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1. clonogenica <ol><li> Preparare una singola sospensioni di cellule di linee cellulari di cancro al seno estrogeno-dipendenti MCF-7 e T47D cellule utilizzando la procedura descritta di seguito <ol><li> Aspirare il mezzo di coltura (DMEM / 10% di calore inattivato siero fetale bovino / glutammina e pen / strep) da un tessuto piatto le colture 10 cm, non confluenti superiore al 50% con MCF-7 o cellule T-47D. Risciacquare con PBS. Incubare con tripsina / EDTA 0,25% 2,21 mM disciolto in DMEM alto glucosio a 37 ° C per 1-4 min. </li><li> Controllare cellule a intervalli di 1 minuto sotto un microscopio a contrasto di fase per garantire una distribuzione singola cellula. Risospendere le cellule con una pipetta 2 ml pipettando su e giù parecchie volte per interrompere il contatto cellula-cellula per ottenere un, lo stato della cella singola quasi invariabile. </li><li> Continuare a incubare cellule in tripsina a 37 ° C per un massimo di 4 minuti se si osserva grumi di cellule dopo soli 2 minuti di incubazione. Non utilizzare queste cellule per studi clonogeniche se rimangono aderenti alla postaach altro dopo 4 minuti di tripsinizzazione perché sarà introdotto errore nella resa numero di colonia. NOTA: Se le cellule sono raggruppate, il numero di colonie formate rifletterà il prodotto di un minor numero di cellule rispetto al numero incubata. Se le cellule sono eccessivamente tripsinizzate, il loro potenziale clonogenico può essere ridotta. </li><li> Preparare una sospensione di cellule singole di 1500 cellule / ml terreno di coltura per 24 pozzetti, o meno, (+ 500 cellule / ml, a seconda del tipo di cellula o numero di passaggio), per diluizioni seriali in un tubo master contenente l&#39;intero volume necessario per tutte le variabili nell&#39;esperimento. NOTA: L&#39;obiettivo è una densità cellulare finale di 800 cellule / cm 2 (gamma di circa 500 a 1.100 cellule / cm 2). L&#39;obiettivo è di produrre circa 100 + 50 colonie, che permette relativamente facile conteggio, impedisce affollamento e permette colonie sufficienti per portare a differenze statistiche significative quando le colonie sono aumentati o diminuiti di perturba sperimentalezioni. </li></ol></li><li> Incubare le cellule a clonogenica densità con procedura descritta di seguito <ol><li> Incubare le cellule in pozzetti quadruplice su 24 piastre fibronectina rivestite pozzetti ad una densità di 1.500 clonogenica cellule / pozzetto da un master singolo tubo sospensione cellulare di 1500 cellule / ml. Triturare il terreno contenente celle con una pipetta da 5 ml mediante l&#39;elaborazione di 3 ml e l&#39;erogazione di 1 ml di mezzo in ognuna delle 2 pozzi. NOTA: fibronectina rivestite piastre devono essere acquistati già rivestito da un fornitore commerciale. Piastre rivestimento esterno di una qualità controllata, risultati processo automatizzato in una superficie irregolare inadatto per questo dosaggio. </li><li> Mescolare la sospensione pipettando su e giù con una pipetta da 5 ml, elabora 3 ml di sospensione cellulare e riempire 2 pozzi con 1 ml ciascuno. Riempire solo 2 pozzetti alla volta da una pipetta. Restituisce il volume rimanente nella pipetta alla sospensione cellulare nel tubo principale, risospendere le cellule ancora pipettando su e giù e redige un altro 3 ml per riempire altri 2 wells con 1 ml ciascuno. NOTA: La continua miscelazione del tubo principale è necessario perché le cellule saranno continuamente sedimentare. Elaborazione di volume sufficiente per riempire solo 2 pozzetti è necessario aggiungere il numero di cellule simili a ciascun pozzetto perché cellule sedimentare nella pipetta pure. </li><li> Lavorare rapidamente per distribuire i grandi volumi di cellule perché le cellule che consentono di sedersi in sospensione a temperatura ambiente e la concentrazione di CO 2 si modula loro clonogeniche potenziali (osservazioni non pubblicate). </li><li> Ottimizzare la distribuzione spaziale delle cellule durante l&#39;atto della loro pipettando nei pozzetti per saggi colonia. Farlo lentamente pipettando la sospensione contenente la concentrazione cellulare finale nel mezzo del pozzo. Non sottoporre il piatto per favorire il movimento prima di cellule si depositano sul fondo. Non agitare la piastra perché miscelazione circolare efficacemente centrifugare le cellule al perimetro delle ben creando alte densità di cellulari e colonie confluenti innumerevoli a 6 giorni. NOTA: Non mescolare se non necessario in quanto questo è meno desiderabile di alcuna miscelazione a tutti dopo l&#39;introduzione del volume con le cellule in sospensione. Se è necessario miscelare cellule, farlo spostando la piastra posteriore e indietro in direzioni perpendicolari mentre poggia su una superficie piana. </li><li> Incubare le cellule