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>
	<li>Induce general anesthesia in mice using an isoflurane vaporizer along with oxygen at a constant flow rate of 1 L per min. The system is self-reliant and includes a precision vaporizer.</li>
	<li>Position animal in a plastic box that has the precision vaporizer and gas/anesthetic system connected to it. Initially set the vaporizer to 2.5 until animal is unconscious, at which point lower the vaporizer to 2.</li>
	<li>At that time, switch the valve line, sealing it to the box and opening it to the nose cone.</li>
	<li>Move the animal to the preparation area, with the nose cone placed properly and connected (for maintenance anesthesia).</li>
	<li>Prepare 5 &#215; 106 BNL 1ME A.7R.1 mouse HCC cells for implantation per mouse. BNL 1ME A.7R.1 should be growing in medium (10% heat inactivated FBS, 1% penicillin/streptomycin and 2 mM L-Glutamine) incubating in a cell culture dish at 37 &#176;C in a humidified incubator with air and 5 % CO2.</li>
	<li>With a syringe having a 20 G needle, implant 100 &#181;l of PBS containing 5 x 106 BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells into a lobe of the livers of BALB/c mice to produce the primary tumors. Post-implantation, house the BALB/c mice in a cage with the same house requirements as pre-implantation.</li>
	<li>At the experimental end point, clinical evidence of hepatocellular carcinoma is observable as significant reduction in body condition score (usually detected after 4 weeks). At this point, there is a likelihood that primary tumors have developed in the liver while metastatic deposits have formed in the lungs. Thereafter, commence the process of the isolation and propagation of circulating tumor cells.</li>
</ol>
2. Collection of Whole Blood for Circulating Tumor Cell Isolation
NOTE: Properly sanitized the surgery area and make sure it is clutter-free. Autoclave all surgical instruments or soak in a disinfectant according to manufacturer's recommendation
<ol>
	<li>Sacrifice mice by humane euthanasia at experimental end-point (clinical evidence of tumor development). Euthanization of mice should involve CO2 asphyxiation. Observe the mice properly to confirm anesthesia is correctly distributed. Carbon dioxide should be released slowly into the chamber over a period of 5 - 10 min, causing respiratory arrest. Follow by intracardiac exsanguination and cervical dislocation.5</li>
	<li>Use a 25 G needle attached to a 1 ml syringe for collection of whole blood. Heparanize the syringe with 0.01 ml heparin. Puncture skin immediately with the needle inferior to the xyphisternum at approximately 45&#176; angle. Advance needle slowly to puncture the heart, and then lower the angle of the needle. Steadily hold the needle and syringe in place, and draw out 500 &#181;l - 1,000 &#181;l of whole blood from the heart.</li>
	<li>Remove the syringe and needle and transfer the blood into a pre-labelled 1.5 ml pyrogen-free tube. Centrifuge the whole blood collected into 1.5 ml pyrogen-free tube at 1,167 x g for 5 min. 
	NOTE: The centrifugation separates the whole blood sample into three layers: the bottom or hematocrit layer consisting of red blood cells, an intermediate thin layer of buffy coat, and a top plasma layer.</li>
	<li>Remove the plasma carefully by pipetting and collecting it into a separate 1.5 ml pyrogen-free tube for further experiments such as detection of cell-free nucleic acids7.</li>
	<li>For isolation of circulating tumor cells (CTCs), collect the buffy coat into a separate 1.5 ml pyrogen-free tube in preparation for red blood cell (RBC) lysis. This is necessary to remove residual RBCs in the buffy coat layer.</li>
</ol>
3. Recipe for Red Blood Cell (RBC) Lysis Buffer
<ol>
	<li>Make a 1 L solution of RBC lysis buffer.</li>
	<li>Make a 500 ml of Ammonium Chloride and 500 ml of Tris. Then mix to create a 1 L solution of RBC lysis buffer with a final concentration of 155 mM of Ammonium Chloride and 10 mM of Tris.</li>
	<li>Buffer the solution to a pH of 7.5. Store the RBC lysis buffer at +2 to +8 &#176;C, and bring to RT immediately prior to use.</li>
</ol>
4. RBC Lysis of Buffy Coat
<ol>
	<li>Add 1 ml of RBC lysis buffer to the sterile pyrogen-free 1.5 ml tube containing the collected buffy coat.</li>
	<li>Mix the contents of the tube by inverting multiple times. Incubate the mixed contents of the tube for 5 min at RT on the bench. Centrifuge the mixed contents for 5 min at 1,167 x g.</li>
	<li>After centrifugation, a whitish pellet will be obtained. Discard the reddish supernatant slowly and carefully into 10% bleach without any disruption to the pellet. If the pellet is not whitish, re-do steps 3.1 - 3.5. Increasing the incubation time in RBC lysis buffer to 10 - 15 min may also be helpful.</li>
	<li>Safely dispose the biological waste once decontamination of biological waste in 10% bleach is complete after 24 hr.</li>
</ol>
5. Propagation in Cell Culture
NOTE: Tumor cells are typically rapidly growing cells white blood cells that are abundant in the original mix of cells seeded into the dish. Following repeated change of medium, and subsequent passages of CTCs, all the white blood cells are removed and a relatively pure population of CTCs remains.
<ol>
	<li>After discarding the supernatant into 10% bleach, wash the whitish pellet in 1 ml phosphate-buffered saline (PBS) at least once, and resuspend in complete culture medium for propagation in cell culture.</li>
	<li>Once wash is completed, add 1 ml of complete BNL 1ME A.7R.1 culture medium (10% heat inactivated FBS, 1% penicillin/streptomycin and 2mM L-Glutamine) to the pellet and resuspend the pellet in the culture medium.</li>
	<li>Seed the resuspended mix of cells into a cell culture dish. Incubate the cell culture dish at 37 &#176;C in a humidified incubator with air and 5% CO2.</li>
	<li>Change the cell culture medium every two to three days. After five to seven days, CTCs will have adhered to the cell culture dish and started proliferating. These cells will remain viable beyond 25 passages and after repeated freezing and thawing cycles5.</li>
</ol>
6. Verification of Hepatocellular Carcinoma Circulating Tumor Cell Line
<ol>
	<li>Perform a polymerase chain reaction (PCR)-based method, to amplify only one specific DNA segment of the mouse &#946;-globin gene to confirm that the novel established CTC cell lines are mouse cells like the original implanted BNL 1ME A.7R.1 HCC line. Method was originally described and validated by Steube et al.5,8</li>
	<li>Perform immunostaining for the hepatocyte-specific marker cAMP responsive element binding protein 3-like 3 (CREB3L3) by using a specific antibody. This will confirm that novel established CTCs are indeed hepatocytes like the original implanted BNL 1ME A.7R.1 cell line.5</li>
</ol>

