Treatment of bone metastases is largely symptomatic and is still an unmet medical need. Current therapies mainly target the late phase of tumor-induced osteoclast activation and hereby inhibit further metastatic growth. This treatment method is, however, less effective in preventing initial tumor engraftment, a process that is supposed to depend on the bone microenvironment. We explored whether bone-derived placental growth factor (PlGF), a homologue of vascular endothelial growth factor-A, regulates osteolytic metastasis. Osteogenic cells secrete PlGF, the expression of which is enhanced by bone-metastasizing breast tumor cells. Selective neutralization of host-derived PlGF by anti-mouse PlGF (αPlGF) reduced the incidence, number, and size of bone metastases, and preserved bone mass. αPlGF did not affect metastatic tumor angiogenesis but inhibited osteoclast formation by preventing the upregulation of the osteoclastogenic cytokine receptor activator of NF-κB ligand in osteogenic cells, as well as by blocking the autocrine osteoclastogenic activity of PlGF. αPlGF also reduced the engraftment of tumor cells in the bone and inhibited their interaction with matrix components in the metastatic niche. αPlGF therefore inhibits not only the progression of metastasis but also the settlement of tumor in the bone. These findings identify novel properties of PlGF and suggest that αPlGF might offer opportunities for adjuvant therapy of bone metastasis. Cancer Res; 70(16); 6537–47. ©2010 AACR.

This article is featured in Breaking Advances, p. 6399

Bone metastases are frequent complications in breast cancer patients and cause considerable morbidity (1, 2). According to current concepts, breast cancer cells that engraft in the bone can remain clinically dormant for several years; however, after a variable length of time, these latent tumor cells achieve full metastatic competence and progress to the “vicious cycle” of the osteolytic outgrowth phase. Tumor cells then stimulate osteoclast activity, in part by upregulating the expression of receptor activator of NF-κB (RANKL), a key osteoclastogenic factor, in osteogenic cells. Consequently, the bone becomes resorbed with the release of bone matrix–embedded growth factors that, in turn, promote tumor growth (3, 4). Antiresorptive therapies slow the progression of bone metastasis but hardly prolong overall survival of cancer patients (5). An alternative approach for bone antimetastatic therapy may rely on targeting the initial phases, including the engraftment of tumor cells in the bone. Little is known, however, on how the bone microenvironment regulates these processes (6, 7).

During colonization, micrometastases require adequate vascularization to grow (8). The key angiogenic factor vascular endothelial growth factor-A (VEGF-A) is produced in metastatic bone lesions (911). Blocking VEGF-A in breast cancer bone metastasis reduces osteolytic lesions (12). However, it remains unknown whether angiogenic factors may create a permissive environment for the initial growth of tumor cells in the bone.

Placental growth factor (PlGF) is a homologue of VEGF, which binds to VEGFR-1 and is expressed by various cell types, including macrophages, (pre)osteoclasts, and tumor cells (13). In humans, three splicing isoforms exist, PlGF-1 to PlGF-3; however, in mice, only a single PlGF-2 isoform is present [further denoted as murine (m)PlGF; refs. 14, 15]. PlGF is redundant for development but contributes to the angiogenic and inflammatory switch in several diseases. Consistent with these observations, loss of PlGF does not affect bone development, but impairs healing of bone fractures (16). VEGFR-1 is implicated in osteoclast differentiation (1719), but the role of PlGF in this process remains incompletely understood.

PlGF also induces growth, survival, and migration of tumor cells (2022). In various malignancies, including breast cancer, the levels of PlGF in plasma and tumors correlate with tumor stage, metastasis, and indirectly with survival (23, 24). Recently, PlGF was suggested to be a survival factor for tumor cells that preferentially form bone metastases (25). Treating mice with the anti-PlGF monoclonal antibody 5D11D4 (αPlGF), which inhibits the binding of mPlGF to VEGFR-1 and neuropilin-1, reduces tumor growth and lymph node metastases (21). Furthermore, we recently showed that genetic and pharmacologic blockage of PlGF inhibits tumorigenesis in several carcinogen-induced or transgenic spontaneous tumor models. However, similar to other antiangiogenic agents or monoclonal antibodies, not all anti-PlGF antibodies were effective and not all tumor models were responsive to anti-PlGF (26, 27).

Based on its biological profile, we explored whether PlGF, produced by the bone microenvironment, might provide a conducive milieu for disseminating and growing tumor cells. We therefore used a chimeric tumor model (human breast tumor cells in nude mice) and αPlGF to selectively neutralize mPlGF, produced by the metastatic microenvironment. Our findings highlight the importance of PlGF in the engraftment of tumor cells in the bone and in regulating osteoclast formation, two key processes in bone metastasis.

Animals

Nude mice (Hsd:Athymic Nude-Foxn1nu; Harlan), C57BL/6J mice (Janvier), and wild-type (WT) and PlGF−/− littermates (20) were housed in our animal facility under conventional conditions. All animal experiments were approved by the ethical committee of K.U. Leuven.

Cell lines

MDA-MB-231 (a gift from T.A. Guise, received in 2005; ref. 4) and B16/F10 cells (American Type Culture Collection; ref. 21) were cultured in DMEM (Invitrogen) supplemented with 10% FCS (Sigma-Aldrich). When indicated, MDA-MB-231 cells fluorescently labeled with CM-DiI (Molecular Probes) according to the manufacturer's instructions (labeling for 5′ at 37°C) or stably transfected with the pEGFP expression vector were injected (see Supplementary section).

