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This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.
Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9–18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.
Malignant pleural mesothelioma (MPM) is a cancer most often associated with the exposure to asbestos fibers1,2,3. Although asbestos has been banned in most Western countries4,5,6, the incidence of MPM is still increasing7,8. Recently, exposure of mice to carbon nanotubes suggests that they may result in significant health risk in humans9,10. The data suggest that exposure to these products may induce chronic inflammation and molecular changes (e.g., loss of tumor-suppressor pathways) that underlie progression to malignant mesothelioma. Currently, multiwall carbon nanotubes are one of the most important products of nanotechnology and are increasingly incorporated in various products such as composites, energy storage materials, medicine, electronics, and environmental remediation materials.
MPM is a cancer with poor prognosis, and most patients die within two years after diagnosis due to a limited efficacy of current treatment modalities11. The choice of the treatment for MPM depends on the cancer stage. For most early-stage MPM (stage 1 and possibly some stage 2 or 3 tumors), the clinical approach is a multimodal therapy including the surgical resection of the tumors, associated to radiotherapy and chemotherapy12. A combined chemotherapy with cisplatin and pemetrexed is indicated for the treatment of most patients diagnosed with advanced locally invasive disease, that is not amenable to surgical resection, or who are otherwise not candidates for curative surgery13,14. There is, therefore, an urgent need to develop more effective treatments for MPM patients. However, there are few validated in vivo animal models that reflect the clinical relevancy of MPM. Several murine MPM models have been developed but most of them do not faithfully recapitulate the complex aspects of the MPM tumor microenvironment15,16,17,18. The use of asbestos-induced MPM in mice, genetically engineered MPM mouse models, or models of syngeneic transplantation of murine MPM cell lines are limited by fundamental phenotypic and functional differences and, consequently, poorly translate new discoveries to the clinic. Other preclinical murine MPM models mostly rely on subcutaneous or peritoneal xenografts of human cell lines in immunodeficient mice. While these models are easy to monitor and provide fundamental data, the microenvironment of these xenografts is not that comparable to human tumors impairing the translational power of most of these preclinical studies17,19. Conversely, orthotopic xenografts better reflect the patient tumor behavior and response to treatment as they are surrounded with a similar microenvironment as the one found in the original tumor site16.
Molecular imaging by [18F]FDG-PET/CT is the method of choice to longitudinally monitor disease progression in patients with MPM20,21. Therefore, resorting to this non-invasive imaging method greatly promotes the translation of preclinical studies to clinical trials16,22. Moreover, it helps to reduce the required number of animals as each animal represents its own control over time.
In this article, we present a reliable orthotopic xenograft MPM model obtained after injection of the human MPM cell line H2052/484 into the pleural cavity of athymic mice. Coupled with [18F]FDG-PET/CT imaging, this model is a valuable and reproducible method to study functional and mechanistic effects of new diagnostic strategies and treatments for human MPM.
All the procedures described below were approved by the institutional animal care and use committee and by the veterinarian state office of Geneva, Switzerland (Authorization GE/106/16). The MPM cell line H2052/484 was established and characterized in our laboratory as detailed in the article of Colin DJ and et al.23. Briefly, H2052/484 cell line was established from a thoracic tumor obtained after an intrapleural injection of NCI-H2052 (ATCC) cells into immunodeficient Nude mice.
1. Experimental Design
2. Preparation of Cells for Implantation
3. Tumor Cell Implantation
4. [18F]FDG-PET/CT Imaging
NOTE: All the procedures described below must be approved by local animal housing and imaging facilities. Make sure that radioactive materials are imported, stored and handled according to local radiation safety rules (e.g., stock solutions activity, shielded hood handling). SPF conditions can be maintained by manipulating animals in a laminar flow hood and by loading them in SPF-compatible scanner bed (Figure 1B, C).
5. Analyses of [18F]FDG-PET/CT Scans
The H2052/484 orthotopic model
Orthotopic MPM models by intra-thoracic injection of cultured cancer cells, especially H2052/484 cells are relatively easy to setup. The different steps described above only require modest cell culture knowledge and the surgery steps are accessible to moderately trained animal experimenters. Nude mice and cells should be manipulated under sterile conditions to maximize the outcome of the implantations. By carefully following this protocol, which involves short anesthe...
This paper describes an original orthotopic model of MPM H2052/484 cells injected in the pleural cavity of athymic mice and a method of monitoring by small animal PET/CT imaging. This model can be implemented with moderate animal handling and surgery skills and displays a very good development rate. It allows a large experimental window of about 10 weeks in untreated mice and non-invasive longitudinal detection of tumors as early as 2 weeks after injection.
Orthotopic models rely on the implan...
The authors have nothing to disclose.
This research was funded by Ligue Genevoise contre le Cancer (to V.S.-B.) and by the Center for Biomedical Imaging (CIBM) of the Universities and Hospitals of Geneva and Lausanne (to D.J.C., O.B. and S.G.).
Name | Company | Catalog Number | Comments |
3-mice bed | Minerve | bed for mice imaging | |
Athymic Nude-Foxn1n nu/nu | Envigo, Huntingdon, UK | 6907F | immunodeficient mouse |
Betadine | Mundipharma Medical Company, CH | 111131 | polyvidone iodine solution |
Dulbecco's Phosphate-Buffered Saline (DPBS) | ThermoFisher Scientific, Waltham, MA, USA | 14190094 | Buffer for cell culture |
Fetal bovine serum (FBS) | PAA Laboratories, Pasching, Austria | A15-101 | cell culture medium supplement |
Insulin syringes | BD Biosciences, San Jose, CA, USA | 324826 | syringe for cell injection |
Penicillin/Streptomycin | ThermoFisher Scientific, Waltham, MA, USA | 15140122 | antibiotics for cell culture medium |
RPMI 1640 | ThermoFisher Scientific, Waltham, MA, USA | 61870010 | basal cell culture medium |
Temgesic (Buprenorphin 0.3 mg/mL) | Alloga SA, CH | 700320 | opioid analgesic product |
Triumph PET/SPECT/CT | Trifoil, Chatsworth, CA, USA | imaging equipment | |
Trypsin | ThermoFisher Scientific, Waltham, MA, USA | 25050014 | enzymatic cell dissociation buffer |
Virkon S 2% | Milian, Vernier, CH | 972472 | disinfectant |
Vivoquant | Invicro, Boston, MA, USA |
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