The authors thank Sara Minemyer and Alexander Comerci for the linguistic revision.

All animal protocols are approved by the Institutional Animal Care and Use Committee and adhered to the National Institutes of Health (NIH) Guidelines. Instruments used for any surgical procedure were thoroughly sterilized.
1. Initial preparation
<ol>
	<li>Before injecting cancer cells into the mouse spleen, autoclave and sterilize all instruments to be used during the procedure.</li>
	<li>Sterilize and/or autoclave a heating pad, surgical gloves, gauze, pairs of scissors, small clamps, vess...

<ol>
	<li>Riihim&#228;ki, M., Hemminki, A., Sundquist, J., Hemminki, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+metastasis+in+colon+and+rectal+cancer.">Patterns of metastasis in colon and rectal cancer.</a> Scientific Reports. 6 (1), 29765 (2016).</li><li>Oki, 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=Recent+advances+in+treatment+for+colorectal+liver+metastasis.">Recent advances in treatment for colorectal liver metastasis.</a> Annals of gastroenterological surgery. 2 (3), 167-175 (2018).</li><li>Wolpin, B. M., Mayer, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systemic+treatment+of+colorectal+cancer.">Systemic treatment of colorectal cancer.</a> Gastroenterology. 134 (5), 1296-1310 (2008).</li><li>van Golen, R. F., Reiniers, M. J., Olthof, P. B., van Gulik, T. M., Heger, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sterile+inflammation+in+hepatic+ischemia/reperfusion+injury:+present+concepts+and+potential+therapeutics.">Sterile inflammation in hepatic ischemia/reperfusion injury: present concepts and potential therapeutics.</a> Journal of Gastroenterology and Hepatology. 28 (3), 394-400 (2013).</li><li>Demicheli, R., Retsky, M. W., Hrushesky, W. J. M., Baum, M., Gukas, I. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+surgery+on+tumor+growth:+a+century+of+investigations.">The effects of surgery on tumor growth: a century of investigations.</a> Annals of Oncology. 19 (11), 1821-1828 (2008).</li><li>Tohme, 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=Neutrophil+Extracellular+Traps+Promote+the+Development+and+Progression+of+Liver+Metastases+after+Surgical+Stress.">Neutrophil Extracellular Traps Promote the Development and Progression of Liver Metastases after Surgical Stress.</a> Cancer Research. 76 (6), 1367-1380 (2016).</li><li>Tseng, W., Leong, X., Engleman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+mouse+model+of+colorectal+cancer.">Orthotopic mouse model of colorectal cancer.</a> Journal of Visualized Experiments. (10), 484 (2007).</li><li>Elkin, M., Vlodavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tail+vein+assay+of+cancer+metastasis.">Tail vein assay of cancer metastasis.</a> Current Protocols in Cell Biology. 19 (19.2), (2001).</li><li>Thalheimer, 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=The+intraportal+injection+model:+A+practical+animal+model+for+hepatic+metastases+and+tumor+cell+dissemination+in+human+colon+cancer.">The intraportal injection model: A practical animal model for hepatic metastases and tumor cell dissemination in human colon cancer.</a> BMC Cancer. 9 (1), 29 (2009).</li><li>Frampas, E., Maurel, C., Thedrez, P., Le Sa&#235;c, P. R. e. m. a. u. d. -., Faivre-Chauvet, A., Barbet, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intraportal+injection+model+for+liver+metastasis:+Advantages+of+associated+bioluminescence+to+assess+tumor+growth+and+influences+on+tumor+uptake+of+radiolabeled+anti-carcinoembryonic+antigen+antibody.">The intraportal injection model for liver metastasis: Advantages of associated bioluminescence to assess tumor growth and influences on tumor uptake of radiolabeled anti-carcinoembryonic antigen antibody.</a> Nuclear Medicine Communications. 32 (2), 147-154 (2011).</li><li>Goddard, E. T., Fischer, J., Schedin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Portal+Vein+Injection+Model+to+Study+Liver+Metastasis+of+Breast+Cancer.">A Portal Vein Injection Model to Study Liver Metastasis of Breast Cancer.</a> Journal of Visualized Experiments. (118), (2016).</li><li>Limani, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+portal+vein+injection+for+the+design+of+syngeneic+models+of+liver+malignancy.">Selective portal vein injection for the design of syngeneic models of liver malignancy.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 310 (9), G682-G688 (2016).</li></ol>

The animal model described in this manuscript is based upon two major approaches. The first is to recognize the ability of cancer cells to localize and proliferate in the liver lobes. The second is to study the effect of hepatic ischemia reperfusion injury influencing the tumor growth and metastases. This model permits the relevant study of liver metastases in the absence of secondary metastases in an immunocompetent mouse. The model is useful in addressing the questions of metastatic efficiency, such as cell survival ex...

