Name: Acellular and Cellular Lung Model to Study Tumor Metastasis (Video) | JoVE Journal
Uploaded: 2018-08-19T14:00:00.000+00:00
Duration: 8 min 31 s
Description: Learn an ex vivo acellular and cellular lung model to study tumor metastasis using rat lung scaffolds. This technique enables isolation of primary tumors, circulating tumor cells, and metastatic lesions for cancer research.

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5 minutes

Min P. Kim received grant support from the Second John W. Kirklin Research Scholarship, American Association for Thoracic Surgery, Graham Research Foundation, Houston Methodist Specialty Physician Group Grant, and Michael M. and Joann H. Cone Research Award. We thank Ann Saikin for the language editing of the manuscript.

The protocols for animal experiments were approved by the Institutional Animal Care and Use Committee at the Houston Methodist&#160;Research Institute and carried out in accordance with all regulations, applicable laws, guidelines, and policies.
1. Rat Lung Harvest
<ol>
	<li>Anesthetize a 4- to 6-week-old male Sprague-Dawley rat by an intraperitoneal (IP) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg) in its flank. Ensure anesthesia by checking for an absence of movement when the hind limb toe is pinched with forceps.&#160;</li>
	<li>After 10 min, shave the chest and abdomen and wipe the skin with a chlorhexidine swab.</li>
	<li>Remove the skin by picking it up with forceps and by incising&#160;it with a disposable scalpel. After opening the thoracic cavity by&#160;incising&#160;with scalpel, perform a bilateral thoracotomy by cutting the anterior side of the rib cage on the left and the right side of the diaphragm with scissors. 
	NOTE: This is a non-survival surgery.</li>
	<li>Hold the rib cage up and inject 2 mL of heparin (1,000 units/mL) into the right ventricle of the beating heart using a 27 G needle. 
	NOTE: This will prevent any formation of blood clots in the lung.</li>
	<li>Completely remove the rib cage with scissors and place an 18 G needle in the left ventricle as a vent, until the blood comes out. Then, inject 15 mL of heparinized phosphate-buffered saline (12.5 units/mL; heparinized PBS) into the right ventricle using a 25 G needle.</li>
	<li>Cut the inferior and superior vena cava, two large veins on the right side of the heart, and flush the lungs with an additional 10 mL of heparinized PBS in the right ventricle using a 27 G needle. 
	NOTE: The inferior vena cava is visualized under the bottom of the heart as a big vessel filled with red blood. Similarly, the superior vena cava is visualized in the upper part of the heart as a big vessel.</li>
	<li>Cut the trachea at the base of the thyroid. Carefully remove the descending aorta at the level of the hemiazygos vein and branches of the aorta at the arch.</li>
	<li>Take out the heart-lung block from the thoracic cavity and the rest of the rat body.</li>
	<li>Perform ventriculotomy by cutting half of the right and left ventricles and place a custom-made prefilled 18 G stainless-steel needle cannula through the right ventricle into the main pulmonary artery (PA).</li>
	<li>Secure the cannula with a 2-0 silk tie and carefully expose the right ventricle and atrium.</li>
	<li>Place a female Luer bulkhead in the left ventricle and secure it with a 2-0 silk tie.</li>
	<li>Flush the heart-lung block with 20 mL of heparinized PBS through the PA cannula and place it in a 50 mL tube containing culture media or heparinized PBS. 
	NOTE: For a cellular model, go to step 3.1 to set up the bioreactor. For an acellular model, proceed to step 2.1 for the decellularization.</li>
</ol>
2. Lung Decellularization
<ol>
	<li>Preparation of the decellularization unit
	<ol>
		<li>Prepare each decellularization chamber/bottle by piercing two holes in the bottle cap and snapping a female Luer in the holes (Figure 1A and 1B). Secure it with a black nylon ring on the other side. Similarly, drill a single hole in another bottle cap for the intravenous set attachment (Figure 1C). 
		NOTE: Figure 1A and 1B are of the same bottle caps from two different sides. Figure 1C is of a different cap with a single hole as stated above in step 2.1.1.</li>
		<li>Attach a 0.5-inch tube (at the pulmonary artery or PA end) and 6-inch tubes (Figure 1J) to a female Luer from inside the bottle caps (Figure 1A and 1B) and attach a male Luer lock at the ends. Set the lock with a 500 mL bottle as the decellularization chamber/bottle.</li>
