Name: utilization of ultrasound guided tissue-directed cellular implantation for the establishment of biologically relevant metastatic tumor xenografts
Uploaded: 2018-05-25T14:30:00
Description: Watch this Scientific Journal Video about Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts at JoVE.com

Dette arbejde fik støtte fra den Robert Wood Johnson Foundation/Amos medicinske fakultet udvikling Program, Taubman Research Institute, og afsnit af Pediatric kirurgi, The University of Michigan. Forfatterne vil gerne takke Kimber Converso-Baran og Dr. Marcus Jarboe bistand med ultralyd injektion procedurer og billedbehandling-platformen. Vi vil gerne takke Paul Trombley for hans hjælp med figur grafik. Vi takker også Institut for radiologi på The University of Michigan til brug af Center for Molekylær Imaging og Tumor Imaging kerne, som er delvist understøttet af omfattende Cancer Center NIH, give P30 CA046592. University of Michigan fysiologi fænotyper kerne, der er delvist understøttet af tilskud fra NIH (OD016502) og Frankel Cardiovascular Center. Celle linje godkendelse blev gjort på IDEXX RADIL Bioresearch faciliteter, Columbia, MO. Vi takker Tammy Stoll, Dr. Rajen Mody og Mott Solid Tumor onkologi Program. Vores patienter og familier er taknemmeligt anerkendt for deres inspiration, mod og løbende støtte til vores forskning.

Alt arbejde blev udført i overensstemmelse med The University of Michigan institutionelle Review Board (HUM 00052430) og er i overensstemmelse med procedurer, der er godkendt af universitetet Udvalget om brug og pleje af dyr (UCUCA). Enhed til laboratoriet af dyr medicin (ULAM) overvågede dyrs pleje.
Alt arbejde blev udført med godkendelse fra The University of Michigan institutionelle Review Board (HUM 00052430) og overholder alle menneskelige forskning etiske udvalg forordninger. Mennesk...

