Name: primary orthotopic glioma xenografts recapitulate infiltrative growth and isocitrate dehydrogenase i mutation
Uploaded: 2014-01-14T13:45:00
Description: Watch this Scientific Journal Video about Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation at JoVE.com

We are particularly indebted to patients at Vanderbilt University Medical Center who provided invaluable research material for the Molecular Neurosurgical Tissue Bank. We thank those who established and maintain the Tissue Bank, Reid C. Thompson MD (principal investigator), Cherryl Kinnard RN (research nurse) and Larry A. Pierce MS (manager). Histological services were performed, in part, by the Vanderbilt University Medical Center (VUMC) Translational Pathology Shared Resource (supported by award 5P30 CA068485 to the Vanderbilt-Ingram Cancer Center). This work was supported by grants to MKC from the NINDS (1R21NS070139), the Burroughs Wellcome Fund and VMC development funds. MKC is supported by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development through grant 1 I01 BX000744-01. The contents do not represent the views of the Department of Veterans Affairs or the United States Government.

1. Preparation of Tumor Cell Suspension
Note: Appropriate institutional approvals for the use of patient material and animals are required to establish and maintain primary orthotopic glioma xenografts. At Vanderbilt University Medical Center, resected tumor material that is in excess of that required for diagnostic purposes is collected with patient consent for a research tissue repository. Specimens are labeled with a randomized 5-digit REDcap database number and all patient-...

