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

Bu eser Robert Wood Johnson Vakfı/Amos tıbbi öğretim geliştirme programı, Taubman Araştırma Enstitüsü ve bölüm, çocuk cerrahisi, The University of Michigan destek aldı. Yazarlar Kimber'la Converso-Baran ve Dr. Marcus Jarboe yardımı ile ultrason enjeksiyon yordamlar ve görüntüleme platformu için teşekkür etmek istiyorum. Paul Trombley şekil grafik ile onun yardım için teşekkür. Ayrıca merkezi kullanım kısmen olarak kapsamlı Kanser Merkezi NIH tarafından desteklenen, tümörü Imaging Core, moleküler görüntüleme ve için için Radyoloji Bölümü, University of Michigan teşekkür P30 CA046592 verin. Üniversitenin NIH (OD016502) ve Frankel dolaşım merkezi hibe fon tarafından kısmen desteklenir Michigan fizyolojisi fenotipleme çekirdek. Hücre satır kimlik doğrulama IDEXX RADIL Bioresearch özellikleri, Columbia, Mo yapıldı Tammy Stoll, Dr Rajen Mody ve Mott Solid tümör Onkoloji Program teşekkür ederim. Bizim hastalar ve aileleri minnetle onların ilham, cesaret ve bizim araştırma devam eden destek için kabul vardır.

Tüm iş The University of Michigan Kurumsal değerlendirme Komitesi (HUM 00052430) uygun olarak yapıldığını ve kullanımı ve bakımı hayvanlar (UCUCA) üniversite Komitesi tarafından onaylanmış yordamlar için uygundur. Birim için laboratuvar, hayvan Tıp (ULAM) hayvan bakımı konusunda çalışmalar yaptı.
Tüm çalışma onayı ile University of Michigan kurumsal İnceleme Kurulu (HUM 00052430) yapıldı ve bütün insan Araştırma Etik Komitesi düzenlemeler için uygundur. ...

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Ultrason destekli implantasyon NB ve ES hücre kanser biyolojide preklinik çalışmaları için güvenilir fare xenografts kurmak için verimli ve güvenli bir yöntemdir. Ultrason destekli başarısı için kritik doku hedefli implantasyon varlığı ve uzmanlık anatomik olarak faiz organı yerelleştirme ve tümör hücrelerinin stereotaksik enjeksiyon ile eğitimli personel durumu olduğunu.
Tümör dokusunun ayrılma açıklanan hasta kaynaklı xenograft modeli geliştirmek çok önemli ...

Hayvan xenograft modelleri roman antikanser terapiler preklinik çalışmaları için gerekli araçları vardır. Subkutan kanat implantasyon hücrelerinin tümör büyümesini izlemek için bir verimli ve kolay erişilebilir sitesi sağlayan standart fare xenografts güveniyor. Subkutan modelleri1metastaz potansiyellerini sınırlayabilir tümör özgü biyolojik özellikleri onların eksikliği dezavantajdır. Gibi sınırlamalar tarafından hangi tümör hücreleri metastatik potansiyel2ile ilgili bir microenvironment sağlayan yerel doku sitelerdeki engrafted orthotopic xenografts kullanımı üstesinden vardır. Orthotopic xenograft modelleri orijinal biyolojik özellikleri korumak ve güvenilir modelleri preklinik ilaç bulma3,4için sağlar. Doku yönetmen implantasyon için kullanılan kanser hücrelerinin kurulan hücre hatları veya hasta kaynaklı hücrelerden hasta tümör vardır. Xenografts kanser hücre satırlarından kurulan hasta türetilmiş xenografts5' e göre birincil tümör üzerinden yüksek genetik sapma sergileyebilirler. Bu göz önüne alındığında, hasta kaynaklı orthotopic xenografts kurulması roman tedavi kanser ilaç bulma gelen test etmek için tercih edilen standart haline gelmiştir.
Pediatrik kanser Nöroblastom (NB), orthotopic xenograft modelleri birincil tümör biyolojisi özetlemek ve metastaz yaymak NB6,7tipik sitelere geliştirmek. NB böbreküstü bezi veya paravertebral sempatik zinciri boyunca gelişir. Orthotopic implantasyonu en yaygın yöntem açık trans-abdominal cerrahi işlemler gerektirir. Tür yöntemler genellikle sıkıcı, yüksek hayvan hastalık ve karmaşık kurtarma dönemleri yoktur. Yüksek çözünürlüklü ultrason son zamanlarda doku yönetmen implantasyonu tümör hücreleri kanser araştırma8,9için birkaç fare modelleri geliştirilmesi için kullanılmıştır. Güvenilir, tekrarlanabilir, verimli ve güvenli ilgili metastatik tümör xenografts10,11kurulması için tekniğidir.
Pediatrik kanser xenografts ultrason destekli hedef organ yerelleştirme ve iğne implantasyon hücre satırları ve hasta kaynaklı tümör hücreleri tarafından kurulması gösterdiği11olduğunu. Teknik fare böbreküstü bezinin hedef NB için kullanılmıştır. Ewing sarkomu (ES) ağırlıklı olarak uyluk kemiği ve pelvik kemik12gibi uzun kemiklerde yaygın olarak görülen bir kemik kanseri var. Olgu sunumları büyüme ağırlıklı olarak kemik kanseri böbrek dokusunda uygulanabilir olup olmadığını belirlemek için bir böbrek alt kapsüler yerde orthotopic implantasyon13için seçildi olduğunu göstermiştir. Tümör hücreleri böbrek alt kapsüler hücre implantasyonu spontan Metastazlari ES14için çalışmaya gelecek vaat eden bir model olarak kullanılmıştır.

