Ringraziamo il Dr. Roger Erickson per il suo sostegno e assistenza alla preparazione del manoscritto. Questo lavoro è stato sostenuto dal National Cancer Institute presso il National Institutes of Health (Grant Numero RO1CA177984, RO1CA138642), progetto del programma VA sul cancro alla prostata (BX001604).

Campioni freschi cancro alla prostata congelato fissati in formalina, inclusi in paraffina (FFPE) o sono stati ottenuti dalla SFVAMC. Campioni di pazienti con cancro alla prostata sottoposti a prostatectomia radicale a SFVAMC. Consenso informato scritto è stato ottenuto da tutti i pazienti e lo studio è stato approvato dal Comitato UCSF per la ricerca umana. In alternativa, tessuti di cancro alla prostata microarray sono stati acquistati da fonti commerciali e utilizzati per le analisi da miRNA ISH. Informazioni clinicopatologica e follow-up per i pazienti affetti da cancro alla prostata analizzati sono stati raccolti. 1. I campioni di tessuto <ol><li> Tagliare campioni di tessuto del cancro della prostata in 10 sezioni micron utilizzando un microtomo e macchia con H &amp; E seguendo il protocollo del produttore. </li><li> Commenta vetrini colorati per l&#39;identificazione del cancro alla prostata foci così come adiacente normale epitelio ghiandolare. Nota: Una scheda certificata patologo dovrebbe rivedere le diapositive H &amp; E macchiato e mark per tumore e aree normali. Utilizzare le diapositive contrassegnate come guide in successive sezioni per miRNA analisi da tumore vs aree normali. </li></ol> 2. Le analisi di espressione di miRNA Quantitative Real-time PCR Nota: Questo flusso di lavoro prevede le seguenti fasi: cattura microdissezione laser, isolamento di RNA totale (compresi i miRNA e mRNA), il saggio dei miRNA maturi utilizzando i saggi di espressione TaqMan MicroRNA come dettagliato nelle sezioni seguenti. <ol><li> RNA Estrazione <ol><li> Isolamento di miRNA da cancro della prostata FFPE tessuti <ol><li> Microdissezione laser (LCM) Nota: Eseguire i passi 2.1.1.1.1- 2.1.1.1.4 in una cappa. <ol><li> Preparare vetrini di tessuto (10 sezioni micron) per LCM. Deparrafinize sezioni di tessuto di immersione in xilene (2x), per 10 minuti ciascuno, quindi reidratare i tessuti incubando i vetrini per 5 minuti ciascuna in etanolo graduata (100%, 95%, 90%, 80%, 70%) seguita da distillata acqua(5 minuti). </li><li> A seguito di reidratazione, sezioni macchia con ematossilina per 30 secondi seguiti da acqua. </li><li> Inserire tessuti in etanolo graduata (70%, 95% 90%, 80%, 70%) (5 min ciascuna) e xilene (5 min). </li><li> Scivoli asciutti in una cappa aspirante e quindi inserire nello strumento LCM per microdissezione. </li><li> Eseguire microdissections con il sistema LCM conformemente alle istruzioni del produttore 24. Usa patologo marcato diapositive come guide per l&#39;identificazione di cancro alla prostata foci così come il tessuto normale adiacente. Utilizzare il raggio laser ad una 10-20 impulsi diametro micron con una potenza di 70-90 mW. </li><li> Cattura aree di interesse con impulsi laser infrarossi sui tappi LCM. Combina le celle da 2-5 tappi per l&#39;estrazione di miRNA per campione. Per garantire l&#39;integrità di RNA estratto, immediatamente elaborare cellule catturate per l&#39;estrazione miRNA. </li><li> In alternativa, le cellule Lisare nel buffer di estrazione miRNA (secondo il protocollo del produttore) e store lisati a -80 ° C. </li></ol></li><li> miRNA Estrazione e analisi da LCM microdissezione tessuti <ol><li> Estratto RNA totale da tessuti FFPE microdissezione utilizzando