A.S. was partially supported by grants from the National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research (5R03DE021741-02) and the National Cancer Institute (1K08CA154963-01A1).&#160;
The Howard Hughes Medical Institute provided funding for M.L. through the HHMI Medical Research Fellows Program.

1. Egg Incubation <ol><li> Erhalten Sie 8 Tage alt spezifischen pathogenfreien befruchteten Eiern. </li><li> Zeigen Eier in rotierenden Eierablage, gestempelt Enden nach oben zeigen, und setzen Sie den Drehkorb in einem Ei-Inkubator. Eier Inkubieren für 48 Stunden bei 36 ° C und 50% Luftfeuchtigkeit. </li></ol> 2. Löschen Sie die CAM und Öffnen der Eier <ol><li> Sammeln Eier aus Inkubator. Platz Eier im Eierregal, gestempelt Seite nach oben und bringen Sie zu Sterilbank. </li><li> Schalten Sie die Zimmer und Kapuze Lichter. </li><li> Halten Sie die gestempelt Ende Ei leicht, um das Ei Candler, um die Gefäßsystem des CAM sowie die Luftsack freizulegen. HINWEIS: Das Ei candler ist ein verwendet, um den sich entwickelnden Embryo und zugehörigen Luftsack und Gefäßsystem zu visualisieren Lichtquelle. </li><li> Legen Sie einen Bleistiftstrich zwischen zwei großen Blutgefäße. </li><li> Schalten Sie das Licht und mit einem sterilen Push-Pin, um ein Loch an der Spitze des Eis über dem Luftsack (gestempelt Ende) und ein Loch an der pe machenNciI Marke. Schieben Sie die Push-Pin den ganzen Weg durch; ein Loch ca. 3 mm tief üblicherweise ausreichend. </li><li> Schalte das Licht aus. Squeeze Sicherheitskugel Drücken der offenen Ende fest gegen das Loch über dem Luftsack. Absaugen, um Luft in das Loch an der Bleistiftstrich zu ziehen, wodurch der CAM an der Bleistiftstrich weg von der Schale fallen. </li><li> Prüfen, ob der Luftsack aus dem Stanz Ende des Eis auf den Bleistift markierte Loch mit der candler bewegt. Wenn nicht, verwenden Sie die Push-Pin beide Löcher etwas tiefer zu machen und dann erneut bewerben Saug mit der Sicherheitskugel. </li><li> Schalten Sie das Licht. Mit dem Ei in einer Hand, schalten Sie den Dremel Rotationswerkzeug mit einer 15/16-Zoll-Trennscheibe angebracht und stellen zwei Querschnitte nur tief genug, um durch die Schale geschnitten, aber nicht tief genug, um den ganzen Weg hinunter zu der gedrückten geschnitten CAM. Stellen die Schnitte etwa 2 cm Länge und 1 cm voneinander entfernt sind. Nehmen Sie die Schnitte breit genug ist, so dass ein Silikonring 1 cm Durchmesser passieren kannaber dünner als die Breite der Standard-Klebeband. </li><li> Als nächstes stellen Sie 1 Längsschnitt zwischen und an den Enden der beiden Querschnitte durch leichtes Berühren der Trennscheibe auf die Schale. </li><li> Schieben einer sterilen Pinzette unter dem Mantelteil und parallel zur Schale. Besorgen Sie sich die Shell-Stück mit der Zange und das ganze Stück sauber zu entfernen. </li><li> Legen Sie Klebeband über dem neu geöffneten Fenster, so dass Sie sicher, dass an einem Ende zum leichteren Entfernen fach über sich selbst. </li><li> Die Eier zurück in Inkubator in der Eierschale (in den sich drehenden Eies Racks) bis zum Zeitpunkt der Impfung. </li></ol> 3. Impfung <ol><li> Halten Basalmembranprotein Gemisch, wie Matrigel, auf Eis in der Haube (um die Polymerisation zu verhindern) und Ort. </li><li> Verwenden 2 mM EDTA, um Zellen zu trennen (4-5 ml pro T75-Flasche) und bringe Kolben für 5 Minuten in Gewebekultur-Inkubator. </li><li> Resuspendieren der abgelösten Zellen in Medien und rechnen mit einer Zählkammer und