<ol>
	<li>Johnson, J. 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=Relationships+between+drug+activity+in+NCI+preclinical+in+vitro+and+in+vivo+models+and+early+clinical+trials.">Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials.</a> Br. J. 84, 1424-1431 (2001).</li><li>Witt Hamer, D. e., C, 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=The+genomic+profile+of+human+malignant+glioma+is+altered+early+in+primary+cell+culture+and+preserved+in+spheroids.">The genomic profile of human malignant glioma is altered early in primary cell culture and preserved in spheroids.</a> Oncogene. 27, 2091-2096 (2008).</li><li>Lee, 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=Tumor+stem+cells+derived+from+glioblastomas+cultured+in+bFGF+and+EGF+more+closely+mirror+the+phenotype+and+genotype+of+primary+tumors+than+do+serum-cultured+cell+lines.">Tumor stem cells derived from glioblastomas cultured in bFGF and EGF more closely mirror the phenotype and genotype of primary tumors than do serum-cultured cell lines.</a> Cancer Cell. 9, 391-403 (2006).</li><li>Pandita, A., Aldape, K. D., Zadeh, G., Guha, A., James, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrasting+in+vivo+and+in+vitro+fates+of+glioblastoma+cell+subpopulations+with+amplified+EGFR.">Contrasting in vivo and in vitro fates of glioblastoma cell subpopulations with amplified EGFR.</a> Genes Chromosomes Cancer. 39, 29-36 (2004).</li><li>Daniel, V. 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=A+primary+xenograft+model+of+small-cell+lung+cancer+reveals+irreversible+changes+in+gene+expression+imposed+by+culture+in+vitro.">A primary xenograft model of small-cell lung cancer reveals irreversible changes in gene expression imposed by culture in vitro.</a> Cancer Res. 69, 3364-3373 (2009).</li><li>Piaskowski, 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=Glioma+cells+showing+IDH1+mutation+cannot+be+propagated+in+standard+cell+culture+conditions.">Glioma cells showing IDH1 mutation cannot be propagated in standard cell culture conditions.</a> Br. J. Cancer. 104, 968-970 (2011).</li><li>Vescovi, A. L., Galli, R., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+tumour+stem+cells.">Brain tumour stem cells.</a> Nat. Rev. 6, 425-436 (2006).</li><li>Sasai, 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=Shh+pathway+activity+is+down-regulated+in+cultured+medulloblastoma+cells:+implications+for+preclinical+studies.">Shh pathway activity is down-regulated in cultured medulloblastoma cells: implications for preclinical studies.</a> Cancer Res. 66, 4215-4222 (2006).</li><li>Shu, Q., 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=Direct+orthotopic+transplantation+of+fresh+surgical+specimen+preserves+CD133++tumor+cells+in+clinically+relevant+mouse+models+of+medulloblastoma+and+glioma.">Direct orthotopic transplantation of fresh surgical specimen preserves CD133+ tumor cells in clinically relevant mouse models of medulloblastoma and glioma.</a> Stem Cells. 26, 1414-1424 (2008).</li><li>Suggitt, M., Bibby, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=50+years+of+preclinical+anticancer+drug+screening:+empirical+to+target-driven+approaches.">50 years of preclinical anticancer drug screening: empirical to target-driven approaches.</a> Clin. Cancer Res. 11, 971-981 (2005).</li><li>Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+tumor+xenografts+as+predictive+preclinical+models+for+anticancer+drug+activity+in+humans:+better+than+commonly+perceived-but+they+can+be+improved.">Human tumor xenografts as predictive preclinical models for anticancer drug activity in humans: better than commonly perceived-but they can be improved.</a> Cancer Biol. Ther. 2, 134-139 (2003).</li><li>Park, C. Y., Tseng, D., Weissman, I. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cell-directed+therapies:+recent+data+from+the+laboratory+and+clinic.">Cancer stem cell-directed therapies: recent data from the laboratory and clinic.</a> Mol. Ther. 17, 219-230 (2009).</li><li>Carlson, B. L., Pokorny, J. L., Schroeder, M. A., Sarkaria, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment,+maintenance+and+in+vitro+and+in+vivo+applications+of+primary+human+glioblastoma+multiforme+(GBM)+xenograft+models+for+translational+biology+studies+and+drug+discovery.">Establishment, maintenance and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery.</a> Curr. Protoc. Pharmacol. Chapter. 14, (2011).</li><li>Hambardzumyan, D., Parada, L. F., Holland, E. C., Charest, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+modeling+of+gliomas+in+mice:+new+tools+to+tackle+old+problems.">Genetic modeling of gliomas in mice: new tools to tackle old problems.</a> Glia. 59, 1155-1168 (2011).</li><li>Sarangi, 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=Targeted+inhibition+of+the+Hedgehog+pathway+in+established+malignant+glioma+xenografts+enhances+survival.">Targeted inhibition of the Hedgehog pathway in established malignant glioma xenografts enhances survival.</a> Oncogene. 28, 3468-3476 (2009).</li><li>Valadez, J. 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=Identification+of+Hedgehog+pathway+responsive+glioblastomas+by+isocitrate+dehydrogenase+mutation.">Identification of Hedgehog pathway responsive glioblastomas by isocitrate dehydrogenase mutation.</a> Cancer Lett. 328, 297-306 (2013).</li><li>Bar, E. 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=Cyclopamine-mediated+hedgehog+pathway+inhibition+depletes+stem-like+cancer+cells+in+glioblastoma.">Cyclopamine-mediated hedgehog pathway inhibition depletes stem-like cancer cells in glioblastoma.</a> Stem Cells. 25, 2524-2533 (2007).</li><li>Ehtesham, 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=Ligand-dependent+activation+of+the+hedgehog+pathway+in+glioma+progenitor+cells.">Ligand-dependent activation of the hedgehog pathway in glioma progenitor cells.</a> Oncogene. 26, 5752-5761 (2007).</li><li>Kelly, J. 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=Oligodendroglioma+cell+lines+containing+t(1;19)(q10;p10.">Oligodendroglioma cell lines containing t(1;19)(q10;p10.</a> Neuro-oncology. 12, 745-755 (2010).</li><li>Quintana, 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=Efficient+tumour+formation+by+single+human+melanoma+cells.">Efficient tumour formation by single human melanoma cells.</a> Nature. 456, 593-598 (2008).</li><li>Shultz, L. 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=Human+lymphoid+and+myeloid+cell+development+in+NOD/LtSz-scid+IL2R+gamma+null+mice+engrafted+with+mobilized+human+hemopoietic+stem+cells.">Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2R gamma null mice engrafted with mobilized human hemopoietic stem cells.</a> J. Immunol. 174, 6477-6489 (2005).</li><li>Singh, S. 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=Identification+of+human+brain+tumour+initiating+cells.">Identification of human brain tumour initiating cells.</a> Nature. 432, 396-401 (2004).</li></ol>

Cultured cell lines, xenografts and genetically engineered mice are the most common methods for modeling gliomas, and there are distinct benefits and limitations for each model system3,13,14. Relevant benefits of primary orthotopic glioma xenografts include infiltrative growth that typifies diffuse gliomas and the retention of genetic alterations and important signaling mechanisms that can be exceedingly difficult to maintain in cultured glioma cells. For example, isocitrate dehydrogenase mutations an...

Malignant gliomas are primary glial tumors of the central nervous system that occur in the brain and occasionally the spinal cord. Gliomas are classified by the World Health Organization (WHO) according to histologic resemblance to astrocytes, oligodendrocytes or ependymal cells and then numerically graded (I to IV) for pathologic features of malignancy. The most common histologic subtypes are astrocytomas, oligodendrogliomas and mixed oligoastrocytomas. Malignant gliomas encompassing WHO grades II to IV are characterized by invasive growth and recalcitrance to current therapies. Each year in the United States, approximately 15,750 individuals are diagnosed with a malignant glioma and an estimated 12,740 patients succumb to this disease. These statistics highlight the particularly lethal nature of malignant gliomas and important need for enhanced therapeutic efficacy.
Cancer models are essential for investigating tumor biology and therapies. Human cancer cell lines represent an important first step for in vitro manipulations and in vivo xenografting studies (secondary xenografts)1. However, standard cancer cell cultures undergo phenotypic and genotypic transformation2-4 that may not be restored in secondary xenografts5. Furthermore, genetic alterations such as isocitrate dehydrogenase (IDH) mutations6, distinct stem cell populations7 and dependency on key signaling pathways8 can be lost in cancer cell cultures. Genomic profiles can be maintained to better extent in cancer sphere culture, though still fail to mirror fully the genotype of primary tumors2,3. Direct orthotopic transplantation negates the influences of in vitro culture, provides a proper microenvironment, and preserves the integrity of tumor-initiating cells9,10. Therefore, primary xenografts represent a powerful and relevant preclinical model for rigorously testing targeted agents to aid in the rational design of future clinical trials5,11,12.

<table border="1" cellpadding="0" cellspacing="0">
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			Phosphate buffered saline
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			<td style="width:142px;">
			Life Technologies
			</td>
			<td style="width:140px;">
			14040-133
			</td>
			<td style="width:97px;">
			
			</td>
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		<tr>
			<td style="width:211px;">
			Papain dissociation system
			</td>
			<td style="width:142px;">
			Worthington Biochemical Corp.
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			<td style="width:140px;">
			LK003150
			</td>
			<td style="width:97px;">
			
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			<td style="width:211px;">
			Trypan blue solution 0.4%
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			<td style="width:142px;">
			Life Technologies
			</td>
			<td style="width:140px;">
			15250061
			</td>
			<td style="width:97px;">
			
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			Ketamine HCl
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			Obtained from institutional pharmacy or local veterinary supply company
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			Xylazine HCl
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			Ketoprofen
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			Ophthalmic ointment
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			Povidone-iodine
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			<td style="width:142px;">
			Fisher Scientific
			</td>
			<td style="width:140px;">
			190061617
			</td>
			<td style="width:97px;">
			
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			<td style="width:211px;">
			Cryopreservation medium and proliferation supplement
			</td>
			<td style="width:142px;">
			StemCell Technologies
			</td>
			<td style="width:140px;">
			05751
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			<td style="width:97px;">
			
