This research was supported by research grants RO1CA109545 and RO1CA086984 to M.S.S. by the National Institutes of Health/National Cancer Institute and by an award from the Leo and Ann Albert Charitable Trust (to M.S.S.).

Tutti gli studi in vivo sono stati approvati dalla University of Notre Dame cura degli animali e del Comitato uso e utilizzati femminili topi C57 / BL6J. 1. murino Ovarian Cancer Cell Culture <ol><li> Rendere il murino terreno di coltura delle cellule del cancro ovarico ID8 come segue: 1 L di Dulbecco Modified Eagle Medium (DMEM) integrato con 4% siero fetale bovino (FBS), 1% penicillina / streptomicina, 5 mg / Insulina ml, 5 mg / ml transferrina e 5 ng / ml di sodio selenite. </li><li> Trasdurre ID8 murine cellule di cancro ovarico 13 per esprimere proteine ​​rosso fluorescente (RFP) utilizzando particelle lentivirali commercialmente acquistati che esprimono RFP e un marcatore di selezione (blasticidin gene). Si noti che la proteina fluorescente verde può essere utilizzato, ma fornisce un segnale di fluorescenza debole. <ol><li> Immediatamente prima di trasduzione, le cellule di coltura per 50 - 70% di confluenza e aggiungere mezzo fresco (senza penicillina / streptomicina), che include 1 ml polibrene (5 mg / ml finaleconcentrazione). </li><li> Delicatamente pipetta RFP-lentivirus (20 ml) goccia a goccia nel piatto e incubare a 37 ° C per 72 ore. Rimuovere medie e incubare con terreno fresco per 24 ore. Iniziare selezione di cellule trasdotte aggiungendo blasticidin al mezzo di coltura (5 mg / ml) e monitor per eritrociti mediante microscopia in fluorescenza (Figura 1). </li></ol></li><li> Coltivare le cellule RFP-tag in media ID8 di semina di circa 10 6 cellule in un piatto di coltura tissutale e la visualizzazione di tutti i giorni utilizzando un microscopio ottico fino a quando il monostrato cellulare è confluenti. </li><li> Quando confluenti, raccogliere le cellule con tripsina (0,25%, 3 min) e riportare il pallone al incubatore a 37 o fino a quando le cellule sono staccati. </li><li> Neutralizzare la tripsina con 6 ml di mezzo di coltura contenente siero ID8. Pipettare su e giù contro il fondo del piatto, poi trasferire in un tubo da 15 ml. Centrifugare a 1200 rpm per 2 minuti, quindi aspirare il mezzo utilizzando un glass pipetta. </li><li> Lavare le cellule con tampone fosfato (PBS) per risospendere delicatamente in 6 ml di caldo PBS e centrifugazione come in 1.5 sopra. Risospendere in PBS ad una concentrazione finale di 5 x 10 6 cellule / ml. Mantenere le cellule calde (37 ° C) fino a iniezione. </li></ol> 2. iniezione intra-peritoneale di cellule ID8 e in vivo Imaging <ol><li> Iniettare ogni topo con 2 mL di soluzione calda ID8-PBS per un totale di 10 milioni di cellule attraverso (ip) iniezione intra-peritoneale. Consentono alle cellule tumorali di crescere in vivo per circa 10 settimane con l&#39;imaging dal vivo ogni settimana. </li><li> Immediatamente dopo l&#39;iniezione e ogni settimana da allora in poi, pesare i topi e poi anestetizzare ogni topo usando isofluorane (portata 2,5% in O 2 flusso di 0,5 l / min). Nota: I topi sono stati pesati, non come una misura quantitativa della massa tumorale, ma piuttosto una misura più generale dell topo progressione fisica durante lo studio. I topi pesanots sono stati confrontati con i propri valori di peso precedenti. <ol><li> Anestetizzare topi mettendo in una camera isofluorane. Mantenere l&#39;anestesia costanti durante la scansione posizionando la testa del mouse nel naso-cono per l&#39;esposizione al 2,5% isofluorane portata durante tutta la procedura. </li></ol></li><li> Usare una crema depilatoria per rimuovere i peli sul torace e dell&#39;addome di ogni topo, come i capelli neri causerà attenuazione del segnale di fluorescenza. I topi sono bagnate con acqua calda dopo applicazione della crema depilatoria e asciugati con carta assorbente RT. </li><li> Mentre anestetizzato, scansione dell&#39;intero corpo di ciascun topo usando un piccolo sistema di imaging ottico animale. Scansione di tutti i topi in una coorte in ogni