Trypanblau. Verwenden Sie zwischen 500.000 to 2.000.000 Zellen zum Pfropfen auf dem CAM, je nach Zelllinie. Optimieren Sie die richtige Zahl in Vorversuchen. </li><li> Maßnahme Volumen für geeignete Zellzahl und Zentrifuge bei 500 xg für 5 min benötigt. </li><li> Überstand verwerfen und dann resuspendieren Zellpellet in 40 μ l PBS ++ und 20 μ l der Basalmembran Mischung. HINWEIS: An diesem Punkt können die Zellen mit Arzneimitteln oder anderen Testmitteln für Experimente benötigt wird durch Resuspendieren in die Zelle / Basalmembran Gemisch Suspension behandelt. Alternativ können Mittel auf die Zelle / Basalmembran Mischung Suspension nach der Transplantation pipettiert werden. </li><li> Abrufen Eierregal mit Eier aus dem Inkubator und Platz in der Sterilbank. </li><li> Peel off Band des Fensters, rufen Sie eine 1 mm Silikonring von der Kappe eines kryogenen Fläschchen mit sterilisierten Pinzette und legen Sie den Ring durch das Fenster. </li><li> Pipettieren Sie die 60 μ l Zell Lösung von PBS ++ und Basalmembran Mixture direkt in das Zentrum des Silikonring auf der CAM ruht. </li><li> Re-Band das Fenster und verschieben Eierablage zurück zum Inkubator. An dieser Stelle seien Sie vorsichtig beim Bewegen der Eier als dem Ring und der Zellsuspension kann leicht verschoben werden. </li><li> Ermöglichen Tumoren, für 5-7 Tage wachsen gelassen. </li></ol> 4. Ernten Tumoren <ol><li> Zeigen under-Pad in der Haube. Vorbereitung 35 x 10 mm Platten und Paraformaldehyd als Container für die Histologie notwendig. Setzen Sie 1 ml PBS ++ je 35 x 10 mm Platte. </li><li> Zeigen BIOHAZARD Beutel in der Haube. Nehmen sterile Dissektion Schere und legen Sie das spitze Ende in das Ei in das Loch in der Nähe des Ende gestempelt. </li><li> Um den gesamten Ei längs geschnitten. Hinweis: Die CAM und der Tumor mit dem Ring sollte auf die obere Hälfte der Schale mit dem Fenster zu halten. </li><li> Verwendung Dissektion Schere und Pinzette, sezieren den Tumor von der CAM und legen Tumor in der 35 x 10 mm Platte. Messen und wiegen Tumoren. Festen Tumoren in paraformaldehyde und sie für die Verwendung in der Histologie und Immunhistochemie Experimenten in Paraffin eingebettet werden. </li></ol> 5. (Optional): Tumor Zelldissoziation <ol><li> Verwenden Sie Dissektion Schere, um den Tumor in der 35 x 10 mm Platten Blatt vor den Mund. Pour Hackfleisch Tumor in ein 15 ml Zentrifugenrohr entfernen und so viel PBS ++ wie möglich durch Pipettieren. </li><li> 2 ml Collagenase (1 mg / ml in PBS ++) biegen Rohr über mehrere Male, und inkubieren Rohr bei 37 ° C für mindestens 30 min (bis zu mehrere Stunden). </li><li> 5 ml Medium in jedes Röhrchen und gründlich resuspendieren durch Auf- und Abpipettieren 20 - 40x. Gründliche Pipettieren ist entscheidend für die komplette Zelle Dissoziation. </li><li> Erlauben Sediment zu begleichen, und den Überstand in ein frisches Röhrchen. Zentrifuge bei 500 × g für 5 min. Überstand entfernen und hinterlässt dissoziierten Zellpellet. HINWEIS: An diesem Punkt kann die Zellpellets für verschiedene Experimente verwendet werden (Durchflusszytometrie Lyse für die Verwendung in Western Blots, die RNA-Isolierung, usw.). Gesamttumorzellzahl kann auch mit einer Zählkammer und Trypanblaufärbung erhalten werden. Beachten, dass kleinere, oval geformte Zellen Hühnerzellen aus dem CAM und sollten nicht als Tumorzellen gezählt. Es sollte eine minimale Anzahl von verunreinigenden Nicht-Tumor (CAM-Zellen), wenn der Tumor vom CAM sorgfältig seziert. </li></ol>

