We would like to acknowledge the veterinary staff at the University of Illinois - Chicago&#39;s Biological Resources Laboratory.

The following protocol follows all requirements and guidelines mandated by the University of Illinois - Chicago. It was reviewed and approved by the local Institutional Animal Care and Use Committee prior to execution.
1. VX2 Hind Limb Tumor Development
<ol>
	<li>Procure the VX2 tumor cell line from the National Cancer Institute Division of Cancer Treatment Diagnosis and Treatment Tumor/Cell Line Repository. 
	Note: At this time, the order catalog can be found at the following link: https://dtp.cancer.gov/repositories/.</li>
	<li>To prepare the cell suspension for injection, place the frozen VX2 sample and methylcellulose medium in lukewarm water until they have thawed.
	<ol>
		<li>Aspirate 0.5&#8210;1 mL of the thawed cell suspension and an equal volume of thawed methylcellulose medium into a 5-mL syringe. 
		Note: It may help to use a 21 or 22 G needle to aspirate the cell suspension and a larger gauge needle to aspirate the methylcellulose medium. Place the syringes on ice.</li>
	</ol>
	</li>
	<li>Prepare the hind limb tumor donor rabbit for inoculation of cell suspension, starting with sedation using 1 mg/kg acepromazine and 0.02 mg/kg buprenorphine given intramuscularly. Shave the inoculation site (hind limb) with clippers and clean the shaved are with an alcohol- or iodine-based agent. 
	Note: Meloxicam can be given at a dose of 0.2 mg/kg subcutaneously for analgesia. Rabbit fur is difficult to shave. It is recommended that the researcher perform multiple passes, first focusing on clearing a bulk of the fur and then pressing the clippers against the skin and going against the grain.</li>
	<li>Attach a 16 G needle to the 5-mL syringe containing the cell suspension and inject 1 mL of the suspension into the belly of the hind limb muscle of the donor rabbit, roughly 1 cm deep. Be sure to guard the sciatic nerve running within a palpable groove along the femur. 
	Note: Fresh VX2 successfully inoculates 88% of subjects while frozen and thawed VX2 is only successful 33% of the time19. 
	CAUTION: The VX2 tumor will grow quickly. Side effects that may be noted as the tumor progresses are: increased respiratory rate, lethargy, decreased alertness, and/or behavioral changes (e.g., unusual aggressiveness). Consequently, recommendations are to monitor the animals closely and to plan for tumor harvest within two weeks after inoculation.</li>
</ol>
2. VX2 Hind Limb Tumor Growth and Harvesting
Note: Assuming successful inoculation, there should be a palpable (3&#8210;4 cm) tumor nodule at the injection site within 2 weeks. Usually, this nodule will be palpable around 1 week; however, it is better to let the tumor grow to allow for sufficient tissue collection. Typically, this nodule will be firm, indurated, and elevated above the level of the muscle. If
<ol>
	<li>Palpate the inoculated area at 2 weeks from inoculation. If no tumor growth is detected at 2 weeks, perform step 1 again. See Figure 1. 
	Note: The presence of tumor can also be assessed via ultrasound. While there have been no studies to assess the difference in tumor growth metrics when propagated from frozen stock as compared to fresh stock, it has been our experience that the frozen stock tumor grows slightly slower and will be detectable after its fresh stock counter-part. Regardless, either method should yield a harvestable tumor by 2 weeks.</li>
	<li>Prepare the hind limb tumor donor for tumor harvest. Anesthetize the rabbit with 45 mg/kg ketamine and 5 mg/kg xylazine. Euthanize with an intravenous (IV) dose of sodium pentobarbital exceeding 390 mg/kg.</li>
	<li>Once the rabbit has been euthanized, begin harvesting the tumor by removing the cutaneous and subcutaneous tissue overlying the tumor nodule using a scalpel with whichever size blade the researcher sees fit. See Figure 2.</li>
	<li>Once the tumor nodule has been identified within the hind limb muscle, remove the tumor en bloc using wide curvilinear margins. For this, cut the tendinous attachment points at the proximal and distal ends of the of the muscle and then trace the scalpel blade along the underlying bone. Bisect the explanted specimen exposing the tumor capsule and necrotic core. See Figure 3. 
	Note: The VX2 tumor has a highly necrotic core. Be sure to use proper eye protection and other personal protective equipment when harvesting the tumor, since any damage to the tumor capsule can result in high pressure ejection of necrotic debris.</li>
	<li>Gently scrape the necrotic core with a blunt object (e.g. forceps, hemostats) to clean the tumor. This should allow for better visualization of the tumor capsule and the transition point between capsule and surrounding muscle. See Figure 4.</li>
	<li>From this sample, harvest several pieces of tumor approximately 1&#8210;2 mm3 and immediately store them in a cup containing room temperature sterile saline. 
	Note: These tumor samples will be used for subsequent intrahepatic implantation.</li>
	<li>Place the remaining tumor in a sterile Petri dish and bathe it with Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM). If this specimen is kept cold on ice, it can be preserved for handling up to 2&#8210;3 h later.</li>
</ol>
3. Liver Tumor Implantation via Laparotomy
<ol>
	<li>Prepare the recipient rabbit for laparotomy. Anesthetize the recipient rabbit using 45 mg/kg ketamine and 5 mg/kg xylazine for induction followed by intubation and maintenance with 1%--3% isoflurane as needed. When the rabbit is anesthetized, shave the surgical site using hair clippers. Clean the incision area&#160;using triplicate wash with betadine scrub, 70% ethanol solution, and betadine solution, and drape the abdomen in a sterile surgical fashion.&#160;</li>
	<li>Using a #15-blade, initiate the laparotomy by making a small downward vertical midline incision through the rabbit&#8217;s skin starting from the xiphoid process. This should be easily palpated and tends to be roughly the size of an American penny. See Figure 5.</li>
	<li>Reflect the skin and identify the linea alba. This should be a reflective white band of tissue traveling inferiorly in the midline. Use blunt dissection to traverse the linea alba and expose the peritoneum. See Figure 6. 
	Note: The peritoneum is easily identified since it will be directly overlying the bowel, which can be seen moving with respiration. 
	CAUTION: The peritoneum tends to be adherent to the underlying bowel due to surface tension. Using blunt trauma to dissect the linea alba helps to dissipate the risk of perforating the underlying bowel.</li>
