Name: isolation and propagation of circulating tumor cells from a mouse cancer model
Uploaded: 2015-10-09T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model at JoVE.com

Work in Dr. Ogunwobi&#8217;s laboratory is supported by a Research Centers in Minority Institutions Program grant from the National Institute on Minority Health and Health Disparities (MD007599) of the National Institutes of Health. The contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the NIMHD or the NIH.

Ethics Statement: All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Hunter College of The City University of New York.
1. Pre-experiment Procedures
<ol>
	<li>Upon arrival to the animal facility, initially house 3 BALB/c mice to a cage with a filtered top and wood chip bedding. Ensure that the mice have free access to food, purina rodent chow, and water. Bedding and food should be changed twice a week while cages should be cleaned once a week.</li>...

<ol>
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A Vision towards the Future.</a> Cancers. 6, 1195-1207 (2014).</li><li>Sheng, 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=Capture,+release+and+culture+of+circulating+tumor+cells+from+pancreatic+cancer+patients+using+an+enhanced+mixing+chip.">Capture, release and culture of circulating tumor cells from pancreatic cancer patients using an enhanced mixing chip.</a> Lab on a chip. 14, 89-98 (2014).</li><li>George, T. J., Ogunwobi, O. O., Sheng, W., Fan, Z. H., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#34;Tissue+is+the+issue&#34;:+circulating+tumor+cells+in+pancreatic+cancer.">&#34;Tissue is the issue&#34;: circulating tumor cells in pancreatic cancer.</a> Journal of gastrointestinal cancer. , (2014).</li><li>Ogunwobi, O. O., Puszyk, W., Dong, H. J., Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+upregulation+of+HGF+and+c-Met+drives+metastasis+in+hepatocellular+carcinoma.">Epigenetic upregulation of HGF and c-Met drives metastasis in hepatocellular carcinoma.</a> PloS one. 8, e63765 (2013).</li><li>Cheng, B., 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=Transparent,+biocompatible+nanostructured+surfaces+for+cancer+cell+capture+and+culture.">Transparent, biocompatible nanostructured surfaces for cancer cell capture and culture.</a> International journal of nanomedicine. 9, 2569-2580 (2014).</li><li>Yu, M., Stott, S., Toner, M., Maheswaran, S., Haber, D. A. <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:+approaches+to+isolation+and+characterization.">Circulating tumor cells: approaches to isolation and characterization.</a> The Journal of cell biology. 192, 373-382 (2011).</li><li>Steube, K. G., Koelz, A. L., Drexler, H. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+verification+of+rodent+cell+lines+by+polymerase+chain+reaction.">Identification and verification of rodent cell lines by polymerase chain reaction.</a> Cytotechnology. 56, 49-56 (2008).</li><li>Pantel, K., Alix-Panabieres, C., Riethdorf, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+micrometastases.">Cancer micrometastases.</a> Nature reviews. 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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=Endoscopic+ultrasound-guided+fine+needle+aspiration+biopsy+of+patients+with+suspected+pancreatic+cancer:+diagnostic+accuracy+and+acute+and+30-day+complications.">Endoscopic ultrasound-guided fine needle aspiration biopsy of patients with suspected pancreatic cancer: diagnostic accuracy and acute and 30-day complications.</a> The American journal of gastroenterology. 98, 2663-2668 (2003).</li><li>O'Toole, 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=Assessment+of+complications+of+EUS-guided+fine-needle+aspiration.">Assessment of complications of EUS-guided fine-needle aspiration.</a> Gastrointestinal endoscopy. 53, 470-474 (2001).</li></ol>

In this study, how to isolate and propagate CTCs from the whole blood of a syngeneic mouse with HCC were described. The objective of this work is to enhance the ongoing studies of the mechanisms of cancer metastasis. 
A factor that contributes to the poor prognosis of many cancers is the lack of timely detection and consequent widespread dissemination of the malignancy3. CTCs originate from primary cancers and spread via the blood stream to distant organs. As such, CTCs are importan...

