombrechtikon, Swit-
zerland), according to the manufacturer’s instructions.
Single strand complementary DNA (cDNA) was syn-
thesised from 2.0 lg of total RNA according to stan-
dard protocols using the SuperScript� First-Strand
Synthesis System for RT-PCR. cDNA was amplified by
PCR to generate products corresponding to messenger
RNA (mRNA) encoding human gene products for
GAPDH, RANKL, OPG and TRAIL (Table 1).
Aliquots of PCR products were fractionated on 1%
agarose gels stained with ethidium bromide. Gel pic-
tures and quantification of signals were obtained after
scanning with AlphaImager 2200 (Alpha Innotech
Corporation, USA) and ImageJ software analysis
(public domain Java image processing program).
Co-culture of PBMCs and breast cancer-derived
fibroblasts/bone stromal cells
Breast cancer-derived fibroblasts, harvested as previ-
ously described, were seeded at 1 · 104 cells per well
Breast Cancer Res Treat (2007) 105:7–16 9
123
onto PBMCs prepared as described above, and sup-
plemented with the following factors:
(1) M-CSF (25 ng/ml)
(2) M-CSF (25 ng/ml) and OPG (500 ng/ml)
(3) M-CSF (25 ng/ml) and RANK:Fc (500 ng/ml)
Parallel co-culture experiments were set up with
human bone stromal cells. All cultures were main-
tained for 24 h, 14 and 21 days. Culture medium and
factors was replenished every 3–4 days.
Effect of breast cancer cells on osteoclast formation
Breast cancer cell conditioned medium (CM) was ob-
tained from cultures of the human breast cancer cell
lines, MDA-MB-231 and MCF-7. This was added to
human PBMCs plated onto glass coverslips and den-
tine slices, prepared as described above, in a 24-well
tissue culture plate containing 1 ml of MEM/FBS,
subjected to one of the following treatments:
(a) 0–50% breast cancer cell CM and M-CSF (25 ng/
ml)
(b) 0–50% breast cancer cell CM, M-CSF (25 ng/ml)
and RANKL (30 ng/ml)
(c) 10% breast cancer cell CM, M-CSF (25 ng/ml)
and anti-human TNF-a antibody (10 lg/ml)
(d) 10% breast cancer cell CM, M-CSF (25 ng/ml)
and RANK:Fc (500 ng/ml)
Cultures on coverslips and dentine slices were
maintained for 14 and 21 days respectively, with cul-
ture medium, CM and factors replenished every 3–
4 days.
Statistical analysis
The extent of lacunar resorption was measured using
an image analysis software (Adobe Photoshop, USA)
as previously [17] described, and expressed as the mean
percentage of surface area resorbed (%SA) ± standard
error of mean (SEM). In order to minimise the effect
of batch-to-batch variation of PBMCs, all resorption
data were normalised and expressed relative to the
response obtained in PBMC cultures incubated with
25 ng/ml M-CSF and 30 ng/ml RANKL (positive con-
trol). Statistical significance was determined using the
unpaired t-test and P values <0.05 were considered
significant.
Results
Characterisation of TAMs isolated from breast
cancer
After 24-h incubation in the presence or absence of
RANKL and M-CSF, TAMs isolated from all eight tu-
mours strongly expressed CD14 (Fig. 1), a macrophage
antigen which is known not to be present on osteoclasts.
These cells were negative for the osteoclast markers,
TRAP and VNR and the breast cancer cell markers,
EMA and cytokeratin. 24-h TAM cultures on dentine
slices, both in the presence and absence of M-CSF and
Table 1 Human primer sequences used in amplification
Primer sequence Size of product(base pairs)
Annealingtemp. (�C)
GAPDH forward 5¢-CAC TGA CAC GTT GGC AGT GG-3¢reverse 5¢-CAT GGA GAA GGC TGG GGC TC-3¢
360 60
OPG forward 5¢- ATG AAC AAG TTG CTG TGC TG-3¢reverse 5¢-GCA GAA CTC TAT CTC AAG GTA-3¢
354 58
RANKL forward 5¢-CAG ATG GAT CCT AAT AGA AT-3¢reverse 5¢-ATG GGA ACC AGA TGG GAT GTC-3¢
324 56
TRAIL forward 5¢-ATC ATG GCT ATG ATG GAG GT-3¢reverse 5¢-AAC TGT AGA AAT GGT TTC CTC-3¢
315 58
Fig. 1 Day 1 TAMs isolated from melanoma strongly expressmacrophage marker CD14. Bar = 50 lm
10 Breast Cancer Res Treat (2007) 105:7–16
123
RANKL, also showed no evidence of lacunar resorp-
tion. The mononuclear cells isolated from these
tumours thus only expressed the phenotypic markers of
macrophages and not osteoclasts or tumour cells.