<ol>
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M., Burroughs, A., Bruix, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatocellular+carcinoma.">Hepatocellular carcinoma.</a> Lancet. 362 (9399), 1907-1917 (2003).</li><li>Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+the+evidence+that+tumors+are+angiogenesis+dependent.">What is the evidence that tumors are angiogenesis dependent.</a> J Natl Cancer Inst. 82, 4-6 (1990).</li><li>Hagedorn, 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=Accessing+key+steps+of+human+tumor+progression+in+vivo+by+using+an+avian+embryo+model.">Accessing key steps of human tumor progression in vivo by using an avian embryo model.</a> Proc Natl Acad Sci U S A. 102 (5), 1643-1648 (2005).</li><li>Dumartin, L., 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=Netrin-1+mediates+early+events+in+pancreatic+adenocarcinoma+progression,+acting+on+tumor+and+endothelial+cells.">Netrin-1 mediates early events in pancreatic adenocarcinoma progression, acting on tumor and endothelial cells.</a> Gastroenterology. 138 (4), 1595-1606 (2010).</li><li>Aguirre-Ghiso, J. A., Estrada, Y., Liu, D., Ossowski, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ERK(MAPK)+activity+as+a+determinant+of+tumor+growth+and+dormancy;+regulation+by+p38(SAPK).">ERK(MAPK) activity as a determinant of tumor growth and dormancy; regulation by p38(SAPK).</a> Cancer Res. 63 (7), 1684-1695 (2003).</li><li>Baroni, T. 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=Ribonomic+and+short+hairpin+RNA+gene+silencing+methods+to+explore+functional+gene+programs+associated+with+tumor+growth+arrest.">Ribonomic and short hairpin RNA gene silencing methods to explore functional gene programs associated with tumor growth arrest.</a> Methods Mol Biol. 383, 227-244 (2007).</li><li>Lopez-Rivera, 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=Inducible+nitric+oxide+synthase+drives+mTOR+pathway+activation+and+proliferation+of+human+melanoma+by+reversible+nitrosylation+of+TSC2.">Inducible nitric oxide synthase drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2.</a> Cancer Res. 74 (4), 1067-1078 (2014).</li><li>Balke, 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=A+short-term+in+vivo+model+for+giant+cell+tumor+of+bone.">A short-term in vivo model for giant cell tumor of bone.</a> BMC Cancer. 11, 241 (2011).</li><li>Balke, 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=Morphologic+characterization+of+osteosarcoma+growth+on+the+chick+chorioallantoic+membrane.">Morphologic characterization of osteosarcoma growth on the chick chorioallantoic membrane.</a> BMC Res Notes. 3, 58 (2010).</li><li>Sys, G. 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=The+in+ovo+CAM-assay+as+a+xenograft+model+for+sarcoma.">The in ovo CAM-assay as a xenograft model for sarcoma.</a> J Vis Exp. (77), e50522 (2013).</li><li>Dohle, D. 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=Chick+ex+ovo+culture+and+ex+ovo+CAM+assay:+how+it+really+works.">Chick ex ovo culture and ex ovo CAM assay: how it really works.</a> J Vis Exp. (33), (2009).</li><li>Ossowski, L., Reich, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+model+for+quantitative+study+of+metastasis.">Experimental model for quantitative study of metastasis.</a> Cancer Res. 40 (7), 2300-2309 (1980).</li><li>Ghanekar, A., Ahmed, S., Chen, K., Adeyi, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cells+do+not+arise+from+tumor-initiating+cells+in+human+hepatocellular+carcinoma.">Endothelial cells do not arise from tumor-initiating cells in human hepatocellular carcinoma.</a> BMC Cancer. 13, 485 (2013).</li><li>Ho, J. 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A.S.&#160;receives&#160;funding from the Advaxis Pharmaceutical company to support an investigator-initiated clinical trial.&#160;

