This protocol allowed to answer an important question in the cancer metastasis field. Including, how tumor cells colonize the lung? How tumor cells remain dormant?
And what induce them to exit dormancy and eventually form metastasis. The main advantage of this protocol is that the dynamic metastatic process can be directly studied in vivo, in real time, in a real microenvironment. This method can provide insight into several pulmonary diseases, but it's particularly useful for understanding the behavior individual tumor cells disseminating to the lung, a common site of metastasis.
To begin with use a 2-0 silk suture to tie a knot at the base of a 22 gauge catheter, leaving 2 inch long tails. Once the mouse is completely anesthetized, move the mouse to the operation table, intubate the mouse, and then secure the intubation catheter by tying the 2-0 silk suture around the mouse's snout. Connect the ventilator to the intubation catheter.
Using paper tape, secure the front cranially and hind limbs caudally to the heated surgical stage. Place a paper tape along the length of the mouse's back to maximize exposure to the surgical field. Lift the skin with the forceps and make an approximately 10 mm circular incision at about 7 mm to the left to the sternum and about 7 mm superior to the subcostal margin.
Excise the soft tissue overlying the ribs. Elevate the sixth and seventh rib using forceps. Use a single blade of the blunt micro dissecting scissors with the rounded side towards the lung to carefully pierce the intercostal muscle between the sixth and seventh ribs for entering the intrathoracic space.
Delicately discharged the compressed air canister at the defect to collapse the lung and separate the lung from the chest wall. Fire the compressed air in short bursts to prevent iatrogenic lung injury. Place the biopsy punch over the cutting tool and carefully maneuver the cutting tool's base through the intercostal incision.
Orient the cutting tool base parallel with the chest wall and punch a 5 mm circular hole through the rib cage. Using a 5-O silk suture, place a purse string stitch, approximately 1 mm from the hole, circumferentially. Next, position the window frame into the chest wall with the edges of the circular defect captured within the windows groove.
Tightly tie the 5-O silk suture to securely lock the implanted window. Apply a steady and gentle stream of compressed air for about 10 to 20 seconds to dry the lung. Use the forceps to grip the window frame by its outside edge and gently lift to ensure separation of the lung from the undersurface of the window frame.
Dispense a thin layer of cyanoacrylate adhesive along the undersurface of the optical window frame. Gently but firmly press the optical window frame onto the lung tissue for 10 to 20 seconds for attachment. Dispense a 5 mm drop of adhesive on a rectangular cover slip.
Use the vacuum pickup tool to pick up a 5 mm cover slip. Dip the undersurface of the cover slip into the adhesive, and then scrape off the excess adhesive three times against the side of the rectangular cover slip to make a very thin layer of adhesive on the cover slip. Carefully position the cover slip at an angle inside the recess at the center of the optical window frame, just above the lung tissue.
Briefly clamp the ventilator to generate positive pressure, hyper inflating the lung. Orient the cover slip parallel to the lung tissue, using the rotating motion to create direct deposition between the lung surface and the undersurface of the cover slip. Maintain gentle pressure for approximately 25 seconds to set the cyanoacrylate adhesive.
Separate the cover slip from the vacuum pickup tool with forceps. Create a purse string stitch, circumferentially, less than 1 mm from the cut edge of the skin incision using 5-O silk suture. Tuck any excess skin underneath the outer rim of the window frame before tying the window tightly with locking knots.
Dispense a small amount of adhesive at the metal glass interface to ensure an airtight seal between the cover slip and window frame. Attach the sterile needle to a 1 ml insulin syringe. Insert the needle below the xiphoid process, advancing toward the left shoulder, entering the thoracic cavity through the diaphragm.
Gently draw back the syringe to remove any residual air from the thoracic cavity. The multiphoton intravital imaging of a single lung region showed that the microvascular could be relocated over three consecutive days. The definable branch point from a single vessel was identified each consecutive day.
The unlabeled erythrocytes created shadows when flowing in larger vessels. The angle of the branch points relative to the vessel can be used to calculate the erythrocyte flow rates. Erythrocyte speed was also quantified by injecting the mouse with fluorescent microspheres and their track lengths were measured divided by the frame acquisition time.
The stationary and flowing microspheres were distinguishable. The fate of the disseminated tumor cells was tracked with serial imaging over several days, using the window for high resolution imaging of the lung. The tumor cell arrived at and lodged in the lung vasculature on day one.
However, the cell was not detected in the lung vasculature on the second and third days, indicating recirculation or extravasation. The culminating steps of metastatic progression in the lung were visualized starting with the tumor cell arrival, the extravasation of tumor cell into the lung parenchyma and proliferation to form macro metastasis. Take care to avoid trauma to the lung during optical window frame placement.
The lung is prone to bleeding, which will later impede cover slip adherence during placement. Other methods that can be performed following this procedure include intravital microscopy, which when employed with a modified approach to microcartography, allows precise relocalization of areas of interest for repeated imaging. This protocol allowed the visualization of the lung in the investigation of a key question about the mechanism of metastasis.
This study carry the potential to rebuild a new treatment for cancer patients.
