Therapeutic Potential of Adult Bone Marrow–Derived Mesenchymal Stem Cells in Prostate Cancer Bone Metastasis Free

Prostate cancer is the second leading cause of cancer deaths in men behind lung cancer in the United States and metastasizes to bone in >70% of the cases during advance stages (1). Bone metastasis causes severe bone pain and pathologic fractures, and shortens life span by significant amount. Most bone metastatic cancers (breast, lung, thyroid, and kidney) generate osteolytic lesions, whereas prostate cancer generates osteoblastic phenotype, with an overall increase in bone volume (2–4). However, the appearance of osteoblastic lesions is preceded and/or accompanied by an osteolytic event, which is required for the establishment and growth of prostate cancer cells in the bone microenvironment (4, 5). The binding of receptor activator of nuclear factor κB ligand (RANKL) to RANK on preosteoclasts or osteoclasts is essential for their maturation and activity (6, 7). Increased expression of RANKL has been observed in osteolytic malignancies, and the inhibition of osteoclastogenesis or metastasis has been considered as an intervention strategy. Osteoprotegerin is a soluble decoy receptor for RANKL and prevents binding of RANKL to RANK, leading to the inhibition of osteoclast activity and bone metastasis (8–10). Osteoprotegerin therefore promises tremendous hope for potential clinical use in the management of osteolytic bone metastasis. Systemic delivery of osteoprotegerin has shown promise as a potential therapy in animal models, limiting hypercalcemia and osteolysis induced by myeloma, breast, lung, or prostate cancer, and reducing tumor establishment in bone (11–17).

Homing of adult bone marrow derived mesenchymal stem cells to the sites of tumor growth is well known besides their ability to self-renew and differentiate into bone, cartilage, fat, and of other tissue types (18). Systemic administration of mesenchymal stem cells in mice has been shown to engraft within the tumor microenvironment in many cancers and thus represent an attractive cellular vehicle for cell therapy and gene therapy (19, 20). Because osteoprotegerin is constitutively produced by mesenchymal stem cells, we speculated that lack of mesenchymal stem cells in sufficient quantities in the bone microenvironment is the reason for the inability to inhibit excess osteoclastogenesis and compensate for bone loss, and overcoming this limitation would provide therapy for osteolytic bone damage. The present study determined the potential of mesenchymal stem cells that were unmodified as compared with that genetically engineered to overexpress osteoprotegerin in bone remodeling following osteolytic damage. Results indicated that naïve mesenchymal stem cells inhibited tumor growth comparable with mesenchymal stem cells overexpressing osteoprotegerin by formation of new bone around the tumor cells and by inhibiting osteoclast activation.

Osteolytic prostate cancer cell line PC3 expressing firefly luciferase was a generous gift from Dr. Kenneth J. Pienta (University of Michigan) and maintained in RPMI-1640 medium (Mediatech, Inc.) supplemented with 10% fetal bovine serum (Mediatech, Inc.) and penicillin/streptomycin (Mediatech, Inc.). Osteoblastic prostate cancer cell line C4-2B was a generous gift from Dr. Marco G. Cecchini, Departments of Urology and Clinical Research, University of Bern, and maintained in T-medium (Invitrogen) supplemented with 10% fetal bovine serum and antibiotics. RAW 264.7 cells were obtained from the American Type Culture Collection and maintained in α-MEM supplemented with 10% fetal bovine serum, 4 mmol/L l-glutamine and antibiotics. Mesenchymal stem cells were maintained in Stem Line medium (Sigma-Aldrich Corp.) supplemented with 10% fetal bovine serum, 4 mmol/L l-glutamine and penicillin/streptomycin. HEK293 cells were purchased from American Type Culture Collection and maintained in DMEM supplemented with 10% newborn calf serum and penicillin/streptomycin. Isolation, purification, and differentiation of mouse mesenchymal stem cells from C57BL/6 mice were carried out as described recently (21). Antibodies for cytokeratin-18 and green fluorescent protein (GFP) were purchased from Abcam Ltd. Secondary immunodetection was done using anti-rat and/or anti-rabbit ABC kits purchased from Vector Laboratories. Total RNA was isolated using Trizol (Invitrogen) and purified using a Qiagen mini kit. iScript cDNA synthesis kit was purchased from Bio-Rad. Primers for reverse transcriptase-PCR analysis were designed using the Primer 3 (version 4.0) software, and oligonucleotides were purchased from Integrated DNA Technologies, Inc. cDNA samples were analyzed in Bio-Rad iCycler (Hercules). Three-dimensional PC3 beads were prepared and supplied by Vivo Biosciences. Proliferation of PC3 cells were determined by Vybrant MTT Cell Proliferation Assay Kit (Molecular Probes, Inc.), as recommended by the manufacturer. Lentivirus encoding short-hairpin RNA (shRNA) constructs for silencing osteoprotegerin were designed and supplied by Sigma-Aldrich Corp. Alkaline phosphatase enzyme activity was measured using a commercial kit (Sigma-Aldrich Corp.), following manufacturer's instructions. Von Kossa staining was done to detect calcium deposition following standard protocols (22).