Mehrere wichtige Schritte in diesem Protokoll wahrscheinlichste Konto für die verbesserte Lebensrate der Embryonen sowie erhöhte Zuverlässigkeit des Tumorwachstums. Fallenlassen der CAM weg von der Schale durch eine Saugwirkung auf den Luftsack ist weniger invasiv als andere bestehende Methoden (mit einer Nadel an Albumin aus dem Ei, stumpf, usw. zu entfernen). Mit einem sterilen Push-Pin, um die beiden kleinen Löcher für dieses Verfahren erforderlich zu schaffen, ist der schwierigste Teil des Protokolls. Mit zu vi...

Das Leberzellkarzinom (HCC) ist die 3. häufigste Ursache der Krebssterblichkeit in der Welt 1. Derzeit werden nur 30% der HCC-Patienten sind für potenziell kurative chirurgische Behandlungen 2 und systemische Chemotherapie nicht wirksam ist 3. Daher besteht ein dringender ungedeckten klinischen Bedarf nach neuen HCC-Therapien, und die Entwicklung von Modellsystemen für eine effiziente Prüfung von neuen Wirkstoffen. Hühnchen-Chorioallantois-Membran (CAM) Assay liefert eine reproduzierbare und kosteneffiziente und schnelle mittleren Durchsatz Verfahren zum Testen potentiellen Antitumormedikamenten in vivo. Das CAM-Assay wurde ausgiebig verwendet, um die Angiogenese 4 studieren. Es wurde auch erfolgreich in einem Tumor-Xenotransplantat-Modell von Krebs entwickelt, einschließlich Glioblastom 5, Bauchspeicheldrüsenkrebs 6, Melanom 7-9 und Osteosarkom 10-11. Sowohl in ovo 12 und ex ovo13 Techniken sind in der Literatur verwendet worden, mit Details variierend von Protokoll zu Protokoll. Eine große Herausforderung für die CAM-Xenograft-Modell ist die relativ hohe Inzidenz von embryonalen Tod nach Manipulation des Ei, mit veröffentlichten Hühnerembryosterblichkeit reicht von 25 bis 50 Prozent 11-14. In diesem Artikel beschreiben wir die Entwicklung eines in-ovo-Xenograft-Modell des HCC, die zuverlässig das Wachstum von dreidimensionalen, vaskularisierten Tumoren, die histologisch ähnlich undifferenziert HCC produziert. Wir haben ein Protokoll zuerst von Ossowski et al. 14 beschrieben angepasst und chick embryonale Überlebensraten von bis zu 93% mit extrem hoher Tumor engraftment erreicht.

<table border="0" cellpadding="0" cellspacing="0" width="1094">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Name of Reagent/ Equipment</td>
			<td style="width:108px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:467px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Premium incubated eggs</td>
			<td style="width:108px;">Charles River</td>
			<td style="width:119px;">N/A</td>
			<td>http://www.criver.com/files/pdfs/avian/av_c_spf_egg_price_list.aspx</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg incubators</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">Hova-Bator 2362N</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Rotating egg trays</td>
			<td style="width:108px;">GQF</td>
			<td style="width:119px;">1611 Automatic Egg Turner</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:401px;">Egg candler</td>
			<td style="width:108px;">Lyon</td>
			<td style="width:119px;">Hi-Power 950-070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Dremel 100 rotary tool with 15/16 cut-off wheel</td>
			<td style="width:108px;">Dremel</td>
			<td style="width:119px;">100-N/7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Sterile forceps, push pin, dissection scissors, Scotch tape</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Matrigel</td>
			<td style="width:108px;">BD Biosciences</td>
			<td style="width:119px;">356234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Cryogenic vials, external thread with silicone washer</td>
			<td style="width:108px;">Corning</td>
			<td style="width:119px;">430659</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:401px;">Collagenase from Clostridium histolyticum</td>
			<td style="width:108px;">Sigma</td>
			<td style="width:119px;">C9891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the in ovo chick chorioallantoic membrane (cam) assay as an efficient xenograft model of hepatocellular carcinoma

