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/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 for the ex vivo 4-D lung model. A delay in administering the heparin injection after the thoracic opening may result in a blood clot in the lungs, which may affect the free flow of the reagents during the decellularization. A proper flushing with heparinized PBS until a colorless fluid comes from the vent is important, especially with the cellular model. The careful cutting of the superior vena cava is important to avoid any harm to the PA. During the decellularization process, it is very important to avoid air bubbles moving to the lung scaffold. The presence of air bubbles may slow down or stop the flow of reagents and contribute to an improper decellularization. Bubbles can be removed by sucking out the bubbles using a 10 - 20 mL syringe. It is very important to make sure the PA cannula is not twisted. During the bioreactor set-up, any twisting in the tubing may result in a pop-up at the connectors and result in a complete mess inside the incubator. To avoid twisting in the cannula, the cannula/needles can be cut and reconnected using biocompatible tubing, which provides the flexibility needed to rotate the cannula without disturbing the connections. Any leakage of culture media inside the incubator may further require a professional cleaning. While creating the metastasis model, careful suturing is required at the trachea bifurcation site. Any puncture of the lungs or trachea may lead to the leaking of cells and inconsistent results.
The acellular and cellular ex vivo 4-D lung models have several advantages over conventional 3D cell cultures to better understand cancer progression, as they provide the opportunity to study the major biological steps in metastasis with an additional dimension of flow. Both models mimic the biology of tumor growth and metastasis and provide an opportunity to collect tumor cells in different phases of cancer growth to better understand the gene signatures, the response to treatments, and the development of drug resistance. Although it is a lung matrix, it has the potential to grow a range of established cell lines and primary cells. The limitation of the ex vivo 4-D model is the requirement of &gt; 10 million cells seeding to study the metastasis. Cells with a low proliferative potential may take a longer time to show the metastatic lesion. In addition, this model requires more aseptic conditions once the cells are seeded.

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 &#8482;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 of the native rat cells present in the lungs, which can be observed by hematoxylin and eosin (H&#38;E) staining showing the absence of cells and by DNA analysis11 showing a reduction in DNA amount (Figure 3D and 3G). The ex vivo 4-D lung model can be used to grow any kind of adherent cells12. Lung cancer, lung fibroblast, breast cancer, and sarcoma cells were grown by seeding through the trachea11,12,13. The metastasis model allows for the collection of primary tumor tissue, CTCs, and metastatic lesions at different time intervals by lobectomy10. The H&#38;E staining of the tissues showed intact vasculature and alveoli in both the cellular and acellular model (Figure 3H and 3I). The acellular 4-D lung model is an add-on model to study the interaction between different components of the microenvironment. The cellular 4-D lung model provides an intact lung microenvironment with no immune cells14. Immune cells can be added to this model through the vasculature to study their effect on tumor growth and the interaction with tumor cells. Reproducibility and quality of the model can be assessed by running the model in duplicate and analyzing the tissue/cells by H&#38;E and immunohistochemistry using cell-specific markers. Figure 4 shows a schematic diagram that represents the major steps involved in creating the ex vivo 4-D lung model.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58145/58145fig1.jpg" /> 
Figure 1: Required items for the decellularization unit of the lung scaffold. (A and B) 500 mL bottle caps are drilled and female Luer connectors are set up with tubing to create the decellularization bottles. (C) Another bottle cap needs only one hole to set up (D) the intravenous set for reagent flows through the pulmonary artery of the lung scaffold. The pump is set up with (F) a pump, (E) a pump head, and (G) a cartridge. (H) Pump tubing with female Luer connectors at each end, (I) connectors, and (J) tubes with male Luer connectors at both ends are required to set up (K) the decellularization unit. (L) The lung scaffolds are washed for 3 - 5 d in a closed-loop system with 1x antibiotics-antimycotic PBS. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58145/58145fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58145/58145fig2.jpg" /> 
Figure 2: Bioreactor set-up for an ex vivo 4-D lung cell culture. (A and B) Bioreactor bottle (500 mL) caps are drilled and three female Luer lock connectors are inserted and fixed with a black nylon ring. (C) 10 ft of oxygenator tube is wrapped around a wire-mesh solenoid with male Luer integral lock rings on both ends. (D) A pulmonary artery cannula and (E) a tracheal cannula are prepared using an 18 G needle and pump tube. (F) A 20 mL syringe is set to the one-way trachea connector to seed the cells in the epithelial space of the lung scaffold. (G) The closed-loop bioreactor is set up inside the cell culture incubator for perfusion and tumor growth. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58145/58145fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58145/58145fig3.jpg" /> 
Figure 3: Representative images of the ex vivo 4-D lung model for metastasis. (A) An intact heart-lung block is harvested from 4- to 6-week-old rat. (B) Histopathology shows the alveoli and pneumocytes. (C) Decellularization leads to the complete removal of the lung cells. (D) Histopathology shows an intact basement membrane with acellular lungs. (E and F) Movat&#39;s pentachrome and elastin stain reveals the presence of collagen, fibrin, and elastin with intact vasculature and bronchus. (G) DNA analysis shows a reduction in DNA content of more than 99%. (H) The cellular lung can be used directly in the bioreactor for cell culture and (I) a histological analysis shows the presence of tumor growth. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58145/58145fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58145/58145fig4.jpg" /> 
Figure 4: Schematic diagram showing the major steps in the creation of the ex vivo 4-D lung model. In step 1, a heart-lung block is harvested from a rat. As per the study requirement, in step 2, the lungs can either be used directly as a cellular model or can be decellularized to remove any native cells. The next step involves setting up the lung model in the bioreactor and cell seeding. Finally, bioreactors are set up in the incubator for the cell culture. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58145/58145fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Acellular and Cellular Lung Model to Study Tumor Metastasis at JoVE.com

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Cancer Research

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 ...

