Anti-Transforming Growth Factor ß Antibody Treatment Rescues Bone Loss and Prevents Breast Cancer Metastasis to Bone

Breast cancer often metastasizes to bone causing osteolytic bone resorption which releases active TGFβ. Because TGFβ favors progression of breast cancer metastasis to bone, we hypothesized that treatment using anti-TGFβ antibody may reduce tumor burden and rescue tumor-associated bone loss in metastatic breast cancer. In this study we have tested the efficacy of an anti-TGFβ antibody 1D11 preventing breast cancer bone metastasis. We have used two preclinical breast cancer bone metastasis models, in which either human breast cancer cells or murine mammary tumor cells were injected in host mice via left cardiac ventricle. Using several in vivo, in vitro and ex vivo assays, we have demonstrated that anti-TGFβ antibody treatment have significantly reduced tumor burden in the bone along with a statistically significant threefold reduction in osteolytic lesion number and tenfold reduction in osteolytic lesion area. A decrease in osteoclast numbers (p = 0.027) in vivo and osteoclastogenesis ex vivo were also observed. Most importantly, in tumor-bearing mice, anti-TGFβ treatment resulted in a twofold increase in bone volume (p<0.01). In addition, treatment with anti-TGFβ antibody increased the mineral-to-collagen ratio in vivo, a reflection of improved tissue level properties. Moreover, anti-TGFβ antibody directly increased mineralized matrix formation in calverial osteoblast (p = 0.005), suggesting a direct beneficial role of anti-TGFβ antibody treatment on osteoblasts. Data presented here demonstrate that anti-TGFβ treatment may offer a novel therapeutic option for tumor-induced bone disease and has the dual potential for simultaneously decreasing tumor burden and rescue bone loss in breast cancer to bone metastases. This approach of intervention has the potential to reduce skeletal related events (SREs) in breast cancer survivors.