The recombinant osteoprotegerin used in this study comprised the ligand-binding domain of human osteoprotegerin (1-201 amino acids) fused to the Fc domain of human IgG. The osteoprotegerin Fc fusion gene was isolated from an adenoviral construct (kindly provided by Dr. Joanne Douglas, University of Alabama at Birmingham) and subcloned into adeno-associated virus (AAV) plasmid under the control of cytomegalovirus/chicken β-actin promoter. Expression of osteoprotegerin Fc as a secreted protein from the AAV plasmid was confirmed by transient transfection into HEK293 cells using Lipofectamine-2000 reagent (Invitrogen, Inc.) and testing the supernatants on SDS-PAGE using a mouse monoclonal antibody against human osteoprotegerin (Chemicon, Inc.).

Mesenchymal stem cells were cultured for 2 d at confluency when conditioned media was collected and centrifuged at 4,000 rpm to pellet any floating cells, and the supernatant was stored at −80°C. RAW 264.7 cells were cultured in six-well culture dishes with 25 ng/mL of RANKL (SIGMA-Aldrich), either in regular medium or mesenchymal stem cells conditioned medium for 8 d. The culture medium was replaced every alternate day. After 8 d, cells were stained for tartrate-resistant acid phosphatase to determine the effect of mesenchymal stem cells conditioned media on osteoclast formation using a leukocyte acid phosphatase kit (SIGMA-Aldrich).

Six-week-old male severe combined immunodeficient (SCID) mice were purchased from the National Cancer Institute–Frederick Animal Production Facility. Maintenance of the animals was carried out following guidelines of the Institutional Animal Care and Use Committee, and all experimental procedures were approved by the Institutional Animal Care and Use Committee and the Occupational Health and Safety Department of the University of Alabama at Birmingham. The mice were acclimatized for a week, following which 10⁵ osteolytic PC3 prostate cancer cells, constitutively expressing luciferase, were implanted in the tibia of right leg (n = 30) in 20 μL PBS. The left tibia served as control and was injected with only PBS. A 3/10-mL (28 gauge) insulin syringe (BD Biosciences) was used for the intratibial injection of the prostate cancer cells under isoflurane anesthesia.

In vivo bioluminescence imaging was conducted in a cryogenically cooled IVIS-100 system (Xenogen Corp.) to detect luciferase expression using living imaging acquisition and analysis software (Xenogen Corp.), as described (11). For each animal, bioluminescence imaging was done before and 4 wk after the initiation of mesenchymal stem cells treatment. The intensity of light emission was represented with a pseudo–color scaling of bioluminescent images. The bioluminescent images were overlaid on black-and-white photographs of the mice collected at the same time. Bioluminescence units were converted as counts per second for each animal, and final counts were divided by the initial counts and were plotted graphically as a measure of tumor growth.

