Vorremmo ringraziare la Nikon Imaging Center presso la Harvard Medical School, in particolare Jennifer Waters, Lara Petrak e salmone Wendy, per la formazione e l&#39;uso delle loro microscopi timelapse. Vorremmo anche ringraziare Rosa Ng e Achim Besser per le discussioni importanti. Questo lavoro è stato supportato dal NIH di Grant 5695837 (da M. Iwanicki) e GM064346 di ACC, da una sovvenzione dal Dott. Miriam e Sheldon G. Adelson Medical Research Foundation (a JSB).

1. Ovarian Cancer Cell Formazione Spheroid <ol><li> RFP-esprimono cellule di cancro ovarico vengono coltivate in terreno di base 10% (una consuetudine mezzo di coltura cellulare contenente una miscela 50:50 di 199 e MCDB105, 10% siero fetale bovino inattivato e 1% pen-strep). Per esprimere RFP in cellule non marcate cancro ovarico, trasfettare le cellule con un plasmide contenente RFP e per selezionare cellule esprimenti RFP. In alternativa, vettori virali possono essere usati per esprimere transitoriamente proteine ​​fluorescenti, o le cellule possono essere pre-incubare con fluorescente rossa cella inseguitore colorante (Invitrogen). </li><li> Prima di formare sferoidi di cancro ovarico, è necessario preparare bassa aderenza e rotonde 96 piastre di coltura fondo. Per produrre i bassa aderenza piastre di coltura, 30μl poli-HEMA (6mg polyhydroxyethylmethacrylate in 1 ml di EtOH 95%) soluzione viene aggiunta a ciascun pozzetto di un pozzo 96 piatto di coltura cellulare Corning. Le piastre a 96 pozzetti vengono incubate a 37 ° C non incubatore umidificato per far evaporare l&#39;etanolo, Leaving una pellicola di poli-HEMA in ciascun pozzetto. Questo poli-HEMA pellicola impedisce alle cellule di attaccarsi al fondo del pozzo, costringendo le cellule a crescere in sospensione 18. [In alternativa, Ultra-Low piastre di coltura Attachment (Corning) può essere usato al posto di piatti rivestiti poly-HEMA.] </li><li> Dopo bassa aderenza piastre di coltura vengono preparati, Tripsinizzare una piastra di cellule tumorali ovariche, pellet delle cellule in una centrifuga da tavolo (Heraeus) a 900 RCF per 3 minuti, aspirare il supernatante e risospendere in terreno di base 10%. </li><li> Contare le cellule utilizzando un emocitometro. </li><li> Regolare la concentrazione di tali cellule che ci sono 100 cellule per 50 microlitri di terreno di base 10%. </li><li> Aggiungere 50 microlitri della sospensione cellulare diluita uniformemente sospeso in ciascun pozzetto della piastra a 96 pozzetti poli-HEMA cultura rivestita. </li><li> Incubare la piastra a 96 pozzetti a 37 ° C incubatore di coltura cellulare per 16 ore (tale quantità di tempo dovrebbe essere aumentata o diminuita a seconda della quantità di tempo necessario peruna linea cellulare particolare per formare sferoidi multicellulari o desiderati condizioni sperimentali) permettere alle cellule del carcinoma ovarico a raggrupparsi, formando un unico sferoide multicellulare in ciascun pozzetto. Alcune cellule tumorali possono apoptosi durante questo periodo, quindi è importante scegliere un momento prima della induzione di apoptosi. </li></ol> 2. Mesoteliali formazione di cellule monostrato <ol><li> In una cappa coltura cellulare, pre-rivestimento dei pozzetti di una ben 6 fondo di vetro piatto MatTek con fibronectina aggiungendo 2 ml di fibronectina 5μg / mL di soluzione salina in ogni pozzetto della piastra e incubando a temperatura ambiente per 30 minuti. La qualità ottica dei fondi di vetro in piatti MatTek consentono immagini ad alta risoluzione microscopico. </li><li> GFP-esprimono cellule mesoteliali vengono coltivate in terreno di base 10%. Tripsinizzare un piatto di cellule mesoteliali, spin down in una centrifuga da tavolo (Heraeus) a 900RPM per 3 minuti, aspirare il surnatante, e risospendere in terreno di base del 