Journal of Cancer 2011, 2 123 Journal of Cancer 2011; 2:123-131 Research Paper In Vivo Imaging of Human Malignant Mesothelioma Grown Orthotopically in the Peritoneal Cavity of Nude Mice Mingqian Feng1, Jingli Zhang1, Miriam Anver2, Raffit Hassan1, Mitchell Ho1 1. Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, 37 Convent Drive, Bethesda, MD 20892, USA; 2. Pathology/Histotechnology Laboratory, SAIC-Frederick, National Cancer Institute, P.O. Box B, Frederick, MD 21702, USA Corresponding author: Dr. Mitchell Ho, Antibody Therapy Unit, Laboratory of Molecular Biology, National Cancer Institute, 37 Convent Drive, Room 5002C, Bethesda, MD 20892-4264. Tel: (301) 451-8727; Fax: (301) 402-1344; E-mail: [email protected] © Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/ licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited. Received: 2011.01.22; Accepted: 2011.03.01; Published: 2011.03.01 Abstract Malignant mesothelioma (MM) causes significant morbidity and mortality in patients. With increasing efforts devoted to developing therapeutics targeting mesothelioma, a xenograft mouse model with in vivo tumor imaging is especially desired for evaluating anti-tumor therapies. In the present study, we fluorescently labeled the NCI-H226 human mesothelioma cell line by a lentiviral vector harboring a luciferase-GFP (Luc/GFP) fusion gene driven by the RNA polymerase II promoter. After single-cell cloning by flow cytometry, a clone (named LMB-H226-GL) that stably expresses high levels of Luc/GFP was obtained. The in vivo tu- morigenicity of Luc/GFP-labeled LMB-H226-GL was determined by using intraperitoneal in- jections of the cells in nude mice. LMB-H226-GL was found to be able to consistently form solid tumors in the peritoneum of mice. Tumor growth and aggressive progression could be quantitated via in vivo bioluminescence imaging. The model exhibited the pathological hall- marks consistent with the clinical progression of MM in terms of tumor growth and spread inside the peritoneal cavity. To evaluate the in vivo efficacy of drugs targeting mesothelioma, we treated mice with SS1P, a recombinant immunotoxin currently evaluated in Phase II clinical trials for treatment of mesothelioma. All the tumor-bearing mice had a significant response to SS1P treatment. Our results showed that this is a well-suited model for mesothelioma, and may be useful for evaluating other novel agents for mesothelioma treatment in vivo. Key words: H226/NCI-H226, mouse xenograft model, in vivo bioluminescence imaging, malignant mesothelioma, mesothelin, monoclonal antibody, immunotoxin, preclinical model Introduction Malignant mesothelioma (MM) is a fatal asbes- Mesothelin is a glycosyl-phosphatidylinositol tos-associated malignancy. It commonly originates (GPI)–anchored cell surface glycoprotein. It was first from the mesothelial cells lining the pleural and peri- identified in 1992 by a mAb generated by the im- toneal cavities, as well as the pericardium and tunica munization of mice with human ovarian carcinoma vaginalis testis [1]. Although combination chemo- cells [3]. The mesothelin gene encodes a 71-kDa pre- therapy with pemetrexed plus cisplatin has been cursor protein that is processed to a 40-kDa mem- shown to be effective, the median overall survival of brane-bound protein and a 31-kDa fragment (a these patients is only 12 months [1]. One of the prom- megakaryocyte-potentiating factor) that is released ising approaches that may improve patient outcome from the cell after furin cleavage [4, 5]. Mesothelin is a involves the use of monoclonal antibodies (mAb) [2]. differentiation antigen whose expression is limited to http://www.jcancer.org Journal of Cancer 2011, 2 124 mesothelial cells lining the body cavity [6, 7]. It is necessary for binding to MUC16 [20]. We have found overexpressed in a variety of cancers including mes- that the MUC16-binding domain can effectively in- othelioma, ovarian cancer, and pancreatic cancer. In hibit heterotypic cancer cell adhesion, indicating that addition, mesothelin is expressed on the surface of the domain is a good candidate for use as a potential lung adenocarcinomas [8, 9]. Recently, our prelimi- antagonist to prevent or treat peritoneal malignant nary studies have shown that mesothelin is a potential tumors. therapeutic target in cholangiocarcinoma (CCA) [10]. Xenograft models are important tools for pre- We have demonstrated strong immunochemical clinical evaluation of SS1P and other an- mesothelin staining in about 30% of the surgically ti-mesothelioma targeted therapies. Recently, Yanag- resected CCA specimens. No mesothelin staining is ihara et al. established an orthotopic implantation present in hepatocelluar carcinoma or normal liver mouse model of pleural mesothelioma [21]. In the tissue. Mesothelin is primarily localized to the cellular present study, we engineered the human mesotheli- plasma membrane and the mature form (~40 kDa) is oma cell