Name: modeling and imaging 3-dimensional collective cell invasion
Uploaded: 2011-12-07T13:00:00
Description: Watch this Scientific Journal Video about Modeling and Imaging 3-Dimensional Collective Cell Invasion at JoVE.com

Finansieringen af ​​denne forskning er fra Cancer Research Storbritannien.

1. Retroviral mærkning af celler med fluorescerende proteiner <ol><li> Plate retrovirale emballage celler (f.eks Phoenix) ud på 0,25 x 10 6 celler pr brønd i en 6-godt fad i 10% føtalt bovint serum (FBS) / DMEM. </li><li> Transfect celler med retrovirale DNA 48 timer senere celler, der bruger Effectene efter fabrikantens anvisninger. </li><li> Skyl brønde to gange med medium 24 timer senere, hvorefter der tilsættes 1,5 ml 10% FBS / DMEM per brønd. </li><li> Saml pakket vir...

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	<li>Olson, M. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+actin+cytoskeleton+in+cancer+cell+motility.">The actin cytoskeleton in cancer cell motility.</a> Clin. Exp. Metastasis. 26, 273-287 (2008).</li><li>Hoelzinger, D. B., Demuth, T., Berens, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autocrine+factors+that+sustain+glioma+invasion+and+paracrine+biology+in+the+brain+microenvironment.">Autocrine factors that sustain glioma invasion and paracrine biology in the brain microenvironment.</a> J. Natl. Cancer. Inst. 99, 1583-1593 (2007).</li><li>Chaffer, C. L., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+perspective+on+cancer+cell+metastasis.">A perspective on cancer cell metastasis.</a> Science. 331, 1559-1564 (2011).</li><li>Hanahan, D., Weinberg, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hallmarks+of+Cancer.">The Hallmarks of Cancer.</a> Cell. 100, 57-70 (2000).</li><li>Francia, G., Cruz-Munoz, W., Man, S., Xu, P., Kerbel, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mouse+models+of+advanced+spontaneous+metastasis+for+experimental+therapeutics.">Mouse models of advanced spontaneous metastasis for experimental therapeutics.</a> Nat. Rev. Cancer. 11, 135-141 (2011).</li><li>Hooper, S., Marshall, J. F., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell+migration+in+three+dimensions.">Tumor cell migration in three dimensions.</a> Methods. Enzymol. 406, 625-643 (2006).</li><li>Croft, D. R., Olson, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulating+the+conversion+between+rounded+and+elongated+modes+of+cancer+cell+movement.">Regulating the conversion between rounded and elongated modes of cancer cell movement.</a> Cancer Cell. 14, 349-351 (2008).</li><li>Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-step+pericellular+proteolysis+controls+the+transition+from+individual+to+collective+cancer+cell+invasion.">Multi-step pericellular proteolysis controls the transition from individual to collective cancer cell invasion.</a> Nat. Cell. Biol. 9, 893-904 (2007).</li><li>Hennigan, R. F., Hawker, K. L., Ozanne, B. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fos-transformation+activates+genes+associated+with+invasion.">Fos-transformation activates genes associated with invasion.</a> Oncogene. 9, 3591-3600 (1994).</li><li>Scott, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LIM+kinases+are+required+for+invasive+path+generation+by+tumor+and+tumor-associated+stromal+cells.">LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells.</a> J. Cell. Biol. 191, 169-185 (2010).</li></ol>

Matrigel invasion analyser har traditionelt været oprettet med celler placeret på et lag af ekstracellulære matrix protein med chemoattractant-induceret motilitet hen imod og gennem et filter i bunden. Invasionsevne blev scoret som en funktion af, hvor mange celler kunne tælles på undersiden af ​​filteret. Selvom stort set at der er lidt forskel med &quot;omvendte&quot; invasionen assay beskrevet ovenfor, er der betydeligt flere oplysninger om processen med invasion, som kan bestemmes ved at visualisere invader...