In vivo mouse model of osteolytic bone metastasis

MDA-MB-231 (105) or B16/F10 (104) cells were injected into the left cardiac ventricle of female nude mice or syngeneic male mice, respectively (5–6-week old; refs. 28, 29). Mice were treated with murine anti-mouse PlGF monoclonal antibody (clone 5D11D4, αPlGF), control IgG1 (50 mg/kg BW), or PBS every alternate day, starting 5 days before tumor inoculation. Radiographs (Agfa) were taken at postinoculation day (PID)-17 and on the day of sacrifice to quantify the total osteolytic area in both femurs and tibias. The presence of osteolytic lesions was evaluated on three-dimensional reconstructions of ex vivo micro-computed tomography (μCT) images (Skyscan 1172, 50 kV, 200 μA, 0.5-mm Al filter, 5-μm pixel size).

Histology and histomorphometry

Bones were isolated at PID-3, PID-7, PID-18, or PID-21, then processed and stained as previously described (16, 30). Tumor burden was quantified on H&E-stained sections, osteoclast parameters were measured after staining for tartrate-resistant acid phosphatase (TRAP) activity (30), and vascularity was determined on CD31-stained sections. The number of CM-DiI–labeled MDA-MB-231 cells was quantified on unstained sections or after staining for fibronectin (Sigma-Aldrich). Detailed description of histomorphometry on images obtained using a Zeiss Axiovert microscope and on confocal images is outlined in the Supplementary section.

Biochemical assays

Serum C-telopeptide levels were measured using the RatLaps enzyme-linked immunosorbent assay (ELISA; Nordic Bioscience Diagnostics) and mPlGF and sRANKL levels by a Quantikine ELISA (R&D Systems).

Coculture of bone marrow stromal cells and MDA-MB-231 cells

Bone marrow cells were isolated from tibias/femurs of 8- to 10-week-old male WT and PlGF−/− mice and seeded in DMEM (10% FCS). Adherent bone marrow stromal cells (BMSC) were replated at 3 × 104 cells/cm2 (day 0), and MDA-MB-231 cells (104 cells/cm2) were added at day 1 either in direct contact with BMSCs or separated by a Transwell insert (0.4-μm pore size, Corning). At days 3 and 6, media were collected and cells were analyzed for gene expression. When indicated, BMSCs (3 × 104 cells/cm2) were transduced with mPlGF-expressing adenoviral vector (PlGF-adv; ref. 22) or control vector at MOI-100. All experiments were done at least three times with duplicates for each condition.

In vitro migration, invasion, and adhesion assays

Migration and invasion of MDA-MB-231 cells was analyzed using untreated or Matrigel-coated (2 μg/insert; BD Biosciences) Transwell inserts (8 μm pore size, Corning), respectively, whereas adhesion was assessed on fibronectin or collagen I coatings. When indicated, MDA-MB-231 cells were pretreated for 2 hours with PD98059, an inhibitor of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway (20 μmol/L; Merck) before the migration assay. These experiments are described in detail in the Supplementary section. Phosphorylation of ERK1/2 in MDA-MB-231 cells in response to PlGF was analyzed by Western blot as previously described (31). Briefly, after an overnight serum starvation (0.1% FCS), cells were treated with PlGF (100 ng/mL) for the indicated time, followed by cell lysis and immunoblotting using anti–phospho-ERK1/2 (pERK) and anti-ERK1/2 antibodies (Cell Signaling Technologies).

In vitro osteoclast assays

Osteoclast formation was investigated using hematopoietic bone marrow cells (32). In brief, bone marrow cells were cultured overnight in αMEM (10% FCS) with 10 ng/mL macrophage-colony stimulating factor (M-CSF; R&D Systems). Nonadherent cells (1.25 × 105/cm2) were replated in αMEM containing 20 ng/mL M-CSF and 100 ng/mL RANKL (Peprotech; day 1). When indicated, αPlGF (250 μg/mL) was added on day 1 or 3. On day 6, cells were stained for TRAP (33) and TRAP-positive multinuclear cells (three or more nuclei; TRAP+ MNC) were counted. The number of osteoclast precursors in the bone marrow was determined by FACS using R-phycoerythrin–conjugated anti-CD11b and FITC-conjugated anti–Gr-1 antibodies (16).

Real-time quantitative reverse transcriptase-PCR

RNA isolation and quantitative reverse transcriptase-PCR (qRT-PCR) procedures are described in the Supplementary section. Briefly, qRT-PCR was done on a 7500 Fast RT-PCR system (Applied Biosystems) using species-specific (human versus murine) primers and the probes listed in Table S1. Gene expression was corrected for hypoxanthine-guanine phosphoribosyltransferase (HPRT) expression. As the homology between mHPRT and hHPRT sequences is 90%, common forward and reverse primers were used in combination with two different MGB probes.

Statistical analysis

Data are presented as mean ± SEM and statistically analyzed using NCSS 2000 software: Statistical significance was analyzed using two-sided two-sample Student's t test (for two groups), ANOVA followed by Fisher's Least Significant Difference (LSD) multiple comparison test (for multiple groups), Fisher's exact test (for proportions), or Wilcoxon rank-sum test (for scoring results). Differences were considered significant at P < 0.05.

