Breast Cancer Res Treat (2007) 105:7–16 DOI 10.1007/s10549-006-9438-y
PRECLINICAL STUDY
RANKL-dependent and RANKL-independent mechanisms of macrophage-osteoclast differentiation in breast cancer Y. S. Lau Æ L. Danks Æ S. G. Sun Æ S. Fox Æ A. 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 multinucleated 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 isolated from primary breast cancers and cultured in the presence and absence of cytokines/growth factors influencing osteoclastogenesis. Extensive TAM-osteoclast 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
Y. S. Lau L. Danks A. Sabokbar N. A. Athanasou (&) Department of Pathology, Nuffield Department of Orthopaedic Surgery, University of Oxford, Nuffield Orthopaedic Centre, Oxford OX3 7LD, UK e-mail:
[email protected] S. G. Sun Department of Orthopaedics, Tangdu Hospital, The Fourth Military Medical University, Xian 710038, China S. Fox Department of Cellular Pathology, John Radcliffe Hospital, Oxford OX3 7DU, UK A. Harris Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK
RANKL-dependent mechanism. Osteoclast formation and lacunar resorption also occurred by a RANKLindependent mechanism when the conditioned medium from breast cancer cells, MDA-MB-231 and MCF7, 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 contribute 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 complication in patients with cancer of the breast. These metastatic 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 precursors 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
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differentiation from these mononuclear precursors requires the presence of macrophage-colony stimulating factor (M-CSF) and involves a receptor-ligand interaction with cells of the osteoblast lineage, which express 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 osteoprotegerin (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 cancer differentiate into osteoclasts are not known. In this study, we have analysed the role of RANKL-dependent and RANKL-independent mechanisms in TAMosteoclast differentiation in breast cancer. We have also examined whether the other major cellular components 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 fibroblasts 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 Research 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
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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 reagents used in reverse transcription and DNA amplification were obtained from Invitrogen (Paisley, UK). All cultures were incubated at 37C 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 obtained 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-Aldrich, 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 factors. 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) directed 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
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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 centrifugation 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 osteoclastassociated 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 monoclonal antibody 23C6 (Serotec, Oxford, UK) directed against the vitronectin receptor (VNR) (an osteoclastassociated 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 ammonium 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 incubation and then at 5–7 day intervals until the cell cultures were confluent. These cultures, containing
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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 spindleshaped cells, were passaged twice before being removed and used for RNA extraction. Both tumour-derived fibroblasts and bone stromal cell cultures were stained for alkaline phosphatase, an osteoblast-associated marker, and immunohistochemically with antibodies directed against prolyl-4-hydroxylase 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, Switzerland), according to the manufacturer’s instructions. Single strand complementary DNA (cDNA) was synthesised from 2.0 lg of total RNA according to standard 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 pictures 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 previously described, were seeded at 1 · 104 cells per well
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Breast Cancer Res Treat (2007) 105:7–16
Table 1 Human primer sequences used in amplification
GAPDH OPG RANKL TRAIL
Primer sequence
Size of product (base pairs)
Annealing temp. (C)
forward 5¢-CAC TGA CAC GTT GGC AGT GG-3¢ reverse 5¢-CAT GGA GAA GGC TGG GGC TC-3¢ forward 5¢- ATG AAC AAG TTG CTG TGC TG-3¢ reverse 5¢-GCA GAA CTC TAT CTC AAG GTA-3¢ forward 5¢-CAG ATG GAT CCT AAT AGA AT-3¢ reverse 5¢-ATG GGA ACC AGA TGG GAT GTC-3¢ forward 5¢-ATC ATG GCT ATG ATG GAG GT-3¢ reverse 5¢-AAC TGT AGA AAT GGT TTC CTC-3¢
360
60
354
58
324
56
315
58
onto PBMCs prepared as described above, and supplemented with the following factors: (1) (2) (3)
M-CSF (25 ng/ml) M-CSF (25 ng/ml) and OPG (500 ng/ml) 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 maintained 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 obtained 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 dentine 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)
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 control). Statistical significance was determined using the unpaired t-test and P values
