BMC Cancer 2006, 6:130 http://www.biomedcentral.com/1471-2407/6/130 Table 3: Growth progression of IST-Mes3/2P cell line after orthotopic implantation in nude mice. Days after implantation Mediastinum nodules Pericardium nodules Parietal Pleura nodules Lungs nodules Diaphragms nodules 54 < 101 < 101 < 101 - 61 <102 <102 <102 - - 68 <103 <103 <103 - - 75 <103 <103 <103 - - 82 10–203 10–203 10–203 <102 <102 89 10–203 10–203 10–203 <102 <102 2 × 106 IST-Mes3/2P cells were injected into the pleural cavity of nude mice. At indicated times mice were sacrificed and tumor nodules were counted, measured, removed and frozen for immunohistochemical analysis. Data shown are mean of 5 mice. 1 Nodule diameter ≤ 1 mm; 2 Nodule diameter = 2–3 mm; 3 Nodule diameter ≥ 3. No nodules were found before the days indicated in the table. ton Dickinson, NJ), monoclonal anti-Ber-EP4 (dilution 1:100; NeoMarkers, Fremont, CA), monoclonal anti- cytokeratin 8 and 18 (dilution 1:50; Biomeda, Foster City, CA), monoclonal anti-EMA (dilution 1:500; NeoMarkers, Fremont, CA). monoclonal anti-mesothelioma (HBME-1) (dilution 1:25; NeoMarkers, Fremont, CA), monoclonal anti-podoplanin (dilution 1:50; Serotec, Dusseldorf, Ger- many). Hematoxylin-and-eosin staining 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), ace- tone/chlorophorm 1/1 (5 min), cold acetone (5 min). Slides were then rinsed with water for10 min, stained with Mayer's Hematoxylin Solution for 10 min (Sigma, St. Louis, MO), rinsed with water for 10 min and then stained with eosin 0.5% for 30 sec (Sigma, St. Louis, MO). Slides were then dehydrated through 95% alcohol (5 min.) and 2 changes of absolute alcohol, 5 min each, cleared in 2 changes of xylene (5 min) and finally mounted with xylene based mounting medium. Results Growth of human MPM cells in nude mice Human MPM cell lines were tested for their ability to form tumors after intrapleural and subcutaneous injection in nude mice. Table I summarizes the tumor take rates of these cell lines. Tab. II, and III summarize the growth rate of these cell lines after orthotopic implantation, whereas Fig. 1. shows their growth rate following subcutaneous implantation. IST-Mes2 cells were unable to grow in nude mice. By contrast, 2 × 106 IST-Mes3 or MMB-1 tumor cells had a take-rate of 100 % when injected into the left pleu- ral cavity. The mean survival time was of 72.5 ± 6.4 days for mice injected with MM-B1 cells; 81.5 ± 21.7 days for mice injected with the IST-Mes3 cells and 69.6 ± 14.5 days for mice injected with the IST-Mes3/2P cells. These mice were sacrificed when moribund, or at different times after cell injection as specified in Tab. I, II, III and IV. Animals injected with the IST-Mes3 cells were analyzed when they became dyspneic because of the tumor growth rate varies from animal to animal. These variations were not observed upon in vitro culture of IST-Mes3 tumor as shown by the analysis of the growth rate of IST-Mes3/2P cells. Tumor cells grew on both sides of parietal and vis- ceral pleura, diaphragm and mediastinum; at later times, tumor invasion into lung parenchyma was observed (Fig. 2, 3). No pneumothorax was evidenced in the mice. Pleu- ral effusions as well as lymph node metastasis were noted only in the later stages of orthotopic growth of MM-B1, IST-Mes3 and IST-Mes3/2P cells. MM-B1 and IST-Mes2 cells did not grow when injected subcutaneously. IST- Mes3 cells started to grow only at forty days after tumor cell injection. This lag phase was independent on the number of cells injected (Fig. 1). Immunohistochemical analysis MPM cells were injected into pleura cavity of nude mice and tumor samples were collected for immunohisto- Table 4: Growth progression of MM-B1 cell line after orthotopic implantation in nude mice. Days after implantation Mediastinum nodules Pericardium nodules Parietal Pleura nodules Lungs nodules Diaphragms nodules 38 < 101 < 101 - - - 45 10–201 10–201 - - - 52 10–202 10–202 - - - 59 10–202 10–202 10–202 10–202 - 66 >203 >203 >203 >203 - 73 >203 >203 >203 >203 - 2 × 106 MM-B1 cells were injected into the pleural cavity of nude mice. At indicated times, mice were sacrificed and tumor nodules were counted, measured, removed and frozen for immunohistochemical analysis. Data shown are mean of 5 mice. 1 Nodule diameter ≤ 1 mm; 2 Nodule diameter 2–3 mm; 3 Nodule diameter ≥ 3 mm. No nodules were found before the days indicated in the table. Page 4 of 7 (page number not for citation purposes)