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			<td style="width:211px;">
			0.2% Heparin sodium salt in PBS
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			<td style="width:142px;">
			StemCell Technologies
			</td>
			<td style="width:140px;">
			07980
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			<td style="width:97px;">
			
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			<td style="width:211px;">
			Penicillin-streptomycin
			</td>
			<td style="width:142px;">
			Life Technologies
			</td>
			<td style="width:140px;">
			15140-122
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			<td style="width:97px;">
			
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			Dimethyl sulfoxide
			</td>
			<td style="width:142px;">
			Sigma-Aldrich
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			<td style="width:140px;">
			D6250-5X10ML
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			<td style="width:97px;">
			
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			<td style="width:211px;">
			NOD.Cg-Prkdcscid I/2rgtm1Wjl/SzJ mice
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			<td style="width:142px;">
			The Jackson Laboratory
			</td>
			<td style="width:140px;">
			005557
			</td>
			<td style="width:97px;">
			NSG mice
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			<td style="width:211px;">
			Anti-human vimentin antibody
			</td>
			<td style="width:142px;">
			Dako
			</td>
			<td style="width:140px;">
			M7020
			</td>
			<td style="width:97px;">
			Use 1:200 to 1:800
			</td>
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			<td style="width:211px;">
			Anti-human IDH1 R132H antibody
			</td>
			<td style="width:142px;">
			Dianova
			</td>
			<td style="width:140px;">
			DIA-H09
			</td>
			<td style="width:97px;">
			Use 1:100 to 1:400
			</td>
		</tr>
	</tbody>
</table>




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			Catalogue Number
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			Comments
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			Centrifuge with swinging bucket rotor
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			Pipetter with dispensing speed control
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			<td style="width:140px;">
			
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			</td>
			<td style="width:97px;">
			
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			<td style="width:210px;">
			Disposable hemocytometer
			</td>
			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			22-600-100
			</td>
			<td style="width:97px;">
			
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			<td style="width:210px;">
			Sterile surgical gloves
			</td>
			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			11-388128
			</td>
			<td style="width:97px;">
			
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			<td style="width:210px;">
			Disposable gown
			</td>
			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			18-567
			</td>
			<td style="width:97px;">
			
			</td>
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			<td style="width:210px;">
			Surgical mask
			</td>
			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			19-120-1256
			</td>
			<td style="width:97px;">
			
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			<td style="width:210px;">
			Tuberculin syringe
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			<td style="width:140px;">
			BD
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			<td style="width:143px;">
			305620
			</td>
			<td style="width:97px;">
			
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			<td style="width:210px;">
			Alcohol pads
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			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			22-246-073
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			<td style="width:97px;">
			
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			<td style="width:210px;">
			Portable electronic scale
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			<td style="width:140px;">
			Fisher Scientific
			</td>
			<td style="width:143px;">
			01-919-33
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			<td style="width:97px;">
			
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			<td style="width:210px;">
			Zoom stereomicroscope
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			<td style="width:140px;">
			
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			Surgical clipper
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			<td style="width:140px;">
			Stoelting
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			<td style="width:143px;">
			51465
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			<td style="width:97px;">
			
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			<td style="width:210px;">
			Scalpel handle
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			<td style="width:140px;">
			Fine Science Tools
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			<td style="width:143px;">
			10003-12
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			<td style="width:97px;">
			
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			<td style="width:210px;">
			Scalpel blades, #10
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			<td style="width:140px;">
			
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			<td style="width:143px;">
			
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			<td style="width:97px;">
			
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			<td style="width:210px;">
			Stereotaxic instrument
			</td>
			<td style="width:140px;">
			Stoelting
			</td>
			<td style="width:143px;">
			51730
			</td>
			<td style="width:97px;">
			
			</td>
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			<td style="width:210px;">
			High-speed drill
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			<td style="width:140px;">
			Stoelting
			</td>
			<td style="width:143px;">
			51449
			</td>
			<td style="width:97px;">
			
