BMC Cancer 2006, 6:130 http://www.biomedcentral.com/1471-2407/6/130 FOirgtuhroeto3pic MPM histology Orthotopic MPM histology. Fig. 3a. Frozen sections of tumors grown into the pleural cavity of nude mice injected with IST-Mes3 cells were stained with an anti-mouse CD31 antibody. Tumors were intensively stained revealing an intri- cate network of microvessels. Bar = 500 µm. Fig. 3b. H&E staining of tumor invading the lung. T: tumor, L: lung. Bar = 100 µm. tions are not observed when ex vivo tumor cells are cul- tured in vitro. The growth rate of MM-B1 cells is reproducible. Tumors develop in all animals with a similar latency and the rate of tumor growth is slow enough to follow the progression of the disease. In this context, a noninvasive imaging methodology would be a useful tool to follow tumor development. IST-Mes2 cells did not grow in nude mice, but, since the epithelioid phenotype is the most common sub-type of mesothelioma, we plan to set up an orthotopic model using different ephitelioid cells. Immunohistochemistry of tumors growing in the nude mice showed that MM-B1 and IST-Mes3 cells maintained the mesothelioma-specific characteristics, thus confirm- ing the validity of the orthotopic model here established. Table 5: Phenotypic characterizzation of MM-B1 and IST-Mes3 cells after orthotopic implantation in nude mice. Marker CEA Calretinin CD15 (LeuM1) Ber-EP4 Cytokeratin 8/18 EMA HBME-1 Podoplanin Cell lines IST-Mes3 - + - - + + + + (membrane) + (membrane) MM-B1 - + - - + + + IST-Mes3 and MM-B1 cells were injected into the pleural cavity of nude mice. At indicated times the mice were sacrificed and tumor nodules were removed and frozen for immunohistochemical analysis of mesothelioma-specific markers. Conclusion Overall, this work describes the biological behavior of human MPM cells injected in the pleural cavity of nude mice. Because of the similarity of this orthotopic model with the human disease, the simplicity of execution and the reproducibility of the results, we propose this model as an useful tool for in vivo MPM studies. Competing interests The author(s) declare that they have no competing inter- ests. Authors' contributions DM performed the orthotopic and subcutaneous implan- tation of cancer cells in nude mice, the autopsy and the immunoassays. AC carried out the cells culture and partic- ipated in the immunohistochemical analysis. GS designed and coordinated the study. PA participated in the coordi- nation of the study and helped to draft the manuscript. SO and RL performed the phenotypic characterization on MPM cells orthotopically implanted in nude mice. All the authors read and approved the final manuscript. Acknowledgements We thanks Roberta Lucciarini, Consuelo Amantini and Maria Rita Rippo for technical advices and help. This work was supported and approved by the Italian Ministry of Health and the University of Camerino (Italy). References 1. Antman KH: Natural history and epidemiology of malignant mesothelioma. Chest 1993, 103:73S-76S. 2. Edwards JG, Swinson DE, Jones JL, Muller S, Waller DA, O'Byrne KJ: Tumor necrosis correlates with angiogenesis and is a predic- tor of poor prognosis in malignant mesothelioma. Chest 2003, 124:1916-1923. 3. Stewart JH, Nguyen DM, Chen GA, Schrump DS: Induction of apoptosis in malignant pleural mesothelioma cells by activa- tion of the Fas (Apo-1/CD95) death-signal pathway. J Thorac Cardiovasc Surg 2002, 123:295-302. 4. Rippo MR, Moretti S, Vescovi S, Tomasetti M, Orecchia S, Amici G, Catalano A, Procopio A: FLIP overexpression inhibits death receptor-induced apoptosis in malignant mesothelial cells. Oncogene 2004, 23:7753-7760. 5. Edwards JG, Cox G, Andi A, Jones JL, Walker RA, Waller DA, O'Byrne KJ: Angiogenesis is an independent prognostic factor in malignant mesothelioma. Br J Cancer 2001, 85:863-868. 6. Catalano A, Romano M, Robuffo I, Strizzi L, Procopio A: Methionine aminopeptidase-2 regulates human mesothelioma cell sur- vival: role of Bcl-2 expression and telomerase activity. Am J Pathol 2001, 159:721-731. 7. Sterman DH, Albelda SM: Advances in the diagnosis, evaluation, and management of malignant pleural mesothelioma. Respirology 2005, 10:266-283. 8. Le Pimpec-Barthes F, Bernard I, Abd Alsamad I, Renier A, Kheuang L, Fleury-Feith J, Devauchelle P, Quintin Colonna F, Riquet M, Jaurand MC: Pleuro-pulmonary tumours detected by clinical and chest X-ray analyses in rats transplanted with mesothelioma cells. Br J Cancer 1999, 81:1344-1350. 9. Colt HG, Astoul P, Wang X, Yi ES, Boutin C, Hoffman RM: Clinical course of human epithelial-type malignant pleural mesothe- lioma replicated in an orthotopic-transplant nude mouse model. Anticancer Res 1996, 16:633-639. 10. Nakataki E, Yano S, Matsumori Y, Goto H, Kakiuchi S, Muguruma H, Bando Y, Uehara H, Hamada H, Kito K, Yokoyama A, Sone S: Novel orthotopic implantation model of human malignant pleural mesothelioma (EHMES-10 cells) highly expressing vascular Page 6 of 7 (page number not for citation purposes)