<ol>
	<li>Ashworth, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+case+of+cancer+in+which+cells+similar+to+those+in+the+tumors+were+seen+in+the+blood+after+death.">A case of cancer in which cells similar to those in the tumors were seen in the blood after death.</a> Australian Medical Journal. 1869, 146-147 (14).</li><li>van de Stolpe, A., den Toonder, J. <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:+What+Is+in+It+for+the+Patient?+A+Vision+towards+the+Future.">Circulating Tumor Cells: What Is in It for the Patient? 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. Clinical oncology. 6, 339-351 (2009).</li><li>Pantel, K., Brakenhoff, R. H., Brandt, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection,+clinical+relevance+and+specific+biological+properties+of+disseminating+tumour+cells.">Detection, clinical relevance and specific biological properties of disseminating tumour cells.</a> Nature reviews. Cancer. 8, 329-340 (2008).</li><li>Cristofanilli, 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=Circulating+tumor+cells,+disease+progression,+and+survival+in+metastatic+breast+cancer.">Circulating tumor cells, disease progression, and survival in metastatic breast cancer.</a> The New England journal of medicine. 351, 781-791 (2004).</li><li>De Giorgi, 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+tumor+cells+and+bone+metastases+as+detected+by+FDG-PET/CT+in+patients+with+metastatic+breast+cancer.">Circulating tumor cells and bone metastases as detected by FDG-PET/CT in patients with metastatic breast cancer.</a> Annals of oncology : official journal of the European Society for Medical Oncology / ESMO. 21, 33-39 (2010).</li><li>Eloubeidi, M. 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>

The authors declare that they have no competing financial interests.

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 important in cancer metastasis and have potential for multiple clinical applications, such as for diagnosis3,9,10. This is supported by data from several clinical studies that have showed that CTCs have a strong correlation with the progression of the disease in various cancer types such as pancreatic, prostate and breast, among others3,11,12. Despite the fact that CTCs may be very useful for cancer diagnosis, isolating and characterizing CTCs are challenging because very few are present among billions of blood cells3,7.
A significance of isolating CTCs using our technique is that it provides a reproducible model which can be optimized for potential utility in a clinical setting. This may ultimately lead to the possibility of using this method to replace current biopsy procedures that are more invasive and associated with side effects3,13. Modifications of this method, such as peripheral intravenous blood collection, would be expected to utilize it in a clinical setting. Of course, it is acknowledged that isolating CTCs from humans is a much more challenging endeavor due to general low yield4,14. But, in many cases such as in pancreatic cancer, histo-pathologic or cytological validation using tissue biopsies is fruitless in spite of multiple invasive attempts4. Isolating CTCs has been described as &#8220;liquid biopsy&#8221; or &#8220;live biopsy&#8221; of a tumor which enables noninvasive cancer diagnosis and real-time monitoring of therapeutic response3. This methodology models the isolation of CTCs in a mouse model. Thus far, only one FDA- cleared assay, such as Veridex CellSearch System, has been approved to detect CTCs. In one study, this technology identified CTCs in only 11 of 26 patients. This system is associated with some inefficiency, limited purity and high cost4. Moreover, it lacks the important feature of being able to collect viable CTCs whether they are EpCAM-expressing or not for extensive analyses using multiple in vitro and in vivo methods.
An important feature of the described method is that it preserves the viability of the isolated CTCs3. Therefore, using this method allows for the subsequent extensive downstream analysis of both the molecular and functional characteristics of CTCs using a variety of techniques. 
In conclusion, a methodology for isolating and propagating CTCs that are viable and can be characterized molecularly and functionally is presented. There is potential that this method can be optimized for clinical applications.

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><tbody><tr><td>Heparin Sodium Salt 1 g</td><td>VWR</td><td>89508-852</td><td></td></tr><tr><td>BTX Tube Micro 1.5 ml Clear NS</td><td>VWR</td><td>89511-254</td><td>1.5 ml&amp;nbsp; pyrogen-free eppendorf Tubes</td></tr><tr><td>Needle Sterile Disp BD 25 G x 1 in</td><td>VWR</td><td>BD305125</td><td>25 G Needle</td></tr><tr><td>Slp Tip SRNG 1 ml 200 each per pack</td><td>VWR</td><td>BD309659</td><td>1 ml syringe tip</td></tr><tr><td>Syringe 1 ml 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 15 x 15 x 5 mm PK 100</td><td>VWR</td><td>25608-924</td><td>Cyromold</td></tr><tr><td>Cryo-oct compund 4 oz</td><td>VWR</td><td>25608-930</td><td>Oct compound</td></tr><tr><td>VWR Slide sprfrst 25 x 75 mm PK72</td><td>VWR</td><td>48311-703</td><td>Slides</td></tr><tr><td>VWR Cover Glass #2 22 x 50 mm</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 2 L</td><td>VWR</td><td>101454-204</td><td>Nuclease Free Water</td></tr><tr><td>Buffer Tris Ultra Pure Grade 500 g</td><td>VWR</td><td>97061-796</td><td>Tris Buffer</td></tr><tr><td>Ammonium Chloride ACS Grade 2, 5 Kg</td><td>VWR</td><td>97062-048</td><td>Ammonium Chloride Buffer</td></tr><tr><td>Falcon Tissue Culture Dish 60 x 15 mm Style polystyrene</td><td>VWR</td><td>353002</td><td>Tissue Culture Dish</td></tr><tr><td>Clorox 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 &amp;amp; magnesium (500 ml)</td><td>Thermo Fischer Scientific</td><td>21-040-CV</td><td>PBS, 1x</td></tr><tr><td>DMEM, 1x&amp;nbsp; with 4.5 g/L glucose &amp;amp; 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 Serum</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>75002411</td><td>13,000 rpm 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 the RBC lysis. This was then followed by another centrifugation step to collect a whitish pellet that was washed in PBS, and resuspended in complete cell culture medium for propagation in cell culture. An issue that may come up is that the pellet obtained may not be clear in color. This problem can be easily resolved by modifying the incubation time in RBC lysis buffer by increasing it to 10 - 15 min, or possibly by performing it additional times. Representative images of CTCs being propagated in culture from three different mice are shown in Figure 1 as compared to BNL 1ME A.7R.1 cells. Rapid proliferation and morphology will help identify if cells seeded are CTCs. However, verification techniques such as PCR and immunostaining described in steps 5.1 - 5.2 are needed to confirm the establishment of a viable CTC line.
<img alt="Figure 1" src="/files/ftp_upload/52861/52861fig1.jpg" /> 
Figure 1. Propagation of Circulating Tumor Cells (CTCs) in Cell Culture from an Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis. Cells were propagated in 15 mm x 60 mm cell culture dishes in BNL 1ME A.7R.1 culture medium. Proliferating tumor cells adhered to dish and are shown from three separate mice. Tumor cells have remained viable through continuous passages. Phase contrast image of CTCOL1MEA081211, CTCOL1MEACTC082911 and CTCOLUF2419 were taken at 40X magnification (objective lens) in comparison to BNL 1ME A.7R.1 cells (40X magnification). <a href="https://www.jove.com/files/ftp_upload/52861/52861fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 1" src="/files/ftp_upload/52861/52861fig2.jpg" /> 
Figure 2. Schematic of the Procedure of Isolating and Propagating Circulating Tumor Cells from a Mouse Cancer Model System. When clinical evidence of hepatocellular carcinoma was observed as significant reduction in body condition score, whole blood was collected from mice. After several centrifugations, RBC lysis, and several wash steps, the CTCs were seeded into cell culture dishes for propagation. Tumor cell proliferation was observed after several days. <a href="https://www.jove.com/files/ftp_upload/52861/52861fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

isolation-propagation-circulating-tumor-cells-from-mouse-cancer

Nanyang Technological University

Research

JoVE Journal

Medicine

12.0K Views.  Hunter College of The City University of New York. 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.

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

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.

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 ...

Cancer Research

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

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

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

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

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common ...

Genetics

Circulating Tumor Cell Lines: an Innovative Tool for Fundamental and Translational Research