Superficial computed tomography scanning of whole skeleton was done on live animals using MicroCAT II (Imtek, Inc.). For the determination of the three-dimensional architecture of the trabecular and cortical bones, mice were sacrificed, and tibia were harvested and analyzed in an advanced micro–computed tomography instrument (μCT 40, Scanco Medical AG). Two scans were done on each tibia, one for whole tibial bone with 16-μm resolution and one for trabecular analysis with a 6-μm resolution. For the whole tibia, the scan was composed of 1,129 slices, with a threshold value of 265. A three-dimensional reconstruction of the images was done with the region of interest consisting of trabecular and cortical areas. The scan of the trabecular bone was done below the growth plate. Each scan consisted of 209 slices, of which 100 were used for the analysis. Regions of interest were drawn on each of the 100 slices, just inside the cortical bone, to include only the trabecular bone and the marrow. Trabecular bone was set to a threshold at 327 to distinguish it from the marrow. The three-dimensional reconstruction was done on the region of interest, which only contained trabecular bone; no cortical bone was present in these regions of interest.

Soft tissues were fixed in 10% neutral buffered-formalin solution for 48 h before embedding in paraffin for histologic analysis. Bone tissues were decalcified in 0.5 mol/L EDTA in Ca²⁺- and Mg²⁺-free Dulbecco's PBS (Cellgro) before embedding in paraffin. Six-micrometer longitudinal serial sections were cut from the femur and tibia, and stained with hematoxylin and eosin or Goldner's trichrome stain to determine the characteristics of tumor growth in the bone and the extent of osteolysis in response to different treatments. Tartrate-resistant acid phosphatase staining was done on bone sections to determine osteoclast activity. Quantitative osteomeasurements of bones were done using an Olympus BX51 microscope and Bioqaunt Image analysis Software (R&M Biometrics).

Mice were sacrificed 4 wk after the mesenchymal stem cells treatments, and tibiae were collected and fresh frozen. Specimens were tested to failure by three-point bending on 858 MiniBionix Materials Testing System (MTS Systems). Stiffness and peak load were calculated from the force displacement data.

Briefly, 6-mm paraffin sections of tibiae were deparaffinized in xylene and hydrated through graded alcohol. Antigen retrieval was done in citrate buffer (pH 6.0), under steam for 20 min. Sections were cooled to room temperature, and endogenous peroxidase was removed using 0.3% H₂O₂ in methanol for 30 min and blocked with 3% goat serum for 30 min. Tissue sections were then incubated with primary antibodies overnight at 4°C. Sections were washed in Phosphate-buffered saline (PBST) with 0.05% Tween-20 and again incubated at room temperature with biotin-conjugated goat anti-rabbit/anti-rat secondary antibody for 2 h. After washing, sections were incubated with streptavidin-conjugated horseradish peroxidase for 1 h at room temperature. After another wash with PBST, immunodetection was done using 3,3′-diaminobenzidine-H₂O₂ (Vector Labs) and counterstained with hematoxylin.

For determination of osteoprotegerin levels secreted by the PC3 cells, mesenchymal stem cells and mesenchymal stem cells–osteoprotegerin cells were cultured separately for 72 h; cell numbers were counted, and 100 μL of culture media was subjected to ELISA (ALPCO Diagnostics), following manufacturer's instructions. Each sample was analyzed in triplicate, and the absorbance was measured in a microplate reader (BioTek Instruments, Inc.).

Data were analyzed by one-way ANOVA. A Tukey test was also applied for multiple comparisons wherever applicable. Values provided are the mean ± SE, and the differences were considered significant if P < 0.05.