			</td>
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			<td style="width:210px;">Drill bit, 0.6 mm</td>
			<td style="width:140px;">Stoelting</td>
			<td style="width:143px;">514552</td>
			<td style="width:97px;"></td>
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			<td style="width:210px;">
			Hamilton syringe
			</td>
			<td style="width:140px;">
			Hamilton
			</td>
			<td style="width:143px;">
			80336
			</td>
			<td style="width:97px;">
			
			</td>
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			<td style="width:210px;">
			Autoclip, 9 mm
			</td>
			<td style="width:140px;">
			BD
			</td>
			<td style="width:143px;">
			427630
			</td>
			<td style="width:97px;">
			
			</td>
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		<tr>
			<td style="width:210px;">
			Circulating water warming pad
			</td>
			<td style="width:140px;">
			Kent Scientific
			</td>
			<td style="width:143px;">
			TP-700
			TP-1215EA
			</td>
			<td style="width:97px;">
			
			</td>
		</tr>
		<tr>
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			Hot bead dry sterilizer
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			Kent Scientific
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			INS300850
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			14101-14
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			14094-11
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			15018-10
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			Dumont forceps
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			11251-30
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			Semimicro spatulas
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			Fisher Scientific
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			14374
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			Mouse brain slicer matrix
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			Zivic Instruments
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			BSMAS002-1
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			Fisher Scientific
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			12-567-501
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</table>

primary orthotopic glioma xenografts recapitulate infiltrative growth and isocitrate dehydrogenase i mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models. To model WHO grades III and IV malignant gliomas in transplantation assays, human tumor cells are xenografted into an orthotopic site, the brain, of immunocompromised mice. In contrast to secondary xenografts that utilize cultured tumor cells, human glioma cells are dissociated from resected specimens and transplanted without prior passage in tissue culture to generate primary xenografts. The procedure in this report details tumor sample preparation, intracranial transplantation into immunocompromised mice, monitoring for tumor engraftment and tumor harvesting for subsequent passage into recipient animals or analysis. Tumor cell preparation requires 2 hr and surgical procedure requires 20 min/animal.

Dissociated glioma cells are transplanted directly into the brains of immunocompromised mice to obtain primary orthotopic xenograft lines. Each tumor specimen is assigned a randomized number prior to transplantation, as part of the deidentification process to remove protected health information. We use a 5-digit REDcap database number for this purpose. Figure 1 illustrates the process and nomenclature for establishing a xenograft line from a glioblastoma (GBM 17182) with isocitrate dehydrogenase 1 (IDH1)...

Watch this Scientific Journal Video about Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation at JoVE.com

Primary Orthotopic Glioma Xenografts Recapitulate Infiltrative Growth and Isocitrate Dehydrogenase I Mutation

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary orthotopic xenografts recapitulate the histopathological and molecular features of malignant glioma subtypes in preclinical animal models.

Malignant gliomas constitute a heterogeneous group of highly infiltrative glial neoplasms with distinct clinical and molecular features. Primary ...