Die Hühner-Chorioallantoismembran (CAM) beginnt am Tag 7 nach der Befruchtung zu entwickeln und reift bis zum Tag 12. Die CAM ist natürlich immungeschwächten und gefäßreiche, so dass es ein ideales System für die Tumorimplantation. Weiterhin enthält der CAM extrazelluläre Matrixproteine ​​wie Fibronektin, Laminin, Kollagen, Integrin alpha (v) beta3 und MMP-2, so dass es ein attraktives Modell zur Tumorinvasion und Metastasenbildung zu untersuchen. Wissenschaftler haben lange Vorteil der Physiologie des CAM durch Verwendung als ein Modell der Angiogenese gemacht. Vor kurzem hat der CAM-Assay wurde geändert, um als ein in vivo-Xenotransplantat-Modellsystem für verschiedene Krebsarten, die die Lücke zwischen Grundlagenarbeit in vitro und komplexere Tierkrebsmodellen überbrückt zu arbeiten. Der CAM-Test ermöglicht die Untersuchung von Tumorwachstum, Anti-Tumor-Therapien und pro-Tumor molekularen Wege in einem biologisch relevanten System, das sowohl kosten- und zeiteffektiv ist. Hier die Entwicklung von CAM xen beschreiben wirograft Modell des hepatozellulären Karzinoms (HCC) mit embryonalen Lebensraten von bis zu 93% und zuverlässigen Tumor nehmen, um das Wachstum von dreidimensionalen, vaskularisierten Tumoren führt.

Repräsentative Abbildungen der wichtigsten Schritte in dem Protokoll werden hier gezeigt. 1A zeigt die Verwendung des candler um den sich entwickelnden Embryo, den Luftsack und das Gefäßsystem des CAM zu visualisieren. 1B-1C zeigen den Vorgang des Fallenlassens der CAM indem die beiden Löcher und dann Anlegen eines Unterdrucks unter Verwendung der Sicherheitskugel und 1D zeigt eine erfolgreich fiel CAM mit einer großen Luftblase unter dem Bleistift markierte Loch. ...

Watch this Scientific Journal Video about The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma at JoVE.com

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

Das<em&gt; In Ovo</em&gt; Chick Chorioallantoismembran (CAM) Assay als effiziente Xenograftmodell des hepatozellulären Karzinoms

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

the-ovo-chick-chorioallantoic-membrane-cam-assay-as-an-efficient

Research

JoVE Journal

Medicine

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

35.0K Aufrufe.  Icahn School of Medicine at Mount Sinai. The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

Serie: The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-Photonen-Imaging der Tumorzellinvasion in einem orthotopen Mausmodell des oralen Plattenepithelkarzinoms

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Forschung

Medizin

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Ein orthotopen Mausmodell des Anaplastic Schilddrüsenkarzinom

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

The In ovo CAM-assay as a Xenograft Model for Sarcoma

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for personalized medicine after stratification, explaining the current interest in developing a reproducible and low-cost xenotransplant model for this disease. The chick chorioallantoic membrane is a natural immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition, it is easily accessed, manipulated and imaged using optical and fluorescence stereomicroscopy. Histology further allows detailed analysis of heterotypic cellular interactions.
This protocol describes in detail the in ovo grafting of the chorioallantoic membrane with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines (Saos-2 and SW1353). The chick survival rates are up to 75%. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior. For localized grafting of single cell suspensions, ECM gel provides significant advantages over inert containment materials. The Ki67 proliferation index is related to the distance of the cells from the surface of the CAM and the duration of application on the CAM, the latter determining a time frame for the addition of therapeutic products.

