Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

We describe a relatively simple method for ex vivo live imaging of the tumor cell-stroma interactions within lung metastasis, utilizing fluorescent reporters in mice. Using spinning-disk confocal microscopy, this technique enables visualization of live cells for at least 4 hr and could be adapted to study other inflammatory lung conditions.

The overall goal of this procedure is to visualize dynamic tumor cell stroma interactions within lung metastases.This method can help answer key questions in the cancer metastasis field, such as how are metastasis established.and what cell types within the micro-environment are involved in this process?The main advantage that this technique offers is that this is a fast and easy method for ex vivo lung imaging that enables visualization of live cells for at least four hours.This technique may have furthered our understanding of mechanisms of action of various targeted therapeutics for treatment of lung metastasis.We first had an idea for this method when we were examining metastasis by lung section imaging and were struggling with extensive cell death as a result of this procedure.To prepare the lungs for ex vivo live imaging, begin by immobilizing the mouse on a polystyrene foam lid covered with a piece of soaker and sterilize the animal with 70%ethanol.Next, using surgical scissors make a transverse epigastric incision through the skin, followed by a similar incision through the peritoneum.Then, moving the dissection board into a vertical position, cut the descending aorta, so that the blood pools into the abdomen and not into the chest cavity.Snip the small opening in the diaphragm to release the vacuum.Then, cut along the rib cage to excise the diaphragm and to visualize the lungs.Using surgical scissors, cut the skin up to the trachea over the rib cage leaving the ribs intact.Then, separate the skin from the rib cage, and remove the surrounding connective tissue to expose the trachea, taking care not to damage the trachea itself.Snip a small opening approximately one millimeter in diameter in the exposed trachea, parallel to the cartilaginous rings as close to the larynx as possible without cutting completely through the trachea.Then, gently insert a 20 gauge needle four to five millimeters into the trachea without any counter-force.The end of the needle should be visible through the trachea.Fill a syringe with 400 microliters of 37 degrees Celsius 2%low melting temperature agarose, and then stabilize the needle with forceps.Keeping the dissection board vertical, slowly instill the entire quantity of warm agarose through the needle into the lungs.Once the lungs are inflated, detach the syringe while keeping the needle inside the trachea and pour approximately 50 milliliters of 20 degrees Celsius PBS over the lungs to help set the agarose.When the agarose has solidified, remove the needle and use forceps to close the trachea to prevent the leakage of any non-solidified agarose.Now, open the chest further by sternotomy and grasp the trachea with forceps.Then, using a pair of surgical scissors, cut through the trachea completely.To remove the lungs, gently pull the trachea up, cut away the connective tissue and esophagus, and pull the lungs completely out of the chest cavity.Immerse the tissue in 37 degrees Celsius RPMI 1640 to wash off the excessive blood.Using scissors and forceps, cut the lobes