TAM-osteoclast differentiation is mediated through
RANKL
In the presence of RANKL and M-CSF, numerous
multinucleated cells, expressing the osteoclast-associ-
ated markers, TRAP and VNR, were formed in 14-day
TAM cultures incubated on glass coverslips (Fig. 2A,
B) in all eight cases. No expression of TRAP or VNR
was seen when either RANKL or M-CSF was omitted.
Scattered mononuclear and multinucleated cells posi-
tive for CD14 were also noted in these cultures, indi-
cating that not all TAMs incubated with RANKL and
M-CSF underwent osteoclast differentiation.
In 21-day TAM cultures on dentine slices incubated
with M-CSF and RANKL, functional evidence of
osteoclast differentiation was noted with the formation
of numerous areas of lacunar resorption in the all
cases; these were largely in the form of multiple com-
pound areas of lacunar excavation on the dentine
surface (Fig. 2C). In the absence of either M-CSF or
soluble RANKL, lacunar resorption was not seen.
Characterisation of breast cancer-derived
fibroblasts and bone stromal cells
After 3–4 passages, the cells which were isolated from
breast cancers consisted almost entirely of spindle-
shaped, fibroblast-like mononuclear cells. These cells
did not stain positively for EMA, cytokeratin, CD45,
CD14, VNR or TRAP, indicating that these cultures
did not contain tumour cells, macrophages or osteo-
clasts; cultured cells were positive for vimentin and
prolyl-4-hydroxylase but negative for alkaline phos-
phatase (Fig. 3A, B). Bone stromal cells cultured from
femoral bone explants showed similar morphological
characteristics and were positive for alkaline
phosphatase. Multinucleated cells did not form in
fibroblast or bone stromal cell cultures and no
resorption was seen on dentine slices on which only
fibroblasts or bone marrow stromal cells were cultured.
Using a semi-quantitative RT-PCR method, signals
generated by mRNA levels of RANKL, OPG and
TRAIL were quantified relative to GAPDH. This
showed that mRNA for RANKL, OPG and TRAIL
was expressed by bone stromal cells and cultured
breast cancer-derived fibroblasts in all cases studied
(Fig. 3C).
Effect of breast cancer-derived fibroblasts
and breast cancer cells on osteoclast formation
Co-cultures of breast cancer-derived fibroblasts and
human monocytes in the presence of M-CSF resulted
in the formation of TRAP+ and VNR+ multinucleated
cells capable of lacunar resorption (Fig. 4). The addi-
tion of OPG or RANK:Fc to these co-cultures abol-
ished osteoclast formation and resorption. As
previously shown [24], co-cultures of human bone
stromal cells and monocytes in the presence of M-CSF,
also resulted in the formation of TRAP+/VNR+ mul-
tinucleated cells capable of lacunar resorption. The
addition of CM from cultured breast cancer-derived
fibroblasts to monocytes did not induce osteoclast
formation.
Cultures of human PBMCs incubated with M-CSF
and CM from the breast cancer cell lines MCF-7 and
MDA-MB-231 resulted in the generation of mononu-
clear and small multinucleated (<4 nuclei) TRAP+ and
VNR+ cells (Fig. 5A, B) capable of forming a few
small round or ovoid lacunar resorption pits (Fig. 5C);
large areas of compound lacunar excavation, as noted
in M-CSF and RANKL-treated positive controls, were
not seen (Fig. 5D). The formation of these resorption
pits was not abolished by the addition of OPG,
RANK:Fc or a neutralising antibody to TNF-a. The
addition of RANKL to human PBMC cultures incu-
bated with M-CSF and CM for breast cancer cell lines
Fig. 2 (A) TRAP positive and (B) VNR positive multinucleatedcells in 14-day TAM cultures in the presence of M-CSF andRANKL and (C) compound areas of lacunar resorption on
dentine slices in 21-day TAM cultures under similar conditions(Toluidine blue staining) (Bars = 50 lm)
Breast Cancer Res Treat (2007) 105:7–16 11
123
MCF-7 and MDA-MB-231 showed a dose-dependent
inhibition of osteoclast formation and lacunar resorp-
tion with formation of fewer TRAP+ multinucleated
cells and fewer resorption pits in 14- and 21-day
cultures respectively (relative to positive control)
(Fig. 6).
