Name: generation of a liver orthotopic human uveal melanoma xenograft platform in immunodeficient mice
Uploaded: 2019-11-06T13:00:00
Description: Watch this Scientific Journal Video about Generation of a Liver Orthotopic Human Uveal Melanoma Xenograft Platform in Immunodeficient Mice at JoVE.com

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>
	<li>Aronow, M. E., Topham, A. K., Singh, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uveal+Melanoma:+5-Year+Update+on+Incidence,+Treatment,+and+Survival+(SEER+1973-2013).">Uveal Melanoma: 5-Year Update on Incidence, Treatment, and Survival (SEER 1973-2013).</a> Ocular Oncology and Pathology. 4 (3), 145-151 (2018).</li><li>Krantz, B. A., Dave, N., Komatsubara, K. M., Marr, B. P., Carvajal, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uveal+melanoma:+epidemiology,+etiology,+and+treatment+of+primary+disease.">Uveal melanoma: epidemiology, etiology, and treatment of primary disease.</a> Clinical Ophthalmology. 11, 279-289 (2017).</li><li>Gragoudas, E. 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=Survival+of+patients+with+metastases+from+uveal+melanoma.">Survival of patients with metastases from uveal melanoma.</a> Ophthalmology. 98 (3), 383-389 (1991).</li><li>Diener-West, M., 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=Development+of+metastatic+disease+after+enrollment+in+the+COMS+trials+for+treatment+of+choroidal+melanoma:+Collaborative+Ocular+Melanoma+Study+Group+Report+No.+26.">Development of metastatic disease after enrollment in the COMS trials for treatment of choroidal melanoma: Collaborative Ocular Melanoma Study Group Report No. 26.</a> Archives of Ophthalmology. 123 (12), 1639-1643 (2005).</li><li>Collaborative Ocular Melanoma Study Group. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+metastatic+disease+status+at+death+in+435+patients+with+large+choroidal+melanoma+in+the+Collaborative+Ocular+Melanoma+Study+(COMS):+COMS+report+no.+15.">Assessment of metastatic disease status at death in 435 patients with large choroidal melanoma in the Collaborative Ocular Melanoma Study (COMS): COMS report no. 15.</a> Archives of Ophthalmology. 119 (5), 670-676 (2001).</li><li>Hidalgo, M., 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=Patient-derived+xenograft+models:+an+emerging+platform+for+translational+cancer+research.">Patient-derived xenograft models: an emerging platform for translational cancer research.</a> Cancer Discovery. 4 (9), 998-1013 (2014).</li><li>Kim, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+orthotopic+and+heterotopic+human+pancreatic+cancer+xenografts+in+immunodeficient+mice.">Generation of orthotopic and heterotopic human pancreatic cancer xenografts in immunodeficient mice.</a> Nature Protocols. 4 (11), 1670-1680 (2009).</li><li>N&#233;mati, F., 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=Establishment+and+characterization+of+a+panel+of+human+uveal+melanoma+xenografts+derived+from+primary+and/or+metastatic+tumors.">Establishment and characterization of a panel of human uveal melanoma xenografts derived from primary and/or metastatic tumors.</a> Clinical Cancer Research. 16 (8), 2352-2362 (2010).</li><li>Wilmanns, 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=Modulation+of+Doxorubicin+sensitivity+and+level+of+p-glycoprotein+expression+in+human+colon-carcinoma+cells+by+ectopic+and+orthotopic+environments+in+nude-mice.">Modulation of Doxorubicin sensitivity and level of p-glycoprotein expression in human colon-carcinoma cells by ectopic and orthotopic environments in nude-mice.</a> International Journal of Oncology. 3 (3), 413-422 (1993).</li><li>Kang, Y., 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=Proliferation+of+human+lung+cancer+in+an+orthotopic+transplantation+mouse+model.">Proliferation of human lung cancer in an orthotopic transplantation mouse model.</a> Experimental and Therapeutic. 1 (3), 471-475 (2010).</li><li>Fichtner, I., 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=Establishment+of+patient-derived+non-small+cell+lung+cancer+xenografts+as+models+for+the+identification+of+predictive+biomarkers.">Establishment of patient-derived non-small cell lung cancer xenografts as models for the identification of predictive biomarkers.</a> Clinical Cancer Research. 14 (20), 6456-6468 (2008).</li><li>Marangoni, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+patient+tumor-derived+breast+cancer+xenografts+for+preclinical+assays.">A new model of patient tumor-derived breast cancer xenografts for preclinical assays.</a> Clinical Cancer Research. 13 (13), 3989-3998 (2007).</li><li>Bergamaschi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+profiling+and+characterization+of+luminal-like+and+basal-like+in+vivo+breast+cancer+xenograft+models.">Molecular profiling and characterization of luminal-like and basal-like in vivo breast cancer xenograft models.</a> Molecular Oncology. 3 (5-6), 469-482 (2009).</li><li>Ho, K. S., Poon, P. C., Owen, S. C., Shoichet, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessel+hyperpermeability+and+pathophysiology+in+human+tumour+xenograft+models+of+breast+cancer:+a+comparison+of+ectopic+and+orthotopic+tumours.">Blood vessel hyperpermeability and pathophysiology in human tumour xenograft models of breast cancer: a comparison of ectopic and orthotopic tumours.