This method can help answer key questions in the tumor biology field about the molecular mechanisms of metastasis. The main advantage of this technique is that the recruitment of tumor cells within a remote organ can be quantitate. Begin by using a non-enzymatic cell dissociation reagent to remove fluorescent protein-expressing Lewis lung carcinoma, or LLC, cells from their culture container according to standard protocols.
Transfer the resulting cell solution into a 15-milliliter conical tube before centrifugation, and centrifuge wash the pellets two times in five milliliters of PBS per wash. After the second wash, filter the cells through a 40-micrometer pore cell strainer, and dilute the suspension to a one times 10 to the six cells per milliliter concentration. Then load the cells into a one-milliliter syringe equipped with a 29-gauge needle, and inject 100 microliters of cells into the tail vein of each eight-to-10-week-old, C57-Black-6, male mouse.
At the appropriate experimental endpoint, use scissors to remove the skin from the ventral surface of each mouse, and cut the ribs and the diaphragm to expose the thoracic cavity. Use a 25-gauge winged infusion needle and tubing to flush 15 milliliters of PBS through the right ventricle, and remove the heart and the thymus. Gripping the trachea with forceps, pull up, dissecting the connective tissues around the trachea, to harvest the lungs.
Then wash the lungs in PBS, and detach one lobe for visualization of the fluorescent cells in situ under a fluorescence microscope. To digest the lung tissue, first dice the caudal lobe with scissors, and place the lung pieces in a 10-milliliter syringe. Close the syringe with the plunger, and aspirate five milliliters of digestion solution into the syringe, moving the plunger in and out of the syringe shaft until the lung tissue is disseminated throughout the solution.
Dispense the lung slurry into a 50-milliliter tube, and digest the tissue on a reciprocal shaker for 15 minutes at 37 degrees Celsius and 150 rpm. At the end of the incubation, triturate the remaining tissue until the lung cells are completely dispersed, and return the tube to the shaker for an additional 30 minutes. Then filter the cells through a 40-micrometer pore strainer, and collect the cells by centrifugation.
To analyze the isolated lung cells by flow cytometry, resuspend the pellet in two milliliters of red blood cell lysis buffer for one minute at room temperature, and collect the cells by centrifugation. Then centrifuge the pellet two times in five milliliters of fresh PBS supplemented with 1%bovine serum albumin per wash. After the second centrifugation, resuspend the pellet in one milliliter of fresh PBS plus BSA, and filter the cells through a new 40-micrometer pore strainer for quantification of the tumor cells by flow cytometry.
The lungs present many GFP-LLC cells two hours after injection, with the vast majority of cells disappearing from the lungs within 24 hours. Note that any fluorescent spots that are detected within the red filter should be excluded from the cell count. To confirm the number of GFP-LLC in the lungs, one of the lobes can be used for flow cytometric analysis as demonstrated.
While attempting this procedure, it's important to remember that a frozen section procedure is a more accurate method for observing the exact location of tumor cells.