line NCI-H226 (named LMB-H226-GL) to expressed at a high level in CCA tissues. We and express very high levels of GFP-luciferase fusion others have shown that mesothelin is shed from tu- proteins (Luc/GFP). Using the resultant mor cells [11, 12]. Given its link to tumor cells, serum LMB-H226-GL cell line, we established an MM model mesothelin has been approved by the U.S. Food and in nude mice. In this model, tumor growth can be Drug Administration for use as a diagnostic bi- precisely measured by non-invasive and very sensi- omarker in MM. tive in vivo bioluminescence imaging. Importantly, the The limited distribution of mesothelin on normal pathological hallmarks of the model were consistent tissues makes it a promising target for antibody-based with the clinical progression of MM in terms of tumor immunotherapy. Pastan and colleagues have devel- growth and spread inside the peritoneal cavity. We oped a recombinant immunotoxin (named SS1P) that further examined the effectiveness of the an- targets mesothelin-expressing tumors. It contains a ti-mesothelin immunotoxin SS1P in our model. We murine SS1 Fv fused to a 38-kDa fragment of Pseudo- found that all of the mice with tumor burdens had monas exotoxin A (PE38) [13]. Two Phase I clinical significant response upon SS1P treatment, as evi- trials of single agent SS1P have been completed at the denced by in vivo imaging and necropsy observance, National Cancer Institute (NCI) , National Institutes which strongly supports the role that SS1P may have of Health (NIH) in Bethesda, MD and is currently as a potent anti-mesothelioma drug candidate. being evaluated in combination with chemotherapy for treatment of patients with mesothelioma [14, 15]. Materials and Methods A chimeric antibody (MORAb-009) containing the Cell lines same murine SS1 Fv specific for mesothelin was also The human mesothelioma cell line NCI-H226 developed and is currently being examined in a Phase was obtained from the American Type Culture Col- II clinical trial for mesothelioma and pancreatic cancer lection (ATCC, Rockville, MD). The cell line was [7, 16]. We have recently generated a high affinity maintained as adherent monolayer cultures in RPMI fully human IgG (named HN1) specific for mesothelin 1640 medium (Invitrogen, Carlsbad, CA) supple- [17]. HN1 kills cancer cells with very strong anti- mented with 10% fetal bovine serum (FBS) (HyClone, body-dependent cell-mediated cytotoxicity. The HN1 Logan, UT), 1% L-glutamine, and 1% penicil- epitope is different from that of SS1. HN1 binds to cell lin-streptomycin (Invitrogen) and incubated in 5% surface-associated mesothelin on human mesotheli- CO2 with a balance of air at 37°C. Cells were har- oma, ovarian cancer, lung adenocarcinoma and pan- vested and the media were changed twice a week. creatic cancer cells. Cells were confirmed to be negative for mycoplasma. Despite such advances in mesothelin-targeted cancer therapy, the biological functions of mesothelin Generation of the LMB-H226-GL cell line remain largely unknown given that there are no ap- The NIH Institutional Biosafety Committee ap- parent abnormalities in mesothelin knockout mice proved the Recombinant DNA Registration Docu- [18]. Nevertheless, mesothelin interacts with mucin ment (RD-08-IX-07) for the following experiment and MUC16 (also known as CA125) [19, 20], indicating animal studies using the LMB-H226-GL cell line. The that mesothelin may play an important role in the lentiviral vectors containing a plasmid (plen- metastatic spread of mesothelin-bearing cancer cells ti4-B1-pPol2-B3-fluc-eGFP-B2) encoding the Luc/GFP in the peritoneal cavity. We have shown that it is were prepared by Viral Technology Laboratory, Ad- primarily the N terminus of the extracellular domain vanced Technology Program, SAIC-Frederick, Inc. as of mesothelin (residues 296–359) that is sufficient and previously described [22]. http://www.jcancer.org Journal of Cancer 2011, 2 125 Lentiviral supernatant was added into a culture D-luciferin (Caliper Life Sciences) in PBS was injected plate containing 20–40% confluent parental NCI-H226 i.p. before imaging. The photometry of the tumor was cells. The plate was incubated at 37°C and the me- calculated by software Living Image 3.1.0, (Caliper dium changed the following day. Two days after in- Life Sciences) and the results were used to generate fection, GFP expression was examined by flow cy- the tumor growth curve. To correlate fluorescence tometry. At day 10, only cells showing the top 5% of intensity and tumor size, tumor dimensions were de- high GFP expression were collected by flow cytome- termined using calipers, and the tumor volume (mm3) try. After single-cell cloning, the highest GFP expres- was calculated by the formula: length x (width) 2 x 0.4. sion clone (LMB-H226-GL) was selected and main- Necropsy and histopathology tained in RPMI 1640 media supplemented with 10% FBS, 1% glutamine, and 1% streptomycin/penicillin.