Total RNA was isolated from mouse mesenchymal stem cells, converted to cDNA, and subjected to real-time PCR analysis. The result indicated the expression of osteoprotegerin mRNA by the mesenchymal stem cells (Fig. 1A). Osteoprotegerin immunostaining was also done on cultured mesenchymal stem cells, which clearly indicated production of osteoprotegerin by the mesenchymal stem cells (Fig. 1B). Ability of mesenchymal stem cells to inhibit osteoclastogenesis was tested in vitro by culturing preosteoclast RAW 264.7 cells in regular medium or mesenchymal stem cells–conditioned medium in the presence of RANKL for 7 days. Tartrate-resistant acid phosphatase staining indicated significant number of osteoclasts in RAW cells cultured in regular medium comparable with RAW cells grown in mesenchymal stem cells conditioned medium (Fig. 1C). Levels of osteoprotegerin produced in culture as protein secreted by PC3 cells, mesenchymal stem cells, and mesenchymal stem cells genetically engineered to overexpress osteoprotegerin are given in Supplementary Fig. S4.

To determine the effect of mesenchymal stem cells in preventing the growth of prostate tumor in the bone, 6-week-old male SCID mice were injected intratibially with 10⁵ osteolytic human prostate cancer cells, PC3, expressing firefly luciferase. The next day, 5 × 10⁵ bone marrow–derived mouse mesenchymal stem cells, which were unmodified or overexpressing osteoprotegerin (mesenchymal stem cells–osteoprotegerin) were injected in the tibia in proximity to the tumor cells. Growth of prostate tumor in the bone was evaluated by bioluminescence imaging 4 weeks after the administration of the mesenchymal stem cells, which indicated almost 90% inhibition of tumor growth in both treatment groups (Fig. 2A and B). Mesenchymal stem cells–osteoprotegerin did not show any advantage over unmodified mesenchymal stem cells in preventing tumor progression.

To test if therapy with mesenchymal stem cells is effective after the tumor has established in the bone microenvironment with high degree of osteolytic damage, PC3 cells were injected in the tibia and allowed to grow for 2 weeks. Then, 5 × 10⁵ mesenchymal stem cells (unmodified or overexpressing osteoprotegerin) were injected in the same location. Tumor progression was evaluated 4 weeks after the treatment, and bioluminescence imaging indicated inhibition of tumor growth in some of the treated mice compared with the untreated ones, although data was not significantly different (P > 0.05) between treated and untreated animals when all the mice were taken into consideration for comparison (Fig. 2C). Similar observations were made when histologic architecture of the tibia was studied for bone loss due to mesenchymal stem cell therapy (data not shown). These data suggest requirement of optimal number of mesenchymal stem cells to prevent osteolysis in prostate cancer bone metastasis at an earlier time.

Micro–computed tomography of the skeleton showed significant loss of bone in the region of implantation of the PC3 cells, whereas complete restoration was observed in the tibiae treated with mesenchymal stem cells and mesenchymal stem cells–osteoprotegerin (Fig. 3A). Tibiae were harvested from the mice, and studies were done to understand the ultrastructure of the tibia. Three-dimensional micro–computed tomography data indicated a significant decline in relative bone volume and trabecular connectivity density in untreated mice compared with age-matched normal mice. Trabecular and cortical bone structures were completely restored in mice with PC3 cells in the tibia by the mesenchymal stem cell therapy (Fig. 3B). It is interesting to note that the relative bone volume and trabecular connectivity density in the treated mice significantly exceeded that observed in the tibiae of normal mice, indicating the effectiveness of the therapy. Bone restoration was highest in mice treated with mesenchymal stem cells–osteoprotegerin, which may be because of the significant inhibition of osteoclastogenesis due to overproduction of osteoprotegerin as compared with unmodified mesenchymal stem cell–treated mice (Fig. 3B; Supplementary Fig. S1A and B). In mesenchymal stem cells–osteoprotegerin–treated mice, the restoration of tibial bone resulted in limitation of marrow space and might compromise important event(s) such as hematopoiesis and hence was excluded in further experiments. Histomorphometry supported the results obtained from bioluminescence imaging and micro–computed tomography analysis. Tartrate-resistant acid phosphatase staining revealed highest number of osteoclasts in the untreated tibia, mainly at the tumor-bone interface, whereas the number and size of osteoclasts were significantly decreased in the tibia of the treated mice. The mesenchymal stem cells– and mesenchymal stem cells–osteoprotegerin–treated mice indicated similar pattern of osteoclast staining (Fig. 3C). Three-point mechanical testing of the tibial bone was done to determine the mechanical strength after treatment. Data indicated similar bone strength in mesenchymal stem cell–treated mice compared with normal mice tibia (Supplementary Fig. S2A and B).

