Name: isolation of circulating tumor cells in an orthotopic mouse model of colorectal cancer
Uploaded: 2017-07-18T13:00:00
Description: Watch this Scientific Journal Video about Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer at JoVE.com

This work was supported by the German Research foundation (WE 3548/4-1) and Roland-Ernst-Stiftung f&#252;r Gesundheitswesen (1/14).

The animal experiments presented here were independently reviewed and permitted by an institutional and a governmental Animal Care and Use Committee and were conducted according to Federation of Laboratory Animal Science Associations (FELASA) guidelines. All possible measures were taken to minimize suffering including anesthesia and analgesia or, if necessary, premature euthanasia.
1. Preparation of Cells and Organoids
NOTE: Use a volume of 20 &#181;L with 10...

<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,+2016.">Cancer statistics, 2016.</a> CA Cancer J Clin. 66 (1), 7-30 (2016).</li><li>Weitz, J., Koch, M., Debus, J., H&#246;hler, T., Galle, P. R., B&#252;chler, M. W. <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.">Colorectal cancer.</a> Lancet. 365 (9454), 153-165 (2005).</li><li>Sch&#246;lch, 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=Circulating+tumor+cells+of+colorectal+cancer.">Circulating tumor cells of colorectal cancer.</a> Cancer Cell Microenviron. 1 (5), (2014).</li><li>Steinert, G., Sch&#246;lch, S., Koch, M., Weitz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biology+and+significance+of+circulating+and+disseminated+tumour+cells+in+colorectal+cancer.">Biology and significance of circulating and disseminated tumour cells in colorectal cancer.</a> Langenbecks Arch Surg. 397 (4), 535-542 (2012).</li><li>Bork, U., 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=Prognostic+relevance+of+minimal+residual+disease+in+colorectal+cancer.">Prognostic relevance of minimal residual disease in colorectal cancer.</a> World J Gastroenterol. 20 (30), 10296-10304 (2014).</li><li>Bork, U., 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=Circulating+tumour+cells+and+outcome+in+non-metastatic+colorectal+cancer:+a+prospective+study.">Circulating tumour cells and outcome in non-metastatic colorectal cancer: a prospective study.</a> Br J Cancer. 112 (8), 1306-1313 (2015).</li><li>Rahbari, 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=Compartmental+differences+of+circulating+tumor+cells+in+colorectal+cancer.">Compartmental differences of circulating tumor cells in colorectal cancer.</a> Ann Surg Oncol. 19 (7), 2195-2202 (2012).</li><li>Rahbari, 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=Metastatic+Spread+Emerging+From+Liver+Metastases+of+Colorectal+Cancer:+Does+the+Seed+Leave+the+Soil+Again?.">Metastatic Spread Emerging From Liver Metastases of Colorectal Cancer: Does the Seed Leave the Soil Again?.</a> Ann Surg. 263 (2), 345-352 (2016).</li><li>Rahbari, 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=Meta-analysis+shows+that+detection+of+circulating+tumor+cells+indicates+poor+prognosis+in+patients+with+colorectal+cancer.">Meta-analysis shows that detection of circulating tumor cells indicates poor prognosis in patients with colorectal cancer.</a> Gastroenterology. 138 (5), 1714-1726 (2010).</li><li>Steinert, 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=Immune+Escape+and+Survival+Mechanisms+in+Circulating+Tumor+Cells+of+Colorectal+Cancer.">Immune Escape and Survival Mechanisms in Circulating Tumor Cells of Colorectal Cancer.</a> Cancer Res. 74 (6), 1694-1704 (2014).</li><li>Sharpless, N. E., Depinho, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mighty+mouse:+genetically+engineered+mouse+models+in+cancer+drug+development.">The mighty mouse: genetically engineered mouse models in cancer drug development.</a> Nat Rev Drug Discov. 5 (9), 741-754 (2006).</li><li>Roper, J., Hung, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Priceless+GEMMs:+genetically+engineered+mouse+models+for+colorectal+cancer+drug+development.">Priceless GEMMs: genetically engineered mouse models for colorectal cancer drug development.</a> Trends Pharmacol Sci. 33 (8), 449-455 (2012).</li><li>Sch&#246;lch, 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=Radiotherapy+combined+with+TLR7/8+activation+induces+strong+immune+responses+against+gastrointestinal+tumors.">Radiotherapy combined with TLR7/8 activation induces strong immune responses against gastrointestinal tumors.</a> Oncotarget. 6 (7), 4663-4676 (2015).</li><li>Sch&#246;lch, S., Rauber, C., Weitz, J., Koch, M., Huber, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TLR+activation+and+ionizing+radiation+induce+strong+immune+responses+against+multiple+tumor+entities.">TLR activation and ionizing radiation induce strong immune responses against multiple tumor entities.</a> Oncoimmunology. 4 (11), e1042201 (2015).</li><li>Sch&#246;lch, 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=Circulating+tumor+cells+exhibit+stem+cell+characteristics+in+an+orthotopic+mouse+model+of+colorectal+cancer.">Circulating tumor cells exhibit stem cell characteristics in an orthotopic mouse model of colorectal cancer.