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. Cultura tridimensionale delle cellule del cancro al seno in membrana basale Matrix (La Tecnica Embedment) <ol><li> Manipolazione Matrigel matrice della membrana basale: Scongelare su ghiaccio notte a 4 ° C. Matrice membrana basale è liquido a bassa temperatura ma solidifica a temperatura ambiente. Mantenere matrice membrana basale su ghiaccio (Figure 1A-B). </li><li> Coprire il piatto confocale No.1 fondo di vetro con 50 ml di matrice membrana basale mediante diffusione della matrice usando la punta di un Pipetman P-200 in un modello a spirale (Figura 1C). Prestare attenzione quando la diffusione della matrice membrana basale per evitare la formazione di bolle d&#39;aria. Allo stesso modo, evitare di diffondere la matrice di chiudere i confini del piatto fondo di vetro per evitare la formazione di menisco. Se matrice membrana movimentazione inesperto scantinato, poi preraffreddato il piatto, P-200 Pipetman e pipette può essere di ghiaccio (in alternativa lasciato una notte in frigorifero a 4 ° C). Questo passo offertetempo supplementare per diffondere la matrice membrana basale prima della solidificazione. Porre la capsula (es) in un incubatore di coltura cellulare (a 37 ° C con 5% CO 2) per almeno 30 minuti per consentire la matrice membrana basale a solidificare (Figura 1D). </li><li> Mentre la matrice è in fase di solidificazione, trypsinize un confluente 100 mm piastra 70-80% delle cellule. Una volta che le cellule hanno iniziato sollevamento fuori della piastra, risospendere in 10 ml di terreno RPMI 1640 contenente il 10% (v / v) di siero fetale bovino (FBS), per inattivare la tripsina (Figura 1E). Poi il trasferimento delle cellule risospese in un tubo da 15 ml (Figura 1F). </li><li> Spin le cellule (presenti nel tubo conico) a 100 xg per 3 minuti in una coltura cellulare centrifuga dedicato (Figure 1G-H). </li><li> Mentre le cellule vengono centrifugati, un&#39;aliquota di 50 ml di matrice in una provetta 1 ml (nota: ogni piatto fondo di vetro viene allocata una provetta, di conseguenza, sel&#39;esperimento richiede 3 piatti, ne consegue che 3 provette da microcentrifuga sono necessari pure), e quindi la provetta in ghiaccio (Figura 1B). </li><li> Aspirare il mezzo dal tubo conico dal punto 1.4, pur lasciando il pellet indisturbato (Figura 1I). Risospendere le cellule (che sono stati filati in giù) in 1 ml di FBS-integrato RPMI (Figura 1J). </li><li> Una volta che le cellule sono contate con un emocitometro (Figura 1K), o un contatore di un&#39;aliquota particella cella 2,5 x 10 4 cellule in provetta e ciliegina sulla torta con opportuno supporto in modo da ottenere il volume totale di 50 microlitri (Figura 1L). </li><li> Mescolare le cellule dal punto 1.7 (25.000 cellule in 50 microlitri) con il tubo per microcentrifuga matrice contenente da passo 1,5 in rapporto 1:1; volume finale sarà di 100 microlitri (Figura 1M). </li><li> Piastra delicatamente 100 ml di matrice: miscela di cellule dal punto 1.8 sulla sPiatto olidified membrana basale matrice rivestita dal punto 1.2 (Figura 1N). Questo permette alle cellule di essere incorporati in matrice membrana basale. </li><li> Trasferire il piatto (es) nell&#39;incubatore coltura cellulare (a 37 ° C con 5% CO 2) e lasciare la matrice: miscela di cellule solidificare per almeno 30 min. </li><li> Una volta che la matrice: miscela di cellule è solidificato, aggiungere 2 ml di media RPMI FBS-integrate per il piatto (Figura 1O) e prendere il piatto indietro l&#39;incubatrice dove verrà