The liver is one of the most common sites for the development of metastatic disease1. Mortality is almost invariably attributable to complications associated with tumor growth in the liver. In patients with metastatic solid tumors in the liver, surgery remains a crucial intervention for disease control and a possible curative approach. However, the vast majority of patients ultimately present with recurrent disease, predominantly in the liver2,3. During hepatic surgery, intraoperative bleeding is common, often necessitating blood transfusion and different technical approaches for control of bleeding, including vascular clamping methods. However, such measures cause hepatic ischemia/reperfusion (I/R) to the liver tissue. The adverse effects of I/R on hepatocellular function have been well documented. The liver I/R insult ignites inflammatory cascades during the restoration of blood flow via inflammatory pathways4. Not only does liver I/R injury contribute to liver failure, but current evidence also shows that I/R injury stimulates tumor cell adhesion, and promotes the incidence of metastases formation and the growth of existing micrometastatic disease5. We have previously reported that surgical stress induces activation of immune cells which not only helps in the growth of the primary tumor, but also facilitates metastases by capturing cancer cells within the circulation6.
Here we describe in detail a technique to establish a liver metastasis mouse tumor model. In this model, we also present a method to induce hepatic ischemia reperfusion injury which acts as a surrogate to the surgical stress present clinically during hepatectomies. The combined methods of cancer injection and hepatic I/R can successfully interpret the development of CRLM in patients who have undergone primary tumor resection.

<table>
	<tbody>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle Medium</td>
			<td>Lonza</td>
			<td>12-614F</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Lonza</td>
			<td>900-108</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamin</td>
			<td>Gibco</td>
			<td>25030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicilin</td>
			<td>Fisher scientific</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Stretomysin</td>
			<td>Fisher scientific</td>
			<td>15-140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>SH3023701</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Hyclone</td>
			<td>sh30042.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture Flask 75cm</td>
			<td>5 Cells Star</td>
			<td>658170</td>
			<td></td>
		</tr>
		<tr>
			<td>15ml PP Conical Tubes</td>
			<td>BioExcell</td>
			<td>41021037</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain</td>
			<td>Giibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Fisherbrand</td>
			<td>1376152</td>
			<td></td>
		</tr>
		<tr>
			<td>Cautry</td>
			<td>Bovie</td>
			<td>AA01</td>
			<td></td>
		</tr>
		<tr>
			<td>Microvascular clamp</td>
			<td>Finescience tools</td>
			<td>18055-03</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Serrefine clamp applicator with lock</td>
			<td>Fine science toosl</td>
			<td>FST-18056-14</td>
			<td></td>
		</tr>
		<tr>
			<td>Spring scissor</td>
			<td>Fine science toosl</td>
			<td>FST-15021-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Vessel Dilator</td>
			<td>Fine science toosl</td>
			<td>FST-00276-13</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic fixator Retraction system</td>
			<td>Fine science toosl</td>
			<td>FST-18200020</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps</td>
			<td>Fine science toosl</td>
			<td>FST-11019-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-Adson Forceps</td>
			<td>Fine science toosl</td>
			<td>FST-11018-12</td>
			<td></td>
		</tr>
		<tr>
			<td>4-0 polypropylene suture</td>
			<td>Ethicon</td>
			<td>K881H</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>Harvard Apparatus</td>
			<td>72-8826</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating Pad</td>
			<td>Fisher scientific</td>
			<td>1443915</td>
			<td></td>
		</tr>
		<tr>
			<td>Clipper</td>
			<td>Oster</td>
			<td>559A</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-Iodine solution</td>
			<td>Medline</td>
			<td>MDS093945</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1ml 25G</td>
			<td>BD safety Glide</td>
			<td>305903</td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin syringe 0.5 ml</td>
			<td>BD insulin Syringes</td>
			<td>32946</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton -Tipped Applicator</td>
			<td>Fisher Scientific</td>
			<td>23-400-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Microscope</td>
			<td>Leica</td>
			<td>LR92240</td>
			<td></td>
		</tr>
		<tr>
			<td>Mycoplasma Elisa Kit</td>
			<td>Roche</td>
			<td>11663925910</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Putney</td>
			<td>#056344</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>NADA</td>
			<td>#139-236</td>
			<td></td>
		</tr>
		<tr>
			<td>ALT strip</td>
			<td>Heska</td>
			<td>15809554</td>
			<td></td>
		</tr>
		<tr>
			<td>AST strip</td>
			<td>Heska</td>
			<td>15809542</td>
			<td></td>
		</tr>
		<tr>
			<td>LDH strip</td>
			<td>Heska</td>
			<td>15809607</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