Time-dependent increase of bone-derived mPlGF during bone metastases

Intracardiac injection of the human breast cancer MDA-MB-231 cells in immune-deficient mice (28, 29) resulted in metastatic osteolytic lesions in the long bones of the hind limbs at PID-21; lung and liver metastases were not detected (not shown; refs. 4, 29). In this model, metastatic tumor cells became detectable as a small tumor mass in the metaphyseal bone region beyond day 10 (Supplementary Fig. S1A), after which tumor growth accelerated robustly. Use of a human (h)PlGF-specific ELISA revealed that MDA-MB-231 tumor cells produced PlGF (29 ± 3 pg/mg protein, secreted during 2 days of serum-free culture). We also monitored mPlGF gene expression in total bone, including bone marrow cells, and in isolated bone marrow cells. By 10 to 14 days after tumor cell injection, when tumor cells had colonized the bone, mPlGF mRNA levels increased in both fractions (Fig. 1A and B), indicating that the presence of tumor cells upregulated mPlGF expression by bone cells. Serum mPlGF and hPlGF levels in tumor-injected and control mice were undetectable at all stages (not shown).

Figure 1.

mPlGF expression increases during bone metastases. A and B, MDA-MB-231 cells, intracardially injected into nude mice, induce bone metastasis. Species-specific qRT-PCR showed increased mPlGF expression (corrected for mHPRT) in total tibia (including cortex, trabeculae, and bone marrow; A) and in bone marrow flushed from the femur (B) during metastatic disease. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus day 0 (n = 6 mice). Coculture of BMSC with MDA-MB-231 cells induced mPlGF mRNA expression (fold increase versus BMSC only; C) and protein secretion (D) in BMSCs. *, P < 0.05; **, P < 0.01 versus BMSC at the same time point. One representative experiment of three is shown. Data are mean ± SEM.

Figure 1.

mPlGF expression increases during bone metastases. A and B, MDA-MB-231 cells, intracardially injected into nude mice, induce bone metastasis. Species-specific qRT-PCR showed increased mPlGF expression (corrected for mHPRT) in total tibia (including cortex, trabeculae, and bone marrow; A) and in bone marrow flushed from the femur (B) during metastatic disease. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus day 0 (n = 6 mice). Coculture of BMSC with MDA-MB-231 cells induced mPlGF mRNA expression (fold increase versus BMSC only; C) and protein secretion (D) in BMSCs. *, P < 0.05; **, P < 0.01 versus BMSC at the same time point. One representative experiment of three is shown. Data are mean ± SEM.

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The possible cellular sources of mPlGF in the bone are the stromal cell population, containing mesenchymal cells, and the osteoblasts. In vitro coculture of BMSCs with MDA-MB-231 cells time-dependently upregulated the mPlGF transcript levels and mPlGF secreted by BMSCs (Fig. 1C and D), but only when both cell types were in direct contact (Supplementary Fig. S2A and B). A comparable increase in mPlGF expression was observed when primary osteoblasts were used instead of BMSCs (Supplementary Fig. S2C). The increase in mPlGF mRNA levels was even more pronounced than the upregulation of stromal cell-derived factor-1 (SDF-1/CXCL12) expression (34), a bone-derived factor promoting bone metastasis (Supplementary Fig. S2D). Thus, increased mPlGF expression in the bone suggests a role for mPlGF in bone metastatic disease.

Inhibition of host-derived mPlGF reduces bone metastasis

We next assessed whether blocking mPlGF affected bone metastatic disease. We therefore treated mice with the anti-mPlGF antibody 5D11D4 (αPlGF; ref. 21) compared with isotypic antibodies (IgG1) or PBS (showing similar results; referred to as controls), starting 5 days before MDA-MB-231 cell inoculation. In vivo radiography and ex vivo μCT analysis revealed that αPlGF prevented the development of osteolytic lesions in 40% of treated mice by PID-21 (Fig. 2A and B; top left). αPlGF also reduced the number of affected bones per mouse in metastasis-positive animals (Fig. 2B, top right). Indeed, 86% of control mice developed metastatic lesions in both tibias and femurs (three or four of a total of four bones), but only 22% of αPlGF-treated mice (P = 0.0014; Wilcoxon rank-sum test; n = 18–22 mice). In addition, αPlGF reduced the total osteolytic area in each mouse by 62%, as quantified in the radiographs (Fig. 2B, bottom, n = 18–22 mice).

Figure 2.

Inhibition of mPlGF reduces bone metastases. A, representative digital radiographs (top) and μCT images of the femoral metaphysis (bottom) showing osteolytic lesions (arrows) at PID-21 in control and αPlGF-treated mice. B, quantification of radiographs showing that αPlGF reduced total bone metastasis incidence (top left), the number of affected long bones (tibias/femurs) per mouse (top right), and the total osteolytic area per animal (bottom). **, P < 0.01 versus control (n = 18–22 mice). C, H&E-stained femoral sections showing no tumor (right) or smaller tumor (middle) in αPlGF-treated compared with control mice (left). T, tumor; bars, 200 μm. Corresponding histomorphometry showing that αPlGF decreased the tumor area (% of total tissue area) in bones with a visible tumor mass. **, P < 0.01 versus control (n = 18–22 mice). D, αPlGF preserved the bone volume [BV/TV, bone volume/total volume (%)] compared with normal mice (without tumor cell injection; top) and decreased the number of animals with cortical destruction (bottom). *, P < 0.05; **, P < 0.01 versus control or normal (n = 10–20 mice). Data are mean ± SEM.