un kit commerciale seguendo le istruzioni del produttore. Nota: La resa di RNA da cattura laser miscrodissection è tipicamente basso. Pertanto, l&#39;RNA viene eluito in volumi bassi (20-30 mL) e utilizzato per la profilazione espressione utilizzando Taqman microRNA saggi di espressione seguito le istruzioni del produttore (anche descritto nella sezione 2.2). Ottimizzazione di ingresso dell&#39;RNA per PCR real time il rilevamento dei miRNA maturi suggerisce che 5 ml di RNA eluizione è ottimale per ogni reazione di trascrizione inversa specifici miRNA-. Come un controllo endogeno, è usato RNU48 / RNU24. Per le reazioni di controllo, 2 microlitri del RNA eluito viene utilizzato per la reazione di trascrizione inversa. </li></ol></li></ol></li><li> Isolamento di miRNA da cancro della prostata congelati tessuti <ol><li> Omogenize congelati tessuti della prostata resezione dalla macinazione dei tessuti in azoto liquido con un mortaio e pestello. Trasferire il tessuto omogeneizzato in una provetta con una spatola raffreddato. Mantenere la malta / pestello e spatola alla stessa temperatura per immersione in azoto liquido tessuto omogeneizzato così può essere facilmente trasferiti. </li><li> Aggiungere guanidina isotiocianato-fenolo commerciale reagente (1 ml / 0,1 g di tessuto) al tessuto omogeneizzato ed incubare a temperatura ambiente per 5 min. Nota: i tessuti omogeneizzate della guanidina commerciale isotiocianato-fenolo reagente possono essere conservati a -80 ° C per diversi mesi. </li><li> Aggiungere cloroformio per l&#39;omogeneizzato (0,2 ml di cloroformio / ml) e si agita energicamente per 30 sec. Incubare i campioni a temperatura ambiente per 3-5 min seguita da centrifugazione a 10.000 xg per 20 min a 4 ° C. </li><li> Trasferire la fase acquosa superiore ad una provetta sterile da 1,5 ml fresco RNasi-free. </li><li> Aggiungere un volume uguale di alcool isopropilico. Mescolare e incubare per 10 min a RT seguita da centrifugazione a 12.000 xg per 20 min a 4 ° C per sedimentare il RNA. </li><li> Aspirare il surnatante e lavare il pellet di RNA con 1 ml di etanolo al 70%. Centrifugare a 12,000 xg per 10 minuti a 4 ° C. </li><li> Con attenzione aspirare il surnatante. Pellet di RNA a secco a temperatura ambiente per 5-10 minuti. Sciogliere RNA in 50-100 acqua priva di nucleasi microlitri. </li><li> Eseguire la quantificazione di RNA utilizzando uno spettrofotometro NanoDrop e determinare l&#39;integrità dell&#39;RNA con un bioanalizzatore. Regolare le concentrazioni di RNA a 10 ng / ml. Utilizzare 10-50 ng RNA per miRNA- reazione cDNA specifico seguito da PCR in tempo reale le analisi (paragrafo 2.2). </li></ol></li></ol></li><li> PCR quantitativa in tempo reale per miRNA analisi di espressione Nota: miRNA maturi Assay utilizzando un protocollo RT-PCR in due fasi come descritto sotto. <ol><li> Reverse trascrizione (RT) <ol><li> Reverse trascrivere cDNA da RNA utilizzando un kit di trascrizione inversa miRNA in conformità con le istruzioni del produttore.Utilizzare 10-50 ng di RNA totale con miRNA primer specifico dal kit MicroRNA Assays e RT. Utilizzare RNU48 / RNU24 / RNU6B come controlli. Diluire cDNA 1: 5 a 1:10 (seconda dall&#39;abbondanza del miRNA analizzati) e utilizzare in tempo reale di rilevamento PCR come descritto nel passaggio seguente. </li></ol></li><li> Real-time PCR per il rilevamento di miRNA maturo <ol><li> Amplifica prodotti di PCR di campioni di cDNA utilizzando i saggi MicroRNA con una master mix universale veloce secondo le istruzioni del produttore. Normalizzare campioni