</a> Oncotarget. 7 (19), 27232-27242 (2016).</li><li>Nanduri, L. K., Garc&#237;a, S., Weitz, J., Sch&#246;lch, 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+Colorectal+Cancer-Derived+Circulating+Tumor+Cells.">Mouse Models of Colorectal Cancer-Derived Circulating Tumor Cells.</a> Med Chem (Los Angeles). 6 (7), 497-499 (2016).</li><li>van Noort, 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=Novel+Drug+Candidates+for+the+Treatment+of+Metastatic+Colorectal+Cancer+through+Global+Inverse+Gene-Expression+Profiling.">Novel Drug Candidates for the Treatment of Metastatic Colorectal Cancer through Global Inverse Gene-Expression Profiling.</a> Cancer Res. 74 (20), 5690-5699 (2014).</li><li>Sato, 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+expansion+of+epithelial+organoids+from+human+colon,+adenoma,+adenocarcinoma,+and+Barrett's+epithelium.">Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett's epithelium.</a> Gastroenterology. 141 (5), 1762-1772 (2011).</li><li>Boj, S. F., 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=Organoid+models+of+human+and+mouse+ductal+pancreatic+cancer.">Organoid models of human and mouse ductal pancreatic cancer.</a> Cell. 160 (1-2), 324-338 (2015).</li><li>Gao, D., 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=Organoid+cultures+derived+from+patients+with+advanced+prostate+cancer.">Organoid cultures derived from patients with advanced prostate cancer.</a> Cell. 159 (1), 176-187 (2014).</li><li>Ito, 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=NOD/SCID/gamma(c)(null)+mouse:+an+excellent+recipient+mouse+model+for+engraftment+of+human+cells.">NOD/SCID/gamma(c)(null) mouse: an excellent recipient mouse model for engraftment of human cells.</a> Blood. 100 (9), 3175-3182 (2002).</li><li>Simon, M. 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=A+comparative+phenotypic+and+genomic+analysis+of+C57BL/6J+and+C57BL/6N+mouse+strains.">A comparative phenotypic and genomic analysis of C57BL/6J and C57BL/6N mouse strains.</a> Genome Biol. 14 (7), R82 (2013).</li><li>Kalish, 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=C57BL/6N+Mice+Are+More+Resistant+to+Ehrlich+Ascites+Tumors+Than+C57BL/6J+Mice:+The+Role+of+Macrophage+Nitric+Oxide.">C57BL/6N Mice Are More Resistant to Ehrlich Ascites Tumors Than C57BL/6J Mice: The Role of Macrophage Nitric Oxide.</a> Med Sci Monit Basic Res. 21, 235-240 (2015).</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), e484 (2007).</li><li>Roper, J., 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=Combination+PI3K/MEK+inhibition+promotes+tumor+apoptosis+and+regression+in+PIK3CA+wild-type,+KRAS+mutant+colorectal+cancer.">Combination PI3K/MEK inhibition promotes tumor apoptosis and regression in PIK3CA wild-type, KRAS mutant colorectal cancer.</a> Cancer Lett. 347 (2), 204-211 (2014).</li><li>Coffee, E. 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=Concomitant+BRAF+and+PI3K/mTOR+blockade+is+required+for+effective+treatment+of+BRAF(V600E)+colorectal+cancer.">Concomitant BRAF and PI3K/mTOR blockade is required for effective treatment of BRAF(V600E) colorectal cancer.</a> Clin Cancer Res. 19 (10), 2688-2698 (2013).</li><li>Belmont, P. J., 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=Resistance+to+dual+blockade+of+the+kinases+PI3K+and+mTOR+in+KRAS-mutant+colorectal+cancer+models+results+in+combined+sensitivity+to+inhibition+of+the+receptor+tyrosine+kinase+EGFR.">Resistance to dual blockade of the kinases PI3K and mTOR in KRAS-mutant colorectal cancer models results in combined sensitivity to inhibition of the receptor tyrosine kinase EGFR.</a> Sci Signal. 7 (351), ra107 (2014).</li><li>Hung, K. E., 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=Development+of+a+mouse+model+for+sporadic+and+metastatic+colon+tumors+and+its+use+in+assessing+drug+treatment.">Development of a mouse model for sporadic and metastatic colon tumors and its use in assessing drug treatment.</a> Proc Natl Acad Sci USA. 107 (4), 1565-1570 (2010).</li><li>Wang, F., Johnson, R. L., Snyder, P. W., DeSmet, M. L., Fleet, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+Inducible,+Large-Intestine-Specific+Transgenic+Mouse+Model+for+Colitis+and+Colitis-Induced+Colon+Cancer+Research.">An Inducible, Large-Intestine-Specific Transgenic Mouse Model for Colitis and Colitis-Induced Colon Cancer Research.</a> Dig Dis Sci. 61 (4), 1069-1079 (2016).</li><li>Xue, Y., Johnson, R., Desmet, M., Snyder, P. W., Fleet, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+a+transgenic+mouse+for+colorectal+cancer+research+with+intestinal+cre+expression+limited+to+the+large+intestine.">Generation of a transgenic mouse for colorectal cancer research with intestinal cre expression limited to the large intestine.</a> Mol Cancer Res. 8 (8), 1095-1104 (2010).</li><li>Tetteh, P. W., 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=Generation+of+an+inducible+colon-specific+Cre+enzyme+mouse+line+for+colon+cancer+research.">Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research.</a> Proc Natl Acad Sci USA. 113 (42), 11859-11864 (2016).</li></ol>

