Er scores throughout. When statistically comparing the HER2 expression of primary tumor and metastases between therapeutic groups, a significantly higher HER2-expression was observed in the trastuzumab-treated group (p = 0.003) 
and the AMD3100-treated group (p = 0.003) compared to the control group. Upon examination of CXCR4expression, a significantly higher expression was observed in the trastuzumab-treated group (p = 0.003) and a higher expression in the AMD3100-treated group (p = 0.065) compared to the control group. The combined therapy group (trastuzumab/AMD3100) neither showed significant HER2-expression or CXCR4-expression differences compared to the control group.CXCR4 and HER2 expression profile of OE19 cells in vivoFirstly, OE19 cells were examined for their expression of CXCR4 and HER2. Not only the HER2-overexpression but also the amplification of its gene is of clinical relevance, thus Her2amplification status was verified. OE19 cells showed 22948146 a strong expression of CXCR4- and HER2-receptors in immunostaining (Figure 3A) as well as an amplification of the Her2-gene in FISH (Figure 3B). MedChemExpress BI 78D3 Semiquantitive mRNA analysis showed expression 
of CXCR4 and Her2 compared with MDA-MB-231 and SKBr-3 cell lines (Figure 3C).Correlation of CXCR4- and HER2-expression in the orthotopic in vivo modelSecondly, primary tumor and metastatic tissues from the orthotopic model were examined for CXCR4- and HER2expression. CXCR4- and HER2-expression was observed in all tumor bearing-tissues, including primary tumor, liver, lung and lymph node metastases (Figure 3D). HER2-expression correlated significantly with CXCR4-expression (correlation efficient 0.490, p,0.01).CXCR4 in HER2-Positive Esophageal CancerCXCR4 in HER2-Positive Esophageal CancerFigure 2. A Significant differences in tumor weights between control and trastuzumab-treated groups (p,0.00), control and combination trastuzumab/AMD3100-treated groups (p,0.00), trastuzumab and AMD3100-treated groups (p = 0.04), and AMD and combination trastuzumab/ AMD3100-treated groups (p = 0.02). Although the effect of AMD3100 on the primary tumor weight was not as relevant as the effect of trastuzumab, a potent effect was achieved by AMD3100 treatment alone, compared to the untreated group. B MRI-based tumor volumetry confirmed the results of tumor weight. The tumor weights at time of autopsy correlated significantly with the volumetric measure by MRI (correlation coefficient: 0.837, p,0.01). C Micrometastases in liver and lung after treatment with AMD3100 and trastuzumab, were analysed by real-time PCR according to the level of human gapdh. AMD3100 and trastuzumab-treated mice showed with a mean delta-ct-value of 22 and 23 strong reductions in lung metastasis of 75 to nearly 100 . Additionally the trastuzumab-treated mice had a strong reduction in liver metastasis represented by a mean delta-ct-value of 23. The AMD3100/trastuzumab combination group had a reduced rate of lung (delta-ct 22), and liver (delta-ct 23) metastasis. D Disseminated tumor cells were detected by cytokeratin and HER2 immunhistochemical staining. Figure 3B shows a bone marrow sample. Human cell with a strong positivity for HER2 is detectable (red). * Due to space limitations, AMD3100 was KDM5A-IN-1 site abbreviated to AMD in Figures 2a and c. doi:10.1371/journal.pone.0047287.gValidation of CXCR4- and HER2-coexpression in human esophageal carcinomaTo further validate the correlation of HER2 and CXCR4 that was found in the in vivo studies, primary tumor ti.Er scores throughout. When statistically comparing the HER2 expression of primary tumor and metastases between therapeutic groups, a significantly higher HER2-expression was observed in the trastuzumab-treated group (p = 0.003) and the AMD3100-treated group (p = 0.003) compared to the control group. Upon examination of CXCR4expression, a significantly higher expression was observed in the trastuzumab-treated group (p = 0.003) and a higher expression in the AMD3100-treated group (p = 0.065) compared to the control group. The combined therapy group (trastuzumab/AMD3100) neither showed significant HER2-expression or CXCR4-expression differences compared to the control group.CXCR4 and HER2 expression profile of OE19 cells in vivoFirstly, OE19 cells were examined for their expression of CXCR4 and HER2. Not only the HER2-overexpression but also the amplification of its gene is of clinical relevance, thus Her2amplification status was verified. OE19 cells showed 22948146 a strong expression of CXCR4- and HER2-receptors in immunostaining (Figure 3A) as well as an amplification of the Her2-gene in FISH (Figure 3B). Semiquantitive mRNA analysis showed expression of CXCR4 and Her2 compared with MDA-MB-231 and SKBr-3 cell lines (Figure 3C).Correlation of CXCR4- and HER2-expression in the orthotopic in vivo modelSecondly, primary tumor and metastatic tissues from the orthotopic model were examined for CXCR4- and HER2expression. CXCR4- and HER2-expression was observed in all tumor bearing-tissues, including primary tumor, liver, lung and lymph node metastases (Figure 3D). HER2-expression correlated significantly with CXCR4-expression (correlation efficient 0.490, p,0.01).CXCR4 in HER2-Positive Esophageal CancerCXCR4 in HER2-Positive Esophageal CancerFigure 2. A Significant differences in tumor weights between control and trastuzumab-treated groups (p,0.00), control and combination trastuzumab/AMD3100-treated groups (p,0.00), trastuzumab and AMD3100-treated groups (p = 0.04), and AMD and combination trastuzumab/ AMD3100-treated groups (p = 0.02). Although the effect of AMD3100 on the primary tumor weight was not as relevant as the effect of trastuzumab, a potent effect was achieved by AMD3100 treatment alone, compared to the untreated group. B MRI-based tumor volumetry confirmed the results of tumor weight. The tumor weights at time of autopsy correlated significantly with the volumetric measure by MRI (correlation coefficient: 0.837, p,0.01). C Micrometastases in liver and lung after treatment with AMD3100 and trastuzumab, were analysed by real-time PCR according to the level of human gapdh. AMD3100 and trastuzumab-treated mice showed with a mean delta-ct-value of 22 and 23 strong reductions in lung metastasis of 75 to nearly 100 . Additionally the trastuzumab-treated mice had a strong reduction in liver metastasis represented by a mean delta-ct-value of 23. The AMD3100/trastuzumab combination group had a reduced rate of lung (delta-ct 22), and liver (delta-ct 23) metastasis. D Disseminated tumor cells were detected by cytokeratin and HER2 immunhistochemical staining. Figure 3B shows a bone marrow sample. Human cell with a strong positivity for HER2 is detectable (red). * Due to space limitations, AMD3100 was abbreviated to AMD in Figures 2a and c. doi:10.1371/journal.pone.0047287.gValidation of CXCR4- and HER2-coexpression in human esophageal carcinomaTo further validate the correlation of HER2 and CXCR4 that was found in the in vivo studies, primary tumor ti.