Fig. 3 (A) Fibroblastsderived form breast cancerstaining positive for vimentinand negative for cytokeratin.Bar = 50 lm. (B) Expressionof RANKL, OPG andTRAIL mRNA by fibroblastsderived from breast cancer.Reverse transcription-polymerase chain reactionproducts were fractionated onagarose gel. Lane 1, positivecontrol (+ctl); lane 2, negativecontrol (–ctl); lanes 3–8,breast cancer fibroblasts from6 patients (F1–6); lanes 9–11,normal bone marrow stromalcells from 3 patients (N1–3)
Fig. 4 (A) TRAP positivemultinucleated cell and (B)lacunar resorption on adentine slice (Toluidine bluestaining) in co-cultures ofbreast cancer-derivedfibroblasts and humanmonocytes incubated in thepresence of M-CSF(Bars = 25 lm)
Fig. 5 (A) TRAP and (B)VNR positive cells in 14-dayhuman monocyte culture inthe presence of M-CSF andMCF-7 conditioned medium.(C) Few small, round or ovoidlacunar resorption pits ondentine slices formed in 21-day human monocyte culturein the presence of M-CSF andMCF-7 conditioned medium,were unlike (D) large,compound lacunar excavationseen in RANKL-treatedmonocyte cultures [Toluidineblue staining (A) and (B)](Bars = 100 lm)
12 Breast Cancer Res Treat (2007) 105:7–16
123
Discussion
Bone metastases in breast cancer are commonly
osteolytic, being associated with marked osteoclastic
bone resorption [3]. These metastatic deposits contain
numerous macrophages (i.e. TAMs) as well as other
cellular components including tumour cells and fibro-
blasts in the tumour stroma. In this study, we have
shown that one means whereby tumour osteolysis is
effected in a breast cancer metastasis to bone is by
TAM-osteoclast differentiation. We found that large
numbers of osteoclasts and numerous lacunar resorp-
tion pits were formed when breast cancer-derived
TAMs were cultured with RANKL and M-CSF. We
also noted that fibroblasts derived from breast cancers,
like human bone stromal cells, expressed RANKL and
were capable of supporting monocyte-osteoclast dif-
ferentiation. These findings suggest that RANKL-in-
duced TAM-osteoclast differentiation most likely
involves an interaction with these cellular components
of a skeletal metastasis. Breast cancer cells did not
stimulate RANKL-induced osteoclast formation but
were found to produce a soluble factor that could in-
duce the formation of osteoclasts from mononuclear
phagocytes by a RANKL-independent mechanism.
Tumor-associated macrophages are a major com-
ponent of the inflammatory cell infiltrate within and
around primary and metastatic tumours [14, 15]. A
relatively high macrophage: tumour cell ratio is seen at
sites of skeletal metastases where osteolysis is occur-
ring rapidly [25–27]. Tumour cells are known to secrete
several factors which induce macrophage recruitment
into tumours, including monocyte chemotactic protein-
1 and M-CSF [28–30]. Proliferation and survival of
TAMs has been related to tumour cell production of
M-CSF, and TAMs are known to express the c-fms
proto-oncogene, which encodes the M-CSF receptor
[28, 31]. Previous studies have shown that murine and
human TAMs, isolated from primary breast carcino-
mas, are capable of differentiation into TRAP+
osteoclastic cells capable of extensive lacunar resorp-
tion [16, 17, 19]. Osteoclast differentiation involves an
interaction between RANK-expressing cells of the
monocyte-macrophage lineage and RANKL-express-
ing bone stromal cells, a process that is inhibited by
OPG [6–8]. In this study, we have shown that TAMs
isolated from primary breast carcinomas are capable of
osteoclast differentiation in the presence of RANKL
and M-CSF. TAMs isolated from carcinomas ex-
pressed the monocyte/macrophage marker CD14 and
were negative for the osteoclast markers TRAP and
VNR; these cells, like human monocytes, differentiate
into TRAP+ and VNR+ multinucleated cells capable
of carrying out lacunar resorption when incubated with
RANKL and M-CSF [32–34]. This is likely to be the
principal means whereby osteoclasts are formed from
TAMs in a skeletal metastasis as the lacunar resorption
seen in RANKL-treated monocyte cultures was
extensive and characterised by the formation of
numerous compound lacunar resorption pits.
A breast cancer metastasis in bone contains not only
tumour cells and TAMs but also connective tissue cells,
including fibroblasts in the tumour stroma and bone
stromal cells/osteoblasts in the bone itself. Bone stro-
mal cells and osteoblasts, as well as some fibroblast
populations, are known to express RANKL and OPG
[35–37]. In this study we have shown that fibroblasts
derived from primary breast carcinomas express
RANKL, OPG and TRAIL; the latter is known to bind
OPG. We found that breast cancer-derived fibroblasts,
like bone stromal cells, supported monocyte-osteoclast
differentiation; this process was abolished by the
addition of OPG and RANK:Fc, indicating that these
stromal cells induced osteoclast formation by a
Fig. 6 % Surface area (SA) resorption formed in human PBMCcultures incubated with M-CSF, RANKL and breast cancer cellline CM relative to positive control (PBMC cultures with M-CSFand RANKL). Error bars denote SEM (n = 5). [*(P < 0.05),**(P < 0.005) and ***(P < 0.0001) denote significant differencein lacunar resorption relative to positive control]
Breast Cancer Res Treat (2007) 105:7–16 13
123
RANKL-dependent mechanism. Differential expres-
sion and release of RANKL, OPG and TRAIL by
tumour fibroblasts and bone stromal cells may play a
role in determining the extent of osteoclast formation
and osteolysis that occurs in metastatic carcinomas.