conservato per il resto dell&#39;esperimento (Figura 1P). </li><li> Cambiare media ogni giorno per un periodo di 5 giorni (o secondo la richiesta per un saggio, la durata del saggio per MDA-MB-231 cellule è di 5 giorni di lunghezza). </li><li> Utilizzando un microscopio ottico, prendere contrasto interferenziale differenziale (DIC) immagini delle MDA-MB-231 colonie sospese in matrice membrana basale (Figura 3A). Immagine 20 aree rappresentative a 10 volte più fine, una volta al giornoper cinque giorni per determinare colonia morfogenesi (Figura 3C). </li><li> Analizzare le immagini ciecamente per determinare colonia di cellule stellate formazione (Figura 3B). Una colonia è considerata stellate se una o più sporgenze dal sferoide di cellule sono percepiti. Per determinare la percentuale di colonie stellate invasive, dividere il numero di colonie stellate per il numero totale di colonie di cellule per immagine acquisita, e poi la media delle colonie stellate percentuali delle 20 immagini per ogni giorno. </li></ol><img alt="Figura 1" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig1highres.jpg" width="500" /> Figura 1. Cultura tridimensionale di MDA-MB-231 cellule di carcinoma mammario in matrice membrana basale (la tecnica incastro). AB) Uno schema del setup sperimentale (eseguita nella cappa). C) Il pozzo del piatto fondo di vetro rivestitocon 50 ml di matrice membrana basale. D) Lavastoviglie (es) posto in un incubatore di coltura cellulare (a 37 ° C con 5% CO 2) per consentire la matrice a solidificare per almeno 30 min. state tripsinizzate E) Cellule. F ) Le cellule sono state risospese in una provetta conica da 15 ml. GH) Cells (presenti nel tubo conico) sono stati centrifugati a 100 xg per 3 minuti in un tessuto cultura centrifuga. I) pellet. J) Cells (che sono stati centrifugati) sono stati risospesi in 1 ml di FBS-integrata mezzo RPMI. K) Le cellule sono state contate con un emocitometro. L) 2,5 x 10 4 cellule aliquotati in provetta e condito con appropriati mezzi di comunicazione in modo da ottenere il volume totale di 50 microlitri. M ) Mescolare cellule dal punto 1.7 (25.000 cellule in 50 microlitri) con il tubo per microcentrifuga matrice contenente da passo 1,5 in rapporto 1:1; volume finale sarà di 100 μ., L N) piastra delicatamente 100 ml di matrice: miscela di cellule dal punto 8 sul piatto matrice rivestite solidificato dal punto 1.2 O) Una volta che la matrice: miscela di cellule è solidificato, aggiungere 2 ml di mezzi di RPMI FBS-integrati a. il piatto. P) porre il recipiente in incubatrice dove sarà memorizzato per il resto dell&#39;esperimento. 2. Esame delle caratteristiche morfogenetiche delle Culture 3D con immunofluorescenza <ol><li> Impostare un vassoio di ghiaccio, fosfato freddo soluzione tamponata (PBS), 20% di acetone: 80% di metanolo (soluzione fissativo), e il 3% albumina sierica bovina (BSA, soluzione bloccante) (Figura 2A) prima di prendere il piatto (es) dall&#39;incubatore. </li><li> Porre la capsula (es) sul vassoio ghiaccio e aspirare media, poi lavare 3x con 2 ml di PBS freddo (Figura 2B). </li><li> Dopo l&#39;ultimo lavaggio PBS viene aspirato, aggiungere 2 ml di 20% acetone: soluzione di metanolo 80% nel piatto (es) (Figura 2C </strong&gt;) per fissare le cellule per 20 min a 4 ° C (sul ghiaccio, o in frigorifero) (Figura 2D). </li><li> Dopo il 20 min di fissazione è terminata, portare i piatti indietro a temperatura ambiente, aspirare il fissativo, e successivamente lavare 3x con PBS. Dopo l&#39;ultimo lavaggio PBS viene aspirato, aggiungere 