Doxorubicin and TGFb Inhibitor Figure 4. Doxorubicin and TGFb treatment generated drug resistant cells with a stem cell phenotype. A and B. The murine 4T1 breast cancer cells were treated with TGFb1 (2 ng/ml) or doxorubicin (25 nM) in the presence or absence of TbRI-KI (500 nM) for 7 days. The medium was changed every alternate day. The treated and non-treated cells were harvested with trypsinization and incubated with an alexafluor 405-conjugated antibody (2 mg/106 cells) against MDR1 or a Phycoerythrin-conjugated antibody against Sca-1. Fluorescence Activated Cell Sorting (FACS) analysis was carried out using FACSAria flow cytometer (Becton Dickinson). Results were expressed as fold increase of the number of cells expressing the surface markers for Sca-1 or MDR1 after treatment with TGFb1 or doxorubicin compared to the untreated cells (A) or as the percentage of MDR1 expressing cells after doxorubicin treatment in the presence or absence of TbR1-KI (B). C. 4T1 cells were treated with or without 2 ng/ml TGFb1 and DOXO (10 nM and 25 nM) for 7 days and cell extracts were used for Western blot analysis with antibody to MDR1. D. 4T1 cells, pre-treated with TGFb1 (5 ng/ml) for seven days, were plated in a 96-well plate and were treated with doxorubicin at two different concentrations (1 nM and 10 nM) in the presence or absence of TGFb1 (5 ng/ml). Relative cell number after 5 days of incubation was obtained by MTT assay. Each data point is the mean6SEM from 4 replicate wells. doi:10.1371/journal.pone.0010365.g004 breast cancer 4T1 cells, which could be reversed by the treatment with a TGFb antagonist TbRI-KI. These results prompted us to test the hypothesis that antagonism of increased TGFb signaling during doxorubicin treatment in vivo may enhance anti-tumor activity of doxorubicin in the murine breast cancer 4T1 and human breast cancer MDA-MB-231 xenograft models. TbRI-KI enhanced the efficacy of doxorubicin to reduce tumor growth and spontaneous lung metastasis by murine breast cancer cells Balb/c mice were injected with 4T1 cells at both inguinal mammary glands and treated immediately once a week with a low or a high dose of doxorubicin (4 mg and 8 mg/kg body weight), or with TbRI-KI (1 mg/kg body weight) every alternate day, either alone or in combination, by i.p. injection. Treatment with TbRI- KI for 21 days moderately inhibited primary tumor growth of 4T1 tumors while treatment with doxorubicin induced a dose- dependent and more noticeable inhibition in comparison to the placebo treatment (Figure 6A). However, much more significant inhibition of tumor growth was obtained when the treatment of TbRI-KI was combined with doxorubicin at both low (DOXO-4) and high doses (DOXO-8) (Figure 6A). In fact, the combination of TbRI-KI and the low dose of doxorubicin produced a similar inhibitory effect to that of the high dose of doxorubicin alone. After the termination of the experiment, metastatic tumor nodules on the surface of the lungs, as shown in Figure 6B, were counted. The metastatic burden on the lungs, as represented by the number of tumor nodules, was moderately, but dose dependently, lowered by doxorubicin treatments. Interestingly, while TbRI-KI was not as effective as doxorubicin in inhibiting orthotopic tumor growth, it was much more effective in blocking lung metastasis than doxorubicin suggesting that metastasis can be independent of primary tumor size (Figure 6C). The combination between TbRI- KI and the higher dose of doxorubicin produced greater inhibition of lung metastasis than either treatment alone (Figure 6C). Thus, combination treatment of doxorubicin and TbRI-KI effectively enhanced the inhibition of both tumor growth and spontaneous lung metastasis of murine 4T1 cells in comparison to the single treatments. TbRI-KI inhibited doxorubicin-induced spontaneous lung metastasis of human breast cancer cells We also utilized the human breast cancer MDA-MB-231 cells expressing enhanced green fluorescence protein (EGFP) to investigate the effects of doxorubicin and/or the TGFb antagonist TbRI-KI treatment on metastasis induced by orthotopic xenograft tumors formed by the MDA-MB-231 cells. The treatment was initiated when the average tumor volume was between 80– 100 mm3. After 21 days of treatment with both i.v. injection of doxorubicin (4 mg/kg/wk) and i.p. injection of the TbRI-KI (1 mg/kg/2days), either alone or in combination, we examined the tumor growth rate and the spontaneous lung metastasis. Similar to the 4T1 tumor model, combination treatment significantly reduced tumor growth in comparison to the single treatments although treatment was started after the initiation of tumor growth (Figure 7A). Most interestingly, examination of the whole lungs revealed that the size of the metastatic colonies was much larger in doxorubicin group than in the control group as observed by EGFP fluorescence imaging (Figure 7B). Further- more, there was increased number of mice with greater than 50 lung metastatic colonies in the doxorubicin-treated group in comparison to the control group (Figure 7C) even though doxorubicin treatment moderately reduced primary tumor growth (Figure 7A) indicating that doxorubicin alone can stimulate PLoS ONE | www.plosone.org 5 April 2010 | Volume 5 | Issue 4 | e10365