<ol>
	<li>Sanmamed, M. F., Chester, C., Melero, I., Kohrt, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+optimal+murine+models+to+investigate+immune+checkpoint+blockers+and+their+combination+with+other+immunotherapies.">Defining the optimal murine models to investigate immune checkpoint blockers and their combination with other immunotherapies.</a> Ann Oncol. 27 (7), 1190-1198 (2016).</li><li>Fidler, I. J., Hart, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+diversity+in+metastatic+neoplasms:+origins+and+implications.">Biological diversity in metastatic neoplasms: origins and implications.</a> Science. 217 (4564), 998-1003 (1982).</li><li>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=Orthotopic+models+of+cancer+for+preclinical+drug+evaluation.">Orthotopic models of cancer for preclinical drug evaluation.</a> Eur J Cancer. 40 (6), 852-857 (2004).</li><li>Killion, J. J., Radinsky, R., Fidler, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Orthotopic+Models+are+Necessary+to+Predict+Therapy+of+Transplantable+Tumors+in+Mice.">Orthotopic Models are Necessary to Predict Therapy of Transplantable Tumors in Mice.</a> Cancer Metastasis Rev. 17 (3), 279-284 (1998).</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 (8), 3364-3373 (2009).</li><li>Khanna, C., Jaboin, J. J., Drakos, E., Tsokos, M., Thiele, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biologically+relevant+orthotopic+neuroblastoma+xenograft+models:+Primary+adrenal+tumor+growth+and+spontaneous+distant+metastasis.">Biologically relevant orthotopic neuroblastoma xenograft models: Primary adrenal tumor growth and spontaneous distant metastasis.</a> In Vivo. 16 (2), 77-85 (2002).</li><li>Stewart, 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=Development+and+characterization+of+a+human+orthotopic+neuroblastoma+xenograft.">Development and characterization of a human orthotopic neuroblastoma xenograft.</a> Dev Biol. 407, 344-355 (2015).</li><li>J&#228;ger, W., 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=Minimally+Invasive+Establishment+of+Murine+Orthotopic+Bladder+Xenografts.">Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts.</a> J. Vis. Exp. (84), e51123 (2014).</li><li>Teitz, T., 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=Preclinical+Models+for+Neuroblastoma:+Establishing+a+Baseline+for+Treatment.">Preclinical Models for Neuroblastoma: Establishing a Baseline for Treatment.</a> PLoS ONE. 6 (4), e19133 (2011).</li><li>Braekeveldt, N., 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=Neuroblastoma+patient-derived+orthotopic+xenografts+retain+metastatic+patterns+and+geno-+and+phenotypes+of+patient+tumours.">Neuroblastoma patient-derived orthotopic xenografts retain metastatic patterns and geno- and phenotypes of patient tumours.</a> International Journal of Cancer. 136 (5), 252-261 (2015).</li><li>Van Noord, R. 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=Tissue-directed+Implantation+Using+Ultrasound+Visualization+for+Development+of+Biologically+Relevant+Metastatic+Tumor+Xenografts.">Tissue-directed Implantation Using Ultrasound Visualization for Development of Biologically Relevant Metastatic Tumor Xenografts.</a> In Vivo. 31 (5), 779-791 (2017).</li><li>Vormoor, 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=Development+of+a+Preclinical+Orthotopic+Xenograft+Model+of+Ewing+Sarcoma+and+Other+Human+Malignant+Bone+Disease+Using+Advanced+In+Vivo+Imaging.">Development of a Preclinical Orthotopic Xenograft Model of Ewing Sarcoma and Other Human Malignant Bone Disease Using Advanced In Vivo Imaging.</a> PLoS ONE. 9 (1), e85128 (2014).</li><li>Hakky, T. S., Gonzalvo, A. A., Lockhart, J. L., Rodriguez, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+Ewing+sarcoma+of+the+kidney:+a+symptomatic+presentation+and+review+of+the+literature.">Primary Ewing sarcoma of the kidney: a symptomatic presentation and review of the literature.</a> Ther Adv Urol. 5 (3), 153-159 (2013).</li><li>Cheng, H., Clarkson, P. W., Gao, D., Pacheco, M., Wang, Y., Nielsen, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+Antibodies+Targeting+CSF1+Impede+Macrophage+Recruitment+in+a+Xenograft+Model+of+Tenosynovial+Giant+Cell+Tumor.">Therapeutic Antibodies Targeting CSF1 Impede Macrophage Recruitment in a Xenograft Model of Tenosynovial Giant Cell Tumor.</a> Sarcoma. 2010, 174528 (2010).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+Hemacytometer+to+Count+Cells.">Using a Hemacytometer to Count Cells.</a> Basic Methods in Cellular and Molecular Biology. , (2018).</li></ol>

Ultralydsvejledt implantation af NB og ES-celler er en effektiv og sikker metode til at oprette pålidelige murine xenografts for prækliniske undersøgelser i kræft biologi. Afgørende for succes af ultralydsvejledt væv-målrettet implantation er tilstedeværelsen og tilgængeligheden af uddannet personale med ekspertise i anatomisk lokalisering orgel af interesse og stereotactic injektion af tumorceller.
Dissociation af tumor væv viste sig for at være et afgørende skridt i udviklingen a...

