Name: mame models for 4d live-cell imaging of tumor: microenvironment interactions that impact malignant progression
Uploaded: 2012-02-17T12:00:00
Description: Watch this Scientific Journal Video about MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression at JoVE.com

Dette arbejde blev støttet, delvist af National Institutes of Health R01 CA131990 (BFS og RRM). Live-cell imaging blev udført i Mikroskopi, Imaging og Cytometry Ressourcer støttet Core, dels ved NIH center tilskud P30 CA22453 til Karmanos Cancer Institute, Wayne State University og den Perinatology Research Branch of the National Institutes of Child Health og Udvikling , Wayne State University.

1. Fremstille DQ-substrater <ol><li> Tillad lyofiliseret DQ-substrater til at opvarme til stuetemperatur, før åbning hætteglas, fremstilles stamopløsning af 1 mg / ml DQ-substrat i deioniseret vand, opdeles i 50 pi aliquoter og opbevares ved 4 ° C. </li></ol> Det kan være nødvendigt at omrøre DQ-substrat i et ultrasonisk vandbad i ~ 5 minutter og opvarmes til 50 ° C for at lette dispergering. <ol start="2"><li> Tø rbM på is natten over ved 4 ° C; rbM skal håndteres på...

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	<li>Sameni, M., Dosescu, J., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+proteolysis:+stromal+and+inflammatory+cells+increase+tumor+proteolysis.">Functional imaging of proteolysis: stromal and inflammatory cells increase tumor proteolysis.</a> Mol. Imaging. 2, 159-175 (2003).</li><li>Jedeszko, C., Sameni, M., Olive, M. B., Moin, K., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+protease+activity+in+living+cells:+from+two+dimensions+to+four+dimensions.">Visualizing protease activity in living cells: from two dimensions to four dimensions.</a> Curr. Protoc. Cell Biol. 39, (2008).</li><li>Jedeszko, C., Victor, B. C., Podgorski, I., Sloane, B. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+hepatocyte+growth+factor+promotes+invasion+of+human+mammary+ductal+carcinoma+in+situ.">Fibroblast hepatocyte growth factor promotes invasion of human mammary ductal carcinoma in situ.</a> Cancer Res. 69, 9148-9155 (2009).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+live-cell+imaging+demonstrates+that+beta1-integrin+promotes+type+IV+collagen+degradation+by+breast+and+prostate+cancer+cells.">Functional live-cell imaging demonstrates that beta1-integrin promotes type IV collagen degradation by breast and prostate cancer cells.</a> Mol. Imaging. 7, 199-213 (2008).</li><li>Li, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p21-activated+kinase+1+coordinates+aberrant+cell+survival+and+pericellular+proteolysis+in+a+three-dimensional+culture+model+for+premalignant+progression+of+human+breast+cancer.">p21-activated kinase 1 coordinates aberrant cell survival and pericellular proteolysis in a three-dimensional culture model for premalignant progression of human breast cancer.</a> Neoplasia. 10, 314-328 (2008).</li><li>Sameni, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+quantifying+the+dynamics+of+tumor+associated+proteolysis.">Imaging and quantifying the dynamics of tumor associated proteolysis.</a> Clin. Exp. Metastasis. 26, 299-309 (2009).</li><li>Cavallo-Medved, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+demonstrates+that+proteases+in+caveolae+of+endothelial+cells+degrade+extracellular+matrix.">Live-cell imaging demonstrates that proteases in caveolae of endothelial cells degrade extracellular matrix.</a> Exp. Cell. Res. 315, 1234-1246 (2009).</li><li>Fukumura, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+microvasculature+and+microenvironment:+novel+insights+through+intravital+imaging+in+pre-clinical+models.">Tumor microvasculature and microenvironment: novel insights through intravital imaging in pre-clinical models.</a> Microcirculation. 17, 206-225 (2010).</li></ol>

Som vist kan MAME cokulturer anvendes til levende celler billeddannelse i realtid af interaktioner mellem de forskellige cellulære bestanddele, der omfatter en brysttumor og mikromiljø. I igangværende undersøgelser i vores laboratorium, har vi brugt MAME cokulturer til at identificere proteolytiske veje er forbundet med overgangen fra DCIS til invasive duktalt karcinom, samt samspillet mellem proteolytiske veje og andre stier, der er involveret i denne overgang som kemokin / cytokin / vækstfaktor veje . Vi viste de...