Introduction
Breast cancer remains the second leading cause of cancerrelated death among women, although in recent years there has been significant advancement in terms of treatment and prevention. This disease poses a serious threat due to the high incidence of metastasis to other organs, such as lung and bone. More than 75% of patients with breast cancer develop osteolytic bone metastasis, leading to tremendous bone loss [1], [2], [3], [4] and resulting in a significant burden on health care cost and poor quality of life for patients. Although currently used anti-resorptive therapies, such as bisphosphonates and denosumab, are successful in reducing further osteolysis, they cannot improve the existing damage in the residual bone [5], [6], [7], [8], [9], [10], [11]. Therefore, the surviving population remains prone to a high risk of skeletal-related events (SREs), such as pathological fracture, spinal cord compression, bone pain and hypocalcaemia [12], [13]. To address this issue, new therapeutic approaches to rescue cancerinduced bone loss are urgently required [14], [15].
Therapeutic approaches involving anti-TGFb present an obvious choice in the rescue of cancer-induced bone loss for several reasons. Bone is the largest reservoir of TGFb in the body and one of the major osteogenic cytokines. Both bone mass and bone matrix properties are regulated by TGFb [16] and genetic manipulation of this pathway has been shown to affect bone mass in several murine models [17], [18], [19], [20]. Normal bone remodeling requires a balance between bone resorption and bone formation. However, in cancer-induced bone disease normal remodeling is interrupted. During the progression of osteolytic breast cancers, an increase in the osteoclastic bone resorption takes place. As a result, an excess of active TGFb is secreted in the bone microenvironment, which in turn mediates a cascade of events that favor the vicious cycle of bone metastasis [21]. Although TGFb is growth inhibitory for normal epithelial cells, it plays a favorable role in late tumor progression [22]. It has been demonstrated that active TGFb signaling is needed for the establishment of bone metastasis [23]. This is in agreement with a study reporting higher plasma levels of TGFb associated with poor prognosis in breast cancer patients [24]. Upon reaching the bone microenvironment, tumor cells are exposed to several growth factors including TGFb which leads to upregulation of Gli2, a hedgehog family transcription factor. In osteolytic breast cancer cells, Gli2 has been shown to regulate the expression of parathyroid hormone related protein (PTHrP), a major osteolytic factor [25]. Intact TGFb signaling in the breast cancer cells is necessary for the PTHrP secretion, suggesting a direct mechanistic link between TGFb and tumor-induced osteolytic bone destruction [23]. In addition to promoting the growth of cancer cells in bone, TGFb increases osteoclast differentiation [26], [27] and suppresses osteoblast differentiation [28]. All of these events contribute to the accelerated bone destruction in the tumorinfested bone, in a TGFb-dependent manner. Increased TGFb production in mice has been implicated in bone fragility and osteoporosis [18], suggesting that blockade of excess TGFb may rescue bone loss. Therefore, anti-TGFb antibody seems a logical approach to rescue bone loss.
Several attempts have been made to develop anti-cancer therapies involving an anti-TGFb approach. Despite the predictable side effects, a number of anti-TGFb compounds have been shown to inhibit primary and metastatic cancer and are in preclinical or clinical trials [29]. In a mouse model of bone cancer, blockade of TGFb signaling in breast cancer cells has been shown to inhibit breast cancer to bone metastasis [23]. Yang et al. reported that lifelong treatment with a soluble TGFb receptor II protects mice against metastasis [30]. A study using 4T1 mouse mammary cancer cells indicated that blocking TGFb signaling systemically reduces metastatic events [31]. Small-molecule inhibitors of transforming growth factor b receptor I (TbR1) have been shown to reduce tumor burden in preclinical models of breast cancer bone metastasis and pulmonary metastasis [32]. Mohammad et al have recently shown that a small molecule inhibitor of TGFb was able to inhibit melanoma bone disease in a preclinical model [33]. Whether these approaches also improve breast cancer-induced bone loss has not yet been reported.
Recently, a small molecule inhibitor of TbR1 kinase was shown to have anabolic and anti-catabolic effects on normal bone formation [34]. In addition, our group recently reported that the anti-TGFb antibody has the potential to increase bone volume in normal mice [35]. These results prompted us to test the efficacy of anti-TGFb antibody in preventing cancer-induced bone disease.
To investigate the effect of anti-TGFb antibody on both tumor burden and bone loss, we obtained a pan-TGFb antibody from Genzyme Corporation that blocks all three isoforms of TGFb. Our in vivo results show that an anti-TGFb antibody (1D11) significantly increased bone mineral density (BMD), trabecular thickness and bone volume, along with significant reduction in tumor burden and osteolytic bone damage in preclinical breast cancer bone metastasis models using both human and murine breast cancer cell lines. In vitro, 1D11 was able to block TGFb induced expression of both Gli2 and PTHrP, which provides a mechanistic explanation of reduced tumor burden in our model. To our knowledge, this is the first demonstration of dual efficacy of an anti-TGFb antibody to both inhibit tumor burden and rescue bone loss in a breast cancer to bone metastasis model [33].

Animals
All procedures were performed with the approval of the Vanderbilt University Institutional Animal Care and Use Committee and in accordance with Federal guidelines. For all in vivo experiments, 4-to 5-week-old female athymic nude mice (for MDA-MB-231 human breast cancer cells) or Balb/C mice (for 4T1 mouse mammary tumor cells) were used.

Study design
Both the anti-TGFb (1D11) and control antibody (13C4), directed against Shigella toxin, were obtained from Genzyme Corporation, MA. To test the efficacy of anti-TGFb antibody 1D11 in the inhibition of bone metastases, we used preclinical models of breast cancer to bone metastases. Mice were inoculated with breast tumor cells into the left cardiac ventricle and were treated with either anti-TGFb antibody (1D11, 10 mg/kg body weight) or control antibody (13C4, 10 mg/kg body weight), starting either one day after tumor cell inoculation (the adjuvant, or metastasis prevention regimen) or 2 weeks after tumor cell inoculation (the established metastasis regimen); in both regimens, treatment frequency was 3 days per week and continued until 4 weeks after tumor cell inoculation. Any mice showing the sign of distress before this period was sacrificed immediately. 1D11 is a murine monoclonal antibody which is able to neutralize all three isoforms of TGFb in vitro [36] and in vivo [36], [37], [38]. This antibody only recognizes the active form of the cytokine. The vehicle used for preparing the antibodies showed no significant difference in the tumor burden in comparison to the controlantibody-treated group during initial experiments and was therefore excluded from these studies (communication with Genzyme Corporation). The outcome measures included quantification of osteolytic bone destruction using X-ray and histology. Additionally, trabecular bone volume and architecture were measured using microCT. Bone quality parameters were measured using Confocal Raman spectroscopy. Tumor burden and osteoclast numbers were quantified by means of histology.