10%. Ilcellule mesoteliali usati qui sono stati già esprimendo GFP quando sono stati ottenuti, ma non marcati cellule mesoteliali può essere prodotto da trasfezione con un plasmide contenente cDNA GFP, o preincubating le cellule in un verde fluorescente cella inseguitore colorante (Invitrogen). </li><li> Dopo l&#39;incubazione di 30 minuti fibronectina (in fase 2.1), lavare i pozzetti del piatto MatTek con 2 ml PBS. </li><li> Aspirare il PBS e la piastra 6 x10 5 cellule mesoteliali per bene in ciascun pozzetto della piastra ben 6 MatTek. Incubare il piatto MatTek a 37 ° C incubatore per colture cellulare notte per consentire le cellule mesoteliali per collegare al piatto e formano un monostrato. </li></ol> 3. Mesoteliali cella Assay Clearance <ol><li> Utilizzare una pipetta per raccogliere gli sferoidi carcinoma ovarico da ben 96 poli-HEMA piastra rivestita. </li><li> Aspirare il mezzo da un pozzetto della piastra ben 6 MatTek contenente un monostrato di cellule mesoteliali. Lavare una volta con 2 ml PBS. Aggiungere tutti gli sferoidi dal 96 pozzetti ptardi per un pozzetto della piastra MatTek (~ 3x il numero di sferoidi che stanno per essere ripreso in considerazione per l&#39;atterraggio sferoidi parte del piatto che non può essere ripreso). </li><li> Porre la capsula MatTek sul palco di un microscopio invertito widefield a fluorescenza in grado di eseguire time-lapse imaging per la durata di almeno 8 ore. Utilizzare uno stadio motorizzato a posizioni più immagini nel piatto, con più eventi di intercalazione sferoidali, in un singolo esperimento. Usiamo un Ti-E Nikon invertito motorizzato widefield fluorescenza time-lapse microscopio integrato con un sistema di messa a fuoco perfetta e bassa [20x-0.75 apertura numerica (NA)] ingrandimento / NA contrasto differenziale di interferenza (DIC) l&#39;ottica, un transilluminatore alogena Nikon con 0,52 NA lunga distanza di lavoro (LWD) condensatore, Nikon veloce (&lt;100 millisecondi tempo di commutazione), filtri di eccitazione e di emissione (GFP Ex 480/40, Em 525/50, RFP-mCherry Ex 575/50 Em 640/50), Sutter veloce trasmessi e epifluorescenza percorso Valvola luce intelligenteTERS, una lineare codificato Nikon stadio motorizzato, un ORCA Hamamatsu-AG raffreddato charge-coupled device (CCD), macchina fotografica, una custom-built camera di incubazione microscopio con la temperatura e CO 2 di controllo, Nikon NIS-Elements AR software versione 3, e un TMC vibrazioni tavolo isolamento. </li><li> Gli sferoidi di cellule di cancro ovarico si depositerà sul fondo del piatto e allegare il monostrato cellulare mesoteliali. Raccogliere GFP, RFP e le immagini di fase di 20 + sferoidali / monostrato interazioni, ogni 10 minuti, per 8 ore. </li><li> Le RFP che esprimono sferoidi di cellule di cancro ovarico si invadere la GFP che esprimono monostrato di cellule mesoteliali la creazione di un buco nel monostrato. Dopo 8 ore, misurare le dimensioni dei fori tracciando i buchi neri nelle immagini GFP usando software Elements (o un altro software adatto come immagine J). Normalizza le dimensioni del foro alla dimensione iniziale sferoide dividendo la dimensione del foro a 8 ore dalla dimensione dello sferoide nell&#39;immagine RFP corrispondente al tempo zero. In questo exampio, la dimensione del foro è stata misurata una volta sola, ma può essere misurata più volte durante l&#39;esperimento otto ore per comprendere meglio le dinamiche di intercalazione. </li></ol> 4. Risultati rappresentativi In questo esempio, abbiamo confrontato la capacità mesoteliali liquidazione dei OVCA433 sferoidi di cellule di cancro ovarico che hanno attenuato l&#39;espressione della Talin-1 per controllare OVCA433 sferoidi. OVCA433 sferoidi di ciascun gruppo sono stati aggiunti ad un piatto MatTek contenente ZT monostrati di cellule