	<li>Once the peritoneum is exposed, carefully dissect through it to enter the peritoneal cavity. The liver can now be identified. To better navigate the abdominal space, extend the midline incision 1&#8210;2 cm inferiorly through the skin, muscle, and peritoneum. 
	Note: Extending the midline incision can be easily and safely done by carefully inserting a curved hemostat into the peritoneal space with the curved tip facing superficially towards the peritoneum. Then open the hemostat slightly and use a blade to cute the tissue between the two arms of the hemostat.</li>
	<li>Identify the left lobe of the liver in order to select a site for tumor implantation. The left lobe is infero-lateral to the medial lobe which sits in the midline.</li>
	<li>Before attempting to draw the liver out of the peritoneal space, place a dry piece of gauze at the inferior aspect of the incision. 
	Note: The gauze will provide an adherent surface to lay the liver on to prevent it from retracting back into the abdomen.</li>
	<li>Using either atraumatic forceps or a piece of wet gauze over the fingers, carefully draw the left lobe of the liver out of the abdomen through the incision and lay it down on the dry gauze placed earlier. See Figure 7. 
	CAUTION: The liver capsule is sensitive and can easily rupture. It is critical to be gentle when handling this organ to prevent capsular bleed, liver bruising, and/or eventual hemoperitoneum. 
	Note: Usually, the liver will declare itself visually upon entering the peritoneal space; however, if the rabbit&#8217;s stomach is distended, the liver can be pushed cranially out of sight. If this is the case, gently lift the abdominal wall using a blunt probe. In this scenario, the liver tends to adhere to the ventral aspect of the diaphragm due to surface tension so carefully separate the liver using a blunt probe and it should detach. Then use atraumatic forceps to draw the liver out.</li>
	<li>At this point, prepare the tumor tissue for implantation and place a piece of wet gauze over the liver to protect it.</li>
	<li>Select a 1&#8210;2 mm3 piece of tumor tissue that was generated during step 2.6 for implantation into the liver. See Figure 8.</li>
	<li>Using a #11-blade, puncture the liver tissue at a 45&#176; angle making a 0.5 cm deep pocket, taking care not to penetrate the dorsal aspect of the liver capsule. Leave the blade in place after making the puncture. See Figure 9.</li>
	<li>Gently lift the blade in a ventral direction to create a small pocket in the liver bed. Take the tumor piece using forceps, place the tumor piece in this pocket and then remove the blade. 
	Note: The blade can be removed prior to inserting the tumor piece, however, the liver will bleed and this can obscure the puncture site making it difficult to identify. 
	CAUTION: Be sure to minimize contact of the tumor with any other structures to prevent unintentional tumor seeding. This can also be avoided by setting aside any tools used to assist with implantation afterwards.</li>
	<li>At this point, place a piece of hemostatic agent, such as gel foam, over the tumor pocket to promote hemostasis and to prevent ejection of the tumor piece.</li>
	<li>Return the liver to the abdomen, once hemostasis is confirmed.</li>
	<li>Close the abdominal wall with 3-0 polydioxanone suture on a taper needle using a simple continuous stitch and close the skin with 4-0 polyglactin 910 sutures on a cutting needle using continuous subcuticular stitch. 
	CAUTION: When closing the abdominal wall, take care not to damage the omentum or other bowel structures in a suture throw. 
	Note: While this tumor will take at least 2 weeks to be definitively radiographically identifiable, it reaches a size of 1.5&#8210;2 cm diameter at 3 weeks of growth. Be careful not to allow the hepatic tumor to grow past 3.5&#8210;4 weeks since the tumor will form an exophytic mass and aggressively spread locally.</li>
</ol>
4. VX2 Tumor Suspension Preparation
<ol>
	<li>Place a 40 &#181;m strainer into the mouth of a 50-mL conical propylene tube. Using the tumor from step 2.7, use a #15-blade to scrape the tumor surface to collect viable tumor and place these scrapings into the strainer.</li>
	<li>Wash the viable tumor scrapings through a strainer using DMEM and then centrifuge the propylene tube at 1600 rpm for 8 min.</li>
	<li>After the centrifuge is complete, remove the supernatant and discard it. Then add methylcellulose to the remaining cell block at a 1:1 volumetric ratio.</li>
	<li>Inject this suspension into a hind limb donor rabbit following steps 1.3&#8210;1.4. Place the remaining suspension into 1 mL aliquots in cryogenic tubes and freeze them in liquid nitrogen for later use.</li>
</ol>
5. Angiographic Utilization of the VX2 Liver Tumor
<ol>
	<li>Prepare the rabbit as described in step 3.1.</li>
	<li>Palpate the femoral groove in the groin. When the groove has been identified, make a 2&#8210;3 cm linear incision along the groove. See Figure 10.</li>
	<li>Using blunt dissection, locate and isolate the femoral bundle containing the femoral vein, artery, and nerve. See Figure 11.</li>
	<li>Again, use blunt dissection to separate the femoral artery from the rest of the structures in the bundle and isolate the artery atop a scalpel handle. See Figure 12.</li>
	<li>With a 3-French introducer kit, utilize the Seldinger technique to gain access with a needle. Insert a guidewire and remove the needle to advance the 3-French sheath into the vessel. See Figure 13. 
	CAUTION: Avoid using excessive force in advancing the 3-French sheath, as this can result in transection of the femoral artery.</li>
	<li>Under fluoroscopic guidance and using a catheter, guidewire, and iohexol contrast agent, select for the celiac trunk &#8212; typically located at the T12 level &#8212; and then advance the catheter into the left hepatic artery via the common hepatic and proper hepatic.</li>
	<li>At this point, administer the agent of choice through the catheter. Once the agent is administered, remove the catheter.</li>
	<li>Using 3-0 silk suture, ligate the femoral artery proximally and distally to the insertion point of the sheath. Be sure to tighten the knot proximal to the sheath as it is withdrawn to prevent bleeding.</li>
	<li>Close the groin incision with 4-0 polyglactin 910 sutures on a cutting needle using a subcuticular stitch.</li>
	<li>Maintain standard post-operative care and monitor animal recovery. Perform euthanasia and necropsy as needed using standard techniques.</li>
</ol>

