This method can help to produce an ideal model that closely mimics the natural progress of bone metastasis of prostate cancer cells in vivo. This technique has the advantage of a bone metastasis and representing the natural bone metastasis process of prostate cancer cells in vivo. This technique benefits the exploration of the molecular mechanisms and investigation of in vivo therapeutic effects for prostate cancer bone metastasis.
On the day of the injection, take 80 to 90%confluent luciferase-labeled PC3 cells cultured in a 10 centimeter cell culture dish and start preparing the cells for injection by washing them two times with cold sterile PBS. Trypsinize the washed cells with 1.5 milliliters of 0.25%trypsin for three minutes, following which quench the trypsin by adding six milliliters of F12 medium containing 10%serum and collect the cells into a 15 milliliter centrifuge tube. Transfer 20 microliters of the collected cell suspension into a cell counting plate and calculate the cell concentration using an automatic cell counter.
Next, centrifuge the cells at room temperature for five minutes at 800 G.Discard the supernatant using a pipette and resuspend the cell pellet in F12 medium to achieve the desired final cell density. Now carry the cells to the surgery room and perform the cell injection within two hours. Keep the cells on ice until injection.
To begin the surgery, place the anesthetized mouse in a supine position and immobilize both upper limbs by perpendicularly stretching them away from the midline. Using surgical adhesive tape, immobilize the mouse from the abdomen down without pressing hard and displacing the internal organs. Disinfect the skin of the thorax using a 70%ethanol swab.
Next, palpate down the middle of the anterior chest wall to locate the mouse xiphoid process in the depression at the lowest end of the mid sternum. Label the most inferior point of the xiphoid process. Then by palpating up from the middle of the anterior chest wall, locate the mouse jugular notch in the central depression at the upper border of the manubrium and mark the most inferior point of the jugular notch.
Now locate and mark the midpoint between the previous two marks, slightly towards the right and just over the heart in the third intercostal space. This is the injection point. Thoroughly mix the cell suspension before loading 200 microliters of the suspension into a one milliliter syringe mounted with a 26 gauge needle.
Leave some air in the syringe between the plunger and the suspension to allow the entry of pulsating blood during the injection. To perform the injection, vertically insert the needle through the injection site. If the needle tip is correctly inserted into the left ventricle, bright red pulsating blood will be visible in the needle hub.
Retry injecting if blood pulsation is not visible or blood is clotting. Once the needle is inserted properly, inject 100 microliters of the cell suspension very slowly over 40 to 60 seconds with a stable hand. After completion of the injection, retract the needle while applying pressure on the injection site with a dry cotton swab for 15 seconds to ensure hemostasis.
Return the mouse to the clean cage and monitor the animal until it completely recovers from anesthesia. Bioluminescence imaging performed 24 hours after injecting luciferase-labeled PC3 cells into the mouse showed signals from the entire body of the animal indicating the presence of cancer cells in the systemic circulation. Metastatic lesions appeared in the hind limbs of the mouse two weeks after the injection.
The metastatic lesions became larger as time progressed and started appearing in other sites. The prostate cancer cell-induced metastatic lesions in the bones were further detected using x-ray imaging which showed bone destruction in the proximal tibia of the animal. The same was also confirmed by micro CT scanning.
The metastatic lesions were further confirmed in paraffin embedded tissues by hematin and eosin staining. Accurate injection site is very important to ensure the success rate of this model. This can be achieved by practice.
