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, vessel dilator, surgical forceps, and a needle holder.</li>
	<li>Prepare post-operative analgesic (0.1 mg/kg of buprenorphine) to be administered after splenectomy and every 12 h for 2 days.</li>
</ol>
2. Cell culture
<ol>
	<li>Ensure that cancer cells are free from mycoplasma contamination by using a mycoplasma ELISA kit.</li>
	<li>Prepare a 500 mL solution of Dulbecco&#39;s Modified Eagle medium (DMEM) culturing medium at 4 &#176;C for the culture of murine colorectal cancer cells (MC38). The culturing media should be supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL of penicillin, 100 &#181;g/mL of streptomycin, 15 mM of HEPES and 200 mM of L-glutamine.</li>
	<li>Culture cancer cells in a DNAse- and RNAse-free flask (75 cm2). Incubate the cell culture in a cell/tissue humidified incubator containing 5% CO2. Maintain temperature at 37 &#176;C.</li>
	<li>Once the proliferating cells reach 90&#8211;100% confluency, aspirate the old media, wash cells with 1x phosphate-buffered saline (PBS) and then treat them with 1x trypsin (0.25%) to detach cells from the flask.</li>
	<li>Collect cells in a 15 mL conical tube and centrifuge for 5 min at 700 x g.</li>
	<li>Aspirate the media and wash with 1x PBS twice by repeated centrifugation.</li>
	<li>Proceed to confirm cell viability by staining cells with trypan blue stain (0.4%).</li>
	<li>Resuspend cells to a concentration of 1 x 106 cells/ 100 &#181;L in 1x PBS. Pipet cells thoroughly to avoid any clumps. Keep cancer cells on ice prior to injection.</li>
</ol>
3. Injecting tumor cells
<ol>
	<li>Anesthetize 8&#8211;12-week-old male (C57BL6) mice by administering ketamine (150 mg/kg) and xylazine (12 mg/kg) intraperitoneally using a 1 mL 25 G (0.5 mm x 16 mm) needle.</li>
	<li>Shave the abdominal skin of the mice using clippers to avoid any postoperative infections.</li>
	<li>Place mice on the magnetic fixator retraction system. Confirm that the mice are completely under the effect of anesthesia by pinching a toe or the tail.</li>
	<li>Add saline drops into the eyes to avoid dryness during the procedure.</li>
	<li>Scrub povidone-iodine solution (7.5%) to the shaved abdominal wall to disinfect the skin before making a surgical incision.</li>
	<li>Initially lift the skin with the toothed forceps and make a midline incision with the help of surgical scissors. Then, lift the abdominal muscle and peritoneum to create a midline incision of approximately 3 cm length (midabdominal to xiphoid process to expose the abdominal contents. Take caution not to extend the incision beyond the xiphoid process to avoid extensive bleeding.</li>
	<li>Place the hemostat at both sides of the incision and under the xiphoid process. Extend the abdomen by pulling the tail downward and taping it. Use the 6-inch sterile cotton tip applicator to separate and expose the spleen from the pancreatic fat tissue.</li>
	<li>Before injection into the spleen, vortex the cancer cells to avoid any cell clumps.</li>
	<li>Use a 0.5 mL 28 G (0.36 mm x 13 mm) insulin syringe for injection. Avoid air bubbles.</li>
	<li>Slowly and carefully inject 100 &#181;L of cells into the tip of the spleen. Place a cotton tip and add gentle pressure to avoid backflow into the abdominal region. A successful injection can be observed by identifying the change in the color of the liver during the injection.</li>
	<li>Moisten a sterile gauze with 1x PBS and place it over the dissected area.</li>
	<li>Transfer the mice onto a heating pad for 15 mins to allow cancer cells to circulate within the system.</li>
	<li>To proceed with surgical ischemia and reperfusion injury, follow steps 5.3-5.10.&#160; 
	NOTE: This procedure is to study the effect of I/R induced establishment of metastatic foci.</li>
</ol>
4.&#160;Splenectomy
<ol>
	<li>To perform a splenectomy, use a hand-held cautery device. Carefully lift the spleen with smooth forceps and cauterize the splenic blood vessels to avoid excessive bleeding. Remove spleen by transecting the vessels at the cauterized section.</li>
	<li>Immediately following the procedure, close the incision in a double layer pattern by first suturing the muscle layer and then the skin. Use 4-0 polypropylene sutures for both the abdominal wall and the skin.</li>
	<li>Before repeating the procedure on another animal, disinfect all instruments by either spraying them with 70% isopropanol or inserting them into a bead bath.</li>
	<li>Place mice back into original cages and look for signs of distress and post-surgical pain.</li>
	<li>Inject post-operative analgesic (Buprenorphine 0.1 mg/kg) every 12 h for 2 days to avoid post-surgical pain.</li>
</ol>
5. Ischemia reperfusion Injury
<ol>
	<li>At 5 days after the first laparotomy, anesthetize mice by administering ketamine (150 mg/kg) and xylazine (12 mg/kg) intraperitoneally using a 1 mL 25 G (0.5 mm x 16 mm) needle. Follow steps 3.3&#8211;3.4.</li>
