The overall goal of this procedure is to inject tumor cells in a manner that will induce tumor cell growth in bone and subsequent bone destruction. This is accomplished by first re bending tumor cells in cold PBS and placing them on ice. Next, an appropriate number of cells is injected into the left cardiac ventricle or into the tibia of an anesthetized mouse.
Then bone tumor growth and bone parameters are longitudinally monitored via multiple imaging techniques. Finally, endpoint quantification of tumor and bone parameters is carried out. Ultimately, a combination of in vivo and ex vivo fluorescence or luminescence 2D radiography 3D.
Micro CT and histology are used to show tumor growth and changes in the bone microenvironment at the cellular and growth structural levels. These methods can help answer key questions in the field of cancer metastasis, such as what factors in the bone allow for metastatic colonization of breast and prostate cancer. Demonstrating the cell preparation will be Alisa Merkel, a research assistant in my laboratory, Maintain cells for less than one week.
That harbor the GFP tagged M-D-A-M-B 2 3 1 clone in DMEM containing 10%FBS and 0.1 milligram per milliliter. Penicillin streptomycin at 37 degrees Celsius in five to 8%Carbon dioxide massage cells at 80 to 90%confluence and replace it to one to 10 dilution. To prepare cells for inoculation trypsin eyes them at 80 to 90%confluence with 0.15%tripsin EDTA using 10 milliliters.
Ice cold DMEM with 10%FBS immediately transfer the cells to a 50 milliliter conical tube and centrifusion for five minutes. Re suspend the pellet in a 50 milliliter conical tube in 25 milliliters. Ice cold PBS without calcium and magnesium and count the cells, repel the cells and resuspend an ice cold PBS at a concentration of 10 to the six cells per milliliter for intracardiac inoculation and 250, 000 cells per milliliter for intra tibial injection.
Four to six week old female immune compromised mice that have been fed a Tela 29 20 x diet five to 10 days before injection are used for these experiments. After anesthetizing the mouse in a chamber with 2.5%isof fluorine, move the mouse under a ventilated hood to a nose cone and position the mouse on its back. Use a 10%povidone iodine solution to wash the chest, followed by 70%Ethanol.
Mark the mouse's chest for injection midway between the sternal notch and top of the xiphoid process and slightly left of the sternum. Next, using a 300 microliter 28 gauge half inch insulin syringe. Draw a small bobble of air to create space between the plunger and meniscus and draw up 100 microliters of cells while holding the skin of the mouse tort with one hand.
Keep the needle upright and insert it successful insertion into the left Cardiac ventricle should result in a distinct bright red pulse of blood in the syringe. At this depth, carefully depress the plunger of the syringe without significant movement of the needle to avoid puncturing the heart or spilling cells into the chest cavity. To reduce dripping of cells into the chest cavity, pull up slightly on the plunger to create a small amount of negative pressure.
Then pull the needle directly outta the chest while being sure to avoid tilting the needle during removal, which can tear the lining of the heart and cause bleeding. Apply gentle pressure over the injection site to reduce bleeding for about one minute. While removing the nose cone, move the mouse to a heating pad until it is fully conscious.
Immediately before the intra tibial procedure, inject the mouse with bup printex. After anesthetizing the mouse and placing it in a nose cone. Clean both legs with 10%povidone iodine, followed by ethanol with a four finger and thumb.
Gently grasp the lateral malleolus, medial malleolus and lower half of tibia. Then bend the leg so that the knee is visible and accessible. Using 70%ethanol, wet the skin to increase the visibility of the underlying patella ligament, which should be visible as a distinct thick white line while firmly grasping the ankle.
Insert a 28 gauge half inch needle under the patella through the middle of the patella ligament and into the anterior intercondylar area at the top of the tibia. Use steady firm pressure with a slight drilling action to carefully guide the needle through the growth plate. Upon penetrance of the tibial growth plate, the needle will encounter markedly less resistance.
Use a gentle lateral movement of the needle to ensure it is in the tibia and through the growth plate. Movement will be limited if the needle is in the proper place within the tibia. Slowly depress the plunger to inject 10 microliters of cell solution.
Little to no resistance should be felt at this point. Then slowly extract the needle. Remove the mouse from anesthesia and keep it on a heating pad until it recovers.
Monitor the mouse two to three times per week. A variety of imaging techniques such as luciferase, GFP fluorescence, and x-ray are carried out on anesthetized mice. The fluorescence indicates areas of tumor.
A estron scan is used to locate lesions. The lesions correlate with the fluorescence images. Further details about imaging techniques can be found in the written portion of this protocol.
21 to 35 days after injection. Sacrifice the mice using an iacuc approved method and store the bones in 4%Formalin perform ex vivo analysis using micro CT and histo morpho with hematin and DSN staining on the isolated bones. Details can be found in the written protocol following intracardiac or intra tibial inoculation of tumor cells.
Mice are monitored weekly by radiography and fluorescence or luminescence. Lesions normally become apparent by x-ray between week two and three. While they may be visible by fluorescence and luminescence as early as week two, depending on the model as shown here, lesions are visible as dark holes in the bone and may become more pronounced with time lesions Were quantified using metamorph analysis of radiographs and fluorescence.
Were quantified using maestro imaging acquisition and analysis software. In this figure, bone loss is quantified with micro CT analysis. Bone structural and mineral data from micro CT measurements is rendered as 3D images of healthy bone and a tumor bearing bone where areas of bone destruction are shown as a void area.
Histo morph geometry was then performed on the samples to determine the tumor burden and bone volume to tissue volume. While attempting these procedures, it's important to be patient and to carefully lay out each mouse, monitor the mice closely. If they start to die, it's likely due to cell clumping and you should prepare new cells immediately.
The worst thing you can do is to rush the procedures or to get frustrated. Remember, with more practice and time, you will get better and faster After their development. These techniques paved the way for cancer researchers to study bone metastasis in vivo.
