This method can be used to answer key questions about the role of the bone marrow micro-environment and the survival of engrafted myeloma tumors. The main advantage of this procedure is that it can be applied to longitudinal anti-drug studies and used to assay multiple biochemical pathways in a non-invasive manner. On the day of the experiment first wash the 8226 Luciferase transfacted cells three times in ice cold PBS at 200 RCF for five minutes per centrifigation and resuspend the pellet in fresh ice cold PBS at a five times ten to the sixth cells per 200 microliters of ice cold PBS per mouse.
Next confirm a lack of response to toe pinch in an anesthetized four to six week old NOG mouse and apply ophthalmic ointment to the animal's eyes. Load the cells into a one milliliter insulin syringe equipped with a 26 gauge needle. Inject the entire volume of cells into the tail vein of the animal, before returning the mouse to it's cage, monitoring until full recovery.
Ten to twenty days post challenge, inject each engrafted animal with 200 microliters of in vivo grade D-luciferin substrate in sterile saline intraperitonealy. Place the anesthetized animals in the supine position in a small animal imaging system within 5 to 10 minutes of the injection. Measure the average radiance in selected regions of interest within the corresponding imaging software.
For targeted therapy of the 8226 Luciferase tumors, After measuring the baseline bioluminescence in each animal as just demonstrated, randomize the animals into treatment groups and treat each mouse with a series of 200 microliter IP injection of Temsirolimus or a saline control. Measure the Luciferase activity two times per week and plot the changes in bioluminescence over time. To measure changes in the tumor metabolism, first remove the food from the home cage of the mice for 24 hours to avoid excess nonspecific radiolabeled Fludeoxyglucose uptake.
The next day dilute 500 to 100 microcuries of 18 Fluorine radiolabeled Fludeoxyglucose probe in sterile saline to a final volume of 100 microliters per mouse and record the time and activity of the probes with a dose calibrator. When the probes are ready place the first anesthetized animal on a heating pad with the head facing away and use a shielded one milliliter insulin syringe equipped with a 26 gauge needle to inject 100 microliters of the probe into the tail vein. Measure the residual radioactivity in the needle and syringe with the dose calibrator and note the activity and time.
Then measure the radiolabeled Fludeoxyglucose activity in selected regions of interest that correspond to the engrafted tumors in a small animal PET CT imaging system. A successful bone marrow tumor engraftment can be confirmed by bioluminescence imaging as demonstrated. Serial imaging of multiple animals can be used to visualize the distribution of the bone marrow engrafted multiple myeloma tumors.
Additionally optical imaging x-ray analysis shows a quick and non-invasive determination of the exact location and distribution of the multiple myeloma tumors within the mouse skeleton. The bioluminescence produced by these engrafted tumor cells can be serially and noninvasively measured to assess changes in tumor growth. Furthermore, the survival of mice treated with the mTOR inhibitor Temsirolimus can be monitored and positron emission and computed tomography analysis for tumor radiolabeled Fludeoxyglucose uptake can be used to demonstrate Temsirolimus mediated changes in glucose metabolism.
While performing this procedure it's important to inject the cells IEV. We've found that failure to inject the cells into the vein results in the formation of non-marrow engrafted tumor cells near the site of injection. Following this procedure, other methods like immunohistochemistry and micro-CT can be used to address questions about the tumors impact on bone architecture and the non-tumor cellular and molecular components of the marrow environment.
