Doxorubicin and TGFb Inhibitor Figure 8. TbRI-KI reduced doxorubicin-induced spontaneous bone metastasis in the murine xenograft model. A. 4T1 cells (0.16106) were inoculated into both inguinal mammary fat pads of syngeneic female balb/c mice. When the average tumor volume reached 60–70 mm3 after 2 weeks, the mice were divided into six groups with similar tumor burden and treated i.p. with PBS (control), TbRI-KI at 1 mg/kg every alternative day, doxorubicin 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. The 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 volume6SEM from 6 mice in DOXO-8 and 5 mice in the rest groups. The bars bearing a different letter are significantly different (P,0.05) by ANOVA. B. After the termination of the experiment, the leg bones (Femora and tibiae) were fixed in 10% neutral buffered formalin, decalcified in 10% EDTA and embedded in paraffin. Sections were stained with H&E, orange G and phloxine. The presence of tumors in the bone marrow was examined microscopically. The incidence of bone metastasis in each group of animals was expressed in percentage of the number of animals with bone metastasis. C. Animals with bone tumor burden were divided into four groups and the results were expressed in percentage of animals with no bone metastasis, with ,20% tibia replaced by tumor, with 20–50% tibia replaced by tumor, and with .50% of tibia replaced by tumor. doi:10.1371/journal.pone.0010365.g008 referred as cancer stem cells (CSCs) and often exhibit phenotypes resembling normal stem cell properties such as expression of stem cell markers, self renewal and multipotency [44]. Metastatic breast cancer cells are notorious for their ability to survive treatment and regenerate tumors. One important feature of CSCs is their resistance to the cytotoxicity of varous chemo- therapeutic agents because of their relatively high expression levels of multiple drug resistance transporters such as ABCB1 (MDR1/PGP, P-glycoprotein) and ABCG2 (MXR/BCRP) [38]. Thus, chemotherapeutics might spare CSC’s and allow recur- rence of more aggressive tumors. Recent data showed that immortalized human and murine breast cancer cell lines contain stem-like cells similar to CSCs isolated from primary tumor tissue with identical cell surface markers, clonogenic and sphere formation potential, and in vivo functional transplantation properties [45]. The fact that EMT can generate stem-like cancer cells [26] suggests that therapeutic-resistant cancer stem cells may be the result of either the transformation of normal stem cells or via the induction of EMT in more differentiated cancer cells. Indeed, we found that treatment with TGFb or doxorubicin increased the number of 4T1 cells expressing stem cell marker Sca-1 and the drug resistant transporter protein MDR1, and with the stem cell property of self-renewal as indicated by mammosphere formation. The 4T1 cells pre-treated with TGFb1 for 7 days showed resistance to the doxorubicin- induced growth inhibition. Our results indicate that by activating TGFb signaling, doxorubicin has the ability to induce EMT and generate tumor cells with stem cell phenotypes. The EMT program is often considered a transient and reversible process. Studies of embryonic development have showed that mesenchymal cells of mesoderm can give rise to epithelial organs, including the kidney and ovary, by undergoing the reverse EMT [46]. The finding that EMT plays a role in the development of cancer stem cells suggests that therapies blocking or reversing the EMT program may sensitize resistant cancer stem cells to conventional cancer therapies. We have evaluated the ability of a small molecule TGFb inhibitor, TbRI-KI, to block the EMT process and subsequent cancer stem cell formation by TGFb1 and doxorubicin treatment in 4T1 cells in vitro. We found that blockade of TGFb signaling significantly reversed the TGFb1- and doxorubicin-mediated EMT process. Restoration of the epithelial phenotypes of the cytoskeleton organization and cytoplasmic localization of the EMT transcrip- tion factor Snail was observed in the presence of the inhibitor. The treatment of TbRI-KI also inhibited TGFb1- and doxorubicin- mediated generation of cells with stem cell phenotypes including the expression of MDR1 and the formation of mammospheres. Thus, all our in vitro experiments indicated potential therapeu- tic benefits of using TbRI-KI to antagonize the doxorubicin- induced TGFb signaling, which has the potential to induce EMT and to generate drug resistant stem-like cancer cells. We designed in vivo experiments utilizing murine breast cancer 4T1 cells and human breast cancer MDA-MB-231 cells to study the synergistic anti-tumor effect of the systemic administration of both doxoru- bicin and TbRI-KI in comparison to single-agent treatments. Our studies revealed that while treatment with either agent alone moderately inhibited tumor growth in both xenograft breast cancer models, the combination of both agents was clearly much more effective in inhibiting tumor growth. In fact, a 50% reduction of doxorubicin dose was sufficient to produce the same tumor growth inhibition as the full dose when TbRI-KI was added to the treatment suggesting that the combination with TGFb antagonists may be an effective strategy in reducing doxorubicin dosage and its unwanted side-effects. PLoS ONE | www.plosone.org 9 April 2010 | Volume 5 | Issue 4 | e10365