punto. Utilizzare i seguenti due step protocollo (Figura 2A): <ol><li> Nel passaggio 1, per osservare fluorescente (RFP), le cellule tumorali, eseguire l&#39;acquisizione multispettrale a raccogliere un totale di 5 immagini utilizzando filtri di eccitazione di 440 nm, 460 nm, 480 nm, 520 nm e 540 nm, ed un filtro di emissione di 600 nm. Utilizzare i seguenti parametri di acquisizione: un&#39;esposizione standard di 15 sec, 2 x 2 binning, FOV di 160 mm, e f arresto di 1.1. </li><li> Al punto 2, per osservare l&#39;intero animale, acquisire un&#39;immagine di riflessione utilizzando un filtro aperto di eccitazione (luce bianca), un filtro di emissione aperto, un&#39;esposizione standard di 0,2 sec con 2 x 2 binning e un FOV di 16 cm. Nota: Il tempo di imaging attuale è ~ 1 min. </li></ol></li></ol> 3. mouse dissezione e Image Acquisition <ol><li> Quando l&#39;imaging dal vivo mostra la presenza di una lunga malattia intraperitoneale o animali accumulo mostra di ascite (come evidenziato da distensione addominale), la perdita o il guadagno di peso corporeo superiore al 20%, letargia o altri segni di sofferenza, eutanasia ogni mouse utilizzando CO 2 anestesia seguita da dislocazione cervicale. </li><li> Tagliare la pelle anteriormente lungo la linea mediana ventrale del mouse, utilizzando dissectio appuntiton forbici, dalla parte superiore delle cosce alla parte inferiore della gabbia toracica. Prestare la massima attenzione a tagliare solo lo strato di pelle (Figura 2BI). separare delicatamente la pelle dal tessuto peritoneale, facendo attenzione a non forare la parete peritoneale. </li><li> Utilizzando forbici dissezione appuntiti, tagliati lateralmente sia lungo la parte inferiore della gabbia toracica e la parte superiore delle cosce per creare due lembi di pelle (Figura 2B ii, iii). </li><li> Posizionare ogni mouse sul lato ventrale (cavità peritoneale rivolta verso il basso) con i lembi cutanei tirato aperta e scansione ogni topo con i suoi organi in situ utilizzando il piccolo sistema di imaging ottico animale (Figura 3A, B). <ol><li> Utilizzare gli stessi parametri di acquisizione di immagini, come descritto nella 2.4.1.-2.4.2. </li></ol></li><li> Continuare a sezionare il mouse con l&#39;estrazione di singoli organi peritoneali tra cui il fegato, omento / pancreas, stomaco, il diaframma, intestino tenue, intestino crasso, a sinistra ea destra del peritoneo, ovaie, kidneys, mesentere e cuscinetto adiposo. <ol><li> Osservare, enumerare, e registrare i tumori visibile in ogni organo. Utilizzare un calibro per misurare il diametro di ciascun tumore. </li><li> Designare tumori meno di 2 mm di diametro più piccolo, tra 2 e 5 mm come media e maggiore di 5 mm più grande. </li></ol></li><li> Posizionare gli organi sul modello di scansione organo (figura 4) che identifica una posizione predeterminata per ogni organo. Utilizzare PBS per mantenere gli organi umido. </li><li> Scansione ogni foglio di organi utilizzando il piccolo sistema di imaging ottico animale (Figura 3C, D). <ol><li> Utilizzare gli stessi parametri di acquisizione di immagini come descritto al punto 2.4. </li></ol></li><li> Dopo la scansione, luogo organi in formaldeide al 10% per consentire la successiva lavorazione per l&#39;esame istologico. </li></ol> 4. Quantificazione del carico tumorale in thImmagini e fluorescenza <ol><li> Al fine di ridurre la quantità di auto-fluorescenza ogni scansione multispettrale, prima unmix i file utilizzando software disponibile con il piccolo sistema di imaging animali. <ol><li> Caricare prima la richiesta di offerta: Nel file di spettrale Vivo, seguito dal Autofluor: Nel file di Vivo rosso nella finestra non miscelati Immagini e selezionare Unmix. </li></ol></li><li> Esportare i file in formato .bip 16 bit .tiff (non in scala). </li><li> Utilizzare il software ImageJ (ex vivo organi e poi il corpo pieno mouse con organi in situ). <ol><li> Utilizzare File&gt; Apri per aprire i file TIFF a 16 bit dei file di modello di scansione organo in ImageJ. </li><li> Sotto Immagine&gt; Regola&gt; Luminosità / Contrasto, scegliere di impostare il minimo valore visualizzato a 0 e il massimo valore visualizzato a 35.353 e poi sottoImmagine&gt; tabelle di ricerca, selezionare Red Hot. Nota: I vari modelli animali saranno richiedono diversi livelli di luminosità, ma è importante per mantenere la luminosità costante durante un determinato studio. </li><li> Utilizzando lo strumento Selezione a mano libera, disegnare una regione a forma libera di selezione interesse (ROI) intorno ad ogni organo e utilizzare lo strumento di misura (CTRL + M) per calcolare la superficie di ciascun organo; registrare la superficie di ogni organo in un foglio di calcolo. </li><li> Con il ROI disegnato attorno ad ogni organo, fate clic destro all&#39;interno del ROI e selezionare Duplica per includere solo l&#39;organo. </li><li> Guardando il singolo organo, sotto Immagine&gt; Regola&gt; Soglia, scegliere di impostare la soglia di immagine ad un più basso livello di soglia del + 1200 ed una soglia di livello superiore della + 1700 per selezionare only regioni più luminose fluorescenti e di controllo per selezionare sfondo scuro; selezionare OK. Nota: Le immagini possono richiedere manipolazione manuale dei cursori di soglia in ImageJ modo che le aree precedentemente brillanti sono selezionati per la fase di analisi, come mostrato sopra. Diversi modelli di malattia saranno richiedono diversi vincoli di soglia, ma è importante per mantenere i livelli di soglia costanti durante un determinato studio. </li><li> Sotto Analizza&gt; Analisi Particelle, modificare la dimensione (pixel ^ 2) a 10-Infinity, scegliere di visualizzare: contorni, di controllo per selezionare solo &quot;Risultati&quot; e fare clic su OK per misurare sia le aree e densità prime integrati di queste regioni luminose, tumori considerati. </li><li> Registrare i valori della superficie e la densità di Raw integrato di ogni selezione del tumore visualizzato nel <sTrong&gt; tabella Risultati nello stesso foglio di calcolo contenente le superfici d&#39;organo. </li><li> Sotto Analizza&gt; Strumenti&gt; Calibrazione Bar, aggiungere una barra di scala di calibrazione per le immagini degli organi. Nota: In questo esempio, questo è stato fatto cambiando la posizione di alto a destra, il colore di riempimento al nero, il colore dell&#39;etichetta di bianco, il numero di etichette a 5, le cifre decimali a 0, la dimensione del carattere a 12 e la Zoom fattore a 4,0 e consentendo testo in grassetto. </li><li> Sotto File&gt; Salva con nome ..., salvare il montaggio come .tiff e .jpeg. </li><li> Utilizzare File&gt; Apri per aprire il file .jpeg degli organi e poi sotto Inserisci&gt; Casella di testo </strong&gt;, aggiungere etichette organo creando una casella di testo sotto ciascuna delle tre file di organi. </li><li> Utilizzare File&gt; Apri per aprire ciascuno dei file TIFF a 16 bit delle scansioni corpo pieno in ImageJ in ordine di numero di mouse. </li><li> Sotto Immagine&gt; Regola&gt; Luminosità / Contrasto, scegliere di impostare il minimo valore visualizzato 0 e il massimo valore visualizzato a + 35353 e poi sotto Immagine&gt; tabelle di ricerca, selezionare Red Hot. </li><li> Sotto File&gt; Salva con nome ..., salvare il montaggio come. Tiff e .jpeg. </li></ol></li></ol> Analisi 5. I dati <ol><li> Aggiungere le aree di selezione tumorali e densità integrati prime, rispettivamente, per ogni organo di ciascun topo e registrare la superficie totale tumorale organo per ogni mouse. </li><li> Normalizzare l&#39;area registrata e integrat primevalori di densità ED per la dimensione di ciascun organo dividendo l&#39;area tumorale totale e valori di densità integrati prime totale dalla corrispondente superficie dell&#39;organo (Tabella 1). </li><li> Calcolare e tracciare la media di entrambe le superfici del tumore normalizzati ei valori di densità integrati prime normalizzati (Figura 5A, B). </li><li> Confrontare questi valori medi a quelli ottenuti contando visivamente i tumori che erano visibili ad occhio nudo durante l&#39;ispezione di ogni organo per lesioni metastatiche. </li></ol>