	<li>Scrub povidone-iodine solution (7.5%) on the shaved abdomen of the mouse to disinfect the skin and perform a midline laparotomy as described above in step 3.6.</li>
	<li>Using two moistened cotton tips, gently move the intestine from the cavity to expose the associated structures, including the portal vein. Dissect the liver hilum free of the surrounding tissue.</li>
	<li>Lift up the median and left lateral lobes against the diaphragm. Separate the quadrate lobe from the left lateral lobe by dissecting the liver hilum with the spring scissors using an operating microscope to allow clear visibility towards the portal triad structure.</li>
	<li>Place a small moist cotton swab between the median lobe and right lateral lobe to create sufficient space for clamping. Using the vessel dilator forceps, carefully pass the 10 cm thread (4.0 polypropylene suture) to lift the portal triad. Occlude all structures in the portal triad (hepatic artery, portal vein, and bile duct) to the left and median liver lobes by placing a microvascular clamp using a micro-serrefine clamp applicator with lock.</li>
	<li>If the lobes do not show significant blanching, readjust the clamp by removing and reapplying. 
	NOTE: If the immediate blanching of the liver does not occur even after readjusting the clamp, carefully consider whether or not to proceed with the I/R.</li>
	<li>Remove the small cotton swab placed between the median and right lateral lobes. Gently replace the intestine into the abdominal cavity. Cover the abdominal wall with a moist gauze (soaked with 1x PBS) and cover with a plastic wrap to minimize evaporative loss.</li>
	<li>Place the mouse on the heating pad and apply the clamp for a period of 60 min.</li>
	<li>Throughout the ischemic interval, seek evidence of ischemia injury by visualizing the pale blanching of the right medial and left medial and lateral lobes.</li>
	<li>Initiate reperfusion by removing the clamps after the 60 min period. 
	NOTE: Evidence of reperfusion can be observed by an immediate color change of the median and left lateral lobes.</li>
	<li>Immediately following reperfusion, close the incision with a double layer suture pattern by first suturing the muscle layer and then the skin. Use the 4-0 polypropylene suture with the help of a needle holder to close the abdominal wall and the skin.</li>
	<li>Before repeating the procedure on another animal, disinfect all instruments by either spraying them with 70% isopropanol or inserting them into a heated bead bath.</li>
	<li>Place mice back into original cages and look for signs of distress and post-surgical pain.</li>
	<li>Inject post-operative analgesic (0.1 mg/kg of buprenorphine) every 12 h for 2 days to avoid post-surgical pain.</li>
	<li>For liver I/R sham mice, perform laparotomy, hilum dissection and abdominal sutures. 
	NOTE: The role of surgical stress influencing the establishment of liver metastases can be investigated through two different experimental designs. The above protocol (Model-1) is used to establish micrometastatic liver disease and study the effect of liver I/R on their growth (Figure 1A). Alternatively, liver I/R and tumor injection can be performed concurrently (Model-2) to study the effect of I/R injury in the establishment of new metastatic foci (Figure 1B). To do this, inject cancer cells into the spleen as described above and allow them to circulate for 15 min. Perform liver I/R or sham surgery after the circulating period for 60 min. Perform lateral splenectomy 60 min later, and then close the laparotomy incision.</li>
</ol>
6. Assessment of operated mice
<ol>
	<li>During the surgical procedure ensure that the mice are under the influence of stage III anesthesia by performing palpebral and corneal reflex test. Additional anesthetic dose must be given upon the signs of reflexes.</li>
	<li>Provide post-operative analgesic (Buprenorphine 0.1mg/kg) right after the surgery and every 12h for 2 days to avoid post surgical pain.</li>
	<li>Allow mice 30&#8211;60 min of recovery time from anesthesia. Constantly monitor mice and do not leave them unattended until complete recovery.</li>
	<li>Look for distress signs such as hunched back, closed eyes, slow movement, and failure to groom. Treat accordingly until mice return to their normal activity.</li>
	<li>Supplimental care including fluids, heat, Yohimbine reversal agent for xylazine, soft paper towel bedding (to avoid aspiration) should be provided after the surgery to improve the recovery period.</li>
</ol>
7. Assessment of liver ischemia reperfusion injury
<ol>
	<li>Immediately after applying the clamp, make sure the pale blanching of the median and left lateral lobes occurs compared to the caudate and quadrate lobes.</li>
	<li>Assess liver ischemia injury by measuring serum alanine transaminase (sALT), serum aspartate transaminase (sAST) and serum lactate dehydrogenase (sLDH) levels. The blood can be drawn from the facial vein to extract serum 3&#8211;6 h after the initiation of reperfusion. Perform liver histology to analyze the percent tumor area within the ischemic lobe.</li>
</ol>

