Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

The overall goal of this video, is to identify the advantages of using Micro CT, for the assessment of tumor development, treatment response, and other experiments in mice models of lung cancer. This method can help answer key questions in the field of lung cancer when using mice model, such as when tumors start to grow, the rate of growth, and the response to therapeutic interventions. This technique enables us to identify which animals should be excluded from our study, while detecting real time changes in their tumor size.

The technique saves time and effort, and markedly reduce the number of mice per study group. From the Micro-CT scanner, access the computer control, and open the software package named RMCT2"Then, start the warm-up cycle. Next, prepare the scanner sample bed, for the mouse, by wrapping it in plastic.

Next, anesthetize the mouse using isoflurane and confirm that it is anesthetized, using a skin pinch. A gentle pinch of a small fold of skin is sufficient. Next, apply an ophthalmic ointment to prevent corneal dryness.

Now place the mouse, dorsal side up, on the sample bed. Hold the head of the mouse and pull the body downward from the lower limbs to stretch and straighten the body symmetrically. Then, route the anesthetic gas to the chamber, and position the gas tube next to animal's nose, to administer continuous anesthetic.

Then, wrap the mouse and sample bed with plastic, to keep the mouse in place. Make certain the mouse is secure because any movement or twist during the scan will result in hazy, unusable images. Now, close the chamber, adjust the system to 90 kilo-volts, and 160 micro-amps, and set the scan time to 4.5 minutes.

Set the image range to 24 millimeters. Then, set the system in synchronous mode for heartbeats. Now, make a new folder in the database for the new images and start the scan.

After completing the scan, move the mouse to an empty cage and observe it until it regains consciousness. Do not return it to its housing mates until it is fully recovered from the anesthesia. Open the serial micro CT image files in image J.Scroll through all the images of each mouse from the neck to the abdomen or vice versa.

Now, using scans of naive wild-type mice, identify the density of different areas and normal anatomical structures in the chest. Scroll up to the whitish abdominal viscera and diaphragm, and identify area through the chest, and up to the neck. Take note of the bony chest cage landmarks, the sternum and vertebrae and ribs.

Then, identify the heart in the front of the chest, and the major blood vessels near the heart and in the mediastinum. The tracheal lumen is a small dark circle at the level of the neck and upper chest, which bifurcates into the right and left main bronchi, and then continues to branch into smaller and smaller bronchi. Note that each bronchus is closely associated with two or three blood vessels.

Now, start examining the scans of un-induced DT mice and identify the presence of any abnormalities. Exclude from the experiment any mice with abnormal pre-induction lung shadows, such as nodules or emphysematous bullae. For this experiment, use mice with normal lung scans that can produce FGF9 induced tumors.

To induce the tumors, provide doxycycline in their chow at 200 parts per million. After 10 weeks, perform a second Micro-CT scan of these mice to confirm the development of tumor nodules in their lungs. Then, split the mice into two groups.

Administer the FGFR blocker, AZD4547, to one group via a gastric tube. Do this for 6 days per week over 10 weeks. Give the other group a placebo.

Follow up the changes in the nodular shadows by performing a third scan four to five weeks into the treatment. Later, at the end of the full 10 week treatment, perform a fourth scan. Then, identify dynamic changes in the tumor nodules, in response to the treatment, by comparing similar positions within the serial scan images at the different time points.

Look for the appearance, or disappearance, of any abnormal shadows. Correctly orienting to the same anatomical position is critical to interpreting the results. Use easily identified anatomical landmarks within the chest, such as the trachea, its bifurcation, the right and left main bronchi, the aorta and large blood vessels, and the diaphragm.

Because of body tilts, bones are less useful landmarks. After euthanizing a mouse, and accessing its neck and lungs, cut away the heart and thymus gland. Insert forceps behind the trachea to separate it from the esophagus.

Next, cannulate the trachea using a G24 cannula, and secure the cannula by knotting a suture placed around the inter-tracheal tip. Now, inflate and fix the lung using ice cold 4%PFA via the cannula with a 25cm column. Then, detach the cannula and tighten the knot on trachea to prevent PFA leakage.

Now, cut off the upper trachea, including its attachment to the larynx. And while pulling the trachea from the suture thread, dissect the trachea and lungs from all attachments. Then, remove the trachea with the lungs en blanc.

Place the tissues into a 15ml tube containing 5ml of room temperature 4%PFA. Leave the lungs in PFA overnight for complete tissue penetration and fixation. Then, process the tissue as usual.

In pre-screening 8-12 week old DT mice for this study, about half showed lung abnormalities in the Micro-CT scan, making them unsuitable for inclusion. For the study, tumor induction was activated for 10 weeks, followed by treatment. Mice that were given the FGFR inhibitor, AZD4547, for 10 weeks exhibited disappearance or reduction in the size of nodules that were visible in the pre-treatment scans.

On the other hand, control mice given placebo showed either no change, an increase in nodule size, and/or the appearance of new nodules. End point histological evaluation of the lungs confirm the accuracy of the findings made with Micro-CT scanning. Mice judged to have clear lungs indeed, had normal lung histology.

Whereas, mice that showed abnormalities, showed various histological changes including bullae and nodules. After 10-12 weeks of induction with doxycycline, multiple adenocarcinoma nodules, with variable sizes and positions, were observed in all animals. Subsequent treatment with 10-12 weeks of an FGFR blocker, showed fewer and smaller nodules.

After watching this video, you should have a good understanding of how to use Micro-CT to assess the development of tumor in mouse lung. And to follow up any changes in these tumors, in response to various experimental procedures. Once mouse start scans can be performed in less than 10 minutes per mouse.

While attempting this procedure, it is important to remember to keep the mouse under deep anesthesia, and securely wrapped to the sample bed to minimize movement during the scans. After its development, this technique paved the way for researchers in the field of lung cancer and cancer stem cells to explore the extent of tumor development, progressional regression, and the setting of different experiments. Don't forget that working with X-Rays can be hazardous.

In standard machine running procedures should always be taken while preforming this procedure.