Cellular inoculation routes for modeling breast cancer bone metastases in mice

Once a mouse has been properly anesthetized and is unresponsive to pinch, place it on a sterile surface in supine position ensuring that the vertebral column is straight. Tape the forelimbs away from the torso at a slightly angled and upward position (Figure 2a). Prior to inoculation, clean the chest of the animal thoroughly with betadine and wipe with an alcohol pad or as per the institutional standard operating procedures. Once the chest has been sterilized, gently place one hand on the chest of the mouse to tighten the skin and mark the top of the sternum and the xyphoid process (distal sternum) with a permanent marker (Figure 2a). Make a third mark in the middle of these two landmarks and slightly to your right (animal's left) just over the heart in the third intercostal space (Figure 2a). This mark identifies the location of the left cardiac ventricle where you will insert the needle for tumor cell inoculation.

Inoculation of breast cancer cells in the left cardiac ventricle of a mouse. (a) Sterilize the chest of an anesthetized mouse and mark the top of the sternum and the xyphoid process (distal sternum) with a permanent marker. Make a 3rd mark in the middle of these two landmarks and slightly to your right (animal's left) just over the heart in the third intercostal space. (b) Prepare the needle by leaving a small air space in the top of the syringe before slowly drawing up the desired volume of cell suspension into the syringe. This air space will permit a small influx of bright red oxygenated blood into the syringe hub when properly inserted into the left cardiac ventricle. (c) Once the needle is correctly positioned in the left cardiac ventricle, inject the cell suspension slowly into the left ventricle being careful not to move the needle or press it deeper into the thoracic cavity.

With intra-cardiac inoculation, prepare the needle by leaving a small air space in the top of the syringe before slowly drawing up the desired volume of cell suspension into the syringe (Figure 2b). This air space will permit a small influx of bright red oxygenated blood into the syringe hub when properly inserted into the left cardiac ventricle. Hold the skin of the mouse tight with one hand and insert the needle perpendicularly into the middle marking (Figure 2c). When the needle has entered the left cardiac ventricle, watch for the pulse of blood to appear in the hub of the needle. The appearance of air bubbles in the needle hub upon insertion indicates that it has likely entered the lungs and will need to be removed and repositioned. If you do not see a red pulse of blood in the needle hub but are confident that you are in the correct location, you can pull up slightly on the syringe plunger to verify your position in the cardiac ventricle. If there is still no visible red pulse, the needle can be slowly and slightly adjusted up or down. When small adjustments are futile, remove and reposition the insertion point completely or set the mouse aside temporarily. Extended anesthesia can cause vasoconstriction and reduce the animal's blood pressure such that the pulse of blood into the needle's hub becomes less noticeable.

Once the needle is correctly positioned in the left cardiac ventricle, inject the cell suspension slowly into the left ventricle being careful not to move the needle or press it deeper into the thoracic cavity. Keep a close eye on the cell suspension in the syringe and do not inject the air bubble at the top of the syringe (Figure 2b). As soon as the cells have been inoculated, quickly remove the needle, apply slight pressure at the injection site for a few seconds and place the mouse on a heating pad until fully awake. Once mice have fully recovered, monitor their behavior for 24 h and watch for potential signs of embolism or distress.

Materials for intra-cardiac inoculation

Anesthetized mouse (immune compromised if using human cells)

Cellular suspension

0.5–1 cc insulin syringe, 27–29G, 0.5 inch

Surgical tape

Betadine and alcohol swabs

Permanent marker

Water recirculating heating pad

Once a mouse has been anesthetized with isoflurane, place it on a sterile surface in supine position and tape the torso of the mouse to the table for stability. Use a heating pad or lamp to dilate the vessels within the tail for 2–3 min in order to facilitate greater ease of inoculation. Alternatively, the tail can be placed in warm water (30–35 °C). Using an alcohol pad, disinfect the tail vigorously, which will also help dilate the vessels within the tail and facilitate inoculation. Keep the tail clinched between the forefinger and thumb and insert the needle (beveled edge facing up) horizontally across the proximal section of the tail into the artery (Figure 3). Once the needle has entered the artery, the opposite hand is used to retract the syringe plunger slightly to inspect for a tight fitting within the artery and inspect the barrel of the syringe for a small amount of blood, which should appear at the needle hub. This will not occur if the needle has not breached the epithelial membrane of the artery or if the syringe has protruded through the artery. Once the syringe is properly seated within the artery, depress the plunger slowly in order to minimize cell lysis resulting from fluid shear. The needle should ideally pierce the artery just once in order for the cell bolus to be delivered in its entirety. However, when small adjustments are futile, remove the needle completely and reposition the insertion point proximally to the initial site of injection. Once mice have fully recovered, monitor their behavior for 24 h and watch for potential signs of embolism, pain, or distress.

Schematic representation of the tail vasculature of a mouse.

Materials for intra-arterial tail inoculation

Anesthetized mouse (immune compromised if using human cells)

Cellular suspension

0.5–1 cc insulin syringe, 27–29G, 0.5 inch

Surgical tape

Betadine and alcohol swabs

Water recirculating heating pad

Place an anesthetized mouse in supine position on a sterile surface and tape the forelimbs and hind limbs away from the torso (Figure 4a). Prior to inoculation, clean the inguinal surface of the animal thoroughly with betadine and wipe with an alcohol pad or as per the institutional standard operating procedures. Using sterile surgical instruments, create a small incision in the skin adjacent to the fourth mammary fat pad (Figures 4b and c). Insert the needle into the fourth mammary gland fat pad and slowly inoculate the tumor cell suspension (Figures 4d and e; Supplementary Video 1). The 4th mammary gland fat pad is located at the intersection of three prominent blood vessels (Figure 4c). As with all techniques, it may be important for investigators to practice and familiarize themselves with the necessary anatomical landmarks prior to initiation of a multi-animal study. After the injection of cells, close the wound with 4–6 unconnected sutures, administer an analgesic as per institutional guidelines and place the mouse on a heating pad until fully awake.