2 ml di 3% BSA al piatto (es) per bloccare per almeno 30 minuti a temperatura ambiente (Figura 2E). </li><li> Durante il periodo di blocco preparare diluizioni di primaria (laminina V 1:100) e anticorpi secondari (ad una diluizione appropriata) disciolti in 3% di siero albumina bovina (BSA) tampone di bloccaggio. Si prega di notare che 400-500 microlitri della soluzione &#39;BSA anticorpo primario +&#39; deve essere aggiunto direttamente sulla matrice: miscela di cellule. </li><li> Una volta che il blocco di 30 min scaduta, aggiungere l&#39;anticorpo primario e incubare per almeno 1 ora a temperatura ambiente (Figura 2F). Si prega di notare che non lavaggi PBS devono essere eseguiti seguendo la 30 min blockinperiodo di g. </li><li> Al termine della fase 2.6, rimuovere la soluzione &#39;BSA + primario di anticorpi&#39;, e lavare 3x con PBS. </li><li> Aggiungere l&#39;anticorpo secondario disciolto in 3% BSA (ad una diluizione appropriata) direttamente sulla matrice: miscela di cellule, coprire i piatti e incubare per 1 ora a temperatura ambiente (figura 2G). </li><li> Rimuovere la soluzione &#39;BSA + anticorpo secondario&#39;, e lavare 3x con PBS. </li><li> Aggiungere 2 ml di Hoechst 33258 (1:10.000) disciolti in PBS per nuclei colorazione, e incubare per 5 min sotto un foglio di alluminio (o coprire con un contenitore opaco per limitare fotoscolorimento) (Figura 2H). </li><li> Una volta che la 5 min di incubazione con Hoechst 33258t sono scaduti, lavare il piatto (es) 5x con PBS. </li><li> Aggiungere mezzo di montaggio direttamente sulla matrice: miscela di cellule nel piatto confocale e coprire con coprioggetto vetro con cautela per evitare indurre eventuali interruzioni per l&#39;integrità delle colonie nel matri membrana basalex: miscela di cellule (Figura 2I). </li><li> Lasciare il piatto (es) per asciugare tutta la notte (o 24 ore) a temperatura ambiente (Figura 2I). Una volta asciutto, il piatto (es) può essere conservato a -20 ° C, ma si raccomanda che essi dovrebbero essere ripreso al più presto. </li><li> Acquisire le immagini utilizzando un microscopio a fluorescenza con opportune lunghezze d&#39;onda laser di anticorpi utilizzati (Figure 3D-E). </li></ol><img alt="Figura 2" fo:content-width="5in" src="/files/ftp_upload/51341/51341fig2highres.jpg" width="500" /> Figura 2. Esame delle culture 3D mediante immunofluorescenza. . A) Uno schema del setup sperimentale B) Dish (es) posto sulla vaschetta del ghiaccio e dei media aspirato; Successivamente le cellule sono state lavate tre volte con 2 ml di PBS freddo C) 2 ml di 20% acetone:. soluzione di metanolo 80% aggiunto nel piatto (es) D) Fissare le cellule per.20 min a 4 ° C (sul ghiaccio, o in frigorifero). E) 2 ml di 3% BSA aggiunto al piatto (es) per bloccare per almeno 30 minuti a temperatura ambiente. F) Una volta che il 30 min di blocco sono scaduti, aggiungere gli anticorpi primari e incubare per almeno 1 ora a temperatura ambiente G) anticorpi secondari disciolti in 3% BSA (ad una diluizione appropriata) sono stati aggiunti direttamente sulla matrice membrana basale:. miscela di cellule; . successivamente piatti coperti e incubate per 1 ora a temperatura ambiente H) 2 ml di Hoechst (1:10.000; disciolto in PBS) aggiunto alla dishe (i) e le cellule incubate per 5 minuti sotto un foglio di alluminio I) Soluzione di montaggio aggiunto direttamente. sulla matrice: miscela di cellule nel piatto confocale e il piatto è coperto con vetrino coprioggetti. Lavastoviglie (es) essicca per una notte (o 24 ore) a temperatura ambiente.

