BMC Cancer 2006, 6:130 http://www.biomedcentral.com/1471-2407/6/130 Table 1: Tumorigenicity of MPM cell lines after orthotopic and subcutaneous implantation in nude mice. Cell line IST-Mes2 IST-Mes3 IST-Mes3/2P MM-B1 Histology Tumorigenicity Implantation method Survival(days) (mean ± SD) Survival (days) (Min/Max) Epithelioid Epithelioid Biphasic Biphasic Biphasic Biphasic Biphasic 0/15 0/15 15/15 15/15 15/15 15/15 0/15 Orthotopic Subcutaneous Orthotopic Subcutaneous Orthotopic Orthotopic Subcutaneous - - 81,5 ± 21,7 Sacrificed after 90 days 69,6 ± 14,5 72,5 ± 6,4 - - - 60/122 - 54/89 68/77 - 2 × 106 MPM cells were injected into the pleural cavity of nude mice. The experiment ended when mice were moribund. Autopsy and histology Mice were sacrificed at different times following tumor cell injection as specified. Tumor nodules were counted, measured with the caliper, removed, immediately snap frozen in liquid nitrogen and stored at -80°C. The presence of metastasis in the visceral organs was macroscopically checked. Immunohistochemistry Microvessel density (MVD) was determined by using the endothelial cell marker CD31. Tumors were placed in OTC compound and snap frozen in liquid nitrogen and stored at -80°C. Frozen section (10–20 µm) were fixed with cold acetone (5 min), acetone/chlorophorm 1/1 (5 min) and cold acetone (5 min). Samples were then rinsed with PBS/Triton 1%, and treated with 3% hydrogen per- oxide in methanol (vol/vol). Slides were incubated in a blocking solution and then overnight at 4°C in a humid- ified chamber with a rat anti-mouse CD31 monoclonal antibody (BD Biosciences Pharmingen, NJ, USA). Thereaf- ter, slides were rinsed with PBS and incubated, first with the blocking solution for 20 min and then with a biotin- conjugated goat anti-rat antibody (Santa Cruz Biotechnol- ogy, CA, USA) for 1 h. Slides were then rinsed with PBS and incubated for 30 min with the Vector Vectastain ABC Kit (Vinci-Biochem, Vinci, FI). After 3 washes with PBS, positive reactions were visualized by incubating the slides for 5 min with stable DAB (Sigma, Italy). Slides were dried and mounted with Universal Mount. For mesothelioma markers, 3–4 µm paraffin tumor sec- tions were float mounted on poly-L lysine coating slides, deparaffinized in xylene and rehydrated in a descending ethanol series. Anti-Ber-EP4 monoclonal antibody required enzymatic pre-treatment of the slides with 0.1% trypsin (Sigma, St. Louis, MO) for 10 min at 37°C; for the other antibodies, slides were not enzimatically pre- treated, but they were placed in 0.1 M citrate buffer (pH 6.0) and boiled for 10 min in a microwave oven at 750 W to enhance antigenicity. Slides were washed in phosphate buffer and incubated for 10 min in 0.3% hydrogen perox- ide to quench endogenous peroxidase activity. Slides were then loaded onto a LabVision automated immunostainer (NeoMarkers, Fremont, CA) and sequentially incubated with intervening washes in PBS for 5 min, with 10% oval- bumin for 15 min to reduce non-specific background staining, primary antibody for 60 min at room tempera- ture, the appropriate biotinylated linking antibody (Neo- Markers, Fremont, CA) for 10 min, peroxidase-labeled streptavidin (NeoMarkers, Fremont, CA) for 10 min, and finally with 3,3'-diaminobenzidine chromogen substrate for 10 min. Slides were then thoroughly rinsed in distilled water and counterstained with Mayer's haematoxylin, dehydrated, cleared in xylene and finally mounted in Entellan. Appropriate positive and negative controls were included for each marker. A panel of eight markers was used. Mouse anti-human antibodies included anti-CEA (dilution 1:100; NeoMarkers, Fremont, CA), polyclonal anti-calretinin (dilution 1:1000; NeoMarkers, Fremont, CA), monoclonal anti-CD15 (Leu-M1) (dilution 1:4; Bec- Table 2: Growth progression of IST-Mes3 cell line after orthotopic implantation in nude mice. Days after implantation N° of animals with tumor nodules1 N° of tumor nodules detected in each animal <56 0/5 - 60 1/5 >20 65 2/5 >20 71 1/5 >20 73 1/5 >20 80 0/5 - 87 1/5 >20 94 1/5 >20 98 2/5 >20 2 × 106 IST-Mes3 cells were injected into the pleural cavity of nude mice. Five animals were sacrificed when they became dyspneic or at indicated times and tumor nodules were counted, measured, removed and frozen for immunohistochemical analysis. Data shown report tumor development in individual animals. Page 3 of 7 (page number not for citation purposes)