OPG is expressed at high concentration in a wide range
of tissues [7]. OPG has broad binding specificity and
binds to TRAIL [38]. RANKL is known to exist in cell
membrane-bound and soluble forms but is mainly a
membrane-bound protein in vivo [7]. In contrast,
RANKL is most abundant in skeletal and lymphoid
tissues. It is likely that the RANKL:OPG ratio in dif-
ferent tissues determines the extent of osteoclast for-
mation and resorption activity.
The other major cellular component of a metastatic
breast carcinoma in bone is, of course, the breast
cancer cells themselves. The expression of osteoclas-
togenic factors in breast cancers is controversial. Tho-
mas et al found that breast cancer cells expressed OPG
and TRAIL but not RANKL, and that they did not
support osteoclast formation in co-culture with marrow
haematopoietic precursors [9]. In contrast, others have
found expression of RANKL and TRAIL in some
breast cancers [39]. If this is the case, then TRAIL may
modulate RANKL-induced osteoclastogenesis. Breast
cancer cells are also known to produce numerous
cytokines and growth factors, such as IL-1, IL-6, TNF-aand PTHrP, which promote RANKL but inhibit OPG
expression by cells of the osteoblast lineage [7]. In this
study, we found that the CM derived from cultured
MCF-7 and MDA-MB-231 breast cancer cells did not
stimulate (and actually inhibited) osteoclast formation
when added to RANKL-treated monocyte cultures,
but that this CM could induce osteoclast formation in
the absence of RANKL. CM-induced inhibition of
RANKL-induced osteoclastogenesis may have been
due to the presence of OPG in the CM; breast cancer
cells are known to produce OPG [9], and this would
effectively have neutralised the osteoclastogenic effect
of the soluble RANKL added to monocyte cultures.
The addition of breast cancer CM alone (i.e. in the
absence of RANKL), however, to monocyte cultures
resulted in the formation of small TRAP+ and VNR+
osteoclastic cells that were capable of lacunar resorp-
tion. This process was not inhibited by OPG or
RANK:Fc, indicating that the soluble osteoclastogenic
factor produced by cultured breast cancer cells acted
by a RANKL-independent mechanism.
In contrast to the numerous large osteoclasts and
extensive areas of lacunar excavation produced when
monocytes and TAMs were cultured with RANKL, the
osteoclasts formed in cultures to which breast cancer
CM was added were small; these cells contained fewer
than 4 nuclei and were associated with the formation of
relatively small areas of lacunar resorption. TNF-a and
other growth factors/cytokines known to induce
RANKL-independent osteoclastogenesis typically
generate small TRAP+ and VNR+ osteoclasts that
form relatively small resorption pits [10–13]. We found
that the addition of a neutralising antibody to TNF-adid not abolish osteoclast formation or lacunar
resorption pit formation in monocyte cultures con-
taining breast cancer CM. As breast cancer cells are
known to produce a large number of cytokines and
growth factors [40], it is possible that one or more of
the humoral factors known to stimulate RANKL-
independent osteoclast formation in this way remained
operative, resulting in the formation of mature func-
tional osteoclasts. We have recently reported that
melanoma cells also secrete a soluble factor (>10 kDa)
which promotes osteoclast formation by a RANKL-
independent mechanism [41].
In summary, we have shown that the cellular com-
ponents of a metastatic breast carcinoma play a role in
promoting the osteoclast formation that is required for
tumour osteolysis. Osteoclasts are formed from TAMs
in breast cancer and this can occur by both RANKL-
dependent and RANKL-independent mechanisms; the
former involves an interaction between RANK-
expressing mononuclear phagocyte osteoclast precur-
sors, which are present in the TAM population, and
RANKL-expressing host bone stromal cells and tu-
mour fibroblasts. Osteoclast formation can also occur
by a RANKL-independent mechanism through breast
cancer cell secretion of one or more soluble osteo-
clastogenic factors. The contribution of RANKL-
dependent and RANKL-independent mechanisms of
pathological bone resorption will need to be taken into
account in devising therapies to treat tumour osteolysis
due to skeletal breast cancer metastasis.
Acknowledgements The authors wish to thank the Frances andAugustus Newman Foundation, Jenny Mays-Smith Skin CancerResearch Fund, Oxfordshire Health Service Research Commit-tee and the Rosetrees Charitable Trust.
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