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Questo articolo fa parte di un numero speciale su Imaging multimodale pre-clinico, sponsorizzato da Bruker Biospin.

In contrasto con gli studi utilizzando cellule di cancro ovarico umano che devono essere condotti in topi immunocompromessi, il protocollo sopra descritto utilizza topi immunocompetenti C57 / BL6 e le cellule di cancro ovarico murini singenici. Mentre questo consente di valutare il ruolo potenziale di infiltrati immunitari in progressione tumorale e metastasi, la presenza di peli scuri sulla superficie addominale rende meno sensibili di imaging. Uso di un depilatoria per rimuovere i capelli prima di imaging migliora l&#...

Cancro ovarico epiteliale (EOC) è la più comune causa di morte per neoplasia ginecologica, con una stima di 21,290 nuove diagnosi negli Stati Uniti nel 2015 e si stima che 14.180 decessi 1. La stragrande maggioranza (&gt; 75%) delle donne con diagnosi di malattia in stadio avanzato (stadio III o IV) caratterizzata da diffuse metastasi intra-peritoneale e prognosi infausta. Recidiva di malattia nella cavità peritoneale dopo chemioterapia di prima linea è comune e rappresenta una delle principali cause di mortalità 2,3. EOC metastatizza da un unico meccanismo che coinvolge sia diretta estensione dal tumore primario agli organi peritoneali vicini così come per dissociazione o spargimento di cellule dalla superficie del tumore primario come singole cellule o aggregati multicellulari. Le cellule sono capannone nella cavità peritoneale, in cui resistono distacco indotta l&#39;apoptosi 4. Accumulo di ascite peritoneale è comune, come le cellule tumorali capannone bloccare il drenaggio linfatico peritoneale e tumors producono fattori di crescita che alterano la permeabilità vascolare. Una porzione di cellule tumorali capannone attaccare alla superficie degli organi peritoneali e strutture compreso intestino, fegato, omento e mesentere, dopo di che ancorano e proliferano per produrre più lesioni secondarie ampiamente diffusi 3,5. metastasi ematogena è raro. Così, gestione clinica comunemente è costituito da chirurgia citoriduttiva tra cui &quot;debulking ottimale&quot;, definita come la resezione di tutto tumore visibile (non importa quanto piccolo). Citoriduzione completa è associato ad un significativo aumento della sopravvivenza totale 6,7 ed è associato con la sfida di identificazione e la rimozione delle lesioni &lt;0,5 cm. modelli piccoli animali hanno dimostrato l&#39;utilità nella ricerca sul cancro ovarico nel migliorare la nostra comprensione della progressione della malattia, nonché l&#39;identificazione di biomarcatori prognostici e la sperimentazione di nuovi chemioterapie o approcci di terapia di combinazione. Come il primariosito di incidenza del cancro ovarico e metastasi è la cavità peritoneale, modelli ortotopici di EOC metastasi coinvolgono l&#39;analisi e la caratterizzazione della malattia intraperitoneale. Anche se ci sono stati i recenti miglioramenti nella capacità di cellule tumorali immagine, anche a livello di singola cellula, esistono ancora notevoli difficoltà nel quantificare il carico tumorale metastatico del EOC. Queste sfide si presentano a causa del numero, le dimensioni e la localizzazione anatomica delle lesioni metastatiche. Inoltre esiste una necessità di cellule tumorali etichetta per distinguerli dai normali cellule ospiti. Studi precedenti hanno utilizzato protocolli di etichettatura a base di anticorpi o trasfezione di cellule tumorali con luciferasi 8,9. Etichettatura diretta fluorescente delle cellule tumorali è stato segnalato da Chishima e collaboratori nel 1997 10. Etichette fluorescenti non richiedono aggiunta di substrato esogeno e di offrire una piacevole specificità delle cellule tumorali, fornendo un mezzo più efficace per tenere traccia delle metastasi del cancro 11,12 </sup&gt;. Qui si descrive un metodo di imaging ottico per l&#39;analisi quantitativa della malattia metastatica utilizzando un modello di xenotrapianto ortotopico singenici composta da proteina fluorescente rossa (RFP) -tagged murine ID8 cellule di cancro ovarico 13 e topi C57 / BL6 immuno-competenti. Abbiamo dimostrato un nuovo metodo di relativa carico tumorale quantificazione combinando in vivo ed ex vivo l&#39;imaging con rimozione del tessuto auto-fluorescenza. Questo approccio ha potenziale utilità in studi volti a valutare l&#39;effetto di specifici genetica, epigenetica o microambientali modifiche e / o modalità di trattamento sulla metastasi organo-specifiche di cancro ovarico.

