Name: quantification of breast cancer cell invasiveness using a three-dimensional (3d) model
Uploaded: 2014-06-11T13:00:00
Description: Watch this Scientific Journal Video about Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model at JoVE.com

This work was conducted with funds to M.B. from the Canadian Institutes of Health Research (CIHR) grant MOP 107972. M.B. is a recipient of a CIHR New Investigator Salary Award. D.C. is a recipient of studentships from the Translational Breast Cancer Research Unit and the CIHR Strategic Training Program in Cancer Research and Technology Transfer, London Regional Cancer Program. C.G. is recipient of studentships from the Translational Breast Cancer Research Unit, London Regional Cancer Program and from the CIHR- Strategic Training Program in Cancer Research and Technology Transfer. SGD.

1.. Tre-dimensionel Culture of Breast Cancer Celler i basalmembranmatrix (indlejringsprocessen Technique) <ol><li> Håndtering Matrigel basalmembranmatrix: optøs på is natten over ved 4 ° C. Basalmembranmatrix er flydende ved lav temperatur, men størkner ved stuetemperatur. Hold basalmembranmatrix på is (figurerne 1A-B). </li><li> Dæk Konfokal No.1 glasbund fad med 50 pi basalmembranmatrix ved at sprede den matrix, med spidsen af en P-200 Pipetman i en spiral mønster (figur 1C).</strong...

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Udviklingen af ​​3D cellekultur teknikker har tilladt forskerne at studere omdannelsen af ​​bryst epitelceller, giver os mulighed for at visualisere de dramatiske morfologiske ændringer. Udover at analysere celle invasion, kan enkelt-eller flercellede brystepitelceller sphæroider bruges til at vurdere ændringer i cellulær adhæsion, spredning, størrelse og basal-apikal polaritet. I modsætning til tidligere beskrevne metoder, hvor cellerne er overlejret med ECM 8, vores metode integrerer celler i...