a RNU48 / RNU24 / controllo RNU6B. Utilizzare il metodo comparativo Ct (ciclo soglia) per calcolare le relative variazioni nell&#39;espressione genica su Fast Real Time PCR sistema. Analizzare ogni campione in triplicato. </li></ol></li></ol></li></ol> Analisi 3. miRNA Espressione di ibridazione in situ (ISH) <ol><li> Pre-trattamento dei tessuti <ol><li> Tagliare 5 sezioni micron da blocchi di tessuto di cancro alla prostata FFPE utilizzando un microtomo. </li><li&gt; Fix sezioni di tessuto incubando i campioni a 56 ° C per 1 ora. Nota: Le diapositive possono essere conservati a temperatura ambiente per diverse settimane per miRNA analisi. </li><li> Deparrafinize tessuti immergendo i vetrini con xilene (2x), per 15 minuti ciascuno. </li><li> Reidratare i tessuti incubando i vetrini per 5 minuti ciascuna in etanolo graduata (100% (2x), 95%, 90%, 80%, 70%) seguito da acqua distillata (5 min). </li><li> Fissare i vetrini con 4% paraformaldeide in PBS a temperatura ambiente per 20 min. </li><li> Lavare i vetrini con PBS (2x) a temperatura ambiente per 5 minuti ciascuna. </li><li> Trattare i vetrini con 10 ug / ml proteinasi K a 37 ° C per 10 min in proteinasi pre-riscaldato tampone K (5 mM Tris-HCL pH 7,4, 1 mM EDTA, 1 mM NaCl). </li><li> Dopo il trattamento proteinasi-K, sciacquare i vetrini con lo 0,2% glicina in PBS per 30 sec. </li><li> Lavare i vetrini in PBS (1x) a temperatura ambiente per 5 min. </li><li> Fissare i vetrini con 4% paraformaldeide in PBS per 15 min. </li><li> Sciacquare i vetrini conPBS per 5 min. </li></ol></li><li> Ibridazione <ol><li> Pre-ibridare i vetrini con soluzione pre-ibridazione per 3-4 ore a 55 ° C in una camera umidificata. Utilizzare tessuto salviettine imbevute di laboratorio nel 50% formammide / 50% 5x SSC per mantenere la camera umidificata. </li><li> Utilizzare 5 &#39;sonde marcate con digossigenina ad una concentrazione di 20-50 nM in tampone di ibridazione. Diluire sonda specifica per miRNA (20-50 nM) e una piccola sonda di controllo U6 RNA nucleare (20 nM), calore a 90 ° C per 4 minuti, posizionarlo su ghiaccio, e aggiungere al ghiaccio tampone di ibridazione freddo (2-4 ng / ml ). </li><li> Rimuovere la soluzione pre-ibridazione, aggiungere soluzione di ibridazione (sonda + tampone di ibridazione, 100 ml per vetrino), incubare per 12-16 ore a temperatura di ibridazione specifica sonda (temperatura di ibridazione = sonda-Tm 21 ° C). Eseguire ibridazioni in una camera umidificata (50% formammide / 50% 5x SSC). </li></ol></li><li> Lavaggio Steps <ol><li> Lavare i vetrini con 2x SSC per 10 minuti a 45 ° C. </li><li> Lavare tscivola con 1,5x SSC per 10 minuti a 45 ° C. </li><li> Lavare i vetrini con 0.2x SSC (2x) a 37 ° C per 20 minuti ciascuno. </li><li> Incubare i vetrini con la soluzione 1x di blocco per 1-2 ore a temperatura ambiente </li></ol></li><li> Rilevamento <ol><li> Incubare i vetrini con 1: 100 PBS diluito anticorpo anti-digossigenina AP-coniugato per 1-4 ore o una notte a 4 ° C. </li><li> Lavare i vetrini con PBS (3x) a temperatura ambiente per 10 minuti ciascuno. </li><li> Lavare le diapositive (2x) con fosfatasi alcalina tampone (100 mM Tris pH 9,5, 50 mM MgCl 2, 100 mM NaCl, 0.1% Tween-20) a temperatura ambiente per 5 minuti ciascuna. </li><li> Incubare i vetrini in substrato BM Viola AP al buio a temperatura ambiente per 1-20 ore. </li><li> Risciacquare i vetrini con PBS contenente 0,1% di Tween-20 e lavaggio (2x) in acqua. </li><li> Montare le diapositive utilizzando un mezzo di montaggio acquoso ed esaminare al microscopio. </li></ol></li></ol>