<ol>
	<li>Rous, P., Beard, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Progression+To+Carcinoma+of+Virus-Induced+Rabbit+Papillomas+(Shope).">The Progression To Carcinoma of Virus-Induced Rabbit Papillomas (Shope).</a> The Journal of Experimental Medicine. 62 (4), 523-548 (1935).</li><li>Kidd, J. G., Rous, 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+transplantable+rabbit+carcinoma+originating+in+a+virus-induced+papilloma+and+containing+the+virus+in+masked+or+altered+form.">A transplantable rabbit carcinoma originating in a virus-induced papilloma and containing the virus in masked or altered form.</a> The Journal of Experimental Medicine. 71 (6), 813-838 (1940).</li><li>Galasko, C. S. B., Muckle, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrasarcolemmal+proliferation+of+the+vx2+carcinoma.">Intrasarcolemmal proliferation of the vx2 carcinoma.</a> British Journal of Cancer. 29 (1), 59-65 (1974).</li><li>Maruyama, H., 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=Sonographic+shift+of+hypervascular+liver+tumor+on+blood+pool+harmonic+images+with+definity:+Time-related+changes+of+contrast-enhanced+appearance+in+rabbit+VX2+tumor+under+extra-low+acoustic+power.">Sonographic shift of hypervascular liver tumor on blood pool harmonic images with definity: Time-related changes of contrast-enhanced appearance in rabbit VX2 tumor under extra-low acoustic power.</a> European Journal of Radiology. 56 (1), 60-65 (2005).</li><li>Horkan, C., 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=Radiofrequency+Ablation:+Effect+of+Pharmacologic+Modulation+of+Hepatic+and+Renal+Blood+Flow+on+Coagulation+Diameter+in+a+VX2+Tumor+Model.">Radiofrequency Ablation: Effect of Pharmacologic Modulation of Hepatic and Renal Blood Flow on Coagulation Diameter in a VX2 Tumor Model.</a> Journal of Vascular and Interventional Radiology. 15 (3), 269-274 (2004).</li><li>Bimonte, 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=Induction+of+VX2+para-renal+carcinoma+in+rabbits:+generation+of+animal+model+for+loco-regional+treatments+of+solid+tumors.">Induction of VX2 para-renal carcinoma in rabbits: generation of animal model for loco-regional treatments of solid tumors.</a> Infectious Agents and Cancer. 11 (1), 1-8 (2016).</li><li>Goldberg, S. N., Gazelle, G. S., Compton, C. C., Mueller, P. R., McLoud, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radio-frequency+tissue+ablation+of+VX2+tumor+nodules+in+the+rabbit+lung.">Radio-frequency tissue ablation of VX2 tumor nodules in the rabbit lung.</a> Academic Radiology. 3 (11), 929-935 (1996).</li><li>Rhee, T. K., 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=Rabbit+VX2+Tumors+as+an+Animal+Model+of+Uterine+Fibroids+and+for+Uterine+Artery+Embolization.">Rabbit VX2 Tumors as an Animal Model of Uterine Fibroids and for Uterine Artery Embolization.</a> Journal of Vascular and Interventional Radiology. 18 (3), 411-418 (2007).</li><li>Parvinian, A., Casadaban, L. C., Gaba, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development,+growth,+propagation,+and+angiographic+utilization+of+the+rabbit+VX2+model+of+liver+cancer:+A+pictorial+primer+and+&amp;#34;how+to&amp;#34;+guide.">Development, growth, propagation, and angiographic utilization of the rabbit VX2 model of liver cancer: A pictorial primer and &amp;#34;how to&amp;#34; guide.</a> Diagnostic and Interventional Radiology. 20 (4), 335-340 (2014).</li><li>Xia, X., 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=Intra-arterial+interleukin-12+gene+delivery+combined+with+chemoembolization:+Anti-tumor+effect+in+a+rabbit+hepatocellular+carcinoma+(HCC)+model.">Intra-arterial interleukin-12 gene delivery combined with chemoembolization: Anti-tumor effect in a rabbit hepatocellular carcinoma (HCC) model.