Inoculation of breast cancer cells in the 4th mammary fat pad of a mouse. (a, b) Place an anesthetized mouse in supine position on a sterile surface and tape the forelimbs and hind limbs away from the torso. Prior to inoculation, clean the inguinal surface of the animal thoroughly with betadine and alcohol. Using sterile surgical instruments, create a small incision in the skin adjacent to the 4th mammary fat pad. (c, d). Insert the needle into the fourth mammary gland fat pad and slowly inoculate the tumor cell suspension. The fourth mammary gland fat pad is located at the intersection of three prominent blood vessels. (e) After the injection of cells, close the wound with 4–6 sutures, administer an analgesic as per institutional guidelines and place the mouse on a heating pad until fully awake.

In order to limit the occurrence of spontaneous tumor metastases to the lungs when using murine-derived cells (for example, 4T1), primary tumors can be surgically resected from anesthetized mice when tumors reach ∼1 cm³. Animals can then be followed for 3–4 additional weeks for the development of bone metastases.

Some laboratories have reported that orthotopic implantation of the human-derived breast cancer cell lines SUM-1315 and MDA-MB-231-IV into immune-compromised mice can elicit spontaneous metastasis to bone when preceded by engraftment of human bone plugs.22,23 In this model, 5 mm bone biopsy cores obtained from human femoral heads (within 2–4 h of removal from the patient) can be implanted under the skin on the posterior surface of the animal prior to tumor cell inoculation in the mammary fat pad. Four weeks after bone plug implantation, the human bone grafts become vascularized and bone marrow resembles that of normal bone.22,23 Orthotopic injection of human-derived tumor cells can then proceed as described, and the human bone plugs are then excised at the termination of the study (∼8–14 weeks) and evaluated for the presence of breast cancer metastases.

Materials for orthotopic inoculation

Anesthetized mouse (immune compromised if using human cells)

Cellular suspension

0.1 cc Hamilton syringe, 25–27G, 0.5 inch

Tape

Betadine and alcohol swabs

Sterile scissors and forceps

Suture

Water recirculating heating pad

Analgesic

Prepare a syringe with the desired tumor cell suspension and set aside until ready to inject. After sterilizing the hind limbs, bend the knee to nearly 90° (Figure 5a). While holding the hind limb between the thumb and index finger, locate the patellar ligament between the knee and the tibia, which should be visible through the skin as a white longitudinal structure. Some laboratories choose to make a 2–3 mm incision through the skin on the knee to more easily visualize the tibia and patellar ligament; however, with practice this may not be necessary. Holding the needle parallel to the tibia in the dominant hand, push the needle through the center of the patellar ligament and into the proximal end of the tibia (Figure 5b). Resistance will be felt once the needle reaches the bone. Twist the needle slightly to drill through the growth plate until the needle can be felt giving way. Once inserted into the bone ∼2–3 mm, attempt to move the needle slightly from side to side. When the needle is in the tibia, it will not be easily moved. If it moves freely from side to side, the needle is most likely embedded primarily in muscle and the insertion will need to be repeated. When the needle is accurately placed inside the marrow cavity of the tibia, inject the cell suspension slowly and then withdraw the needle using the same drilling motion used to enter the bone. In the event that the needle becomes clogged when penetrating or drilling through the top of the tibia, remove the original needle and re-insert a new needle, trying to follow the route created by the first needle. Once complete, use the same technique to inject the contralateral tibia with sterile PBS or desired vehicle to serve as a sham control. Bleeding rarely occurs; however, if blood does appear, apply pressure at the site. Administer an analgesic as per institutional guidelines, place the mouse on a heating pad and monitor until active.

Inoculation of breast cancer cells in the proximal tibia of a mouse. (a) After sterilizing the hind limbs of an anesthetized mouse, bend the knee to nearly 90°. While holding the hind limb between the thumb and index finger, locate the patellar ligament between the knee and the tibia, which should be visible through the skin as a white longitudinal structure. (b) Holding the needle parallel to the tibia in the dominant hand, push the needle through the center of the patellar ligament and into the proximal end of the tibia. Resistance will be felt once the needle reaches the bone. Twist the needle slightly to drill through the growth plate until the needle can be felt giving way. Once inserted into the bone approximately 2–3 mm, inject the cell suspension slowly and then withdraw the needle using the same drilling motion used to enter the bone.

Although cells can be inoculated into the tibia at any age, it is more difficult to penetrate the tibia in older animals once the growth plates have mineralized (>8-week old). On the other hand, very young mice can present injection difficulties due to the small size of the tibia; most laboratories therefore select 4- to 6-week-old mice for the intra-tibial inoculation route. If the mouse strain selected is furred, shave or wet the hair on the hind limb prior to the inoculation in order to better visualize the patellar ligament and other landmarks in the knee.

Materials for intra-tibial inoculation

Anesthetized mouse (immune compromised if using human cells)

Cellular suspension

0.1 cc Hamilton syringe, 25–27G, 0.5 inch

Betadine and alcohol swabs

Water recirculating heating pad

Analgesic