Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Our method enables monitoring the increase in metastatic involvement of inguinal lymph nodes by measuring the increase in their volume. This uninvasive method is very well-tolerated, therefore, multiple imaging sessions can be scheduled on the same experimental animal over the course of two weeks. This method is ideal for assessing the impact of pharmacological treatment on metastatic disease.

We applied this method to the Braf/Pten melanoma model, but in principle, it can be adapted to assess the efficacy of various drugs in a variety of preclinical mouse models of metastatic cancer. Demonstrating the procedure will be Dr.Marianna Vitiello, a post-doc from my laboratory, and Dr.Claudia Kusmic, fellow researcher at IFC-CNR. For melanoma induction, apply three microliters of five-millimolar 4-hydroxytamoxifen on approximately one square centimeter of shaved skin of the upper back for three days in a row.

Six weeks after skin painting, use calipers to measure the volume of the primary tumor, and eventually, of subcutaneous metastases. Then, use ultrasound imaging to measure the volume of the inguinal lymph nodes. After analyzing the primary tumor and lymph nodes by visual inspection, excise them for histological studies.

After confirming anesthesia, transfer the animal to a heated board of the ultra-high-frequency ultrasound imaging station, holding the animal in a supine position. Adjust the board temperature using a rectal probe lubricated with petroleum jelly to maintain the mouse's body temperature in the physiological range. Coat the fore and hind paws with conductive paste and tape them to the ECG plate electrodes embedded in the board.

Check that the physiological parameters are correctly acquired and displayed. Remove hair from both inguinal areas by applying a depilatory agent and coat them with an acoustic coupling medium. Then, clamp the ultra-high-frequency ultrasound linear probe into a specialized 3D motor embedded in the imaging station, allowing automated and stepwise movement of the probe.

Properly orient and adjust the position of the ultrasound probe to obtain short-access images of the inguinal lymph node, and place the region-of-interest the focal zone. Set the Scan Distance between two and five millimeters, Step Size to 44 micrometers with an outcome of 46 to 114 scan steps per lymph node slice, and an acquisition time of one to three minutes per animal. Scan the entire volume of the inguinal lymph node as a sequence of 2D B-mode images, acquiring images at multiple levels by linear movement of the transducer with step sizes on a micrometer scale to generate 3D data in terms of respiration and cardiac-gated cine loops.

After imaging, digitally store the acquired images in raw format for further offline analyses. For post-processing of ultrasound images, open the dataset of the 3D inguinal lymph node images, select multi-slice method and press the Start button to visualize both the current frames and thumbnails of all frames corresponding to each image captured during the 3D acquisition. Select the thumbnail of the first frame where the lymph node is evident to load it into the contouring view.

In the contouring view, left-click on the mouse to drop points along the border of the lymph node. Once the desired number of points has been set, right lick to complete the contour. After the first contour is completed, use the thumbnail view to select the next image for contouring.

If required, skip over several images between contours to reduce the number of manual traces needed for each 3D volume. Repeat this process until the entire volume has been outlined, then, click on Finish. While in the 3D mode window workspace, click on the volume measurement icon beneath the image display area to visualize the numerical value of the lymph node volume, and activate the surface view with the 3D rendering of the lymph node volume.

Treating mice with 4-hydroxytamoxifen induces CreER enzyme activity, causing a switch at the genomic level from wild-type Braf to mutated Braf V600E, while Pten is lost. Four weeks after 4-hydroxytamoxifen treatment, primary tumors reach a volume of 50 to 100 cubic millimeters and their growth can be measured by calipers for an additional two weeks, thereby showing a gradual increase in pigmentation observed in the inguinal lymph nodes. The melanin deposits are invariably due to the presence of metastasized melanoma cells, as confirmed by immunohistochemical staining of the melanoma antigen MelanA and Braf V600E.

At the end of the segmentation phase, all sections showed the overlay of the external contour of the lymph node. The 3D renderings of a right inguinal lymph node, analyzed at four, five, and six weeks after 4-HT treatment are shown here. The increase in volume of the left and right inguinal lymph nodes of the same animal over time was also quantified.

It's recommended to couple ultrasound imaging with appropriate immunohistochemical staining, so that the presence of cancer cells is confirmed at the molecular level. Besides lymph nodes, the volume of subcutaneous metastasis can also be monitored over time using ultrasound imaging. If the two-weeks timeframe is too short to appreciate the effects of the drug under study, select a more peripheral induction site or a less tumor-prone genotype.