TGF-b Inhibition in Myeloma nodules for 2 days. 5TGM1 cells were analyzed by flow cytometry with dual staining of BrdU and 7-AAD as described in ‘‘Materials and methods’’. Cells were gated corresponding to cell cycle distribution. C. RPMI8226 cells were cultured alone or cocultured with MC3T3-E1 cells with or without mineralization in quadruplicate for 3 days in the absence or presence of melphalan at 20 mM (left) or dexamethasone at 10 mM (right). Viable RPMI8226 cells were counted. doi:10.1371/journal.pone.0009870.g005 subcutaneously in SCID mice. INA6 cell-derived human soluble IL-6 receptor was detected as a marker for MM tumor growth 4 weeks after the inoculation of INA6 cells in all the SCID-rab mice, which demonstrates that the rabbit bone microenvironment allows the growth of INA6 cells. Because sera from mice orally treated with the TGF-b type I receptor kinase inhibitor Ki26894 effectively inhibited most of TGF-beta-induced reporter activi- ty,[29] we treated INA6-bearing SCID-rab mice with an oral administration of Ki26894 according to procedures described before.[29] We analyzed MM cell growth and the formation of bone lesions in vehicle- or Ki26894-treated mice 6 weeks after the inoculation of INA6 cells. In vehicle-treated mice, the levels of human soluble IL-6 receptor,[27] a marker for human myeloma tumor burden, in mouse sera were substantially increased (Figure 7A); radiolucent osteolytic lesions were observed in the implanted rabbit bones (Figure 7B). In histological analyses, MM cells were tightly packed in the bone marrow cavity of the rabbit bones while bone trabeculae decreased in size (Figure 7C). However, in Ki26894-treated mice, serum levels of soluble IL-6 receptor remained low (Figure 7A) and only marginal bone destruction was observed on X-ray radiography (Figure 7B). The MM cells markedly decreased in number and formed only small foci in rabbit bones, while bone trabeculae remained without apparent loss of size and structure, and cuboid cells lined the surface of bone, namely osteoblasts (Figure 7C). For quantitative histomorphometric analyses, we measured bone volume per tissue volume (BV/TV) and osteoblast surface per bone surface (Ob.S/ BS) of rabbit bones in MM-bearing SCID-rab mice as the indices of bone destruction and osteoblast function, respectively. Both BV/TV and Ob.S/BS were markedly suppressed in vehicle- treated mice. However, both indices retained significantly higher in Ki26894-treated mice, suggesting the protection of bone in vivo by TGF-b inhibition. We also measured tumor per tissue volume (tumor/TV) to assess a tumor burden of the rabbit bones in MM- bearing SCID-rab mice. Tumor/TV was markedly reduced in Ki26894-treated mice (Figure 7D), which is consistent with the serum levels of soluble human IL-6R, a surrogate marker for a human MM tumor burden, in MM-bearing SCID-rab mice. These results suggested that TGF-b inhibition can suppress MM cell growth within the bone marrow while preventing bone destruction and loss. Discussion BMP[30,31] and canonical Wnt pathways[32,33] are among the predominant signaling pathways stimulating OB differentia- tion. A canonical Wnt pathway in OBs is suppressed by soluble Wnt antagonists elaborated by MM cells,[5,6,7] stromal cells and OBs.[8,9] The present study demonstrated that TGF-b plays an important role in arresting the differentiation of stromal cells into mature OBs and that TGF-b suppresses BMP-2 signaling (Figure 3D). In osteolytic lesions in MM which enhance the release and activation of TGF-b, a BMP as well as canonical Wnt signaling pathway in stromal cells and OBs appears to be suppressed, causing severe suppression of OB differentiation. Interestingly, a blockade of TGF-b antagonized the suppressive effects of MM cell conditioned media (Figure 3A) and bone marrow plasma from MM patients (Figure 3B), and was able to release stromal cells from differentiation arrest to achieve terminal OB differentiation. Although the mechanism whereby TGF-b inhibits OB differentiation still remains unclear, we found at least in our experimental conditions that TGF-b inhibition markedly enhances the phosphorylation of Smad1 to potentiate BMP-2 signaling without affecting the canonical Wnt pathway in OB precursor cells suppressed by MM cells (Figures 3C-D). Therefore, the potentiation of BMP-2 signaling at least in part contributes to the restoration of OB differentiation caused by the inhibition of TGF-b. In the present study, TGF-b inhibition was shown to facilitate terminal OB differentiation in parallel with suppression of MM cell growth and survival. A reverse correlation between OB differentiation and MM tumor growth has recently been reported in patients with MM treated with the proteasome inhibitor bortezomib. Serum levels of bone-specific ALP were found to be elevated after treatment with bortezomib, which were inversely correlated with a reduction in tumor burden.[34,35,36] Such bone anabolic effects of bortezomib and their correlation with tumor regression were further demonstrated in MM animal models.[37] MM growth inhibition associated with OB differentiation was also observed in MM animal models treated with other anabolic agents such as anti-DKK1 antibody[38] and lithium chloride[39] as well as with enforced expression of Wnt3a within bone.[40] Together with our results with TGF-b inhibitors, these observations suggest that anti-MM activity emerges with OB differentiation, and that MM cells may protect themselves from such OB-mediated growth suppression by inhibiting the terminal differentiation of OBs. We found that treatment with the TGF-b type I receptor kinase inhibitor Ki26894 in MM-bearing SCID-rab mice suppressed MM cell growth within the bone marrow while preventing bone destruction and loss (Figure 7). In vivo effects of TGF-b inhibition have also been studied with the TGF-b type I receptor kinase inhibitor SD-208, and shown to increase bone mass in mammary tumor-bearing mice (Mohammad KS, Stebbins E, Kingsley L, et al. J Bone Miner Metab. 2008;23 abstract F275) as well as normal mice.[41] Pharmacological blockade of TGF-b action by the anti- TGF-b monoclonal antibody 1D11 has also been demonstrated to reduce serum M-protein levels as well as improve bone volume and strength in 5TGM1-bearing mouse MM models (Hart AJ, Fowler JA, Lwin ST, et al. J Bone Miner Metab. 2008;23 abstract F293). Together with these in vivo results, our study demonstrates that TGF-b appears to be an important therapeutic target in MM bone lesions. However, because TGF-b inhibitors including SB431542 and Ki26894 did not show direct cytotoxic effects on MM cells (Figure 7E), the combination of TGF-b inhibitors with cytoreductive chemotherapeutic agents or bortezomib may further improve the therapeutic efficacy against MM. Stromal cells together with OCs create a MM niche in the bone marrow to promote MM cell growth and protect MM cells from spontaneous and drug-induced apoptosis. Stromal cells confer potent drug resistance to blunt the efficacy of anti-MM agents.[14,15,16] Importantly, terminally differentiated OBs potentiate cytotoxic effects of melphalan and dexamethasone (Figure 5C), suggesting that mature OBs can increase the susceptibility of MM cells to anti-MM agents to overcome the drug resistance mediated by stromal cells. These results are consistent with a hypothesis that induction of OB differentiation PLoS ONE | www.plosone.org 9 March 2010 | Volume 5 | Issue 3 | e9870