<table border="0" cellpadding="0" cellspacing="0" width="863">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Dulbecco&#39;s Modified Eagle Medium</td>
			<td style="width:183px;">Corning</td>
			<td style="width:117px;">10-014-CM</td>
			<td style="width:143px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fetal bovine serum</td>
			<td>Gibco</td>
			<td>10437-028</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">penicillin/streptomycin</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:420px;">Insulin-transferrin-sodium selenite media supplement</td>
			<td>Sigma</td>
			<td>I-1884</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bruker Xtreme small animal imaging system</td>
			<td>Bruker Corp.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bruker Multispectral software</td>
			<td>Bruker Corp</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">lentiviral particles with Red fluorescent protein</td>
			<td>GenTarget, Inc.</td>
			<td>LVP023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">trypsin for cell culture</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Corning</td>
			<td>21-040-CM</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">depilatory cream (such as Nair Hair Remover Lotion)</td>
			<td>purchases from drugstore&#160;</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ImageJ software&#160;</td>
			<td>http://imagej.nih.gov/ij/&#160;</td>
			<td></td>
			<td>free download</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dissecting tools (forceps)</td>
			<td>Roboz Surgical Instrument</td>
			<td>&#160;RS 5130</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">dissecting tools (Scissors)</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS 5910</td>
			<td></td>
		</tr>
	</tbody>
</table>

quantitation of intra-peritoneal ovarian cancer metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Il meccanismo metastatico del cancro ovarico è caratterizzato da altamente diffusa metastasi intraperitoneale composto da numerose lesioni di varie dimensioni, tra cui più di piccole dimensioni (&lt;2 mm) lesioni. Pertanto, l&#39;uso di cellule tumorali RFP marcato (Figura 1) e l&#39;imaging ottico fornisce un metodo alternativo per conteggio manuale e la misurazione delle dimensioni della lesione. Lo sviluppo della massa tumorale nel tempo può essere determinata sett...