Figure 2.

Inhibition of mPlGF reduces bone metastases. A, representative digital radiographs (top) and μCT images of the femoral metaphysis (bottom) showing osteolytic lesions (arrows) at PID-21 in control and αPlGF-treated mice. B, quantification of radiographs showing that αPlGF reduced total bone metastasis incidence (top left), the number of affected long bones (tibias/femurs) per mouse (top right), and the total osteolytic area per animal (bottom). **, P < 0.01 versus control (n = 18–22 mice). C, H&E-stained femoral sections showing no tumor (right) or smaller tumor (middle) in αPlGF-treated compared with control mice (left). T, tumor; bars, 200 μm. Corresponding histomorphometry showing that αPlGF decreased the tumor area (% of total tissue area) in bones with a visible tumor mass. **, P < 0.01 versus control (n = 18–22 mice). D, αPlGF preserved the bone volume [BV/TV, bone volume/total volume (%)] compared with normal mice (without tumor cell injection; top) and decreased the number of animals with cortical destruction (bottom). *, P < 0.05; **, P < 0.01 versus control or normal (n = 10–20 mice). Data are mean ± SEM.

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αPlGF reduces the size of bone metastases and preserves bone mass

Histology of control mice revealed massive tumors, obliterating the bone marrow and replacing most of the cancellous bone (Fig. 2C, left), whereas αPlGF resulted in undetectable (Fig. 2C, right) or small tumors (Fig. 2C, center). Histomorphometry (restricted to tumor-bearing mice) revealed a significant reduction in the tumor area after αPlGF (Fig. 2C, graph). Also, αPlGF preserved trabecular bone volume (bone volume/total volume) to similar levels as in non–tumor-bearing mice, whereas bone loss was substantial in control mice (Fig. 2D, top). Moreover, in 16 of 20 bones of control mice, tumor cells had spread through the cortical bone, whereas the cortex was intact in 8 of 12 bones in αPlGF-treated mice (Fig. 2D, bottom, also see Fig. 2C).

The effect of αPlGF on bone metastasis was validated in a second model, in which B16/F10 melanoma cells were intracardially injected in syngeneic C57BL/6 mice. Also in this model, αPlGF inhibited the formation of bone metastases: by histology, two of six αPlGF-treated mice remained free of skeletal metastases, whereas all control mice developed bone lesions (six of six). Moreover, αPlGF reduced the tumor area by 70% (Supplementary Fig. S3A and B).

Breast tumor cells also metastasize to the lungs. To evaluate the role of host-derived mPlGF in this process, we i.v. injected GFP+MDA-MB-231 cells, as this experimental procedure results in the formation of metastatic nodules selectively in lungs (35). Notably, αPlGF tended to reduce the number and significantly decreased the total area of metastatic nodules in the lung at PID-28 (Supplementary Fig. S4A and B). Also, mPlGF expression in the lungs progressively increased during metastasis (Supplementary Fig. S4C), and coculture with MDA-MB-231 cells increased mPlGF expression in pulmonary fibroblasts (Supplementary Fig. S4D). Thus, the role of mPlGF in metastasis of breast tumor cells is not restricted to bone alone, and mPlGF regulates bone metastasis of breast tumors as well as of melanoma.

Angiogenesis in bone metastases is not altered by αPlGF

The therapeutic effect of αPlGF on bone metastases prompted us to investigate the underlying mechanisms. Although PlGF regulates tumor angiogenesis in other models (2022), αPlGF did not affect the microvessel area (CD31+ area) or density (vessels per tumor area) in metastatic lesions at a stage when large osteolytic tumors had formed in control mice (Fig. 3A). Also at PID-7, when tumor cells had lodged in the bone, vessel density and intercapillary distance in the metaphysis were comparable in control and αPlGF-treated mice (Fig. 3B and C). The apparent lack of inhibition of tumor angiogenesis by αPlGF may relate to the fact that αPlGF does not inhibit hPlGF (produced by tumor cells; see Discussion).

Figure 3.

αPlGF does not affect angiogenesis in the metastatic tumor or bone environment. A, CD31+ staining at PID-18. Bar, 100 μm. Quantification of total CD31+ area (top) and vessel density (bottom) in bone metastases of control (n = 11) and αPlGF-treated mice (n = 5). Quantification of blood vessel density (B) and intercapillary distance (C) on CD31-stained tibia sections at PID-7. Normal mice are treated without tumor cell injection (n = 6). Data are mean ± SEM.

Figure 3.

αPlGF does not affect angiogenesis in the metastatic tumor or bone environment. A, CD31+ staining at PID-18. Bar, 100 μm. Quantification of total CD31+ area (top) and vessel density (bottom) in bone metastases of control (n = 11) and αPlGF-treated mice (n = 5). Quantification of blood vessel density (B) and intercapillary distance (C) on CD31-stained tibia sections at PID-7. Normal mice are treated without tumor cell injection (n = 6). Data are mean ± SEM.

