In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

The systemic effects of primary tumors are now recognized to play a significant role in the process of metastatic dissemination. However, this is often missed in experimental metastasis assays. This model aims to provide researchers with a way to evaluate this effect.

This protocol includes evaluation of spontaneous metastasis after the surgical removal of a primary tumor, which is clinically relevant. Mainly, it also takes advantage of bioluminescence imaging to monitor lung metastasis outgrowth in real time. This experimental setup can be extended to evaluate pharmacological or genetic interventions for breast cancer in the neoadjuvant setting, as well as the relevance of specific signaling pathways in the metastatic process.

To begin, harvest the cells by aspirating the cell culture medium and wash with sterile PBS. Next, incubate with two milliliters of 0.25%Trypsin-EDTA solution for about two to three minutes at 37 degrees Celsius until the cells detach and then wash with eight millimeters of complete medium to quench the reaction. After aspirating the supernatant and washing the cells with PBS, resuspend the cells in PBS in the calculated volume necessary to dilute cells to 6 million cells per milliliter.

Then transfer the cell suspension to 1.5-milliliter microcentrifuge tubes, and keep on ice until ready for injection. Shave the hair on the abdomen of the anesthetized mouse using electric clippers, and then place it in a supine position. Next, clean the prepared abdominal region using 70%ethanol and povidone-iodine solution.

Using scissors, make a small midline incision through the abdominal skin at the level of the fourth mammary tissue, exposing but not penetrating through the underlying peritoneum. Then hold the skin away from the peritoneum using forceps and separate the skin away from the peritoneum with sterile saline-dipped cotton swabs, moving laterally to expose the right mammary fat pad, and repeat on the left side to expose the left mammary fat pad. After resuspending the E0771 cell suspension using a manual pipette, transfer 100 microliters to a new 1.5-milliliter microcentrifuge tube.

Then add an equal volume of basement membrane matrix solution and mix well, taking care not to introduce bubbles. Afterward, keep the tube on ice. Draw 100 microliters of cell suspension into a 28-gauge 0.5-milliliter U-100 insulin syringe, and keep on ice.

Using forceps, lift the skin and gently grab and expose the left mammary fat pad. Next, inject 50 microliters of cell suspension into the mammary fat pad and wait for three to five seconds prior to removing the syringe. Then release the skin from the forceps, allowing it to return naturally to its normal position, and close the skin incision, applying skin staples.

To monitor the growth of the orthotopic breast tumor lesion, measure the primary tumor length and width using calipers three times per week. After anesthetizing, administer 40 microliters of meloxicam subcutaneously for pain control, and then place the mouse in a supine position. Next, remove prior surgical staples if necessary, and clean the abdomen with 70%ethanol and povidone-iodine solution.

Using scissors, make a small midline incision through the abdominal skin at the level of the fourth mammary tissue, exposing but not penetrating through the underlying peritoneum. Then remove the orthotopic tumor by cutting through the normal mammary tissue, located proximally and distally to the tumor using scissors. Discard the tumor tissue into a biohazard bag while repeating the procedure with the contralateral tumor.

Next, close the surgical site using one to three staples and transfer the mouse to a clean recovery cage with a warm heating pad underneath. Inject the animals with 100 microliters of D-luciferin solution using a retroorbital injection by inserting the needle in the medial canthus of the eye at a 45-degree angle from the nose until bony resistance is felt, ensuring that the needle is placed within the retroorbital venous sinus. Confirm successful injection by the lack of flush-back of any liquid upon delivery and wait two minutes prior to imaging.

While waiting, transfer the animals to the nose cones located inside the bioluminescent imager in a supine position. To measure the photon flux, create a square ROI for each animal depicted in the image from under the ROI tool's dropdown menu by clicking the square ROI button. Reposition the automatically-generated ROI over the thorax of each animal by clicking and dragging with the mouse.

And then click the measure ROI's button. Make a midline incision below the xiphoid process, cutting through the skin, musculature, and peritoneum to expose the lower part of the thoracic cavity using scissors until the diaphragm is visible. Afterward, puncture the diaphragm to collapse the lungs and then cut through the diaphragm.

Cut through the rib cage on the right and left side, and then use a hemostat to grab the xiphoid process and move the rib cage out of the way, exposing the heart and lungs. Next, snip the right atrium using scissors. Then perfuse the animal with 10 milliliters of ice-cold PBS through the left ventricle, and assess the completeness of perfusion by confirming that fluid flowing from the right atrium turns clear and that the liver turns a pale yellow color.

Next, identify the trachea and insert a 22-gauge needle syringe with three milliliters of 4%paraformaldehyde, holding it parallel to the trachea. Then deliver the solution at a slow pace until the lungs have fully inflated. Continue gently holding the trachea.

Snip it with scissors above the forceps and start carefully lifting the tissue while removing all the connective tissue. Next, dissect the heart away from the lungs. Afterward, place lung tissue in 4%paraformaldehyde in PBS overnight and store at four degrees Celsius for fixation.

Utilizing C57 black 6 mice, significant lung metastasis bioluminescence signal is typically detected around two weeks following primary tumor resection and can be followed over time, although some variation can be found depending on the luciferase reporter type and mouse strain. The ex vivo bioluminescence imaging of the lung tissue at the endpoint provides accurate and sensitive quantification of lung metastatic burden that can be plotted to compare with other treatments. Lung nodule counting under the stereoscope and hematoxylin and eosin histological analysis can be utilized to complement the quantification studies.

Slightly delaying needle withdrawal after mammary fat pad injection allows the matrix solution to gel, which is key to preventing leakage of the cell suspension and the development of extra-mammary tumors. We are now utilizing this technique in combination with various genetic knockout and knockin mouse models to further study the metastatic cascade, particularly the development of the pre-metastatic niche.