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>

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	<li>Siegel, R. L., Miller, K. D., Jemal, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+Statistics,+2017.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+Cancer+Statistics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+and+CDX+models+as+a+tool+for+preclinical+drug+development.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+for+tumor+metastasis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+don't+we+get+more+cancer?+A+proposed+role+of+the+microenvironment+in+restraining+cancer+progression.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lung+cancer+cells+grown+in+an+ex+vivo+3D+lung+model+produce+matrix+metalloproteinases+not+produced+in+2D+culture.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+cells+from+a+4-dimensional+lung+cancer+model+are+resistant+to+cisplatin.">Circulating tumor cells from a 4-dimensional lung cancer model are resistant to cisplatin.</a> The Journal of Thoracic and Cardiovascular Surgery. 148 (3), 1056-1063 (2014).</li><li>Mishra, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profile+of+A549+cells+from+tissue+of+4D+model+predicts+poor+prognosis+in+lung+cancer+patients.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+vivo+four-dimensional+lung+cancer+model+mimics+metastasis.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+lung+cancer+cells+grown+on+acellular+rat+lung+matrix+create+perfusable+tumor+nodules.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+cells+form+primary+tumors+on+ex+vivo+four-dimensional+lung+model.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Lung+Fibroblasts+Inhibit+Non-Small+Cell+Lung+Cancer+Metastasis+in+Ex+Vivo+4D+Model.">Human Lung Fibroblasts Inhibit Non-Small Cell Lung Cancer Metastasis in Ex Vivo 4D Model.</a> The Annals of Thoracic Surgery. 100 (4), 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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+cell+and+non+small+cell+lung+cancer+form+metastasis+on+cellular+4D+lung+model.">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

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...

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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.

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 ...

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7.5K Views.  Houston Methodist Hospital Research Institute. 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.

Video: Acellular and Cellular Lung Model to Study Tumor Metastasis

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

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

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

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

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

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.

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into mouse tail veins and colonize in the lungs, thereby, resembling the last steps of tumor cell spontaneous metastasis: survival in the circulation, extravasation and colonization in the distal organs. From a therapeutic point of view, the experimental metastasis model is the simplest and ideal model since the target of therapies is often the end point of metastasis: established metastatic tumor in the distal organ. In this model, tumor cells are injected i.v into mouse tail veins and allowed to colonize and grow in the lungs. Tumor-specific CTLs are then injected i.v into the metastases-bearing mouse. The number and size of the lung metastases can be controlled by the number of tumor cells to be injected and the time of tumor growth. Therefore, various stages of metastasis, from minimal metastasis to extensive metastasis, can be modeled. Lung metastases are analyzed by inflation with ink, thus allowing easier visual observation and quantification.

An experimental lung metastasis and CTL immunotherapy mouse model for analysis of tumor cells-T cell interaction in vivo.

Experimental metastasis mouse model is a simple and yet physiologically relevant metastasis model. The tumor cells are injected intravenously (i.v) into ...

Immunology and Infection

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

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

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.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

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

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

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.

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

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

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

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.

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

A Permanent Window for Investigating Cancer Metastasis to the Lung

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as the bone, brain, and lung. Although extensively studied, the mechanistic details of this process remain poorly understood. While common imaging modalities, including computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI), offer varying degrees of gross visualization, each lacks the temporal and spatial resolution necessary to detect the dynamics of individual tumor cells. To address this, numerous techniques have been described for intravital imaging of common metastatic sites. Of these sites, the lung has proven especially challenging to access for intravital imaging owing to its delicacy and critical role in sustaining life. Although several approaches have previously been described for single-cell intravital imaging of the intact lung, all involve highly invasive and terminal procedures, limiting the maximum possible imaging duration to 6-12 h. Described here is an improved technique for the permanent implantation of a minimally invasive thoracic optical Window for High-Resolution Imaging of the Lung (WHRIL). Combined with an adapted approach to microcartography, the innovative optical window facilitates serial intravital imaging of the intact lung at single-cell resolution across multiple imaging sessions and spanning multiple weeks. Given the unprecedented duration of time over which imaging data can be gathered, the WHRIL can facilitate the accelerated discovery of the dynamic mechanisms underlying metastatic progression and numerous additional biologic processes within the lung.

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.

Metastasis, accounting for ~90% of cancer-related mortality, involves the systemic spread of cancer cells from primary tumors to secondary sites such as ...

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

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple opportunities for therapeutic intervention, and the ability to accurately model them in mice is critical to evaluate their effects. Here, a step-by-step protocol is presented for the establishment of orthotopic primary breast tumors and the subsequent monitoring of the establishment and growth of metastatic lesions in the lung using in vivo bioluminescence imaging. This methodology allows for the evaluation of treatment or its biological effects along the entire range of metastatic development, from primary tumor escape to outgrowth in the lungs. Breast orthotopic tumors are generated in mice via injection of a luciferase-labeled cell suspension in the 4th mammary gland. Tumors are allowed to grow and disseminate for a specific amount of time and are then surgically resected. Upon resection, spontaneous lung metastasis is detected, and the growth over time is monitored using in vivo bioluminescence imaging. At the desired experimental endpoint, lung tissue can be collected for downstream analysis. The treatment of established, clinically evident metastasis is critical to improve outcomes for stage IV cancer patients, and it can be evaluated through tail vein models of experimental lung metastasis. However, metastatic dissemination occurs early in breast cancer, and many patients have latent, subclinical disseminated disease after surgery. Utilization of spontaneous models such as this one provides the opportunity to study the whole spectrum of the disease, especially the systemic effects driven by treatment of the primary tumor such as pre-metastatic niche priming, and evaluate treatments on dormant and subclinical disease after surgery.

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

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.

Metastasis remains the primary cause of cancer-related death. The succession of events that characterize the metastatic cascade presents multiple ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

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Chapters in this video

Lung Tumor Cell Recruitment Assay

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

Experimental Metastasis and CTL Adoptive Transfer Immunotherapy Mouse Model

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

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

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

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

A Permanent Window for Investigating Cancer Metastasis to the Lung

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

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis