We would like to thank Pascal DuBois for proof-reading the article. The authors would also like to thank the personnel of the Animal Facility at Hunter College of The City University of New York. This work was supported by a NIH RCMI grant to Hunter College.

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.
Note: Eight BALB/c mice were used in this experimental procedure. Five BALB/c mice were implanted with 5 X 106 BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells to produce the primary tumors. Three BALB/c mice with no implantations were used as controls. BNL 1ME A.7R.1 is a murine hepatocellular carcinoma cell line that was derived by chemical transformation from the murine non-tumorigenic hepatocyte cell line BNL CL.2. BNL 1ME A.7R.1 was obtained from the American Type Culture Collection (ATCC).
1. Pre-surgery
<ol>
	<li>Ensure that mice for all survival surgeries are healthy. To ensure that mice are healthy, inspect signs of dehydration by tenting of the skin. Do not use mice with any evidence of disease or distress in these experiments.</li>
	<li>Weigh mice to ensure they are not underweight. Ensure that the weight is at least 16-20 grams.</li>
	<li>Prepare 5&#215;106 BNL 1ME A.7R.1 mouse HCC cells for implantation per mouse:
	<ol>
		<li>Aspirate media from a 10 ml&#160;60% - 70% confluent cell culture dish of BNL 1ME A.7R.1 mouse HCC cells. Wash twice with 5 ml&#160;PBS. Add 2 ml&#160;of trypsin to the dish and incubate at 37 &#176;C in a humidified incubator with air and 5% CO2 for 5 min.</li>
		<li>Subsequently, pipette 10 &#181;l trypsinized cells into a manual hemocytometer for cell counting under a microscope at magnification of 10X. Count the total number of cells found in the four large corner squares and multiply the dilution factor by the total number of cells. Then, divide by the number of squares counted and multiply by 106 for the cell concentration (cells per ml).</li>
		<li>Using the calculation, collect 5 million cells per mouse in sterile 1.5 ml&#160;microcentrifuge tubes. Label 1.5 ml&#160;microcentrifuge tubes.</li>
	</ol>
	</li>
	<li>Resuspend the 5&#215;106 BNL 1ME A.7R.1 mouse HCC cells in 100 &#181;l of phosphate buffered saline (PBS). Keep these cells on ice till implantation in surgery.</li>
	<li>Perform activities required for preparation of mice for surgery such as anesthesia induction and hair clipping away from sterile equipment and materials to avoid contamination.</li>
	<li>Induce general anesthesia in mice by inhalation of isoflurane using an isoflurane vaporizer along with oxygen at a constant concentration flow rate of 1 L per min&#160;(approx. 2.1%). Note: The system is self-reliant, and includes a precision vaporizer, an oxygen supply and a charcoal canister that is linked to a breathing circuit needed for gas/anesthetic scavenging.</li>
	<li>Ensure that anesthesia is adequate to prevent the animal from undergoing pain during the surgery. Perform toe-pinch and observe animal for non-responsiveness to confirm anesthesia.</li>
	<li>At that time, switch the valve line, seal to the box and open to the nose cone.</li>
	<li>As soon as the mouse is anesthetized, apply a tiny amount of an ophthalmic ointment to the corneas as a preventive measure from drying and damage.</li>
	<li>Subsequently, move the animal to the preparation area, with the nose cone properly placed and connected (for maintenance anesthesia). Prepare the site of the surgical incision for surgery by depilation at this preparation area.</li>
	<li>Ensure that the area of surgery is at a location free of all other activity.</li>
	<li>Ensure that the work surface is sturdy and appropriately sanitized, unsoiled and clutter-free before the surgery. Place a sterile piece of linen or surgical tray over it.</li>
	<li>Ensure that the operative field is available to only persons carrying out the surgery. Ensure that the surgical room is cleaned and swept with an appropriate cleaner and sprayed with an appropriate disinfectant.</li>
	<li>Ensure that it is also positioned away from doors and windows as air currents can spread dust leading to contamination of the site.</li>
	<li>Perform the survival surgery with sterile instruments. Either soak instruments in a disinfectant according to manufacturer&#8217;s recommendation or autoclave. Use basic sterile supplies that are ready for use.</li>
	<li>Separate instruments according to function to help prevent contamination of sterile areas.</li>
	<li>Shave approximately 1 inch around the site of incision (midline incision) to access the liver of the animal. Remove the hair from the animal away from the site of surgery.</li>
	<li>After shaving, scrub the area with a soapy disinfectant (betadine surgical scrub) and wash the excess disinfectant away with sterile PBS solution. Perform this several times, with the entire scrub process lasting approximately 5 min. Remove and discarded the used gloves.</li>
	<li>Afterward, change to protective clothing such as surgical gown, scrub top, and face mask. Next, sanitize hands with proper scrub solution before putting on surgical gloves. Note: This is a necessary step to prevent shedding and contaminating the surgical site with potential contaminants.</li>
</ol>
2. Surgery
<ol>
	<li>Provide an external heat source for warmth throughout the entire period of anesthesia. The animal may experience some hypothermia because anesthesia causes disturbances of thermoregulation; therefore, maintain the body temperature.</li>
	<li>Lay the mouse on its back throughout the surgery.</li>
	<li>Fix the fore limbs, hind limbs and tail of the mouse to the surgery table with pieces of surgical tapes.</li>
	<li>Make a straight line incision immediately inferior to the xyphisternum.</li>
	<li>Use a sterile self-retaining surgical retractor to expose the liver.</li>
	<li>With a syringe having a 20G needle, slowly and carefully inject 100 &#181;l of PBS containing 5&#215;106 BNL 1ME A.7R.1 mouse HCC cells into the right lobe of the liver.</li>
	<li>After removing the needle, apply a sterile cotton swab for at least 1 min&#160;to stop any potential bleeding due to high vascularity of the liver.</li>
	<li>Close the subcutaneous tissue with an absorbable suture followed by skin closure using surgical clips. Subsequently, clean the wound once again with betadine scrub. Note: This will ensure the closure of the surgical wound.</li>
	<li>Subcutaneously give 100 &#181;l of normal saline.</li>
</ol>
3. Post-surgery
<ol>
	<li>Immediately after surgery, transfer the mouse to a clean cage with a post-surgery identification card to notify caretakers of the mouse condition. Provide food and water. Note: In our experience, a mouse that has just undergone surgery will fare best if housed alone with wet food and water for the initial 24-hr&#160;period post-surgery.</li>
	<li>Keep the animal warm with a heat lamp that generates heat not surpassing 38 &#186;C. Use a thermometer to measure the temperature until the mouse is fully recovered.</li>
	<li>House the animal in a cage preferably with paper bedding to ensure comfort.</li>
	<li>After initial recovery, monitor the animal for signs of discomfort, bleeding, pain and distress. Give buprenorphine (0.05 -0.1 mg/kg) to relieve pain. Closely monitor the activity, behavior and appearance, as well as water and food consumption for approximately 2 hr. If ambulatory, move the animal back to the area for regular normal housing of mice and continue to monitor at 6-12 hr&#160;intervals for 24 hr&#160;post-surgery. Perform a weight check if desired.</li>
	<li>However if the animal is having issues recuperating, take appropriate supportive care actions. If mice are unable to recover, humanely euthanize mice via CO2 asphyxiation and cervical dislocation. Follow approved institutional guidelines for euthanasia.</li>
	<li>Post-surgery, check for clinical evidence of HCC which is usually observable as significant reduction in body condition score. Note: At this point, a primary tumor has likely developed in the liver and metastatic deposits have likely developed in the lungs.</li>
	<li>At this point, sacrifice all mice (experimental and control) involved in the experiment by humane euthanasia. Subject the mice to carbon dioxide asphyxiation: very slowly release carbon dioxide into the chamber over a period of 5 -10 min. This will cause respiratory arrest.</li>
</ol>

<ol>
	<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>Ogunwobi, O. O., 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=Therapeutic+and+prognostic+importance+of+epithelial-mesenchymal+transition+in+liver+cancers:+insights+from+experimental+models.">Therapeutic and prognostic importance of epithelial-mesenchymal transition in liver cancers: insights from experimental models.</a> Critical Reviews in Oncology/Hematology. 83, 319-328 (2012).</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>Ogunwobi, O. O., 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=Hepatocyte+growth+factor+upregulation+promotes+carcinogenesis+and+epithelial-mesenchymal+transition+in+hepatocellular+carcinoma+via+Akt+and+COX-2+pathways.">Hepatocyte growth factor upregulation promotes carcinogenesis and epithelial-mesenchymal transition in hepatocellular carcinoma via Akt and COX-2 pathways.</a> Clinical and Experimental Metastasis. 28, 721-731 (2011).</li><li>Fujiwara, 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=A+novel+animal+model+for+in+vivo+study+of+liver+cancer+metastasis.">A novel animal model for in vivo study of liver cancer metastasis.</a> World Journal of Gastroenterology : WJG. 18, 3875-3882 (2012).</li><li>Wirtz, D., Konstantopoulos, K., Searson, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+physics+of+cancer:+the+role+of+physical+interactions+and+mechanical+forces+in+metastasis.">The physics of cancer: the role of physical interactions and mechanical forces in metastasis.</a> Nature Reviews Cancer. 11, 512-522 (2011).</li><li>Heindryckx, F., Colle, I., Van Vlierberghe, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+mouse+models+for+hepatocellular+carcinoma+research.">Experimental mouse models for hepatocellular carcinoma research.</a> International Journal of Experimental Pathology. 90, 367-386 (2009).</li><li>Zhang, L., 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=An+in+vivo+mouse+model+of+metastatic+human+thyroid+cancer.">An in vivo mouse model of metastatic human thyroid cancer.</a> Thyroid Official Journal of the American Thyroid Association. 24, 695-704 (2014).</li><li>Menezes, M. 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=Genetically+engineered+mice+as+experimental+tools+to+dissect+the+critical+events+in+breast+cancer.">Genetically engineered mice as experimental tools to dissect the critical events in breast cancer.</a> Advances in Cancer Research. 121, 331-382 (2014).</li><li>Ogunwobi, O. O., Wang, T., Zhang, L., 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=Cyclooxygenase-2+and+Akt+mediate+multiple+growth-factor-induced+epithelial-mesenchymal+transition+in+human+hepatocellular+carcinoma.">Cyclooxygenase-2 and Akt mediate multiple growth-factor-induced epithelial-mesenchymal transition in human hepatocellular carcinoma.</a> Journal of Gastroenterology and Hepatology. 27, 566-578 (2012).</li><li>Hanahan, D., Weinberg, 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+hallmarks+of+cancer.">The hallmarks of cancer.</a> Cell. 100, 57-70 (2000).</li><li>Chambers, A. F., Groom, A. C., MacDonald, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissemination+and+growth+of+cancer+cells+in+metastatic+sites.">Dissemination and growth of cancer cells in metastatic sites.</a> Nature Reviews Cancer. 2, 563-572 (2002).</li><li>Kim, M. Y., 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=Tumor+self-seeding+by+circulating+cancer+cells.">Tumor self-seeding by circulating cancer cells.</a> Cell. 139, 1315-1326 (2009).</li><li>Stolpe, A., Pantel, K., Sleijfer, S., Terstappen, L. W., den Toonder, J. M. <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+cell+isolation+and+diagnostics:+toward+routine+clinical+use.">Circulating tumor cell isolation and diagnostics: toward routine clinical use.</a> Cancer Research. 71, 5955-5960 (2011).</li><li>Saad, F., Pantel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+current+role+of+circulating+tumor+cells+in+the+diagnosis+and+management+of+bone+metastases+in+advanced+prostate+cancer.">The current role of circulating tumor cells in the diagnosis and management of bone metastases in advanced prostate cancer.</a> Future Oncology. 8, 321-331 (2012).</li></ol>

The authors declare that they have no competing financial interests.

In this article an in-depth description of the method is given that was recently reported by Ogunwobi and colleagues of successful establishment of an orthotopic syngeneic mouse model of HCC metastasis (Figure 2)3. The take rate of tumors for this procedure is generally high. We have previously observed a tumor take rate of 100%3. However, the take rate can be variable depending on the competence of the investigator. In a recent experiment performed by a new graduate student under training, the tumor take rate was as low as 40%. Although most experiments are terminated at 4 - 6 weeks due to decline in body condition score, we believe from experience with subcutaneous implantation of the same cells into the same mice that primary tumors may be established by 2 - 3 weeks. We usually expect to see one tumor nodule from a successful experiment. And although we expect lung metastasis to develop between 4 - 6 weeks, it is conceivable that tumor in the liver may take place much earlier.
Good surgical techniques and aseptic conditions are essential to the success of this survival surgery method. Mice used for survival surgery should be healthy. Weighing pre and post-surgery is a good practice. The average pre-surgery weights of the mice used in or most recent experiment was 18 grams, and at the experimental end-point (63 days post surgical implantation) their average weight was only 22 grams. A gain of only 4 grams of weight during that time period is likely because there was some decline in weight gain due to systemic illness from the tumor. We would have had a more objective assessment of this had we measured mouse weight once or twice weekly throughout the experiment. This is recommended best practice.
As summarized in Figure 2, mice should be adequately anesthetized before the surgery and throughout the period of surgery to avoid pain and distress. The area should be free of all clutter, and access should only be available to those performing the surgery. Both the surgery area as well as the instruments used should all be sterile.
A vital step for the success of implantation is the preparation of the BNL 1ME A.7R.1 mouse HCC cells to be implanted. During the preparation, it is crucial to avoid contamination of the collection of the cells from confluent cell cultures dishes. These steps should be performed in a cell culture hood under sterile conditions. A cell count should be also done to ensure 5&#215;106 BNL 1ME A.7R.1 mouse HCC cells have been collected in a sterile 1.5 ml&#160;microcentrifuge tube per mouse that is resuspended in 100 &#181;l phosphate buffered saline (PBS). Trypan blue staining can also be optionally performed to confirm the viability of the cells. These cells should be kept on ice no longer than 2 hr before implantation in livers of mice.
The implantation of the cancer cells should be handled with care due to the delicate nature of the liver (Figure 2). During injection of the cancer cells, avoid too deep a puncture that may result in the needle going through the entire liver. It is essential to inject the cancer cells slowly and carefully. On the other hand, too shallow a puncture with the needle prior to injection will likely cause spillage of the cancer cells injected out into the peritoneal cavity. Also, after removing the needle, there is always a potential of the organ bleeding out due to its high vascularity. Therefore, applying a sterile cotton swab for at least 1 min&#160;is recommended. Care and attention will ensure the highest rate of implantation success. The animal should also be provided an external source of heat throughout the procedure as hypothermia is a major concern causing disturbances of thermoregulation during anesthesia. Post-surgery, the animal should be monitored for any signs of discomfort or pain at which point appropriate care should be administered. If the mouse is unable to recuperate from the surgery, it should be humanely euthanized.
As cancer metastasis is the primary cause of fatality from cancer, it is crucial to delineate the mechanisms of hematogenous dissemination2-4. Novel circulating tumor cells were established from this orthotopic syngeneic mouse model of HCC metastasis3. Moreover, the CTCs derived from this model consistently show increased tumor initiation and metastatic capability3. The circulating tumor cells isolated using this method may prove to be very important as they help to unravel specific molecular factors, markers and possible therapeutic targets of cancer metastasis2-4. This methodology has already identified that there is increased upregulation of hepatocyte growth factor (HGF) and its receptor, c-Met in circulating tumor cells as compared to the primary tumor cells of HCC3. These results build on previous work demonstrating that an important aspect of tumor metastasis is the cellular process of epithelial-mesenchymal transition2-4,10. Further characterizing circulating tumor cells using this novel model will increase our understanding of the intricate mechanisms of factors that promote the process thus leading to the development of more effective management of HCC1-3,10.
Currently, the most widely used animal tumor models are mouse xenograft tumor models wherein human tumor cells are implanted into immune deficient mice. The model described in this article has important advantages because it is syngeneic and is, therefore, suitable for studying the role of the immune system in tumor metastasis3. This model has proven to be highly reproducible for isolating, culturing, and studying of viable circulating tumor cells3. It, therefore, has tremendous potential for evaluating clinical applications like screening, diagnosis, prognostication, therapy monitoring and discovery of novel therapeutic strategies. A limitation of the methodology described above is that a significant portion of BNL 1ME A.7R.1 mouse HCC cells implanted into the mice will be incapable of surviving and thus only a small portion of the cells will be viable and adept at introducing tumors at secondary sites3. We account for this limitation by initial implantation of 5&#215;106 BNL 1ME A.7R.1 mouse HCC cells per mouse.
In conclusion, this orthotopic syngeneic mouse model of metastatic solid organ cancer provides a good platform for elucidating the biological mechanisms underlying the metastatic capability of circulating tumor cells in the blood of cancer patients3. This in turn will likely shed novel insights into the mechanisms of cancer metastasis because cancer metastasis occurs primarily via hematogenous spread11-13. Novel insights thus gained into the mechanisms of cancer metastasis will be critical to the development of novel beneficial clinical applications3,14,15.

Hepatocellular carcinoma (HCC) is one of the most lethal cancers, with poor prognosis and low life expectancy. Nearly all cancer-related deaths are due to the metastatic spread of the disease from the originating organ to additional distant organs2-6.HCC progression is a complex process. Therefore, being able to model the tumor microenvironment that is naturally found in metastatic HCC in animal models can prove to be a successful and useful way to reveal relevant mechanisms in humans.7Unfortunately, the mechanisms of cancer metastasis are still largely unclear. Therefore, there is a need to establish animal models that will enable us to elucidate the underlying molecular mechanisms of metastases of cancers such as HCC3,5.
Mouse model systems are a very useful approach for delineating mechanisms and evaluating novel strategies for treatment of metastatic human cancers5,8. The various mouse model systems that presently exist are a testament to efforts of researchers to correctly depict the complexity of the disease5,8,9.
Ogunwobi and colleagues recently used a survival surgical approach to demonstrate establishment of a novel orthotopic syngeneic mouse model for the study of metastasis in HCC3. Their work established a &#8220;patient-like&#8221; mouse model that recapitulates features of aggressive and metastatic HCCs3. They further demonstrated that this mouse model system can be used to study the biology of circulating tumor cells, and that this holds potential for gaining novel insights into the mechanisms of cancer metastasis3.
The aim of this paper is to describe in detail the methodology used in establishing this &#8220;patient-like&#8221; orthotopic syngeneic mouse model of HCC metastasis3. The methodology of how to implant BNL 1ME A.7R.1 mouse HCC cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c3 using survival surgery will be described. Unlike other mouse xenograft tumor models where human tumor cells are implanted into immune deficient mice, this system is syngeneic and is, therefore, suitable for studying the role of the immune system in tumor metastasis3,5,8. This approach will likely gain widespread use for studying the mechanisms of metastasis in solid organ cancers.

<table><tbody><tr><td>Micro Dissecting Tweezer&amp;nbsp;</td><td>Roboz</td><td>(RS-5040)</td><td>Tip 0.20 x 0.12 mm</td></tr><tr><td>Graefe Micro Dissecting Forceps, serrated curved tip</td><td>Roboz</td><td>(RS-5111)</td><td>1 X 2 teeth, curved tip width 0.6 mm</td></tr><tr><td>Micro Dissecting Retractor-Agricola (3 by 3 prongs)</td><td>Roboz</td><td>(RS-6501)</td><td>Blunt 3 X 3 prongs, depth 4 mm, spread 25 mm</td></tr><tr><td>Micro Dissecting Retractor-Goldstein (3 by 3 prongs)</td><td>Roboz</td><td>(RS-6503)</td><td>Blunt, 3 X 3 prongs, depth 4 mm, spread 19 mm</td></tr><tr><td>Jameson Caliper (Measures tumors)</td><td>Roboz</td><td>(RS-6466)</td><td>80 mm/3 inch scale, chrome plated&amp;nbsp;</td></tr><tr><td>Micro Dissecting Scissors, large ring sissors, straight and sharp</td><td>Roboz</td><td>(RS-5852)</td><td>23 mm blades, length 4 inches, flat shanks and large rings</td></tr><tr><td>Scalpel with blades, for delicate dissecting procedures</td><td>Roboz</td><td>(RS-9861-36)</td><td></td></tr><tr><td>Scalpel handle&amp;nbsp;</td><td>Roboz</td><td>(RS-9884)</td><td>Solid</td></tr><tr><td>Littauer Stitch Sissors</td><td>Roboz</td><td>(RS-7074)</td><td>Length 4.5 inches</td></tr><tr><td>Brown needle holder, for easy suture tying</td><td>Roboz</td><td>(RS-7960)</td><td>Convex jaw, fine serrations</td></tr><tr><td>Reflex 7MM wound clips with reflex 7 clip applier</td><td>Roboz</td><td>(RS-9262)</td><td>safe, secure alternative method of wound closure</td></tr><tr><td>Instrument tray and lid</td><td>Roboz</td><td>RT-1350S</td><td></td></tr><tr><td>Mini-clipper with detachable blade</td><td>Roboz</td><td>(RS-5903)</td><td></td></tr><tr><td>Germinator 500 (the Germ Terminator) Dry Sterilizer</td><td>Roboz</td><td>Ds-400, Ds-401, DS-501</td><td>For fast decontamination of micro-dissecting instruments.&amp;nbsp;&amp;nbsp; Instruments decontaminate within 15 sec.&amp;nbsp;</td></tr><tr><td>Microinjection needles</td><td>VWR</td><td>BD305125</td><td>25G Needle</td></tr></tbody></table>

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 livers of Balb/c mice were implanted with 5&#215;106 BNL 1ME A.7R.1 mouse HCC cells. Clinical evidence of HCC development was observable 63 days post-surgery. Consequently, mice were humanely euthanized. Necropsy was performed on the euthanized mice. The lungs and liver were resected and a careful gross examination was performed to identify macroscopic tumors. In one mouse, a superficial tumor was observed on the surface of the liver with some attachment to the abdominal wall (Figure 1A). In a second mouse, an abnormally-looking liver was observed. The abnormality very strongly looks like a superficial tumor (Figure 1B). As expected, mice in the control group with no implantation of BNL 1ME A.7R.1 HCC cells developed no tumors, as evidenced by a liver (Figure 1D) and lungs (Figure 1E) from a control mouse. Also, the lungs from the mice with tumors looked somewhat abnormal, displaying areas of hemorrhagic necrosis (Figure 1C). Based on previous experience, these lungs may harbor metastatic deposits3.
<img alt="Figure 1" src="/files/ftp_upload/52858/52858fig1.jpg" /> 
Figure 1. Gross examination of tumor and metastasis. Balb/c mice were implanted with 5 X 106 BNL 1ME A.7R.1 tumor cells and euthanized 63 days later. Representative liver tumors observed were photographed and shown here. (A and B) both show superficial liver tumors. (C) is lungs from tumor cells implanted mice. (D) displays liver of a control mouse. (E) indicates lungs of a control mouse. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52858/52858fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52858/52858fig2.jpg" /> 
Figure 2. Schematic of the methodology of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model of hepatocellular carcinoma metastasis. Once anesthetized, mouse liver is implanted with 5 X 106 BNL 1ME A.7R.1 mouse hepatocellular carcinoma (HCC) cells. When clinical evidence of HCC is observable, animal is sacrificed by humane euthanasia (subjected to carbon dioxide asphyxiation, followed by percutaneous intracardiac exsanguination and cervical dislocation). <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52858/52858fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis at JoVE.com

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

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Universidad San Sebastian

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JoVE Journal

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12.2K Views.  Hunter College of the City University of New York. 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 “patient-like” orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

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

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

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

Cancer Research

A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast cancer metastasis, and results in poor outcomes with median survival rates of only 4.8 - 15 months. Current rodent models of breast cancer metastasis, including primary tumor cell xenograft and spontaneous tumor models, rarely metastasize to the liver. Intracardiac and intrasplenic injection models do result in liver metastases, however these models can be confounded by concomitant secondary-site metastasis, or by compromised immunity due to removal of the spleen to avoid tumor growth at the injection site. To address the need for improved liver metastasis models, a murine portal vein injection method that delivers tumor cells firstly and directly to the liver was developed. This model delivers tumor cells to the liver without complications of concurrent metastases in other organs or removal of the spleen. The optimized portal vein protocol employs small injection volumes of 5 - 10 &#956;l, &#8805; 32 gauge needles, and hemostatic gauze at the injection site to control for blood loss. The portal vein injection approach in Balb/c female mice using three syngeneic mammary tumor lines of varying metastatic potential was tested; high-metastatic 4T1 cells, moderate-metastatic D2A1 cells, and low-metastatic D2.OR cells. Concentrations of &#8804; 10,000 cells/injection results in a latency of ~ 20 - 40 days for development of liver metastases with the higher metastatic 4T1 and D2A1 lines, and &#62; 55 days for the less aggressive D2.OR line. This model represents an important tool to study breast cancer metastasis to the liver, and may be applicable to other cancers that frequently metastasize to the liver including colorectal and pancreatic adenocarcinomas.

A surgical procedure was developed to deliver mammary tumor cells to the murine liver via portal vein injection. This model permits investigation of late stages of liver metastasis in a fully immune competent host, including tumor cell extravasation, seeding, survival, and metastatic outgrowth in the liver.

Breast cancer is the leading cause of cancer-related mortality in women worldwide. Liver metastasis is involved in upwards of 30% of cases with breast ...

A Syngeneic Mouse Model of Metastatic Renal Cell Carcinoma for Quantitative and Longitudinal Assessment of Preclinical Therapies

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of diagnosis. Metastatic RCC (mRCC) is incurable, with a median survival time of only 18 months. Immune-based interventions (e.g., interferon (IFN) and interleukin (IL)-2) induce durable responses in a fraction of mRCC patients, and multikinase inhibitors (e.g., sunitinib or sorafenib) or anti-VEGF receptor monoclonal antibodies (mAb) are largely palliative, as complete remissions are rare. Such shortcomings in current therapies for mRCC patients provide the rationale for the development of novel treatment protocols. A key component in the preclinical testing of new therapies for mRCC is a suitable animal model. Beneficial features that recapitulate the human condition include a primary renal tumor, renal tumor metastases, and an intact immune system to investigate any therapy-driven immune effector responses and the formation of tumor-induced immunosuppressive factors. This report describes an orthotopic mRCC mouse model that has all of these features. We describe an intrarenal implantation technique using the mouse renal adenocarcinoma cell line Renca, followed by the assessment of tumor growth in the kidney (primary site) and lungs (metastatic site).

Implementation of an orthotopic model of renal cell carcinoma in immunocompetent mice affords the investigator a clinically-relevant system defined by the presence of a primary renal tumor and lung metastases in the same animal. This system can be used to preclinically test a variety of treatments in vivo.

Renal cell carcinoma (RCC) affects &#62; 60,000 people in the United States annually, and ~ 30% of RCC patients have multiple metastases at the time of ...

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

Biology

An Oncogenic Hepatocyte-Induced Orthotopic Mouse Model of Hepatocellular Cancer Arising in the Setting of Hepatic Inflammation and Fibrosis

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded elucidation of the underlying mechanisms and development of innovative immunotherapeutic strategies. To develop an ideal animal model recapitulating human HCC, immunocompetent male C57BL/6J mice first receive a carbon tetrachloride (CCl4) injection to induce liver fibrosis, then receive histologically-normal oncogenic hepatocytes from young male SV40 T antigen (TAg)-transgenic mice (MTD2) by intra-splenic (ISPL) inoculation. Androgen generated in recipient male mice at puberty initiates TAg expression under control of a liver-specific promoter. As a result, the transferred hepatocytes become cancer cells and form tumor masses in the setting of liver fibrosis/cirrhosis. This novel model mimics human HCC initiation and progression in the context of liver fibrosis/cirrhosis and reflects the most typical features of human HCC including immune dysfunction.

Here, we describe the development of a clinically relevant murine model of liver cancer recapitulating the typical immune features of hepatocellular cancer (HCC).

The absence of a clinically relevant animal model addressing the typical immune characteristics of hepatocellular cancer (HCC) has significantly impeded ...

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

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

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

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

Non-Invasive PET/MR Imaging in an Orthotopic Mouse Model of Hepatocellular Carcinoma

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and evaluate novel therapeutic approaches. Non-invasive whole-body imaging using positron emission tomography (PET) provides critical insights into the in vivo characteristics of tissues at the molecular level in real-time. We present here a protocol for orthotopic HCC xenograft creation with and without hepatic artery ligation (HAL) to induce tumor hypoxia and the assessment of their tumor metabolism in vivo using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG) PET/magnetic resonance (MR) imaging. Tumor hypoxia could be readily visualized using the hypoxia marker [18F]FMISO, and it was found that the [18F]FMISO uptake was higher in HCC mice that underwent HAL than in the non-HAL group, whereas [18F]FDG could not distinguish tumor hypoxia between the two groups. HAL tumors also displayed a higher level of hypoxia-inducible factor (HIF)-1&#945; expression in response to hypoxia. Quantification of HAL tumors showed a 2.3-fold increase in [18F]FMISO uptake based on the standardized value uptake (SUV) approach.

Here, we present a protocol to create orthotopic hepatocellular carcinoma xenografts with and without hepatic artery ligation and perform non-invasive positron emission tomography (PET) imaging of tumor hypoxia using [18F]Fluoromisonidazole ([18F]FMISO) and [18F]Fluorodeoxyglucose ([18F]FDG).

Preclinical experimental models of hepatocellular carcinoma (HCC) that recapitulate human disease represent an important tool to study tumorigenesis and ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

An Orthotopic Mouse Model of Spontaneous Breast Cancer Metastasis

Metastasis is the primary cause of mortality of breast cancer patients. The mechanism underlying cancer cell metastasis, including breast cancer metastasis, is largely unknown and is a focus in cancer research. Various breast cancer spontaneous metastasis mouse models have been established. Here, we report a simplified procedure to establish orthotopic transplanted breast cancer primary tumor and resultant spontaneous metastasis that mimic human breast cancer metastasis. Combined with the bioluminescence live tumor imaging, this mouse model allows tumor growth and progression kinetics to be monitored and quantified. In this model, a low dose (1 x 104 cells) of 4T1-Luc breast cancer cells was injected into BALB/c mouse mammary fat pad using a tuberculin syringe. Mice were injected with luciferin and imaged at various time points using a bioluminescent imaging system. When the primary tumors grew to the size limit as in the IACUC-approved protocol (approximately 30 days), mice were anesthetized under constant flow of 2% isoflurane and oxygen. The tumor area was sterilized with 70% ethanol. The mouse skin around the tumor was excised to expose the tumor which was removed with a pair of sterile scissors. Removal of the primary tumor extends the survival of the 4T-1 tumor-bearing mice for one month. The mice were then repeatedly imaged for metastatic tumor spreading to distant organs. Therapeutic agents can be administered to suppress tumor metastasis at this point. This model is simple and yet sensitive in quantifying breast cancer cell growth in the primary site and progression kinetics to distant organs, and thus is an excellent model for studying breast cancer growth and progression, and for testing anti-metastasis therapeutic and immunotherapeutic agents in vivo.

An orthotopic breast cancer primary tumor model and surgical removal of primary tumor to extend mouse life to generate spontaneous metastasis are described. The tumor growth and progression are monitored and quantified by luciferase fluorescence imaging.