Doxorubicin and TGFb Inhibitor Figure 5. Abrogation of doxorubicin induced mammosphere formation by TbRI-KI. Murine 4T1 cells were treated with doxorubicin (25 nM) in the presence or absence of TbRI-KI for 6 days. The cells with or without treatment were harvested after trypsinization and filtered through 40 mm strainer to obtain a single-cell suspension. Cells were seeded (1,000 cells/100 ml/well) in a 96-well ultra-low attachment plate (Costar) and mammospheres were counted after 7 days of incubation. A. Representative pictures of mammospheres obtained from cells with or without doxorubicin treatment and also after doxorubicin treatment in the presence of TbRI-KI. B. The number of mammospheres obtained from 1,000 cells in triplicate wells with or without treatments were counted and expressed as mean6SEM. ‘‘*’’ indicates significant difference from the control value, P,0.05. doi:10.1371/journal.pone.0010365.g005 metastasis. Treatment with TbRI-KI also moderately inhibited tumor growth, however a statistically significant reduction of tumor volume was only observed with the treatment of both doxorubicin and TbRI-KI in comparison to that of the control group (Figure 7A). More significantly, the combination treatment remarkably inhibited lung metastasis as evidenced by reduced lung metastatic colonies both in colony size (Figure 7B) and number (Figure 7C9). The size of lung metastatic colonies in TbRI-KI group was similar to that in the combination treatment group (picture not shown). The fact that the number of mice with 1–20 lung metastases in the combination treatment group was still higher than that in TbRI-KI alone group suggests that TbRI-KI treatment did not completely inhibit doxorubicin-induced lung metastasis. Thus, our study indicates that the TGFb inhibitor has the potential to inhibit doxorubicin-induced lung metastasis. TbRI-KI reduced doxorubicin-induced spontaneous bone metastasis in a murine xenograft model Breast cancer metastasizes to multiple sites and frequently to the skeleton, resulting in poor prognosis. In our previous study we found that systemic administration of the TbRI-KI can inhibit bone metastasis of human cancer cells in an intra-cardiac injection model of bone metastasis [22]. Orthotopic tumors formed by 4T1 cells have been shown to induce spontaneous bone metastasis [41]. However, we failed to detect tumor cells in the sections of femora and tibiae from either the control or the treated mice in our experiment described in Figure 6. We suspected that the experiment duration of three weeks might not be long enough for metastatic 4T1 cells to form tumors in bony tissue. Therefore, we performed another set of experiment with the 4T1 cells, in which the treatment with doxorubicin and/or TbRI-KI was PLoS ONE | www.plosone.org 6 April 2010 | Volume 5 | Issue 4 | e10365