</a> Acta Radiologica. 54 (6), 684-689 (2013).</li><li>Gaba, R. C., 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=Ethiodized+oil+uptake+does+not+predict+doxorubicin+drug+delivery+after+chemoembolization+in+VX2+liver+tumors.">Ethiodized oil uptake does not predict doxorubicin drug delivery after chemoembolization in VX2 liver tumors.</a> Journal of Vascular and Interventional Radiology. 23 (2), 265-273 (2012).</li><li>Choi, Y. H., 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=Novel+Intraarterial+Therapy+for+Liver+Cancer+Using+Ethylbromopyruvate+Dissolved+in+an+Iodized+Oil.">Novel Intraarterial Therapy for Liver Cancer Using Ethylbromopyruvate Dissolved in an Iodized Oil.</a> Academic Radiology. 18 (4), 471-478 (2011).</li><li>Ma, H. L., Xu, Y. F., Qi, X. R., Maitani, Y., Nagai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superparamagnetic+iron+oxide+nanoparticles+stabilized+by+alginate:+Pharmacokinetics,+tissue+distribution,+and+applications+in+detecting+liver+cancers.">Superparamagnetic iron oxide nanoparticles stabilized by alginate: Pharmacokinetics, tissue distribution, and applications in detecting liver cancers.</a> International Journal of Pharmaceutics. 354 (1-2), 217-226 (2008).</li><li>Wang, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+tumors:+Monitoring+embolization+in+rabbits+with+VX2+tumors+-Transcatheter+intraarterial+first-pass+perfusion+MR+imaging.">Liver tumors: Monitoring embolization in rabbits with VX2 tumors -Transcatheter intraarterial first-pass perfusion MR imaging.</a> Radiology. 245 (1), 130-139 (2007).</li><li>Bimonte, 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=Radio-frequency+ablation-based+studies+on+VX2rabbit+models+for+HCC+treatment.">Radio-frequency ablation-based studies on VX2rabbit models for HCC treatment.</a> Infectious Agents and Cancer. 11 (1), (2016).</li><li>Gaba, R., Obeid, M., Khabbaz, R., Garcia, K., Schachtschneider, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+Animal+Models+for+Liver+Cancer.">Translational Animal Models for Liver Cancer.</a> American Journal of Interventional Radiology. 2 (2), 1-8 (2018).</li><li>Kuszyk, B. 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=Local+tumor+recurrence+following+hepatic+cryoablation:+radiologic-histopathologic+correlation+in+a+rabbit+model.">Local tumor recurrence following hepatic cryoablation: radiologic-histopathologic correlation in a rabbit model.</a> Radiology. 217 (2), 477-486 (2000).</li><li>Geschwind, J., 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=Chemoembolization+of+Liver+Tumor+in+a+Rabbit+Model+Assessment+of+Tumor+Cell+Death+with+Diffusion-Weighted+MR+Imaging+and+Histologic+Analysis.">Chemoembolization of Liver Tumor in a Rabbit Model Assessment of Tumor Cell Death with Diffusion-Weighted MR Imaging and Histologic Analysis.</a> Journal of Vascular and Interventional Radiology. 11 (10), 1245-1255 (2000).</li><li>Virmani, 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=Comparison+of+Two+Different+Methods+for+Inoculating+VX2+Tumors+in+Rabbit+Livers+and+Hind+Limbs.">Comparison of Two Different Methods for Inoculating VX2 Tumors in Rabbit Livers and Hind Limbs.</a> Journal of Vascular and Interventional Radiology. 19 (6), 931-936 (2008).</li><li>Seldinger, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Catheter+replacement+of+the+needle+in+percutaneous+arteriography:+A+new+technique.">Catheter replacement of the needle in percutaneous arteriography: A new technique.</a> Acta Radiologica. 49 (SUPPL 434), 47-52 (2008).</li><li>Schook, L. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genetic+porcine+model+of+cancer.">A genetic porcine model of cancer.</a> PLoS One. 10 (7), e0128864 (2015).</li></ol>