Migration og invasion af individuelle eller kollektive celler er to kendetegnende for kræft, og der kræves til metastatisk spredning af kræftceller 1-4. Evnen af ​​cancerceller til at indlede metastase afhænger af deres evne til at migrere og invadere de tilgrænsende væv ved hjælp af invadopodia at nedbryde basalmembranen af ​​cellerne. Invadopodia er dynamiske actin-rige matrixnedbrydning fremspring, der muliggør nedbrydning af den ekstracellulære matrix gennem frigivelse af matrix-nedbrydende proteaser 5. Kræftcelle invasion involverer nedbrydningen af matrix efterfulgt af migrationen af de kræftceller, og det er ledsaget af en reorganisering af den tre-dimensionelle (3D) matrix miljø 2. Således at trænge gennem matrixen, skal en celle ændre sin form og interagerer med den ekstracellulære matrix (ECM) 2. Vedligeholdelsen af ​​brystvæv integritet afhænger stramt controlled vævsarkitekturen siden celle-ECM og celle-celle adhæsion vejkryds påvirke genekspression og forstyrrelse af epitelial polaritet kan føre til udbrud af kræft 6-10. Men de fleste in vitro migration og invasion assays, såsom Transwell kammer assays eller sår-scratch analyser er to-dimensionelle (2D) og dermed disse forsømmer de indviklede interaktioner mellem celler og deres tilstødende miljø 3,6,8,11-14. Betydelige morfologiske og funktionelle forskelligheder, herunder variationer i cellulær morfologi, cellulær differentiering, celle-matrix sammenvoksninger og genekspression mønstre er blevet opdaget ved dyrkning af celler i 3D-kulturer, der er almindeligt mangler i 2D-analyser 2,6,8,11. Således anvendelser af 3D-analyser er signifikant fordel i opridset en mere fysiologisk in vivo tilstand, der fører til en bedre oversættelse banebrydende resultater inden for grundforskning til klinikken 6-10. Det bør imidlertid bemærkes,På trods af de mange fordele, der opnås ved anvendelse af 3D-kulturer, kan denne model ikke fange alle de komplekse in vivo tumor mikromiljø, der omfatter forskellige celletyper. Det er imidlertid muligt at inkorporere stromaceller i 3D modeller (for eksempel fibroblaster, leukocytter og makrofager) til at studere virkningen af tumor-stroma interaktioner på kræft celleadhæsion og invasion 15-17. Breast epitelceller i kultur vokser mest effektivt, når ECM-proteiner, såsom laminin og kollagen er til stede. Med dette kendte, har et kommercielt tilgængeligt matrixblandingen stammer fra Engelbreth-Holm-Swarm (EHS) murin tumor og er kendt som Matrigel basalmembranmatrix 2,8. Der er etableret en række teknikker til at dyrke epitelceller som 3D kolonier i basalmembranmatrix 2,8. 3D basalmembranmatrix model er effektiv til oprettelse af både maligne og non-maligne bryst cellervækst, der ligner det, der sker i in vivo miljø 18,19. MCF10A celler er ikke-maligne brystepitelceller celler. Når der dyrkes i basalmembranmatrix, udviser disse celler in vivo træk af normale bryst celler og underkastes kontrolleret celleproliferation, celledifferentiering polarisering, og apoptose at etablere lumen plads 8,12,20. Desuden forekomsten af cellekerner fra MCF10A celler danner acini i 3D kulturer mere ligner dem af brystepitelceller celler i væv end dem dyrket i monolag 21.. Undersøgelser foretaget af Bissell og kolleger var de første til at afsløre, at maligne bryst celler kan skelnes fra ikke-maligne bryst celler, når de dyrkes i en laminin-rige omgivelser, da de maligne celler vise en meget uorganiseret fænotype, øget spredning, nedsat celle-til- celleadhæsion, forøget ekspression af mesenchymale markører og en stigning i antallet af invasive struktur dannet 3,6,22. Abnormiteter i cellen miljø kan påvirke tumordannelse 20. 3D kultur metode kan bruges til effektivt at studere kommunikation, der opstår mellem tumorceller og deres omgivende miljø og bestemme, hvordan protein udtryk påvirkninger såsom kommunikation 14,20,21,23. Denne artikel giver en detaljeret metode til at vokse MDA-MB-231 brystkræftceller i 3D kulturer at analysere invasiv og undersøge tabet af epitelial morfologi ved hjælp af en epitelial markør laminin, en komponent af cellen basalmembran 18,19,24, 25.. De detaljerede procedurer giver evnen til præcist og reproducerbart kvantificere stellate (invasive) strukturdannelse af enhver invasiv cancer celle og er ikke begrænsende til de fælles brystcancercellelinjer (såsom MDA-MB-231, Hs578T, MCF-7 eller T47D ). Således kan denne analyse fungere som en platform for at vurdere, hvordan protein-ekspression i celler eller behandling med pro-eller anti-invasive forbindelser regulerer ekstracellulær matrix nedbrydning ved enkelt eller flere celler.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Name of Material/ Equipment</td>
			<td style="width:265px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:355px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.5 mL tubes</td>
			<td>VWR</td>
			<td>CA10011-700</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">100-mm culture dish BD353003</td>
			<td>VWR</td>
			<td>CABD353003</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15 mL Falcon tube</td>
			<td>VWR</td>
			<td>CA21008-918</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1 mL filtered tips</td>
			<td>VWR</td>
			<td>10011-350</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-016</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20 uL filtered tips</td>
			<td>VWR</td>
			<td>22234-008</td>
			<td>Sterile, disposable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35-mm glass-bottomed Confocal No.1 culture dishes&#160;</td>
			<td style="width:265px;">MatTek Corporation</td>
			<td>P35G-1.0-14-C</td>
			<td>Precooled before use</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine serum albumin (BSA)</td>
			<td>BioShop</td>
			<td>ALB003.100</td>
			<td>Used at 3% for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 488 Goat Anti-Mouse IgG (H+L) Antibody, highly cross-adsorbed</td>
			<td>Life Technologies</td>
			<td>&#160;A11029</td>
			<td>1:250 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Alexa Fluor 568 Goat Anti-Rabbit IgG (H+L) Antibody</td>
			<td>Life Technologies</td>
			<td>&#160;A11011</td>
			<td>1:1200 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Anti-Beta-Catenin&#160;</td>
			<td>BD Transduction Laboratories</td>
			<td>610153</td>
			<td>Mouse-monoclonal; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td>Sigma</td>
			<td>F1051</td>
			<td>Used at 10% (v/v)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hoechst 33258, Pentahydrate (bis-Benzimide) - 10 mg&#8260;mL Solution in Water</td>
			<td>Life Technologies</td>
			<td>H3569</td>
			<td>Used at 0.1% (1:10000 dilution)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">InVivo Analyzer Suite&#160;</td>
			<td style="width:265px;">Media Cybernetics</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kisspeptin-10 (KP-10)</td>
			<td style="width:265px;">EZ Biolabs</td>
			<td style="width:119px;">PT0512100601</td>
			<td>Used at 100nM&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-Laminin&#160;</td>
			<td>Cedarlane</td>
			<td>AB19012(CH)</td>
			<td>Rabbit-polyclonal full length human; Used at 1:100 for IF</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LSM-510 META laser scanning microscope&#160;</td>
			<td style="width:265px;">Zeiss</td>
			<td style="width:119px;"></td>
			<td>Used at 63X objective; oil immersion lens</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Matrigel phenol red free (BD356237)</td>
			<td style="width:265px;">VWR</td>
			<td>CACB356237</td>
			<td>&#160;Lot No.2180819; 10.4 mg/mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus IX-81 microscope&#160;</td>
			<td style="width:265px;">Olympus</td>
			<td style="width:119px;"></td>
			<td>Used for 3D culture imaging (DIC images at 10x and 40x)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td>Antibiotic (added to media; used at 0.01%)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10 mL pipet&#160;</td>
			<td>VWR</td>
			<td>CA53300-523</td>
			<td>Sterile, disposeable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI 1640 Medium with Glutamine</td>
			<td>Life Technologies</td>
			<td>11875-119</td>
			<td>Used for culturing of MDA-MB-231 cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.25% Trypsin-EDTA (1X), Phenol Red</td>
			<td>Life Technologies</td>
			<td>25200-072</td>
			<td>Used to trypsinize MDA-MB-231 cells</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">MEGM (bullet kit): MEBM (CC3151)+Single quots (CC4136)</td>
			<td style="width:265px;">Lonza</td>
			<td style="width:119px;">CC-3150</td>
			<td>Used for culturing of MCF10A cells</td>
		</tr>
	</tbody>
</table>

