Name: advanced animal model of colorectal metastasis in liver: imaging techniques and properties of metastatic clones
Uploaded: 2016-11-30T12:30:00
Description: Watch this Scientific Journal Video about Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones at JoVE.com

We would like to thank Dr. Geoffrey L. Greene (University of Chicago) for the Luc2-tdTomato plasmid and the HCT116 cell line, Mr. Ani Solanki (Animal Resource Center) for the mice management, and Dr. Lara Leoni for the assistance with the DLIT. Quantifications of fluorescent and luminescent intensities were performed in the Integrated Small Animal Imaging Research Resource at the University of Chicago on an IVIS Spectrum (PerkinElmer, Hopkinton, MA). This work was supported by the Virginia and D.K. Ludwig Fund for Cancer Research, the Lung Cancer Research Foundation (LCRF), the Prostate Cancer Foundation (PCF), and the Cancer Center Support Grant (P30CA014599). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Chicago (Protocol # 72213-09) and performed under sterile conditions.
1. Preparations
<ol>
	<li>Make 500 mL of medium for the culture of HCT116 tumor cells: Dulbecco&#39;s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin.</li>
	<li>Autoclave the instruments to be used for the spleen injection model, including 3 - 4 surgical towels,...

<ol>
	<li>Fong, Y., Fortner, J., Sun, R. L., Brennan, M. F., Blumgart, L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clinical+score+for+predicting+recurrence+after+hepatic+resection+for+metastatic+colorectal+cancer:+analysis+of+1001+consecutive+cases.">Clinical score for predicting recurrence after hepatic resection for metastatic colorectal cancer: analysis of 1001 consecutive cases.</a> Ann. Surg. 230 (3), 309-318 (1999).</li><li>Pawlik, T. M., 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=Effect+of+surgical+margin+status+on+survival+and+site+of+recurrence+after+hepatic+resection+for+colorectal+metastases.">Effect of surgical margin status on survival and site of recurrence after hepatic resection for colorectal metastases.</a> Ann. Surg. 241 (5), 715-722 (2005).</li><li>Park, J. H., Watt, D. G., Roxburgh, C. S., Horgan, P. G., McMillan, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colorectal+Cancer,+Systemic+Inflammation,+and+Outcome:+Staging+the+Tumor+and+Staging+the+Host.">Colorectal Cancer, Systemic Inflammation, and Outcome: Staging the Tumor and Staging the Host.</a> Ann. Surg. 263 (2), 326-336 (2016).</li><li>Veen, T., 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=Long-Term+Follow-Up+and+Survivorship+After+Completing+Systematic+Surveillance+in+Stage+I-III+Colorectal+Cancer:+Who+Is+Still+at+Risk.">Long-Term Follow-Up and Survivorship After Completing Systematic Surveillance in Stage I-III Colorectal Cancer: Who Is Still at Risk.</a> J. Gastrointest. Cancer. 46 (3), 259-266 (2015).</li><li>Siegel, R., 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=Cancer+treatment+and+survivorship+statistics.">Cancer treatment and survivorship statistics.</a> CA Cancer J. Clin. 62 (2), 220-241 (2012).</li><li>O'Connell, J. B., Maggard, M. A., Ko, C. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Colon+cancer+survival+rates+with+the+new+American+Joint+Committee+on+Cancer+sixth+edition+staging.">Colon cancer survival rates with the new American Joint Committee on Cancer sixth edition staging.</a> J. Natl. Cancer Inst. 96 (19), 1420-1425 (2004).</li><li>House, M. G., 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=Survival+after+hepatic+resection+for+metastatic+colorectal+cancer:+trends+in+outcomes+for+1,600+patients+during+two+decades+at+a+single+institution.">Survival after hepatic resection for metastatic colorectal cancer: trends in outcomes for 1,600 patients during two decades at a single institution.</a> J. Am. Coll. Surg. 210 (5), 744-752 (2010).</li><li>Smakman, N., Martens, A., Kranenburg, O., Borel Rinkes, I. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Validation+of+bioluminescence+imaging+of+colorectal+liver+metastases+in+the+mouse.">Validation of bioluminescence imaging of colorectal liver metastases in the mouse.</a> J. Surg. Res. 122 (2), 225-230 (2004).</li><li>Rajendran, S., 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=Murine+bioluminescent+hepatic+tumour+model.">Murine bioluminescent hepatic tumour model.</a> J. Vis. Exp. (41), (2010).</li><li>Oshima, G., 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=Imaging+of+tumor+clones+with+differential+liver+colonization.">Imaging of tumor clones with differential liver colonization.</a> Sci. Rep. 5 (10946), (2015).</li><li>Liu, H., 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=Cancer+stem+cells+from+human+breast+tumors+are+involved+in+spontaneous+metastases+in+orthotopic+mouse+models.">Cancer stem cells from human breast tumors are involved in spontaneous metastases in orthotopic mouse models.