Animalske xenograft modeller er uundværlige redskaber til prækliniske undersøgelser af roman anticancer behandlingsformer. Standard murine xenografts stole på subkutan flanke implantation af celler, giver en effektiv og let tilgængelig hjemmeside overvågning tumorvækst. Ulempen ved subkutan modeller er deres manglende tumor-specifikke biologiske karakteristika, som kan begrænse deres potentiale til at metastaserer1. Sådanne begrænsninger er overvundet ved hjælp af orthotopic xenografts i hvilke tumor celler er aflægger på native væv websteder, giver en relevant mikromiljø med metastatisk potentielle2. Orthotopic xenograft modeller bevare oprindelige biologiske funktioner og give pålidelige modeller for prækliniske drug discovery3,4. Kræftcellerne udnyttet til væv-instrueret implantation er enten etablerede cellelinier eller patient-afledte celler fra patientens tumorer. Xenografts etableret fra kræft cellelinjer kan udstille høj genetisk afvigelse fra den primære tumor i forhold til patientens afledte xenografts5. Da dette, er etablering af patient-afledt orthotopic xenografts blevet den foretrukne standard for afprøvning roman therapeutics i kræft drug discovery.
I pædiatriske kræft neuroblastoma (NB), orthotopic xenograft modeller sammenfatte primær tumor biologi og udvikle metastaser til typiske lokaliteter af NB sprede6,7. NB udvikler i binyrerne eller langs paravertebral sympatisk kæde. De mest almindelige metoder til orthotopic implantation kræver åben trans-abdominal kirurgiske procedurer. Sådanne metoder er ofte kedelige, høj animalsk sygelighed og komplekse fredninger. Høj opløsning ultralyd er for nylig blevet udnyttet til væv-instrueret implantation af tumorceller i udviklingen af flere murine modeller for kræft forskning8,9. Teknikken er pålidelige, reproducerbar, effektiv og sikker for etablering af relevante metastatisk tumor xenografts10,11.
Etablering af pediatric cancer xenografts ved ultralyd-styrede målet orgel lokalisering og nål implantation af cellelinjer og patient-afledte tumorceller er påvist11. Teknikken blev udnyttet til NB målrettet mod murine binyrerne. Ewings sarkom (ES) er overvejende en ossøse kræft, almindeligvis ses i de lange knogler som femur og bækken knogler12. Case rapporter har vist, for at afgøre om vækst af en overvejende ossøse kræft er muligt i nyre væv, en nyre sub kapsulær placering blev valgt til orthotopic implantation13. Renal sub kapsulær celle implantation af tumorceller har været udnyttet som en lovende model til at studere spontan metastaser til ES14.