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The authors have no financial disclosures.

In questo articolo, si descrive un flusso di lavoro semplificato per miRNA profilatura in FFPE archiviati o cancro alla prostata congelato tessuti clinici freschi. Nel cancro della prostata, diversi studi suggeriscono un ruolo importante dei microRNA nel cancro alla prostata iniziazione, la progressione e la metastasi. Tuttavia, risultati contrastanti sono spesso ottenuti su uno specifico miRNA 22 in quanto l&#39;estrazione miRNA e analisi metodi differiscono ampiamente. In considerazione degli elementi di pr...

Il cancro della prostata è un tumore maligno maschio comunemente diagnosticato che è una delle principali cause di mortalità per cancro correlati tra gli uomini. Negli Stati Uniti, si stima che 220,800 nuovi casi e 27.540 decessi saranno presentate nel 2015 1. Il cancro della prostata è una malattia eterogenea con malattia molto variabile corso- tumori possono essere indolenti o molto aggressivo. Una sfida importante nel cancro della prostata gestione clinica è rappresentata dal inadeguatezza dei metodi attualmente in uso / biomarcatori per lo screening della malattia, la diagnosi, la prognosi e il trattamento 2. Metodi di screening attuali includono l&#39;antigene prostatico specifico (PSA) e un esame rettale digitale (DRE) seguito da biopsie prostatiche 3. Antigene prostatico specifico (PSA) è il biomarcatore cancro alla prostata più diffuso che ha rivoluzionato in modo significativo la gestione clinica e miglioramento dei tassi di sopravvivenza 4. Tuttavia, a causa dei limiti intrinseci di PSA compreso lack di specificità, screening di PSA-ha causato oltre diagnosi e sopra il trattamento della malattia. In considerazione di ciò, intensi sforzi sono orientati verso la ricerca di alternative biomarcatori del cancro alla prostata, in particolare quelli che possono prevedere l&#39;aggressività della malattia e guidare le decisioni terapeutiche migliori 4,5. Nel corso degli ultimi anni, microRNA (miRNA) sono emersi come promettenti alternative biomarcatori del cancro alla prostata. I microRNA (miRNA) costituiscono una classe evolutivamente conservata di piccoli RNA non codificanti che sopprimono l&#39;espressione genica livello post-trascrizionale tramite interazioni sequenza-specifiche con le regioni 3&#39;non tradotte (UTR) del target mRNA affini. Si stima che il&gt; 60% di mRNA sono conservate bersagli di miRNA 6. geni miRNA sono localizzati nelle regioni intergeniche o all&#39;interno introni o esoni di proteine ​​/ non-codificanti proteine ​​geni 7. Questi geni sono preferenzialmente trascritti dalla RNA polimerasi II in primamiRNA ry (pri-miRNA, lunga diversi kilobasi) che ad anello a forma di gambo strutture secondarie forma a gomito. Questi pri-miRNA vengono trasformati in miRNA precursori (pre-miRNA, lungo 60-75 nucleotidi) che vengono esportati al citoplasma e successivamente trattati in miRNA maturi (lunghi 18-25 nucleotidi) 8-10. miRNA regolano processi cellulari chiave, tra cui la proliferazione, lo sviluppo, la differenziazione e l&#39;apoptosi 11. Gli studi suggeriscono una disregolazione diffuso di profili di espressione dei miRNA in diversi tumori umani tra cui il cancro alla prostata 12-15. Sono stati riportati i profili di espressione di miRNA ad essere ampiamente disregolazione nel cancro della prostata primari e metastatici. MiRNA Altered sono stati collegati con la progressione del cancro alla prostata, l&#39;aggressività e la ricorrenza mettendo in evidenza il potenziale prognostico di miRNA 12,14,16-19. Un crescente corpo di evidenze indicano che i miRNA svolgono ruoli importanti meccanicistici nell&#39;iniziazione cancro alla prostata, lo sviluppo, il progressoioni e metastasi. Nel complesso, miRNA stanno emergendo come biomarcatori alternativi promettenti per la diagnosi del cancro alla prostata e la prognosi in grado di distinguere tra tessuti e gli aiuti normali e tumorali nella stratificazione di tumori della prostata 12. Inoltre, miRNA sono obiettivi importanti per lo sviluppo di terapie efficaci contro il cancro alla prostata 20. A causa delle loro piccole dimensioni e la resistenza all&#39;attività RNasi endogena, miRNA sono biomarcatori stabili