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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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	<li>Braun, S., 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=Cytokeratin-positive+cells+in+the+bone+marrow+and+survival+of+patients+with+stage+I,+II,+or+III+breast+cancer.">Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or III breast cancer.</a> N Engl J Med. 342 (8), 525-533 (2000).</li><li>Benoy, I. H., 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=Relative+microvessel+area+of+the+primary+tumour,+and+not+lymph+node+status,+predicts+the+presence+of+bone+marrow+micrometastases+detected+by+reverse+transcriptase+polymerase+chain+reaction+in+patients+with+clinically+non-metastatic+breast+cancer.">Relative microvessel area of the primary tumour, and not lymph node status, predicts the presence of bone marrow micrometastases detected by reverse transcriptase polymerase chain reaction in patients with clinically non-metastatic breast cancer.</a> Breast Cancer Res. 7 (2), R210-R219 (1186).</li><li>Wapnir, I. L., Barnard, N., Wartenberg, D., Greco, R. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+inverse+relationship+between+microvessel+counts+and+tumor+volume+in+breast+cancer.">The inverse relationship between microvessel counts and tumor volume in breast cancer.</a> Breast J. 7 (3), 184-188 (2001).</li><li>Wyckoff, J. B., 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=A+critical+step+in+metastasis:+in+vivo+analysis+of+intravasation+at+the+primary+tumor.">A critical step in metastasis: in vivo analysis of intravasation at the primary tumor.</a> Cancer Res. 60 (9), 2504-2511 (2000).</li><li>Phadke, P. 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=Kinetics+of+metastatic+breast+cancer+cell+trafficking+in+bone.">Kinetics of metastatic breast cancer cell trafficking in bone.</a> Clin Cancer Res. 12 (5), 1431-1440 (2006).</li><li>Braun, S., 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=Lack+of+an+effect+of+adjuvant+chemotherapy+on+the+elimination+of+single+dormant+tumor+cells+in+bone+marrow+of+high+risk+breast+cancer+patients.">Lack of an effect of adjuvant chemotherapy on the elimination of single dormant tumor cells in bone marrow of high risk breast cancer patients.</a> J Clin Onc. 18 (1), 80-86 (2000).</li><li>Korah, R., Boots, 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=Integrin+&#945;5&#946;1+promotes+survival+of+growth-arrested+breast+cancer+cells:+an+in+vitro+paradigm+for+breast+cancer+dormancy+in+bone+marrow.">Integrin &#945;5&#946;1 promotes survival of growth-arrested breast cancer cells: an in vitro paradigm for breast cancer dormancy in bone marrow.</a> Cancer Res. 64 (13), 4514-4522 (2004).</li><li>Najmi, S., Korah, R., Chandra, R., Abdellatif, 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=Flavopiridol+blocks+integrin-mediated+survival+in+dormant+breast+cancer+cells.">Flavopiridol blocks integrin-mediated survival in dormant breast cancer cells.</a> Clin Cancer Res. 11 (5), 2038-2046 (2005).</li><li>Braun, S., 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=A+pooled+analysis+of+bone+marrow+micrometastasis+in+breast+cancer.">A pooled analysis of bone marrow micrometastasis in breast cancer.</a> N England J Med. 353 (8), 793-802 (2005).</li><li>Dent, 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=Triple-negative+breast+cancer:+clinical+features+and+patterns+of+recurrence.">Triple-negative breast cancer: clinical features and patterns of recurrence.</a> Clin Cancer Res. 13 (15 Part 1), 4429-4434 (2007).</li><li>Kim, R. S., 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=Dormancy+signatures+and+metastasis+in+estrogen+receptor+positive+and+negative+breast+cancer.">Dormancy signatures and metastasis in estrogen receptor positive and negative breast cancer.</a> PLoS One. 7 (4), e35569 (2012).</li><li>Hanin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+the+invisible:+how+mathematical+models+uncover+tumor+dormancy,+reconstruct+the+natural+history+of+cancer,+and+assess+the+effects+of+treatment.">Seeing the invisible: how mathematical models uncover tumor dormancy, reconstruct the natural history of cancer, and assess the effects of treatment.</a> Adv in Exp Med &amp; Biol. 734, 261-282 (2013).</li><li>Wilkie, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+mathematical+models+of+cancer-immune+interactions+in+the+context+of+tumor+dormancy.">A review of mathematical models of cancer-immune interactions in the context of tumor dormancy.