Cell culture
The human cancer cell line MDA-MB-231 was obtained from ATCC (American Type Culture Collection), and a bone metastatic variant generated and reported previously by our group [39] was used for all in vitro and in vivo studies. The murine mammary cell line 4T1 had previously been obtained from another investigator [40] and used in a cardiac injection model within our group [41]. Both cell lines were maintained in DMEM (Invitrogen, Carlsbad, CA) containing 10% Fetal Bovine Serum (FBS: Hyclone Laboratories, Logan, UT) and 1% penicillin/streptomycin (Mediatech). Cells were cultured in a 37uC atmosphere of 5% CO 2 and 95% O 2 using standard tissue culture techniques.

Intracardiac bone metastasis model
MDA-MB-231 or 4T1 cells were trypsinized, washed and then resuspended in ice-cold sterile PBS at a final concentration of 1610 6 /ml. Four-to 5-week-old female nude (for MDA-MB-231cell injection) or Balb/C (for 4T1cell injection) mice were anesthetized using a ketamine/xylazine mixture. Mice were positioned ventral side up, and tumor cells were injected into the left cardiac ventricle using a percutaneous approach with a 27gauge needle attached to a 1 ml syringe, as described previously [42], [23]. Correct injection position in the left ventricle was confirmed by the appearance of bright red blood at the hub of the needle in a pulsatile fashion. Each mouse received 1610 5 cells in a 100-ml volume (resuspended in PBS) which was injected slowly over 1 minute. For the adjuvant, or metastasis prevention regimen, mice were treated starting 1 day after tumor cell inoculation. For established metastasis protocol, mice were imaged until visible lesion detection (approximately two weeks) and treatment was started at that point and was continued for another two weeks. All mice were imaged weekly and sacrificed 4 weeks post-tumor inoculation. Any mice showing signs of distress prior to 4 weeks were sacrificed immediately.

Radiographic analysis of bone lesions
Beginning 1 week after tumor cell inoculation, tumor-bearing animals were subjected to radiographic imaging. In brief, mice were sedated using ketamine/xylazine and placed in a prone position. X-ray images were then taken at 35 kVp for 8 s using a digital radiography system (Faxitron LX-60). Images were saved and lesion area and lesion numbers were evaluated using image analysis software (Metamorph, Molecular Devices, Inc.). Data presented are the average of lesion area and lesion numbers per mouse in each treatment group.

Bone histology and histomorphometry
After sacrifice, hind limbs (tibiae and femora) from each mouse were harvested, fixed in 10% neutral-buffered formalin (Fisher Scientific) for 48 h and stored in 70% ethanol for further processing. Following microCT analysis, the tibiae and femora were decalcified in 10% EDTA for two weeks and embedded in paraffin using an automated tissue processor for histological analysis. Mid-sagittal sections (5 mm) of tibiae or femora were stained with hematoxylin, orange G (Sigma) and phloxine B (Sigma). Separate sections were also stained for TRAPC activity for visualization of osteoclasts. Histomorphometric analysis of tumor burden, osteoclast numbers and osteoblast numbers was conducted on digital micrographs (1006) using an image quantification software (Metamorph, Molecular Devices, Inc) software. Tumor burden, defined as area occupied by tumor within the medullary region, was calculated. Osteoclasts numbers per area of trabecular bone surface was measured in a blinded fashion in the TRAP stained mid section of long bones using Metamorph software.