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Lo sviluppo delle tecniche di coltura cellulare 3D ha permesso ai ricercatori di studiare la trasformazione di cellule epiteliali mammarie, che ci permette di visualizzare i drammatici cambiamenti morfologici. Oltre ad analizzare invasione cellulare, sferoidi epiteliali mammarie singole o multicellulari possono essere usate per valutare i cambiamenti nella adesione cellulare, la proliferazione, la dimensione e basale-apicale polarità. In contrasto con metodologie precedentemente riportati in cui le cellule vengono sovr...

Migrazione e l&#39;invasione delle cellule individuali o collettivi sono due caratteristiche di cancro, e necessari per la diffusione metastatica delle cellule tumorali 1-4. La capacità delle cellule di cancro di avviare metastasi dipende dalla loro capacità di migrare e invadere nel tessuto limitrofo utilizzando invadopodia a degradare la membrana basale delle cellule. Invadopodia sono sporgenze degradazione matrice ricca di actina dinamiche che consentono la degradazione della matrice extracellulare attraverso il rilascio di proteasi matrice degradazione 5. Invasione cellula tumorale attraverso la degradazione della matrice e la migrazione delle cellule tumorali e questo è accompagnato da una riorganizzazione dell&#39;ambiente matrice tridimensionale (3D) 2. Così, per penetrare attraverso la matrice, una cella deve trasformare la sua forma e interagire con la matrice extracellulare (ECM) 2. Il mantenimento dell&#39;integrità del tessuto mammario dipende strettamente controlled architettura tissutale da giunzioni cellula-ECM e cellula-cellula adesione influenzano l&#39;espressione genica e la rottura della polarità epiteliale può provocare l&#39;insorgenza del cancro 6-10. Tuttavia, la maggior vitro migrazione e l&#39;invasione test in quali transwell saggi camera o saggi ferita-graffio sono bidimensionali (2D) e quindi queste trascurano le interazioni complesse tra le cellule e il loro ambiente adiacente 3,6,8,11-14. Diversità morfologiche e funzionali significativi, in particolare le variazioni di morfologia cellulare, differenziazione cellulare, adesioni cellula-matrice e modelli di espressione genica sono stati rilevati da coltura di cellule in colture 3D che sono comunemente carenti in 2D saggi 2,6,8,11. Così, utilizza delle analisi 3D sono significativamente utile ricapitolare in un più fisiologico in condizioni vivo, portando ad una migliore definizione di risultati innovativi nella ricerca di base alla clinica 6-10. Tuttavia, va notato, Nonostante i numerosi vantaggi ottenuti con l&#39;uso di colture 3D, questo modello non può catturare tutte le complessità del microambiente tumorale in vivo che comprende vari tipi di cellule. Tuttavia, è possibile incorporare cellule stromali nei modelli 3D (ad esempio, fibroblasti, leucociti e macrofagi) studiare l&#39;effetto delle interazioni tumore stromale sull&#39;adesione cellula tumorale e invasione 15-17. Cellule epiteliali mammarie in coltura crescono in modo più efficace quando le proteine ​​ECM come laminina e collagene sono presenti. Con questa nota, una miscela di matrice disponibile in commercio è stata derivata da Engelbreth-Holm-Swarm (EHS) tumore murino ed è noto come matrice della membrana Matrigel seminterrato 2,8. Un certo numero di tecniche sono state stabilite a crescere cellule epiteliali come colonie 3D a matrice membrana basale 2,8. Il piano seminterrato modello a matrice membrana 3D è efficace per stabilire delle cellule del seno sia maligno e non malignocrescita, simile a quello che sta succedendo nell&#39;ambiente in vivo 18,19. Cellule MCF10A sono cellule epiteliali mammarie non maligne. Quando coltivate in matrice membrana basale, queste cellule mostra a tratti vivo delle cellule normali del seno e sottoporsi controllata la proliferazione cellulare, la polarizzazione delle cellule, apoptosi e per stabilire lo spazio lume 8,12,20. Inoltre, la comparsa di nuclei di cellule di MCF10A formano acini in culture 3D avvicinano maggiormente a quelle delle cellule epiteliali mammarie in tessuto da quelle coltivate in monostrato 21. Gli studi di Bissell e colleghi sono stati i primi a rivelare che le cellule mammarie maligne possono essere differenziate da cellule della mammella non-maligne quando coltivate in un ambiente ricco di laminina, poiché le cellule maligne mostrano un fenotipo altamente disorganizzata, un aumento della proliferazione, diminuzione cellula- adesione cellulare, aumento dell&#39;espressione di marcatori mesenchimali e un aumento del numero di struttura invasiva formano 3,6,22. Anomalie del l&#39;ambiente cellulare possono influenzare la formazione del tumore 20. Il metodo di coltura 3D può essere usato per studiare efficacemente la comunicazione che avviene tra le cellule tumorali e l&#39;ambiente circostante e determinare come influenze espressione proteica tale 14,20,21,23 comunicazione. Questo articolo fornisce una metodologia dettagliata per crescere MDA-MB-231 cellule di carcinoma mammario in culture 3D per analizzare invasività, e per studiare la perdita di morfologia epiteliale utilizzando un marcatore epiteliale laminina, un componente della membrana basale cellulare 18,19,24, 25. Le procedure dettagliate consentono di quantificare con precisione e riproducibile stellato (invasive) formazione della struttura da qualsiasi cellula di cancro invasivo e non è limitante per le linee cellulari del cancro al seno comuni (come MDA-MB-231, Hs578T, MCF-7, o T47D ). Pertanto, questo test può servire come piattaforma per valutare come espressione della proteina in cellule o trattamento con pro o anti-composti invasive regolano la degradazione della matrice extracellulare, da cellule singole o multiple.