che possono essere facilmente rilevati nei tessuti fissati in formalina 21 e 22 biopsie prostatiche. Inoltre, i profili di espressione di miRNA sono stati confrontati in tessuti congelati e fissati in formalina e sono stati trovati per essere fortemente correlato 21. Tuttavia, miRNA profiling in cancro alla prostata tessuti clinici è spesso difficile a causa della eterogeneità del tumore, gli errori di campionamento, contaminazione stromale ecc Lo sviluppo dei miRNA come biomarcatori efficaci per il cancro alla prostata pesantemente relies sul loro rilevamento accurato nei tessuti clinici. Qui si descrive un flusso di lavoro semplificato utilizzato nel nostro laboratorio per miRNA profilatura in FFPE archiviati o cancro alla prostata congelato campioni clinici freschi. Ci avvaliamo di una combinazione di quantitativa real-time PCR e ibridizzazione in situ per miRNA le analisi di campioni clinici, con l&#39;ex producendo maggiori informazioni quantitative e la seconda per visualizzare l&#39;espressione differenziale delle potenziali biomarcatori miRNA in una vasta gamma di tessuti. All&#39;interno di questo flusso di lavoro, ottimizziamo le metodologie esistenti per l&#39;estrazione miRNA da FFPE e tessuti della prostata congelati, espressione analizza da Taqman-sonda basato miRNA RT-PCR e miRNA in situ tecnica di ibridazione con acido nucleico bloccato (LNA) -based sonde 23. Sonde basato LNA-offrono una maggiore sensibilità e specificità rispetto al DNA o RNA sonde based e consente il rilevamento affidabile di tutte le sequenze di miRNA, indipendentemente dal loro contenuto GC e permettono anche discriminazione delle famiglie miRNA. Il nostro protocollo miRNA ISH ottimizzato può essere applicato alle diapositive di cancro alla prostata di tessuto o microarray di tessuti di cancro alla prostata (TMA), con quest&#39;ultimo che offre il potenziale di accelerare miRNA biomarcatore scoperta.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems&#160;</td>
			<td>RM2255</td>
			<td></td>
		</tr>
		<tr>
			<td>Arcturus Autopix for LCM</td>
			<td>Arcturus/ Life Technologies</td>
			<td>LCM1621/LCM1110</td>
			<td>Alternatively, Arcutus Xt system from Life Technolgies can be used.&#160;</td>
		</tr>
		<tr>
			<td>CapSure Macro LCM Caps</td>
			<td>Life Technologies</td>
			<td>LCM0211</td>
			<td></td>
		</tr>
		<tr>
			<td>miRNeasy FFPE Kit&#160;</td>
			<td>Qiagen</td>
			<td align="right">217504</td>
			<td></td>
		</tr>
		<tr>
			<td>7500 Fast Real Time PCR System&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4351106</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman MicroRNA Reverse Transcription kit&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4366596</td>
			<td></td>
		</tr>
		<tr>
			<td>Taqman Fast Universal PCR master mix&#160;</td>
			<td>Applied Biosystems/ Life Technologies</td>
			<td align="right">4352042</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG labeled LNA probe for U6</td>
			<td>Exiqon</td>
			<td>99002-01</td>
			<td></td>
		</tr>
		<tr>
			<td>BM Purple AP substrate</td>
			<td>Roche</td>
			<td align="right">11442074001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pre-hybridization solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-1</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-2</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Blocking solution&#160;</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-8</a></td>
			<td></td>
		</tr>
		<tr>
			<td>AP-conjugated anti-digoxigenin antibody</td>
			<td>Biochain</td>
			<td><a href="http://www.biochain.com/coa/K21910XXCA.pdf">K2191050-7</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Aqueous mounting media&#160;</td>
			<td>Vector Laboratories&#160;</td>
			<td>H-5501&#160;&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol (guanidine isothiocyanate-phenol reagent)&#160;</td>
			<td>Life Technologies</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/15596018">15596-018</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Harris hematoxylin</td>
			<td>Statlab</td>
			<td>SL200</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin&#160;</td>
			<td>Statlab</td>
			<td>SL201&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

