Name: implantation and monitoring by pet/ct of an orthotopic model of human pleural mesothelioma in athymic mice
Uploaded: 2019-12-21T10:00:00
Description: Watch this Scientific Journal Video about Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice at JoVE.com

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.).

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.
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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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>3-mice bed</td>
			<td>Minerve</td>
			<td></td>
			<td>bed for mice imaging</td>
		</tr>
		<tr>
			<td>Athymic Nude-Foxn1n nu/nu</td>
			<td>Envigo, Huntingdon, UK</td>
			<td>6907F</td>
			<td>immunodeficient mouse</td>
		</tr>
		<tr>
			<td>Betadine</td>
			<td>Mundipharma Medical Company, CH</td>
			<td>111131</td>
			<td>polyvidone iodine solution</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate-Buffered Saline (DPBS)</td>
			<td>ThermoFisher Scientific, Waltham, MA, USA</td>
			<td>14190094</td>
			<td>Buffer for cell culture</td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>PAA Laboratories, Pasching, Austria</td>
			<td>A15-101</td>
			<td>cell culture medium supplement</td>
		</tr>
		<tr>
			<td>Insulin syringes</td>
			<td>BD Biosciences, San Jose, CA, USA</td>
			<td>324826</td>
			<td>syringe for cell injection</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ThermoFisher Scientific, Waltham, MA, USA</td>
			<td>15140122</td>
			<td>antibiotics for cell culture medium</td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>ThermoFisher Scientific, Waltham, MA, USA</td>
			<td>61870010</td>
			<td>basal cell culture medium</td>
		</tr>
		<tr>
			<td>Temgesic (Buprenorphin 0.3 mg/mL)</td>
			<td>Alloga SA, CH</td>
			<td>700320</td>
			<td>opioid analgesic product</td>
		</tr>
		<tr>
			<td>Triumph PET/SPECT/CT</td>
			<td>Trifoil, Chatsworth, CA, USA</td>
			<td></td>
			<td>imaging equipment</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>ThermoFisher Scientific, Waltham, MA, USA</td>
			<td>25050014</td>
			<td>enzymatic cell dissociation buffer</td>
		</tr>
		<tr>
			<td>Virkon S 2%</td>
			<td>Milian, Vernier, CH</td>
			<td>972472</td>
			<td>disinfectant</td>
		</tr>
		<tr>
			<td>Vivoquant</td>
			<td>Invicro, Boston, MA, USA</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

implantation and monitoring by pet/ct of an orthotopic model of human pleural mesothelioma in athymic mice

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&#8211;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.

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...

Watch this Scientific Journal Video about Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice at JoVE.com

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

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. ...

