Gli autori ringraziano Chalid Ghadban per la sua eccellente assistenza tecnica, Andreas Wodarz e Stephan Heermann per la loro consulenza tecnica in materia confocale e microscopia time-lapse. Non c&#39;è conflitto di interessi per qualsiasi degli autori. Questo lavoro è finanziato dal Consiglio di ricerca tedesco (DFG) in Project 2 Forschergruppe 942 (FOR942 BI 703/3-1), dai Dres. Bayer-Stiftung (Baden Württembergischer Krebspreis, Germania) e dal Programma di Ricerca della Facoltà di Medicina, Georg-August-Universität Göttingen, Germania.

Questo nuovo modello è un adattamento di un approccio fetta cerebrale dell&#39;ippocampo organotipica pubblicato in precedenza 9-12. Modifiche e aggiunte sono state introdotte per ottimizzare le interazioni cellula-tessuti tumorali cerebrali e garantire la riproducibilità. Lo studio è stato esaminato e approvato dal comitato etico locale. Gli animali sono stati trattati con cura secondo le linee guida per la cura degli animali presso l&#39;Medicina dell&#39;Università di Göttingen. Il organotipica fetta cervello co-coltura può essere suddivisa in due fasi. Primo passo è la preparazione della fetta cervello organotipica. Fase due comprende la preparazione delle cellule tumorali e deposizione nel modello di co-coltura. 1. Organotipica Cervello Slice <ol><li> Preparare il mezzo dissezione costituito mezzo minimo essenziale (MEM), supplementato con 0,2 mM glutammina, 100 U / ml di penicillina, 100 mg / ml di streptomicina, e 4,5 mg / ml di glucosio. </li><li> Decapitare i topi da qualsiasi ceppo di topi tra giorno postnatalesei e otto (P6-8). </li><li> Rimuovere il cervello rapidamente dal cranio in condizioni asettiche e trasferirlo medio dissezione ghiacciata. </li><li> Rimuovere il polo frontale e cervelletto da tutta la sezione cervello. </li><li> Fissare e stabilizzare il cervello su un palco con colla crio e il 5% agarosio. </li><li> Tagliare sezioni cerebrali orizzontalmente per uno spessore di 350 micron utilizzando un vibratome. </li><li> Raccogliere 4-6 intere sezioni di cervello da un unico cervello di topo, a seconda della specie e dell&#39;età. </li><li> Preparare il terreno di coltura costituito da 50% MEM, soluzione salina bilanciata di Hanks 25% &#39;(HBSS), 25% siero di cavallo normale (NHS), glutammina 0,2 mM, 100 U / ml di penicillina, 100 mg / ml di streptomicina (Sigma, Monaco, Germania), e 4,5 mg / ml glucosio. </li><li> Mettere ogni fetta cerebrale organotipica su un micron policarbonato transwell inserto membrana 0.4 in una piastra sei pozzetti con 1 ml di mezzo di coltura nel pozzo inferiore. </li><li> Cultura organotipica fettine di cervello durante la notte in un humidified un&#39;atmosfera con 5% di CO 2 a 37 ° C incubatore. </li></ol> 2. Il modello Slice coculture <ol><li> Incorporare 10 5 di tumore GFP-trasfettate o di altre cellule (ad esempio cellule MCF-7-GFP) in 20 microlitri matrice del gel, composto del 15% RPMI medie e l&#39;85% gel ECM. </li><li> Posizionare il mix matrice MCF-7-gel in un distanziatore metallico sterile (diametro 3,8 millimetri) direttamente adiacente alla regione corticale del cervello fetta organotipica e incubare per 2 ore. </li><li> Rimuovere il distanziale e consentire al sferoide tumore 3D per co-coltura con la fetta organotipica per 24-96 ore. </li><li> Cambiare il terreno di coltura ogni altro giorno. </li></ol> 3. Immunofluorescenza degli astrociti e microglia nel cervello Organotipica Slice coculture <ol><li> Fissare il organotipica fetta cervello co-coltura con paraformaldeide 4% per 8 ore a 4 ° C. </li><li> Lavare la co-coltura fetta con PBST (PBS con 0,5% Triton X-100) per 5 min. </li><li> Bloccare le samples con siero normale di capra in PBST (1,20) a temperatura ambiente per 1 ora. </li><li> Macchiare gli astrociti incubando la co-coltura fetta cervello con fibrillare gliale anti-acido proteina monoclonale (GFAP, 1:200 in PBST) per 36 ore a 4 ° C, seguita da capra anti-topo-TRITC (1:100 in PBST) colorazione per 1 ora a temperatura ambiente. </li><li> Lavare i campioni con PBST tre volte per 5 min. </li><li> Colorare le cellule microgliali con ILB 4 Alexa Fluor 647 (1:100 in PBST) per 1 ora a temperatura ambiente. </li><li> Controcolorare la fetta coculture cervello con DAPI (1:1.000) per 3 minuti a temperatura ambiente. </li><li> Montare e coprioggetto la fetta coculture cervello con mezzo di montaggio DAKO fluorescente. </li><li> Valutare il grado di invasione tumorale sulla base della seguente sistema di punteggio: 0 = nessuna delle celle; + &lt;1/3; + + = 1/3 - 2/3; + + + ≥ 2/3 delle cellule invase (prima misurare la lunghezza della sezione di contatto tra la spina tumore e fetta, quindi misurare il frazionamenton di contatto rilevabile dalle cellule invasori). </li></ol> 4. Immagini dal vivo di interazione tra gliali e cellule tumorali <ol><li> Eseguire l&#39;esperimento sotto un microscopio invertito Leica DMI 6000B a 10 volte più lente di ingrandimento e una camera CCD 350 FX Leica DFC. </li></ol>