<table border="0" cellpadding="0" cellspacing="0" width="1053">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Mice</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td style="width:295px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NOD-SCID</td>
			<td>Charles River</td>
			<td>394</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NSG</td>
			<td>The Jackson Laboratory</td>
			<td>5557</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cell Line&#160;</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">NB</td>
			<td style="width:231px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">IMR-32</td>
			<td style="width:231px;">ATCC</td>
			<td>CCL-127</td>
			<td>Established human neuroblastoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">SH-SY5Y</td>
			<td style="width:231px;">ATCC</td>
			<td style="width:232px;">CRL-2266</td>
			<td>Established human neuroblastoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">SK-N-Be2</td>
			<td style="width:231px;">ATCC</td>
			<td style="width:232px;">CRL-2271</td>
			<td>Established human neuroblastoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">ES</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TC32&#160;</td>
			<td style="width:231px;">COGcell.ORG</td>
			<td style="width:232px;"></td>
			<td>Established human Ewing&#39;s Sarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">A673</td>
			<td style="width:231px;">COGcell.ORG</td>
			<td style="width:232px;"></td>
			<td>Established human Ewing&#39;s Sarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CHLA-25</td>
			<td style="width:231px;">COGcell.ORG</td>
			<td style="width:232px;"></td>
			<td>Established human Ewing&#39;s Sarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">A4573</td>
			<td style="width:231px;">COGcell.ORG</td>
			<td style="width:232px;"></td>
			<td>Established human Ewing&#39;s Sarcoma cell line</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cell Line media</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">RPMI</td>
			<td style="width:231px;">Life Technologies</td>
			<td style="width:232px;">11875-093</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Matrigel</td>
			<td style="width:231px;">BD BioSciences</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Dissociation</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Dissection Tools</td>
			<td style="width:231px;">KentScientific</td>
			<td style="width:232px;">INSMOUSEKIT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Human Tumor Dissociation Kit&#160;</td>
			<td style="width:231px;">MACS Miltenyi Biotec</td>
			<td style="width:232px;">130-095-929</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">gentleMACS dissociator</td>
			<td style="width:231px;">MACS Miltenyi Biotec</td>
			<td>130-093-235</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">gentleMACS C tubes</td>
			<td style="width:231px;">MACS Miltenyi Biotec</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Cell Strainer</td>
			<td style="width:231px;">Corning</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Luciferase Tagging</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Lenti-GFP1 virus</td>
			<td style="width:231px;">University of Michigan, Vector Core</td>
			<td style="width:232px;"></td>
			<td>Luciferase Virus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Steady Glo-Luciferase Assay Kit</td>
			<td style="width:231px;">Promega</td>
			<td>E2510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Bioluminescence Imaging</td>
			<td style="width:231px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Ivis Spectrum Imaging System</td>
			<td style="width:231px;">PerkinElmer</td>
			<td>124262</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">D-Luciferin</td>
			<td style="width:231px;">Promega</td>
			<td>E160X</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Anesthetic</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Inhaled Isoflurane&#160;</td>
			<td style="width:231px;">Piramal Critical Care Inc</td>
			<td style="width:232px;">66794-0017-25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Ultrasound Guided Injection</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Vevo 2100 High Resolution Imaging</td>
			<td style="width:231px;">Vevo</td>
			<td style="width:232px;">2100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Hamilton Syringes (27 gauge needle)</td>
			<td style="width:231px;">Hamilton</td>
			<td style="width:232px;">80000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">22 Gauge Angiocatheter</td>
			<td style="width:231px;">BD Biosciences</td>
			<td style="width:232px;">381423</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Optical ointment</td>
			<td style="width:231px;">Major Pharmaceuticals</td>
			<td style="width:232px;">301909</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Nair</td>
			<td style="width:231px;">Church &#38; Dwight Co</td>
			<td style="width:232px;"></td>
			<td style="width:295px;">Hair Removal agent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">Aquasonic 100 Ultrasound Transmission gel</td>
			<td style="width:231px;">Parker</td>
			<td style="width:232px;"></td>
			<td>Ultrasound gel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Histology</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">CD99</td>
			<td style="width:231px;">DAKO</td>
			<td>M3601</td>
			<td>Primary Antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Tyrosine Hydroxylase</td>
			<td style="width:231px;">Sigma-Aldrich</td>
			<td>T2928</td>
			<td>Primary Antibody</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Secondary HRP-Polymer antibody</td>
			<td style="width:231px;">Biocare</td>
			<td>BRR4056KG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Miscelleneous</td>
			<td style="width:231px;"></td>
			<td style="width:232px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">10 mL Pipettes</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">13-676-10J</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">5 mL Pipettes</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">13-676-10H</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">1.5 mL Microcentrifuge tubes</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">P1000 pipette</td>
			<td style="width:231px;">Eppendorf</td>
			<td style="width:232px;">3120000062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">P200 pipette</td>
			<td style="width:231px;">Eppendorf</td>
			<td style="width:232px;">3120000054</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">P1000 pipette tips</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">21-375E</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">P200 pipette tips</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">21-375D</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Portable pipette aid</td>
			<td style="width:231px;">Drummond</td>
			<td>4-000-101</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">digital animal Weighing Scale&#160;</td>
			<td style="width:231px;">KentScientific</td>
			<td style="width:232px;">SCL-1015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Calipers</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">06-664-16</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">6well low attachment plates</td>
			<td style="width:231px;">Corning</td>
			<td style="width:232px;">07-200-601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">10 cm Tissue Culture Treated Dishes</td>
			<td style="width:231px;">Fisher Scientific</td>
			<td style="width:232px;">FB012924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">Polybrene</td>
			<td style="width:231px;">Sigma-Aldrich</td>
			<td style="width:232px;">TR-1003-G</td>
			<td></td>
		</tr>
	</tbody>
</table>

utilization of ultrasound guided tissue-directed cellular implantation for the establishment of biologically relevant metastatic tumor xenografts

