Patient characteristics.

<p>Patient characteristics.</p

PGK1 and CXCR4 expression, proliferation and inhibition of CXCR4 of neuroblastoma cell lines.

<p>Kelly (<b>A</b>) and SH-EP Tet-21/N (<b>B</b>) neuroblastoma cells were immunostained for PGK1 and CXCR4 expression (<b>Immunohistochemistry</b>). Both cell lines show a positivity for CXCR4 and react to treatment with 20 µg AMD3100 with an inhibition of proliferation (<b>MTT-assay</b>), although only SH-EP Tet-21/N cells reach a significant level of growth reduction. On examination of PGK1 protein expression levels (<b>Western blot</b>) after 48 h of CXCR4 receptor inhibition, treatment with 20 µg AMD3100 leads to a downregulation of PGK1 protein (45 kDa) in SH-EP Tet-21/N but not in Kelly cells. Tubulin (55 kDa) served as control.</p

Overall Survival.

<p>For Kaplan-Meier survival analysis patients were grouped according to positive and negative PGK1 expression. All patients that died during the follow-up period showed positive PGK1 expression, while none of the PGK1 negative patients died. Overall survival of neuroblastoma patients with a PGK1 negative expression was significantly better than that of PGK1 positive patients (<i>p = 0.003</i>).</p

Metastatic sites.

<p>Metastatic sites.</p

Patient collective.

<p>Characteristic of 202 patients that were evaluated for CXCR4 and HER2 expression.</p

Figure 3

<p><b>A</b> CXCR4-expression of OE19 cells determined by fluorescence immunostaining (IgG1-control) <b>B</b> Confirmation of <i>Her2</i>-amplification determined by fluorescence in situ hybridization (red: <i>Her2</i>-gene loci, green reference <i>CENT-17</i>-loci) <b>C </b><i>CXCR4</i> and <i>HER-2</i> mRNA-expression analysis of esophageal cancer cell line OE19 compared to MDA-MB-231 and SKBr-3 cell lines and null control (nc). <b>D</b> CXCR4 and HER2 expression level analysis determined by immunostaining in primary tumor, liver, lung and lymph node. Representative images are shown from the tissues of an untreated animal (magnification ×100). <b>E</b> Intensity of HER2- and CXCR4-expression was scored in primary tumor and metastases. Positivity-scores of primary tumor and respective metastases were matched to evaluate the occurrence of and correlation of primary tumor expression and that of its respective metastases between the therapeutic groups. Trastuzumab treatment led to an absence of metastases and thus could not be included. <i>* Due to space limitations, AMD3100 was abbreviated to AMD in </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047287#pone-0047287-g003" target="_blank"><i>Figure 3e</i></a><i>.</i></p

HER2- and CXCR4-receptor expression.

<p>Expression summary of HER2 and CXCR4 in human esophageal carcinoma patients with positive correlation (p = 0.036).</p><p>For simplified presentation high receptor expression in this table <b>is indicated by (+), all other expression levels by (−).</b></p

Figure 2

<p><b>A</b> 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>B</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). <b>C</b> Micrometastases in liver and lung after treatment with AMD3100 and trastuzumab, were analysed by real-time PCR according to the level of human <i>gapdh</i>. AMD3100 and trastuzumab-treated mice showed with a mean delta-ct-value of −2 and −3 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 −3. The AMD3100/trastuzumab combination group had a reduced rate of lung (delta-ct −2), and liver (delta-ct −3) metastasis. <b>D</b> Disseminated tumor cells were detected by cytokeratin and HER2 immunhistochemical staining. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047287#pone-0047287-g003" target="_blank">Figure 3B</a> shows a bone marrow sample. Human cell with a strong positivity for HER2 is detectable (red). <i>* Due to space limitations, AMD3100 was abbreviated to AMD in </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047287#pone-0047287-g002" target="_blank"><i>Figures 2a and c</i></a><i>.</i></p

Mean Body Weight, Tumor Weight and Volume of Mice.

<p>Summary of mean body weights of mice at the beginning of treatment and at the termination of the experiment. No significant differences between treatment groups were seen. Tumor weights and tumor volumes are summarized for each treatment group. A positive correlation of tumor weight and volume was noted (correlation coefficient: 0.837, p<0.01).</p

Figure 1

<p><b>A</b> Effect of trastuzumab and ADM3100 treatment on proliferation (%) of OE19 cells compared to control in the lactate-dehydrogenase assay. Receptor inhibition leads to reduced proliferation of cells. It shows a significant reduction of cell proliferation under HER2- and CXCR4-receptor inhibition after treatment with trastuzumab (p = 0.005) as well as with AMD3100 (p = 0.02) compared to the untreated control. <b>B</b> Microscopic evaluation shows dose-dependent effect of SDF-1α-stimulated cell migration on OE19 cells. <b>C</b> A relevant effect of SDF-1α on cell migration is observed at 250 ng/ml compared to unstimulated cells (control).</p