<ol>
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Non abbiamo nulla da rivelare.

Precedenti indagini istologiche di metastasi cerebrali hanno dimostrato rapidi e drastici cambiamenti delle cellule gliali residenti, in particolare di astrociti e microglia 13. Per studiare questi cambiamenti e le interazioni con le cellule di carcinoma, questo sistema di co-coltura romanzo è adatto. Altri campi di ricerca già hanno esperienza di lunga data con organotipica fettine di cervello di ippocampo. Un vantaggio è che i organotipica culture fetta dell&#39;ippocampo del cervello sono vitali ed effettivamente conservato per giorni o settimane, che lo rende adatto per esperimenti a lungo termine. Dall&#39;introduzione di Stoppini del sistema fetta dell&#39;ippocampo organotipica nel 1991, è stato ampiamente utilizzato, per esempio nella ricerca di malattie degenerative. Così, questo sistema di co-coltura innovare rappresenta una modifica di un approccio consolidata con una gamma di applicazioni in biologia tumorale 9,11. La modifica ci ha offerto un modello riproducibile per valutare il grado di invasione tumorale afterwards e colture cresciute dal metodo di interfaccia sono ideali per esperimenti che richiedono una struttura tridimensionale. Diverse tecniche sono state usate per coculture fettine ippocampali organotipiche con altre cellule. Questi includono un sistema indiretto tra macrofagi e la fetta cervello organotipica 14, e un co-coltura diretta di due sezioni diverse dalla regione dell&#39;ippocampo 15. Aggregati di glioma sono stati co-coltivate con fettine di cervello di 16. Questi modelli possono essere utilizzati per analizzare gli eventi cellulari e molecolari delle fettine di cervello, ma non consentono, il contatto fisiologico diretto tra le cellule tumorali, microglia e il parenchima cerebrale. Inoltre, questo metodo consente l&#39;osservazione di microglia, senza contaminazione di midollo osseo periferico monociti / macrofagi. L&#39;uso di CCR2 e CX3CR1 modello di topo transgenico è un miglioramento critico dovuto al fatto che è difficile distinguere la microglia residente di invadere monocitiin base alle loro proprietà simili 17,18,19. Anche se l&#39;iniezione intracerebrale di cellule di carcinoma permette di indagare la progressione del tumore, non si può dire molto sul fatto che le cellule macrofago-come circondate provengono dalla popolazione microglia cerebrale residenti o dal midollo osseo periferico monociti / macrofagi 17. Il team di Kettenmann introdotto un cervello modello fetta organotipica che ha coinvolto inoculando cellule di glioma in fettine di cervello con un micromanipolatore 20. Tuttavia, gliomi maligni primari differiscono per molti aspetti da carcinomi metastatici. In primo luogo, gliomi maligni sono di origine mesenchimale, non metastasi al di fuori del sistema nervoso, e migrano / invadere le cellule come singole senza confine tra tumore e tessuto cerebrale. Per contro, la crescita infiltrante è una caratteristica tipica patologico, e che tali carcinomi solito migrare / invadere come coorti. In secondo luogo, carcinomi spesso cercano di ricostruire le strutture epiteliali del cervello, mentre le cellule glialicercare di separare il tumore dal tessuto cerebrale da una (pseudo) capsula. Considerando le caratteristiche biologiche e morfologiche, glioma maligno e metastasi dei carcinomi non sono realmente comparabili. Per queste ragioni, abbiamo modificato e sviluppato un sistema di co-coltura dove non ci iniettiamo, ma co-coltura di una spina di cellule di carcinoma adiacente la fetta cervello. Inoltre, abbiamo osservato microglia e l&#39;accumulo di astrociti al confine della spina tumore, il che significa che la microglia entrare la spina tumore e potrebbe essere facilmente individuata mediante microscopia in campo chiaro e confermato successivamente mediante microscopia confocale. Le cellule tumorali invadono nella fetta cervello, che è paragonabile alla reale situazione in vivo e ad un&#39;osservazione di Baumert e colleghi, che hanno trovato una zona infiltrazione nel 63% dei casi autoptici con metastasi cerebrali 21. A causa della mancanza di perfusione di sangue, ci sono solo residenti macrofagi / microglia in questa cultura. Inoltre, a causa della mcellule Issing T e, di conseguenza, assente allo-reattività, cellule di carcinoma umane potrebbero essere utilizzati per co-coltura anche con fettine di cervello di topi immunocompetenti o ratti (NMRI, B6, o Wistar). Questo potrebbe servire come alternativa al modello di topo nudo. Da 4-5 fette possono essere ottenuti da ciascun topo, sono richiesti significativamente ridotto numero di animali, in confronto ai modelli iniezione esistenti. Inoltre, gli animali non soffrono per un lungo periodo di malattia metastatica e non sottoposti a procedure operative ripetutamente 22. Con questo sistema di co-coltura, abbiamo dimostrato l&#39;attivazione della microglia dalle cellule tumorali e la capacità di promuovere l&#39;invasione delle cellule del cancro. Inoltre, questa è la prima volta, a nostra conoscenza, sono stati trovati per trasportare attivamente cellule di carcinoma 23 microglia. Nonostante tutti questi vantaggi, il sistema di co-coltura ha, infatti, anche limitazioni. E &#39;ancora in vitromodellare mancano le fasi di metastasi prima della colonizzazione. A causa della mancanza di perfusione, non è possibile studiare la stravaso. Così, il modo alternativo di studiare la stravaso è di utilizzare il modello in vivo iniezione o il sistema di camera di Boyden modificata con matrice extracellulare, HUVEC e astrociti per imitare la barriera emato-encefalica 22,24. Il metodo coculture fetta cervello è ancora un modello affidabile e riproducibile con molti vantaggi e un potenziale per un&#39;ampia varietà di applicazioni, come l&#39;analisi di colonizzazione, in particolare con un focus sul ruolo delle cellule residenti. La combinazione con altre tecniche consolidate (come l&#39;immunoistochimica, microscopia confocale e microscopia time-lapse) sostiene la ricerca di interazioni dirette cellula-cellula. Si tratta di una alternativa semplice e complementazione di altre tecniche e offre l&#39;accesso alla ricerca di spunti e gli effetti come imposte dal microambiente metastatico.

