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

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.