Watch this Scientific Journal Video about Quantitation of Intra-peritoneal Ovarian Cancer Metastasis at JoVE.com

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

La quantificazione di intra-peritoneale del cancro ovarico metastasi

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

quantitation-of-intra-peritoneal-ovarian-cancer-metastasis

Research

JoVE Journal

Medicine

10.8K Views.  University of Notre Dame. Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Video: Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Experimental Metastasis Assay

Metastasis is the leading cause of death in cancer patients. To understand the mechanism of metastasis, an experimental metastasis assay was established using immunodeficient mice. This article delineates the procedures involved in this assay, including sample preparation, intravenous injection, and culturing cells from lung metastases. Briefly, a pre-determined number of human cancer cells were prepared in vitro and directly injected into the circulation of immunodeficient mice through their tail veins. A small number of cells survive the turbulence in the circulation and grow as metastases in internal organs, such as lung. The injected mice are dissected after a certain period. The tissue distribution of metastases is determined under a dissecting microscope. The number of metastases in a specific tissue is counted and it directly correlates with the metastatic ability of the injected cancer cells. The arisen metastases are isolated and cultured in vitro as cell lines, which often show enhanced metastatic abilities than the parental line when injected again into immunodeficient mice. These highly metastatic derivatives become useful tools for identifying genes or molecular pathways that regulate metastatic progression.

This article describes the procedures of an experimental metastasis assay that is used to determine the metastatic potential of human cancer cell lines.

Metastasi di analisi sperimentale

Metastasis is the leading cause of death in cancer patients.  To understand the mechanism of metastasis, an experimental metastasis assay was established ...

Ricerca

Medicina

Quantitative Analysis of Cancer Metastasis using an Avian Embryo Model

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex process that can involve many tissue types, span variable time periods, and often occur deep within organs, making it difficult to investigate and quantify. In addition, the efficacy of the metastatic process is influenced by multiple steps in the metastatic cascade making it difficult to evaluate the contribution of a single aspect of tumor cell behavior. As a consequence, metastasis assays are frequently performed in experimental animals to provide a necessarily realistic context in which to study metastasis. Unfortunately, these models are further complicated by their complex physiology. The chick embryo is a unique in vivo model that overcomes many limitations to studying metastasis, due to the accessibility of the chorioallantoic membrane (CAM), a well-vascularized extra-embryonic tissue located underneath the eggshell that is receptive to the xenografting of tumor cells (figure 1). Moreover, since the chick embryo is naturally immunodeficient, the CAM readily supports the engraftment of both normal and tumor tissues. Most importantly, the avian CAM successfully supports most cancer cell characteristics including growth, invasion, angiogenesis, and remodeling of the microenvironment. This makes the model exceptionally useful for the investigation of the pathways that lead to cancer metastasis and to predict the response of metastatic cancer to new potential therapeutics. The detection of disseminated cells by species-specific Alu PCR makes it possible to quantitatively assess metastasis in organs that are colonized by as few as 25 cells. Using the Human Epidermoid Carcinoma cell line (HEp3) we use this model to analyze spontaneous metastasis of cancer cells to distant organs, including the chick liver and lung. Furthermore, using the Alu-PCR protocol we demonstrate the sensitivity and reproducibility of the assay as a tool to analyze and quantitate intravasation, arrest, extravasation, and colonization as individual elements of metastasis.

Using quantitative PCR, we demonstrate how the well-established chick CAM model can be used to quantitatively analyze the metastasis of human tumor cells to distant organs.

L&#39;analisi quantitativa delle metastasi del cancro utilizzando un modello di aviaria Embryo

During metastasis cancer cells disseminate from the primary tumor, invade into surrounding tissues, and spread to distant organs. Metastasis is a complex ...