Quantitative microCT
MicroCT analysis was performed in the Vanderbilt University Institute of Small Animal Imaging. To assess the effect of anti-TGFb treatment on the architecture and structure of bone in tumor bearing mice, long bones were used. Micro-computed Xray tomography (MicroCT) was used to measure trabecular bone volume within the metaphysis of the tibia and trabecular bone volume, architecture and density in the metaphysis of the femur. The long axis of each specimen was aligned with the scanning axis. One hundred slices from the proximal tibia were scanned at a 12-mm resolution (mCT40 Scanco Medical, Switzerland). The region of interest was trabeculae within the proximal metaphysis of the tibia (0.24 to 1.20 mm) below the growth plate. Images were acquired using 55 kV, 114 mA, 300-ms integration, and 500 projections per 180u rotation. Contiguous cross sectional images of the entire metaphyseal region were acquired. Following reconstruction, the bone tissue was segmented from air or soft tissue using a threshold of 270 per thousand (or 438.7 mgHA/cm 3 ), a Gaussian noise filter of 0.8 and support of 2. Standard architectural characteristics such as trabecular bone volume (BV/TV), trabecular thickness (Tb.Th*), trabecular number (Tb.N*), connectivity density (Conn.D) and mean volumetric density of the mineralized tissue (Tb.TMD or mBMD) were calculated using the Scanco evaluation software.

Raman micro-Spectroscopy
To determine whether neutralizing TGFb in the tumor-bone microenvironment affected composition at the tissue level, we collected Raman spectra from the cortex of the tibial metaphysis. Chemical bonds naturally absorb a small amount of energy from a laser photon as they vibrate (known as Raman scattering of light). In Raman spectroscopy, the spectrum of reflected light (shift in wave number) is collected after the laser excites the bonds in a tissue at a given wavelength, which for these purposes is nearinfrared (785 nm). This spectrum then characterizes the physiochemical properties of the tissue. Using the confocal Raman microscope (Renishaw Inc., Ramanscope Mark III) with a spatial resolution of 2-5 mm and a spectral resolution of 1 cm 21 , we acquired nine spectra from polished sections of embedded tibia (,150 mm below growth plate) and quantified the intensities of key peaks related to mineral (v 1 phosphate and Type-B carbonate) and collagen (proline ring). Thus, mineral-to-collagen ratio, type-B carbonate substitution and crystallinity were the averages of v 1 phosphate/proline, v 1 phosphate/carbonate, and the inverse of the full width at half the maximum of v 1 phosphate per bone.

Osteoclastogenesis assay
Mouse long bones were flushed with PBS, resuspended by pipeting, and strained through a cell strainer (BD Biosciences, 40 mM). Mononuclear cells were isolated from resuspended bone marrow using Histopaque 1077 (Sigma), following manufacturers' instructions. Cells were plated in alpha-MEM media supplemented with 10% fetal bovine serum, RANKL (100 ng/ml) and MCSF (30 ng/ml) to support osteoclast formation. Both reagents were obtained from R&D systems. Two treatment groups were used, one treated with isotype antibody (13C4, 25 ug/ml) and other treated with anti-TGFb antibody (1D11, 25 ug/ml). TRAP staining was performed using Leukocyte Acid Phosphatese kit (Sigma) and number of osteoclasts per field was counted under microscope.

Osteoblast differentiation assay
Primary cultures of calvarial osteoblasts were prepared using a modified sequential collagenase/trypsin digestion method [43]. Briefly, calvaria were removed from 3-to 4-day-old C57Black 6 mice, cleaned free from soft tissue, washed for 10 min with PBS containing 0.025% trypsin, and digested with type-IV Collagenase p (1 mg/ml; source: Clostridium histolyticum, Roche) and 0.025% trypsin for 30 min at 37uC in HBSS with gentle agitation. The procedure was repeated twice, with a 1-h digestion followed by a 30-min digestion using above mentioned concentration of collagenase p and trypsin. The cells from the second and third digestions were collected and centrifuged at 25006g for 10 min. The supernatant was aspirated and discarded, and the pellet was resuspended in alpha-MEM containing 10% fetal bovine serum. The culture was kept undisturbed for at least 2 days. At confluence, cells were trypsinized using the standard procedure and plated in 24 well plates for osteoblast differentiation assay. Cells were cultured in alpha-MEM containing 2.5% fetal bovine serum for a further 3 weeks in the presence of 5 mM beta glycerophosphate (Sigma) and 100 ug/ml L-ascorbic acid (Sigma) either in the presence of isotype control antibody (13C4, 25 mg/ ml) or anti-TGFb antibody (1D11, 25 mg/ml). 6 wells were dedicated to each treatment group. Media containing ascorbic acid and beta glycerophosphate was changed every 2 days until mineralized nodules (approximately 15-28 days) were formed. Mineralized matrix formation was detected by means of Von Kossa staining and quantified using Metamorph image analysis software.