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αPlGF inhibits bone resorption during osteolytic metastasis

Because PlGF regulates bone remodeling during fracture repair (16), αPlGF might reduce osteolytic metastases by blocking bone resorption. Histomorphometry of TRAP+ osteoclasts showed that αPlGF reduced osteoclast surface and numbers at the tumor-bone interface at PID-18 (Fig. 4A). αPlGF completely prevented the tumor-induced increase in serum levels of C-telopeptide, a marker of osteoclast activity (Fig. 4B, top), and of soluble RANKL (sRANKL; ref. 36), observed in control mice (Fig. 4B, bottom).

Figure 4.

αPlGF reduces osteoclastogenesis and bone resorption during bone metastases. A, TRAP-stained femur sections showing abundant osteoclasts at the tumor (T)-bone interface at PID-18 in control, but not in αPlGF-treated mice. Bar, 50 μm. αPlGF reduced osteoclast surface (OcS/BS; left graph) and osteoclast number (OcN/BS; right graph) at the tumor-bone interface. *, P < 0.05 versus control (n = 5–10). B, serum C-telopeptide (CTX; top) and sRANKL levels (bottom) at PID-21. Normal mice are non–tumor-injected mice. *, P < 0.05 versus normal (n = 5–7). C, TRAP staining and corresponding histomorphometric quantification of OcS/BS on tibia sections at PID-7 in control and αPlGF-treated mice with or without tumor injection (normal). Bar, 100 μm. *, P < 0.05 versus normal (n = 5–6). D, FACS analysis of osteoclast precursors (CD11b+/Gr-1−/low) in bone marrow isolated at PID-7 (n = 5–6 mice). Data are mean ± SEM.

Figure 4.

αPlGF reduces osteoclastogenesis and bone resorption during bone metastases. A, TRAP-stained femur sections showing abundant osteoclasts at the tumor (T)-bone interface at PID-18 in control, but not in αPlGF-treated mice. Bar, 50 μm. αPlGF reduced osteoclast surface (OcS/BS; left graph) and osteoclast number (OcN/BS; right graph) at the tumor-bone interface. *, P < 0.05 versus control (n = 5–10). B, serum C-telopeptide (CTX; top) and sRANKL levels (bottom) at PID-21. Normal mice are non–tumor-injected mice. *, P < 0.05 versus normal (n = 5–7). C, TRAP staining and corresponding histomorphometric quantification of OcS/BS on tibia sections at PID-7 in control and αPlGF-treated mice with or without tumor injection (normal). Bar, 100 μm. *, P < 0.05 versus normal (n = 5–6). D, FACS analysis of osteoclast precursors (CD11b+/Gr-1−/low) in bone marrow isolated at PID-7 (n = 5–6 mice). Data are mean ± SEM.

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Notably, αPlGF blunted osteoclast activation already at early stages of bone metastasis. Indeed, by PID-7, the TRAP+ osteoclast surface was higher in control tumor-bearing than in non–tumor-injected animals; relevantly, this response was completely abrogated in αPlGF-treated, tumor-injected mice (Fig. 4C). The blockage of osteoclast accumulation by αPlGF was, however, not due to a difference in the number of CD11b+/Gr-1−/low osteoclast precursors in the bone marrow (Fig. 4D), indicating that αPlGF blocked mainly osteoclast differentiation. Thus, αPlGF inhibits osteoclast differentiation and activity already at an early phase in response to tumor cell metastasis.

αPlGF inhibits osteoclastogenesis

To study the role of mPlGF in osteoclast differentiation, we treated bone marrow osteoclast precursors with M-CSF and RANKL. αPlGF reduced the formation of TRAP+ multinuclear osteoclasts (Fig. 5A) when administered at the start of the culture, but was ineffective at later stages (day 3, Fig. 5A, graph). Interestingly, mPlGF mRNA and protein levels increased during osteoclastogenesis (Fig. 5B), suggesting that PlGF is an autocrine signal for osteoclast differentiation; VEGFR-1 expression was, however, not changed (not shown).

Figure 5.

PlGF is crucial during osteoclastogenesis. A, TRAP staining of cultured bone marrow hematopoietic cells treated with M-CSF and RANKL in the presence of αPlGF or IgG1 (control). Bar, 200 μm. αPlGF decreased the formation of osteoclasts (TRAP+ MNC) when added at day 1, but not from day 3 onward. ***, P < 0.001 versus control (n = 3). B, PlGF mRNA (left) and protein (right) levels increased during early osteoclastogenesis (M-CSF + RANKL–treated cultures at days 2 and 4) compared with M-CSF–treated cultures. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus M-CSF (n = 3). C, RANKL/OPG gene expression in WT and PlGF−/− BMSCs cultured with or without MDA-MB-231 cells. **, P < 0.01 versus BMSC of the same genotype; §, P < 0.05 versus WT BMSC + MDA-MB-231. One experiment (with n = 3) of three is shown. D, RANKL/OPG gene expression in WT BMSCs 3 days after transduction with mPlGF-adenoviral vector (PlGF-adv) or empty vector (control). *, P < 0.05 versus control. One experiment (with n = 3) of two is shown. Data are mean ± SEM.

Figure 5.