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic effect is lacking and the exact mechanisms of its action are unclear. Here, we report an animal model that allows for direct testing of the effects of tumor hypoxia on ES dissemination and investigation into the underlying pathways involved. This approach combines two well-established experimental strategies, orthotopic xenografting of ES cells and femoral artery ligation (FAL), which induces hindlimb ischemia. Human ES cells were injected into the gastrocnemius muscles of SCID/beige mice and the primary tumors were allowed to grow to a size of 250 mm3. At this stage either the tumors were excised (control group) or the animals were subjected to FAL to create tumor hypoxia, followed by tumor excision 3 days later. The efficiency of FAL was confirmed by a significant increase in binding of hypoxyprobe-1 in the tumor tissue, severe tumor necrosis and complete inhibition of primary tumor growth. Importantly, despite these direct effects of ischemia, an enhanced dissemination of tumor cells from the hypoxic tumors was observed. This experimental strategy enables comparative analysis of the metastatic properties of primary tumors of the same size, yet significantly different levels of hypoxia. It also provides a new platform to further assess the mechanistic basis for the hypoxia-induced alterations that occur during metastatic tumor progression in vivo. In addition, while this model was established using ES cells, we anticipate that this experimental strategy can be used to test the effect of hypoxia in other sarcomas, as well as tumors orthotopically implanted in sites with a well-defined blood supply route.

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

This manuscript describes the development of an animal model that allows for the direct testing of the effects of tumor hypoxia on metastasis and the deciphering the mechanisms of its action. Although the experiments described here focus on Ewing sarcoma, a similar approach can be applied to other tumor types.

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

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. ...

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

Intracarotid Cancer Cell Injection to Produce Mouse Models of Brain Metastasis

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. Recently, impressive advances in novel therapies have dramatically prolonged survival and improved quality of life for many cancer patients. Sadly, incidence of brain metastatic recurrences is fast rising, and all current therapies are merely palliative. Hence, good experimental animal models are urgently needed to facilitate in-depth studies of the disease biology and to assess novel therapeutic regimens for preclinical evaluation. However, the standard in vivo metastasis assay via tail vein injection of cancer cells produces predominantly lung metastatic lesions; animals usually succumb to the lung tumor burden before any meaningful outgrowth of brain metastasis. Intracardiac injection of tumor cells produces metastatic lesions to multiple organ sites including the brain; however, the variability of tumor growth produced with this model is large, dampening its utility in evaluating therapeutic efficacy. To generate reliable and consistent animal models for brain metastasis study, here we describe a procedure for producing experimental brain metastasis in the house mouse (Mus musculus) via intracarotid injection of tumor cells. This approach allows one to produce large number of brain metastasis-bearing mice with similar growth and mortality characteristics, thus facilitating research efforts to study basic biological mechanisms and to assess novel therapeutic agents.

Brain metastasis has become an urgent unmet medical need as its incidence has increased while therapeutic options have remained palliative. Creating experimental animal models of brain metastasis via intracarotid arterial injection of cancer cells facilitates mechanistic studies of the disease biology and evaluation of novel intervention regimens.

Metastasis, the spread and growth of malignant cells at secondary sites within a patient&#39;s body, accounts for &#62; 90% of cancer-related mortality. ...

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new tumors at distant sites. CTCs are found in the bloodstream of patients as single cells (single CTCs) or as multicellular aggregates (CTC clusters and CTC-white blood cell clusters), with the latter displaying a higher metastatic ability. Beyond enumeration, phenotypic and molecular analysis is extraordinarily important to dissect CTC biology and to identify actionable vulnerabilities. Here, we provide a detailed description of a workflow that includes CTC immunostaining and micromanipulation, ex vivo culture to assess&#160;proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, for instance, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading us to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single CTCs. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.

Here, we present an integrated workflow to identify phenotypic and molecular features that characterize circulating tumor cells (CTCs). We combine live immunostaining and robotic micromanipulation of single and clustered CTCs with single cell-based techniques for downstream analysis and assessment of metastasis-seeding ability.

Blood-borne metastasis accounts for&#160;most cancer-related deaths and involves circulating tumor cells (CTCs) that are successful in establishing new ...

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 ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

Zebrafish Model of Neuroblastoma Metastasis

Zebrafish has emerged as an important animal model to study human diseases, especially cancer. Along with the robust transgenic and genome editing technologies applied in zebrafish modeling, the ease of maintenance, high-yield productivity, and powerful live imaging altogether make the zebrafish a valuable model system to study metastasis and cellular and molecular bases underlying this process in vivo. The first zebrafish neuroblastoma (NB) model of metastasis was developed by overexpressing two oncogenes, MYCN and LMO1, under control of the dopamine-beta-hydroxylase (d&#946;h) promoter. Co-overexpressed MYCN and LMO1 led to the reduced latency and increased penetrance of neuroblastomagenesis, as well as accelerated distant metastasis of tumor cells. This new model reliably reiterates many key features of human metastatic NB, including involvement of clinically relevant and metastasis-associated genetic alterations; natural and spontaneous development of metastasis in vivo; and conserved sites of metastases. Therefore, the zebrafish model possesses unique advantages to dissect the complex process of tumor metastasis in vivo.

This paper introduces the method of developing, characterizing, and tracking in real-time the tumor metastasis in zebrafish model of neuroblastoma, specifically in the transgenic zebrafish line with overexpression of MYCN and LMO1, which develops metastasis spontaneously.