<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 authors disclose no conflicts of interest that pertain to this work.

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 ...

murine-model-metastatic-liver-tumors-setting-ischemia-reperfusion

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Cancer Research

11.4K Views.  University of Pittsburgh. 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.

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

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. ...

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 ...

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis and to identify novel therapeutic targets. To investigate molecular events that drive the progression from primary cancer to metastasis, we have developed a bitransgenic mouse model, RIP-Tag; RIP-tva. In this mouse model, the rat insulin promoter (RIP) drives the expression of the SV40 T antigen (Tag) and the receptor for subgroup A avian leukosis virus (tva) in pancreatic &#946; cells. The mice develop pancreatic neuroendocrine tumors with 100% penetrance through well-defined stages that are similar to human tumorigenesis, with stages including hyperplasia, angiogenesis, adenoma, and invasive carcinoma. Because RIP-Tag; RIP-tva mice do not develop metastatic disease, genetic alterations that promote metastasis can be identified easily. Somatic gene transfer into tva-expressing, proliferating pancreatic &#946; premalignant lesions is achieved through intracardiac injection of avian retroviruses harboring the desired genetic alteration. A titer of &#62;1 x 108 infectious units per ml is considered appropriate for in vivo infection. In addition, avian retroviruses can infect cell lines derived from tumors in RIP-Tag; RIP-tva mice with high efficiency. The cell lines can also be used to characterize the metastatic factors. Here we demonstrate how to utilize this mouse model and cell lines to assess the functions of candidate genes in tumor metastasis.

Identification and Characterization of Metastatic Factors by Gene Transfer into the Novel RIP-Tag; RIP-tva Murine Model

We present a protocol to demonstrate a novel somatic gene transfer system utilizing RIP-Tag; RIP-tva mouse model to study the function of genes in metastasis. The avian retroviruses are delivered intracardiacally to ensure gene transfer into pre-malignant, noninvasive lesions of pancreatic &#946; cells in adult mice.