		<li>Connect one end of two 2 ft tubes (which are connected with a male Luer lock) to either ends of the pump tube (Figure 1H), which is attached to the pump head (Figure 1E and 1F) by a cartridge (Figure 1G) with a female Luer lock (Figure 1I).</li>
		<li>Connect the other ends of the 2 ft tubes (from step 2.1.3) each to a female Luer lock head on the decellularization chamber/bottle cap (Figure 1L).</li>
		<li>Set up a 250 mL heparinized PBS bottle (with 200 mL of PBS) by hanging an inverted bottle on a stand with the proximal end of an intravenous set (Figure 1D) inserted in the bottle cap and the distal end replacing the 2 ft tube connected to the decellularization chamber/bottle at the PA end (Figure 1B). Connect this 2 ft tube to the discard bottle. 
		NOTE: The bottles are set in cardboard, as shown in Figure 1K. The intravenous tubes come out of the hanging bottles to dispense reagents in the lung scaffold.</li>
	</ol>
	</li>
	<li>Lung decellularization process
	<ol>
		<li>Run the heparinized PBS at a physiologic perfusion pressure of 30 mm Hg (Figure 1K) until it flows freely in the decellularization chamber/bottle and, then, attach the heart-lung block to the PA end through the PA cannula.</li>
		<li>Keep running the pump continuously, so that any excess buffer/solution inside the decellularization chamber/bottle gets discarded.</li>
		<li>Run the heparinized PBS through the PA for 15 min at a perfusion pressure of 30 mm Hg for the initial wash (Figure 1K).</li>
		<li>Replace the heparinized PBS bottle with 0.1% sodium dodecyl sulfate (SDS) in deionized water and perfuse the lung for 2 h for the decellularization.</li>
		<li>After the decellularization, perfuse deionized autoclaved water through the lung scaffold for 15 min.</li>
		<li>Then, perfuse 1% non-ionic detergent in deionized water for 10 min.</li>
		<li>Replace the decellularization chamber/bottle with 500 mL of autoclaved PBS supplemented with 1x antibiotics (penicillin-streptomycin amphotericin), keeping the hanging lung intact with the cap inside the bottle.</li>
		<li>Discard the intravenous set, put the 2 ft tube attached to the discarding container back to the pulmonary artery end of the decellularization chamber, and run the pump at 6 mL/min (Figure 1L).</li>
		<li>Perfuse the lungs for 72 h and keep changing the PBS with antibiotics every 12 h. 
		NOTE: The acellular lung is ready for immediate use. It can be stored at -80 &#730;C until needed. If pink spots or blood clots can be visualized clearly in the lung lobes, discard the lung. It is possible to randomly test for DNA or histopathology. A complete acellular lung will not show cells and 99.9% of the DNA will be washed off. 
		NOTE: When working with a cellular lung model, skip the decellularization. The harvested lung (step 1) can be directly set in the bioreactor.</li>
	</ol>
	</li>
</ol>
3. Bioreactor Set-up
<ol>
	<li>Prepare the bioreactor bottle and the items required for the set-up.
	<ol>
		<li>Drill three holes in one 500 mL bottle cap at an equal distance and fix a female Luer in all three holes, using black nylon rings (Figure 2A and 2B). 
		NOTE: In two holes, the Luer lock ends face the outside, while one end faces the inside of the bottle.</li>
		<li>Cut a 6 in pump tube, a 2 in tube, a 6 in tube, a 1.5 ft tube, a 2 ft tube, and a 10 ft oxygenator tube per bioreactor.</li>
		<li>Cut an 18 G stainless-steel needle and, again, connect the ends with biocompatible tubing for a tracheal cannula.</li>
		<li>Wrap the oxygenator tube, with Luer lock connectors at the ends, on a solenoid wire mesh using autoclave tapes (Figure 2C).</li>
		<li>Autoclave the above-mentioned items and keep them in UV light for 10 min.</li>
	</ol>
	</li>
	<li>Set up the pump inside the regular cell culture incubator (37 &#176;C, 5% CO2) with proper spacing between the trays to easily fit a 500 mL bottle.</li>
	<li>Spray 70% ethanol on the pump cartridge and connect the pump tube with female Luer lock connectors on both ends to the pump.</li>
	<li>Set up the oxygenator wrapped around the solenoid wire mesh inside the cell culture incubator by connecting one end (the outflow) of the oxygenator to the pump tube and the other end with the 1.5 ft tube in the bioreactor.</li>