mesoteliali. Sei sferoidi di ogni gruppo sono stati ripresi ogni 10 minuti per otto ore (Figura 4, Movie 3, Movie 4). I fori prodotti nella monostrato dai sferoidi diffusione sono state misurate e sei posizioni di ciascun gruppo sono stati mediati. La figura 4 mostra che l&#39;area di clearance media creato da Talin 1 sferoidi atterramento era significativamente più piccola rispetto alla superficie media creato da sferoidi di controllo, suggerendo che Talin è necessario per la loro liquidazione mesoteliali da OVCA433 carcinoma ovarico sferoidi. <img alt="Figura 1" src="/files/ftp_upload/3888/3888fig1.jpg" /> Figura 1. Cancer Metastasi ovarica. Tumori ovarici primari sviluppare sia dall&#39;epitelio di superficie ovarico o tube di Falloppio. Le cellule tumorali / cluster staccarsi dal tumore primario e raccogliere nella cavità peritoneale. Le cellule tumorali possono quindi aggregare per formare sferoidi multicellulari. Sferoidi quindi allegare ai monostrati di cellule mesoteliali che rivestono la cavità peritoneale. Le cellule mesoteliali sono esclusi da sotto lo sferoide allegata cancro ovarico, consentendo gli sferoidi di accedere alla membrana basale sottostante. Movie 1. Metastasi del cancro ovarico. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_1_Lower_Quality.mp4">Clicca qui per vedere il film</a> . <img alt="Figura 2" src="/files/ftp_upload/3888/3888fig2.jpg" /> Figura 2. </strong&gt; Linea mesoteliale cellule della superficie del tessuto umano peritoneale e sono esclusi da sotto gli impianti di cellule di cancro ovarico. <img alt="Figura 3" src="/files/ftp_upload/3888/3888fig3.jpg" /> Figura 3. Assay Clearance mesoteliali. Sferoidi carcinoma ovarico formate incubando 100 RFP cellule esprimenti carcinoma ovarico per pozzetto in una poli-HEMA rivestita 96 piatto fondo del pozzetto rotondo coltura a 37 ° C per 16 ore. Poly-HEMA impedisce alle cellule di attaccarsi alla piastra di coltura, permettendo alle cellule di rimanere in sospensione e aderire l&#39;una all&#39;altra a formare un unico cluster per pozzetto. Monostrati di cellule mesoteliali sono preparati mediante placcatura 6x10 5 cellule mesoteliali per pozzetto in una fibronectina rivestito 6 ben piatto MatTek e incubando la piastra a 37 ° C per 16 ore. Gli sferoidi sono poi trasferiti al piatto MatTek con il monostrato mesoteliali e le due popolazioni cellulari vengono esposte ogni 10 minuti per 8 ore utilizzando un legame Ti-E Nikon Inverted motorizzato widefield fluorescenza time-lapse microscopio e software Elements. Movie 2. Saggio Clearance mesoteliali. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_2_lower_quality.mp4">Clicca qui per vedere il film</a> . <img alt="Figura 4" src="/files/ftp_upload/3888/3888fig4.jpg" /> Attenuazione Figura 4. Di Talin uno espressione in OVCA433 sferoidi possono diminuire la capacità mesoteliali clearance. OVCA433 sferoidi (rosso) con e senza l&#39;espressione attenuata di Talin 1 sono stati autorizzati a cui attaccarsi e invadere in un monostrato ZT mesoteliali (verde). Le due popolazioni cellulari sono stati ripresi ogni 10 minuti per 8 ore utilizzando una Ti-E Nikon invertito motorizzato widefield fluorescenza time-lapse microscopio e software elementi. Il grafico mostra che Talin 1 attenuazioneriduce significativamente la clearance delle cellule mesoteliali (plot Quantile con barre verdi i mezzi). Movie 3. Controllo OVCA433 sferoidi (rosso) che invadono in un monostrato mesoteliali (verde). <a href="http://www.jove.com/files/ftp_upload/3888/Movie_3.mov">Clicca qui per vedere il film</a> . Attenuazione Movie 4. Di Talin uno espressione in OVCA433 sferoidi (rosso) diminuisce mesoteliali (verde) capacità di clearance. <a href="http://www.jove.com/files/ftp_upload/3888/Movie_4.mov">Clicca qui per vedere il film</a> .