Co-culture assay
Ex vivo co-culture assay was done using mouse calverial osteoblasts and adult mouse bone marrow mononuclear cells. Calverial osteoblasts were isolated from 3-4 days old pups following the method described previously [43] and cultured in 6 well tissue culture plates until confluent. After these cells were confluent, bone marrow mononuclear cells were isolated from normal mice and plated on top of the osteoblast layer. The coculture system was treated with either control antibody (13C4, 25 mg/ml) or anti-TGFb antibody (1D11, 25 mg/ml) every other day for 7-10 days. Cells were fixed and stained for assessment of mature osteoclasts formation using Leucocyte Acid Phosphatase kit (Sigma) according to manufacturer's instruction and mature osteoclasts (red) were scored using microscope.

Quantitative real-time PCR
Total RNA was extracted using RNeasy Mini Kit (QIAgen) according to the manufacturer's instruction. cDNA was synthesized using SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) and random hexamers from 2 mg of total RNA per manufacturer's instructions. cDNA (2 mg) was used for quantitative real-time PCR using the Real MasterMix (Eppendorf, Hamburg, Germany) and 0.5 mL of prepared cDNA per manufacturer's instructions. Real-time PCR was done in triplicate using the Real Plex Machine (Eppendorf) with the following cycling conditions: 95uC for 15 seconds, 58uC for 30 seconds, and 68uC for 30 seconds. Normalization was done using 18S as an internal control.

Statistical Considerations
The data are presented using box plots showing the quartiles along with the raw data, plotted separately for each group and for each outcome. Wilcoxon rank-sum tests and Kruskal-Wallis tests were used to test the null hypotheses of no difference in the distribution of the outcomes among the treatment groups. All analyses were performed using R version 2.11.1. In vivo results presented are from the 4 week treatments; however, the two week treatment showed similar outcome.

Anti-TGFb antibody treatment reduces tumor burden in bone
Using two preclinical mouse models of breast cancer to bone metastases, we have assessed the efficacy of the anti-TGFb antibody 1D11 in reducing tumor burden. Female nude mice (4 weeks old) were inoculated with MDA-MB-231 cells via the intracardiac route. Mice were treated with either control antibody (13C4) or anti-TGFb antibody (1D11), either from one day after tumor cell inoculation (the adjuvant, or metastasis prevention regimen) or 2 weeks after tumor cell inoculation (the established metastases regimen), as described in the Materials and methods section. Following 4-weeks of treatment, anti-TGFb treatment significantly reduced the tumor burden in the long bones (p value = 0.001; Figure 1b) and only microscopic small foci of tumor cells were observed in most mice treated with 1D11 (Figure 1a, white line indicates area occupied by tumor). Following 2-weeks treatment (established metastases protocol), a similar but less dramatic effect was observed (p value = 0.016; Figure 1c).
To test whether this treatment was effective in other bone metastases models, female Balb/C mice was inoculated with 4T1 murine mammary breast cancer cells and mice were treated one day after tumor cell inoculation and continued to be treated for 4 weeks. Tumor burden was significantly reduced in mice treated with anti-TGFb antibody compared to the isotype control group (p = 0.03, Figure 1d).