</a> Adv in Exp Med &amp; Biol. 734, 201-234 (2013).</li><li>Barkan, D., Green, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+system+to+study+tumor+dormancy+and+the+switch+to+metastatic+growth.">An in vitro system to study tumor dormancy and the switch to metastatic growth.</a> J Vis Exp. (54), e2914 (2011).</li><li>Barrios, J., 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=Dual+FGF-2+and+intergrin+&#945;5&#946;1+signaling+mediate+GRAF-induced+RhoA+inactivation+in+a+model+of+breast+cancer+dormancy.">Dual FGF-2 and intergrin &#945;5&#946;1 signaling mediate GRAF-induced RhoA inactivation in a model of breast cancer dormancy.</a> Cancer Microenvironment. 2 (1), 33-47 (2009).</li><li>Ganapathy, V., 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=Luminal+breast+cancer+metastasis+is+dependent+on+estrogen+signaling.">Luminal breast cancer metastasis is dependent on estrogen signaling.</a> Clin &amp; Exp Metastasis. 29 (5), 493-509 (2012).</li><li>Barkan, D., 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=Inhibition+of+metastatic+outgrowth+from+single+dormant+tumor+cells+by+targeting+the+cytoskeleton.">Inhibition of metastatic outgrowth from single dormant tumor cells by targeting the cytoskeleton.</a> Cancer Res. 68 (15), 6241-6250 (2008).</li><li>Ghajar, C. M., 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=The+perivascular+niche+regulates+breast+tumour+dormancy.">The perivascular niche regulates breast tumour dormancy.</a> Nat Cell Biology. 15 (7), 807-817 (2013).</li><li>Marshall, J. C., 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=Effect+of+inhibition+of+the+lysophosphatidic+acid+receptor+1+on+metastasis+and+metastatic+dormancy+in+breast+cancer.">Effect of inhibition of the lysophosphatidic acid receptor 1 on metastasis and metastatic dormancy in breast cancer.</a> J Nat Cancer Inst. 104 (17), 1306-1319 (2012).</li><li>Almog, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+and+regulatory+pathways+involved+in+persistence+of+dormant+micro-tumors.">Genes and regulatory pathways involved in persistence of dormant micro-tumors.</a> Adv in Exp Med &amp; Biol. 734, 3-17 (2013).</li><li>Wang, H., 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=Basic+FGF+causes+growth+arrest+in+MCF-7+human+breast+cancer+cells+while+inducing+both+mitogenic+and+inhibitory+G1+events.">Basic FGF causes growth arrest in MCF-7 human breast cancer cells while inducing both mitogenic and inhibitory G1 events.</a> Cancer Res. 57 (9), 1750-1757 (1997).</li><li>Fenig, E., 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=Role+of+transforming+growth+factor+beta+in+the+growth+inhibition+of+human+breast+cancer+cells+by+basic+fibroblast+growth+factor.">Role of transforming growth factor beta in the growth inhibition of human breast cancer cells by basic fibroblast growth factor.</a> Breast Cancer Res Treat. 70 (1), 27-37 (2001).</li><li>Fenig, E., 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=Basic+fibroblast+growth+factor+confers+growth+inhibition+and+Mitogen-activated+Protein+Kinase+activation+in+human+breast+cancer+cells.">Basic fibroblast growth factor confers growth inhibition and Mitogen-activated Protein Kinase activation in human breast cancer cells.</a> Clinical Cancer Research. 3 (1), 135-142 (1997).</li><li>Coleman-Krnacik, S., Rosen, J. 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><tbody><tr><td>MCF-7 cells</td><td>ATCC</td><td>HTB-22</td><td></td></tr><tr><td>T47D cells&amp;nbsp;</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&amp;nbsp;basic</td><td>R&amp;amp;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&amp;nbsp;</td><td>CalBiochem</td><td>19-142</td><td>Chenical PI3K inhibitor</td></tr><tr><td>C3 transferase&amp;nbsp;</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&amp;nbsp;</td><td>Santa Cruz Biotechnology</td><td>129830-28-2</td><td></td></tr><tr><td>BODIPY FL-Phallacidin (green)&amp;nbsp;</td><td>Molecular Probes</td><td>B607</td><td>Fluorochrome for fibrillar actin staining</td></tr><tr><td>BODIPY FL-Rhodamine phalloidin (red)&amp;nbsp;</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&amp;nbsp;</td><td>Molecular Probes</td><td>R37114</td><td></td></tr><tr><td>ProLong Gold Antifade Mountant with DAPI&amp;nbsp;</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 ...