This method can help to answer key questions related to the development, treatment and diagnosis of human pleural mesothelioma. The main advantage of this reliable pre-clinical orthotopic model is that it replicates the human disease progression and pathology in a microenvironment close to the one found in patients with pleural mesothelioma. Therefore, this invaluable model is of high significance and the use of non-invasive molecular imaging allows longitudinal monitoring in line with the three R concept.Prior to starting prepare the anesthesia system and surgical area in a laminar flow hood by spraying all surfaces with disinfectant. Place the micro-isolated SPF cages in the disinfected flow hood. Place a heating pad, povidone-iodine solution 30 gauge Hamilton syringe, gauze and cotton swabs surgery instruments and micropipettes and tips in the laminar flow hood.Once the mouse is properly anesthetized subcutaneously inject 0.05 milligrams per kilogram Buprinorphine for post-operative pan relief. Then place the mouse on its right side on top of the heating pad and clean the surgical area with povidone-iodine solution and make a 5 millimeter incision of the skin. Clear the surrounding fat and muscles with blunt scissors to expose the ribs.Homogenize the cell suspension and load 50 microliters into the Hamilton syringe making sure to avoid air bubbles. Wipe the needle with 70%alcohol prior to each injection. Slowly inject the cells into the pleural cavity between the sixth and seventh ribs holding the needle at an angle of 30 degrees and two to three millimeters under the intercostal muscles.Keep the needle just under the ribs to avoid injecting into the lungs. It should be visible through the muscles. When finished, close the wound with three to four absorbable sutures and store the mouse in a warmed environment until it wakes up.When performing implantation it is very important to correctly position the needle to limit the depth of the needle penetration. Use a heating chamber heating pad, or infrared lamp to prewarm the mice at 30 degrees celsius for 30 minutes prior to the injection of fluor-18(FDG)Using a dose calculator prepare three to four megabecquerel doses of fluor-18(FDG)in 150 to 200 microliters of saline in 1 milliliter insulin syringes. Make sure to record all times of radioactivity dose measurements injections, and PET scans in order to calculate standard uptake values or SUVs.Weigh the mice then intravenously inject the fluor-18(FDG)After the injection leave the mice awake in warm conditions for 45 minutes. Then load the mice on the scanner bed transfer the bed to a scanner and subject the animals to a CT scan centered on the lungs. Move the bed to the PET subsystem insert the acquisition one hour after the fluor-18(FDG)injection for a duration of 15 minutes.Then remove the mice from the imaging chamber and allow them to recover in their cage keeping them in an area dedicated to radioactive decay. Prior to image analysis reconstruct CT and PET scans as described in the manuscript. Calibrate the images by scanning a phantom cylinder and automatically co register the scans according to the built in software solution.To analyze the images load the CT data as a reference by clicking on the open data icon. Then load the PET data as input by clicking on the append data icon. Adjust the color scale so CT and PET to contrast images for visual inspection.Select the 3D ROI tool from the drop down menu click on add ROI, and name the file lungs. Click on segmentation algorithms and neighborhood thresholding then define input as background and image as ref. Enter min and max according to mouse lung density values.Inspect the 3D rendered lungs by clicking the VTK icon. Then click show table icon and retrieve the volume in the generated table. To analyze the fluor-18(FDG)in tumors convert PET images to SUV by selecting arithmetics from the drop-down menu.Select scalar multiplicity then use NP one as selected and set the becquerel per milliliter to SUV factor as scalar. Finally select 3D ROI tool from the drop-down menu and click add ROI and name the file tumors. Click on 3D paint mode and sphere uncheck 2D only and adjust the size of the shape to surround the tumors.Click on show table icon and retrieve the SUV max value from the generated table. The 3D renderings from the CT scans give an overview of MPM tumor localization and allow for calculation of lung volumes. Lung volume measurements decrease significantly over time after the injection of intrapleural tumors.The PET scan provides valuable information about the metabolic status of MPM tumors. The tumors were distinguishable two weeks after grafting and fluor-18(FDG)uptake was quantified by extracting SUVs which were positively correlated with the number of days post injection. Furthermore, lung volumes and fluor-18(FDG)avidity correlate with each other with an R squared of 0.6 which supports the strength of these measurements for monitoring MPM orthotopic tumor development.Following this procedure other methods such as histology, immunohistochemistry, and flow cytometry can be performed in order to respond to orthobiological questions such as the proliferation state and phenotypic characteristic of tumors and of the microenvironment. To conclude, these pre-clinical techniques pave the way for researchers to explore new diagnostic and treatment strategy of pleural mesothelioma. Furthermore, the use of molecular imaging warrants rapid translation of new findings to the clinic.

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JoVE Journal

Immunology and Infection

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Comprehensive &amp; Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

Comprehensive & Cost Effective Laboratory Monitoring of HIV/AIDS: an African Role Model

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical ...

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Induction of Graft-versus-host Disease and In Vivo T Cell Monitoring Using an MHC-matched Murine Model

Graft-versus-host disease (GVHD) is the limiting barrier to the broad use of bone marrow transplant as a curative therapy for a variety of hematological ...

Recurrent Herpetic Stromal Keratitis in Mice, a Model for Studying Human HSK

Herpetic eye disease, termed herpetic stromal keratitis (HSK), is a potentially blinding infection of the cornea that results in over 300,000 clinical ...

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

An In vitro Model to Study Heterogeneity of Human Macrophage Differentiation and Polarization

Monocyte-derived macrophages represent an important cell type of the innate immune system. Mouse models studying macrophage biology suffer from the ...

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Optimized Protocols for Mycobacterium leprae Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. ...

A Semi-automated Approach to Preparing Antibody Cocktails for Immunophenotypic Analysis of Human Peripheral Blood

Immunophenotyping of peripheral blood by flow cytometry determines changes in the frequency and activation status of peripheral leukocytes during disease ...

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo Cell Trafficking by PET/CT

Murine Lymphocyte Labeling by 64Cu-Antibody Receptor Targeting for In Vivo  Cell Trafficking by PET/CT

This protocol illustrates the production of 64Cu and the chelator conjugation/radiolabeling of a monoclonal antibody (mAb) followed by murine lymphocyte ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

An IL-8 Transiently Transgenized Mouse Model for the In Vivo Long-term Monitoring of Inflammatory Responses

Airway inflammation is often associated with bacterial infections and represents a major determinant of lung disease. The in vivo determination of the ...