mirna expression analyses in prostate cancer clinical tissues

Una sfida importante nel cancro della prostata (PCa) gestione clinica è rappresentata dal inadeguatezza di biomarcatori attualmente utilizzati per lo screening delle malattie, la diagnosi, la prognosi e il trattamento. Negli ultimi anni, microRNA (miRNA) sono emersi come biomarcatori alternativi promettenti per la diagnosi del cancro alla prostata e la prognosi. Tuttavia, lo sviluppo di miRNA come biomarcatori efficaci per il cancro alla prostata si basa molto sul loro rilevamento accurato nei tessuti clinici. miRNA analisi nel cancro alla prostata campioni clinici è spesso difficile a causa della eterogeneità del tumore, gli errori di campionamento, contaminazione stromale ecc L&#39;obiettivo di questo articolo è quello di descrivere un flusso di lavoro semplificato per miRNA analizza FFPE archiviati o cancro alla prostata congelato campioni clinici freschi utilizzando una combinazione di quantitativa real-time PCR (RT-PCR) e ibridazione in situ (ISH). All&#39;interno di questo flusso di lavoro, ottimizziamo le metodologie esistenti per l&#39;estrazione miRNA da FFPE e congelato tissu prostataes e espressione analisi da Taqman-sonda basato miRNA RT-PCR. Inoltre, si descrive un metodo ottimizzato per ISH analizza il rilevamento formiRNA in tessuti prostatici con acido nucleico bloccato (LNA) - sonde based. Il nostro protocollo miRNA ISH ottimizzato può essere applicato alle diapositive di cancro alla prostata di tessuto o microarray di tessuti di cancro alla prostata (TMA).

Profilo di espressione di miR-203 nel cancro della prostata primario LCM campioni clinici mediante RT-PCR analisi (Figura 1) RT-PCR analisi di espressione relativa miR-203 in LCM prostata primaria tessuti tumorali e le regioni adiacenti normali appaiati è stata effettuata come descritto in Saini et al. 15 RNU48 è stato utilizzato come controllo. La tabella seguente riassume l&#39;espressione relativa miR-203 nei tessuti tumorali del cancro alla prostata...

Watch this Scientific Journal Video about miRNA Expression Analyses in Prostate Cancer Clinical Tissues at JoVE.com

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

Analisi miRNA nel tumore alla prostata tessuti cliniche

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

mirna-expression-analyses-in-prostate-cancer-clinical-tissues

Research

JoVE Journal

Medicine

10.8K Views.  University of California San Francisco. Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

Video: miRNA Expression Analyses in Prostate Cancer Clinical Tissues

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Valutazione della captazione nanoparticelle nei tumori in tempo reale utilizzando intravitale Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Ricerca

Medicina

Cell Population Analyses During Skin Carcinogenesis

Cancer development is a multiple-step process involving many cell types including cancer precursor cells, immune cells, fibroblasts and endothelial cells. Each type of cells undergoes signaling and functional changes during carcinogenesis. The current challenge for many cancer researchers is to dissect these changes in each cell type during the multiple-step process in vivo. In the last few years, the authors have developed a set of procedures to isolate different cell populations during skin cancer development using K14creER/R26-SmoM2YFP mice. The procedure is divided into 6 parts: 1) generating appropriate mice for the study (K14creER+ and R26-SmoM2YFP+ mice in this protocol); 2) inducing SmoM2YFP expression in mouse skin; 3) preparing mouse skin biopsies; 4) isolating epidermis from skin; 5) preparing single cells from epidermis; 6) labeling single cell populations for flow cytometry analysis. Generation of sufficient number of mice with the right genotype is the limiting step in this protocol, which may take up to two months. The rest of steps take a few hours to a few days. Within this protocol, we also include a section for troubleshooting. Although we focus on skin cancer, this protocol may be modified to apply for other animal models of human diseases.

Carcinogenesis is a process involving interactions between cancer cells and the cancer microenvironment. To dissect the molecular events, one needs to isolate different cell populations at different stages during cancer development. Using a mouse model for basal cell carcinoma, we describe a protocol for cell analyses during carcinogenesis.

Analisi popolazione cellulare Durante carcinogenesi della pelle

Cancer development is  a multiple-step process involving many cell types including cancer precursor  cells, immune cells, fibroblasts and endothelial ...