Anti-TGFb antibody reduced PTHrP and Gli2 expression in breast cancer cells
It has been reported that TGFb can upregulate the expression of Gli2, a hedgehog signaling molecule which is one of the driving factors of osteolytic bone metastasis, we have tested whether treatment with 1D11 might suppress Gli2 expression. As anticipated, TGFb-induced expression of Gli2 was decreased when MDA-MB-231 tumor cells were treated with 1D11 (Wilcoxon rank-sum p-value for TGFb versus TGFb and 1D11 is 0.005) (Figure 1e). This might be one of the mechanisms by which anti-TGFb antibody 1D11 inhibited osteolytic bone damage in our model. Gli2 is also known to increase the secretion of parathyroid hormone-related protein (PTHrP), another major osteolytic factors, in a TGFb dependent process. Inhibition of PTHrP can prevent tumor induced bone destruction, therefore, we tested whether by neutralizing excess TGFb, 1D11 may also decrease PTHrP expression in the tumor cells. Using real time PCR, we found that 1D11 significantly reduced TGFb-induced expression of PTHrP in the MDA-MB-231 cells (Wilcoxon ranksum p-value for TGFb versus TGFb and 1D11 is 0.005) (Figure 1f).

Anti-TGFb antibody treatment reduces osteolytic lesions in MDA-MB-231 cardiac injection model
Advanced metastatic breast cancer in patients leads to severe osteolytic damage. MDA-MB-231 cells injected via the left cardiac ventricle of female nude mice give rise to comparable metastatic lesions, which can be quantified using X-ray image analysis. Radiographic image analysis in MDA-MB-231 tumor-bearing mice treated with either control antibody (13C4, 10 mg/kg, Figure 2a, left panel, arrow indicating osteolytic damage) or anti-TGFb antibody (1D11, 10 mg/kg, Figure 2a, right panel) has indicated that the average number of osteolytic lesions was reduced by more than three-fold (Figure 2b; p,0.001) and the average lesion area was reduced ten-fold in the anti-TGFb treatment group when compared with control (Figure 2c; p,0.001).

Anti-TGFb antibody treatment reduces osteoclast numbers in tumor-bearing mice
Increased bone resorption in the tumor-bearing bone has been associated with increased osteoclast numbers. Since, X-ray imaging has revealed that treatment with anti-TGFb antibody resulted in fewer osteolytic lesions and overall smaller osteolytic area, we have anticipated that, anti-TGFb treatment may reduce number of osteoclasts in vivo. Mouse long bones from both control antibody and anti-TGFb antibody treatment groups were stained with TRAP stain. As anticipated, histological analysis revealed that the number of TRAP-positive osteoclasts per millimeter of bone surface was significantly lower in the group treated with anti-TGFb antibody (1D11) compared to the isotype control (13C4) (Figure 3a; p = 0.027).
Bone marrow microenvironment is a complex multicellular system. The differentiation of osteoclasts is also regulated by osteoblasts. Therefore, fewer osteoclasts in vivo may be a direct effect of the treatment on the osteoblast precursor cells (bone marrow mononuclear cells) or mediated via osteoblasts.
In an attempt to test the direct effect of anti-TGFb treatment on osteoclastogenesis, mononuclear cells were isolated from mouse bone marrow and subjected to osteoclast differentiation, either in the presence control antibody (13C4) or anti-TGFb antibody (1D11) (Figure 3b). As anticipated, TGFb significantly increases the number of TRAP-positive osteoclasts and treatment with 1D11 significantly reduced TGFb-mediated osteoclast formation (p = 0.024, compared between TGFb versus TGFb and 1D11 treatment).
By secreting both OPG and RANKL, osteoblasts maintain the homeostasis of osteoclasts in bone microenvironment [44], [45], [46], [47], [48]. Since TGFb has also been reported to alter the RANKL/OPG ratio, we asked whether the TGFb neutralizing antibody interfered with osteoblast-induced osteoclastogenesis. Mouse calvarial osteoblasts and mouse bone marrow mononuclear cells were co-cultured in the presence of the control antibody or anti-TGFb antibody (3c, representative images. Arrow indicates presence of TRAP positive osteoclasts). The anti-TGFb treatment resulted in almost fivefold reduction the number of osteoclasts (Figure 3d, Wilcoxon rank-sum p value = 0.006). The result of the ex vivo assay are in agreement with the in vivo data and reinforce the notion that TGFb plays a critical role in tumor-induced bone resorption at least in part through the induction of osteoclastogenesis.