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an urgent need to understand the mechanisms behind metastasis development, and thus to propose efficient treatments for advanced cancer. Metastatic cancers are hard to treat, as biopsies are invasive and inaccessible. Recently, there has been considerable interest in liquid biopsies including both cell-free circulating deoxyribonucleic acid (DNA) and circulating tumor cells from peripheral blood and we have established several circulating tumor cell lines from metastatic colorectal cancer patients to participate in their characterization. Indeed, to functionally characterize these rare and poorly described cells, the crucial step is to expand them. Once established, circulating tumor cell (CTC) lines can then be cultured in suspension or adherent conditions. At the molecular level, CTC lines can be further used to assess the expression of specific markers of interest (such as differentiation, epithelial or cancer stem cells) by immunofluorescence or cytometry analysis. In addition, CTC lines can be used to assess drug sensitivity to gold-standard chemotherapies as well as to targeted therapies. The ability of CTC lines to initiate tumors can also be tested by subcutaneous injection of CTCs in immunodeficient mice.
Finally, it is possible to test the role of specific genes of interest that might be involved in cancer dissemination by editing CTC genes, by short hairpin ribonucleic acid (shRNA) or Crispr/Cas9. Modified CTCs can thus be injected into immunodeficient mouse spleens, to experimentally mimic part of the metastatic development process in vivo.
In conclusion, CTC lines are a precious tool for future research and for personalized medicine, where they will allow prediction of treatment efficiency using the very cells that are originally responsible for metastasis.

Culturing CTCs allows a deeper functional characterization of cancer, through assaying specific marker expression, and assessing drug resistance and the ability to colonize the liver among other possibilities. Overall, CTC culture could be a promising clinical tool for personalized medicine to improve patient outcome.

Metastasis is a leading cause of cancer death. Despite improvements in treatment strategies, metastatic cancer has a poor prognosis. We thus face an ...

Guided Tumor Inoculation and Multimodal Imaging in Mice

A Dorsal Skinfold Window Chamber Tumor Mouse Model for Combined Intravital Microscopy and Magnetic Resonance Imaging in Translational Cancer Research

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the tumor microenvironment and its response to therapy. These imaging modalities have micron-scale resolution but have limited use in the clinic due to their shallow penetration depth into tissue. More clinically applicable imaging modalities such as CT, MRI, and PET have much greater penetration depth but have comparatively lower spatial resolution (mm scale). 
To translate preclinical intravital imaging findings into the clinic, new methods must be developed to bridge this micro-to-macro resolution gap. Here we describe a dorsal skinfold window chamber tumor mouse model designed to enable preclinical intravital and clinically applicable (CT and MR) imaging in the same animal, and the image analysis platform that links these two disparate visualization methods. Importantly, the described window chamber approach enables the different imaging modalities to be co-registered in 3D using fiducial markers on the window chamber for direct spatial concordance. This model can be used for validation of existing clinical imaging methods, as well as for the development of new ones through direct correlation with "ground truth" high-resolution intravital findings. 
Finally, the tumor response to various treatments-chemotherapy, radiotherapy, photodynamic therapy-can be monitored longitudinally with this methodology using preclinical and clinically applicable imaging modalities. The dorsal skinfold window chamber tumor mouse model and imaging platforms described here can thus be used in a variety of cancer research studies, for example, in translating preclinical intravital microscopy findings to more clinically applicable imaging modalities such as CT or MRI.

Author Spotlight: Integrating High-Resolution Intravital Imaging and MRI to Enhance Stereotactic Body Radiation Therapy Planning

Translation of Intravital microscopy findings is challenged by its shallow depth penetration into tissue. Here we describe a dorsal window chamber mouse model that enables co-registration of intravital microscopy and clinically applicable imaging modalities (e.g., CT, MRI) for direct spatial correlation, potentially streamlining clinical translation of intravital microscopy findings.

Preclinical intravital imaging such as microscopy and optical coherence tomography have proven to be valuable tools in cancer research for visualizing the ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Intracecal Tumor Cell Injection: A Technique to Deliver Cancer Cells for Establishing Orthotopic Colorectal Cancer Mouse Model

Source: Kochall, S., et al. <a href="https://www.jove.com/v/55357"> Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer </a> J. Vis. Exp. (2017)
The video describes the technique for intracecal injection of cancerous cells to develop an orthotopic mouse model with highly uniform tumors. Therefore, the orthotopic mouse model allows realistic simulation of metastasis, providing a valuable tool to study the phenotype changes during tumor growth and dissemination.

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

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