quantification of breast cancer cell invasiveness using a three-dimensional (3d) model

Det er nu velkendt, at den cellulære og væv mikromiljø er kritiske regulatorer, der påvirker tumor initiering og progression. Desuden er den ekstracellulære matrix (ECM) er påvist at være en kritisk regulator af celle adfærd i kultur og homeostase in vivo. Den nuværende tilgang til dyrkning af celler på to-dimensionelle (2D), plastoverflader resulterer i forstyrrelse og tab af komplekse interaktioner mellem celler og deres mikromiljø. Gennem brug af tre-dimensionelle (3D) kultur assays, er betingelserne for celle-mikromiljø vekselvirkning etableret ligner in vivo mikromiljø. Denne artikel giver en detaljeret metode til at vokse brystcancerceller i en 3D-basalmembran proteinmatrix, eksemplificering potentiale 3D kultur i vurderingen af ​​celleinvasion i det omgivende miljø. Desuden diskuterer vi, hvordan disse 3D assays har potentiale til at undersøge tabet af signalering Molecbundmoduler der regulerer epitelial morfologi ved immunfarvning procedurer. Disse undersøgelser hjælpe til at identificere vigtige mekanistiske detaljer i de processer, der regulerer invasion, der er nødvendige for spredningen af ​​brystkræft.

Et eksempel på MDA-MB-231-celler invaderer i 3D matrix er vist i figur 3C. Cellerne er indlejret i matricen (dag 1), og begynder at danne invasive (stjerneformige) strukturer på dag 3, og helt invadere matrixen ved dag 5 (figur 3C). Tælles Antallet af stjerneformige dannede kolonier, og udtrykt som en procentdel af det samlede antal kolonier pr fad (invasive og non-invasive). Derudover, da målingerne er færdig dagligt i fem dage, satsen for invasion kan også evalueres. <p clas...

Watch this Scientific Journal Video about Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model at JoVE.com

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

Denne artikel indeholder detaljerede metoder til anvendelse af tre-dimensionelle (3D) assays at kvantificere brystkræft celle invasion. Konkret har vi diskutere de procedurer, der kræves for at oprette sådanne assays, kvantificering og dataanalyse, samt metoder til at undersøge tabet af membranintegritet der opstår, når celler invadere.

Kvantificering af Breast Cancer Cell invasiv Ved hjælp af en tre-dimensionel (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

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Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

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