The work depicted in this report was supported by a research grant from Guerbet LLC.

The first critical step in the VX2 tumor methodology is successful propagation of a tumor in the hind limb of a donor rabbit. Refer to the first paragraph in the &#34;Representative Results&#34; section for more information regarding this step.
The next critical step is ensuring that the viable tumor capsule is properly identified. Not only will this be necessary for tumor suspension preparation, but it is also important for selecting and generating tumor pieces for hepatic implantation. The demarcation between viable tumor and surrounding muscle tissue is annotated in Figure 4. If the incorrect tissue sample is scraped during suspension preparation, subsequent hind limb propagation will fail. If this occurs during hepatic implantation, tumor growth in the liver will not occur. This will not be apparent until angiography.
During the hepatic implantation process, care should be taken when approaching the left lobe of the liver. Oftentimes, if the liver is readily apparent when entering the peritoneum, it is actually the medial lobe of the liver that operators are observing. Implantation into the medial lobe of the liver presents a handful of issues for angiographic use. The first is the medial lobe&#39;s anatomic relationship with the spine. A medial lobe tumor can often be obfuscated by the spine on fluoroscopy making confirmation and treatment of the tumor difficult. Additionally, the gastroduodenal artery is more often associated with the vasculature supplying the medial lobe. This increases the risk of non-target embolization of the gut and can potentially lead to bowel ischemia/infarction and possible death of the animal. As stated earlier, this will not qualify as a failure; however, it does warrant more care during visualization and treatment.
The final critical step is successful and stable femoral artery access. As seen in Figure 12, the femoral artery should be isolated atop a scalpel blade handle. While this is mostly done to allow the researcher to more accurately perform the Seldinger technique20, it should be maintained throughout the procedure. This is because removing the scalpel handle once the sheath has been introduced can cause unintended motion of the sheath within the vasculature leading to sheath occlusion and possible damage of the vasculature and surrounding structures. If the sheath becomes dislodged during the procedure, apply pressure in the femoral groove proximal to the access site in order to stop the bleeding, and the artery can then be ligated. Do not attempt to reinsert the sheath. The researcher can attempt to gain access from the contralateral femoral artery.
While the VX2 platform is a robust model in current use for translational research regarding HCC, it does have relevant shortcomings. The primary weakness of this model is that its disease state does not mimic that of a human HCC. The tumor induced is not pathologically similar to human HCC nor is the non-cirrhotic liver parenchymal microenvironment. Moreover, the VX2 tumor shows substantial internal necrosis, which excludes this model from use for treatment efficacy studies. Some alternative models include rodent models such as mice, rats, and woodchucks or larger animal models such as pigs and primates.16,21 These alternatives all provide different advantages and disadvantages; however, in the opinion of the authors, for angiographic utilization and cost efficacy, the VX2 rabbit remains dominant.

The rabbit VX2 tumor model has played a role in experimental oncology since its development in 19351,2. This tumor is a virus-induced anaplastic squamous cell carcinoma characterized by hypervascularity, rapid growth, and easy propagation in skeletal muscle3,4. While the rabbit VX2 tumor model has been used to investigate a multitude of cancers5,6,7,8; the focus of this paper is liver cancer9.
The purpose of the method described is to present a model for primary liver cancer, or hepatocellular carcinoma (HCC), that can be used by Interventional Radiologists for translational research. It can be used for pharmacokinetic studies, therapeutic investigations, and ablative method testing10,11,12,13,14,15.
The method detailed herein yields multiple advantages over other models within the same sphere such as rodent models like rats, mice, and woodchucks, or larger models like primates16. One of the primary benefits is the rapid and reliable tumor growth which allows researchers to establish an active tumor line within a month of first hind limb propagation17. Additionally, this tumor has straightforward sonographic visibility and a hypervascular periphery which allows for both transarterial locoregional treatments and ablative therapies. Finally, and most importantly, the size of the rabbit vasculature permits feasible and technically easy utilization of vascular instrumentation18.

