TGF-b Inhibition in Myeloma expressed as means +/2 SD. *, ,0.05. B. CD138-positive MM cells and CD138-negative non-MM bone marrow cells were immunomagnetically isolated from bone marrow aspirates of MM patients. The cells were cultured alone or co-cultured in triplicate with MC3T3-E1 cells with or without mineralization. After culturing for 3 days, the MM and non-MM bone marrow cells were harvested and viable cell numbers were counted. Data were expressed as means +/2 SD. *, p,0.05. doi:10.1371/journal.pone.0009870.g004 BMP-2 signaling by TGF-b without affecting canonical Wnt signaling down-regulated by MM cells. Terminally differentiated OBs suppress MM cell growth and survival To determine whether induction of OB differentiation in stromal cells affects the MM cell growth-promoting activity of stromal cells, we examined MM cell growth and survival in cocultures with MC3T3-E1 cells or bone marrow stromal cells isolated from a patient with MM with or without induction of terminal OB differentiation. Terminally differentiated OBs forming mineralized nodules derived from MC3T3-E1 cells or primary stromal cells markedly reduced the viability of MM cell lines, while untreated MC3T3-E1 cells or primary stromal cells without OB differentiation did not (Figure 4A). We next determined whether or not the suppression of growth and survival by terminally differentiated OBs is specific to MM cells. CD138- positive MM cells and CD138-negative non-MM cells were immunomagnetically isolated from bone marrow aspirates from MM patients, and cocultured with untreated or terminally differentiated MC3T3-E1 cells. The viability of CD138-positive primary MM cells was substantially decreased after coculturing with MC3T3-E1 cells with mineralized nodules, while CD138- negative normal hematopoietic cells remained intact (Figure 4B). MC3T3-E1 cells without OB differentiation showed suppressive effects on neither CD138-positive nor negative cells. Thus, terminally differentiated OBs suppress the growth and survival of MM cells but not normal hematopoietic cells in the bone marrow. Terminally differentiated OBs induce MM cell apoptosis with G1 arrest We next investigated the effects of terminally differentiated MC3T3-E1 cells on the induction of apoptosis and cell cycle distribution of MM cells. Viable 5TGM1 MM cells slightly increased in number when cocultured with untreated MC3T3-E1 cells (Figure 5A, middle). However, a large number of 5TGM1 MM cells underwent apoptosis with positive staining of annexin V when cocultured with differentiated MC3T3-E1 cells with mineralized nodules (Figure 5A, right). Untreated MC3T3-E1 cells increased the number of 5TGM1 cells in S phase (Figure 5B, middle). In contrast, 5TGM1 cells in S phase almost completely disappeared, and those in G0/G1 and sub-G0/G1 substantially increased in number when cocultured with MC3T3-E1 cells with mineralized nodules (Figure 5B, right). These results demonstrate that terminally differentiated OBs induce MM cell apoptosis with G1 arrest. Because stromal cells with suppressed OB differentiation in MM confer drug resistance in MM cells, we next determined whether the induction of terminal OB differentiation can reverse the protective effects of stromal cells on MM cell viability against anti- MM chemotherapeutic agents, melphalan and dexamethasone. Untreated MC3T3-E1 cells attenuated the cytotoxic effects of melphalan (Figure 5C left) and dexamethasone (Figure 5C right) on RPMI8226 MM cells. In contrast, differentiated MC3T3-E1 cells with mineralized nodules did not show protective effects on MM cells and further enhanced the cytotoxicity of these agents against MM cells. These results demonstrate that the induction of terminal OB differentiation can abolish the stromal cell-induced resistance against anti-MM agents, and that the susceptibility of MM cells to anti-MM agents can be reinforced by enhancing the terminal differentiation of OBs. Enhancement of OB maturation by TGF-b inhibition facilitates the suppression of MM cell growth To determine whether TGF-b can expedite OB differentiation, we next sequentially analyzed the effects of inhibiting TGF-b on the formation of mineralized nodules by MC3T3-E1 preosteo- blastic cells. SB431542 facilitated OB differentiation as evidenced by the enhanced development of mineralized nodules at day 6, earlier than that with BMP-2 alone (Figure 6A). Addition of TGF- b almost completely suppressed the BMP-2-induced formation of mineralized nodules even at day 12. However, with SB431542 the modulation resumed as early as day 6 even in the presence of TGF-b. These results demonstrate that TGF-b inhibition expedites OB maturation even in a TGF-b-rich milieu. We next determined whether enhancement of OB maturation by TGF-b inhibition can suppress MM cell growth at earlier time points. After MC3T3-E1 cells had achieved different stages of OB differentiation through culturing with or without BMP-2 and/or SB431542 for 3, 6 and 9 days, they were washed and subsequently cocultured with 5TGM1 MM cells for 3 days. ALP activity in MC3T3-E1 cells was already enhanced at day 3 in the presence of BMP-2 (Figure 6B, upper panels), while no mineralized nodules were observed (Figure 6B, middle panels). Despite the elevation of ALP activity at day 3, 5TGM1 MM cell growth was not suppressed when the MM cells were cocultured with MC3T3-E1 cells at this stage of differentiation (Figure 6B, lower panels). In contrast, suppression of MM cell growth was observed in parallel with the development of mineralized nodules by cocultured MC3T3-E1 cells after 6 and 9 days in the presence of BMP-2 with or without SB431542, and correlated well with the levels of mineralization. These results suggest that terminally differentiated mature OBs with mineralized nodules have the ability to suppress MM cell growth and survival, and that TGF-b inhibition can expedite the differentiation of OBs to suppress MM cell growth and survival. TGF-b inhibition suppresses MM cell growth and formation of bone destructive lesions in MM-bearing SCID-rab mice To evaluate in vivo the effects of TGF-b inhibition on both MM cell growth and the formation of bone destructive lesions, we first established MM-bearing animal models developing a bone disease. Because most MM cell lines grow rapidly and disseminate extraosseously in SCID mice, we utilized an IL-6 or stromal cell-dependent human MM cell line, INA6, which has been shown to hardly grow subcutaneously but grow within a human fetal bone implanted in SCID mice (SCID-hu mice).[27] SCID-rab mice have been developed to substitute for SCID-hu mice to recapitulate MM expansion within the bone marrow and formation of a bone disease.[28] Therefore, we generated SCID- rab MM models using INA6 cells. INA6 cells were inoculated directly into the bone marrow cavity in rabbit bones implanted PLoS ONE | www.plosone.org 7 March 2010 | Volume 5 | Issue 3 | e9870