Anti-TGFb antibody treatment increases bone volume and improves bone architecture in breast cancer to bone metastasis
We have used either MDA-MB-231 cells and injected those in the left ventricle of nude mice or 4T1 cells and injected those in Balb/C mice for assessing the efficacy of anti-TGFb treatment in cancer induced bone disease. Representative 3D images of mice tibiae from both 13C4 (left panel) and 1D11 (right panel) treatment groups are shown in figure 4a, from the experiment where MDA-MB-231 cells were injected in nude mice. MicroCT analysis of tibia from mice bearing MDA-MB-231 human breast cancer cells in bone following intra-cardiac injection demonstrated that a 4-week treatment with anti-TGFb antibody 1D11 resulted in an approximately 5-fold increase in the overall bone volume when compared with the isotype control 13C4 treated group (Figure 4b; p,0.001). A similar but less dramatic effect was observed with murine 4T1 mammary cancer cells injected into syngeneic Balb/C mice via intra-cardiac route (Figure 4c; p,0.036). Further analysis using quantitative microCT showed that treatment with 1D11 resulted in a greater number of trabeculae, in thicker trabeculae, and in higher connectivity density (lack of fenestrations) of the trabecular bone, suggesting that suppression of TGFb improves trabecular architecture in the presence of a tumor (Table 1). However, bone mineral density (mBMD or Tb.TMD) of the trabeculae was unchanged.

Anti-TGFb antibody increases osteoblast differentiation in vitro
To investigate whether increased bone volume and improved architecture observed in anti-TGFb antibody-treated animals are a reflection of increased osteoblast differentiation, an ex vivo osteoblast differentiation assay was performed. Mouse calverial osteoblasts were isolated and cultured in presence of either isotype control (Figure 4d

Anti-TGFb antibody treatment increases mineral-tocollagen ratio in tumor-bearing animals
Confocal Raman Spectroscopy revealed that inhibiting TGFb signaling with 1D11 increased the mineral-to-collagen ratio in the metaphyseal cortex of the tibia ( Table 2). Of note, 1D11 did not affect the Type-B carbonate substitution, a measure of mineral distortion. Moreover, it did not affect crystallinity. This suggests that the suppression of TGFb increased the rate of mineral accumulation in the organic matrix but not the structure of mineral crystals themselves. We anticipate this to be a reflection of improvement of osteoblastic activity with anti-TGFb treatment.