a 37 ° C 5% di CO 2 senza cambiamento dei media per 6 giorni. Il piccolo numero di cellule in un pozzo dopo 6 giorni non influisce significativamente la composizione nutritiva o citochina né il pH del mezzo originale. </li><li> Progettare il decorso del test nel modo seguente: incubare celle su fibronectina rivestite substrati del giorno 1. Sostituire medio esistente con 1 ml di media fresco o mezzo fresco contenente 10 ng / ml il giorno 0. cellule macchia il giorno 6, come di seguito 2 FGF- in 1.4). Condurre eventuali perturbazioni sperimentali il giorno 3, come di seguito al punto 1.3). </li></ol></li><li> Perturbazioni set up sperimentale di molecolare di segnalazione o di Adesione Molecole <ol><li> Il giorno 3, aggiungere 10056; l di una soluzione contenente 10 volte della concentrazione finale previsto dell&#39;agente perturbante al mezzo 1 ml nei pozzetti. Non mescolare. Continuare ad incubare le cellule a 37 ° C, 5% CO 2 per altri 3 giorni. NOTA: Agenti perturbante può includere una varietà di inibitori e agenti di blocco delle molecole di adesione, recettori o altre proteine ​​di superficie, gli inibitori di vie intracellulari di segnalazione, molecole, fattori, cofattori o proteine ​​strutturali, che possono svolgere un ruolo nel sostenere lo stato dormiente. </li><li> Colonie Stain il giorno 6, come segue. </li></ol></li><li> Colonie Stain <ol><li> Cellule Macchia dopo 6 giorni in coltura con un fresco 0,1% viola cristallo nel 2% di etanolo / 10 mM borato di sodio (pH 9,0) soluzione. Aspirare e aggiungere la soluzione al cristalvioletto uno ml a ciascun pozzetto per 20 min. </li><li> Lavare le piastre immergendole con le aperture e rivolti verso il basso con un angolo acuto in un secchio di ghiaccio traboccante di rubinetto in esecuzione continuadell&#39;acqua nel lavandino. Inclinare la piastra per un angolo orizzontale, una volta sott&#39;acqua, bene l&#39;apertura verso il basso, e poi inclinare indietro ad angolo acuto quando si toglie in un unico movimento fluido delicato. </li><li> Ripetere l&#39;immersione 2 o 3 volte fino a quando l&#39;acqua sul fondo dei pozzetti non è blu. Lavaggi vigorosi possono rimuovere le cellule o colonie che sono meno aderente causa di intervento sperimentale, che aumenta notevolmente l&#39;errore nel conteggio e dati. </li><li> Lastre secche durante la notte mettendo a testa in giù sul asciugamani sul banco accanto ai loro corrispondenti coperture etichettati. </li></ol></li><li> Conta Colonie <ol><li> Contare il numero di crescita e dormienti colonie in ogni bene dopo 6 giorni di incubazione, colorazione e asciugatura. Contare le colonie in modo ottimale a 40X con lo zoom in un microscopio a contrasto di fase invertito. Contare colonie di&gt; 30 cellule come cresce e colonie di 12 o meno celle, con l&#39;aspetto morfologico di dimensioni molto grandi rispetto alle cellule in crescita, grande Expacitoplasma nded con ampio citoplasma al nucleo rapporti, mostrati in figura 1 7,15. NOTA: Gruppi di 13-29 cellule non sono normalmente considerati come non sono molto frequenti nei saggi di dormienza semplici. Essi possono essere conteggiati se perturbazioni spostano il potenziale di crescita sia cellule in crescita o dormienti. Questi risultati saranno poi bisogno di essere correlato con significato biologico. </li></ol></li></ol> 2. clonogenica Incubazione per immunofluorescenza Studies <ol><li> Regolare il numero di cellule a circa 7,500-8,000 cellule / pozzetto in 6 pozzetti, per corrispondere alla cella i numeri / superficie analoga a 24 esperimenti così. </li><li> Posizionare un giro, sterile, fibronectina rivestite fodera in ogni base ben di 6 piatti e per studi di imaging prima dell&#39;aggiunta delle cellule. Cellule Pipettare in 3 ml di volume in ciascun pozzetto 2 pozzetti alla volta a concentrazioni delineati, come descritto per gli esperimenti clonogeniche sopra in 1.2). </li><li> Incubare le celluleper 6 giorni, come sopra in 1.2.4 e 1.2.5. Aggiungere fattori perturbanti il ​​giorno 3 a 10x concentrazioni in 300 volumi microlitri, come descritto nelle procedure di saggio colonia in 1.3). </li><li> Cellule Stain il giorno 6 con anticorpi per cellulare molecole di adesione come integrine α4, α5, α6, β1, β3, per esempio, di adesione focale molecole complesse FAK, paxillina e vinculina, per esempio, proteine ​​coinvolte nella motilità quali α-tubulina, per esempio, di segnalazione membri pathway come fosfo-Akt, fosfo-ERK, fosfo-p38, fosfo-JNK, per esempio, o qualsiasi altra proteina che è la destinazione di ricerca per il suo ruolo in dormienza, utilizzando tecniche standard per la diretta o indiretta immunofluorescenza. <ol><li> Rimuovere i vetrini con una pinza giorno 6. Fissare in acetone / metanolo 1: 1 a -20 ° C per 20 min e aria secca. Un fissativo alternativa, ad esempio paraformaldeide, possono