BMC Cancer 2006, 6:130 http://www.biomedcentral.com/1471-2407/6/130 Background Malignant pleural mesothelioma is a tumor of the pleura mainly caused by exposure to asbestos fibers. MPM diag- nosis regards about 2500 persons every year in the United States [1] and the prognosis is poor despite the therapies currently used, including surgery, radiotherapy and chem- otherapy. Because of the inefficacy of the conventional treatments, novel therapeutic strategies are under investigation, with particular attention devoted to agents capable of inhibit- ing the angiogenesis or inducing tumor cell apoptosis [2- 7]. Tumor angiogenesis, apoptosis and metastasis, strictly depend on the site of tumor development; therefore a human-like animal model is an important tool for study- ing new approaches for MPM treatment. A number of evidences indicate that orthotopic models of tumor growth are more valuable as compared to those in which the tumor mass grows subcutaneously [8]. In regard to MPM, subcutaneous implantation of human cancer cells in immunodeficient mice results in tumor growth at the injection site and no metastatic dissemina- tion, whereas human MPM growth in humans is associ- ated with regional tumor spreading and lung invasion [9]. Presently, human MPM cells have been grow orthotopi- cally in mice upon transplantation of tumor masses or tumor cell suspensions following surgery [10]. In these models however, surgery can interfere with the tumor growth [8] and the early stages of tumor development cannot be easily explored. Finally, results may be not so accurate due to implantation of potentially different tumor samples in different experimental groups [9]. Over- all, the aim of this work was to establish a new orthotopic tumor model by injecting human MPM cells directly into the pleural cavity of nude mice. This model does not need surgical operations, can be easily performed and more importantly can mimic tumor development in humans. Thus it can represent an useful tool for studying human MPM biology and assessing the efficacy of novel thera- pies. Methods Cell lines Human malignant pleura mesothelial cell lines were established as previously described [11,12]. Three distinct cell lines with two different phenotypes, biphasic (MM-B1 and IST-Mes3) and epithelioid (IST-Mes2) were used between the eighth and twelfth passage in culture. Cells were maintained in RPMI-1640 medium and 10% fetal bovine serum, 1% L-glutamine and 1% penicillin-strepto- mycin, (Euroclone, Devon, UK) at 37°C in a humidified incubator in an atmosphere of 5% CO2 in air. Animals Athymic male nude mice nu/nu (Harlan, Italy), 6 week- old were used. Mice were kept in laminar-flow cages in standardized environmental conditions. Sterilized food (Harlan, Italy) and water were supplied ad libitum. Subcutaneous implantation of MPM cells in nude mice Cells were harvested at near confluence with trypsin/ EDTA solution. Only cell suspensions with a viability of >90% as assessed by trypan blue exclusion assay, were used. Two × 106 MPM cells in 100 µl of Ca++ and Mg++ free Hank's balanced salt solution (HBSS) were injected subcutaneously on the left lateral chest wall near to the axilla. Tumor growth was monitored twice a week using a caliper. 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 diameter, respectively. Orthotopic implantation of MPM cells in nude mice Mice were anaesthetized with Tiletamine chlorohydrate and Zolazepam chlorohydrate and placed in position of right lateral decubitus. A 27 gauge needle of a 1000 µl syringe was advanced approximately through the fourth intercostal space for about 5 mm, into the left pleural cav- ity, and two × 106 tumor cells suspended in 100 µl of HBSS were injected. The site of injection in the chest and the precise depth of the needle tip required to reach the pleural space were previously determined by injecting cre- syl violet. A primary tumor cell line IST-Mes3/2P (IST- Mes3/2nd Passage) was established from a IST-Mes3 tumor grown orthotopically. Tumors were grounded into small pieces in RPMI-1640 and digested with trypsin/ EDTA solution. The cell suspension was then plated in a 75 ml culture flask and the following day, non adherent cells were removed. Cells were maintained in culture, and 2 × 106 cells were injected into the pleural cavity of nude mice when devoid of fibroblasts or endothelial cells. Therapeutic procedures Four groups of animals were used. The first group of ani- mals were used to analyze the tumorigenicity of MPM cell lines after orthotopic or subcutaneous implantation in nude mice. Animals were then injected with MPM cells as reported in Table 1. The second, third and fourth groups were used to analyze the growth rate of IST-Mes3, IST-Mes3/2P and MM-B1 cells respectively after orthotopic implantation in mice. Page 2 of 7 (page number not for citation purposes)