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>Hank&#39;s balanced salt solution (HBSS)</td>
			<td>Gibco, Darmstadt,Germany</td>
			<td>24020</td>
			<td></td>
		</tr>
		<tr>
			<td>Minimum essential medium (MEM)</td>
			<td>Gibco, Darmstadt , Germany</td>
			<td>32360</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640</td>
			<td>PAA Laboratories Inc., C&#246;lbe, Germany</td>
			<td>E15-840</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (PBS)</td>
			<td>Pan Biotech, Aidenbach, Germany</td>
			<td>P04-36500</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal horse serum (NHS)</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>16050-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>10091148</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>B. Braum, Melsungen, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine-Penicillin-Streptomycin solution</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>G1146</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>Leica VT1200S</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>Leica SM 2000R</td>
			<td></td>
		</tr>
		<tr>
			<td>Polycarbonate membrane</td>
			<td>BD Falcon, Heidelberg,Germany</td>
			<td>353090</td>
			<td>transwell membrane insert (0.4 &#956;m pore size)</td>
		</tr>
		<tr>
			<td>ECM gel</td>
			<td>Trevigen, R&#38;D, Wiesbaden-Nordenstadt, Germany</td>
			<td>3432-005-01</td>
			<td>Basement membrane extract</td>
		</tr>
		<tr>
			<td>Metallic spacer</td>
			<td>Kig GmbG, Kirkel, Germany</td>
			<td>DIN 433</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal microscope</td>
			<td>Zeiss, G&#246;ttingen, Germany</td>
			<td>LSM 510</td>
			<td></td>
		</tr>
		<tr>
			<td>Time-lapse microscope and camera</td>
			<td>Leica, Wetzlar, Germany</td>
			<td>DMI 6000B microscope and a DFC 350 FX CCD camera</td>
			<td></td>
		</tr>
		<tr>
			<td>Anatomy microscope</td>
			<td>Zeiss, G&#246;ttingen, Germany</td>
			<td>Stemi SV11</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Merck, Darmstadt, Germany</td>
			<td>1.04005.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse monoclonal anti-glial fibrillary acidic protein (GFAP) antibody</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>G3893</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat anti-mouse IgG, F(ab&#39;)2- TRITC</td>
			<td>Santa Cruz, Heidelberg, Germany</td>
			<td>SC3796</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolectin GS-IB4 from Griffonia simplicifolia, Alexa Fluor 647 conjugate</td>
			<td>Invitrogen, Karlsruhe, Germany</td>
			<td>132450</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI (4&#39;,6&#39;-diamidino-2-phenylindole dihyfrochloride)</td>
			<td>Sigma, Steinheim, Germany</td>
			<td>D8417</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent mounting medium</td>
			<td>DAKO, Glostrup, Denmark</td>
			<td>S3023</td>
			<td></td>
		</tr>
	</tbody>
</table>

coculture system with an organotypic brain slice and 3d spheroid of carcinoma cells