	<li>Set up the bioreactor bottle in a biosafety cabinet with aseptic conditions.
	<ol>
		<li>Attach a 3-way stopcock (yellow) to the head of one of the female Luers to control the flow through the PA.</li>
		<li>Connect a one-way stopcock (blue) to another female Luer lock bulkhead for cell seeding through the trachea (Figure 2F).</li>
		<li>Connect 2 in tubing on the outside (white) and 6 in tubing on the inside of the bottle to circulate the media out. Fit a male Luer lock to a 2 ft tube and attach a female Luer lug style Tee to provide accessibility to add or remove anything.</li>
		<li>Attach a one-way stopcock to one of the openings of the three-way connector (female Luer lug style Tee).</li>
		<li>Attach a male Luer lock and a female Luer lock to 2 ft tubes.</li>
		<li>Add 200 mL of RPMI1640 with 10% FBS and 1% antibiotics cell culture medium (varies as per cell requirement) and transfer the bioreactor to the incubator. Connect the oxygenator tube to the two open ends (one-way stopcock and Tee connector) to make it a closed-loop bioreactor.</li>
	</ol>
	</li>
	<li>Pre-run the pump at 6 mL/min inside the cell culture incubator with cell culture media to fill the oxygenator and tubes, so that no air bubbles exist in the tubing.</li>
	<li>Once the tubes are filled with media, close the stopcock and remove the bioreactor bottle from the incubator by disconnecting the oxygenators from both ends of the bioreactor bottle.</li>
	<li>Put the bioreactor bottle in the biosafety cabinet, pass culture media through the stopcock, and attach the PA cannula of the lung scaffold (acellular or cellular) to the stopcock through the male Luer connector. 
	NOTE: Here it is possible to use the acellular model (following the decellularization process) or a freshly harvested heart-lung block (i.e., the cellular model, as it retains the native rat cells).</li>
	<li>Pass culture media through a one-way stopcock to remove any air and tie the tracheal cannula by a silk thread to the trachea.</li>
	<li>Make sure to avoid any twisting of the PA and trachea. Close the bioreactor bottle cap with the lung scaffold inside and, again, aseptically transfer the bioreactor to the incubator and start the pump at a 6 mL/min speed.</li>
	<li>Run culture media for 10 - 15 min to make sure all lobes get inflated and the lung looks in good shape (Figure 2G).</li>
</ol>
4. Metastasis Model and Cell Seeding
NOTE: Proceed to cell seeding with the above lung model for a primary tumor growth study. Modify the lung scaffold (acellular or cellular) as follows for the metastatic model.
<ol>
	<li>Take out the bioreactor with the lung scaffold from the incubator and put it in the biosafety cabinet.</li>
	<li>Use a Luer lock syringe to pass 5 mL of culture media through the trachea and open the bioreactor cap with the hanging lung scaffold (Figure 2F).</li>
	<li>At this step, one more person is required to help in the modification of the lung to a metastatic model.</li>
	<li>Get silk 2-0 ready and use angular forceps to make a pass through the trachea at the bifurcation point.</li>
	<li>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 media. 
	NOTE: Once the trachea is tied at the bifurcation point, cells seeded through the trachea will automatically be directed to seed to the left lobes. It can be tested before seeding the cells by pushing culture media through the trachea. In the metastatic model, the media will move to the left lobes only, which will inflate, and not to the right lobes10.</li>
	<li>Set a 20 mL Luer lock syringe on top of the tracheal cannula and add ATCC lung cancer cells (A549, H1299) in 50 mL of RPMI1640 complete culture media (Figure 2F).</li>
	<li>Perform the cell seeding in the biosafety cabinet. Add 15 mL of cells in the syringe and let it pass through the lung lobes by gravity. Add the rest of the cells before any air bubbles pass through.</li>
	<li>In acellular lung seeding, collect the perfused media dripping down in the bioreactor bottle and let it pass through the trachea again 3x. 
	NOTE: The perfused media sometimes has the cancer cells seeded. Therefore, for efficient seeding, put back the media again in the trachea through the syringe.</li>
	<li>Once seeded, remove the syringe, wait for 15 min, add fresh media, and return the bioreactor back to an incubator. Remove any air bubbles in the tubing.</li>
	<li>Start the pump to perfuse the media at 6 mL/min.</li>
</ol>