essere utilizzati, se necessario. Cellule permeabilize con 0,1% Triton X-100, 0,1% di citrato di sodio per 2 min fo il rilevamento di antigeni intracellulari. Lavare le cellule con PBS. </li><li> Per la colorazione immunofluorecence indiretta, scivoli blocco per 1 ora a temperatura ambiente con il 5% BSA o con 10% di siero preimmune dalla specie in cui si genera l&#39;anticorpo secondario. </li><li> Incubare per una notte a 4 ° C con anticorpi primari a cellula molecole di adesione, molecole complesse focali, segnalando membri pathway o qualsiasi altra proteina che è l&#39;obiettivo di ricerca per il suo ruolo in dormienza diluito per diluizioni specifiche consigliate dal produttore in PBS 0,1% TRITON X -100. </li><li> Lavare 3 volte con PBS. Incubare coprioggetto con anticorpi fluoroforo-coniugato a temperatura ambiente per 2 ore. Come esempio, Alexa Fluor 488 asino anti-topo IgG anticorpo può essere utilizzato per rilevare anticorpi primari monoclonali murini. Mount coprioggetto lato della cella verso il basso su vetrini utilizzando un agente antifade con Dapi. Sigillare i perimetri con smalto. </li><li> Per immunofluorescenza diretta, effettuare la BSAil blocco di cui sopra al punto 2.4.2), incubare con anticorpo primario fluoroforo coniugato notte a 4 ° C. Lavare 3 volte con PBS. Incubare i vetrini con un agente antifade e Dapi e sigillare con smalto. </li><li> Vassoi diapositive Coprire con un foglio di alluminio e conservare a 4 ° C per l&#39;imaging e la fotografia in qualsiasi momento fino a diverse settimane più tardi. Visualizzare e fotografia cellule utilizzando qualsiasi sistema di imaging microscopici fluorescenza dotato di una fotocamera a 1,000x ingrandimenti. </li><li> Per fibrillare actina colorazione, i vetrini blocco in 1% di albumina di siero bovino (BSA) per 30 min e incubare in BODIPY FL-Phallacidin (verde) o Rodamina phalloidin (rosso) a temperatura ambiente per 20 min. Aggiungi un agente antifade e sigillare come sopra. </li></ol></li></ol> 3. clonogenica incubazione per studi molecolari <ol><li> Western Blot <ol><li> Incubare ER + di cancro al seno cellule MCF-7 o T47D a densità clonogenici di 20.000 cellule / 60 mm Piastra e 50.000 cellule mm piastra / 100 su fibronecstagnato piastre a 37 ° C in 5% CO 2. </li><li> Incubare le cellule a densità leggermente superiori di 75.000 cellule / 100 millimetri piatto per studi molecolari richiedono quantità mg per le proteine ​​da lisati. Usa fino a dieci targhe per punto sperimentale per raccogliere sufficienti proteine ​​per studi molecolari con macchie occidentali o per l&#39;isolamento di RNA per Northern blot. </li><li> Numero delle cellule in gel a base di carico è un complemento necessario per confrontare l&#39;espressione della proteina in cellule di crescita e dormienti molto diverse dimensioni. Raccogliere le cellule per trypsinizing e contare una aliquota in un emocitometro nello 0,2% Trypan Blue per la preparazione di lisati per Western blot invece di raschiare via le cellule con una sola lama bordo. </li><li> Cellule centrifuga a 10.000 xg per 2 minuti, togliere i media per aspirazione, aggiungere 200 microlitri tampone di lisi, sonicazione le cellule in tampone di lisi e determinare la concentrazione di proteine. </li><li> Calcolare la quantità di proteina per cellula dividendo la resa di proteine ​​per il numero di celluleche hanno generato tale importo. Caricare il lisato in ogni pozzetto di gel di poliacrilammide separati che rappresenta sia) quantità equivalenti di proteine ​​e b) le quantità di proteine ​​che rappresentano il numero di cellule equivalenti. Almeno 25 ug di proteine ​​deve essere caricato in ciascun pozzetto. NOTA: Questo permetterà il confronto tra crescita e dormienti cellule, che sono significativamente differenti in termini di dimensioni e di proteine ​​(Figura 1). </li></ol></li><li> Citometria A Flusso <ol><li> Raccogliere le cellule da 100 lastre mm da tripsinizzazione, come in 3.1 e analizzare con la fluorescenza delle cellule attivi standard (FACS) i protocolli utilizzando primaria o secondaria colorazione immunofluorescenza per antigeni intracellulari o extracellulari, come indicato in 2.4. NOTA: Rimozione cellule da piastre di coltura tissutale mediante tripsinizzazione non influenza la concentrazione di proteine ​​di membrana come determinato mediante etichettatura anticorpo. </li></ol></li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+temporal+and+spatial+gene+expression+of+fibroblast+growth+factor+family+members+during+mouse+mammary+gland+development.">Differential temporal and spatial gene expression of fibroblast growth factor family members during mouse mammary gland development.</a> Molecular Endocrinology. 8 (2), 218-229 (1994).