I pazienti con metastasi cerebrali di carcinomi hanno una prognosi sfavorevole. Tuttavia, il processo nel sito metastatico è appena stata studiata, in particolare, il ruolo del (stromali) cellule residente. Studi in carcinomi primari dimostrano l&#39;influenza del microambiente sulla metastasi, anche sulla prognosi 1,2. Soprattutto i macrofagi associati al tumore (TAM) migrazione di sostegno, l&#39;invasione e la proliferazione 3. È interessante notare che i maggiori siti bersaglio di metastasi possiedono macrofagi tessuto-specifici, come le cellule di Kupffer nel fegato o microglia nel SNC. Inoltre, i siti metastatici possiedono anche altre cellule tessuto-specifici, come astrociti. Recentemente, gli astrociti sono state dimostrate per favorire la proliferazione e la persistenza di cellule tumorali 4,5. Pertanto, le funzioni di questi tipi di cellule tessuto-specifiche sembrano essere molto importante nel processo di metastasi cerebrali 6,7. Nonostante queste osservazioni,Tuttavia, fino ad ora non c&#39;è idoneo modello in vivo / in vitro disposizione di visualizzare direttamente reazioni gliali durante la formazione di metastasi cerebrale, in particolare mediante microscopia in campo chiaro. Recente in vivo imaging dal vivo delle cellule di carcinoma dimostrato il loro comportamento colonizzazione cerebrale 8. Tuttavia, questo metodo è molto laboriosa, costosa e tecnicamente complessa. Inoltre, questi tipi di esperimenti sugli animali sono limitate a piccole serie e sono dotati di una notevole sollecitazione per gli animali (per impiantazione della lastra di vetro, iniezione di cellule tumorali, anestesia ripetitivo e fissazione a lungo termine). Inoltre, l&#39;imaging in vivo è finora limitata alla visualizzazione delle cellule di carcinoma, mentre non sono ancora stati illustrati interazioni con cellule residenti. Infine, ricerche di cellule di carcinoma umani all&#39;interno animali immunocompetenti sono impossibili 8. Per queste ragioni, abbiamo stabilito un sistema di co-coltura consipungiglione di una fetta cervello organotipica mouse e cellule epiteliali incorporato in matrigel (sfera di cellule 3D). Le sfere di cellule di carcinoma in 3D sono stati collocati direttamente accanto al bordo fetta cervello al fine di indagare l&#39;invasione del tessuto cerebrale adiacente. Questo ci permette di visualizzare i cambiamenti morfologici e le interazioni tra le cellule gliali e cellule di carcinoma di fluorescenza e anche mediante microscopia in campo chiaro. Dopo l&#39;esperimento di co-coltura, il tessuto cerebrale o sferoidi cellulari 3D possono essere raccolti e utilizzati per ulteriori analisi molecolari (ad es qRT-PCR, IHC, o immunoblot) nonché per indagini mediante microscopia confocale. Questo metodo può essere applicato per monitorare gli eventi all&#39;interno di un tessuto cerebrale vivente per giorni senza effetti deleteri verso le sezioni di cervello. Il modello permette anche la soppressione selettiva e sostituzione di cellule residenti da cellule da un tessuto donatore per determinare l&#39;impatto distinto di un dato genotipo. Infine, il modello di co-coltura è un&#39;alternativa praticabilein vivo approcci durante il test manipolazioni farmacologiche mirate.