<ol><li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cancer+Statistics%2C+2017%5BTitle%5D)+AND+%22CA%3A+A+Cancer+Journal+for+Clinicians%22%5BJournal%5D)">Cancer Statistics, 2017.</a> CA: A Cancer Journal for Clinicians. 67 (1), 7-30 (2017).</li>
<li>Torre, L. A., Siegel, R. L., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Lung+Cancer+Statistics%5BTitle%5D)+AND+%22Advances+in+Experimental+Medicine+and+Biology%22%5BJournal%5D)">Lung Cancer Statistics.</a> Advances in Experimental Medicine and Biology. 893, 1-19 (2016).</li>
<li>Lallo, A., Schenk, M. W., Frese, K. K., Blackhall, F., Dive, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+tumor+cells+and+CDX+models+as+a+tool+for+preclinical+drug+development%5BTitle%5D)+AND+%22Translational+Lung+Cancer+Research%22%5BJournal%5D)">Circulating tumor cells and CDX models as a tool for preclinical drug development.</a> Translational Lung Cancer Research. 6 (4), 397-408 (2017).</li>
<li>Yang, S., Zhang, J. J., Huang, X. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+for+tumor+metastasis%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Mouse models for tumor metastasis.</a> Methods in Molecular Biology. 928, 221-228 (2012).</li>
<li>Bissell, M. J., Hines, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Why+don%27t+we+get+more+cancer%3F+A+proposed+role+of+the+microenvironment+in+restraining+cancer+progression%5BTitle%5D)+AND+%22Nature+Medicine%22%5BJournal%5D)">Why don't we get more cancer? A proposed role of the microenvironment in restraining cancer progression.</a> Nature Medicine. 17 (3), 320-329 (2011).</li>
<li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics%5BTitle%5D)+AND+%22Nature+Reviews+Cancer%22%5BJournal%5D)">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nature Reviews Cancer. 11 (2), 135-141 (2011).</li>
<li>Mishra, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+lung+cancer+cells+grown+in+an+ex+vivo+3D+lung+model+produce+matrix+metalloproteinases+not+produced+in+2D+culture%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Human lung cancer cells grown in an ex vivo 3D lung model produce matrix metalloproteinases not produced in 2D culture.</a> PloS One. 7 (9), e45308(2012).</li>
<li>Vishnoi, M., Mishra, D. K., Thrall, M. J., Kurie, J. M., Kim, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Circulating+tumor+cells+from+a+4-dimensional+lung+cancer+model+are+resistant+to+cisplatin%5BTitle%5D)+AND+%22The+Journal+of+Thoracic+and+Cardiovascular+Surgery%22%5BJournal%5D)">Circulating tumor cells from a 4-dimensional lung cancer model are resistant to cisplatin.</a> The Journal of Thoracic and Cardiovascular Surgery. 148 (3), discussion 1063-1064 1056-1063 (2014).</li>
<li>Mishra, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+profile+of+A549+cells+from+tissue+of+4D+model+predicts+poor+prognosis+in+lung+cancer+patients%5BTitle%5D)+AND+%22International+Journal+of+Cancer.+Journal+International+du+Cancer%22%5BJournal%5D)">Gene expression profile of A549 cells from tissue of 4D model predicts poor prognosis in lung cancer patients.</a> International Journal of Cancer. Journal International du Cancer. , (2013).</li>
<li>Mishra, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Ex+vivo+four-dimensional+lung+cancer+model+mimics+metastasis%5BTitle%5D)+AND+%22The+Annals+of+Thoracic+Surgery%22%5BJournal%5D)">Ex vivo four-dimensional lung cancer model mimics metastasis.</a> The Annals of Thoracic Surgery. 99 (4), 1149-1156 (2015).</li>
<li>Mishra, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+lung+cancer+cells+grown+on+acellular+rat+lung+matrix+create+perfusable+tumor+nodules%5BTitle%5D)+AND+%22The+Annals+of+Thoracic+Surgery%22%5BJournal%5D)">Human lung cancer cells grown on acellular rat lung matrix create perfusable tumor nodules.</a> The Annals of Thoracic Surgery. 93 (4), 1075-1081 (2012).</li>
<li>Pence, K. A., Mishra, D. K., Thrall, M., Dave, B., Kim, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Breast+cancer+cells+form+primary+tumors+on+ex+vivo+four-dimensional+lung+model%5BTitle%5D)+AND+%22Journal+of+Surgical+Research%22%5BJournal%5D)">Breast cancer cells form primary tumors on ex vivo four-dimensional lung model.</a> Journal of Surgical Research. 210, 181-187 (2017).</li>
<li>Mishra, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+Lung+Fibroblasts+Inhibit+Non-Small+Cell+Lung+Cancer+Metastasis+in+Ex+Vivo+4D+Model%5BTitle%5D)+AND+%22The+Annals+of+Thoracic+Surgery%22%5BJournal%5D)">Human Lung Fibroblasts Inhibit Non-Small Cell Lung Cancer Metastasis in Ex Vivo 4D Model.</a> The Annals of Thoracic Surgery. 100 (4), discussion 1174 1167-1174 (2015).</li>
<li>Mishra, D. K., Miller, R. A., Pence, K. A., Kim, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+cell+and+non+small+cell+lung+cancer+form+metastasis+on+cellular+4D+lung+model%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">Small cell and non small cell lung cancer form metastasis on cellular 4D lung model.</a> BMC Cancer. 18 (1), 441(2018).</li>
</ol>