Præklinisk afprøvning af anticancer behandlingsformer bygger på relevante xenograft modeller, der efterligner de medfødte tendenser af kræft. Fordele ved standard subkutane flanke modeller omfatter proceduremæssige lethed og evnen til at overvåge tumor progression og svar uden invasive billeddannelse. Sådanne modeller er ofte uforenelige i translationel kliniske forsøg og har begrænset biologisk relevante egenskaber med lav hang til at producere metastase, da der mangler en native mikromiljø. I sammenligning, har orthotopic xenograft modeller på native tumor websteder vist sig at efterligne tumor mikromiljø og kopiere vigtige sygdomskendetegn som metastatisk spredning. Disse modeller kræver ofte trættende kirurgiske procedurer med langvarig bedøvelsesmiddel tid og recovery perioder. For at løse dette, har kræftforskere for nylig udnyttet ultralydsvejledt injektion teknikker for at etablere kræft xenograft modeller for prækliniske forsøg, som giver mulighed for hurtig og pålidelig etablering af væv-instrueret murine modeller. Ultralyd visualisering giver også en invasiv metode for langsgående vurdering af tumor engraftment og vækst. Her, beskrive vi metoden til ultralydsvejledt injektion af kræftceller, udnytter binyrerne for NB og renal sub kapsel til ES. Denne minimalt invasiv metode overvinder kedelig åben kirurgi implantation af kræftceller i væv-specifikke steder for vækst og metastase, og aftager morbid fredninger. Vi beskriver udnyttelse af både etablerede cellelinier og patient afledte cellelinjer til orthotopic injektion. Pre-Made kommercielle kits er tilgængelige for tumor dissociation og luciferase tagging af celler. Injektion af cellesuspension ved hjælp af billede-vejledning giver et minimalt invasive og reproducerbare platform for oprettelsen af prækliniske modeller. Denne metode er udnyttet til at skabe pålidelige prækliniske modeller for andre kræftformer såsom blære, leveren og bugspytkirtlen eksemplificere sin uudnyttet potentiale for mange cancer modeller.

Anvendelse af de procedurer, der er fremlagt, blev ultralydsvejledt implantering af NB celler i binyrerne gjort i en dedikeret procedure værelse udstyret med en opvarmet kirurgisk bord. Arm og mund puder var lagt til overvågning af murine hjertet aktivitet (figur 1A). Dyret forblev bedøvede under isofluran ved hjælp af næsen kegle indånding. Ved hjælp af en høj opløsning ultralydssonde, blev venstre nyre identificeret med binyrerne bare kranie til ny...

Watch this Scientific Journal Video about Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts at JoVE.com

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Vi præsenterer her, en protokol for at udnytte ultralydsvejledt injektion af neuroblastoma (NB) og Ewings sarkom (ES) celler (etableret cellelinjer og patient-afledte tumorceller) på biologisk relevante websteder til at oprette pålidelige prækliniske modeller for kræft forskning.

Udnyttelse af ultralyd guidede væv-instrueret cellulære Implantation for etablering af biologisk relevante metastatisk Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

