Name: development and angiographic use of the rabbit vx2 model for liver cancer
Uploaded: 2019-01-07T14:00:00
Description: Watch this Scientific Journal Video about Development and Angiographic Use of the Rabbit VX2 Model for Liver Cancer at JoVE.com

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: htt...

<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 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 d...

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...

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 ...

This pre-clinical liver tumor model can help answer key questions about local regional therapy in the fields of interventional radiology and interventional oncology. The main advantage of this technique, is that it results in a reliable tumor induction by orthotopic allograft implantation. For implementation of the tumor, first confirm a lack of response to pedal reflex.Once confirmed, the animal subject is placed under anesthesia. An appropriate level of anesthesia is confirmed, and the abdomen is sterilely prepared and draped. After anaesthetisation, use a number 15 blade to make a three to four centimeter vertical, midline skin incision starting from the xiphoid process.Retract the skin to identify the linea alba, a reflective white band of tissue that travels inferiorly along the midline. And use both blunt and sharp dissection to traverse the linea alba, exposing the peritoneum, taking care not to perforate the underlying bowel tissue. Carefully dissect through the abdominal cavity tissue, to locate the liver, using a haemostat to extend the midline incision one to two centimeters inferiorly through the skin, muscle and peritoneum as necessary.Identify the left lobe, which is inferolateral to the medial lobe that sits in the midline. And place a dry piece of gauze at the inferior aspect of the incision to prevent the liver from retracting back into the abdomen. Place a new piece of wet gauze over the extracted liver.Select a one to two cubic millimeter tumor tissue piece, with forceps. And use a number 11 blade to puncture the liver tissue at a 45 degree angle to a 0.5 centimeter deep pocket, taking care not to penetrate the dorsal aspect of the liver capsule. Gently lift the blade in ventrally to create a small pocket in the liver bed, and place the tumor into the pocket.Place a piece of haemostatic agent over the tumor pocket to promote haemostasis, and to prevent ejection of the tumor piece. And, after confirming haemostasis, return the liver to the abdominal cavity. Then use a three zero polydioxanone suture on a taper needle, in a simple continuous stitch to close the abdominal wall.And four zero polyglactin 910 sutures on a cutting needle in a continuous, sub-cuticular stitch to close the skin. To confirm a successful tumor placement, palpate the femoral groove in the groin, and make a two to three centimeter linear incision along the groove. Using blunt dissection, locate and isolate the femoral bundle containing the femoral vein, artery and nerve, and use blunt dissection to separate the femoral artery from the rest of the structures in the bundle.Isolate the artery atop a scalpel handle, and use the Seldinger technique, and a three French introducer kit to introduce a needle into the vessel. Insert a guide wire into the artery. And remove the needle, to carefully advance the three French sheath into the vessel.Remove the dilator and guide wire from the sheath to complete vascular access. Under fluoroscopic guidance, insert the catheter into the celiac trunk at the T 12 level, and advance the catheter into the left hepatic artery, via the common hepatic, and proper hepatic arteries. When the catheter reaches the left hepatic artery, inject contrast agent to confirm the presence of a hypervascular tumor, which can then be treated with an intra-arterial therapy of choice.And remove the catheter. Using three zero silk suture, ligate the femoral artery proximally and distally to the insertion point of the sheath, taking care to tighten the knot proximal to the sheath, as it is withdrawn to prevent bleeding. Then use four zero polyglactin 910 sutures, on a cutting needle in a subcuticular stitch to close the groin incision.Three to four weeks after implantation, the tumor can be visualized macroscopically on the left quadricep of the rabbit. The use of angiography and the injection of a contrast agent as demonstrated, can be used to confirm a successful tumor propagation after placement. Upon necropsy, a successfully implanted tumor should be readily visible.Since its development, this technique has paved the way for researchers in the fields of interventional radiology and interventional oncology to explore liver tumor local regional therapy in a small animal model.

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

Research

JoVE Journal

Cancer Research

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 ...