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 percent of women with ovarian cancer develop new metastases, and the recurrence is often fatal2. It is, therefore, necessary to understand how secondary metastases arise in order to develop better treatments for intermediate and late stage ovarian cancer. Ovarian cancer metastasis occurs when malignant cells detach from the primary tumor site and disseminate throughout the peritoneal cavity. The disseminated cells can form multicellular clusters, or spheroids, that will either remain unattached, or implant onto organs within the peritoneal cavity3 (Figure 1, Movie 1). 
All of the organs within the peritoneal cavity are lined with a single, continuous, layer of mesothelial cells4-6 (Figure 2). However, mesothelial cells are absent from underneath peritoneal tumor masses, as revealed by electron micrograph studies of excised human tumor tissue sections3,5-7 (Figure 2). This suggests that mesothelial cells are excluded from underneath the tumor mass by an unknown process. 
Previous in vitro experiments demonstrated that primary ovarian cancer cells attach more efficiently to extracellular matrix than to mesothelial cells8, and more recent studies showed that primary peritoneal mesothelial cells actually provide a barrier to ovarian cancer cell adhesion and invasion (as compared to adhesion and invasion on substrates that were not covered with mesothelial cells)9,10. This would suggest that mesothelial cells act as a barrier against ovarian cancer metastasis. The cellular and molecular mechanisms by which ovarian cancer cells breach this barrier, and exclude the mesothelium have, until recently, remained unknown. 
Here we describe the methodology for an in vitro assay that models the interaction between ovarian cancer cell spheroids and mesothelial cells in vivo (Figure 3, Movie 2). Our protocol was adapted from previously described methods for analyzing ovarian tumor cell interactions with mesothelial monolayers8-16, and was first described in a report showing that ovarian tumor cells utilize an integrin &#x2013;dependent activation of myosin and traction force to promote the exclusion of the mesothelial cells from under a tumor spheroid17. This model takes advantage of time-lapse fluorescence microscopy to monitor the two cell populations in real time, providing spatial and temporal information on the interaction. The ovarian cancer cells express red fluorescent protein (RFP) while the mesothelial cells express green fluorescent protein (GFP). RFP-expressing ovarian cancer cell spheroids attach to the GFP-expressing mesothelial monolayer. The spheroids spread, invade, and force the mesothelial cells aside creating a hole in the monolayer. This hole is visualized as the negative space (black) in the GFP image. The area of the hole can then be measured to quantitatively analyze differences in clearance activity between control and experimental populations of ovarian cancer and/ or mesothelial cells. This assay requires only a small number of ovarian cancer cells (100 cells per spheroid X 20-30 spheroids per condition), so it is feasible to perform this assay using precious primary tumor cell samples. Furthermore, this assay can be easily adapted for high throughput screening.

In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis

The mesothelial clearance assay described here takes advantage of fluorescently labeled cells and time-lapse video microscopy to visualize and quantitatively measure the interactions of ovarian cancer multicellular spheroids and mesothelial cell monolayers. This assay models the early steps of ovarian cancer metastasis.

In vitro test Clearance mesoteliali che modella i primi passi di metastasi del cancro ovarico

Ovarian cancer is the fifth leading cause of cancer related deaths in the United States1. Despite a positive initial response to therapies, 70 to 90 ...

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 ...

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  ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Metodo per ottenere primarie cellule tumorali ovariche da campioni solidi

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

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

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

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

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

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

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Modello murino per l&#39;imaging non invasivo per rilevare e monitorare Ovarian Cancer Ricorrenza

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, invasive, and chemoresistant tumor cells. Therefore, CSCs are an attractive population of cells to target therapeutically. CSCs are predicted to contribute to a number of types of malignancies including those in the blood, brain, lung, gastrointestinal tract, prostate, and ovary. Isolating and enriching a tumor cell population for CSCs will enable researchers to study the properties, genetics, and therapeutic response of CSCs. We generated a protocol that reproducibly enriches for ovarian cancer CSCs from ovarian cancer cell lines (SKOV3 and OVCA429). Cell lines are treated with 20 &#181;M cisplatin for 3 days. Surviving cells are isolated and cultured in a serum-free stem cell media containing cytokines and growth factors. We demonstrate an enrichment of these purified CSCs by analyzing the isolated cells for known stem cell markers Oct4, Nanog, and Prom1 (CD133) and cell surface expression of CD177 and CD133. The CSCs exhibit increased chemoresistance. This method for isolation of CSCs is a useful tool for studying the role of CSCs in chemoresistance and tumor relapse.

Cancer stem cells (CSCs) are&#160;implicated in tumor relapse due to chemoresistance. We have optimized a protocol for selection and expansion of CSCs from ovarian cancer cell lines. By treating cells with the chemotherapeutic cisplatin and culturing&#160;in a stem cell promoting media we enrich for non-adherent CSC cultures.

Arricchimento per chemioresistente Ovarian Cancer Stem Cells da linee cellulari umane

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

In Arricchimento vitro di Ovarian Cancer Cells-tumorali avvio

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...