The goal of this method is to establish biologically relevant cancer orthotopic xenografts for preclinical studies. Patient-derived neuroblastoma cells are injected into the murine adrenal gland by ultrasound guidance without open surgery or prolonged recovery. This method can help answer critical questions in cancer biology and in translational research, such as studies of tumor evolution, therapeutic responses in the native microenvironment and metastases.Such a knowledge would greatly improve the reliability of preclinical studies and facilitate drug discovery. The main advantage of this technique is that it is patient-derived, tissue-directed, efficient, and reliable in producing an orthotopic xenographed model for cancer therapy research. Demonstrating the critical steps in the procedure will be Sahiti Chukkapalli, a senior technician and our lab manager, along with Tina Thomas, a research fellow in our laboratory.Generate a single, patient-derived cancer cell suspension from tumor tissue using a tumor dissociation kit. Begin by transferring approximately half a gram of tumor tissue to a 100 millimeter cell culture dish containing five milliliters of enzyme supplemented RPMI buffer. Then, use scissors and tissue forceps to mince the tumor into small pieces of two to four millimeters.Pipette the tumor mixture into a dissociater tube and close the tube. Invert the tube, attach onto the sleeve of a tissue dissociater, and dissociate the tissue using the appropriate program. After dissociating, incubate the tumor cell suspension on a rotating rack at 37 degrees Celsius for one hour.Triturate the cell suspension every 15 minutes during the incubation. Following the incubation, transfer the cell suspension to a new 50 milliliter conical tube and add 10 milliliters of RPMI. Then, centrifuge the cell suspension at 314g for five minutes.After the centrifugation, remove the supernatant and suspend the pellet in five milliliters of RPMI. Pass the cell suspension through a 40 micron cell strainer and collect the strained solution into a fresh, 50 milliliter tube. After washing the strainer with five milliliters of RPMI media, centrifuge the cell suspension at 314g for five minutes to collect a pellet.Count the cells using a hemocytometer. Then, suspend the pellet in RPMI to obtain a final concentration of four times 10 to the five cells per 10 microliter volume. Transfer five microliters of this cell suspension per injection to a fresh tube, and the same volume of basement membrane matrix to make 10 microliters of cell solution for each injection and place it on ice.Transfer the mouse to the imaging platform with the abdominal side down. Fit and secure the nose cone to maintain isoflurane anesthesia. Begin the implantation procedure using hair removal lotion and a shaver to depilate the back and flank of a properly anesthetized six to eight week old immune-deficient NSG mouse.Apply optical ointment over the animal's eyes to prevent drying. Then, tape the mouse in place to prevent any inadvertent movement. Next, use ultrasound visualization to identify the murine liver, vena cava, spleen, left kidney, and adjacent left adrenal gland.Load a chilled Hamilton syringe, fitted with a small bore needle, with 10 microliters of cell solution. Then, under ultrasound guidance, gently inserted a chilled 22-gauge catheter through the skin and back muscle, directly into the left adrenal gland to provide a channel for cellular injection. Remove the needle and leave the catheter in place.Visualization of the adrenal gland throughout the insertion of the catheter, along with the needle and its trajectory, is imperative to ensure minimal injury to surrounding structures and organs and lower murine morbidity. Then, guide the syringe through the catheter positioned into the center of the adrenal gland. As the syringe is guided through the catheter, it is important to maintain catheter stability and watch the needle advance.Note that the needle itself extends approximately two millimeters beyond termination of the catheter, its position easily seen on ultrasound. Inject the cells into the targeted adrenal tissue. Leave the needle in place for one to two minutes to allow the basement membrane matrix to set.After the basement membrane matrix has set, slowly remove the needle, followed by the removal of the catheter. Finally, place the mouse into a recovery cage until it regains sufficient consciousness to maintain sternal recumbency before returning to the home cage. Ultrasound imaging monitors in vivo tumor progression.This image shows an ultrasound image of the adrenal gland, one week following injection. Here, luminescence imaging of a neuroblastoma injected mouse one week post-injection, shows readings not indicative of tumor engraftment. After two weeks, ultrasound imaging shows tumor engraftment and progression to an area of approximately 40 square millimeters.Bioluminescence at two weeks post-injection showed radiance measuring 10 to the seventh, which is suggestive of cell uptake and tumor growth correlating with ultrasound findings. Ultrasound imaging eight weeks post-injection showed continued tumor growth with an area measurement of greater than 100 square millimeters. Again, bioluminescence confirmed the ultrasound findings with increased radiance levels of 10 to the nine.3-D ultrasound imaging of the tumor showed a volume greater than 100 cubic millimeters, the baseline that our laboratory utilizes for initiation of preclinical therapeutic trials. Excised tumor grossly measured greater than one centimeter in size, correlating with ultrasound measurements and luminescence signals. This media provides demonstration of how to successfully establish adrenal orthotopic xenographs with patient-derived neuroblastoma cells, utilizing ultrasound guidance.Once mastered, this technique can be done in less than 12 minutes per injection if it is performed properly.

Research

JoVE Journal

Cancer Research

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