Outcome of the previous experiment suggests that the therapeutic effects of mesenchymal stem cells are not highly apparent when administered at the advanced stages of tumor-induced osteolytic bone lesion. It is likely that, at a later stage, the number of tumor cells in the tibia outnumbered the input mesenchymal stem cells. Mesenchymal stem cell therapy did not influence the prostate tumor growth in the bone in a negative manner, even at later stages. This prompted us to study the interaction between the mesenchymal stem cells and the PC3 cells in the bone in vivo. PC3 cells (10⁵) were injected in the tibia and allowed to grow for 1 week for detectable tumors when 5 × 10⁵ mesenchymal stem cells were injected in the same site. Mesenchymal stem cells used here were derived from a GFP transgenic mouse. Mice were sacrificed 1 week after the injection of mesenchymal stem cells, and tibiae were harvested and subjected to histomorphometry. Results showed formation of new bone surrounding the tumor nests (Fig. 4A). When analyzed under polarized light, this newly formed bone comprised of randomly oriented collagen fibers, called woven bone and characteristic to fracture healing and prostate cancer bone metastasis in humans (Fig. 4A and B). Human epithelial cell marker cytokeratin-18 immunostaining was done to identify the prostate tumors in the bone (Fig. 4C, c), which also exposed a multiple layers of fibroblast-like cells arranged in concentric circles, separating the tumor cells from the newly formed bone, which also negatively stained for cytokeratin-18. The outermost layer of these fibroblast-like cells was often seen to be embedded or being transformed into the newly formed bone (Fig. 4C, a and c). These cells stained positively for GFP, which indicated new bone formation in the treated mice formed predominantly from exogenously administered mesenchymal stem cells (Fig. 4C, d). Formation of new bone around the tumor cells resulted in restricting the growth of prostate tumor cells in the tibia. No such woven bone formation was noticed in tibia injected with mesenchymal stem cells in the absence of PC3 cells, suggesting bone formation in the PC3 injected tibia is triggered by the prostate tumor cells (Fig. 4A). Reverse transcriptase-PCR analysis of mRNA isolated from mesenchymal stem cells obtained from a coculture experiment with PC3 cells for 10 days showed no significant upregulation of osteogenic genes in the mesenchymal stem cells, indicating that the differentiation of mesenchymal stem cells toward an osteoblastic lineage may have been driven by osteolysis because of enhanced osteoclastogenesis (Fig. 5A and B). In fact, tartrate-resistant acid phosphatase staining indicated intense osteoclast activity at the tumor-bone interface (Fig. 4C, b).

We compared the growth kinetics of highly osteolytic PC3 cells and the osteoblastic C4-2B cells in the tibia of SCID mice. The rate of tumor progression was significantly lower in the tibia injected with C4-2B cells as compared with PC3 cells (Fig. 6A and B). Although PC3 cells induced severe osteolysis by 1 month after inoculation, C4-2B cells produced osteoblastic events by 2 months after administration. There was a gradual switch from osteoblastic to osteolytic phenotype when C4-2B cells were allowed to grow for 6 months in the tibia (Fig. 6C). These observations indicate that the growth kinetics of the cancer cells in the bone might be the determining factor favoring osteoblastic or osteolytic outcome.

