Doxorubicin and TGFb Inhibitor Figure 6. TGFb inhibitor enhances the efficacy of doxorubicin treatment to reduce tumor growth and spontaneous lung metastasis of murine breast cancer cells. A. Murine breast cancer 4T1 cells (0.16106) were inoculated at the both inguinal mammary fat pad of syngeneic Balb/c mice and divided into six groups. Animals were treated with PBS (Placebo), TbRI-KI at 1 mg/kg every alternative day, doxorubicin (DOXO) at 4 mg/kg (DOXO-4) or 8 mg/kg (DOXO-8) once per 7 days, or the combination of TbRI-KI and DOXO-4 or DOXO-8 through i.v. injection starting at the day of tumor cell inoculation. Tumor sizes were measured twice weekly with a caliper, and the tumor volumes were calculated with the equation V = (L6W2)60.5, where L is length and W is width of a tumor. Experiment was terminated after 3 wks of treatment and the final tumor volume (sum of two tumors per mouse) was used for calculating mean tumor volume 6 SEM from 6 mice in Placebo and 5 mice in the rest of the groups. B and C. After the termination of the experiment, lungs were excised and fixed in Bouin’s solution (Sigma) to count surface lung metastasis, followed by paraffin embedding for histological H & E staining. Representative picture of lungs without and with metastatic nodules are shown with corresponding histological identification of the nodules by H & E staining (B). The visible surface lung nodules were counted. ‘‘*’’ denotes a significant difference (P,0.005) from the corresponding control with a Student t test (C). doi:10.1371/journal.pone.0010365.g006 delayed until the orthotopic xenograft tumors reached an average size of 60–70 mm3. Doxorubicin was administered by i.v. injection and TbRI-KI by i.p. injection for 21 days. This combination treatment inhibited the tumor growth in comparison to the single treatments (Figure 8A). Tibia specimens in all treatment groups were analyzed for the incidence of bone metastasis, tumor burden and osteolysis indicated by the loss of trabecular bone after histological staining. Metastatic 4T1 tumors in different specimens were seen at different areas of tibia, often away from the trabecular bone area. Therefore, osteolysis as reflected by loss of trabecular bone could not be compared between the placebo and the treated mice with bone metastasis. More metastatic tumors in the tibia were seen after single treatment with either dose of doxorubicin than treatment with TbRI-KI, which moderately reduced the incidence of metastasis in comparison to the placebo treatment group (Figure 8B). More significantly, the metastasis incidence was reduced by 50–60% when animals were treated with both doxorubicin and TbRI-KI (Figure 8B). We also compared the percent incidence of different levels of the tibial tumor burden among the treatment groups. We recorded the levels of tumor burden as: no bone metastasis, ,20% tibia replaced by tumor, 20–50% tibia replaced by tumor, and .50% of tibia replaced by tumor. The percentage of specimens with higher tumor burden (.50%) was evident only in the placebo or doxorubicin-treated groups but not in the TbRI-KI-treated or the combination treatment groups (Figure 8C). About 70% animals were free of bone metastasis after the combination treatment in comparison to about 24% and 32% in the low and high dose doxorubicin treatment group respectively (Figure 8C). These results demon- strated that doxorubicin treatment alone increased the bone metastasis incidence and bone tumor burden which were remarkably reduced by the simultaneous treatment with a TGFb inhibitor. Although the combination treatment after tumors were formed and growing was effective in inhibiting bone metastasis, its effect on lung metastasis was much less obvious because the majority of lungs in each treatment group were covered with metastatic nodules making it difficult to count the nodules. Thus, our results indicate that early treatment with the combination of doxorubicin and TbRI-KI will likely be much more effective than delayed treatment in blocking lung metastasis. Discussion In this study, we have evaluated the potential therapeutic benefit of using a small molecule TGFb signaling inhibitor with doxorubicin in animal models of breast cancer. Doxorubicin, which is a frequently used chemotherapeutic agent against metastatic breast cancer, has been reported to induce acute and chronic toxicities and immunosuppression. In addition, we found in our study that doxorubicin can activate TGFb signaling in cancer cells, which has the potential to accelerate malignant tumor progression. Thus, novel treatment options are needed to reduce dosages of doxorubicin and consequently unwanted toxicities while enhancing its therapeutic efficacy in treating metastatic cancer. An elevated plasma level of TGFb has been correlated to the progression of various types of malignancies including metastatic breast cancer progression [9] and survival [42]. Persistent high levels of circulating TGFb predicted early metastatic recurrences in the liver of patients with colorectal cancer after curative resection [43]. In addition to the carcinogenesis-associated PLoS ONE | www.plosone.org 7 April 2010 | Volume 5 | Issue 4 | e10365