PlGF is crucial during osteoclastogenesis. A, TRAP staining of cultured bone marrow hematopoietic cells treated with M-CSF and RANKL in the presence of αPlGF or IgG1 (control). Bar, 200 μm. αPlGF decreased the formation of osteoclasts (TRAP+ MNC) when added at day 1, but not from day 3 onward. ***, P < 0.001 versus control (n = 3). B, PlGF mRNA (left) and protein (right) levels increased during early osteoclastogenesis (M-CSF + RANKL–treated cultures at days 2 and 4) compared with M-CSF–treated cultures. *, P < 0.05; **, P < 0.01; ***, P < 0.001 versus M-CSF (n = 3). C, RANKL/OPG gene expression in WT and PlGF−/− BMSCs cultured with or without MDA-MB-231 cells. **, P < 0.01 versus BMSC of the same genotype; §, P < 0.05 versus WT BMSC + MDA-MB-231. One experiment (with n = 3) of three is shown. D, RANKL/OPG gene expression in WT BMSCs 3 days after transduction with mPlGF-adenoviral vector (PlGF-adv) or empty vector (control). *, P < 0.05 versus control. One experiment (with n = 3) of two is shown. Data are mean ± SEM.

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Because tumor cells induce the expression of the osteoclastogenic factor RANKL in BMSCs and osteoblasts (37), we investigated whether mPlGF also regulates RANKL expression and cocultured WT or PlGF-deficient (PlGF−/−) BMSCs with MDA-MB-231 cells. As expected, when WT BMSCs were cocultured, RANKL mRNA levels increased, whereas expression of osteoprotegerin (OPG), a soluble decoy receptor of RANKL, decreased, resulting in a significantly increased ratio of RANKL/OPG (Fig. 5C). These changes were considerably curtailed when BMSCs from PlGF−/− mice were cocultured, mainly because of a smaller increase in RANKL expression (6-fold, P < 0.05, WT versus PlGF−/−). These data suggest that RANKL production by BMSCs is regulated by PlGF in a cell autonomous way. Indeed, transduction of BMSCs with a mPlGF-expressing adenoviral vector significantly increased RANKL mRNA levels compared with cells transduced with a control vector (Fig. 5D); this increase was paralleled by upregulated mPlGF levels in the culture medium (control: 79 ± 3 pg/mL versus PlGF-adv: 1,117 ± 17 pg/mL; P < 0.001; n = 4). These results suggest that interaction of metastatic tumor cells with BMSCs upregulates mPlGF in BMSCs, which promotes RANKL expression. In addition, autocrine signaling of mPlGF during osteoclastogenesis reinforces osteoclast formation.

Initial evidence that bone-derived PlGF supports engraftment of tumor cells

Although the inhibitory effect of αPlGF on osteoclastogenesis already explains, in part, the reduced growth of osteolytic lesions in αPlGF-treated mice, we noticed that 11 of 18 αPlGF-treated mice did not develop any bone metastases at all (Fig. 2B), indicating that initial growth of tumor cells was abrogated. Because mPlGF was expressed in bone cells (Fig. 1A), we explored whether mPlGF also regulated bone engraftment of MDA-MB-231 tumor cells expressing VEGFR-1 (ref. 38; our data). When analyzing fluorescently (CM-DiI)–labeled MDA-MB-231 cells in the metaphysis, the primary site of tumor engraftment, we observed that fewer CM-DiI–labeled cells were present in αPlGF-treated mice compared with control mice at PID-3 (Fig. 6A; n = 5–6 mice).

Figure 6.

mPlGF mediates the early phases of bone metastasis. A, confocal imaging of a bone section showing that CM-Dil–labeled (red) MDA-MB-231 cells at PID-3 primarily localized between the trabeculae (trab) of the metaphysis. Cell nuclei are visualized by 4′,6-diamidino-2-phenylindole (blue). Bar, 10 μm. αPlGF reduced the number of CM-DiI–labeled tumor cells in both femurs and tibias. *, P < 0.05 versus control (n = 5–6 mice). B, confocal imaging of a metaphyseal bone section showing that CM-DiI–labeled tumor cells (red) at PID-3 localized adjacent to fibronectin-positive staining (green) and blood vessels (BV). As a control, the inset shows that few CM-Dil–labeled MDA-MB-231 cells were detected at fibronectin-poor sites. Quantification of CM-DiI–labeled tumor cells bordering fibronectin deposition (% of the total number of CM-DiI–labeled cells). C, adhesion of MDA-MB-231 cells on fibronectin (top left) or collagen I (bottom left) and migration and invasion (center) were stimulated by PlGF and blocked by αPlGF. PlGF-induced migration was inhibited by blocking ERK1/2 activation (PD98059; top right); Western blot shows increased phosphorylation of ERK1/2 by PlGF (bottom right). *, P < 0.05; **, P < 0.01 versus control; §, P < 0.05; §§, P < 0.01 versus PlGF. One experiment (with n = 5) out of three is shown. Data are mean ± SEM. D, model of the role of locally produced PlGF in osteolytic bone metastasis. PlGF secreted by osteogenic cells favors tumor cell engraftment in the bone microenvironment by stimulating their invasion, migration, and adhesion. Surviving tumor cells form micrometastases and induce PlGF secretion in osteogenic cells. Increased PlGF levels advance the switch to the osteolytic phase: PlGF stimulates osteoclastogenesis by enhancing RANKL expression in BMSCs and by signaling directly on osteoclast precursors in a positive feedback loop. As a consequence of these pleiotropic actions of PlGF during the early and late phases of bone metastasis, αPlGF treatment significantly reduces osteolytic bone metastases.