</a> Proc. Natl. Acad. Sci. U. S. A. 107 (42), 18115-18120 (2010).</li><li>Wang, X. M., 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=Integrative+analyses+identify+osteopontin,+LAMB3+and+ITGB1+as+critical+pro-metastatic+genes+for+lung+cancer.">Integrative analyses identify osteopontin, LAMB3 and ITGB1 as critical pro-metastatic genes for lung cancer.</a> PLoS One. 8 (2), e55714 (2013).</li><li>Fidler, I. J., Kripke, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastasis+results+from+preexisting+variant+cells+within+a+malignant+tumor.">Metastasis results from preexisting variant cells within a malignant tumor.</a> Science. 197 (4306), 893-895 (1977).</li><li>Yachida, S., 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=Distant+metastasis+occurs+late+during+the+genetic+evolution+of+pancreatic+cancer.">Distant metastasis occurs late during the genetic evolution of pancreatic cancer.</a> Nature. 467 (7319), 1114-1117 (2010).</li><li>Khodarev, N. N., 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=STAT1+pathway+mediates+amplification+of+metastatic+potential+and+resistance+to+therapy.">STAT1 pathway mediates amplification of metastatic potential and resistance to therapy.</a> PLoS One. 4 (6), e5821 (2009).</li><li>Langley, R. R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell-organ+microenvironment+interactions+in+the+pathogenesis+of+cancer+metastasis.">Tumor cell-organ microenvironment interactions in the pathogenesis of cancer metastasis.</a> Endocr. Rev. 28 (3), 297-321 (2007).</li><li>Lussier, Y. A., 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=Oligo-+and+polymetastatic+progression+in+lung+metastasis(es)+patients+is+associated+with+specific+microRNAs.">Oligo- and polymetastatic progression in lung metastasis(es) patients is associated with specific microRNAs.</a> PLoS One. 7 (12), e50141 (2012).</li><li>Lussier, Y. A., 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=MicroRNA+expression+characterizes+oligometastasis(es).">MicroRNA expression characterizes oligometastasis(es).</a> PLoS One. 6 (12), e28650 (2011).</li><li>Calon, A., 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=Dependency+of+colorectal+cancer+on+a+TGF-beta-driven+program+in+stromal+cells+for+metastasis+initiation.">Dependency of colorectal cancer on a TGF-beta-driven program in stromal cells for metastasis initiation.</a> Cancer Cell. 22 (5), 571-584 (2012).</li><li>Vanharanta, S., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origins+of+metastatic+traits.">Origins of metastatic traits.</a> Cancer Cell. 24 (4), 410-421 (2013).</li><li>Khodarev, N. N., Roizman, B., Weichselbaum, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+pathways:+Interferon/Stat1+Pathway:+Role+in+the+tumor+resistance+to+genotoxic+stress+and+aggressive+growth.">Molecular pathways: Interferon/Stat1 Pathway: Role in the tumor resistance to genotoxic stress and aggressive growth.</a> Clin. Cancer Res. 18 (11), 3015-3021 (2012).</li><li>Li, C., 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=Interferon-stimulated+gene+15+(ISG15)+is+a+trigger+for+tumorigenesis+and+metastasis+of+hepatocellular+carcinoma.">Interferon-stimulated gene 15 (ISG15) is a trigger for tumorigenesis and metastasis of hepatocellular carcinoma.</a> Oncotarget. 5 (18), 8429-8441 (2014).</li><li>Cespedes, M. V., 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=Orthotopic+microinjection+of+human+colon+cancer+cells+in+nude+mice+induces+tumor+foci+in+all+clinically+relevant+metastatic+sites.">Orthotopic microinjection of human colon cancer cells in nude mice induces tumor foci in all clinically relevant metastatic sites.</a> Am. J. Pathol. 170 (3), 1077-1085 (2007).</li><li>Tseng, W., Leong, X., Engleman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+mouse+model+of+colorectal+cancer.">Orthotopic mouse model of colorectal cancer.</a> J. Vis. Exp. (10), (2007).</li><li>Soares, K. C., 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=A+preclinical+murine+model+of+hepatic+metastases.">A preclinical murine model of hepatic metastases.</a> J. Vis. Exp. (27), e51677 (2014).</li><li>Evans, J. P., 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=From+mice+to+men:+Murine+models+of+colorectal+cancer+for+use+in+translational+research.">From mice to men: Murine models of colorectal cancer for use in translational research.</a> Crit. Rev. Oncol. Hematol. 98, 94-105 (2016).</li></ol>

The animal model presented in the current report is based on two major approaches. First, in order to ensure the ability to observe metastatic clones with different propensities to colonize and proliferate in the liver, a panel of highly heterogeneous monoclonal cell lines was established, rather than an established unfractionated cancer cell line12,13. The monoclonal approach to metastasis development is justified by recent genomic data14 and was successfully used previously to model the metastatic...

