Mages as analyzed by IVIS Lumina imaging system at different time points after intracardiac injection of Title Loaded From File MDA-MB-231 (BR) cells. (D) Average count of quantum dot labeled MDA-MB-231 (BR) 10781694 tumor cells in mice brain of control andSuppression of Brain Metastasis by PEITCPEITC (10 mmol) treated groups. Values are Title Loaded From File represented as means6SEM. * P,0.05, statistically different when compared with control. At least 150 brain sections from each group were analyzed. doi:10.1371/journal.pone.0067278.gCell CultureHuman breast carcinoma cell lines MDA-MB-231 (BR) Luc2 and the MDA-MB-231 (BR) cells with HER2 overexpression were kindly provided by Dr. Patricia Steeg (NIH, Bethesda, MD, USA) and Dr. Quentin Smith (Texas Tech University Health Sciences Center, Amarillo, TX, USA). These cells were maintained in DMEM supplemented with 10 FBS and 5 PSN [10]. The HER2 overexpressing cells MDA-MB-231 (HH) were maintained in the medium described above in the presence of 300 mg/ml zeocin. All the cells used in this study were within twenty passages after receipt or resuscitation. The cells were maintained and passaged in culture as described by us previously [39].the brains were analyzed by counting quantum dots in a blinded manner (Fig. 1A). Based on the average counts of quantum dots in each mouse brain, our results showed about 50 reduction in brain metastasis of breast cancer cells in PEITC treated group, as compared to controls (Fig. 1D). These results suggest the potential of PEITC in blocking the metastasis of breast cancer cells to brain by inhibiting the seeding capability of metastatic cells.PEITC Suppresses the Growth of Metastasized Breast TumorsAfter observing the reduced metastasis in PEITC treated mice, next step was to see whether PEITC could suppress the growth of metastasized tumors in brain. Two weeks after the breast tumor cells were lodged in the brain and started growing as indicated by the reappearance of luminescence in the brain, mice were treated with 10 mmol PEITC by oral gavage every day for 25 days (Fig. 2A). Our results reveal that the luminescence in the brain was relatively less in mice treated with PEITC, as compared to the control mice over the period of time (Fig. 2A). At the end of the experiment, tumor growth in the brain was suppressed by almost 50?5 by PEITC treatment (Fig. 2B), indicating the tumor growth suppressive effects of PEITC. Interestingly, luminescence was also observed at places other than the brain in the mice indicating that tumor cells metastasized to other sites. However, PEITC treatment substantially blocked the growth of these minor metastatic tumors as well (Fig. 2A). To evaluate the mechanism of the overall inhibitory effects of PEITC on the growth of metastatic breast tumors in the brain, brain sections from control and PEITC treated mice were examined by immunofluorescence. Our results show significantly reduced expressions of HER2 (by 90 ), EGFR (by 50 ) and VEGF (by 60 ) in the brain sections of PEITC treated mice as compared to control mice (Fig. 3). Based on these observations, it is imperative that PEITC not only can block the metastasis but also can suppress the growth of metastasized breast tumors.Transwell Cell Invasion AssayCell invasion was performed according to manufacturer’s instructions in Boyden’s Transwell chamber with 8.0 mm pore size filters (BD Biosciences, San Jose, California, USA) and as described by us earlier [40]. Briefly, cells were serum starved overnight and collected after.Mages as analyzed by IVIS Lumina imaging system at different time points after intracardiac injection of MDA-MB-231 (BR) cells. (D) Average count of quantum dot labeled MDA-MB-231 (BR) 10781694 tumor cells in mice brain of control andSuppression of Brain Metastasis by PEITCPEITC (10 mmol) treated groups. Values are represented as means6SEM. * P,0.05, statistically different when compared with control. At least 150 brain sections from each group were analyzed. doi:10.1371/journal.pone.0067278.gCell CultureHuman breast carcinoma cell lines MDA-MB-231 (BR) Luc2 and the MDA-MB-231 (BR) cells with HER2 overexpression were kindly provided by Dr. Patricia Steeg (NIH, Bethesda, MD, USA) and Dr. Quentin Smith (Texas Tech University Health Sciences Center, Amarillo, TX, USA). These cells were maintained in DMEM supplemented with 10 FBS and 5 PSN [10]. The HER2 overexpressing cells MDA-MB-231 (HH) were maintained in the medium described above in the presence of 300 mg/ml zeocin. All the cells used in this study were within twenty passages after receipt or resuscitation. The cells were maintained and passaged in culture as described by us previously [39].the brains were analyzed by counting quantum dots in a blinded manner (Fig. 1A). Based on the average counts of quantum dots in each mouse brain, our results showed about 50 reduction in brain metastasis of breast cancer cells in PEITC treated group, as compared to controls (Fig. 1D). These results suggest the potential of PEITC in blocking the metastasis of breast cancer cells to brain by inhibiting the seeding capability of metastatic cells.PEITC Suppresses the Growth of Metastasized Breast TumorsAfter observing the reduced metastasis in PEITC treated mice, next step was to see whether PEITC could suppress the growth of metastasized tumors in brain. Two weeks after the breast tumor cells were lodged in the brain and started growing as indicated by the reappearance of luminescence in the brain, mice were treated with 10 mmol PEITC by oral gavage every day for 25 days (Fig. 2A). Our results reveal that the luminescence in the brain was relatively less in mice treated with PEITC, as compared to the control mice over the period of time (Fig. 2A). At the end of the experiment, tumor growth in the brain was suppressed by almost 50?5 by PEITC treatment (Fig. 2B), indicating the tumor growth suppressive effects of PEITC. Interestingly, luminescence was also observed at places other than the brain in the mice indicating that tumor cells metastasized to other sites. However, PEITC treatment substantially blocked the growth of these minor metastatic tumors as well (Fig. 2A). To evaluate the mechanism of the overall inhibitory effects of PEITC on the growth of metastatic breast tumors in the brain, brain sections from control and PEITC treated mice were examined by immunofluorescence. Our results show significantly reduced expressions of HER2 (by 90 ), EGFR (by 50 ) and VEGF (by 60 ) in the brain sections of PEITC treated mice as compared to control mice (Fig. 3). Based on these observations, it is imperative that PEITC not only can block the metastasis but also can suppress the growth of metastasized breast tumors.Transwell Cell Invasion AssayCell invasion was performed according to manufacturer’s instructions in Boyden’s Transwell chamber with 8.0 mm pore size filters (BD Biosciences, San Jose, California, USA) and as described by us earlier [40]. Briefly, cells were serum starved overnight and collected after.