We are thankful to M. Ohara, K. Saito, and M. Terai, for reviewing the manuscript. The authors acknowledge critical review for editorial and English assistance of this manuscript by Dr. R. Sato at Fox Chase Cancer Center. The work described herein was supported by the Bonnie Kroll Research Fund, the Mark Weinzierl Research Fund, the Eye Melanoma Research Fund at Thomas Jefferson University, The Osaka Community Foundation, and JSPS KAKENHI Grant Number JP 18K15596 at Osaka City University. Studies in Dr. A. Aplin&#39;s laboratory were supported by NIH grant R01 GM067893. This project was also funded by a Dean&#39;s Transformative Science Award, a Thomas Jefferson University Programmatic Initiative Award.

Patients enrolled in the study should provide written consent allowing the use of discarded surgical samples for research purposes and genetic studies, according to an Institutional Review Board-approved protocol. This protocol was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and approved by the Institutional Animal Care and Use Committee (IACUC).
1. Collection of Fresh Patient-derived Tumor...

<ol>
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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=Real-time+monitoring+of+rare+circulating+hepatocellular+carcinoma+cells+in+an+orthotopic+model+by+in+vivo+flow+cytometry+assesses+resection+on+metastasis.">Real-time monitoring of rare circulating hepatocellular carcinoma cells in an orthotopic model by in vivo flow cytometry assesses resection on metastasis.</a> Cancer Research. 72 (10), 2683-2691 (2012).</li><li>Jacob, D., Davis, J., Fang, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Xenograftictumor+modelsinmiceforcancer+research,+atechnical+review.">Xenograftictumor modelsinmiceforcancer research, atechnical review.</a> Gene Therapy and Molecular Biology. 8, 213-219 (2004).</li><li>Ahmed, S. 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The current orthotopic xenograft models are labor-intensive, time-consuming, and expensive to create. Orthotopic tumor xenograft mouse models for liver cancer were established more than two decades ago19,20,21. However, this technique is complicated and requires use of special equipment, such as a micro-needle holder and 6-0 to 8-0 fine sutures under a microscope. Tumor and normal liver tissue must be sewn up carefully so that t...

Uveal melanoma is the most common intraocular malignant tumor among adults in the western world. During the past 50 years, the incidence of uveal melanoma (5.1 cases per million) has remained stable in the United States1,2. Uveal melanoma arises from melanocytes in the iris, ciliary body, or choroid, and it is an extremely lethal disease when it develops metastasis. The death rate of patients with uveal melanoma metastasis was 80% at 1 year and 92% at 2 years after initial diagnosis of the metastases. The time between diagnosis of metastases and death is typically short, less than 6 months, without regards to therapy3,4. The cancer spreads through the blood and tends to dominantly metastasize to the liver (89-93%)4,5. An effective mouse model is urgently needed for further investigation of liver-metastatic uveal melanoma. For translational research, there is a clear demand to generate a liver-localized metastatic uveal melanoma mouse model.
Patient-derived tumor xenograft (PDX) mouse models are expected to provide individualized medicine strategies. These models might be predictive of clinical outcomes, be useful for preclinical drug evaluation, and be used for biological studies of tumors6. Representative PDX models are ectopically tumor-implanted xenograft mice, which have tumor at subcutaneous sites. Most researchers can do surgery for subcutaneous implantation without special practice7,8. They can also monitor subcutaneous tumors easily. Although subcutaneous PDX models became popular in the research phase, they have some hurdles in moving to practical use. Subcutaneous implantation forces patient-derived tumors to engraft at a different microenvironment from the tumor origin, so that it leads to engraftment failure and slow tumor growth 9,10,11,12,13,14. Orthotopic engraftment may be a more ideal and rational approach for a PDX model because it uses the same organ as the original tumor15,16.
Recently, we developed protocols for surgical orthotopic implantation techniques of patient-derived liver-metastatic uveal melanoma tumors and needle injection techniques with a cultured human liver-metastatic uveal melanoma cell line in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice17,18. The protocols result in consistently high technical success rates. We also established CT scanning techniques that are useful to detect interior liver tumors, and we developed re-implantation of cryopreserved tumors in the PDX platform. We found that uveal melanoma tumor xenograft models maintain the characteristics of the original patient liver tumor, including their histopathological and molecular features. Together, these techniques provide a better platform for liver orthotopic tumor models for uveal melanoma in translational research.

