Name: chick heart invasion assay for testing the invasiveness of cancer cells and the activity of potentially anti-invasive compounds
Uploaded: 2015-06-06T15:00:00
Description: Watch this Scientific Journal Video about Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds at JoVE.com

We thank Marleen De Meulemeester for demonstrating the assay technique in the video film. B. I. R. is a Postdoctoral Research Fellow of the Research Foundation &#8211; Flanders (FWO &#8211; Vlaanderen). L.M.M. is a recipient of an Emmanuel van der Schueren grant from the Flemish League against Cancer (Vlaamse Liga tegen Kanker).

<img alt="Figur 1" src="/files/ftp_upload/52792/52792fig1.jpg" /> Figur 1. Skematisk oversigt over de forskellige assay trin. <a href="https://www.jove.com/files/ftp_upload/52792/52792fig1large.jpg" target="_blank">Klik her for at se en større version af dette tal.</a> 1. Fremstilling af fordyrket Heart Fragmenter (PHF&#39;er) <ol><li> Inkuber et befrugtet æg chick ved 37 ° C i 9 dage. Fuldføre denne inkubation inden en dato, ...

<ol>
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Under forberedelsen af ​​PHF&#39;er kan fragmenterne ikke ophold i suspension, men holde sig til karvæggen; dette kan overvindes ved forøgelse af volumenet af dyrkningsmediet. Hvis antallet af PHF&#39;er er for lavt, og deres størrelse er for stor, sænke lydstyrken af ​​dyrkningsmediet. Svigt i testcellerne at aggregere kan skyldes udsving i temperatur eller til mikrobiel infektion. Alternativt kan en manglende evne til at aggregere som en iboende egenskab af cellerne. Under fastgørelse af aggregaterne til ...

Invasion er kendetegnende for maligne tumorer. Denne aktivitet ikke blot fører til ødelæggelse af omgivende væv, men er også impliceret i metastase-dannelse. Da kræftpatienter dør af invasion og metastase, og effektive anti-invasive behandlinger er stadig knappe, laboratorieanalyser, der efterligner invasion af tumorceller er blevet udviklet. Målet med chick hjerte-assayet er at tilbyde en relevant organkultur metode til at studere tumor invasion i tre dimensioner. Assayet kan skelne mellem invasive og ikke-invasive celler og gør det muligt at undersøge virkningerne af testforbindelserne på tumor invasion. Rationalet bag brugen af analysen er selve begrebet, at tumorer er økosystemer, hvor de neoplastiske celler kontinuerligt interagerer med deres stroma (værtsceller og ekstracellulære matrix), og at gennem disse molekylære interaktioner invasion er finjusteret 1. Så i assayet tumorceller konfronteres med Living embryonale chick hjerte-fragmenter 2, der tjener ikke kun som substrater for invasion af tumorcellerne, men også som en kilde til forskellige typer af stromale celler og matrixelementer. Chick hjerte indeholder myocytter, fibroblaster og endotelceller, og den extracellulære matrix er sammensat af laminin, fibronectin og forskellige typer af collagen. På denne måde, det tredimensionale organkultur teknik omfatter mange cellulære og molekylære interaktioner impliceret i invasionen af ​​patientens tumorer. Den største fordel ved chick hjerte-assayet er gennemførelsen af ​​stromale virkninger. Dette aspekt er mere komplet end i andre invasion assays in vitro, der er baseret på tumorcelleinvasion i ikke-levende geler sammensat af basalmembran 3 eller interstitiel matrix 4 molekyler. Begrebet konfrontation mellem tumorceller og normale levende vært væv som findes i orgel kultur eksperimenter er blevet indført af flereforfattere, herunder Wolff og Schneider i Frankrig 5, easty og easty i Det Forenede Kongerige 6, og Schleich i Tyskland 7. To tekniske fordele ved chick hjerte invasion assay over citerede metoder er, at mængden af ​​fragmenterne let kan standardiseres, og at de forbliver kontraktile, som giver funktionelle integritet overvågning under organkultur. Endvidere er aviære embryoner foretrækkes, fordi de let kan udskæres fra den sterile indhold af ægget. Assayet har begrebsmæssige lighed med chick Chorioallantois membranassay 8 ved at tilbyde en kompleks stromal omkring til tumorcellerne. Assayet har med succes været anvendt til at skelne mellem invasive og ikke-invasive celle-varianter af de samme humane tumorer såsom i MCF-7 (mamma) 9 og HCT-8 (colon) 10 cellelinje familier. Teknikken er nyttig til at teste potentielt anti-invasive forbindelser samt <sup&gt; 11,12. Som yderligere forklaret, kan det bruges til etablering af struktur-aktivitet relationer små organiske molekyler. Assayet giver dog ikke hensyn til bidraget af immunologiske celler til invasion cancer. Det skal understreges, at teknikken ikke kan betragtes som en high-throughput analysesystem, på grund af det store antal manipulationer, de begrænsede antal analysekørsler (højst 30 kulturer) og den lange turn-around tid (ca. 1 måned).