Patients with liver metastases from primary colorectal cancers (CRC) are characterized by a poor prognosis. The 5-year survival rate for primary nonmetastatic CRC (stages I - III) is estimated as 75 - 88%3,4, while patients with liver metastases (stage IV) have a 5-year survival rate of only 8 - 12%5,6. However, metastatic patients represent a heterogeneous group, presenting with different numbers of metastases and different recurrence times. Clinical observations indicate that the number of metastases (which may be proportional to colonizing ability or frequency of colonization) and the size of any single metastasis (proportional to the local growth rate) are independent prognostic factors1,7. In other words, the success of metastatic clones colonizing the liver depends on two major properties: their ability to grow and their ability to disseminate and survive in the liver microenvironment.
The design of successful clinical models with the capability of capturing and quantifying the properties of metastatic clones can drastically improve our understanding of liver metastasis biology and provide an effective tool for the design of potential therapeutic approaches. Models of experimental liver metastasis have been previously reported8,9, but neither of them provided the ability to quantitatively capture and describe properties of individual metastatic clones both in vivo and ex vivo.
Here, we present a new, advanced model of liver metastasis that includes the generation of tumor clones with different liver colonization efficiencies and growth properties. We employed a combination of dual-labeling of cancer cells with luciferase and tdTomato fluorescent protein with the generation of monoclonal cell lines that have intrinsic differences in metastatic capacity. In this experimental model, the data indicate that the development of liver metastases can be described in terms of colonization frequency and doubling time (Td), which is consistent with clinical observations. The quantitative nature of this model makes it easily adoptable for drug discovery and diagnostic purposes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>IVIS Spectrum In Vivo Imaging System</td>
			<td>Caliper Life Sciences</td>
			<td>124262</td>
			<td>In vivo imaging system</td>
		</tr>
		<tr>
			<td>LivingImage 4.0 Software</td>
			<td>Caliper Life Sciences</td>
			<td>128165</td>
			<td>Imaging software</td>
		</tr>
		<tr>
			<td>VAD-MGX Research Anesthetic Machine</td>
			<td>Vetamac</td>
			<td>VAD-MGX</td>
			<td>Inhalation anesthesia machine</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965-118</td>
			<td>Cell culture reagents</td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190250</td>
			<td>Cell culture reagents</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin, liquid (10,000 units penicillin;10,000 &#956;g streptomycin)</td>
			<td>Invitrogen</td>
			<td>15140163</td>
			<td>Cell culture reagents</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>ThermoFisher</td>
			<td>24020117</td>
			<td>Cell culture reagents</td>
		</tr>
		<tr>
			<td>Buprenex Injection (0.3mg/mL)</td>
			<td>Reckitt Benckiser Healthcare Ltd.</td>
			<td>12496-0757-5</td>
			<td>Buprenorphine hydrochloride</td>
		</tr>
		<tr>
			<td>Gemini Cautery System</td>
			<td>Braintree Scientific</td>
			<td>GEM 5917</td>
			<td>Hand-held cautery for splenectomy</td>
		</tr>
		<tr>
			<td>Micro Clip; Straight; 70 Grams Pressure; 1.5mm Clip Width; 10mm Jaw Length</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS-5426</td>
			<td>Hemoclip: Hemostasis instruments after spleen injection</td>
		</tr>
		<tr>
			<td>D-luciferin, potassium salt</td>
			<td>Goldbio Technology</td>
			<td>LUCK-1G</td>
			<td>Luciferin potassium salt</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium</td>
			<td>Gibco</td>
			<td>31985062</td>
			<td>Reduced Serum Medium</td>
		</tr>
		<tr>
			<td>TC20 Automated Cell Counter</td>
			<td>BIO-RAD</td>
			<td>1450102</td>
			<td>Automatic cell counter</td>
		</tr>
		<tr>
			<td>JMP10 software&#160;</td>
			<td>SAS Institute</td>
			<td></td>
			<td>Data analysis software</td>
		</tr>
		<tr>
			<td>BD FACSAria II cell sorter</td>
			<td>BD Biocsiences</td>
			<td></td>
			<td>Cell sorter</td>
		</tr>
	</tbody>
</table>

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 goal of this experiment was to establish a consistent and easily reproducible animal model with the potential for the serial quantification of the in vivo metastatic tumor burden and for the estimation of the colonizing frequency and growth kinetics of developing liver metastases. Figures 2-6, with legends, are provided from our previous publication under a Creative Commons CC-BY license10.
...

Watch this Scientific Journal Video about Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones at JoVE.com

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

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. ...

Research

JoVE Journal

Cancer Research

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

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 ...

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 ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

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 ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

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

Micromanipulation of Circulating Tumor Cells for Downstream Molecular Analysis and Metastatic Potential Assessment (Video) | JoVE

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

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury (Video) | JoVE

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

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

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

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

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres

Discovery of Driver Genes in Colorectal HT29-derived Cancer Stem-Like Tumorspheres (Video) | JoVE

Cancer stem cells play a vital role against clinical therapies, contributing to tumor relapse. There are many oncogenes involved in tumorigenesis and the ...