Discussion
Despite major advancement in the treatment and prevention of early stage breast cancers, a large number of patients remain at risk of developing painful osteolytic bone metastases [3]. Although current anti-resorptive therapies using bisphosphonates are successful in preventing further bone resorption, they cannot repair the previously damaged bone. This leaves the patients with a high risk of pathological fracture and an increased morbidity and mortality. Thus, there is an urgent need for therapies directed at rescuing bone loss. Anti-TGFb antibodies have been reported to reduce metastatic tumor burden related to breast cancers [31], [49]. Our data presented herein confirms these results in two preclinical breast cancers to bone metastases models and extends those to demonstrate that anti-TGFb treatment increases mineralized matrix formation by osteoblasts as well as increases bone mass in preclinical bone metastasis models. Therapeutic approaches with a potential to maintain normal osteoblast activities and reducing osteoclastic bone resorption represent a novel paradigm.
While several laboratories have tested the efficacy of the anti-TGFb antibody against inhibition of tumor burden, much less has been reported on the possible efficacy of this agent toward new bone formation. In addition to histology and microCT analysis, we have used cutting-edge biochemical techniques to analyze the  composition of the bone in the tumor-bearing animals. We have previously reported that in normal murine bone, anti-TGFb treatment increases the number of osteoblasts and decreases the number of osteoclasts, thereby increasing the overall bone mass [35]. In agreement with this, similar findings were noted by other groups using a small-molecule inhibitor of TGFb receptor kinase, SD208, suggesting that blocking excess TGFb is overall beneficial to the bone [33,50]. Although the number of osteoblasts and osteoclasts is critical in maintaining bone remodeling, healthy bone formation also depends on the normal functioning of these cell types. The amount of bone is not always the exact measure of whether bone tissue is healthy and capable of normal load bearing involved in everyday activities. Of note, patients suffering from osteolytic bone damage often present with pathological fracture at the time of diagnosis. The suppression of TGFb signaling affected tissue-level properties, namely bone resorption and mineralization. By studying TGFb1 transgenic mice, Balooch et al. [16] found that decrease in tissue modulus is a function of increased TGFb signaling. Likewise, treating young mice (4 weeks of age) with a pharmacological inhibitor of the TGFb type I receptor (TbRI)  week old female nude mice and 4T1 cells were injected in 4-5 week old female Balb/C mice. Mice were treated either with control antibody (13C4, 10 mg/kg) or anti-TGFb antibody (1D11, 10 mg/kg) for 4 weeks, starting 1 day after tumor cell inoculation. Trabecular bone volume in the tibial metaphysis of tumor-bearing mice was analyzed by microCT. kinase for 6 weeks increased both the degree of mineralization, as determined by X-ray tomography, and elastic modulus of the tibia cortex, as determined by nanoindentation [33]. Similarly, the current study using tumor-bearing animals found that TGFb suppression increased the Raman-derived measure of mineralization (mineral-to-collagen ratio). This suggests that an anti-TGFb antibody may prevent, if not reverse, the negative effect of tumors on bone quality. Our finding using tumor-bearing animals has revealed that anti-TGFb treatment modulated both osteoclast and osteoblast cell compartments, making this therapy more appealing for rescuing bone loss in osteolytic tumor models. We have demonstrated that anti-TGFb antibody treatment inhibits osteoclast formation in vivo. In addition, a direct negative effect on osteoclast formation was demonstrated using bone marrow mononuclear cells. Of note, we have previously reported that in normal bone, there is almost 50% decrease in number of osteoclasts in 1D11-treated mice [35]. In addition to the direct effect on both osteoblasts and osteoclasts, osteoblast-mediated osteoclastogenesis was also inhibited using this approach. Much focus has been given to develop therapies directed to inhibition of osteoclastic bone resorption to prevent osteolytic bone damage. In osteolytic bone disease, osteoblast differentiation is often suppressed [51]. It has been reported that TGFb modulates osteoblast differentiation [52]. Using an ex vivo assay, we have demonstrated that 1D11 antibody treatment increases mineralized matrix formation by calvarial osteoblasts, compared to the control antibody, which may likely contribute to an increase in the bone mass. In addition to make new bones, osteoblasts also maintain the homeostasis of osteoclast formation in the bone. Using a co-culture assay system, we have also demonstrated osteoblast-mediated osteoclastogenesis was inhibited by anti-TGFb treatment. This emphasizes an indirect yet very important role for osteoblasts in affecting osteolytic bone damage.
Data presented here exploits the concept of intervention of osteolytic bone damage by decreasing osteoclastic resorption and increasing osteoblastic differentiation simultaneously.
In agreement with previously reported anti-tumor efficacy of 1D11, we have also shown that tumor burden has been significantly reduced in both 4-week and 2-week treatment regimen (data not shown). As a possible mechanism of reduced tumor burden in the bone, 1D11 was able to inhibit TGFbmediated upregulation of Gli2 and PTHrP in MDA-MB-231 cells.
Since the vicious cycle of bone metastasis is driven by multiple cell types in the bone [21], an effective therapy should target all these components to successfully cure the disease. We conclude that, in the two preclinical models used in this study, treatment with an anti-TGFb antibody preserved bone volume and architecture, decreased tumor lesion number and size, and decreased osteoclast numbers. The overall effect of 1D11 appears to be partly on tumor cells and partly on the bone microenvironment, resulting in both improvement of bone volume and reduction in skeletal metastasis. We suggest that an approach to neutralize excess TGFb might be a promising therapy for the treatment of patients with breast cancer metastasis to bone and may be successful in reducing bone related complications.