Lung metastasis is prevalent among the patients with metastatic breast cancer [47]. About 60% to 74% of patients who die of breast carcinoma have lung metastasis [48]. In several animal models, TGFb has been shown to promote lung metastasis [49,50]. Since Doxorubicin is widely used as an adjuvant chemotherapeutic agent in metastatic breast cancer with limited response rate (4) and enhanced lung metastasis in an animal model [32], we also examined the lungs of the animals for the presence of metastatic colonies in the placebo and treatment groups. We observed moderate reduction of lung metastasis after doxorubicin treatment which was significantly improved by combinational therapy with TbRI-KI in the murine 4T1 xenograft model. In the human breast cancer xenograft model, our results indicated that doxorubicin alone can stimulate lung metastasis with larger metastatic colonies which can be effectively blocked by the simultaneous treatment with TbRI-KI. Since smaller sized pulmonary metastasis has been shown to be significantly associated with longer survival [47], our results suggest that administration of TbRI-KI along with doxorubicin may have the potential to improve the survival rate of doxorubicin-treated metastatic breast cancer patients. Metastases to bone are frequently seen pathological lesions in advanced breast cancer, which causes debilitating skeletal complications such as osteolysis, intractable bone pain and pathologic fractures [51]. Most importantly, once breast tumor cells metastasize to bone marrow, mortality increases to 70% in comparison to 40% in the breast cancer patients with no tumor cells in their bone marrow [52,53]. Therefore, prevention and treatment of bone metastasis is important for the improvement of survival in breast cancer patients. In our study, we observed that mice bearing orthotopic 4T1 tumors showed an increased incidence of spontaneous bone metastasis as well as greater bone tumor burden after doxorubicin treatment in comparison to the placebo treatment. Both incidence of tumor cells and tumor volume in the bone marrow were reduced remarkably after simultaneous treatment with TbRI-KI even though TbRI-KI treatment alone was only moderately effective in reducing bone metastasis incidence and tumor burden. Thus, the combination therapy appears to produce a synergistic inhibition of bone metastasis in this model system. In summary, our study suggests that doxorubicin at certain dosages can promote breast cancer invasion and metastasis in part through its ability to activate TGFb signaling, which in turn generates stem-like cancer cells with increased drug resistance through an EMT process. Our study also suggests that administration of TGFb antagonists during chemotherapy may sensitize carcinoma cells, in part through the inhibition of EMT, to the anti-tumor and anti-metastatic activities of chemotherapy resulting in increased therapeutic efficacy and reduced dosage of chemotherapeutics and their unwanted side effects. Materials and Methods Ethics statement All animal experiments were conducted following appropriate guidelines. They were approved by the Institutional Animal Care and Use Committee and monitored by the Department of Laboratory Animal Resources at the University of Texas Health Science Center at San Antonio. Cell lines Human breast cancer cell line MDA-MB-231 and murine breast cancer 4T1 cell line was originally obtained from the American Type Tissue Culture Collection (ATCC). These cell Doxorubicin and TGFb Inhibitor lines were cultured in McCoy’s 5A medium supplemented with pyruvate, vitamins, amino acids, antibiotics, and 10% fetal bovine serum (FBS) as described previously [54]. Western Blotting The murine 4T1 and human MDA-MB-231 breast cancer cells were treated with doxorubicin (25 nM) in the presence or absence of TGFb1 (2 ng/ml) and/or TbRI-KI (500 nM) for 7 days. The medium was changed every alternate day with the addition of doxorubicin and TGFb1 each time. The cell lysates were used in the western blotting analysis as described previously [14]. Antibody to the Phospho-Smad2 was from Cell signaling, to vimentin was from Sigma, and to Phospho-Smad3 and MDR1 was from Santa Cruz Biotechnology. The TbRI-KI used in our study was previously reported to be an ATP competitive inhibitor of the TGFb RI kinase [55,56]. The compound, [3-(pyridine-2yl)- 4-(4-quinonyl)]-1H pyrazole, was synthesized according to the procedure described by Sawyer et al. [55]. In vitro cell migration and invasion assays Cell migration and invasion assays were performed in 24-well Boyden Chambers with 8-mm pore polycarbonate membranes (Becton Dickinson Labware, Rockville, MD) for migration assays and with matrigel coated membranes for invasion assay. For migration assay, 40,000 murine breast cancer 4T1 cells and human breast cancer MDA-MB-231 cells in serum free medium were seeded in the upper chamber and treated with doxorubicin (100 nM) or TGFb1 (5 ng/ml) and incubated for 6 hours and 24 hours respectively. Lower chamber contained the medium with 1% serum as chemoattractant. After incubation, cells were removed from the upper surface of the chamber membrane with a cotton swab and the membrane was then stained for the migratory cells at the lower surface of the chamber with Hema 3 stain kit (Fisher Scientific, Houston, TX) according to the manufacturer’s protocol. The stained cells were counted under a microscope. For invasion assay using matrigel coated chambers, 50,000 murine 4T1 cells and human MDA-MB-231 cells in serum free medium were used and treated similarly as described above with doxorubicin or TGFb1 for 18 hours and 24 hours respec- tively. Lower chamber contained the complete medium with 10% serum as chemoattractant. Invasive cells were stained and counted as described above. Mammosphere culture Murine 4T1 cells were treated with doxorubicin (25 nM) for 6 days in the presence or absence of TbRI-KI (500 nM). The medium with doxorubicin was changed every alternate day. The cells with and without treatment were harvested after trypsiniza- tion with 0.05% trypsin and filtered through a 40-mm strainer to obtain a single-cell suspension. Cells were seeded (1000 cells/ 100 ul/well) in a 96 well ultra-low attachment plate (Costar) in DMEM-F12 medium supplemented with 2% B27 (Gibco), which is a serum replacement that excludes constituents that lead to cell differentiation and 20 ng/ml EGF, 20 ng/ml bFGF, 0.5 mg/ml hydrocortisone, 5 mg/ml insulin and gentamycin/amphotericin-B (Lonza). Mammospheres were counted after 5–7 days of incubation. Flow Cytometry The murine 4T1 breast cancer cells were treated with doxorubicin (25 nM) in the presence or absence of TGFb1 (2 ng/ml) and/or TbRI-KI (500 nM) for 7 days. The medium was changed every alternate day with the addition of doxorubicin and PLoS ONE | www.plosone.org 10 April 2010 | Volume 5 | Issue 4 | e10365