</a> BMC Cancer. 12, 579 (2012).</li><li>Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+orthotopic+xenografts:+better+mimic+of+metastasis+than+subcutaneous+xenografts.">Patient-derived orthotopic xenografts: better mimic of metastasis than subcutaneous xenografts.</a> Nature Reviews Cancer. 15 (8), 451-452 (2015).</li><li>Rubio-Viqueira, B., Hidalgo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+in+vivo+xenograft+tumor+model+for+predicting+chemotherapeutic+drug+response+in+cancer+patients.">Direct in vivo xenograft tumor model for predicting chemotherapeutic drug response in cancer patients.</a> Clinical Pharmacology &amp; Therapeutics. 85 (2), 217-221 (2009).</li><li>Ozaki, 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=Establishment+and+Characterization+of+Orthotopic+Mouse+Models+for+Human+Uveal+MelanomaHepatic+Colonization.">Establishment and Characterization of Orthotopic Mouse Models for Human Uveal MelanomaHepatic Colonization.</a> American Journal of Pathology. 186 (1), 43-56 (2016).</li><li>Kageyama, 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=Establishment+of+an+orthotopic+patient-derived+xenograft+mouse+model+using+uveal+melanomahepatic+metastasis.">Establishment of an orthotopic patient-derived xenograft mouse model using uveal melanomahepatic metastasis.</a> Journal of Translational Medicine. 15 (1), 145 (2017).</li><li>Fu, X. Y., Besterman, J. M., Monosov, A., Hoffman, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+of+human+metastatic+colon+cancer+in+nude+mice+orthotopically+constructed+by+using+histologically+intact+patient+specimens.">Models of human metastatic colon cancer in nude mice orthotopically constructed by using histologically intact patient specimens.</a> Proceedings of the National Academy of Sciences of the United States of America. 88 (20), 9345-9349 (1991).</li><li>Rashidi, 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=An+orthotopic+mouse+model+of+remetastasis+of+human+colon+cancer+liver+metastasis.">An orthotopic mouse model of remetastasis of human colon cancer liver metastasis.</a> Clinical Cancer Research. 6 (6), 2556-2561 (2000).</li><li>Fan, Z. 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. U., 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=Generation+of+subcutaneous+and+intrahepatic+human+hepatocellular+carcinoma+xenografts+in+immunodeficient+mice.">Generation of subcutaneous and intrahepatic human hepatocellular carcinoma xenografts in immunodeficient mice.</a> Journal of Visualized Experiments. 25 (79), e50544 (2013).</li><li>Kim, M., 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=Generation+of+humanized+liver+mouse+model+by+transplant+of+patient-derived+fresh+human+hepatocytes.">Generation of humanized liver mouse model by transplant of patient-derived fresh human hepatocytes.</a> Transplantation Proceedings. 46 (4), 1186-1190 (2014).</li><li>Lavender, K. J., Messer, R. J., Race, B., Hasenkrug, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+bone+marrow,+liver,+thymus+(BLT)+humanized+mice+on+the+C57BL/6+Rag2(-/-)&#947;c(-/-)CD47(-/-)+background.">Production of bone marrow, liver, thymus (BLT) humanized mice on the C57BL/6 Rag2(-/-)&#947;c(-/-)CD47(-/-) background.</a> Journal of Immunological Methods. 407, 127-134 (2014).</li><li>Boll, 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=Micro-CT+based+experimental+liver+imaging+using+a+nanoparticulate+contrast+agent:+a+longitudinal+study+in+mice.">Micro-CT based experimental liver imaging using a nanoparticulate contrast agent: a longitudinal study in mice.</a> PLoS One. 6 (9), e25692 (2011).</li><li>Zhao, 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=Global+gene+expression+profiling+confirms+the+molecular+fidelity+of+primary+tumor-based+orthotopic+xenograft+mouse+models+of+medulloblastoma.">Global gene expression profiling confirms the molecular fidelity of primary tumor-based orthotopic xenograft mouse models of medulloblastoma.</a> Neuro-Oncology. 14 (5), 574-583 (2012).</li><li>Rubio-Viqueira, 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=An+in+vivo+platform+for+translational+drug+development+in+pancreatic+cancer.Clinical.">An in vivo platform for translational drug development in pancreatic cancer.Clinical.</a> Cancer Research. 12 (15), 4652-4661 (2006).</li><li>Siolas, D., Hannon, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patient-derived+tumor+xenografts:+transforming+clinical+samples+into+mouse+models.">Patient-derived tumor xenografts: transforming clinical samples into mouse models.</a> Cancer Research. 73 (17), 5315-5319 (2013).</li><li>Alkema, N. G., 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=Biobanking+of+patient+and+patient-derived+xenograft+ovarian+tumour+tissue:+efficient+preservation+with+low+and+high+fetal+calf+serum+based+methods.">Biobanking of patient and patient-derived xenograft ovarian tumour tissue: efficient preservation with low and high fetal calf serum based methods.</a> Scientific Reports. 6 (5), 14495 (2015).</li></ol>