Watch this Scientific Journal Video about An In Vitro Dormancy Model of Estrogen-sensitive Breast Cancer in the Bone Marrow: A Tool for Molecular Mechanism Studies and Hypothesis Generation at JoVE.co

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

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

Sviluppo di un modello preclinico umano di osteoclastogenesi dai monociti del sangue periferico co-coltivati con linee cellulari di cancro al seno

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

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Ricerca sul cancro

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 ...

Medicina

Three-Dimensional Bone Extracellular Matrix Model for Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with diagnostic or prognostic difficulties. OS comprises genotypically and phenotypically heterogeneous cancer cells. Bone microenvironment elements are proved to account for tumor heterogeneity and disease progression. Bone extracellular matrix (BEM) retains the microstructural matrices and biochemical components of native extracellular matrix. This tissue-specific niche provides a favorable and long-term scaffold for OS cell seeding and proliferation. This article provides a protocol for the preparation of BEM model and its further experimental application. OS cells can grow and differentiate into multiple phenotypes consistent with the histopathological complexity of OS clinical specimens. The model also allows visualization of diverse morphologies and their association with genetic alterations and underlying regulatory mechanisms. As homologous to human OS, this BEM-OS model can be developed and applied to the pathology and clinical research of OS.

The bone extracellular matrix (BEM) model for osteosarcoma (OS) is well established and shown here. It can be used as a suitable scaffold for mimicking primary tumor growth in vitro and providing an ideal model for studying the histologic and cytogenic heterogeneity of OS.

Modello di matrice extracellulare tridimensionale dell'osso per Osteosarcoma

Osteosarcoma (OS) is the most common and a highly aggressive primary bone tumor. It is characterized with anatomic and histologic variations along with ...

Models of Bone Metastasis

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible for significant morbidity and mortality1. Thus, there is a significant need to understand the molecular mechanisms controlling the establishment, growth and activity of tumors in bone. Several in vivo models have been established to study these events and each has specific benefits and limitations. The most commonly used model utilizes intracardiac inoculation of tumor cells directly into the arterial blood supply of athymic (nude) BalbC mice. This procedure can be applied to many different tumor types (including PC-3 prostate cancer, lung carcinoma, and mouse mammary fat pad tumors); however, in this manuscript we will focus on the breast cancer model, MDA-MB-231. In this model we utilize a highly bone-selective clone, originally derived in Dr. Mundy's group in San Antonio2, that has since been transfected for GFP expression and re-cloned by our group3. This clone is a bone metastatic variant with a high rate of osteotropism and very little metastasis to lung, liver, or adrenal glands. While intracardiac injections are most commonly used for studies of bone metastasis2, in certain instances intratibial4 or mammary fat pad injections are more appropriate. Intracardiac injections are typically performed when using human tumor cells with the goal of monitoring later stages of metastasis, specifically the ability of cancer cells to arrest in bone, survive, proliferate, and establish tumors that develop into cancer-induced bone disease. Intratibial injections are performed if focusing on the relationship of cancer cells and bone after a tumor has metastasized to bone, which correlates roughly to established metastatic bone disease. Neither of these models recapitulates early steps in the metastatic process prior to embolism and entry of tumor cells into the circulation. If monitoring primary tumor growth or metastasis from the primary site to bone, then mammary fat pad inoculations are usually preferred; however, very few tumor cell lines will consistently metastasize to bone from the primary site, with 4T1 bone-preferential clones, a mouse mammary carcinoma, being the exception 5,6.
This manuscript details inoculation procedures and highlights key steps in post inoculation analyses. Specifically, it includes cell culture, tumor cell inoculation procedures for intracardiac and intratibial inoculations, as well as brief information regarding weekly monitoring by x-ray, fluorescence and histomorphometric analyses.

Animal models are frequently utilized to study cancer metastasis to bone. In this protocol we will describe two common methods of tumor inoculation for bone metastasis studies and briefly describe some of the analyses utilized to monitor and quantify these models.

Modelli di metastasi ossee

Bone metastases are a common occurrence in several malignancies, including breast, prostate, and lung. Once established in bone, tumors are responsible ...

Orthotopic Transplantation of Breast Tumors as Preclinical Models for Breast Cancer

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. Immortalized cell lines are easy to grow and genetically modify to study molecular mechanisms, yet the selective pressure from cell culture often leads to genetic and epigenetic alterations over time. Patient-derived xenograft (PDX) models faithfully recapitulate the heterogeneity and drug response of human breast tumors. PDX models exhibit a relatively short latency after orthotopic transplantation that facilitates the investigation of breast tumor biology and drug response. The transplantable genetically engineered mouse models allow the study of breast tumor immunity. The current protocol describes the method to orthotopically transplant breast tumor fragments into the mammary fat pad followed by drug treatments. These preclinical models provide valuable approaches to investigate breast tumor biology, drug response, biomarker discovery and mechanisms of drug resistance.

Patient-derived xenograft (PDX) models and transplantable genetically engineered mouse models faithfully recapitulate human disease and are preferred models for basic and translational breast cancer research. Here, a method is described to orthotopically transplant breast tumor fragments into the mammary fat pad to study tumor biology and evaluate drug response.

Trapianto ortotopico di tumori al seno come modelli preclinici per il cancro al seno

Preclinical models that faithfully recapitulate tumor heterogeneity and therapeutic response are critical for translational breast cancer research. ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

Isolamento e valutazione funzionale di cellule staminali di cancro al seno umano da campioni di cellule e tessuti

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

Sferoidi epiteliali ed endoteliali mammari come potenziale modello funzionale in vitro per la ricerca sul cancro al seno

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

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

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

Profiling di microRNA regolati dagli estrogeni in cellule di carcinoma mammario

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

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 ...

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

Metodi per la coltura di femore umano espianti di tessuto di studio sul carcinoma della mammella cellulare colonizzazione della nicchia metastatico

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

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