The authors have nothing to disclose.

The ex vivo 4-D lung provides an opportunity to study tumor growth and metastasis in a laboratory set-up. A native lung matrix is a complex system that provides support to normal tissue and maintains cell-cell interactions, cell-matrix interactions, cellular differentiation, and tissue organization. It provides an opportunity to add any tumor microenvironment components to study their effects on tumor growth and the interaction with other cells. 
The lung harvest is the critical step ...

Cancer metastasis is the culprit behind most cancer-related deaths and poses the ultimate challenge in the effort to fight cancer. The overall goal of this method is to design a protocol for a four-dimensional (4-D) cell culture which has a dimension of flow, in addition to the three-dimensional (3-D) cell growth. It represents the three distinct phases of the metastasis process [i.e., the primary tumor, circulating tumor cells (CTCs), and metastatic lesions].
Over the past three decades, scientists around the world yielded an unparalleled wealth of information to understand the mechanisms underlying the metastatic progression in different cancers that improved the prospect of a cure or progression-free survival. The clinical management of some cancers, such as breast cancer, improved significantly1; however, some cancers, such as lung cancer, still have a poor survival2. In vitro and in vivo animal models have been instrumental in generating new insights into the mechanisms that underpin the development of the disease. In the last few years, cell line-derived xenografts (CDX) and patient-derived xenografts (PDX) have been of more interest as they preserve many relevant features of the primary human tumor3, such as growth kinetics, histological features, behavioral characteristics, and the response to therapy. However, each model has its limitations to understand the mechanism of CTC formation and metastasis to a distant organ4,5,6.
Recently, we developed a 4-D ex vivo lung cancer model by utilizing the concept of organ reengineering and perfusion-based cell culture. It mimics the human lung cancer growth by forming perfusable tumor nodules that grow over time with a similar human cancer-secreted protein production7. It represents the gene expression signature that predicts poor survival in patients with cancer and also shows a therapeutic response by tumor regression upon cisplatin treatment8,9. The lung model was further modified so that it can form metastatic lesions. The CTCs develop from a primary tumor and intravasate into the vasculature and extravasate into the contralateral&#160;lung to form metastatic lesions10. Gene expression studies suggest a distinct expression profile of the primary tumor, the CTCs, and the metastatic lesions, and the upregulation of the subset of genes required for the phenotype10. This metastatic process occurs due to the presence of biologic conditions seen in patients with cancer. The advantage of this model is the presence of a natural matrix and architecture, and a perfusion of nutrients that leads to the formation of tumor nodules. In addition, it also provides an opportunity to study the effects of different components of tumor microenvironment or drugs on tumor progression over time. This model can be used to grow a range of cancer cells (lung cancer, breast cancer, sarcoma etc.) in a laboratory set-up.