<table>
	<tbody>
		<tr>
			<td>MethoCult (Methycellulose)</td>
			<td>Stemcell Technologies</td>
			<td>M3134</td>
			<td></td>
		</tr>
		<tr>
			<td>VX2 Cell Line</td>
			<td>NCI</td>
			<td>VX-2</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL Syringe</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr>
			<td>16-Gauge Needle</td>
			<td>BD</td>
			<td>305197</td>
			<td></td>
		</tr>
		<tr>
			<td>22-Gauge Needle</td>
			<td>BD</td>
			<td>305155</td>
			<td></td>
		</tr>
		<tr>
			<td>Hair Clippers</td>
			<td>Wahl</td>
			<td>41870-0438</td>
			<td></td>
		</tr>
		<tr>
			<td>Foam Insulated Box</td>
			<td>Mr. Box Online</td>
			<td>10 x 10 x 4</td>
			<td></td>
		</tr>
		<tr>
			<td>Acepromazine</td>
			<td>Henry Schein</td>
			<td>003845</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Par</td>
			<td>42023-179-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam</td>
			<td>Henry Schein</td>
			<td>049755</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol Pads</td>
			<td>Covidien</td>
			<td>5033</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Henry Schein</td>
			<td>056344</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Akorn</td>
			<td>59399-110-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Pentobarbital (Fatal-Plus)</td>
			<td>Vortech</td>
			<td>9373</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Petri Dish</td>
			<td>Thermo Fisher</td>
			<td>172931</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11965092</td>
			<td></td>
		</tr>
		<tr>
			<td>Saline</td>
			<td>Baxter</td>
			<td>2F7124</td>
			<td></td>
		</tr>
		<tr>
			<td>15-Blade</td>
			<td>Steris</td>
			<td>02-050-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel Handle x 2</td>
			<td>Steris</td>
			<td>22-2381</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved Hemostat</td>
			<td>WPI</td>
			<td>501288</td>
			<td></td>
		</tr>
		<tr>
			<td>Atraumatic Forceps</td>
			<td>Sklar</td>
			<td>52-5077</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze</td>
			<td>Medline</td>
			<td>NON21430LF</td>
			<td></td>
		</tr>
		<tr>
			<td>11-Blade</td>
			<td>Steris</td>
			<td>02-050-011</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgicel</td>
			<td>Ethicon</td>
			<td>1951</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 PDS / Taper</td>
			<td>Ethicon</td>
			<td>Z305H</td>
			<td></td>
		</tr>
		<tr>
			<td>4 - 0 Vicryl / Cutting</td>
			<td>Ethicon</td>
			<td>J392H</td>
			<td></td>
		</tr>
		<tr>
			<td>40 micron strainer</td>
			<td>BD</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tube</td>
			<td>Thermo Fisher</td>
			<td>339652</td>
			<td></td>
		</tr>
		<tr>
			<td>plastic pipette</td>
			<td>Thomas Scientific</td>
			<td>HS206371B</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Sorvall</td>
			<td>75004240</td>
			<td></td>
		</tr>
		<tr>
			<td>1.40mL Tubes (Internal Thread)</td>
			<td>Micronic</td>
			<td>MP32131-Z20</td>
			<td></td>
		</tr>
		<tr>
			<td>3-F VSI Micro-HV Introducer Kit</td>
			<td>Vascular Solutions</td>
			<td>Custom Order (P15180391)</td>
			<td></td>
		</tr>
		<tr>
			<td>.018 45-degree angle glidewire</td>
			<td>Terumo</td>
			<td>RG*GA1818SA</td>
			<td></td>
		</tr>
		<tr>
			<td>Direxion bern-shape microcatheter</td>
			<td>Boston Scientific</td>
			<td>M001195230</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnipaque</td>
			<td>GE</td>
			<td>Y510</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

