Acellular and Cellular Lung Model to Study Tumor Metastasis

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Transcript

This method can help answer key questions related to tumor progression. The ex vivo acellular and cellular lung model allows the formation of primary tumor that grows over time and forms circulating tumor cells and metastatic lesions. The main advantage of this technique is that it allows for study of cancer progression in an abbreviated time and growth of different types of cancer cells.

The implications of this technique extend to our personalized therapy as tumor cells from patients can be used in the model to assess the efficacy of therapeutic drugs including immune cells. Begin with a deeply anesthetized rat. Shave the chest and abdomen, and wipe the skin with a chlorhexidine swab.

After opening the thoracic cavity, perform a bilateral thoracotomy by cutting the anterior side of the rib cage on the left in the right side of the diaphragm. Hold the rib cage up and inject two milliliters of 1000 units per milliliter heparin into the right ventricle of the beating heart using a 27 gauge needle to prevent formation of blood clots in the lung. After removing the rib cage, place an 18 gauge needle in the left ventricle as a vent to allow blood out, then use a 25 gauge needle to inject 15 milliliters of 12.5 units per milliliter of heparinized PBS into the right ventricle.

Next, cut the trachea at the base of the thyroid and carefully remove the descending aorta at the level of the hemiazygos vein and branches of the aorta at the arch. After removing the heart-lung block from the thoracic cavity, cut away half of the right and left ventricles and then place a custom made 18 gauge stainless steel needle cannula through the right ventricle into the main pulmonary artery. Secure the cannula with the 2-0 silk tie and carefully expose the right ventricle and atrium.

Begin by running heparinized PBS at a physiologic perfusion pressure of 30 millimeters mercury until it flows freely into the bottle of the decellularization unit, then attach the heart-lung block to the decellularization unit at the pulmonary artery end through the pulmonary artery cannula. After 15 minutes of washing with heparinized PBS, replace the heparinized PBS bottle with one containing 0.15%sodium dodecyl sulfate and deionized water, and perfuse the lung for two hours for decellularization. After the decellularization, perfuse deionized autoclaved water through the lung scaffold for 15 minutes followed by 1%non-ionic detergent in deionized water for 10 minutes.

Finally, replace the bottle with 500 milliliters of autoclaved PBS supplemented with antibiotics. Run the pump at six milliliters per minute and perfuse the lungs for 72 hours after which time the acellular lung is ready for immediate use. Spray 70%ethanol on the pump cartridge and set up the oxygenator by connecting the outflow end of the oxygenator to the pump tube and the other end to a 1.5 foot tube in the bioreactor.

Set up the bioreactor bottle in a biosafety cabinet under aseptic conditions. Attach a three way stopcock to the head of a female Luer lock to control the flow through the pulmonary artery, then connect a one way stopcock to another female Luer lock bulkhead for cell seeding through the trachea. Connect two inch tubing on the outside and six inch tubing on the inside of the bottle to circulate the medium.

Prepare the tubing to connect the bioreactor bottle to the oxygenator. Fit a male Luer lock to a two foot tube and attach a female Luer lug style T to provide accessibility, and attach a male Luer lock and a female Luer lock to two foot tubes. Add cell culture medium and transfer the bioreactor to the incubator.

Connect the oxygenator tube to the two open ends to make it a closed loop bioreactor. Prerun the pump at six milliliters per minute to fill the oxygenator and tubes with culture medium so that no air bubbles exist in the tubing. Once the tubes are filled with medium, close the stopcock and disconnect the oxygenators from both ends of the bioreactor bottle.

Put the bioreactor bottle in the biosafety cabinet, pass the culture medium through the stopcock, and attach the pulmonary cannula of the lung scaffold. If working with the cellular lung model, skip the decellularization. The harvested lung can be dyadically set in the bioreactor.

Pass culture medium through the one way stopcock again to remove any air and tie the tracheal cannula by a silk thread to the trachea, then close the bioreactor bottle cap. Transfer the bioreactor to the incubator and start the pump. Run culture medium for 10 to 15 minutes to make sure all lobes get inflated and the lung looks in good shape.

Modify the acellular or cellular lung scaffold as follows for the metastatic model. Open the bioreactor cap with the hanging lung scaffold and use a Luer lock syringe to pass five milliliters of culture medium through the trachea. Use angular forceps to pass 2-0 silk through the trachea at the bifurcation point.

Tie the trachea going to the right lung at the bifurcation point and check the free flow of media through the trachea to the left lung by passing culture medium. Prepare a 50 milliliter volume of lung cancer cells such as A549 or H1299 in RPMI 1640 complete culture media at a concentration of two to three million cells per milliliter. Set a 20 milliliter Luer lock syringe on top of the tracheal cannula and add 15 milliliters of cells to the syringe.

Allow the cell suspension to pass through the lung lobes by gravity. Add the rest of the cells before any air bubbles pass through. Once seeded, remove the syringe and wait for 15 minutes, then add fresh medium to the bioreactor.

Return it to the incubator and, after removing any air bubbles, perfuse the lung scaffold at six milliliters per minute. Histopathology of an intact lung shows the alveoli and pneumocytes. As seen here, decellularization leads to the complete removal of the lung cells, and histopathology of acellular lungs reveals an intact basement membrane.

Movat's pentachrome and elastin stain of a decellularized lung reveals the presence of collagen, fibrin, and elastin with intact vasculature and bronchus. DNA analysis of the decellularized lung shows a reduction in DNA content of more than 99%The acellular lung can be used directly in the bioreactor for cell culture. A histological analysis shows the presence of tumor growth.

While attempting this procedure, it is important to remember to inject the heparin in the right ventricle of the heart. After this development, this technique paved the way for researchers in the field of cancer research to explore the genetic and epigenetic alterations leading to metastasis of tumor cells in the lung cancer.

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Journal (Cancer Research)

Journal (Cancer Research)

Acellular and Cellular Lung Model to Study Tumor Metastasis

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