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ringer&#39;s salt solution</td>
			<td>Braun</td>
			<td>CEO123</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-agar</td>
			<td>Becton Dickinson</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-(hydroxymethyl)-aminomethaan Analar</td>
			<td>VWR</td>
			<td>103157P</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA : albumin from bovine serum, cohn V fract.</td>
			<td>Sigma</td>
			<td>A4503-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM-Rega 3</td>
			<td>Life Technologies&#160;</td>
			<td>19993013</td>
			<td></td>
		</tr>
		<tr>
			<td>Gyrotory shakers</td>
			<td>New Brunswick Scientific</td>
			<td>G10 and G33</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 50 ml</td>
			<td>Novolab</td>
			<td>92717</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dishes</td>
			<td>Novolab</td>
			<td>68516</td>
			<td></td>
		</tr>
		<tr>
			<td>Iridectomy scissors</td>
			<td>Rumex</td>
			<td>11-0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Macroscope with calibrated ocular grid</td>
			<td>Wild</td>
			<td>157702</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>International Medical products</td>
			<td>8599956</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma</td>
			<td>230251-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris&#39; hematoxylin</td>
			<td>Sigma</td>
			<td>HHS32-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslipping resin (Tissue-Tek)</td>
			<td>Sakura</td>
			<td>4494</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin melting apparatus</td>
			<td>GFL</td>
			<td>1052</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome for paraffin sectioning</td>
			<td>Reichert</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stppers for Erlemeyer flasks with gas in- and outlets</td>
			<td>Novolab</td>
			<td>2602421 and 260243</td>
			<td></td>
		</tr>
		<tr>
			<td>Diaminobenzidine</td>
			<td>Sigma</td>
			<td>D8001</td>
			<td></td>
		</tr>
		<tr>
			<td>REAX rocking table</td>
			<td>Heidolph</td>
			<td>54131</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue dishes</td>
			<td>VWR</td>
			<td>NUNC142475</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophtalmological enucleation spoon</td>
			<td>Rumex</td>
			<td>16-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp forceps</td>
			<td>Rumex</td>
			<td>4-111T</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>Nickel-Electro LTD</td>
			<td>7112</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 5 ml</td>
			<td>Novolab</td>
			<td>211901</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides washed and degreased</td>
			<td>International Medical products</td>
			<td>8613908</td>
			<td></td>
		</tr>
	</tbody>
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chick heart invasion assay for testing the invasiveness of cancer cells and the activity of potentially anti-invasive compounds

Målet med chick hjerte-assayet er at tilbyde en relevant organkultur metode til at studere tumor invasion i tre dimensioner. Assayet kan skelne mellem invasive og ikke-invasive celler, og muliggør undersøgelse af virkningerne af testforbindelserne på tumor invasion. Kræftceller - enten som aggregater eller enkelte celler - konfronteres med fragmenter af embryonale chick hjerte. Efter organkultur i suspension i et par dage eller uger konfronterende kulturer fikseret og indlejret i paraffin for histologisk analyse. Det tredimensionale samspil mellem cancerceller og det normale væv derefter rekonstrueret fra serielle snit farvet med hematoxylin-eosin eller efter immunhistokemisk farvning for epitoper i hjertevævet eller står cancerceller. Assayet er i overensstemmelse med den seneste koncept, invasion kræft er resultatet af molekylære interaktioner mellem cancerceller og deres tilstødende stromale vært elementer (myofibroblaster, endothelial celler, ekstracellulære matrixkomponenter, etc.). Her er denne stromale miljø tilbydes cancercellerne som et levende væv fragment. Understøttende aspekter til relevansen af ​​analysen er flere. Invasion i analysen er i overensstemmelse med kriterierne i invasion kræft: progressiv erhverv og udskiftning i tid og rum af værtens væv, og invasivitet og ikke-invasiv in vivo af de står over for celler generelt korrelerer med resultatet af analysen. Endvidere invasionen mønster af celler in vivo, som defineret af patologer, afspejles i de histologiske billeder i assayet. Kvantitative struktur-aktivitet relation (QSAR) analyse af de opnåede resultater med talrige potentielt anti-invasive organiske kongenermønstre forbindelser tillod undersøgelse af struktur-aktivitet relationer for flavonoider og chalconer og kendte anti-metastatiske lægemidler anvendt i klinikken (fx mikrotubulus inhibitorer ) inhiberer invasion i assayet samt. However, er analysen ikke hensyn til immunologiske bidrag til invasion kræft.

De histologiske sektioner som vist i figur 5 viser det endelige resultat af en række succesfulde assays. Lyden histologi af kulturerne indikerer levedygtige celler og tillader at fortolke interaktionen mellem tumorceller og normale væv. Endvidere kan iagttages nogen immunreaktion fra det normale væv, hvilket bekræfter den korrekte alder kyllingefostre anvendt f.eks før immunafstødning systemet udvikles. Der er ingen bakterier, synlige, hvilket indikerer fravær af (brutto) kontaminering under kultur perio...

Watch this Scientific Journal Video about Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds at JoVE.com

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

Her præsenterer vi en protokol til at studere invasionen af ​​tumorceller i levende normale væv fragmenter i tre dimensioner. Denne organkultur teknik anvendes hovedsagelig til at teste potentielt anti-invasive lægemidler in vitro.

Chick Heart Invasion Assay til Test af invasiv af kræftceller og aktiviteten af ​​Potentielt Anti-invasive forbindelser

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

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A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

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En modificeret<em&gt; In vitro</em&gt; Invasion Assay at bestemme rolle Hormoner, cytokiner og / eller vækstfaktorer ved mediering Cancer Cell Invasion

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Chick Heart Invasion Assay til Test af invasiv af kræftceller og aktiviteten af ​​Potentielt Anti-invasive forbindelser

Chick Heart Invasion Assay til Test af invasiv af kræftceller og aktiviteten af Potentielt Anti-invasive forbindelser