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

Microarray identificazione dei singoli HERV Loci Expression: Domanda di Biomarker Discovery in Cancro alla prostata

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Un ortotopico Modello murino di Human Prostate Cancer Metastasi

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

Isolamento di cancro cellule staminali da campioni umani di cancro alla prostata

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

Ex Vivo risposta al trattamento di tumori primari e / o associati per metastasi preclinici e sviluppo clinico di Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Studiare cancro del pancreas staminali caratteristiche cellulari per lo sviluppo di nuove strategie di trattamento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Prostate Cancer Patient Derived Xenograft Models from Circulating Tumor Cells

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, biologic studies, and personalized medicine strategies. Circulating tumor cells (CTC) play a critical role in tumor metastasis and have been isolated from patients with several tumor types. Recently, CTCs have been used to generate PDX experimental models of breast and prostate cancer. This manuscript details the method for the generation of prostate cancer PDX models from CTCs developed by our group. Advantages of this method over conventional PDX models include independence from surgical sample collection and generating experimental models at various disease stages. Density gradient centrifugation followed by red blood cell lysis and flow cytometry depletion of CD45 positive mononuclear cells is used to enrich CTCs from peripheral blood samples collected from patients with metastatic disease. The CTCs are then injected into immunocompromised mice; subsequently generated xenografts can be used for functional studies or harvested for molecular characterization. The primary limitation of this method is the negative selection method used for CTC enrichment. Despite this limitation, the generation of PDX models from CTCs provides a novel experimental model to be applied to prostate cancer research.

This manuscript details a method used to generate prostate cancer patient derived xenografts (PDXs) from circulating tumor cells (CTCs). The generation of PDX models from CTCs provides an alternative experimental model to study prostate cancer; the most commonly diagnosed tumor and a frequent cause of death from cancer in men.

Generazione di cancro alla prostata del paziente Derivato modelli di xenotrapianto di cellule tumorali circolanti

Patient derived xenograft (PDX) models are gaining popularity in cancer research and are used for preclinical drug evaluation, biomarker identification, ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Lavorare con tessuti umani per Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Exosomal miRNA Analysis in Non-small Cell Lung Cancer (NSCLC) Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective tyrosine kinase inhibitors (TKIs). To optimize the use of these TKIs, the discovery of new biomarkers for early detection and disease progression is mandatory. These plasma-isolated exosomes can be used as a non-invasive and repeatable way for the detection and follow-up of these biomarkers. One ml of plasma from 12 NSCLC patients, with different mutations and treatments (and 6 healthy donors as controls), were used as exosome sources. After RNAse treatment, in order to degrade circulating miRNAs, the exosomes were isolated with a commercial kit and resuspended in specific buffers for further analysis. The exosomes were characterized by western blotting for ALIX and TSG101 and by transmission electron microscopy (TEM) analysis, the standard techniques to obtain biochemical and dimensional data of these nanovesicles. Total RNA extraction was performed with a high yield commercial kit. Due to the limited miRNA-content in exosomes, we decided to perform retro-transcription PCR using an individual assay for each selected miRNA. A panel of miRNAs (30b, 30c, 103, 122, 195, 203, 221, 222), all correlated with NSCLC disease, were analyzed taking advantage of the remarkable sensitivity and specificity of Real-Time PCR analysis; mir-1228-3p was used as endogenous control and data were processed according to the formula 2-&#916;&#916;ct 13. Control values were used as baseline and results are shown in logarithmic scale.

Exosomal miRNA Analysis in Non-small Cell Lung Cancer NSCLC Patients' Plasma Through qPCR: A Feasible Liquid Biopsy Tool

This protocol describes the feasibility to perform miRNA profiling in exosomes, released in plasma of NSCLC patients, through a commercial exosome isolation kit with Proteinase K and RNAse treatments, in order to avoid circulating miRNAs contamination and evaluate their biomarker features in NSCLC.

Exosomal Analisi miRNA in non a piccole cellule del cancro del polmone (NSCLC) dei pazienti plasma attraverso qPCR: Un fattibile Liquido biopsia Strumento

The discovery of alterations in the EGFR and ALK genes, amongst others, in NSCLC has driven the development of targeted-drug therapy using selective ...