<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

Preklinik antikanser terapiler test kanser doğuştan gelen eğilimleri taklit ilgili xenograft modellerde dayanır. Yordam kolaylığı ve yeteneği monitör tümör ilerleme ve yanıt invaziv görüntüleme olmadan standart subkutan kanat modelleri avantajları şunlardır. Modelleri gibi çoğu kez translasyonel klinik deneylere tutarsız ve yerel bir microenvironment eksikliği olduğu biyolojik ilgili özellikleri metastaz, üretmek için düşük meyil ile sınırlı. Buna karşılık, orthotopic xenograft modelleri yerli tümör sitelerdeki tümör microenvironment taklit ve uzak metastatik yaymak gibi önemli hastalık özellikleri çoğaltmak için gösterilmiştir. Bu modeller genellikle sıkıcı cerrahi anestezik zaman ve kurtarma takmaktan ile gerektirir. Bu sorunu çözmek için kanser araştırmacılar son zamanlarda kanser xenograft modeller için doku Yönetmen: fare modelleri hızlı ve güvenilir kurulması için sağlar preklinik deneyler kurmak için ultrason destekli enjeksiyon teknikleri kullanılan. Ultrason görselleştirme de boyuna değerlendirme tümör engraftment ve büyüme için invaziv bir yöntem sağlar. Burada, böbreküstü bezi NB ve böbrek alt kapsülü kullanmak için ES için kanser hücrelerinin ultrason destekli enjeksiyon yöntemi açıklanmaktadır. Bu minimal invaziv yaklaşım sıkıcı açık cerrahi implantasyon kanser hücrelerinin büyüme ve metastaz doku özel konumlarda üstesinden gelir ve marazi kurtarma dönemleri azaliyor. Biz kurulan hücre satırları ve hasta türetilmiş hücre satırları orthotopic enjeksiyon için kullanımını açıklar. Tümör ayrılma ve luciferase hücrelerinin etiketleme için önceden hazırlanmış ticari kitleri mevcuttur. Görüntü Kılavuzu kullanarak hücre süspansiyon enjeksiyon preklinik modellerinin oluşturulması için minimal invaziv ve tekrarlanabilir bir platform sağlar. Bu yöntem, mesane, karaciğer ve pankreas delinmemiş potansiyeline çok sayıda kanser modelleri için örnekli gibi diğer kanserler için güvenilir preklinik modelleri oluşturmak için kullanılmaktadır.

Sunulan yordamları kullanarak, ultrason destekli implantasyon NB hücrelerinin böbrek üstü bezi içine ısıtılmış bir cerrahi tablo ile donatılmış bir özel yordamın Oda yapıldı. Kol ve ayak pedleri fare kalp etkinlik (Şekil 1A) izlemek için yerleştirildi. Hayvan imzalat burun konisi inhalasyon kullanarak isoflurane altında kaldı. Bir yüksek çözünürlüklü ultrason sonda kullanarak, sol böbrek böbrek (Şekil 1B</...

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

Burada, biyolojik ilgili sitelerdeki kanser için güvenilir preklinik modelleri oluşturmak için bir protokol ultrason destekli enjeksiyon Nöroblastom (NB) ve Ewing sarkomu (ES) hücre yararlanmak için (hücre satırları ve hasta kaynaklı tümör hücreleri kurulu) mevcut araştırma.

Ultrason kullanımı doku Yönetmen: hücresel implantasyon biyolojik ilgili metastatik tümör Xenografts kurulması için güdümlü

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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