Metastatic cancer accounts for 90% of deaths in patients with solid tumors. There is an urgent need to better understand the drivers of cancer metastasis ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

Practical Considerations in Studying Metastatic Lung Colonization in Osteosarcoma Using the Pulmonary Metastasis Assay

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung colonization in osteosarcoma (OS) by fluorescence microscopy. This article provides a detailed description of the protocol, and discusses examples of obtaining image data on metastatic growth using widefield or confocal fluorescence microscopy platforms. The flexibility of the PuMA model permits researchers to study not only the growth of OS cells in the lung microenvironment, but also to assess the effects of anti-metastatic therapeutics over time. Confocal microscopy allows for unprecedented, high-resolution imaging of OS cell interactions with the lung parenchyma. Moreover, when the PuMA model is combined with fluorescent dyes or fluorescent protein genetic reporters, researchers can study the lung microenvironment, cellular and subcellular structures, gene function, and promoter activity in metastatic OS cells. The PuMA model provides a new tool for osteosarcoma researchers to discover new metastasis biology and assess the activity of novel anti-metastatic, targeted therapies.

The goal of this article is to provide a detailed description of the protocol for the pulmonary metastasis assay (PuMA). This model permits researchers to study metastatic osteosarcoma (OS) cell growth in lung tissue using a widefield fluorescence or confocal laser-scanning microscope.

The pulmonary metastasis assay (PuMA) is an ex vivo lung explant and closed cell culture system that permits researchers to study the biology of lung ...

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Development and Angiographic Use of the Rabbit VX2 Model for Liver Cancer

The rabbit VX2 tumor is an animal model commonly utilized for translational research regarding hepatocellular carcinoma (HCC) in the field of Interventional Radiology. This model employs an anaplastic squamous cell carcinoma that is easily and reliably propagated in the skeletal muscle of donor rabbits for eventual harvest and allograft implantation into the liver of na&#239;ve recipients. This tumor graft rapidly grows within the liver of recipient rabbits into an angiographically identifiable tumor characterized by a necrotic core surrounded by a viable hypervascular capsule. The physical size of the rabbit anatomy is sufficient to facilitate vascular instrumentation allowing for the application and testing of various interventional techniques. Despite these benefits, there exists a paucity of technical resources to act as a concrete reference for researchers working with the model. Herein, we present a comprehensive visual outline for the technical aspects of development, growth, propagation, and angiographic utilization of the rabbit VX2 tumor model for use by novice and experienced researchers alike.

The goal of this article is to provide a primer for the development and use of the VX2 carcinoma rabbit model for liver cancer.

The rabbit VX2 tumor is an animal model commonly utilized for translational research regarding hepatocellular carcinoma (HCC) in the field of ...

Magnetic Resonance Imaging Assessment of Carcinogen-induced Murine Bladder Tumors

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) carcinogen are advantageous over cell line-based models because they closely replicate the genomic profiles of human tumors, and, unlike cell models and xenografts, they provide a good opportunity for the study of immunotherapies. However, bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Described here is a BBN mouse model and protocol to evaluate bladder cancer tumor burden in vivo using a fast and reliable magnetic resonance (MR) sequence (true FISP). This method is simple and reliable because, unlike ultrasound, MR is operator-independent and allows for the straightforward post-acquisition image processing and review. Using axial images of the bladder, analysis of regions of interest along the bladder wall and tumor allow for the calculation of bladder wall and tumor area. This measurement correlates with ex vivo bladder weight (rs= 0.37, p = 0.009) and tumor stage (p = 0.0003). In conclusion, BBN generates heterogeneous tumors that are ideal for evaluation of immunotherapies, and MRI can quickly and reliably assess tumor burden prior to randomization to experimental treatment arms.

Murine bladder tumors are induced with the N-butyl-N-(4-hydroxybutyl) nitrosamine carcinogen (BBN). Bladder tumor generation is heterogeneous; therefore, an accurate assessment of tumor burden is needed before randomization to experimental treatment. Here we present a fast, reliable MRI protocol to assess tumor size and stage.

Murine bladder tumor models are critical for the evaluation of new therapeutic options. Bladder tumors induced with the N-butyl-N-(4-hydroxybutyl) ...

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 ...