<table border="1"><tbody><tr><th> Navn på reagenset </th><th> Firma </th><th> Katalog nummer </th></tr><tr><td> DMEM (Dulbecco ændrede Eagle er Medium) </td><td> GIBCO </td><td> 21969 </td></tr><tr><td> Føtalt bovint serum </td><td> PAA </td><td> A15-101 </td></tr><tr><td> Penicillin Streptomycin </td><td> GIBCO </td><td> 15140 </td></tr><tr><td> 200 mm L-Glutamin (100x) </td><td> GIBCO </td><td> 25050-032 </td></tr><tr><td> Puromycin </td><td> Sigma-Aldrich </td><td> P8833 </td></tr><tr><td> 0,05% Trypsin EDTA </td><td> GIBCO </td><td> 25300 </td></tr><tr><td> Polybrene </td><td> Sigma-Aldrich </td><td> AL-118 </td></tr><tr><td> Lipofectamine 2000 Reagens </td><td> Invitrogen </td><td> 11668019 </td></tr><tr><td> 6.5mm Transwells, 8,0 μm porestørrelse </td><td> Corning </td><td> 3422 </td></tr><tr><td> Komplet Matrigel </td><td> BD Biosciences </td><td> 354234 </td></tr> &lt;tr&gt; <td> Calcein AM </td><td> Invitrogen </td><td> C1430 </td></tr><tr><td> RNase </td><td> Qiagen </td><td> 19101 </td></tr><tr><td> Propidium Iodid </td><td> Sigma-Aldrich </td><td> P4864 </td></tr><tr><td> Konfokal microcope </td><td> Leica </td><td> SP2MP </td></tr></tbody></table>

modeling and imaging 3-dimensional collective cell invasion

Et kendetegn ved kræft malignitet er invasion og metastasering 1. I visse kræftformer (e. g. Gliom 2), lokal invasion ind i det omgivende raske væv er den egentlige årsag til sygdom og død. For andre kræftformer (e. g. Bryst-, lunge, osv..), Er det processen med metastaser, hvor tumorceller flytter fra en primær tumor masse, kolonisere distal steder og i sidste ende bidrage til organsvigt, som i sidste ende fører til sygelighed og dødelighed 3. Det er blevet anslået, at invasion og metastasering er ansvarlige for 90% af alle kræftdødsfald 4. Som følge heraf har der været intense interesse i at identificere de molekylære processer og kritiske protein mediatorer af invasion og metastasering med henblik på at forbedre diagnostik og behandling 5. En udfordring for kræft forskerne er at udvikle invasion assays, at tilstrækkeligt svarer til in vivo-situationentil muliggør nøjagtig sygdom modellering 6. To-dimensionelle celle motilitet assays er kun informativ om ét aspekt af invasion og ikke tager hensyn til ekstracellulære matrix (ECM) protein remodeling, der er også et kritisk element. For nylig har forskning forfinet vores forståelse af tumor celle invasion og afslørede, at de enkelte celler kan bevæge sig ved runde eller aflange tilstande 7. Derudover har der været større anerkendelse af det bidrag kollektive invasion, hvor celler invaderer i tråde, plader og klynger, især i stærkt differentierede tumorer, der opretholder epitel egenskaber, til spredning af kræft 8. Vi præsenterer et raffineret metode 9 for at undersøge bidrag kandidat proteiner til kollektive invasion 10. I særdeleshed, ud fra et teknisk adskilte puljer af celler til at udtrykke forskellige fluorescerende proteiner, er det muligt at molekylemæssigt dissekere de aktiviteter og proteins kræves i førende celler versus dem, der kræves i følgende celler. Brugen af ​​RNAi giver molekylære værktøj til eksperimentelt adskille de processer, der er involveret i enkelte celle invasion samt i forskellige positioner af kollektive invasion. I denne procedure, er blandinger af fluorescens-mærkede celler belagt på bunden af ​​en Transwell indsætte tidligere fyldt med Matrigel ECM protein, derefter lov til at invadere &quot;opad&quot; gennem filteret og ind i Matrigel. Genopbygning af Z-serien Billedstakke, fremstillet ved konfokal billedbehandling, i tre-dimensionelle repræsentationer giver mulighed for visualisering af kollektivt invaderende tråde og analyse af repræsentationen af ​​fluorescens-mærkede celler i ledende versus følgende positioner.

Watch this Scientific Journal Video about Modeling and Imaging 3-Dimensional Collective Cell Invasion at JoVE.com

Modeling and Imaging 3-Dimensional Collective Cell Invasion

Modeller af tumor celle invasion i tre-dimensionelle ekstracellulære matrix bedre afspejler de<em&gt; In vivo</em&gt; Situation end to-dimensionelle motilitet assays. Brug matrix invasion analyser kombineret med konfokal scanning af fluorescens-mærkede celler, detaljerede oplysninger om invasion tilstande og de forskellige bidrag af førende versus følgende celler kan opnås.

Modellering og Imaging 3-Dimensional Collective Cell Invasion

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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