Monitoring of Nanodrug Accumulation in Murine Breast Cancer Metastases

Our research focuses on treating breast cancer metastases using image guided therapeutics on an iron oxide nanoparticle platform. The goal is to develop effective therapies directed specifically at metastatic tissues, which are the main causes of cancer-related death. It can be challenging to determine whether systemic therapeutics have reached the desired tissue without sacrificing animals.

Our nanoparticle can be imaged using MRI and optical imaging modalities, allowing us to monitor drug delivery and accumulation in living animals. Current therapies for metastatic breast cancer are not specific to metastases, have variable efficacy, and cause undesirable side effects. Our nanoparticle platform specifically targets the metastatic niche and allows for non-invasive monitoring of its delivery with no evidence of adverse effects.

Previously, we used our nanoparticle platform to target a driver of metastasis, miR-10b, and were able to arrest metastasis and growth of metastases. With an eye on clinical translation, we are investigating the pharmacodynamics of this formulation to optimize the treatment regimen and maximize efficacy. To begin, place the frozen Matrigel at 4 degrees Celsius for 24 hours to allow the matrix extract to liquefy.

After determining the total number of cells needed for the study, trypsinize cells and wash with PBS. Centrifuge the cells at 200 G for 5 minutes. Then re-suspend the pellet in 500 microliters of chilled PBS to create the cell stock.

Now, dilute the total number of cells to 40 times 10 to the power of 6 cells per milliliter. Then add an equal volume of the chilled matrix extract to achieve a final concentration of 1 times 10 to the power of 6 cells per 50 microliters. Keep the mixture on ice to prevent the extract from solidifying before implantation.

Transfer the anesthetized mouse to a nose cone on a heating pad. After confirming the surgical plane of anesthesia, apply ophthalm ointment to protect the eyes from corneal drying. Then clean the skin near the injection site with an alcohol wipe and allow it to dry for a few seconds.

Induce at mammary gland number four to minimize signal overlap between the primary tumor and common metastasis sites. Now, pipette the cell stock up and down to re-suspend the cells. Draw 50 microliters of the ice cold cell suspension into an insulin syringe with a 29 gauge needle.

Insert the needle bevel directly below the nipple of the desired mammary gland, parallel to the body of the mouse, and inject the cells at a steady, slow rate. Leave the needle in the skin for at least 5 seconds after completing the injection to allow the Matrigel to solidify and prevent leakage. Afterwards, move the mouse to a clean cage on a warming pad for recovery and supervise until it is fully ambulatory and can maintain sternal recumbency.

To monitor tumor growth and metastasis development, inject 150 milligrams per kilogram body weight of luciferin intra peritoneally into the anesthetized metastatic breast cancer mouse model. Return the mice to their cage on a warming pad to allow them to awaken and metabolize the luciferin. Then, image the mice using the imaging system scanner, starting approximately 10 minutes post-injection with luciferin.

Image up to 5 mice together in a supine position, ensuring their entire bodies are included within the field of view guide markings and oriented as straight as possible. Use clear tape to secure their arms to better visualize the axillary lymph nodes. Now, in the imaging system software, set the Exposure to Auto, Binning to Medium, FStop to 1, Excitation to Block, Emission to Open, FOV to D, and Height to 1.50 for bioluminescence imaging.

When imaging the primary tumor, which typically produces a strong signal due to its superficial location, set the Exposure to Auto. If monitoring for metastases, carefully cover the primary tumor with black electrical tape and manually set Exposure to 300 seconds to capture any faint signals. To begin, weigh the metastatic breast cancer mice, as the Nanodrug dosage is based on body weight.

Prepare an insulin syringe with a 29 gauge needle filled with 10 milligrams of iron Nanodrug per kilogram of mouse body weight. Then submerge the anesthetized animal's tail in warm water at 30 to 35 degrees Celsius for 30 seconds to dilate the tail veins. After that, wipe excess water from the tail and clean the injection site with a 70%alcohol wipe.

Now, insert the needle bevel up into the lateral tail vein approximately halfway down the tail. Pull back the plunger slightly to confirm placement with the flashback of blood into the needle. Upon successful insertion, inject the Nanodrug steadily at a slow rate of approximately 5 to 10 seconds for a 40 microliter injection.

Confirm successful injection by the lack of solution pooling under the skin of the tail near the injection site, and by the darkening of the vein from the dark nanoparticle solution. Hold pressure over the injection site with gauze and remove the needle. Maintain pressure for approximately 30 seconds until the bleeding stops.

To collect metastasis samples, begin by imaging the mice using bioluminescence imaging. After collecting the metastases, place the collected tissues in a Petri dish. In the imaging system software, set the Exposure to Auto, Binning to Medium, FStop to 1, Excitation to Block, Emission to Open, FOV to D, and Height to 1.50 for bioluminescence imaging.

For fluorescence imaging, set Exposure to Auto, Binning to Medium, FStop to 1, Excitation to 675, Emission to 720, Lamp Level to High, FOV to D, Height to 1.50. Then image the mouse carcass with BLI to determine if there is any remaining cancer tissue worth collecting. Next, rinse the collected cancer tissues in PBS.

To collect tissues for microscopy or quantitative reverse transcription polymerase chain reaction, embed them in optimal cutting temperature compound and store them at minus 80 degrees Celsius until ready for processing. To collect tissues for inductively coupled plasma optical emission spectroscopy, tare a scale using an empty 1.7 milliliter tube. Place the tissue in the tube and record its weight.

After freezing the tissue, store it at minus 80 degrees Celsius until ready for processing. Cryosection the optimal cutting temperature embedded fresh frozen samples onto microscopy slides at 10 micrometers thickness. Adjust the chamber and specimen holder temperatures between minus 20 degrees Celsius and minus 15 degrees Celsius, depending on the tissue type.

Fix the tissue sections onto the slides by submerging them in 4%paraformaldehyde solution for 15 minutes. Carefully rinse the slides with PBS. Then mount cover slips onto the slides using a medium containing DAPI to visualize tissue architecture.

Use a fluorescence microscope to examine the tissue sections for Cy5.5 fluorescence, which indicates Nanodrug delivery. Confirm that the fluorescence signal is not background noise by comparing it to a negative control sample from a non injected animal. The Nanodrug treated mice showed significant bioluminescent signals in lung metastases one week after administration, confirming the presence of metastases in lung tissues.

Fluorescence imaging confirmed the accumulation of Cy5.5 from the Nanodrug only in the metastatic lung tissues of treated mice.