primary-orthotopic-glioma-xenografts-recapitulate-infiltrative-growth

Research

JoVE Journal

Medicine

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

Orthotopic Small Bowel Transplantation in Rats

Small bowel transplantation has become an accepted clinical option for patients with short gut syndrome and failure of parenteral nutrition (irreversible intestinal failure). In specialized centers improved operative and managing strategies have led to excellent short- and intermediate term patient and graft survival while providing high quality of life 1,3. Unlike in the more common transplantation of other solid organs (i.e. heart, liver) many underlying mechanisms of graft function and immunologic alterations induced by intestinal transplantation are not entirely known6,7. Episodes of acute rejection, sepsis and chronic graft failure are the main obstacles still contributing to less favorable long term outcome and hindering a more widespread employment of the procedure despite a growing number of patients on home parenteral nutrition who would potentially benefit from such a transplant. The small intestine contains a large number of passenger leucocytes commonly referred to as part of the gut associated lymphoid system (GALT) this being part of the reason for the high immunogenity of the intestinal graft. The presence and close proximity of many commensals and pathogens in the gut explains the severity of sepsis episodes once graft mucosal integrity is compromised (for example by rejection). To advance the field of intestinal- and multiorgan transplantation more data generated from reliable and feasible animal models is needed. The model provided herein combines both reliability and feasibility once established in a standardized manner and can provide valuable insight in the underlying complex molecular, cellular and functional mechanisms that are triggered by intestinal transplantation. We have successfully used and refined the described procedure over more than 5 years in our laboratory 8-11. The JoVE video-based format is especially useful to demonstrate the complex procedure and avoid initial pitfalls for groups planning to establish an orthotopic rodent model investigating intestinal transplantation.

Small bowel transplantation has become an accepted treatment option for patients with irreversible intestinal failure. Our experimental model of orthotopic small bowel transplantation in rats serves as a reliable tool to address underlying immunologic and inflammatory processes that complicate intestinal transplantation.

Small  bowel transplantation has become an accepted clinical option for patients with  short gut syndrome and failure of parenteral nutrition ...

Orthotopic Liver Transplantation in Rats

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat liver transplantation in 1979 by Kamada et al.3, this model has remained the gold standard despite various proposed alternative techniques4. Nevertheless, its broader use is limited by its steep learning curve5.
In this video paper, we show a simple and easy-to-establish revision of Kamada's two-cuff technique. The suprahepatic vena cava anastomosis is performed manually with a running suture, and the vena porta and infrahepatic vena cava anastomoses are performed utilizing a quick-linker cuff system6. Manufacturing the quick-linker kit is shown in a separate video paper.

We present an easy-to-establish revision of the classical two-cuff technique for orthotopic liver transplantation in rat.

Clinical progress in the field of liver transplantation has been largely supported by animal models1,2. Since the publication of the first orthotopic rat ...

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers patients only modest benefits, as most patients do not survive more than five years following diagnosis due to glioma relapse 1,2. The discovery of cancer stem cells in human brain tumors holds promise for having an enormous impact on the development of novel therapeutic strategies for glioma 3. Cancer stem cells are defined by their ability both to self-renew and to differentiate, and are thought to be the only cells in a tumor that have the capacity to initiate new tumors 4. Glioma relapse following radiation therapy is thought to arise from resistance of glioma stem cells (GSCs) to therapy 5-10. In vivo, GSCs are shown to reside in a perivascular niche that is important for maintaining their stem cell-like characteristics 11-14. Central to the organization of the GSC niche are vascular endothelial cells 12. Existing evidence suggests that GSCs and their interaction with the vascular endothelial cells are important for tumor development, and identify GSCs and their interaction with endothelial cells as important therapeutic targets for glioma. The presence of GSCs is determined experimentally by their capability to initiate new tumors upon orthotopic transplantation 15. This is typically achieved by injecting a specific number of GBM cells isolated from human tumors into the brains of severely immuno-deficient mice, or of mouse GBM cells into the brains of congenic host mice. Assays for tumor growth are then performed following sufficient time to allow GSCs among the injected GBM cells to give rise to new tumors-typically several weeks or months. Hence, existing assays do not allow examination of the important pathological process of tumor initiation from single GSCs in vivo. Consequently, essential insights into the specific roles of GSCs and their interaction with the vascular endothelial cells in the early stages of tumor initiation are lacking. Such insights are critical for developing novel therapeutic strategies for glioma, and will have great implications for preventing glioma relapse in patients. Here we have adapted the PoRTS cranial window procedure 16and in vivo two-photon microscopy to allow visualization of tumor initiation from injected GBM cells in the brain of a live mouse. Our technique will pave the way for future efforts to elucidate the key signaling mechanisms between GSCs and vascular endothelial cells during glioma initiation.