The In ovo CAM-assay as a Xenograft Model for Sarcoma

The in ovo chorioallantoic membrane (CAM) is grafted with fresh sarcoma-derived tumor tissues, their single cell suspensions, and permanent and transient fluorescently labeled established sarcoma cell lines. The model is used to study graft- (viability, Ki67 proliferation index, necrosis, infiltration) and host (fibroblast infiltration, vascular ingrowth) behavior.

Die<em&gt; In ovo</em&gt; CAM-Assay als Xenograftmodel für Sarkom

Sarcoma is a very rare disease that is heterogeneous in nature, all hampering the development of new therapies. Sarcoma patients are ideal candidates for ...

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.

Generierung von subkutanen und Intrahepatische Menschenleberzellkarzinom Xenografts in immundefizienten Mäusen

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

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Exzitotoxische Stimulation der Hirn Microslices als<em&gt; In-vitro-</em&gt; Modell der Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Confocal Time Lapse Imaging as an Efficient Method for the Cytocompatibility Evaluation of Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The objective of this study was to use 3D CLSM time lapse confocal imaging to assess the in vitro biocompatibility of dental composites. This method provides an accurate and sensitive indication of viable cell rate in contact with dental composite extracts. The ELS extra low shrinkage, a dental composite used for direct restoration, has been taken as example. In vitro assessment was performed on cultured primary human gingival fibroblast cells using Live/Dead staining. Images were obtained with the FV10i confocal biological inverted system and analyzed with the FV10-ASW 3.1 Software. Image analysis showed a very slight cytotoxicity in the presence of the tested composite after 5 hours of time lapse. A slight decrease of cell viability was shown in contact with the tested composite extracts compared to control cells. The findings highlighted the use of 3D CLSM time lapse imaging as a sensitive method to qualitatively and quantitatively evaluate the biocompatibility behavior of dental composites.

The most studied aspect of the biocompatibility of dental composites is their cytotoxicity 3D CLSM time lapse imaging combined with fluorescent signal quantification allows sensitive evaluation of the cell fate over time. Utilizing this method could be an efficient tool to assess the cytocompatibility of dental composites.

Konfokale Zeitraffer Imaging als eine effiziente Methode für die Bewertung der Cytocompatibility Dental Composites

It is generally accepted that in vitro cell material interaction is a useful criterion in the evaluation of dental material biocompatibility. The ...

A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

The metastatic spread of cancer is the major cause of cancer-related deaths. We provide an in-depth description of our survival surgery methodology for establishing a &#8220;patient-like&#8221; orthotopic syngeneic mouse model system for studying the mechanisms of metastasis in solid organ tumors.

Ein &quot;Patient-Like&quot; Orthotope Syngene Maus-Modell der Leberzellkarzinom Metastasen

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

Designing CAD/CAM Surgical Guides for Maxillary Reconstruction Using an In-house Approach

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this technique is expensive and available in only limited areas of the world, we developed a novel CAD/CAM surgical guide using an in-house approach. By using the CAD software, the maxillary resection area and cutting planes and the fibular cutting planes and angles are determined. Once the resection area is decided, the necessary faces are extracted using a Boolean modifier. These superficial faces are united to fit the surface of the bones and thickened to stabilize the solids. Not only the cutting guides for the fibula and maxilla but also the location arrangement of the transferred bone segments is defined by thickening the superficial faces. The CAD design is recorded as .stl files and three-dimensionally (3-D) printed as actual surgical guides. To check the accuracy of the guides, model surgery using 3-D-printed facial and fibular models is performed. These methods may be used to assist surgeons where commercial guides are not available.

Methods for designing a computer-aided design/computer-aided manufacturing (CAD/CAM) surgical guide are shown. Cutting planes are separated, united, and thickened to easily visualize the necessary bone transfer. These designs can be three-dimensional printed and checked for accuracy.