Despite their preclinically proven activity in subcutaneous mouse models, the great majority of novel compounds fail in clinical trials and never reach the clinic.11 This obvious insufficiency of subcutaneous mouse models to accurately simulate the biology and growth patterns of tumors has led to the development of orthotopic mouse models based on the injection of tumor cells directly into the original organ.
Orthotopic mouse models are able to simulate the biology and ...

Colorectal cancer (CRC) is one of the most common causes of cancer death in western countries.1 While the primary tumor can often be resected, the occurrence of distant metastases dramatically worsens the prognosis and often leads to death.2,3 The biological correlate of metastasis is circulating tumor cells (CTCs), which detach from the tumor, survive in circulation, attach to the epithelium in the target organ, invade the organ and ultimately outgrow to new lesions.4 Although CTCs are known to be of prognostic relevance,5,6,7,8,9 their biology is only partly understood as a result of their extreme rarity in CRC.10
Mouse models are a powerful tool to study various aspects of cancer biology. Classical, subcutaneous tumor models are produced by subcutaneous injection of tumor cells into recipient mice, which can be either immunocompetent (if syngeneic murine tumor cells are used) or immunodeficient. Subcutaneous tumor models are inexpensive and produce data fast; their end-point tumor growth can be easily and non-invasively measured. However, 88% of new compounds that have demonstrated antitumor activity in such models fail in clinical trials.11 This is in part due to interspecies differences between humans and mice; however, a large part of this failure is due to the low predictive value of subcutaneous mouse models.
Orthotopic mouse models, in which the tumor cells are injected into the organ of origin and thus grow in their original microenvironment, are therefore increasingly used in cancer research.11,12,13,14 Orthotopic models do not only simulate local tumor growth conditions; as a result of the anatomically correct site of tumor growth, orthotopic mouse models also allow realistic simulation of metastasis and are therefore used to study CTC biology8,15,16 or their response to different treatments in CRC.13,17
A major disadvantage of orthotopic mouse models is their technical complexity. Depending on the organ in which the cells are to be injected, the learning curve until the experimenter is able to induce reproducible tumors is rather long. This especially applies to colorectal cancer models, as the tumor cells need to be injected into the bowel wall, which often results in perforation, tumor cell leakage or endoluminal tumor cell loss. This article is intended to describe the method of cell preparation from primary tissue samples, 2D cell lines and 3D organoid culture and their injection into the cecum of mice. The technique described here leads to highly uniform tumors and, depending on the tumor biology of the cell line used for injection, reproducible formation of distant metastases and CTCs in the recipient mice.15