When looking at Figure 1, it is clear that the quadricep of the rabbit is enlarged. Additionally, multiple small discrete nodules, typically correlating with tumor growth through the fascia, are visible. Upon palpation, the injected limb should appear than the non-injected limb. If a researcher requires more definitive assurance of tumor presence, ultrasound imaging can be used to identify the tumor embedded in the muscle. If a tumor is not detected, the hind limb should be re-injected with a tumor cell suspension.
In order to confirm successful vascular access, blood return into the sheath is observed on aspiration as seen in Figure 13D. If vascular access was unsuccessful, attempted aspiration will yield air in the sheath or present with significant resistance when pulling the plunger of the syringe.
For liver tumor growth, there are two ways to confirm successful propagation: angiographically and on necropsy. On angiography, identification of the tumor may occur immediately as is the case in Figure 14A where the tumor draws blood supply directly from the common hepatic artery. It may also take some time in cases where the tumor is lateral as is the case in Figure 14D. If the tumor is not readily visible after injection of contrast into the common hepatic artery, the researcher should attempt to inject contrast into left and right hepatic arteries in order to improve the chances of highlighting the tumor. It may also help to look for aberrant arteries traveling laterally towards the distal edge of the liver as seen in Figure 14C. On necropsy, the tumor should be readily visible as seen in Figure 15B (compare to Figure 15A).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58600/58600fig1.jpg" /> 
Figure 1: Rabbit hind limb. Shaved rabbit hind limb with mass indicative of tumor growth. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58600/58600fig2.jpg" /> 
Figure 2: Exposed hind limb. The same limb as shown Figure1 with overlying skin reflected revealing a large area of hypervascularity and discoloration distinct from the surrounding muscle representing the location of the tumor (white dotted line). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58600/58600fig3.jpg" /> 
Figure 3: Tumor removed en bloc and bisected. (A) Tumor and surrounding muscle removed en bloc. (B) Tumor has been bisected to reveal its capsular wall (arrowheads) and necrotic core. Tumor process can be seen in both halves as well as some necrotic debris. &#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58600/58600fig4.jpg" /> 
Figure 4: Tumor capsule. Arrowheads denote a piece of tumor (T), which is delineated from adjacent muscle (M) by the tumor capsule (dashed white line).&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58600/58600fig5.jpg" /> 
Figure 5: Exposed xiphoid process. Skin and underlying muscle have been reflected to allow for visualization of the xiphoid process (black arrow) and gut (white arrow). The white star denotes the cranial direction. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/58600/58600fig6.jpg" /> 
Figure 6: Linea alba. Overlying skin and fascia have been reflected to allow for visualization of the linea alba (black arrow) running in a cranial to caudal direction. This area is avascular and provides for blood-loss free access of the peritoneal space. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" class="xfigimg" src="/files/ftp_upload/58600/58600fig7.jpg" /> 
Figure 7: Lobe of liver outside peritoneum. This image shows a lobe of the liver that was gently extracted from the peritoneal space and placed on a piece of gauze. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" class="xfigimg" src="/files/ftp_upload/58600/58600fig8.jpg" /> 
Figure 8: Post-processed tumor piece for implantation. A piece of tumor processed to the appropriate size for implantation placed next to the tip of a #11-blade for scale. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<img alt="Figure 9" class="xfigimg" src="/files/ftp_upload/58600/58600fig9.jpg" /> 
Figure 9: Creating a pocket in the liver for tumor implantation. An #11-blade is inserted to the appropriate depth in the extracted lobe of the liver. This will create an appropriately sized pocked for the implantation of the tumor piece from Figure 8. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" class="xfigimg" src="/files/ftp_upload/58600/58600fig10.jpg" /> 
Figure 10: The femoral groove and initial incision. (A) Palpation of the hind limb allows for visualization of the femoral groove (white dotted line). (B) Initial incision in the hind limb made along the femoral groove. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 11" class="xfigimg" src="/files/ftp_upload/58600/58600fig11.jpg" /> 
Figure 11: Identification of the femoral bundle. Blunt dissection of the initial incision reveals femoral vein (black arrow). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig11large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 12" class="xfigimg" src="/files/ftp_upload/58600/58600fig12.jpg" /> 
Figure 12: Dissection of femoral bundle and isolation of femoral artery. (A) Dissection of the femoral bundle allows us to individually distinguish (from left to right) the femoral vein (FV), femoral artery (FA), and femoral nerve (FN). (B) The femoral artery isolated on a scalpel handle. Note the blood column allowing for distinction from the femoral nerve. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig12large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 13" class="xfigimg" src="/files/ftp_upload/58600/58600fig13.jpg" /> 
Figure 13: Vascular access. (A) A guidewire (G) is advanced into the femoral artery (FA) through the access needle (N) which was previously inserted into the femoral artery. (B) A sheath (S) and dilator (D) are advanced over the guidewire (G) into the femoral artery (FA). (C) Sheath (S) and dilator are advanced fully into the femoral artery (FA) up to the sheath hub. (D) Sheath is secured with silk after the dilator and guidewire have been removed. Aspiration yields blood (black arrow) in the sheath. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig13large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 14" class="xfigimg" src="/files/ftp_upload/58600/58600fig14.jpg" /> 
Figure 14: Angiographic imaging of hepatic tumor. (A) Catheter tip (white arrow) delivering contrast directly into artery feeding the tumor (white star). (B) Catheter tip (white arrow) delivering contrast into distal left hepatic artery and moderate contrast uptake by lateral tumor (white star). (C) Further contrast injection into tumor from B demonstrating an aberrant artery (white line) traveling from the catheter (white arrow) to the tumor (white star). (D) The tumor from B after further contrast uptake. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig14large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 15" class="xfigimg" src="/files/ftp_upload/58600/58600fig15.jpg" /> 
Figure 15: Rabbit liver. (A) A healthy rabbit liver showing the left medial lobe (white star) overlying the left lateral lobe (black star). (B) A rabbit liver with a fully developed hepatic tumor (white arrow). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58600/58600fig15large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Development and Angiographic Use of the Rabbit VX2 Model for Liver Cancer at JoVE.com

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

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 ...

development-angiographic-use-rabbit-vx2-model-for-liver

Nanyang Technological University

Research

JoVE Journal

Cancer Research

11.3K Views.  University of Illinois. The goal of this article is to provide a primer for the development and use of the VX2 carcinoma rabbit model for liver cancer.