<table>
	<tbody>
		<tr>
			<td>Materials, tissues and animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 tank</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryomedium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Exitron nano 12000 (Alkaline earth metal-based nanoparticle contrast agent)</td>
			<td>Miltenyl Biotec</td>
			<td>130-095-700</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS 1X, with calcium &#38; magnesium</td>
			<td>Corning</td>
			<td>21-020-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Human liver metastatic uveal melanoma cell line</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Human uveal melanoma tissue in the liver</td>
			<td></td>
			<td></td>
			<td>All tissue handling should be done in a Biosafety Level 2 hood. Be careful when working with human tissue; always use gloves and avoid direct skin contact. Assume patients may have been infected with HIV or other highly transmissible organisms. Do not process samples known to carry infections.</td>
		</tr>
		<tr>
			<td>Iodine</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>Purdue Products</td>
			<td>67618-150-17</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher scientific</td>
			<td>A416-1</td>
			<td>Avoid direct contact to skin and eye and inhalation of anesthetic agent.</td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel HC</td>
			<td>BD</td>
			<td>354248</td>
			<td></td>
		</tr>
		<tr>
			<td>NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice</td>
			<td>Jackson Lab</td>
			<td>5557</td>
			<td>4 to 8 weeks old</td>
		</tr>
		<tr>
			<td>PBS 1X, without calcium and magnesium</td>
			<td>Corning</td>
			<td>21-031-CM</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile alcohol prep pad (70% isopropyl alcohol)</td>
			<td>Nice-Pak products</td>
			<td>B603</td>
			<td></td>
		</tr>
		<tr>
			<td>4% paraformaldehyde phosphate buffer solution</td>
			<td>Wako</td>
			<td>163-20145</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethyl alcohol solution</td>
			<td>Fisher Scientific</td>
			<td>04-355-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbable hemostat</td>
			<td>Johnson and Johnson</td>
			<td>63713-0019-61</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Body weight measure</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cautery</td>
			<td>Bovie Medical</td>
			<td>MC-23009</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell counter</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuzer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swab</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo freezing container</td>
			<td>NALGENE</td>
			<td>5100-0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryotube</td>
			<td>SARSTEDT</td>
			<td>72.379</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved scissors</td>
			<td>World Precision Instruments</td>
			<td>503247</td>
			<td></td>
		</tr>
		<tr>
			<td>Curved ultrafine forceps</td>
			<td>World Precision Instruments</td>
			<td>501302</td>
			<td></td>
		</tr>
		<tr>
			<td>Fabric sheet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Freezer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>F/AIR Filter Canister</td>
			<td>Harvard Apparatus</td>
			<td>600979</td>
			<td></td>
		</tr>
		<tr>
			<td>Heating pad</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer</td>
			<td>Artisan Scientific</td>
			<td>66317-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Liquid nitrogen jar</td>
			<td>Thermo Fisher Scientific</td>
			<td>2123</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro-CT scan</td>
			<td>Siemens</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needle holder</td>
			<td>World Precision Instruments</td>
			<td>501246</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes</td>
			<td>Fisher Scientific</td>
			<td>FB0875713</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Spray bottle</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile hood</td>
			<td></td>
			<td></td>
			<td>Biosafety level 2 cabinet</td>
		</tr>
		<tr>
			<td>Sterile No.11 scalpel</td>
			<td>AD Surgical</td>
			<td>A300-11-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight forceps</td>
			<td>World Precision Instruments</td>
			<td>14226</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical drape</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tail vein restrainer</td>
			<td>Braintree Scientific</td>
			<td>TV-150-STD</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>1 ml TB syringe with 27-gauge needle</td>
			<td>BD</td>
			<td>309623</td>
			<td></td>
		</tr>
		<tr>
			<td>1.7 ml tube</td>
			<td>Bioexpress</td>
			<td>C-3260-1</td>
			<td></td>
		</tr>
		<tr>
			<td>5-0 PDO Suture</td>
			<td>AD Surgical</td>
			<td>S-D518R13</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL conical tubes</td>
			<td>AZER SCIENTIFIC</td>
			<td>ES-9152N</td>
			<td></td>
		</tr>
		<tr>
			<td>27-gauge needle</td>
			<td>BD</td>
			<td>780301</td>
			<td></td>
		</tr>
		<tr>
			<td>27-gauge needle</td>
			<td>Hamilton</td>
			<td>7803-01</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL conical tubes</td>
			<td>AZER SCIENTIFIC</td>
			<td>ES-9502N</td>
			<td></td>
		</tr>
		<tr>
			<td>50 &#181;l micro syringe</td>
			<td>BD</td>
			<td>80630</td>
			<td></td>
		</tr>
		<tr>
			<td>50 &#181;l micro syringe</td>
			<td>Hamilton</td>
			<td>7655-01</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mL container</td>
			<td>Fisher Scientific</td>
			<td>12594997</td>
			<td></td>
		</tr>
		<tr>
			<td>200&#956;l tip</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of a liver orthotopic human uveal melanoma xenograft platform in immunodeficient mice