</li><li>Plath, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+localization+of+members+of+the+fibroblast+growth+factor+family+in+the+bovine+mammary+gland.">Expression and localization of members of the fibroblast growth factor family in the bovine mammary gland.</a> J Dairy Science. 81 (10), 2604-2613 (1998).</li><li>Lavandero, S., Chappuzeau, A., Sapag-Hagar, M., Oka, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+and+in+vitro+evidence+of+basic+fibroblast+growth+factor+action+in+mouse+mammary+gland+development.">In vivo and in vitro evidence of basic fibroblast growth factor action in mouse mammary gland development.</a> FEBS letters. 439 (3), 351-356 (1998).</li><li>Sinowatz, F., Schams, D., Plath, A., Kolle, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+and+localization+of+growth+factors+during+mammary+gland+development.">Expression and localization of growth factors during mammary gland development.</a> Adv in Exp Med &amp; Biol. 480, 19-27 (2000).</li><li>Korah, R., Sysounthone, V., Scheff, E., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+FGF-2+promotes+differentiation+in+T47-D+breast+cancer+cells.">Intracellular FGF-2 promotes differentiation in T47-D breast cancer cells.</a> Biochem Biophys Res Comm. 277 (1), 255-260 (2000).</li><li>Korah, R., Das, K., Lindy, M. E., Hameed, M., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-ordinate+loss+of+FGF-2+and+laminin+5+expression+during+neoplastic+progression+of+mammary+duct+epithelium.">Co-ordinate loss of FGF-2 and laminin 5 expression during neoplastic progression of mammary duct epithelium.</a> Human Pathology. 38 (1), 154-160 (2007).</li><li>Wieder, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+basic+fibroblast+growth+factor+in+MCF-7+human+breast+cancer+cells:+lack+of+correlation+between+inhibition+of+cell+growth+and+MAP+kinase+activation.">Overexpression of basic fibroblast growth factor in MCF-7 human breast cancer cells: lack of correlation between inhibition of cell growth and MAP kinase activation.</a> J. Cellular Physiology. 177, 411-425 (1998).</li><li>Brunner, G., Gabrilove, J., Rifkin, D. B., Wilson, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phospholipase+C+release+of+basic+fibroblast+growth+factor+from+human+bone+marrow+cultures+as+a+biologically+active+complex+with+a+phosphatidylinositol-anchored+heparan+sulfate+proteoglycan.">Phospholipase C release of basic fibroblast growth factor from human bone marrow cultures as a biologically active complex with a phosphatidylinositol-anchored heparan sulfate proteoglycan.</a> J Cell Biol. 114 (6), 1275-1283 (1991).</li><li>Wilson, E. L., Rifkin, D. B., Kelly, F., Hannocks, M. J., Gabrilove, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+stimulates+myelopoiesis+in+long-term+human+bone+marrow+cultures.">Basic fibroblast growth factor stimulates myelopoiesis in long-term human bone marrow cultures.</a> Blood. 77 (5), 954-960 (1991).</li><li>Gupta, P., McCarthy, J. B., Verfaillie, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stromal+fibroblast+heparan+sulfate+is+required+for+cytokine-mediated+ex+vivo+maintenance+of+human+long-term+culture-initiating+cells.">Stromal fibroblast heparan sulfate is required for cytokine-mediated ex vivo maintenance of human long-term culture-initiating cells.</a> Blood. 87 (8), 3229-3236 (1996).</li><li>Chatterjee, M., van Golen, K. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Farnesyl+transferase+inhibitor+treatment+of+breast+cancer+cells+leads+to+altered+RhoA+and+RhoC+GTPase+activity+and+induces+a+dormant+phenotype.">Farnesyl transferase inhibitor treatment of breast cancer cells leads to altered RhoA and RhoC GTPase activity and induces a dormant phenotype.</a> Int J Cancer. 129 (1), 61-69 (2011).</li><li>Korah, R., Sysounthone, V., Golowa, Y., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+fibroblast+growth+factor+confers+a+more+differentiated+phenotype+in+MDA-MB-231+human+breast+cancer+cells.">Basic fibroblast growth factor confers a more differentiated phenotype in MDA-MB-231 human breast cancer cells.</a> Cancer Res. 60 (3), 733-740 (2000).</li><li>Dontu, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+propagation+and+transcriptional+profiling+of+human+mammary+stem/progenitor+cells.">In vitro propagation and transcriptional profiling of human mammary stem/progenitor cells.</a> Genes &amp; Dev. 17 (10), 1253-1270 (2003).</li><li>Tivari, S., Lu, H., Dasgupta, T., Wieder, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactivation+of+dormant+estrogen-dependent+breast+cancer+micrometastases+by+bone+marrow+stromal+injury+in+an+in+vitro+model+of+dormancy.">Reactivation of dormant estrogen-dependent breast cancer micrometastases by bone marrow stromal injury in an in vitro model of dormancy.</a> Proceedings of the 105th Annual Meeting of the American Association for Cancer Research. , (2014).</li></ol>