All wildtype (C57BL6) mice (n = 20) were subjected to the liver metastases model using the protocol described above. All injected mice with or without ischemia reperfusion injury survived until the date of sacrifice. The schematic diagram Figure 1A of a cancer-injected liver illustrates the clamping of the portal triad (hepatic artery, portal vein, and bile duct) which induces a partial liver ischemic (70%) insult towards the median and left lateral lobes. An increase in the number of liver ...

Watch this Scientific Journal Video about Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury at JoVE.com

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

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 ...

Experimental and clinical evidence show that liver ischemia reperfusion can increase the formation of metastatic foci by circulating cancer cells and the growth of micrometastatic disease. This reproducible murine model permits the relevant study of both the establishment of metastases and the growth of pre-existing tumor within the liver. Liver surgery, bleeding, and septic shock are all associated with liver ischemia reperfusion and can affect the growth of metastatic disease.These techniques allow the study of this phenomena in an effort to better diagnose and prevent the detrimental effect of ischemia reperfusion on tumor growth in a clinical setting. After confirming a lack of response to toe pinch in an anesthetized eight to 12-week old male C57 black 6 mouse, disinfect the exposed abdominal wall skin with a povidone-iodine scrub and use toothed forceps to lift the skin. Using a surgical scalpel, make a midline incision in the skin and lift the abdominal muscle and peritoneum to facilitate the creation of a three-centimeter midline incision to expose the abdominal contents.After placing a hemostat at both sides of the incision and under the xiphoid process, pull and tape the tail downward to extend the abdomen and use a six-inch sterile cotton tip applicator to separate and expose the spleen from the pancreatic fat tissue. Next, vortex the cancer cell suspension of interest to dissociate any cell clumps and load the cells into a 0.5-milliliter insulin syringe equipped with a 28-gauge needle. Slowly and carefully inject 100 microliters of cells into the tip of the spleen, placing a cotton tip adjacent to the side of the needle puncture to absorb any possible tumor spillage into the peritoneal cavity while adding gentle pressure to stabilize the spleen during injection.A successful injection will be evident by a change in the color of the liver. When all of the cells have been delivered, place a piece of PBS-soaked sterile gauze over the dissected area and place the mouse onto a heating pad for 15 minutes to allow the cancer cells to circulate within the system. To induce an ischemia and reperfusion injury, use two moistened cotton tips to gently move the intestine from the cavity to expose the portal vein and place the mouse under a dissecting microscope.Lift the median and left lateral liver lobes up against the diaphragm. Place a small moist cotton swab between the median lobe and the right lateral lobe to create sufficient space for clamping and carefully pass a vessel dilator forceps behind the portal triad and, using the vessel dilator forceps, use a 10-centimeter piece of 4-0 polypropylene suture to pass behind and lift the portal triad. Use a micro-serrifine clamp applicator with a lock to place a microvascular clamp onto the left and median liver lobes to occlude all of the structures in the portal triad.When blanching is observed, remove the 4-0 polypropylene suture and small cotton swab and carefully return the intestine to the abdominal cavity. Cover the abdominal wall with a new piece of PBS-soaked gauze and cover the gauze with plastic wrap to minimize the evaporative loss. Then place the mouse back onto the heating pad for 60 minutes.Throughout the ischemic interval, visualize the pale blanching of the right medial, left medial, and lateral lobes to confirm evidence of the ischemia injury. At the end of the injury period, remove the clamp. To perform a splenectomy, carefully lift the spleen with smooth forceps and use a handheld cautery device to cauterize the splenic blood vessels to avoid excessive bleeding.Transect the vessels at the cauterized section to remove the spleen and immediately use 4-0 polypropylene sutures to close the muscle and skin incisions in a double-layer suture pattern. Then place the mouse back into its home cage with monitoring until full recovery. Surgical stress significantly increases the amount of pre-established micrometastases within the liver.This difference is observed even two weeks after ischemia reperfusion compared to non-ischemia reperfused mice. The liver ischemia reperfusion and tumor cell injection can be affected by room temperature and cell viability, so take care to control these variables. This technique facilitates the study of the effects of surgical stress and liver injury on the growth of metastatic disease.This will help researchers to better understand the phenomenon of surgery-induced tumor growth.

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

Abstract