Name: molecular profiling of the invasive tumor microenvironment in a 3-dimensional model of colorectal cancer cells and ex vivo fibroblasts
Uploaded: 2014-04-29T14:00:00
Description: Watch this Scientific Journal Video about Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts at JoVE.com

MB er støttet af tilskud fra en MRC fællesskab. KP og AHM understøttes af tilskud fra Wessex Medical Research og Cancer Research UK / RCS (England) (C28503/A10013). Vi er taknemmelige for den støtte fra University of Southampton Histochemistry Research Unit.

1.. Etablering af Primary fibroblastkulturer fra Colon Eksplantater <ol><li> Få prøver af normal human colon slimhinde væv direkte fra kirurgisk operationsstue, og den suspenderes i 7-10 ml PBS suppleret med 100 enheder / ml penicillin, 100 ug / ml streptomycin og 0,25 ug / ml Fungizone. </li><li> I laboratoriet anbringes prøven i centrum af en 10-cm vævskulturskål og vask 3 gange med PBS / Pen-strep / Fungizone. Aspirér ikke efter den sidste vask. </li><li> Med steril pincet og skalpel skæres vævet i små st...

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	<li>Nystrom, M. L., Thomas, G. J., Stone, M., Mackenzie, I. C., Hart, I. R., Marshall, J. F. <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+quantitative+method+to+analyse+tumour+cell+invasion+in+organotypic+culture.">Development of a quantitative method to analyse tumour cell invasion in organotypic culture.</a> J Pathol. 205 (4), 468-475 (2005).</li><li>Zhang, 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=miR-153+supports+colorectal+cancer+progression+via+pleiotropic+effects+that+enhance+invasion+and+chemotherapeutic+resistance.">miR-153 supports colorectal cancer progression via pleiotropic effects that enhance invasion and chemotherapeutic resistance.</a> Cancer Res. , (2013).</li><li>Bullock, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pleiotropic+actions+of+miR-21+highlight+the+critical+role+of+deregulated+stromal+microRNAs+during+colorectal+cancer+progression.">Pleiotropic actions of miR-21 highlight the critical role of deregulated stromal microRNAs during colorectal cancer progression.</a> Cell Death Dis. 20 (4), (2013).</li><li>Jenei, V., Nystrom, M. L., Thomas, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+invasion+in+an+organotypic+model.">Measuring invasion in an organotypic model.</a> Methods Mol Biol. 769, 223-232 (2011).</li><li>De Wever, O., Demetter, P., Mareel, M., Bracke, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+myofibroblasts+are+drivers+of+invasive+cancer+growth.">Stromal myofibroblasts are drivers of invasive cancer growth.</a> Int J Cancer. 123 (10), 2229-2238 (2008).</li><li>Fries, K. 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=Evidence+of+fibroblast+heterogeneity+and+the+role+of+fibroblast+subpopulations+in+fibrosis.">Evidence of fibroblast heterogeneity and the role of fibroblast subpopulations in fibrosis.</a> Clin Immunol Immunopathol. 72 (3), 283-292 (1994).</li><li>Moutasim, K. 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=Betel-derived+alkaloid+up-regulates+keratinocyte+alphavbeta6+integrin+expression+and+promotes+oral+submucous+fibrosis.">Betel-derived alkaloid up-regulates keratinocyte alphavbeta6 integrin expression and promotes oral submucous fibrosis.</a> J Pathol. 223 (3), 366-377 (2011).</li><li>Winter, J., Jung, S., Keller, S., Gregory, R. I., Diederichs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Many+roads+to+maturity:+microRNA+biogenesis+pathways+and+their+regulation.">Many roads to maturity: microRNA biogenesis pathways and their regulation.</a> Nat Cell Biol. 11 (3), 228-234 (2009).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+miRNA+expression+patterns+using+total+RNA+extracted+from+matched+samples+of+formalin-fixed+paraffin-embedded+(FFPE)+cells+and+snap+frozen+cells.">Comparison of miRNA expression patterns using total RNA extracted from matched samples of formalin-fixed paraffin-embedded (FFPE) cells and snap frozen cells.</a> BMC Biotechnol. 7 (36), (2007).</li><li>Szafranska, A. 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=Accurate+molecular+characterization+of+formalin-fixed,+paraffin-embedded+tissues+by+microRNA+expression+profiling.">Accurate molecular characterization of formalin-fixed, paraffin-embedded tissues by microRNA expression profiling.</a> J Mol Diagn. 10 (5), 415-423 (2008).</li></ol>

Her beskriver vi en fremgangsmåde til specifikt at isolere og karakterisere tumorceller, som har erhvervet evnen til at invadere cancer associeret stroma i de tidligste stadier af metastatisk progression i en 3-dimensionel co-kultur konstruktionen af ​​epitel og stromaceller. Vævsmodeller består af CRC epitelceller sammenstillet med en syntetisk stroma indeholdende ex vivo humane colon fibroblaster blev anvendt til at undersøge CRC invasion i tre dimensioner. Denne fysiologi...