Projektierung CAD/CAM-Bohrschablonen für den Oberkiefer Wiederaufbau mit einem hauseigenen Ansatz

Computer-aided design/computer-assisted manufacturing (CAD/CAM) is now being evaluated as a preparative technique for maxillofacial surgery. Because this ...

Isometric Contractility Measurement of the Mouse Mesenteric Artery Using Wire Myography

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and drugs. It is widely used in many studies on the physiological functions of vascular smooth muscle, the pathogenesis of vascular diseases such as hypertension, and the development of smooth muscle relaxant drugs. The mouse is a widely used model animal with a large pool of disease models and genetically modified strains. We introduced this method to measure the isometric contraction of mouse mesenteric artery in detail. A 1.4-mm segment of mouse mesenteric resistance artery was isolated and mounted on a myograph chamber by passing two steel wires through its lumen. After equilibration and normalization steps, the vessel segment was potentiated by a high-K+ solution twice prior to the contraction assay. As an example of the application of this method in drug development, we measured the relaxant effect of a novel natural substance, neoliensinine, isolated from a Chinese herb, embryos of the lotus seed (Nelumbo nucifera Gaertn.) on mouse mesenteric arteries. The vessel segments mounted on the myograph chamber were stimulated with a high-K+ solution. When the force tension reached a stable sustained phase, cumulative doses of neoliensinine were added to the chamber. We found that neoliensinine had a dose-dependent relaxant effect on smooth muscle contraction, thus suggesting that it bears potential activity against hypertension. In addition, as the vessel segment can survive at least 4 hours after mounting and maintain contractility induced by the high-K+ solution for many times, we suggest that the wire myograph system may be used for the time-consuming process of drug screening.

The wire myograph technique is used to investigate vascular smooth muscle functions and screen new drugs. We report a detailed protocol for measuring the isometric contractility of the mouse mesenteric artery and for screening new relaxants of vascular smooth muscle.

Messung der isometrischen Kontraktilität der Maus mesenterialen Arterie mit Draht Myographie

The wire myograph technique is used to assess the contractility of vascular smooth muscles in response to depolarization, GPCR agonists/inhibitors and ...

Predicting Treatment Response to Image-Guided Therapies Using Machine Learning: An Example for Trans-Arterial Treatment of Hepatocellular Carcinoma

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this study was to develop a method to predict response to intra-arterial treatment prior to intervention.
The method provides a general framework for predicting outcomes prior to intra-arterial therapy. It involves pooling clinical, demographic and imaging data across a cohort of patients and using these data to train a machine learning model. The trained model is applied to new patients in order to predict their likelihood of response to intra-arterial therapy.
The method entails the acquisition and parsing of clinical, demographic and imaging data from N patients who have already undergone trans-arterial therapies. These data are parsed into discrete features (age, sex, cirrhosis, degree of tumor enhancement, etc.) and binarized into true/false values (e.g., age over 60, male gender, tumor enhancement beyond a set threshold, etc.). Low-variance features and features with low univariate associations with the outcome are removed. Each treated patient is labeled according to whether they responded or did not respond to treatment. Each training patient is thus represented by a set of binary features and an outcome label. Machine learning models are trained using N - 1 patients with testing on the left-out patient. This process is repeated for each of the N patients. The N models are averaged to arrive at a final model.
The technique is extensible and enables inclusion of additional features in the future. It is also a generalizable process that may be applied to clinical research questions outside of interventional radiology. The main limitation is the need to derive features manually from each patient. A popular modern form of machine learning called deep learning does not suffer from this limitation, but requires larger datasets.

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. A method for predicting response to these therapies is proposed. The technique uses pre-procedural clinical, demographic, and imaging information to train machine learning models capable of predicting response prior to treatment.

Vorhersage der Behandlungserfolg für bildgestützte Therapien mit maschinellem lernen: ein Beispiel für eine Trans-arterielle Behandlung des hepatozellulären Karzinoms

Intra-arterial therapies are the standard of care for patients with hepatocellular carcinoma who cannot undergo surgical resection. The objective of this ...