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

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Patient-derived Orthotopic Xenograft Models for Human Urothelial Cell Carcinoma and Colorectal Cancer Tumor Growth and Spontaneous Metastasis

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and colorectal cancer (CRC). More than 50% of patients with muscle-invasive UCC, despite curative therapy for clinically-localized disease, will develop metastases and die within 5 years, and metastatic CRC is a leading cause of cancer-related deaths in the US. Xenograft models that consistently mimic UCC and CRC metastasis seen in patients are needed. This study aims to generate patient-derived orthotopic xenograft (PDOX) models of UCC and CRC for primary tumor growth and spontaneous metastases under the influence of LN stromal cells mimicking the progression of human metastatic diseases for drug screening. Fresh UCC and CRC tumors were obtained from consented patients undergoing resection for HG-UCC and colorectal adenocarcinoma, respectively. Co-inoculated with LN stromal cell (LNSC) analog HK cells, luciferase-tagged UCC cells were intra-vesically (IB) instilled into female non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice, and CRC cells were intra-rectally (IR) injected into male NOD/SCID mice. Tumor growth and metastasis were monitored weekly using bioluminescence imaging (BLI). Upon sacrifice, primary tumors and mouse organs were harvested, weighed, and formalin-fixed for Hematoxylin and Eosin and immunohistochemistry staining. In our unique PDOX models, xenograft tumors resemble patient pre-implantation tumors. In the presence of HK cells, both models have high tumor implantation rates measured by BLI and tumor weights, 83.3% for UCC and 96.9% for CRC, and high distant organ metastasis rates (33.3% detected liver or lung metastasis for UCC and 53.1% for CRC). In addition, both models have zero mortality from the procedure. We have established unique, reproducible PDOX models for human HG-UCC and CRC, which allow for tumor formation, growth, and metastasis studies. With these models, testing of novel therapeutic drugs can be performed efficiently and in a clinically-mimetic manner.

This protocol describes the generation of patient-derived orthotopic xenograft models by intra-vesically instilling high-grade urothelial cell carcinoma cells or intra-rectally injecting colorectal cancer cells into non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice for primary tumor growth and spontaneous metastases under the influence of lymph node stromal cells, which mimics the progression of human metastatic diseases.

Cancer patients have poor prognoses when lymph node (LN) involvement is present in both high-grade urothelial cell carcinoma (HG-UCC) of the bladder and ...