Supportato dal Dipartimento della Difesa Grants DAMD17-01-C-0343 e DAMD17-03-1-0524, la Commissione di Stato del New Jersey su Cancer Research 02-1140-CCR-E0 e Ruth Estrin Goldberg Memorial per la Ricerca sul Cancro (RW)

Il nostro modello è composto da diversi elementi chiave della dormienza nel midollo osseo. Consiste di estrogeni cellule sensibili, che sono del tipo destinato a rimanere sospeso nel osseo per prolungati periodi 10, si compone di fibronectina, un elemento strutturale chiave del midollo, FGF-2, un fattore di crescita abbondantemente sintetizzato dal stroma del midollo osseo e pesantemente depositato nella matrice extracellulare del midollo osseo 31,32 e l&#39;incubazione delle cellule con densità ...

Le cellule tumorali del seno metastatizzano al midollo osseo prima che la malattia è rilevabile 1, non appena i piccoli tumori si sviluppano vasi sanguigni 2,3. Il processo metastatico è rapido ma inefficiente. Le cellule entrano i nuovi vasi sanguigni rapidamente a milioni al giorno 4 ma pochi sopravvivono il viaggio in organi distanti 5. Tuttavia, alcuni sopravvivono micrometastasi nel midollo e può essere trovato come cellule singole o piccoli grumi di cellule in aspirati di midollo osseo da pazienti di nuova diagnosi 1. Queste cellule resistono chemioterapia adiuvante, che viene somministrato per lo scopo di eliminarli 6. Questa resistenza è dotato, sostanzialmente, dalla sopravvivenza segnalazione avviata da interazioni con il midollo microambiente osseo 7,8. Micrometastasi possono essere trovati in circa un terzo delle donne con carcinoma mammario localizzato e rappresentano un indicatore indipendente di sopravvivenza quando analizzato da analisi univariata 9. Alcuni micrometastases sono la crescita iniziata, ma i modelli di ricorrenza dipendono dal tipo di cellulare. I pazienti con carcinoma mammario triplo negativo tendono a ripresentarsi tra 1 a 4 anni, suggerendo scarso controllo sullo stato dormiente. Altri tipi di cellule, tra cui ER / PR + cellule, possono rimanere dormienti per un massimo di 20 anni, con un costante, continuo tasso di recidiva 10. Mentre le differenze di dormienza firme espressione genica tra ER- linee di cellule del seno e tumori ER + e riflettono diversi potenziali dormienza 11, le interazioni con il midollo osseo stroma probabilmente rappresentano un contributo significativo alla dormienza. Lo studio di dormienza in vivo è estremamente difficile perché micrometastasi sono rari e vengono oltrepassati dalle cellule ematopoietiche di oltre 10 6 -fold. Quindi, i modelli interessati devono essere generati che forniscono dati in vitro che possono suggerire meccanismi e generare ipotesi verificabili in vivo. Un certo numero di modelli dormienza, compreso mathemodelli matico 12,13, modelli in vitro 7,8,14,15, modelli in vivo di xenotrapianto 16, combinazioni di in vitro e modelli di xenotrapianto 17,18 e tumorali e metastasi spontanee modelli 19, hanno dato qualche informazione in dormienza cellula tumorale 20 . Ognuno di questi modelli hanno i loro limiti e sono di se stessi in primo luogo utile per generare ipotesi per quanto riguarda la segnalazione molecolare e interazioni che governano dormienza da testare in modelli più biologicamente rilevanti. Con l&#39;obiettivo generale di definire i meccanismi molecolari di dormienza, le interazioni con il microambiente che si traduce in arresto del ciclo, ridifferenziazione e resistenza terapeutica e dei meccanismi che provocano recidiva + cellule ER, abbiamo sviluppato un modello in vitro che fornisce selezionato elementi rilevanti del stromale microambiente 7. Questo modello, mentre relativamente scarsa nella sua componenti, è sufficientemente robusto per permettere agli investigatori di ricavare i meccanismi molecolari specifici che influenzano le funzioni significative di dormienza. Questi esperimenti generano ipotesi che possono essere testati direttamente in vivo. Il modello si basa su alcuni elementi chiave che abbiamo dimostrato di essere rilevanti in dormienza. Essi comprendono l&#39;uso di cellule di carcinoma mammario estrogeno-dipendenti, coltura di cellule ad una densità clonogenico dove loro interazione è principalmente con il substrato e componenti solubili del mezzo, un substrato fibronectina e la presenza del fattore di crescita basico dei fibroblasti (FGF-2) nel mezzo. Abbiamo caratterizzato i meccanismi che regolano il sistema in vitro, compresa l&#39;induzione di arresto del ciclo cellulare da FGF-2 21, mediata attraverso TGFβ 22, la sopravvivenza di segnalazione attraverso PI3 chinasi e ERK 7,8 8 e differenziazione morfogenica ad un fenotipo epiteliale, che dipendeva inattivazione RhoA, integrina45; 5β1 upregulation e legatura dei stromale fibronectina per la sopravvivenza 7,15 (Figura 1). Gli effetti in vitro del ciclo cellulare di FGF-2 in cellule MCF-7 cominciano a concentrazioni almeno un log inferiore a 10 ng / ml 21,23. Il razionale era basato sul controllo temporale del FGF-2 governa mammaria morfogenesi duttale, espansione ciclica e recessione in un certo numero di sistemi di mammifero 24-27. Abbiamo dimostrato che FGF-2 induce la differenziazione, tra cui morfogenesi duttale in coltura 3D 28, e che l&#39;espressione di FGF-2 sono generalmente perso con trasformazione maligna dei tumori umani 29. L&#39;espressione di FGR1 rimasto intatto nei carcinomi mammari intervistati 29 e MCF-7 cellule continuare ad esprimere tutte le 4 recettori FGF 30. Nel contesto di dormienza, FGF-2 è esportato da e pesantemente depositato sul midollo osseo stroma 31,32 dove funziona nella conservazione di cellule staminali ematopoietiche 33. Noi demonstrated che FGF-2 induce uno stato dormiente in ER + di cancro al seno cellule coltivate su substrati di fibronectina, anche abbondanti nel midollo, dove induce la differenziazione morfogenica 7. Nel modello, le cellule del cancro al seno sono la crescita inibita, inattivare Rho A attraverso la RhoGap GRAF, ridifferenziare ad un fenotipo epiteliale e ri-express integrine α5β1 lost con la progressione maligna. Si legano fibronectina attraverso l&#39;integrina α5β1 e attivano la sopravvivenza segnalando che rendono resistenti alla terapia citotossica 7,8,15 (Figura 1). L&#39;inibizione della Rho GTPasi classe è stato dimostrato in precedenza per indurre un fenotipo dormiente 34. Qui ci illustrerà le procedure specifiche che permetteranno ai ricercatori di stabilire il modello e studiare i meccanismi molecolari e cellulari specifiche che disciplinano dormienza di cellule ER + di cancro al seno. Negli esperimenti qui presentati per illustrare l&#39;utilizzo del modello, Abbiamo mirato il percorso PI3K (Figura 1B) con un inibitore di Akt e un inibitore della PI3K e tutti i membri della famiglia Rho (Figura 1B) con un inibitore pan-Rho e Rho chinasi (ROCK) inibitore.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>MCF-7 cells</td>
			<td>ATCC</td>
			<td>HTB-22</td>
			<td></td>
		</tr>
		<tr>
			<td>T47D cells&#160;</td>
			<td>ATCC</td>
			<td>HTB-123</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 24 Well Clear Flat Bottom TC-Treated Multiwell Plate</td>
			<td>Corning</td>
			<td>354411</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 60 mm Culture Dishes</td>
			<td>Corning</td>
			<td>354403</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat Fibronectin 100 mm Culture Dishes</td>
			<td>Corning</td>
			<td>354451</td>
			<td></td>
		</tr>
		<tr>
			<td>BD BioCoat 22x22mm #1 Glass Coverslip with a uniform application of human fibronectin</td>
			<td>Corning</td>
			<td>354088</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well tissue culture plate</td>
			<td>CellTreat</td>
			<td>229106</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco Modified Eagle Medium High Glucose 10X Powder</td>
			<td>Corning Life Sciences</td>
			<td>50-013-PB</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat Inactivated, Fetal Bovine Serum</td>
			<td>Serum Source International</td>
			<td>FB02-500HI</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin/2.21 mM EDTA</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution, 100X</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine, 100x, Liquid</td>
			<td>Corning</td>
			<td>25-005-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Human FGF&#160;basic</td>
			<td>R&#38;D Systems</td>
			<td>234-FSE-025</td>
			<td></td>
		</tr>
		<tr>
			<td>Akt Inhibitor (1L6-Hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-O-octadecyl-sn-glycerocarbonate)</td>
			<td>CalBiochem</td>
			<td>124005</td>
			<td></td>
		</tr>
		<tr>
			<td>LY294002&#160;</td>
			<td>CalBiochem</td>
			<td>19-142</td>
			<td>Chenical PI3K inhibitor</td>
		</tr>
		<tr>
			<td>C3 transferase&#160;</td>
			<td>Cytoskeleton</td>
			<td>CTO3</td>
			<td>Inhibits RhoA, RhoB, and RhoC, but not related GTPases such as Cdc42 or Rac1</td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride&#160;</td>
			<td>Santa Cruz Biotechnology</td>
			<td>129830-28-2</td>
			<td></td>
		</tr>
		<tr>
			<td>BODIPY FL-Phallacidin (green)&#160;</td>
			<td>Molecular Probes</td>
			<td>B607</td>
			<td>Fluorochrome for fibrillar actin staining</td>
		</tr>
		<tr>
			<td>BODIPY FL-Rhodamine phalloidin (red)&#160;</td>
			<td>Molecular Probes</td>
			<td>R415</td>
			<td>Fluorochrome for fibrillar actin staining</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Donkey anti-Mouse IgG Antibody, ReadyProbes Reagent&#160;</td>
			<td>Molecular Probes</td>
			<td>R37114</td>
			<td></td>
		</tr>
		<tr>
			<td>ProLong Gold Antifade Mountant with DAPI&#160;</td>
			<td>Molecular Probes</td>
			<td>P-36931</td>
			<td></td>
		</tr>
	</tbody>
</table>