The cancer research community has known of the existence of circulating tumor cells (CTCs) since first being observed by Thomas Ashworth in 18691. Since then, CTCs have been shown to be important in tumor metastasis and disease progression2-5. Today, solid tumors are a major cause of morbidity and mortality worldwide. CTCs are rare cells that originate from primary tumors and travel through the blood stream to different organs of which only a small fraction ultimately develop into metastasis2-5. Notably, there is positive correlation between tumor size and CTC number3,4. 
An understanding of CTC biology can contribute to the search for targeted therapy. Furthermore, CTCs may have diagnostic applications. To achieve these potential clinical applications, one needs to overcome some current challenges to studying CTCs. One challenge is related to the fact that CTCs may be present as single cells or as clusters and they may even be able to change their phenotype in response to the blood microenvironment2. Moreover, detection can be very challenging, in part, due to the low count of CTCs (a few to hundreds per milliliter) among one billionhematologic cells per milliliterin the blood6. Nevertheless, in recent years, research into the potential clinical applications of CTCs from solid organ cancers has intensified.
Despite these efforts, the challenges of studying and understanding the role of CTCs persist due to the rarity of CTCs and the inadequacy of the technological tools currently available. Despite these challenges, the tremendous potential for clinical applications continues to be an incentive to pursue research into the role of CTCs in cancer metastasis.
We were recently successful in isolating and propagating in cell culture CTCs from an orthotopic syngeneic mouse model of hepatocellular carcinoma (HCC) metastasis5. The purpose of the current paper is to describe in detail all aspects of the successful methodology. The significance of this methodology lies in the fact that this approach may be modified in order to successfully isolate and propagate in culture human CTCs, thus enhancing the possibility of in vitro studies of CTC biology. 
There are multiple potential clinical applications for the use of CTCs. CTCs may be useful for prognosis, response monitoring, screening, dynamic monitoring of tumor molecular alterations, and personalized therapy4. Therefore, a better understanding of the biology of CTCs has high potential for clinical impact.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Heparin Sodium Salt 1G</td>
			<td>VWR</td>
			<td>89508-852</td>
			<td></td>
		</tr>
		<tr>
			<td>BTX Tube Micro 1.5mL Clear NS</td>
			<td>VWR</td>
			<td>89511-254</td>
			<td>1.5mL&#160; pyrogen-free eppendorf Tubes</td>
		</tr>
		<tr>
			<td>Needle Sterile Disp BD 25GX1IN</td>
			<td>VWR</td>
			<td>BD305125</td>
			<td>25G Needle</td>
		</tr>
		<tr>
			<td>Slp Tip SRNG 1ml 200 each per pack</td>
			<td>VWR</td>
			<td>BD309659</td>
			<td>1mL syringe tip</td>
		</tr>
		<tr>
			<td>Syringe 1ml leur lok Pk 100</td>
			<td>VWR</td>
			<td>BD309628</td>
			<td>1 mL syringe</td>
		</tr>
		<tr>
			<td>VWR Forceps Tissue 6</td>
			<td>VWR</td>
			<td>82027-446</td>
			<td>Forceps</td>
		</tr>
		<tr>
			<td>Cyromold intrm 15X15X5MM PK 100</td>
			<td>VWR</td>
			<td>25608-924</td>
			<td>Cyromold</td>
		</tr>
		<tr>
			<td>Cryo-oct compund 4oz</td>
			<td>VWR</td>
			<td>25608-930</td>
			<td>Oct compound</td>
		</tr>
		<tr>
			<td>VWR Slide sprfrst 25X75MM PK72</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td>Slides</td>
		</tr>
		<tr>
			<td>VWR Cover Glass #2 22X5oMM OZ</td>
			<td>VWR</td>
			<td>48-382-128</td>
			<td>Cover Glass</td>
		</tr>
		<tr>
			<td>VWR Slide Box True North Fm Pu</td>
			<td>VWR</td>
			<td>89140-278</td>
			<td>Slide Box</td>
		</tr>
		<tr>
			<td>Super HT PAP Pen</td>
			<td>VWR</td>
			<td>89427-058</td>
			<td>PAP pen</td>
		</tr>
		<tr>
			<td>Water RNASe and DNAse free 2L</td>
			<td>VWR</td>
			<td>101454-204</td>
			<td>Nuclease Free Water</td>
		</tr>
		<tr>
			<td>Buffer Tris Ultra Pure Grade 500G</td>
			<td>VWR</td>
			<td>97061-796</td>
			<td>Tris Buffer</td>
		</tr>
		<tr>
			<td>Ammonium Chloride ACS Grade 2&#160; 5KG</td>
			<td>VWR</td>
			<td>97062-048</td>
			<td>Ammonium Chloride Buffer</td>
		</tr>
		<tr>
			<td>Falcon Tissue Culture Dish 60 x 15mm Style polystyrene</td>
			<td>VWR</td>
			<td align="right">353002</td>
			<td>Tissue Culture Dish</td>
		</tr>
		<tr>
			<td>Clorox&#174; Germicidal Bleach, Regular</td>
			<td>VWR</td>
			<td>89501-620</td>
			<td>Clorox Bleach</td>
		</tr>
		<tr>
			<td>PBS, 1X (Phosphate-Buffered Saline) without calcium &#38; magnesium (500mL)</td>
			<td>Thermo Fischer Scientific</td>
			<td>21-040-CV</td>
			<td>PBS, 1X</td>
		</tr>
		<tr>
			<td>DMEM, 1X&#160; with 4.5 g/L glucose &#38; L-glutamine without sodium pyruvate</td>
			<td>Thermo Fischer Scientific</td>
			<td>10-017-CV</td>
			<td>DMEM 1X media for BNL 1ME A.7R.1 cells</td>
		</tr>
		<tr>
			<td>Fetal Bovine Seru8m</td>
			<td>Thermo Fischer Scientific</td>
			<td>35-010-CV</td>
			<td>FBS</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin Solution, 100X</td>
			<td>Thermo Fischer Scientific</td>
			<td>30-002-Cl</td>
			<td>Penicillin Streptomycin</td>
		</tr>
		<tr>
			<td>Sorvall Biofuge pico</td>
			<td>Thermo Fischer Scientific</td>
			<td align="right">75002411</td>
			<td>13000rpm Centrifuge</td>
		</tr>
	</tbody>
</table>

isolation and propagation of circulating tumor cells from a mouse cancer model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

The methodology described here has shown that CTCs can be isolated. Mice were humanely euthanized at experimental end-point. The process involved carbon dioxide asphyxiation, intra-cardiac exsanguination, and cervical dislocation. A schematic of key steps of the procedure are illustrated in Figure 2. Whole blood samples collected by intra-cardiac exsanguination were processed using the protocol described above. After which, centrifugation was used to determine the buffy coat layer that was subjected to t...

Watch this Scientific Journal Video about Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model at JoVE.com

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

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