To test the significance of osteoprotegerin in this process, a mesenchymal stem cell line was generated wherein osteoprotegerin expression was silenced using a lentivirus producing shRNA targeting (Supplementary Fig. S3). Osteoprotegerin-silenced mesenchymal stem cells failed to differentiate into osteoblast in vitro, as determined by von Kossa staining (Fig. 1D), and no significant new bone formation was observed in the tibia when these mesenchymal stem cells were tested against PC3 cells in a similar experiment mentioned in the previous section (Fig. 4A). Moreover, conditioned medium from osteoprotegerin-silenced mesenchymal stem cells failed to inhibit osteoclastogenesis when RAW cells were cultured for 8 days in the presence of RANKL (Fig. 1C), suggesting the requirement of osteoprotegerin for osteoblast differentiation and inhibition of osteoclastogenesis.

To test the effects of mesenchymal stem cells on the prostate cancer cells, PC3 cells were cocultured in a three-dimensional matrix with mesenchymal stem cells in the presence or absence of bone marrow conditioned medium for 3 days. MTT assay was done to determine cell proliferation. Addition of naïve mesenchymal stem cells to the coculture did not alter PC3 cell proliferation compared with PC3 cultured only in bone marrow conditioned medium. An increase in cell proliferation was noted only when PC3 cells were cultured in bone marrow conditioned media along with mesenchymal stem cells overexpressing osteoprotegerin (P < 0.001; Fig. 5C), supporting that osteoprotegerin is also a survival factor for the prostate cancer cells (23). This suggests that the inhibitory effect of mesenchymal stem cells on the growth of prostate cancer in vivo is an indirect effect and is mediated by the inhibition of osteoclastogenesis and differentiation into osteoblasts.

Results of the present study indicate the therapeutic potential of unmodified mesenchymal stem cells in inhibiting the growth of prostate tumor in the bone and prevention of bone loss. Mesenchymal stem cells did not induce direct apoptosis of tumor cells, instead the inhibition of tumor growth in the bone was mediated by new bone formation around them. Although significant therapeutic advantage can be obtained in osteolytic bone metastasis using this approach, absence of a direct killing mechanism may help the metastasis re-establish itself in the course of time. The beneficial effects of mesenchymal stem cells can thus be further amplified by modifying them ex vivo to express tumoricidal genes besides retaining their ability to differentiate into bone. A recent study used mesenchymal stem cells expressing urokinase-type plasminogen antagonist amino-terminal fragment and showed inhibition of tumor growth by inhibiting angiogenesis prevented bone loss (24). Most studies indicated that mesenchymal stem cells promote tumor growth by participating in tumor stroma formation and establishment of premetastatic niche. Our in vitro studies showed an increase in proliferation rate of prostate cancer cells only when cocultured with the mesenchymal stem cells overexpressing osteoprotegerin. However, the same mesenchymal stem cells overexpressing osteoprotegerin tested provided a therapeutic effect in vivo. Hence, the observed in vitro effects could be attributed only to secretory proteins in the coculture system and may not include other events in the tumor microenvironment. Mesenchymal stem cells inhibited the growth of prostate tumor in the bone in vivo by laying down new bone around the cancer cells, which slowed their rapid growth. Other factors such as cell contact in vivo may have also played important roles in addition to the effects of osteoprotegerin in increasing osteogenesis.

Although the difference in tumor volume between unmodified mesenchymal stem cells (mesenchymal stem cells–GFP) and mesenchymal stem cells overexpressing osteoprotegerin was not statistically significant, there was an observable difference in the mesenchymal stem cells–GFP group, which showed less tumor growth compared with mesenchymal stem cells–osteoprotegerin group. Because osteoprotegerin may serve as a survival factor for tumor cells, this observation also indicates the importance of regulated expression of osteoprotegerin for restoration of bone damage following osteolytic bone metastasis. Furthermore, identification of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binding and RANK-binding domains on osteoprotegerin would allow genetic modifications in osteoprotegerin to abolish TRAIL binding.