In primo luogo, tutte le cellule di carcinoma utilizzate (umane: MCF-7 e murine: 410,4) invasero la fetta cervello di topo organotipica. L&#39;invasione era quindi specie-indipendenti (Figura 1A), che indica una vasta gamma di applicazioni in modo specie-indipendente. Inoltre, microglia e astrociti accumulati all&#39;interfaccia come precedentemente descritto in vivo e in 13 campioni di pazienti. Time-lapse imaging per un periodo prolungato di tempo ha rivelato non solo la vitalità della fetta, ma anche offerto una buona piattaforma per osservare le interazioni cellulari. Con esperimenti di time-lapse, cellule microgliali sono stati registrati per entrare nella sfera cellulare, mentre in 3D, a sua volta, le cellule tumorali hanno invaso anche la fetta cervello (Figure 1B1-B2). Inoltre, abbiamo precedentemente dimostrato la capacità di microglia trasportare cellule di carcinoma da un meccanismo ancora enigmatico perciò aiutare nell&#39;invasione carcinoma. Utilizzando tecniche di etichettatura immunofluorescenza, abbiamodescritto co-localizzazione delle cellule tumorali e cellule stromali (es microglia e astrociti), sia nel tessuto cerebrale e nella spina delle cellule tumorali (Figure 1C-D), suggerendo una stretta interazione tra queste cellule durante il processo di invasione. Risultati analoghi sono stati ottenuti quando fettine di cervello di topo sono state co-coltivate sia con umano (figure 1C1-C3) o murina (Figure 1D1-D3) cellule di carcinoma - mostra la vasta gamma di applicazioni per lo studio delle cellule provenienti da specie diverse, genotipi e ceppi. <img alt="Figura 1" src="/files/ftp_upload/50881/50881fig1.jpg" fo:content-width="4.75in" fo:src="/files/ftp_upload/50881/50881fig1highres.jpg" /> Figura 1. Cervello fetta modello di co-coltura con uno sferoide 3D ​​di topo e cellule di carcinoma umano. A) Quantificazione del cancro invasione delle cellule in organotipica intere co-colture fetta cervello con il cancro al seno del mouse410.4 linea cellulare o una linea cellulare di carcinoma mammario umano MCF-7. I dati rappresentano la percentuale del grado di invasione cellulare in fetta cervello in ciascun gruppo con n ≥ 38. Non vi era alcuna differenza significativa tra i due gruppi (test di Kruskal-Wallis). B) time-lapse immagini di organotipica intere co-colture fetta cervello con le cellule MCF-7-GFP e immagini rappresentative di day2 (B1) e il giorno 5 (B2) sono stati mostrato. immagini di microscopia CD) confocale hanno mostrato coorti di carcinomi, astrociti e microglia. Doppia colorazione di astrociti (anti-GFAP-TRITC, rosso) e microglia (ILB4-Alexa Fluor 647, violetto) dell&#39;intera co-coltura con la fetta cervello 350 micron di spessore organotipico e la spina cellula tumorale GFP-transfettate (verde): 3D- MCF-7-GFP (C1-C3) e 3D-410.4-GFP (D1-D3). Trattini bianchi che il bordo della fetta cervello e la parte anteriore invasione tumorale. Barre di scala rappresentano 50 micron. Microglia, astrociti unnd tumori colocalizations (C1 e D1), astrociti-tumori colocalizations (C2 e D2) e colocalizations microglia-tumorali (C3 e D3) nella co-coltura fetta. <a href="https://www.jove.com/files/ftp_upload/50881/50881fig1highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita</a>

