The overall goal of these experiments is to determine to role of pulmonary alveolar macrophages in cancer metastasis using a syngenaic model of breast cancer. This method can answer some of the key questions in tumor immunology and cancer metastasis fields, such as why lungs are one of the most common target are hepathogenic cancer metastasis. The main advantage of this method that it uses very well-established breast cancer model with very specific method of the pulmioary alveolar microphages.
Demonstrating this procedure will be Surya Kumari Vadrevu, a research associate in my laboratory. On the day of tumor cell injection, first place a shaved, anesthetized mouse on a surgical pad. Next, aspirate one time 10 to the fifth tumor cells in 100 microliters of PBS into a zero point five milliliter insulin syringe equipped with a 29-gauge needle.
Then use the thumb and index finger to lift the skin near the second and third nipple, and insert the needle under the skin into the mammary fat pad just below the third nipple. Slowly subcutaneously inject the cells to form a bubble under the skin, and return the animal to its cage with monitoring until it is fully recovered. Four to five days post inoculation, to measure the tumors, palpagt the tumor site and use a caliper to use both the largest and smallest diameters of the tumors to determine the tumor volume.
To image the injected 41GFP cells, place the anesthetized mouse on a movable stage inside the imager, and image the tumor site by fluorescence microscopy. In a laminer airflow biosafety cabinet, six days after tumor cell injection, vortex the liposomes to evenly distribute the particles, and aspirate 60 microliters of the suspension into a sterile pipette tip. Place the tip near the nostril of the anesthetized tumor-inoculated animal, and slowly release five microliters of the liposome solution, allowing the mouse to inhale the drop.
It is critical to administer the liposome solution slowly while maintaining the appropriate level of anesthesia to allow the animal to breath regularly during the delivery. When the entire 60 microliters has been administered, allow the mouse to fully recover, and return the animal to its cage. To count and score the lung surface metastases, place the lungs under the dissection microscope and focus the objective until a clear view of the lung surface has been obtained.
Then manually count the metasteses on the anterior and posterior sides of both lungs, and image the tumors by fluorescence microscopy. After counting and imaging the metastases, use surgical scissors and forceps to mince the lung and transfer the tissue pieces into a 15-milliliter conical tube containing three milliliters of digestion buffer. Dye test the fragments on a rotating shaker in a 37 degrees Celsius incubator for 40 minutes, and then triterate the slurry to dissociate the tissue.
Next, filter the tissue suspension through a 40-micron strainer to remove any clumps, pressing the larger pieces through the strainer with a syringe plunger as necessary. When a single-cell suspension has been achieved, collect the cells by centrifugation and lyse the red blood cells with ACK buffer for 10 minutes at room temperature. Then, wash the pellet two times in eight milliliters of RPMI.
After the second centrifugation, we suspend the cells in three milliliters of complete RPMI medium for counting, and spin down the cells again. Now dilute the cells to a one times 10 to the sixth cells per 100 microliters of FAX buffer concentration, and transfer 100 microliter eloquotes of cells into individual wells of a V-bottom, 96-well plate. Collect the cells in the bottom of the wells by centrifugation, then flip over the plate to let the buffer drain out.
Block the cells with 100 microliters of CD16 CD32 FC block for 15 minutes at four degrees Celsius, and centrifuse the plate again. Then flip the plate to remove the supernatant, as just demonstrated, and add the antibody cocktail of interest to the appropriate wells. After 30 minutes at four degrees Celsius, pellet the cells by centrifugation, followed by a wash with 200 microliters of FAX buffer.
Then re-suspend the pellets in 200 microliters of fresh PBS and stain the samples with viability dye for 20 minutes at four degrees Celsius. At the end of the incubation, wash the cells in another 200 microliters of PBS, and fix the samples in 100 microliters of one percent paraformaldehyde for storage at four degrees Celsius. Immediately prior to their analysis, transfer the cell suspensions to polypropelene flow cytometry tubes.
The injection of 4T1GFP tumor cells into the mammary fat pad leads to the formation of mouse tumors that recapitulate the metastatic spread of human breast cancer, as evidenced by the rapidly forming metastases observed within the lungs. The stable transfection of 4T1 cells with GFP facilitates the tracking of metastasizing tumor cells and the quantification of the metastatic burden. Routine hastology, in conjunction with digital pathology algorithms also provide a good tool for quantifying and verifying metastases.
Further, multi-color flow cycometric analysis allows the characterization of even rare cell populations, such as CD11b negative, CD11c F80 positive alveolar macrophages. The depletion of which, biclotrenate can be confirmed by the absence of these CD11c F480 positive cells in the dissociated lungs of lyposome-treated animals. After watching this video, you should have a good understanding of how to inject tumor cells into the mammary fat pad, monitor tumor growth and metastasis, and deplete alveolar macrophages, and prepare lung cells for flow cycometric analysis.