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more representative models to study human cancers in immunodeficient mice than traditional established human cell lines in vitro. Recently, orthotopically implanted patient-derived tumor xenograft (PDX) models in mice have been developed to better replicate features of patient tumors. A liver orthotopic xenograft mouse model is expected to be a useful cancer research platform, providing insights into tumor biology and drug therapy. However, liver orthotopic tumor implantation is generally complicated. Here we describe our protocols for the orthotopic implantation of patient-derived liver-metastatic uveal melanoma tumors. We cultured human liver metastatic uveal melanoma cell lines into immunodeficient mice. The protocols can result in consistently high technical success rates using either a surgical orthotopic implantation technique with chunks of patient-derived uveal melanoma tumor or a needle injection technique with cultured human cell line. We also describe protocols for CT scanning to detect interior liver tumors and for re-implantation techniques using cryopreserved tumors to achieve re-engraftment. Together, these protocols provide a better platform for liver orthotopic tumor mouse models of liver metastatic uveal melanoma in translational research.

Surgical orthotopic implantation using the liver pocket method can transplant human liver metastatic uveal melanoma tumor into the mouse liver with a high success rate of 80% within six months. The xenograft tumor engrafts in the liver as a solitary tumor without daughter nodules (Figure 1 and Figure 3A). The surgical orthotopic injection technique into the liver using microneedles successfully engrafted cultured human liver-meta...

Watch this Scientific Journal Video about Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice at JoVE.com

Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice

Orthotopic human liver metastatic uveal melanoma xenograft mouse models were created using surgical orthotopic implantation techniques with patient-derived tumor chunk and needle injection techniques with cultured human uveal melanoma cell lines.

In recent decades, subcutaneously implanted patient-derived xenograft tumors or cultured human cell lines have been increasingly recognized as more ...

Most patient-derived tumor animal models are subcutaneously implanted, preventing the evaluation of important features of tumors and of the drug efficacy. Housing the implanted tumors within the liver facilitated this analysis without the risk of too much deviations occurring within the liver. Before beginning the procedure, spray all of the materials and instruments with 70%ethyl alcohol.Confirm a lack of response to toe pinch in the anesthetized animal. Place the mouse in the supine position on a heating pad, and wet the fur with additional 70%ethyl alcohol. Spread the fur to visualize the skin below the left subcostal area.Remove hair, and apply iodine to be absorbed into the skin. Place a sterile surgical drape with a two-centimeter hole over the mouse, and use curved ultra fine forceps to lift the abdominal skin. Make a one-centimeter transverse left subcostal incision into the tented skin, and insert curved scissor tips into the incision to separate the peritoneum from the skin.Retract the scissors without opening the blades, and locate the liver under the peritoneum by its dark reddish color. Use the curved scissors to make a one-centimeter transverse incision in the peritoneum. Using the other hand, insert the edge of a cotton swab beneath the left liver lobe, and roll the swab downward to push up the liver.Lift the swab to transfer the liver onto a nonwoven, absorbent fabric sheet. Holding a sterile number 11 scalpel blade parallel to the surface, press the blade horizontally into the liver to make a five-millimeter deep by five-millimeter wide incision in the parenchyma while softly pressing the incision site with a cotton swab. Roll the cotton swab upward to open the incision site.Use curved ultra fine forceps to implant a one-millimeter cube of tumor tissue into the pocket. Retract the forceps while rolling the cotton swab in the reverse direction while pressing down, and remove the swab. Immediately place an absorbable hemostat on the incision site, and confirm hemostasis.Next use blunt ended forceps to peel the liver from the fabric sheet, and return the liver to the abdominal cavity. Then close the peritoneum with a 5-0 absorbable suture and a double ligature, and close the skin with a 5-0 absorbable suture and a triple ligature. The xenograft tumor engrafts in the liver as a solitary tumor without daughter nodules.Surgical orthotopic injection into the liver using microneedles can result in a successful engraftment of a cluster of cultured human liver metastatic uveal melanoma cells within the liver. Using this liver orthotopic patient-derived tumor model, the efficacy of the drugs injected directly into the liver can be tested with similar expected outcomes to those observed in clinical patients.

generation-liver-orthotopic-human-uveal-melanoma-xenograft-platform

Research

JoVE Journal

Cancer Research

8.5K Views.  Osaka City University Graduate School of Medicine. La maggior parte dei modelli animali tumorali derivati dal paziente sono impiantati per via sottocutanea, impedendo la valutazione di importanti caratteristiche dei tumori e dell'efficacia del farmaco. L'alloggiamento dei tumori impiantati all'interno del fegato ha facilitato questa analisi senza il rischio di troppe deviazioni che si verificano all'interno del fegato. Prima di iniziare la procedura, spruzzare tutti i materiali e gli strumenti con alcol etilico al 70%.Confermare la man...