Watch this Scientific Journal Video about Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells at JoVE.com

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Il organotipico coculture fetta cervello con cellule di carcinoma consente la visualizzazione cambiamenti morfologici mediante fluorescenza e campo chiaro (video) microscopia durante il processo di carcinoma invasione delle cellule del tessuto cerebrale. Questo modello di sistema permette anche approcci di scambio cellulare e di rifornimento e offre una vasta gamma di manipolazioni e analisi.

Co-coltura di sistema con un Organotipica cervello Slice e 3D Spheroid delle cellule di carcinoma

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

coculture-system-with-an-organotypic-brain-slice-3d-spheroid

Research

JoVE Journal

Medicine

20.5K Views.  University of Göttingen.  Il organotipico coculture fetta cervello con cellule di carcinoma consente la visualizzazione cambiamenti morfologici mediante fluorescenza e campo chiaro (video) microscopia durante il processo di carcinoma invasione delle cellule del tessuto cerebrale. Questo modello di sistema permette anche approcci di scambio cellulare e di rifornimento e offre una vasta gamma di manipolazioni e analisi. 

Video: Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Mammary Transplantation of Stromal Cells and Carcinoma Cells in C57BL/6J Mice

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular through transplantation of stromal cells and epithelial cells in the mammary gland of mice. Current transplantation models often involve the use of immunocompromised mice due to the different genetic backgrounds of stromal cells and epithelial cells. Extracellular matrices are often used to embed the two different cell types for consistent cell-cell interactions, but involve the use of Matrigel or rat tail collagen, which are immunogenic substrates. The lack of functional T cells from immunocompromised mice prevents accurate assessment of stromal cells on mammary tumor progression in vivo, with important implications on drug development and efficacy. Moreover, immunocompromised mice are costly, hard to breed and require special care conditions. To overcome these obstacles, we have developed an approach to orthotopically transplant stromal cell and epithelial cells into mice from the same genetic background to induce consistent tumor formation. This system involves harvesting normal, carcinoma associated fibroblasts, PyVmT mammary carcinoma cells and collagen from donor C57BL/6J mice. The cells are then embedded in collagen and transplanted in the inguinal mammary glands of female C57BL/6J mice. Transplantation of PyVmT cells alone form palpable tumors 30-40 days post transplantation. Endpoint analysis at 60 days indicates that co-transplantation with fibroblasts enhances mammary tumor growth compared to PyVmT cells transplanted alone. While cells and matrix from C57BL/6J mice were used in these studies, the isolation of cells and matrix and transplantation approach may be applied towards mice from different genetic backgrounds demonstrating versatility. In summary, this system may be used to investigate molecular interactions between stromal cells and epithelial cells, and overcomes critical limitations in immunocompromised mouse models.

In this report, we demonstrate a system to isolate and culture donor cells from the mouse mammary gland, and orthotopically transplant these cells in recipient mice to analyze stromal: epithelial interactions during mammary tumor development.

Trapianto mammaria di cellule stromali e cellule di carcinoma in topi C57BL/6J

The influence of stromal cells, including fibroblasts on mammary tumor progression has been well documented through the use of mouse models, in particular ...

Ricerca

Medicina

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Un modello di mouse ortotopico di anaplastico della tiroide Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

Valutazione del cancro ovarico Spheroid attaccamento e l&#39;invasione delle cellule mesoteliali in tempo reale

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

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 ...