<table border="1"><tbody><tr><td> Reagent / Udstyr </td><td> Firma </td><td> Katalognr </td><td> Kommentarer </td></tr><tr><td> Rekonstitueret basalmembran (Cultrex) </td><td> Trevigen </td><td> 3445-005-01 </td><td> Sammenlignes med Matrigel (BD Biosciences) </td></tr><tr><td> Kollagen I </td><td> Samhørighedsfonden Laboratories </td><td> 5005-B </td><td></td></tr><tr><td> DQ-substrater (Kollagen I, IV) </td><td> Invitrogen </td><td> DQ-col I-D12060 DQ-col IV D12052 </td><td></td></tr><tr><td> 22 mm plastik dækglas </td><td> Fisher Scientific Co </td><td> 12-547 </td><td> Skær i halve, lade i 70% ethanol i 10 minutter og luft-tørre før brug. </td></tr><tr><td> Cellekulturmedium </td><td> Lonza </td><td> MEBM-PRF CC-3153 MEGM CC-4136 </td><td> Phenolrødt-frit </td></tr><tr><td> ibiTreat μ-Dish </td><td> Ibidi </td><td> 80136 </td><td></td></tr><tr><td> Volocity software </td><td> Perkin-Elmer </td><td> Version 5,5 </td><td> Andre mærker af imaging software kan bruges til at generere 3D-rekonstruktioner og film. </td></tr></tbody></table>

mame models for 4d live-cell imaging of tumor: microenvironment interactions that impact malignant progression

Vi har udviklet 3D cokultur modeller, som vi kalder MAME (m ammary en rchitecture og m icroenvironment e ngineering), og brugte dem til live-cell imaging i real-tid celle: celle interaktioner. Vores overordnede mål var at udvikle modeller, der opsummere arkitektur preinvasive brystlæsioner at studere deres progression til en invasiv fænotype. Specifikt har vi udviklet modeller til at analysere interaktioner mellem præ-maligne brystepithelceller cellevarianterne og andre celletyper i tumormikromiljøet, som er blevet impliceret i at øge eller reducere progressionen af ​​preinvasive brystepithelceller celler til invasive duktale carcinomer. Andre celletyper undersøgt til dato, er myoepitelcellerne, fibroblaster, makrofager og blod-og lymfekar mikrovaskulære endotelceller. Ud over de Mame modeller, der er udformet til at rekapitulere de cellulære interaktioner inden for brystet underudviklingen af ​​kræft, har vi udviklet sammenlignelige modeller for udviklingen af ​​prostatakræft. Her viser vi de nærmere vilkår for oprettelse af 3D cokulturer sammen med brugen af live-cell imaging og et funktionelt proteolyse analyse til at følge overgang cokulturer af bryst duktalt carcinoma in situ (DCIS) celler og fibroblaster til en invasiv fænotype over tid, i dette tilfælde i tyve tre dage i kultur. Den MAME cokulturer består af flere lag. Fibroblaster er indlejret i det nederste lag af type I collagen. Den, der er placeret et lag af rekonstitueret basalmembran (rbM), som DCIS celler podes. En endelig øverste lag af 2% rbM er inkluderet, og efterfyldes med hver ændring af medier. At afbilde proteolyse forbundet med progression til en invasiv fænotype, anvender vi farvestof-bratkølede (DQ) fluorescerende matrixproteiner (dq, collagen I blandet med lag af kollagen I og DQ-collagen IV blandes med det midterste lag af rBM) og observere levende kulturer med konfokal mikroskopi. Optiske sektioner er fanget, behandlet og rekonstrueret i 3D med Volocity visualisering software. I løbet af 23 dage i MAME cokulturer, prolifererer DCIS cellerne og smelter sammen til store invasive strukturer. Fibroblaster migrere og bliver indarbejdet i disse invasive strukturer. Fluorescerende proteolytiske fragmenter af collagener findes i association med overfladen af ​​DCIS strukturer intracellulært, og også dispergeret i den omgivende matrix. Lægemidler, der target proteolytisk, chemokin / cytokin og kinase pathways eller modifikationer i den cellulære sammensætning cokulturer kan reducere invasivitet, hvilket antyder, at Mame modeller kan anvendes som prækliniske skærme for nye terapeutiske metoder.

Watch this Scientific Journal Video about MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression at JoVE.com

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

Vi har udviklet 3D cokultur modeller for live-cell imaging i real-tid interaktioner mellem bryst tumorceller og andre celler i deres mikromiljø, der påvirker progression til en invasiv fænotype. Disse modeller kan tjene som prækliniske skærme for lægemidler til at målrette paracrine-inducerede proteolytiske, chemokin / cytokin og kinase veje involveret i invasiv.

Mame Modeller for 4D live-cell imaging af tumor: mikromiljø interaktioner, Impact malignt Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

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