<table border="0" cellpadding="0" cellspacing="0" width="1062">
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			<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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>
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		<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

È ormai noto che il microambiente cellulare e tissutale sono regolatori critici che influenzano iniziazione e la progressione tumorale. Inoltre, la matrice extracellulare (ECM) è stato dimostrato per essere un regolatore critico del comportamento delle cellule in coltura e omeostasi in vivo. L&#39;attuale approccio di coltura di cellule in bidimensionale (2D), superfici plastiche risultati del disturbo e perdita di complesse interazioni tra le cellule e il loro microambiente. Attraverso l&#39;uso di tridimensionale (3D) saggi di coltura, sono stabilite le condizioni per l&#39;interazione cellula-microambiente simile microambiente in vivo. Questo articolo fornisce una metodologia dettagliata per far crescere le cellule del cancro al seno in una matrice 3D di proteine ​​della membrana basale, esemplificando il potenziale della cultura 3D nella valutazione di invasione delle cellule nell&#39;ambiente circostante. Inoltre, si discuterà di come questi test 3D hanno il potenziale per esaminare la perdita di segnalare molecduli che regolano la morfologia epiteliale da immunoistochimica procedure. Questi studi aiutano a identificare importanti dettagli meccanicistici nei processi che regolano l&#39;invasione, necessarie per la diffusione del cancro al seno.

Un esempio dei MDA-MB-231 cellule invasione nel matrice 3D è illustrato in Figura 3C. Le celle sono incorporate in una matrice (giorno 1), e cominciano a formare invasive (stellate) strutture dal giorno 3, e completamente invadono nella matrice da 5 ° giorno (Figura 3C). Il numero di colonie formate stellate sono contati, ed espresso come percentuale del numero totale di colonie per piastra (invasive e non invasive). Inoltre, poiché le misurazioni vengono effettuate giornalmente per ...

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

Questo articolo fornisce metodologie dettagliate per l&#39;utilizzo di tridimensionali (3D) test per quantificare l&#39;invasione delle cellule di cancro al seno. In particolare, discuteremo le procedure necessarie per configurare tali analisi, quantificazione e analisi dei dati, così come i metodi per esaminare la perdita dell&#39;integrità della membrana che si verifica quando le cellule invadono.

Quantificazione del cancro al seno Cellula Invasività Utilizzando una tridimensionale (3D) Modello

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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Research

JoVE Journal

Medicine

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

15.7K Views.  University of Western Ontario.  Questo articolo fornisce metodologie dettagliate per l'utilizzo di tridimensionali (3D) test per quantificare l'invasione delle cellule di cancro al seno. In particolare, discuteremo le procedure necessarie per configurare tali analisi, quantificazione e analisi dei dati, così come i metodi per esaminare la perdita dell'integrità della membrana che si verifica quando le cellule invadono. 

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

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

In Imaging bioluminescenza in vivo di Dinamica Tumore ipossia di metastasi cerebrali cancro al seno in un modello murino

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Ricerca

Medicina

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multi-modal Imaging di angiogenesi in un modello di ratto Nudo di metastasi ossee cancro al seno utilizzando la risonanza magnetica, tomografia computerizzata volumetrica e Ultrasuoni

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Eterotipica tridimensionale<em&gt; In Vitro</emModellazione&gt; di stromale-epiteliali interazioni Durante iniziazione e la progressione del cancro ovarico

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrini umani cella linee tumorali come un modello tridimensionale per lo studio dei diritti dell&#39;uomo e terapia dei tumori neuroendocrini

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

A Three-dimensional Tissue Culture modello per studiare Primario Osso Osseo e le sue Malignità

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Avvio del carcinoma metastatico della mammella dalla Targeting del duttale con Adenovirus-Cre: A Novel modello di topo transgenico di cancro al seno

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Profiling di microRNA regolati dagli estrogeni in cellule di carcinoma mammario

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Tre Culture dimensionali: strumento per studiare normale acinose Architettura vs trasformazione maligna delle cellule mammarie

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

Tridimensionale Co-cultura Modello di tumore stromale-Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensionale (3D) Tumore Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...