Patient-derived Heterogeneous Xenograft Model of Pancreatic Cancer Using Zebrafish Larvae as Hosts for Comparative Drug Assessment

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and basic cancer researches. Generations of PDX models in traditional host mice are time-consuming and only working for a small proportion of samples. Recently, zebrafish PDX (zPDX) has emerged as a unique host system, with the characteristics of small-scale and high efficiency. Here, we describe an optimized methodology for generating a dual fluorescence-labeled tumor xenograft model for comparative chemotherapy assessment in zPDX models. Tumor cells and fibroblasts were enriched from freshly-harvested or frozen pancreatic cancer tissue at different culture conditions. Both cell groups were labeled by lentivirus expressing green or red fluorescent proteins, as well as an anti-apoptosis gene BCL2L1. The transfected cells were pre-mixed and co-injected into the 2 dpf larval zebrafish that were then bred in modified E3 medium at 32 &#176;C. The xenograft models were treated by chemotherapy drugs and/or BCL2L1 inhibitor, and the viabilities of both tumor cells and fibroblasts were investigated simultaneously. In summary, this protocol allows researchers to quickly generate a large amount of zPDX models with a heterogeneous tumor microenvironment and provides a longer observation window and a more precise quantitation in assessing the efficiency of drug candidates.

This protocol describes optimization procedures in a virus-based dual fluorescence-labeled tumor xenograft model using larval zebrafish as hosts. This heterogeneous xenograft model mimics the tissue composition of pancreatic cancer microenvironment in vivo and serves as a more precise tool for assessing drug responses in personalized zPDX (zebrafish patient-derived xenograft) models.

Patient-derived tumor xenograft (PDX) and cell-derived tumor xenograft (CDX) are important techniques for preclinical assessment, medication guidance and ...

A Melanoma Patient-Derived Xenograft Model

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture vessels versus in vivo in human patients. This is due to the bottleneck selection of clonal populations of melanoma cells that can robustly grow in vitro in the absence of physiological conditions. Further, responses to therapy in two-dimensional tissue cultures overall do not faithfully reflect responses to therapy in melanoma patients, with the majority of clinical trials failing to show the efficacy of therapeutic combinations shown to be effective in vitro. Although xenografting of melanoma cells into mice provides the physiological in vivo context absent from two-dimensional tissue culture assays, the melanoma cells used for engraftment have already undergone bottleneck selection for cells that could grow under two-dimensional conditions when the cell line was established. The irreversible alterations that occur as a consequence of the bottleneck include changes in growth and invasion properties, as well as the loss of specific subpopulations. Therefore, models that better recapitulate the human condition in vivo may better predict therapeutic strategies that effectively increase the overall survival of patients with metastatic melanoma. The patient-derived xenograft (PDX) technique involves the direct implantation of tumor cells from the human patient to a mouse recipient. In this manner, tumor cells are consistently grown under physiological stresses in vivo and never undergo the two-dimensional bottleneck, which preserves the molecular and biological properties present when the tumor was in the human patient. Notable, PDX models derived from organ sites of metastases (i.e., brain) display similar metastatic capacity, while PDX models derived from therapy naive patients and patients with acquired resistance to therapy (i.e., BRAF/MEK inhibitor therapy) display similar sensitivity to therapy.

Patient-derived xenograft (PDX) models more robustly recapitulate melanoma molecular and biological features and are more predictive of therapy response compared to traditional plastic tissue culture-based assays. Here we describe our standard operating protocol for the establishment of new PDX models and the characterization/experimentation of existing PDX models.

Accumulating evidence suggests that molecular and biological properties differ in melanoma cells grown in traditional two-dimensional tissue culture ...

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. 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 ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...

A Robust Discovery Platform for the Identification of Novel Mediators of Melanoma Metastasis

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was established using robust, reproducible in vivo models and standardized methods to identify novel players in melanoma metastasis. This approach allows for data inference at specific experimental stages to precisely characterize a gene's role in metastasis. Models are established by introducing genetically modified melanoma cells via intracardiac, intradermal, or subcutaneous injections into mice, followed by monitoring with serial in vivo imaging. Once preestablished endpoints are reached, primary tumors and/or metastases-bearing organs are harvested and processed for various analyses. Tumor cells can be sorted and subjected to any of several 'omics' platforms, including single-cell RNA sequencing. Organs undergo imaging and immunohistopathological analyses to quantify the overall burden of metastases and map their specific anatomic location. This optimized pipeline, including standardized protocols for engraftment, monitoring, tissue harvesting, processing, and analysis, can be adopted for patient-derived, short-term cultures and established human and murine cell lines of various solid cancer types.

This article describes a workflow of techniques employed for testing novel candidate mediators of melanoma metastasis and their mechanism(s) of action.

Metastasis is a complex process, requiring cells to overcome barriers that are only incompletely modeled by in vitro assays. A systematic workflow was ...