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

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Development and Maintenance of a Preclinical Patient Derived Tumor Xenograft Model for the Investigation of Novel Anti-Cancer Therapies

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor pieces are injected subcutaneously into athymic nude mice (immunocompromised, T cell deficient) to create a bank of tumors and subsequently are passaged into different generations of mice in order to maintain these tumors from patients. Importantly, cellular heterogeneity of the original tumor is closely emulated in this model, which provides a more clinically relevant model for evaluation of drug efficacy studies (single agent and combination), biomarker analysis, resistant pathways and cancer stem cell biology. Some limitations of the PDTX model include the replacement of the human stroma with mouse stroma after the first generation in mice, inability to investigate treatment effects on metastasis due to the subcutaneous injections of the tumors, and the lack of evaluation of immunotherapies due to the use of immunocompromised mice. However, even with these limitations, the PDTX model provides a powerful preclinical platform in the drug discovery process.

Utilizing patient-derived tumors in a subcutaneous preclinical model is an excellent way to study the efficacy of novel therapies, predictive biomarker discovery, and drug resistant pathways. This model, in the drug development process, is essential in determining the fate of many novel anti-cancer therapies prior to clinical investigation.

Patient derived tumor xenograft (PDTX) models provide a necessary platform in facilitating anti-cancer drug development prior to human trials. Human tumor ...

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 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 ...

Evaluation of the Feasibility, Safety, and Accuracy of an Intraoperative High-intensity Focused Ultrasound Device for Treating Liver Metastases

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% of patients. Surgery has been widely used in association with radiofrequency, cryotherapy, or microwaves to expand the number of treatments performed with a curative intent. Nevertheless, several limitations have been documented when using these techniques (i.e., a traumatic puncture of the parenchyma, a limited size of lesions, and an inability to monitor the treatment in real-time).High-intensity focused ultrasound (HIFU) technology can achieve precise ablations of biological tissues without incisions or radiation. Current HIFU devices are based on an extracorporeal approach with limited access to the liver. We have developed a HIFU device designed for intraoperative use. The use of a toroidal transducer enables an ablation rate (10 cm3&#183;min-1) higher than any other treatment and is independent of perfusion.
The feasibility, safety, and accuracy of intraoperative HIFU ablation were evaluated during an ablate-and-resect prospective study. This clinical phase I and IIa study was performed in patients undergoing hepatectomy for liver metastases. The HIFU treatment was performed on healthy tissue scheduled for resection.Liver metastases measuring less than 20 mm will be targeted during phase IIb (currently ongoing). This set-up allows the real-time evaluation of HIFU ablation while protecting participating patients from any adverse effects related to this new technique.
Fifteen patients were included in phase I&#8211;IIa and 30 HIFU ablations were safely created within 40 s and with a precision of 1&#8211;2 mm. The average dimensions of the HIFU ablations were 27.5 x 21.0 mm2, corresponding to a volume of approximately 7.5 cm3. The aim of the ongoing phase IIb is to ablate metastases of less than 20 mm in diameter with a 5 mm margin.

Here, we present an ablate-and-resect prospective study to evaluate the feasibility, safety, and accuracy of intraoperative high-intensity focused ultrasound ablation in patients undergoing hepatectomy for liver metastases.

Today, the only potentially curative option in patients with colorectal liver metastases is surgery. However, liver resection is feasible in less than 20% ...

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

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 ...

Multidimensional Coculture System to Model Lung Squamous Carcinoma Progression

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions contribute to tissue architectural changes observed during tumorigenesis remains challenging due to the lack of appropriate models. In this protocol, we describe the generation of a 3D coculture model using a LUSC primary cell culture known as TUM622. TUM622 cells were established from a LUSC patient-derived xenograft (PDX) and have the unique property to form acinar-like structures when seeded in a basement membrane matrix. We demonstrate that TUM622 acini in 3D coculture recapitulate key features of tissue architecture during LUSC progression as well as the dynamic interactions between LUSC cells and components of the tumor microenvironment (TME), including the extracellular matrix (ECM) and cancer-associated fibroblasts (CAFs). We further adapt our principal 3D culturing protocol to demonstrate how this system could be utilized for various downstream analyses. Overall, this organoid model creates a biologically rich and adaptable platform that enables one to gain insight into the cell-intrinsic and extrinsic mechanisms that promote the disruption of epithelial architectures during carcinoma progression and will aid the search for new therapeutic targets and diagnostic markers.

An in vitro model system was developed to capture tissue architectural changes during lung squamous carcinoma (LUSC) progression in a 3-dimensional (3D) co-culture with cancer-associated fibroblasts (CAFs). This organoid system provides a unique platform to investigate the roles of diverse tumor cell-intrinsic and extrinsic changes that modulate the tumor phenotype.

Tumor-stroma interactions play a critical role in the development of lung squamous carcinoma (LUSC). However, understanding how these dynamic interactions ...

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...