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Un sistema organotipica High Throughput per la caratterizzazione di droga sensibilità di primaria multicampo cellule del mieloma

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Un tumore cerebrale / organotipica fetta sistema di co-coltura per lo Studio microambiente tumorale e mirata terapie farmacologiche

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

Un Cancer Cell Saggio sferoide valutare Invasion in un Ambiente 3D

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

A Human Glioblastoma Organotypic Slice Culture Model for Study of Tumor Cell Migration and Patient-specific Effects of Anti-Invasive Drugs

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor invasion into surrounding brain parenchyma represents an enduring therapeutic challenge. To develop anti-migration therapies for GBM, model systems that provide a physiologically relevant background for controlled experimentation are essential. Here, we present a protocol for generating slice cultures from human GBM tissue obtained during surgical resection. These cultures allow for ex vivo experimentation without passaging through animal xenografts or single cell cultures. Further, we describe the use of time-lapse laser scanning confocal microscopy in conjunction with cell tracking to quantitatively study the migratory behavior of tumor cells and associated response to therapeutics. Slices are reproducibly generated within 90 min of surgical tissue acquisition. Retrovirally-mediated fluorescent cell labeling, confocal imaging, and tumor cell migration analyses are subsequently completed within two weeks of culture. We have successfully used these slice cultures to uncover genetic factors associated with increased migratory behavior in human GBM. Further, we have validated the model&#39;s ability to detect patient-specific variation in response to anti-migration therapies. Moving forward, human GBM slice cultures are an attractive platform for rapid ex vivo assessment of tumor sensitivity to therapeutic agents, in order to advance personalized neuro-oncologic therapy.

Current ex vivo models of glioblastoma (GBM) are not optimized for physiologically relevant study of human tumor invasion. Here, we present a protocol for generation and maintenance of organotypic slice cultures from fresh human GBM tissue. A description of time-lapse microscopy and quantitative cell migration analysis techniques is provided.

Un modello di cultura organica fetta di Glioblastoma umano per lo studio della migrazione delle cellule tumorali e degli effetti specifici del paziente di farmaci antiinvasivi

Glioblastoma (GBM) continues to carry an extremely poor clinical prognosis despite surgical, chemotherapeutic, and radiation therapy. Progressive tumor ...

Symmetric Bihemispheric Postmortem Brain Cutting to Study Healthy and Pathological Brain Conditions in Humans

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena described in those patients for whom a brain autopsy has been requested. Although the advancements of biomedical sciences and neuroimaging have revolutionized the neuropsychiatric field, they have also generated the misleading idea that brain autopsies have only a confirmatory value. This false idea created a drastic reduction of autopsy rates and, consequently, a reduced possibility to perform more detailed and extensive neuropathological investigations, which are necessary to comprehend numerous normal and pathological aspects yet unknown of the human brain. The traditional inferential method of correlation between observed neuropsychiatric phenomena and corresponding localization/characterization of their possible neurohistological correlates continues to have an undeniable value. In the context of neuropsychiatric diseases, the traditional clinicopathological method is still the best possible methodology (and often the only available) to link unique neuropsychiatric features to their corresponding neuropathological substrates, since it relies specifically upon the direct physical assessment of brain tissues. The assessment of postmortem brains is based on brain cutting procedures that vary across different neuropathology centers. Brain cuttings are performed in a relatively extensive and systematic way based on the various clinical and academic contingencies present in each institution. A more anatomically inclusive and symmetric bi-hemispheric brain cutting methodology should at least be used for research purposes in human neuropathology to coherently investigate, in depth, normal and pathological conditions with the peculiarities of the human brain (i.e., hemispheric specialization and lateralization for specific functions). Such a method would provide a more comprehensive collection of neuropathologically well-characterized brains available for current and future biotechnological and neuroimaging techniques. We describe a symmetric bi-hemispheric brain cutting procedure for the investigation of hemispheric differences in human brain pathologies and for use with current as well as future biomolecular/neuroimaging techniques.

Organized brain cutting procedures are necessary to correlate specific neuropsychiatric phenomena with definitive neuropathologic diagnoses. Brain cuttings are performed differently based on various clinico-academic contingencies. This protocol describes a symmetric bihemispheric brain cutting procedure to investigate hemispheric differences in human brain pathologies and to maximize current and future biomolecular/neuroimaging techniques.

Simmetrica Bihemispheric Postmortem cervello taglio per studiare sano e patologico del cervello Condizioni di esseri umani

Neuropathologists, at times, feel intimidated by the amount of knowledge needed to generate definitive diagnoses for complex neuropsychiatric phenomena ...

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.
This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 &#181;m thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.
OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.