Figure 6.

mPlGF mediates the early phases of bone metastasis. A, confocal imaging of a bone section showing that CM-Dil–labeled (red) MDA-MB-231 cells at PID-3 primarily localized between the trabeculae (trab) of the metaphysis. Cell nuclei are visualized by 4′,6-diamidino-2-phenylindole (blue). Bar, 10 μm. αPlGF reduced the number of CM-DiI–labeled tumor cells in both femurs and tibias. *, P < 0.05 versus control (n = 5–6 mice). B, confocal imaging of a metaphyseal bone section showing that CM-DiI–labeled tumor cells (red) at PID-3 localized adjacent to fibronectin-positive staining (green) and blood vessels (BV). As a control, the inset shows that few CM-Dil–labeled MDA-MB-231 cells were detected at fibronectin-poor sites. Quantification of CM-DiI–labeled tumor cells bordering fibronectin deposition (% of the total number of CM-DiI–labeled cells). C, adhesion of MDA-MB-231 cells on fibronectin (top left) or collagen I (bottom left) and migration and invasion (center) were stimulated by PlGF and blocked by αPlGF. PlGF-induced migration was inhibited by blocking ERK1/2 activation (PD98059; top right); Western blot shows increased phosphorylation of ERK1/2 by PlGF (bottom right). *, P < 0.05; **, P < 0.01 versus control; §, P < 0.05; §§, P < 0.01 versus PlGF. One experiment (with n = 5) out of three is shown. Data are mean ± SEM. D, model of the role of locally produced PlGF in osteolytic bone metastasis. PlGF secreted by osteogenic cells favors tumor cell engraftment in the bone microenvironment by stimulating their invasion, migration, and adhesion. Surviving tumor cells form micrometastases and induce PlGF secretion in osteogenic cells. Increased PlGF levels advance the switch to the osteolytic phase: PlGF stimulates osteoclastogenesis by enhancing RANKL expression in BMSCs and by signaling directly on osteoclast precursors in a positive feedback loop. As a consequence of these pleiotropic actions of PlGF during the early and late phases of bone metastasis, αPlGF treatment significantly reduces osteolytic bone metastases.

Close modal

To provide initial mechanistic insight why mPlGF regulated tumor cell engraftment, we determined whether the CM-DiI–labeled tumor cells lied adjacent to fibronectin, a matrix component that has been implicated in the formation of the premetastatic niche (39). Immunostaining revealed that αPlGF reduced the association of CM-DiI–labeled cells with fibronectin (Fig. 6B). This defect was not attributable to reduced mRNA expression of fibronectin in the bone by αPlGF (not shown); rather, PlGF increased the adhesion of MDA-MB-231 cells to fibronectin, whereas αPlGF abolished this effect (Fig. 6C, top left). Adhesion of MDA-MB-231 cells to collagen (Fig. 6C, bottom left) and to BMSC-produced matrix, a more physiologic and adhesive substrate (Supplementary Fig. S5), as well as migration and invasion (Fig. 6C, center), were all increased by PlGF, whereas αPlGF blocked these effects. The migratory response of MDA-MB-231 cells to PlGF was inhibited by blocking ERK1/2 signaling (Fig. 6C, top right) and (albeit less prominently) by blocking Akt (not shown). Consistent with these results, PlGF increased the level of active (phosphorylated) ERK1/2 (Fig. 6C, bottom right), but not of active Akt (not shown).

Our results suggest a model in which αPlGF inhibits at least two key steps of osteolytic bone metastasis (Fig. 6D): first, PlGF blockage reduces the engraftment of tumor cells in the bone microenvironment; second, it inhibits the activation of osteoclasts by tumor cells.

αPlGF hinders the early phases of bone metastasis

A critical step in bone metastasis is the lodging and engraftment of cancer cells in the bone marrow, as underscored by clinical studies (40, 41). Here, we provide in vivo evidence that bone-derived PlGF promotes the engraftment of tumor cells in the bone. Indeed, PlGF is constitutively expressed at low levels in the bone, and blocking its activity by αPlGF reduces the number of tumor cells in the bone early after inoculation. Tumor cells can survive in the metastatic environment by interacting with extracellular matrix components (42). We show that PlGF stimulates the adhesion of tumor cells to the BMSC-produced matrix; collagen I, the most abundant bone matrix protein; and to fibronectin, expressed by BMSCs and osteoblasts (43, 44). Upon lodging in the bone, breast tumor cells may spread (migrate) throughout the bone marrow to the metastatic niche before establishing expansive growth. Notably, migration and invasion of breast tumor cells was also increased by PlGF (this study; ref. 45). These data together can explain why, after αPlGF, fewer tumor cells were detected adjacent to fibronectin, an important mediator of the premetastatic niche in lung metastasis (39) and which can activate dormant tumor cells (46). Hence, PlGF participates in creating a conducive microenvironment for disseminating tumor cells to engraft in the bone and to enhance their survival (25) and metastatic competence (Fig. 6D).