Imaging Glioma Initiation In Vivo Through a Polished and Reinforced Thin-skull Cranial Window

By combining a polished and reinforced thin-skull (PoRTS) cranial window and glioblastoma (GBM) cell injection, we can observe glioma initiation and growth from injected GBM cells in the brain of a live mouse longitudinally.

Glioma is the one of the most lethal forms of human cancer. The most effective glioma therapy to date-surgery followed by radiation treatment-offers ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Fetal Echocardiography and Pulsed-wave Doppler Ultrasound in a Rabbit Model of Intrauterine Growth Restriction

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ultrasound and fetal echocardiography. Increasingly, these imaging modalities are being employed clinically to examine cardiac function and assess wellbeing in utero, thereby guiding timing of birth decisions. Here, we used a rabbit model of IUGR that allows analysis of cardiac function in a clinically relevant way. Using isoflurane induced anesthesia, IUGR is surgically created at gestational age day 25 by performing a laparotomy, exposing the bicornuate uterus and then ligating 40-50% of uteroplacental vessels supplying each gestational sac in a single uterine horn. The other horn in the rabbit bicornuate uterus serves as internal control fetuses. Then, after recovery at gestational age day 30 (full term), the same rabbit undergoes examination of fetal cardiac function. Anesthesia is induced with ketamine and xylazine intramuscularly, then maintained by a continuous intravenous infusion of ketamine and xylazine to minimize iatrogenic effects on fetal cardiac function. A repeat laparotomy is performed to expose each gestational sac and a microultrasound examination (VisualSonics VEVO 2100) of fetal cardiac function is performed. Placental insufficiency is evident by a raised pulsatility index or an absent or reversed end diastolic flow of the umbilical artery Doppler waveform. The ductus venosus and middle cerebral artery Doppler is then examined. Fetal echocardiography is performed by recording B mode, M mode and flow velocity waveforms in lateral and apical views. Offline calculations determine standard M-mode cardiac variables, tricuspid and mitral annular plane systolic excursion, speckle tracking and strain analysis, modified myocardial performance index and vascular flow velocity waveforms of interest. This small animal model of IUGR therefore affords examination of in utero cardiac function that is consistent with current clinical practice and is therefore useful in a translational research setting.

We describe examination of fetal cardiac function with contemporary functional fetal echocardiography and fetoplacental Doppler ultrasound using the VisualSonics VEVO 2100 microultrasound in a surgically induced model of intrauterine fetal growth restriction in a rabbit.

Fetal intrauterine growth restriction (IUGR) results in abnormal cardiac function that is apparent antenatally due to advances in fetoplacental Doppler ...

Bioluminescent Orthotopic Model of Pancreatic Cancer Progression

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%. New therapeutic options are critically needed and depend on improved understanding of pancreatic cancer biology. To better understand the interaction of cancer cells with the pancreatic microenvironment, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression. Luciferase-tagged pancreatic cancer cells are resuspended in Matrigel and delivered into the pancreatic tail during laparotomy. Matrigel solidifies at body temperature to prevent leakage of cancer cells during injection. Primary tumor growth and metastasis to distant organs are monitored following injection of the luciferase substrate luciferin, using in vivo imaging of bioluminescence emission from the cancer cells. In vivo imaging also may be used to track primary tumor recurrence after resection. This orthotopic model is suited to both syngeneic and xenograft models and may be used in pre-clinical trials to investigate the impact of novel anti-cancer therapeutics on the growth of the primary pancreatic tumor and metastasis.

Improved understanding of pancreatic cancer biology is critically needed to enable the development of better therapeutic options to treat pancreatic cancer. To address this need, we demonstrate an orthotopic model of pancreatic cancer that permits non-invasive monitoring of cancer progression using in vivo bioluminescence imaging.

Pancreatic cancer has an extremely poor five-year survival rate of 4-6%.  New therapeutic options are critically needed and depend on improved ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...