BMC Cancer 2006, 6:130 http://www.biomedcentral.com/1471-2407/6/130 SFuigbucurtean1eous growth of MPM cells Subcutaneous growth of MPM cells. Subcutaneously growth of MPM cells in nude mice. = IST-Mes3 (2 × 106 cells injected), ■ = IST-Mes3 (4 × 106 cells injected), ▲ = IST-Mes2 (2 × 106 cells injected), ● = MM-B1 (2 × 106 cells injected). Tumor volume was calculated using the formula: V (mm3) = (D × d2)/2, where d (mm) and D (mm) are the smallest and largest perpendicular tumor diameters, respec- tively. chemical analysis. CD31 staining revealed that tumor mass was highly vascularized (Fig. 3). For the phenotypic characterization of tumor mass, a panel of eight markers was used (Tab. 5). Recent findings showed that Mesothe- lin and TTF-1 have a limited value in assisting in the diag- nosis of Mesothelioma and therefore were not used [13,14]. CEA and BerEP4 are adenocarcinoma specific markers and therefore are nor expressed by mesotheli- oma. Calretinin and HBME-1 are positive markers shared PFlieguurraelM2PM nodules Pleural MPM nodules. IST-Mes3 cells (2 × 106 in 100 µl of HBSS) were injected into the left pleural cavity of nude mice. Tumor masses completely invaded the thoracic and visceral pleural and the lungs. The irregular growth of this cell line is evidenced by a major bulk of tumor in the right chest. by both epithelioid and biphasic variants of mesotheli- oma, whereas CD15 is a negative marker. Surface expres- sion of the epithelial membrane antigen (EMA) is a marker that discriminates between reactive proliferation of mesothelial cells and malignant mesothelioma. The epithelia of biphasic mesotheliomas show strong reactiv- ity for Cytokeratin 8/18, whereas Podoplanin is the most recently recognized marker for epithelioid mesothelio- mas. Discussion New strategies for MPM treatment, which include inhibi- tion, of angiogenesis, induction of tumor cell apoptosis, gene therapy and vaccines [15], need clinically represent- ative animal models to test new drugs and explore tumor biology. In this work we established a non invasive orthotopic model of human MPM by injecting the cancer cells directly into the pleural cavity of nude mice. The charac- teristics of tumor growth resemble those observed in tumor-bearing patients, with colonization of parietal and visceral pleura, diaphragm, mediastinum and, at later stages, lung parenchyma. We analyzed the tumorigenicity of three different human MPM cell lines, which exhibited different biological behaviors in vivo. Interestingly, the growth of MM-B1 cells seems to be site-specific as they did not form tumor masses when injected subcutaneously. Differently, the IST-Mes3 cells grow subcutaneously with a latency which does not depend on the number of cells injected (Fig. 1). This resembles some human situations in which cancer cells start to grow exponentially and form manifest tumor masses only after a long period of time from the acquisition of the tumorigenic phenotype. As previously reported, neo-angiogenesis is an important process in MPM and protocols to inhibit this process are currently under investigation. Our model can be useful to study angiogenesis in MPM as tumors displayed high den- sity of micro-vessels. IST-Mes3 tumor grew with a latency of 60 days and this period remained substantially con- stant after tumor passage in mice. All the cell lines tested were unable to form metastases in the visceral organs, likely due to lack of time for tumor dissemination as the rapid spreading of the tumor cells into the pleural cavity dramatically resulted in mouse death. Among the cell lines tested, the MM-B1 cells seem to be the more appro- priate for drug evaluation. In fact, as shown in Tab. II, the IST-Mes3 cells grow very rapidly to follow the progression of the disease, colonizing the entire pleural cavity and dra- matically affecting the physical conditions of the mice which become cachectic and dyspneic, and the latency of tumor takes varies from animal to animal. The results obtained with the IST-Mes3/2P cell show that these varia- Page 5 of 7 (page number not for citation purposes)