The present study also highlights the formation of woven bone in osteoblastic metastases (25–27). In our study, the therapeutic effect of the mesenchymal stem cells is initially imparted by the woven bone formation surrounding the tumor nests. Formation of woven bone also characterizes osteoblastic metastases in prostate cancer and normal-to-fracture healing. Roudier et al. (2) did a detailed histomorphic analysis of bone samples obtained from prostate cancer patients with bone metastasis and observed equal representation of osteoblastic and osteolytic components. They also showed that the woven bone formation in the osteoblastic metastases originated from the skeletal mesenchymal stem cells. The pattern of woven bone formation in our experiment truly matches the histologic pattern observed in patient with prostate cancer bone metastasis, as reported by Roudier et al. (2). We speculate that the presentation of woven bone in prostate cancer bone metastasis is an endogenous therapeutic response by the resident mesenchymal stem cells rather than a pathologic outcome. Continuous presence of tumor cells in the bone induces osteolysis, which in turn signals the mesenchymal stem cells to initiate osteoblastogenesis and manifest the formation of woven bone as part of an early therapeutic response. As we compared growth kinetics of highly osteolytic PC3 cells versus osteoblastic C4-2B cells in the tibia of SCID mice, the rate of tumor growth is significantly lower in tibia injected with C4-2B cells compared with PC3 cells. Therefore, it is likely that low turnover of C4-2B cells provides a therapeutic window for the endogenous mesenchymal stem cells to induce bone formation to make up for the lost bone due to initial osteolytic event required by the cancer cells to colonize in the bone microenvironment. At this point, we are unable to comment on other factors responsible for the slow in vivo turnover of the C4-2B cells. When mice were sacrificed 6 months after the implantation of the C4-2B cells, all the lesions in the tibiae were of osteolytic nature. Determination of osteoblastic and/or osteolytic phenotype may be dependent on factors such as growth kinetics of the cancer cells and phenotypic changes of the cancer cells, and events such as hypoxia in the tumor microenvironment. Hypoxia is known to promote osteolytic bone metastasis and suppresses osteoblast differentiation (28); therefore, slower turnover of C4-2B cells may have delayed the onset of hypoxia compared to PC3 cells, which may have delayed the formation of osteolytic lesions. Nyambo et al. (29) reported that osteoprotegerin produced by the bone marrow stromal cells inhibits TRAIL-induced apoptosis of the tumor cells and favors the growth of prostate cancer in vitro. Our in vitro coculture assay showed that the bone marrow microenvironment and osteoprotegerin produced by the mesenchymal stem cells favored PC3 cell proliferation. Interestingly, mesenchymal stem cells or mesenchymal stem cells–osteoprotegerin imparted therapeutic benefits when applied in vivo. Based on these findings, we conclude that primary mesenchymal stem cells has the potential to provide therapeutic advantage in limiting the establishment of prostate cancer in the bone at an early stage by the virtue of its ability to differentiate into bone and inhibit osteoclastogenesis. These data signify that relatively abundant amounts of mesenchymal stem cells in the tumor microenvironment can provide therapeutic effects by activating osteoprotegerin and/or other factors through interactions with prostate cancer cells. Because the amount of mesenchymal stem cells in the bone microenvironment is extremely low (1 in 10⁸ cells), strategies to endogenously mobilize and proliferate mesenchymal stem cells upon bone metastasis of osteolytic cancers may provide significant therapy and reduce morbidity and mortality encountered in late-stage prostate cancer patients. The aim of the present study was to determine potential of adult bone marrow–derived mesenchymal stem cells for the prevention of cancer osteolysis. Based on the limitations of the model used, it is imperative that additional studies should be done with other osteolytic bone metastasis models having the bone defect in the entire skeleton in immunocompetent animals before translating the findings for humans.

No potential conflicts of interest were disclosed.

We thank Dr. Maria Johnson and Xingsheng Li for the excellent technical assistance in micro–computed tomography measurements.