an in vitro dormancy model of estrogen-sensitive breast cancer in the bone marrow: a tool for molecular mechanism studies and hypothesis generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant cells with their microenvironment, their rarity and the overwhelming excess of hematopoietic cells. Towards this end, we developed an in vitro 2D clonogenic model of dormancy of estrogen-sensitive breast cancer cells in the bone marrow. The model consists of a few key elements necessary for dormancy. These include 1) the use of estrogen sensitive breast cancer cells, which are the type likely to remain dormant for extended periods, 2) incubation of cells at clonogenic density, where the structural interaction of each cell is primarily with the substratum, 3) fibronectin, a key structural element of the marrow and 4) FGF-2, a growth factor abundantly synthesized by bone marrow stromal cells and heavily deposited in the extracellular matrix. Cells incubated with FGF-2 form dormant clones after 6 days, which consist of 12 or less cells that have a distinct flat appearance, are significantly larger and more spread out than growing cells and have large cytoplasm to nucleus ratios. In contrast, cells incubated without FGF-2 form primarily growing colonies consisting of >30 relatively small cells. Perturbations of the system with antibodies, inhibitors, peptides or nucleic acids on day 3 after incubation can significantly affect various phenotypic and molecular aspects of the dormant cells at 6 days and can be used to assess the roles of membrane-localized or intracellular molecules, factors or signaling pathways on the dormant state or survival of dormant cells. While recognizing the in vitro nature of the assay, it can function as a highly useful tool to glean significant information about the molecular mechanisms necessary for establishment and survival of dormant cells. This data can be used to generate hypotheses to be tested in vivo models.