αPlGF impairs osteoclast formation

Osteoclast activation induced by contact with growing micrometastases is a key step in the subsequent osteolytic phase of bone metastasis (37). Our study shows that PlGF participates as a bone-derived cytokine in osteoclast formation (Fig. 6D). Indeed, PlGF stimulated osteoclast formation, indirectly through upregulation of RANKL expression in BMSCs, as well as directly as an osteoclastogenic factor. Interestingly, PlGF is one of the few molecules known thus far to be released by differentiating preosteoclasts and to function in an autocrine amplification loop.

PlGF may promote osteoclastogenesis through VEGFR-1 signaling in osteoclast precursors (this study and ref. 47). Indeed, PlGF can (partially) rescue the impaired osteoclastogenesis of M-CSF–deficient mice (op/op mice), but only when these mice expressed a signaling-competent VEGFR-1 (18). Overall, the role of PlGF in osteoclast formation is consistent with previous reports that PlGF activates macrophages, which share a similar lineage as osteoclasts (20, 48).

αPlGF does not affect (tumor) angiogenesis during bone metastasis

Angiogenesis promotes tumor growth and metastasis. Treatment of mice with the anti-hVEGF antibody bevacizumab inhibits further osteolysis and tumor growth, presumably by blocking angiogenesis (12). PlGF regulates angiogenesis in primary tumors (2022, 27), and the anticancer activity of αPlGF is partially due to its antiangiogenic effects (21, 27). In the present study, αPlGF did not affect vessel density or size in the metastatic tumor, although differences in vessel function cannot be excluded. These findings are not necessarily in conflict with published data (2022) because αPlGF only neutralizes mPlGF and not the angiogenic factors produced by human tumor cells. An inhibitory role for supraphysiologic PlGF levels on tumor growth has been suggested (49), but this effect has been attributed to the formation of VEGF/PlGF heterodimers, which thereby indirectly inhibits the formation of proangiogenic VEGF/VEGF homodimers.

Locally produced PlGF facilitates metastasis

αPlGF reduced bone metastasis by blocking mPlGF, which is most likely locally produced in the bone upon tumor cell contact rather than being systemically increased (Fig. 6D). Indeed, metastasis to sites other than bone was exceptional after intracardiac tumor injection, suggesting that αPlGF unlikely inhibits tumor cell dissemination from distant sites to the bone. Also, mPlGF levels in the bone progressively increased during the development of bone metastases, whereas serum mPlGF levels remained undetectable and mPlGF mRNA levels in lung tissue were not altered (not shown), arguing for an in situ role of PlGF in the bone. We show that BMSCs and osteoblasts produce increased levels of PlGF upon direct tumor cell contact. Obviously, we cannot exclude the possibility that other cell types, such as endothelial cells and osteoclasts, known to produce PlGF, also contribute to the release of PlGF in the bone microenvironment.

The prometastatic activity of PlGF is not restricted to the bone only. Indeed, mPlGF levels progressively increased during lung metastatic disease, and PlGF also increased the adhesion of breast tumor cells to the matrix produced by lung fibroblasts. Furthermore, αPlGF reduced the number and size of metastatic nodules in the lung. It is also likely that the mechanisms underlying the metastatic activity of PlGF will be tissue-specific. Indeed, initial data using the i.v. versus intracardiac injection models suggest that breast tumor cells do not seem to localize preferentially at fibronectin-rich sites in the lungs (not shown), whereas they do so in the bone. Furthermore, PlGF promotes the osteolytic phase of bone metastases by regulating osteoclast formation, whereas PlGF has been reported to enhance lung metastases by recruiting BMDCs (50). In fact, recent genetic and pharmacologic studies illustrated that the anticancer therapeutic potential of PlGF blockage was highly tissue-specific (27). Characterization of these tissue-specific effects of PlGF warrants further investigation.

αPlGF for treatment of bone metastasis?

The finding that PlGF regulates the engraftment of metastatic breast tumor cells (this study), together with observations that PlGF could be a survival factor for breast tumor cells during the initial latent phase (25) and promotes osteoclast differentiation during the late outgrowth phase (this study), might warrant further exploration of αPlGF as a complementary treatment of bone metastases in breast cancer; however, this outstanding question remains to be addressed in the future. Current therapies, such as bisphosphonates, diminish metastasis-induced bone resorption by targeting osteoclast activity (5, 51). Targeting a multitasking cytokine as PlGF might perhaps offer novel therapeutic opportunities. As in previous studies (21, 27), we did not observe toxicity signs of αPlGF (not shown). Furthermore, additional studies will be required to assess the therapeutic potential and underlying mechanisms of αPlGF in metastasis of additional types of cancer to the bone or other organs.

Peter Carmeliet is named as the inventor on patent application that is partially based on the findings described in this paper.

We thank K. Moermans, N. Smets, I. Stockmans, M. Tjwa, A. Vanden Bosch, S. Baret, A. Manderveld, B. Vanwetswinkel, S. Vinckier (K.U. Leuven) for technical assistance; and G. Marchal and A. Similon (Radiology, UZ Gasthuisberg) for sharing X-ray equipment.

Grant Support: Fund for Scientific Research-Flanders (FWO; G.0229.04, G.0500.08), “Stichting tegen Kanker” (SCIE2006-31), and Center of Excellence (Mosaic, EF/05/08; G. Carmeliet); and Long-Term Structural Methusalem Funding by the Flemish Government and projects funded by FWO and Interuniversitaire Attractiepolen (P. Carmeliet). C. Maes is a postdoctoral fellow of the FWO.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data