<table border="0" cellpadding="0" cellspacing="0" width="1294">
	<tbody>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">Cell culture Media and Components</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td style="width:457px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Advanced DMEM F12</td>
			<td style="width:297px;">Invitrogen</td>
			<td style="width:119px;">12634010</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">HEPES (1 M)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">15630056</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Glutamax-I Supplement (200 mM)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">35050038</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Penicillin/Streptomycin (PenStrep)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">15140122</td>
			<td style="width:457px;">DMEM/ F12 +++ medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">DMEM</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">61965026</td>
			<td>basic medium of 2D cell lines (DMEM/10%FCS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Fetal Calf Serum (FCS)</td>
			<td style="width:297px;">BIOCHROM AG</td>
			<td style="width:119px;">S 0115</td>
			<td>basic medium of 2D cell lines (DMEM/10%FCS)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">TrypLE Express enzymatic dissociation buffer</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">12604021</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Matrigel basement membrane matrix (BMM, phenol red free)</td>
			<td style="width:297px;">CORNING B.V. Life Sciences</td>
			<td style="width:119px;">356231</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">14190169</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Trypsin-EDTA (0,25%, Phenol-Red)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">25200072</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">6-/48-well plates with lid</td>
			<td style="width:297px;">CORNING</td>
			<td style="width:119px;">3516/3548</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">cell culture flask 75cm&#178;, 250 mL</td>
			<td style="width:297px;">VWR International GmbH</td>
			<td style="width:119px;">734-2066</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">cell culture flask 150cm&#178;, 600 mL</td>
			<td style="width:297px;">Corning B.V. Life Sciences</td>
			<td style="width:119px;">355001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Eppendorf tubes 1,5 mL / 2 mL</td>
			<td style="width:297px;">Sarstedt AG &#38; Co.</td>
			<td style="width:119px;">72.706.400/ 72.695.400</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">15 ml, 50 ml centrifuge tubes</td>
			<td style="width:297px;">Greiner-Bio-One GmbH</td>
			<td style="width:119px;">188271/227270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">TC10 Counting Slides (for TC20 Counting Machine)</td>
			<td style="width:297px;">Bio-Rad Laboratories GmbH</td>
			<td style="width:119px;">1450016</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Pasteur pipettes (glass, 150 mm)</td>
			<td style="width:297px;">Fisher Scientific GmbH</td>
			<td style="width:119px;">11546963/ FB50251</td>
			<td>thinly pulled by using a bunsen burner</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS Dissociator</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MACSmix Tube Rotator</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-090-753</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS C Tubes</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-093-237</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Human Tumor Dissociation Kit</td>
			<td style="width:297px;">Miltenyi Biotec</td>
			<td>130-095-929</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Falcon 70&#181;m Cell Strainer</td>
			<td style="width:297px;">Corning B.V. Life Sciences</td>
			<td>352350</td>
			<td>for primary tumor tissue preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">Surgical Equipment</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Sevoflurane</td>
			<td style="width:297px;">AbbVie Germany GmbH &#38; Co. KG</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Medical oxygen</td>
			<td style="width:297px;">Air Liquide Medical GmbH</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Buprenorphine</td>
			<td style="width:297px;">Temgesic</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Bepanthen - opthalmic ointment</td>
			<td style="width:297px;">Bayer Vital GmbH</td>
			<td style="width:119px;">10047757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Normal saline 0.9% (E154)</td>
			<td style="width:297px;">Serumwerk Bernburg AG</td>
			<td style="width:119px;">10013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Aqua ad injectabilia</td>
			<td style="width:297px;">Braun</td>
			<td style="width:119px;">235144</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">1 mL Syringe (without dead volume) - Injekt-F SOLO</td>
			<td style="width:297px;">Braun/neoLab</td>
			<td style="width:119px;">194291661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">30G injection needle</td>
			<td style="width:297px;">BECTON DICKINSON</td>
			<td style="width:119px;">304000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">cellulose swabs</td>
			<td style="width:297px;">Lohmann &#38; Rauscher Deutschland</td>