<html> Gli esperimenti sono stati condotti per ricapitolare il test. L&#39;andamento temporale dell&#39;esperimento è mostrato in Figura 2A. Le cellule vengono incubate a densità clonogenica giorno -1, si aggiunge FGF-2 in un mezzo fresco al giorno 0 e le cellule sono coltivate fino al giorno 6, quando sono macchiati e le colonie sono contate. Eventuali perturbazioni al sistema vengono somministrati il giorno 3 in 100 volumi microlitri a 10x concentrazioni finali desiderato. Figura 2B ...

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An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo.

Un<em&gt; In Vitro</em&gt; Dormienza Modello di estrogeno-sensibili cancro al seno nel midollo osseo: Uno strumento per gli studi molecolari del meccanismo e ipotesi Generation

The study of breast cancer dormancy in the bone marrow is an exceptionally difficult undertaking due to the complexity of the interactions of dormant ...

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An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

8.2K Views.  Rutgers New Jersey Medical School. We developed an in vitro model of dormancy in the bone marrow for estrogen-sensitive breast cancer cells. The goal of this protocol is to demonstrate use of the model for the study of the molecular and cellular biology of dormancy and for generation of hypotheses for subsequent testing in vivo. 

Video: An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Un In Vitro Sistema per studio dormienza tumorale e l&#39;interruttore di crescita metastatica

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

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Medicina

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotone Imaging di invasione delle cellule tumorali in un modello di mouse ortotopico di carcinoma orale a cellule squamose

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

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 ...

