Name: acellular and cellular lung model to study tumor metastasis
Uploaded: 2018-08-19T14:00:00
Description: Watch this Scientific Journal Video about Acellular and Cellular Lung Model to Study Tumor Metastasis at JoVE.com

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 ...

<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 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 &#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 o...

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 ...

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