Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

This study shows that manual guided ultrasound is well-suited to monitor the development of uterine pathology in an induced mouse model of endometrial cancer. Ultrasound imaging is noninvasive and thus allows for longitudinal studies of individual mice across days and weeks. This allows for the capture of individual variation and ultimately for fewer animals to be used for study.

This method can be readily applied to monitor pathologic changes and other mirroring models, including those for neoplasia and chronic disease of the urinary and digestive tract. Begin setting up the ultrasound machine for imaging the uterus or ovary by selecting a transducer or probe with a range of 32 to 56 megahertz. Go to the Applications tab from the home screen and select Mouse Small Abdomen Mode.

Then click on Scan to return to the home screen and wait for a live image to be displayed. Next, select B Mode from the options on the left toolbar before clicking on More Controls option to view additional tools for image refinement, such as image gain and depth or to adjust the clip acquisition settings, such as the number of frames per second. Once the image settings are selected, return to the home screen by clicking on B Mode.

Place a three to 10-milliliter syringe filled with a sterile isotonic fluid solution between a heating pad and an absorbent pad for several minutes to preheat it to 35 to 40 degrees Celsius. If the machine does not have a warmer, place a bottle of ultrasound gel on the heating pad. To inject one to two milliliters of solution into the peritoneal cavity of a mouse, secure the mouse by the scruff in one hand, exposing the ventrum.

Then hold the mouse at approximately a 20-degree angle with the nose pointed to the floor. Insert a 25-gauge tuberculin syringe needle through the skin and abdominal wall of the caudal right quadrant of the abdomen. To avoid injection into the vasculature or the GI tract, pull back on the syringe plunger with minimal pressure to look for blood.

Position the mouse in ventral recumbency on the absorbent pad over a heating pad before securely placing a rodent nose cone over the mouse's nose and muzzle. Place a small amount of pre-warmed ultrasound gel abaxial to the spine on either side of the anesthetized mouse between the last rib and pelvis. Put a small amount of gel on the ultrasound probe.

Next, place the probe parallel to the vertebrae with the front of the probe on the cranial side. With a mouse in ventral recumbency, slowly scan the area for the kidney landmark. With the kidney in view, pull the probe caudal to find the ovary, which is a slightly hyperechoic oval to round structure within a very hyperechoic ovarian fat pad, bordered cranial-ventrally by the kidney and dorsal-laterally by the dorsal abdominal wall.

Improve the image contrast by adjusting the signal gain using the slider at the bottom of the control screen. To improve the imaging of the kidney, apply pressure to the contra-lateral abdomen using a finger. Vary the pressure and angle from parallel to the spine to approximately 20 degrees ventral.

Once the organ of interest is in view, capture a video by clicking on Save Clip or Start Recording. Once done, click Stop Recording to retain images at a preset number of frames. Hold the probe caudally until the ovary is in the most cranial aspect of the field of view.

Vary the probe pressure and angle to view and image the uterus. Repeat video and frame collection for each organ of interest. Find the uterus running longitudinally along the dorsal abdominal wall with the lateral leg musculature in view.

Monitor the tissue for a peristaltic motion to differentiate the intestinal loops from the uterine stationary horns. Place the mouse in dorsal recumbency to collect the images from a ventral approach. Ensure the eye lubrication is sufficient and the muzzle is secured in the nose cone.

Apply a small amount of pre-warmed ultrasound gel to the ventral abdomen before applying the probe at the midline and cranial of the pubis to locate the bladder as a hypoechoic landmark. To find the uterine horns, pull the probe lateral to the bladder. Apply light digital pressure from either or both sides of the mouse.

Once the uterine horns are in the field of view, hold the probe perpendicular to the mouse and scan both sides of the abdomen to capture transverse views of both horns. Rotate the probe to capture sagittal views. After the ultrasound, wipe the mouse with a paper towel before returning it to its cage to recover.

Once the mouse is fully awake after two to five minutes, return the mouse to the animal room. The ultrasound image comparison of the mouse abdominal cavity before and after the saline injection showed the improved visualization of the abdominal organs, including the uterus and ovaries after the saline injection. A ventral approach was used to study the cross-sections of the uterine horns adjacent to the hypoechoic bladder.

Immunohistochemistry with antibodies directed against Arid1a and Pten was used to study the adequate tumor suppressor protein loss accomplished in the uterine epithelial cells compared to the neighboring stromal tissue. Ultrasound images of a mouse uterus with early pathology changes were detected after three weeks of doxycycline treatment, showing the hypoechoic and dilated lumen of the uterus. In the gross pathology and histopathology study of the same uterus, a smaller but dilated thin-walled uterus and early endometrial hyperplasia with the preservation of the distinct lumen were observed respectively.

The mice euthanized at later time points showed larger uterus with a smaller lumen and a thickened wall. When learning to use manual placement of probes, excessive pressure or rapid movement can make organ identification difficult. Use gentle, deliberate motions and focus on identifying the organs which are landmarks first.

At study endpoint, mice are sacrificed and tissue is collected to confirm gene knockdown and characterize tissue changes. This technique allows investigators to monitor intraabdominal lesions over time. And in our case, we used it to monitor the uterine cancer progression in the iPad model, which we then confirmed by histopathology.