Screening for Melanoma Modifiers using a Zebrafish Autochthonous Tumor Model

Genomic studies of human cancers have yielded a wealth of information about genes that are altered in tumors1,2,3. A challenge arising from these studies is that many genes are altered, and it can be difficult to distinguish genetic alterations that drove tumorigenesis from that those arose incidentally during transformation. To draw this distinction it is beneficial to have an assay that can quantitatively measure the effect of an altered gene on tumor initiation and other processes that enable tumors to persist and disseminate. Here we present a rapid means to screen large numbers of candidate melanoma modifiers in zebrafish using an autochthonous tumor model4 that encompasses steps required for melanoma initiation and maintenance. A key reagent in this assay is the miniCoopR vector, which couples a wild-type copy of the mitfa melanocyte specification factor to a Gateway recombination cassette into which candidate melanoma genes can be recombined5. The miniCoopR vector has a mitfa rescuing minigene which contains the promoter, open reading frame and 3'-untranslated region of the wild-type mitfa gene. It allows us to make constructs using full-length open reading frames of candidate melanoma modifiers. These individual clones can then be injected into single cell Tg(mitfa:BRAFV600E);p53(lf);mitfa(lf)zebrafish embryos. The miniCoopR vector gets integrated by Tol2-mediated transgenesis6 and rescues melanocytes. Because they are physically coupled to the mitfa rescuing minigene, candidate genes are expressed in rescued melanocytes, some of which will transform and develop into tumors. The effect of a candidate gene on melanoma initiation and melanoma cell properties can be measured using melanoma-free survival curves, invasion assays, antibody staining and transplantation assays.

A rapid way to screen for melanoma modifiers using a zebrafish autochthonous tumor model is presented. It takes advantage of the miniCoopR vector which allows for expression of candidate melanoma genes in melanocytes. A method to obtain melanoma-free survival curves, an invasion assay, a protocol for antibody staining of scale melanocytes and a melanoma transplantation assay are described.

Screening per Modificatori melanoma utilizzando un modello Zebrafish Tumore autoctone

Genomic studies of  human cancers have yielded a wealth of information about genes that are altered  in tumors1,2,3. A challenge arising from these ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Utilizzando quantitativa real-time PCR per la determinazione cellulare attecchimento dei donatori in un modello murino competitivo trapianto di midollo osseo

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Eccitotossica stimolazione del cervello Microslices come<em&gt; In vitro</em&gt; Modello di Corsa

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Femorale aspirazione del midollo osseo in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

An Ex vivo Model to Study Hormone Action in the Human Breast

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial cells tend to lose hormone receptor expression. Widely used hormone receptor positive breast cancer cell lines are of limited relevance to the in vivo situation. Here, we describe an ex vivo model to study hormone action in the human breast. Fresh human breast tissue specimens from surgical discard material such as reduction mammoplasties or mammectomies are mechanically and enzymatically digested to obtain tissue fragments containing ducts and lobules and multiple stromal cell types. These tissue microstructures kept in basal medium without growth factors preserve their intercellular contacts, the tissue architecture, and remain hormone responsive for several days. They are readily processed for RNA and protein extraction, histological analysis or stored in freezing medium. Fluorescence activated cell sorting (FACS) can be used to enrich for specific cell populations. This protocol provides a straightforward, standard approach for translational studies with highly complex, varied human specimens.

An Ex vivo Model to Study Hormone Action in the Human Breast

We have developed a novel ex vivo model to study hormone action in the human breast. It is based on tissue microstructures isolated from surgical breast tissue specimens which preserve tissue architecture, intercellular interactions, and paracrine signaling.

Un<em&gt; Ex vivo</em&gt; Modello per studiare Hormone azione in seno umano

The study of hormone action in the human breast has been hampered by lack of adequate model systems. Upon in vitro culture, primary mammary epithelial ...

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of women are alive 5 years after diagnosis. The omentum is a preferred site of HGSC metastasis formation. Despite the clinical importance of this microenvironment, the contribution of omental adipose tissue to ovarian cancer progression remains understudied. Omental adipose is unusual in that it contains structures known as milky spots, which are comprised of B, T, and NK cells, macrophages, and progenitor cells surrounding dense nests of vasculature. Milky spots play a key role in the physiologic functions of the omentum, which are required for peritoneal homeostasis. We have shown that milky spots also promote ovarian cancer metastatic colonization of peritoneal adipose, a key step in the development of peritoneal metastases. Here we describe the approaches we developed to evaluate and quantify milky spots in peritoneal adipose and study their functional contribution to ovarian cancer cell metastatic colonization of omental tissues both in vivo and ex vivo. These approaches are generalizable to additional mouse models and cell lines, thus enabling the study of ovarian cancer metastasis formation from initial localization of cells to milky spot structures to the development of widespread peritoneal metastases.

In Vivo and Ex Vivo Approaches to Study Ovarian Cancer Metastatic Colonization of Milky Spot Structures in Peritoneal Adipose

We outline a protocol that implements both in vivo and ex vivo approaches to study ovarian cancer colonization of peritoneal adipose tissues, particularly the omentum. Furthermore, we present a protocol to quantitate and analyze immune cell-structures in the omentum known as milky spots, which promote metastases of peritoneal adipose.

In vivo ed ex vivo approcci per studiare il cancro ovarico metastatico colonizzazione delle strutture Lattea Spot in peritoneale adiposo

High-grade serous ovarian cancer (HGSC), the cause of widespread peritoneal metastases, continues to have an extremely poor prognosis; fewer than 30% of ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

Utilizzo del saggio soft Agar Colony Formazione per identificare inibitori di tumorigenicità in cellule di carcinoma mammario

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...