			<td style="width:119px;">13356</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Micro-Adson Forceps</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">11018-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Iris Scissor - ToughCut</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">14058-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Olsen-Hegar Needle Holder</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">12002-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">AutoClip Kit</td>
			<td style="width:297px;">FST - Fine Science Tools</td>
			<td style="width:119px;">12020-00</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">PDS Z1012H 6/0 C1 (surgical suture)</td>
			<td style="width:297px;">Johnson &#38; Johnson Medical GmbH</td>
			<td style="width:119px;">Z1012H</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Table Top Research Anesthesia Machine w/O2 Flush and a Sevoflurane Vaporizer</td>
			<td style="width:297px;">Parkland Scientific</td>
			<td style="width:119px;">V3000PS/PK</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">UltraMicro Pump with Micro4 Controller</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">UMP3-4</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Footswitch for SYS-Micro4 Controller</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">15867</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Three-axis Manual Micromanipulator</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">M325</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Magnetic Stand for Micromanipulator</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">M10</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Steel Base Plate for M10 Magnetic Stand</td>
			<td style="width:297px;">World Precision Instruments</td>
			<td style="width:119px;">5479</td>
			<td>equipment for highly controlled orthotopic injection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Hot Plate 062</td>
			<td style="width:297px;">Labotect</td>
			<td style="width:119px;">13854</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Isis - Hair shaver</td>
			<td style="width:297px;">AESCULAP - Braun</td>
			<td style="width:119px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Binocular Surgical Microscope</td>
			<td style="width:297px;">Parkland Scientific</td>
			<td style="width:119px;">VS-2Z</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">CTC isolation</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">EDTA</td>
			<td style="width:297px;">Roth</td>
			<td>8040.1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Density gradient medium &#8211; Ficoll</td>
			<td>StemCell - Lymphoprep</td>
			<td>7801</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Alexa Fluor 488 anti-human CD326 (EpCAM) Antibody clone 9C4</td>
			<td>BioLegend</td>
			<td>324210</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:421px;">Alexa Fluor 488 anti-mouse CD326 (EpCAM) Antibody clone G8.8</td>
			<td>BioLegend</td>
			<td style="width:119px;">118210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Petri Dish, &#248; 60 x 15 mm, 21 cm&#178;, Vent</td>
			<td>Greiner bio-one</td>
			<td>628102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fluorescence Cell Culture Microscope</td>
			<td style="width:297px;">Leica</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Transferman 4r Micromanipulator</td>
			<td style="width:297px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CellTram Air</td>
			<td style="width:297px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td style="width:457px;">aspiration pump connected to the micromanipulator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dmz Universal Microelectrode Puller</td>
			<td style="width:297px;">Dagan Corporation</td>
			<td style="width:119px;"></td>
			<td>required for the manufacturing of micro capillaries for single cell aspiration</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Prism Glass Capillaries</td>
			<td style="width:297px;">Dagan Corporation</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">PAP pen</td>
			<td style="width:297px;">Abcam</td>
			<td style="width:119px;">ab2601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">14190169</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Fetal Calf Serum (FCS)</td>
			<td style="width:297px;">BIOCHROM AG</td>
			<td style="width:119px;">S 0115</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Penicillin/Streptomycin (PenStrep)</td>
			<td style="width:297px;">Life Technologies GmbH</td>
			<td style="width:119px;">15140122</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">EDTA</td>
			<td style="width:297px;">Roth</td>
			<td>8040.1</td>
			<td>picking buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Name</td>
			<td style="width:297px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:421px;">Immunohistochemistry</td>
			<td style="width:297px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">Purified anti-human CD326 (EpCAM) antibody clone 9C4</td>
			<td>BioLegend</td>
			<td style="width:119px;">324201</td>
			<td>EpCAM immunohistochemistry (cf, fig 2C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:421px;">HRP rabbit anti-mouse IgG</td>
			<td style="width:297px;">Abcam</td>
			<td style="width:119px;">ab97046</td>
			<td>EpCAM immunohistochemistry (cf, fig 2C)</td>
		</tr>
	</tbody>
</table>