Organotypiske co-kulturer er væv-manipuleret modeller som støtte i at rekonstruere in vivo tumor mikromiljø i 3 dimensioner ved at sætte maligne epitelceller og stromacellerne i et kollagen gel indeholder væsentlige ekstracellulære matrixkomponenter 1-3. Hovedsagelig tænkt som en metode til måling af tumor invasion, organotypics mindske afhængigheden af in vivo dyremodeller, og undgå manglerne i andre in vitro-teknikker såsom Transwell assays, hvor invaderende celler er tvunget kunstigt ind i en mono-spredt tilstand 2-4. Inddragelsen af stromaceller i disse modeller, såsom fibroblaster, afspejler den vigtige rolle tumormikromiljøet i reguleringen malign invasion og metastase 3-4, men den fænotypiske heterogenitet af stroma er sådan, at for at maksimere den fysiologiske relevans af organotypisk modeller, orgel specifik, anatomisk korrekte ex vivofibroblaster bør medtages så vidt muligt 3,6. Organotypics er en alsidig platform til at studere samspillet mellem tumor og stromaceller og anvendes i stigende grad på nye måder at undersøge indvirkningen på tumor invasion af kemiske inhibitorer og målrettede gen 7,8 ombygninger. Studere den invasive tumor margin er en særdeles spændende mulighed. I organotypiske modeller etableret colorectal cancer (CRC)-cellelinjer producerer typisk et godt stratificeret epitel lag og i tværsnit, celler, der har erhvervet evnen til at invadere den ekstracellulære matrix (ECM) kan let ses. I lyset af den etablerede mikro-topografisk heterogenitet tumor og tumorassocierede stromale celler in vivo, udvinde disse celler ved hjælp af Laser mikrodissektion og studere dem i isolation, kan afsløre vigtige biologiske indsigter vedrørende oprindelsen af kolorektal cancer metastaser, og hvordan det kan være mere effektivt målrettet. Jegn følgende metode, alle patienter, som doneret vævsprøver forudsat skriftligt informeret samtykke, og undersøgelsen blev godkendt af de institutioner, regionale videnskabsetiske komité.

<table border="0" cellpadding="0" cellspacing="0" width="634">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Colorectal cancer cell lines - example shown SW480</td>
			<td style="width:132px;">ATCC</td>
			<td style="width:119px;">ATCC CCL-228</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagen</td>
			<td style="width:132px;">BD Biosciences</td>
			<td style="width:119px;">354265</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Matrigel</td>
			<td style="width:132px;">BD Biosciences</td>
			<td style="width:119px;">354234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nylon membrane</td>
			<td style="width:132px;">Merck Millipore</td>
			<td style="width:119px;">VVLP01300</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Metal grid</td>
			<td style="width:132px;">The Mesh Company</td>
			<td>WSS20-A4</td>
			<td>themeshcompany.com</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laser microdissection platform</td>
			<td style="width:132px;">Leica Microsystems</td>
			<td style="width:119px;">Leica AS LMD</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Membrane mounted slides</td>
			<td style="width:132px;">Molecular devices</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cresyl Violet</td>
			<td style="width:132px;">Merck Millipore</td>
			<td style="width:119px;">1052350025</td>
			<td></td>
		</tr>
	</tbody>
</table>

molecular profiling of the invasive tumor microenvironment in a 3-dimensional model of colorectal cancer cells and ex vivo fibroblasts

Invaderende kolorektal cancer (CRC) celler har erhvervet evnen til at bryde fri fra deres søster celler, infiltrere stroma, og remodel den ekstracellulære matrix (ECM). Karakteriserer biologi dette fænotypisk særskilt gruppe af celler væsentligt kunne forbedre vores forståelse af tidlige hændelser i løbet af metastatisk kaskade. Tumor invasion er en dynamisk proces lettes ved tovejs interaktioner mellem ondartet epitel og cancer associeret stroma. For at undersøge celle-responser på tumorstroma-interface-vi kombineret organotypisk co-kultur og laser mikro-dissektion teknikker. Organotypiske modeller, hvor vigtige stromale bestanddele, som f.eks fibroblaster 3-dimentioanally co-dyrket med kræft epitelceller, er meget manipuleres eksperimentelle værktøjer, der giver invasion og kræft-stroma interaktion, der skal undersøges i nær-physiological forhold. Laser mikrodissektion (LMD) er en teknik, som indebærer kirurgisk dissektion og udvinding af de forskellige lag i tumorvæv, med micron niveau præcision. Ved at kombinere disse teknikker med genomisk, transkriptom og epigenetisk profilering vi sigter mod at udvikle en dybere forståelse for de molekylære karakteristika af invaderende tumorceller og omgivende stromavæv, og dermed potentielt afsløre nye biomarkører og muligheder for udvikling af lægemidler i CRC.

Vi har anvendt den ovennævnte fremgangsmåde til flere kombinationer af CRC-cellelinier og stromaceller. Et eksempel præsenteres her, er SW480 epitelial CRC cellelinjer med primære humane ex vivo colon fibroblaster. 3-dimensionelle co-kulturer er konstrueret (figur 1), underkastet mikroskopi og laseropfangnings-mikrodissektion (figur 2), og analyseret ved microRNA (miRNA) profilering (figur 3) til sammenligning af differentielt udtrykte miRNA mellem celler p...

Watch this Scientific Journal Video about Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts at JoVE.com

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

Molekylær profilering af laser mikrodissekeres celler og stroma fra syntetiske, væv-manipuleret kolorektal cancer modeller repræsenterer en ny og manipuleres tilgang til at karakterisere og dissekere den særprægede biologi på grænsefladen mellem tumorceller ved den invasive front og cancer associeret stromaceller.

Molekylær profilering af Invasiv tumormikromiljøet i en 3-dimensionel model af kolorektal cancer celler og<em&gt; Ex vivo</em&gt; Fibroblaster

Invading colorectal cancer (CRC) cells have acquired the capacity to break free from their sister cells, infiltrate the stroma, and remodel the ...

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Medicine

Molecular Profiling of the Invasive Tumor Microenvironment in a 3-Dimensional Model of Colorectal Cancer Cells and Ex vivo Fibroblasts

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We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

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The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

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