Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The orthotopic transplantation of mouse lung adenocarcinoma cells into syngeneic graft recipients allows the study of tumorigenesis in the presence of a fully active immune system under physiological conditions. Of course, the tumors develop into an immunocompetence mice and this is much more physiological that if you perform a transplant in immunodeficient mice. Gene editing of the tumor cells before a transplantation makes this model a direct and time-saving approach for studying the impact of genetic factors on tumor growth and gene expression profiles.

Compared to inducible autochthonous lung tumor models, this method avoids the extensive breeding of mice, and therefore represents a refinement of previous lung cancer mouse models. Before beginning the procedure, use scissors to remove the end of a catheter needle and push the catheter completely over the end of the needle. After confirming a lack of response to toe pinch, apply ointment to the eyes of an anesthetized, synergetic eight to 12 week old mouse and hook the upper incisors over a suture string across an intubation platform with the chest perpendicular to the suture.

Place a fiber optic cable between the front limbs to illuminate the chest and carefully open the mouth using disinfected flat forceps to extract the tongue. Look for the emission of white light to locate the larynx, epiglottis, and arytenoid cartilages. Once the opening of the trachea is clearly visible, gently slide the catheter into the trachea, up to but not beyond the bifurcation, to guarantee an even distribution of the lung adenocarcinoma cells within the lungs.

Take care that the insertion of the catheter is very smooth. If you feel any resistance, remove the catheter and expose the trachea again to avoid injuring the mouse. Quickly remove the needle from the catheter and check whether the white light can be observed shining through the catheter.

To confirm a proper placement of the catheter within the trachea, attach a one milliliter syringe of water to the catheter. The water in the syringe should move rapidly up and down in time with the breathing. Before delivering the cell suspension, warm the cells by hand and load 50 microliters of cells into the catheter.

As soon as the suspension is aspirated, attach an empty one milliliter syringe to the catheter and dispense 300 microliters of air into the catheter to ensure a complete distribution of the tumor cells within the lungs. Then gently remove the catheter and place the mouse on a heat pad with monitoring until full recumbency. After the appropriate experimental endpoint, soak the carcass in 70%ethanol and secure the animal to a dissection board.

Make a ventral midline incision and gently invert the skin to expose the thoracic wall muscles and the abdominal organs. Use scissors to puncture the diaphragm and to cut the ribs, exposing the thoracic cavity. Cut a small opening in the left ventricle and use a 27 gauge needle to perfuse the lung three times through the right ventricle with six to eight milliliters of ice cold PVS per infusion.

After the last perfusion, the lungs should be completely clear of blood and appear white. After harvesting, use scissors to mince the lung tissue into small pieces. Transfer the lung fragments into a two milliliter microcentrifuge tube containing 1.5 milliliters of lung digestion buffer for a 30 to 60 minute incubation at 37 degrees Celsius, with shaking.

At the end of the digestion, transfer the cell suspension through a 70 micron cell strainer into a 50 milliliter tube and use the end of a sterile 10-milliliter syringe plunger to press any remaining tissue fragments through the filter. Rinse the strainer with 15 milliliters of PVS, supplemented with 2%FCS. And collect the cells by centrifugation.

Re-suspend the pellet in one milliliter of ammonium chloride potassium lysis buffer for a five minute incubation at room temperature and centrifuge the cells again. Then re-suspend the white blood cell pellet in one milliliter of PVS supplemented with 2%FCS and stain the cells for flow cytometric analysis according to the experimental protocol. As expected, the survival of the recipient mice is correlated to the number of engrafted cells.

And the lungs harvested at the time of death demonstrate an even distribution of tumor nodules throughout all of the lobes. When comparing sections from lungs harboring autochthonous tumors, with tumors following orthotopically transplanted lung tumor cells, no morphological differences are observed. Flow cytometric analysis of PD-L1 expression of lung cells isolated three weeks after orthotopic tumor cell transplantation into immunocompetent mice, as just demonstrated, reveals up-regulation in PD-L1 cell surface marker expression, compared to in-vitro cultured lung tumor cells.

Following orthotopic transplantation, other analytical methods can be applied, including immunohistochemical analysis, and mRNA profiling to address additional experimental questions.