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 tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

The successful and reproducible generation of colorectal tumors in this model critically depends on accurate injection of the cells without spillage or leakage. If this is achieved, this model is extremely reliable and very rarely results in artificial peritoneal dissemination. The growth kinetics of the tumors as well as their dissemination patterns depend on the biology of the used organoids and cells.15 While HCT116 cells reliably metastasize to the liver in thi...

Watch this Scientific Journal Video about Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer at JoVE.com

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

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

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

The overall goal of this microsurgical technique is to inject colorectal tumor cells into the cecal wall to induce reproducible and metastatic colorectal tumors in mice. This method can answer key questions in the field of circulating tumor cell biology, its main advantages are the highly reproducible and uniform tumors, allowing reliable stage prediction and circulating tumor cell prediction. Begin by using atraumatic forceps to carefully exteriorize the cecum of a six to eight week old immunocompromised mouse.Placing the blind end of the cecum on the abdomen so that the pouch points cranially. For the intra-cecal injection, mount a standard one milliliter syringe equipped with a 30 gauge canella and load it with the tumor cells onto a micro injection pump that is mounted on a micro manipulator. Next, use the atraumatic forceps to carefully grasp the tip of the cecum and use a second forceps, moistened with warm saline to gently smooth the cecum with downward strokes.Now move the animal under a binocular surgical microscope and with the canella directly parallel to and above the cecum, use two atraumatic forceps to carefully stretch the tissue at both ends of the exteriorized end of the cecum. Slowly pull the cecum over the canella, taking care not to perforate the entire belt of wall or the serosa beyond the initial point of penetration and place the canella above the blood vessels and under the thin translucent membrane inside belt of wall. When the canella is in place, use a footswitch to inject 20 microliters of cells over a period of 20 seconds.Between the thin translucent serosa lining, above the intramural blood vessels and the muscularis. When all of the cells have been injected, carefully remove the canella and place a dry swap under the cecum. To prevent artificial peritoneal dissemination, lyse any leaked cells with a thorough distilled water rinse and gently return the cecum to the abdominal cavity.Close the abdominal wall with rapidly absorbable running sutures, in the skin with surgical wound clips. Then place the mouse on a heating mat set to 38 degrees celsius with monitoring until full recumbency. To isolate the circulating tumor cells, at the appropriate experimental end point, fill 15 milliliter conical tubes with five milliliters of density gradient medium per tube and carefully transfer whole blood samples collected from tumor bearing animals onto the density gradient layers.Separate the cells by centrifugation, and carefully recover the interface containing the mononuclear cells. Pipette the mononuclear cells into a new 15 milliliter tube for a second centrifugation. Followed by tube washes in PBS.After the second wash, re-suspend the pellets in 200 microliters of PBS, supplemented with EDTA and add four microliters of anti amp camp antibody to each sample, for 20 minute incubation on ice in the dark. Next, use a hydrophobic barrier pen to draw an approximately one centimeters circle in one sterile six centimeter petri dish per sample and add 700 microliters of picking buffer to each circle, followed by 50 microliters of cell suspension. Use a microscope to check the density of the cells, and allow the samples to settle for about five minutes.Then screen the drop of cells for amp camp positivity and use the micromanipulator to transfer the cells of interest into 15 microliters of the appropriate buffer for the intended subsequent downstream analysis. The use of colorectal cells in this model, reliably results in more abund mice within 35 days of tumor cell injection, with the primary tumors measuring about 10 millimeters in size, and with liver, and lung metastasis, and circulating tumor cells, almost invariably present. Further, the circulating tumor cells can be easily isolated for downstream analysis.Once mastered and if performed properly, this technique can be performed within 10 minutes, facilitating the formation of uniform and metastatic tumors that can both be used for therapeutic and molecular studies. Limitations of the model include, its dependency on cell lyse, which have their own limitations, as well as the immunodeficiency of the mice, both of which, can be overcome by the use of mutant colorectal cancer cells. Due to its simplicity, this model maybe a valuable addendum to the currently available models of colorectal cancer.

Research

JoVE Journal

Cancer Research

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Capture and Release of Viable Circulating Tumor Cells from Blood

We demonstrate a method for size based capture of viable circulating tumor cell (CTC) from whole blood, along with the release of these cells from chip ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Isolation of Human Endothelial Cells from Normal Colon and Colorectal Carcinoma - An Improved Protocol

Primary cells isolated from human carcinomas are valuable tools to identify pathogenic mechanisms contributing to disease development and progression. In ...

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

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

Culture, Manipulation, and Orthotopic Transplantation of Mouse Bladder Tumor Organoids

The development of advanced tumor models has long been encouraged because current cancer models have shown limitations such as lack of three-dimensional ...

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the ...