<table><tbody><tr><td>Sprague Dowley rat</td><td>Harlan</td><td>206M</td><td>Male</td></tr><tr><td>Chlorhexidine swab</td><td>Prevantics, NY, USA</td><td>NDC 10819-1080-1</td><td></td></tr><tr><td>Heparin</td><td>Sagent Pharmaceuticals, Schaumburg, IL, USA</td><td>NDC 25021-400-10</td><td></td></tr><tr><td>18-gauge needle</td><td>McMaster Carr, USA</td><td>75165A249</td><td></td></tr><tr><td>2-0 silk tie</td><td>Ethicon, San Angelo, TX, USA</td><td>A305H</td><td></td></tr><tr><td>Masterflex L/S pump</td><td>Cole-Parmer, Vernon Hills, IL, USA</td><td>EW-07554-80</td><td></td></tr><tr><td>Masterflex L/S pump head</td><td>Cole-Parmer, Vernon Hills, IL, USA</td><td>EW-07519-05</td><td></td></tr><tr><td>Masterflex L/S pump cartridge</td><td>Cole-Parmer, Vernon Hills, IL, USA</td><td>EW-07519-70</td><td></td></tr><tr><td>Tygon Tube</td><td>Cole-Parmer, Vernon Hills, IL, USA</td><td>14171211</td><td></td></tr><tr><td>MasterFlex Pump tube</td><td>Cole-Parmer, Vernon Hills, IL, USA</td><td>06598-16</td><td></td></tr><tr><td>Female luer lock connectors</td><td>Cole-Parmer, Vernon Hills, IL, USA</td><td>45508-34</td><td>75165A249</td></tr><tr><td>Male luer lock connectors</td><td>Cole-Parmer, Vernon Hills, IL, USA</td><td>45513-04</td><td></td></tr><tr><td>black nylon ring</td><td>Cole-Parmer, Vernon Hills, IL, USA</td><td>EW-45509-04</td><td></td></tr><tr><td>Intravenous set</td><td>CareFusion</td><td>41134E</td><td></td></tr><tr><td>Sodium Dodecyl Sulfate (SDS)</td><td>Fisher Scientific</td><td>CAS151-21-3</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>X100-1L</td><td></td></tr><tr><td>Antibiotics</td><td>Gibco</td><td>15240-062</td><td></td></tr><tr><td>Silicone oxygenator</td><td>Cole-Parmer, Vernon Hills, IL, USA</td><td>ABW00011</td><td>Saint-GoBain-</td></tr><tr><td>Wire mesh</td><td>1164610105</td><td>Lowes</td><td>New York Wire</td></tr><tr><td>Female luer Lug Style TEE</td><td>Cole-Parmer, Vernon Hills, IL, USA</td><td>45508-56</td><td></td></tr><tr><td>Male luer integral lock ring to 200series Barb</td><td>Cole-Parmer, Vernon Hills, IL, USA</td><td>45518-08</td><td></td></tr><tr><td>Female luer thread style coupler</td><td>Cole-Parmer, Vernon Hills, IL, USA</td><td>45508-22</td><td></td></tr><tr><td>Clave connector</td><td>ICU Medical</td><td>11956</td><td></td></tr><tr><td>Hi-Flo &amp;trade;4-way Stopcock w/swivel male luer lock</td><td>smith Medical</td><td>MX9341L</td><td></td></tr><tr><td>MasterFlex Pump tube</td><td>Cole-Parmer, Vernon Hills, IL, USA</td><td>06598-13</td><td>for cannula</td></tr></tbody></table>

acellular and cellular lung model to study tumor metastasis

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

The lung harvested from rat maintains the intact vasculature and alveoli11 (Figure 3A and 3B). Upon decellularization, the extracellular matrix components of an acellular lung, such as collagen, fibronectin, and elastin, are preserved11 (Figure 3C, 3D, 3E, and 3F). The decellularization leads to a complete removal o...

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.

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

acellular-and-cellular-lung-model-to-study-tumor-metastasis

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This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

Lung Tumor Cell Recruitment Assay

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.

Ex vivo Live Imaging of Lung Metastasis and Their Microenvironment

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Here, we present a protocol for the surgical implantation of a permanently indwelling optical window for the murine thorax, which enables high-resolution, intravital imaging of the lung. The permanence of the window makes it well-suited to the study of dynamic cellular processes in the lung, especially those that are slowly evolving, such as metastatic progression of disseminated tumor cells.

A Permanent Window for Investigating Cancer Metastasis to the Lung

The manuscript describes a methodology for the establishment as well as longitudinal growth monitoring of spontaneous lung metastasis from orthotopically-injected breast tumors, amenable to intervention at all stages of the metastatic cascade.

In Vivo Imaging to Measure Spontaneous Lung Metastasis of Orthotopically-injected Breast Tumor Cells

Learn an ex vivo acellular and cellular lung model to study tumor metastasis using rat lung scaffolds. This technique enables isolation of primary tumors, circulating tumor cells, and metastatic lesions for cancer research.

Acellular and Cellular Lung Model to Study Tumor Metastasis

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