And Tissue Staining Kit, using the HRP-AEC-System, from R DSystems (Minneapolis, MN, USA). Sections were counterstained with Mayer’s hematoxylin solution (Merck). Tumor tissue was identified by hematoxylin eosin (HE) staining. Immunostaining was scored by one pathologist (U.R.) and a second independent examiner (A.D.).following a four-step scale (0,1,2,3) according to the manufacturer’s directionsFluorescence in situ hybridization (FISH)HER2 FISH analysis was performed using the HER2FISH pharmDxTM Kit (DAKO, 1676428 Glostrup, Denmark) according to the manufacturers protocol.Materials and Methods Cell Line, cell proliferation, and cell migration in vitroThe human cell line OE19 (European Collection of Cell Cultures (ECACC), Health Protection Agency, Wiltshire, UK) was cultured in RPMI1560 medium (Biochrome KG, Berlin, Germany) as previously described [25]. Cell proliferation was measured using the LDH Cytotoxicity Kit (PromoKine, Heidelberg, Germany). 50000 OE19 cells were seeded into a 24-well plate and grown overnight. AMD3100 (Sigma-Aldrich, Munich, Germany) was supplemented to the culture medium and cell vitality was analysed after 48 hours. Tumour cell migration through a microporous membrane was assessed based on the Boyden chamber principle. Cells were incubated with culture medium for 90 min, and then plated onto the top chamber. Culture medium containing 500 ng/ml of recombinant human SDF-1a (R D Systems, Mineapolis, USA) was added into the lower chamber. The plate was incubated at 37uC, 5 CO2 for 18 hrs. The migrated cells were stained using DAPI (Sigma-Aldrich, Munich, Germany) and counted under a fluorescence microscope (Carl Zeiss, Jena, Germany).Detection of micrometastasesTotal RNA was isolated from liver and lung samples with an RNA isolation kit (60940-34-3 Qiagen, Hilden, Germany) and reverse transcribed with a high-capacity cDNA reverse-transcription kit (Applied Biosystems). Micrometastases were detected by mRNA expression of the human gapdh gene by real-time PCR analysis. Results were normalised using 18S RNA expression of the tissue samples. PCR primers (TaqMan Gene Expression Assay Gapdh human Hs99999905_m1, Partnumber 4351370, TaqMan Gene Expression Assay 18S Hs99999901_s1) and TaqMan Universal PCR Mastermix were obtained (Applied Biosystems). Micrometastases data are presented as delta-ct-values.Detection of disseminated tumor cells in bone marrowBone marrow was sampled from the femur of mice at the time of sarifice and isolated by density gradient as previously described [39]. Slides with bone marrow cells were immunocytochemically assessed for disseminated tumor cells using the monoclonal antihuman anticytokeratin antibody AE1/AE3 (Dako, Glostrup, Denmark) labeled with fluorochrome FITC and anti-HER2 monoclonal antibody NCL-CB11 (Novocastra Reagents and Antibodies, Leica Microsystems, Wetzlar, Germany) according to the manufacturers protocols. After staining, slides were covered with Vectashield Mounting Medium containing Dapi (Vector Laboratories, Burlingame, CA).Tumor model and therapeutic treatmentNMRI/nu (U.S. Naval Medical Research Institute) mice were
obtained from Charles River Deutschland (Sulzfeld, Germany) at 10 weeks of age. All animal Alprenolol custom synthesis procedures were performed in accordance with a protocol approved by the Behorde fur ??Wissenschaft und Gesundheit (Freie und Hansestadt Hamburg, Germany). The esophageal carcinoma implantation model was obtained as previously described [25,37,38]. Mice were weighed and examined for tumor.And Tissue Staining Kit, using the HRP-AEC-System, from R DSystems (Minneapolis, MN, USA). Sections were counterstained with Mayer’s hematoxylin solution (Merck). Tumor tissue was identified by hematoxylin eosin (HE) staining. Immunostaining was scored by one pathologist (U.R.) and a second independent examiner (A.D.).following a four-step scale (0,1,2,3) according to the manufacturer’s directionsFluorescence in situ hybridization (FISH)HER2 FISH analysis was performed using the HER2FISH pharmDxTM Kit (DAKO, 1676428 Glostrup, Denmark) according to the manufacturers protocol.Materials and Methods Cell Line, cell proliferation, and cell migration in vitroThe human cell line OE19 (European Collection of Cell Cultures (ECACC), Health Protection Agency, Wiltshire, UK) was cultured in RPMI1560 medium (Biochrome KG, Berlin, Germany) as previously described [25]. Cell proliferation was measured using the LDH Cytotoxicity Kit (PromoKine, Heidelberg, Germany). 50000 OE19 cells were seeded into a 24-well plate and grown overnight. AMD3100 (Sigma-Aldrich, Munich, Germany) was supplemented to the culture medium and cell vitality was analysed after 48 hours. Tumour cell migration through a microporous membrane was assessed based on the Boyden chamber principle. Cells were incubated with culture medium for 90 min, and then plated onto the top chamber. Culture medium containing 500 ng/ml of recombinant human SDF-1a (R D Systems, Mineapolis, USA) was added into the lower chamber. The plate was incubated at 37uC, 5 CO2 for 18 hrs. The migrated cells were stained using DAPI (Sigma-Aldrich, Munich, Germany) and counted under a fluorescence microscope (Carl Zeiss, Jena, Germany).Detection of micrometastasesTotal RNA was isolated from liver and lung samples with an RNA isolation kit (Qiagen, Hilden, Germany) and reverse transcribed with a high-capacity cDNA reverse-transcription kit (Applied Biosystems). Micrometastases were detected by mRNA expression of the human gapdh gene by real-time PCR analysis. Results were normalised using 18S RNA expression of the tissue samples. PCR primers (TaqMan Gene Expression Assay Gapdh human Hs99999905_m1, Partnumber 4351370, TaqMan Gene Expression Assay 18S Hs99999901_s1) and TaqMan Universal PCR Mastermix were obtained (Applied Biosystems). Micrometastases data are presented as delta-ct-values.Detection of disseminated tumor cells in bone marrowBone marrow was sampled from the femur of mice at the time of sarifice and isolated by density gradient as previously described [39]. Slides with bone marrow cells were immunocytochemically assessed for disseminated tumor cells using the monoclonal antihuman anticytokeratin antibody AE1/AE3 (Dako, Glostrup, Denmark) labeled with fluorochrome FITC and anti-HER2 monoclonal antibody NCL-CB11 (Novocastra Reagents and Antibodies, Leica Microsystems, Wetzlar, Germany) according to the manufacturers protocols. After staining, slides were covered with Vectashield Mounting Medium containing Dapi (Vector Laboratories, Burlingame, CA).Tumor model and therapeutic treatmentNMRI/nu (U.S. Naval Medical Research Institute) mice were obtained from Charles River Deutschland (Sulzfeld, Germany) at 10 weeks of age. All animal procedures were performed in accordance with a protocol approved by the Behorde fur ??Wissenschaft und Gesundheit (Freie und Hansestadt Hamburg, Germany). The esophageal carcinoma implantation model was obtained as previously described [25,37,38]. Mice were weighed and examined for tumor.