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Il &quot;test Liquidazione mesoteliale&quot; qui presentata usa microscopia time-lapse per monitorare le interazioni di sferoidi multicellulari di cancro ovarico e monostrati di cellule mesoteliali, in grande dettaglio spaziale e temporale. In precedenza, diversi gruppi 8-14 aveva usato saggi endpoint per dimostrare che le cellule tumorali ovariche attaccarsi e invadere monostrati di cellule mesoteliali. Questo saggio è unico in quanto utilizza celle fluorescente di distinguere le cellule tumorali da cellule...

<table border="1"><tbody><tr><td> Reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Comments </td></tr><tr><td> OVCA433 Ovarian Cancer Cells </td><td></td><td></td><td> Regalo da Dr. Dennis Slamon </td></tr><tr><td> ZT cellule mesoteliali </td><td></td><td></td><td> Regalo da Dr. Tan Ince </td></tr><tr><td> Medium 199 </td><td> Gibco </td><td> 19950 </td><td></td></tr><tr><td> MCDB105 </td><td> Cella Applications Inc. </td><td> 117-500 </td><td></td></tr><tr><td> -FBS inattivato al calore </td><td> Gibco </td><td> 10082 </td><td></td></tr><tr><td> Pen-Strep </td><td> Gibco </td><td> 15070 </td><td></td></tr><tr><td> Piastre da 96 pozzetti </td><td> Corning Costar </td><td> 3799 </td><td></td></tr><tr&gt; <td> Polyhydroxyethylmethacrylate (poly-HEMA) </td><td> Sigma Aldrich </td><td> 192066-25G </td><td> Per poli-HEMA soluzione sciogliere 6mg poli-HEMA polvere in 1 ml di 95% EtOH </td></tr><tr><td> EtOH </td><td> Pharmco-aaper </td><td> 111ACS200 </td><td> Diluire al 95% in DH 2 0 </td></tr><tr><td> Coltura cellulare cappuccio </td><td> Nuaire </td><td> NU-425-300 </td><td></td></tr><tr><td> Tissue incubatore per colture </td><td> Thermo Scientific </td><td> 3110 </td><td></td></tr><tr><td> incubatore di poly-HEMA piastre </td><td> Labline Instruments </td><td> Imperial III 305 </td><td></td></tr><tr><td> Centrifuga da tavolo </td><td> Heraeus </td><td> 75003429/01 </td><td></td></tr><tr><td> 6 pozzetti con fondo di vetro piatto </td><td> MatTek corp. </td><td> P06G-1.5-20-F </td><td></td></tr><tr><td> Fibronectina </td><td> Sigma </td><td> F1141-1MG </td><td></td></tr><tr><td> PBS </td><td> Cellgro </td><td> 21-040-CV </td><td></td></tr><tr><td> Timelapse microscopio: </td><td></td><td></td><td></td></tr><tr><td> Microscopio </td><td> Nikon </td><td></td><td> Ti-E motorizzato invertito a fluorescenza time-lapse microscopio integrato con un sistema di messa a fuoco perfetta </td></tr><tr><td> Lente </td><td> Nikon </td><td></td><td> 20X-0.75 apeture numerica </td></tr><tr><td> Alogena transilluminatore </td><td> Nikon </td><td></td><td> 0,52 NA lunga distanza di lavoro del condensatore </td></tr><tr><td> Eccitazione ed emissione filtri </td><td> Chroma filtri passa singoli Nikon custodia </td><td></td><td> GFP Ex 480/40, Em 525/50 RFP-mCherry Ex 575/50 Em 640/50 </td></tr><tr><td> Trasmessi e Epifluoresce percorso della luce </td><td> Sutter </td&gt; <td></td><td> Persiane Smart </td></tr><tr><td> Linear-encoded stage motorizzato </td><td> Nikon </td><td></td><td></td></tr><tr><td> Raffreddato charged-coupled fotocamera del dispositivo </td><td> Hamamatsu </td><td> ORCA-AG </td><td></td></tr><tr><td> Microscopio camera di incubazione con la temperatura e CO 2 di controllo </td><td> custom-built </td><td></td><td></td></tr><tr><td> Isolamento delle vibrazioni tavolo </td><td> TMC </td><td></td><td></td></tr><tr><td> NIS-Elements software </td><td> Nikon </td><td></td><td> Versione 3 </td></tr></tbody></table>

in vitro mesothelial clearance assay that models the early steps of ovarian cancer metastasis

Il cancro ovarico è la quinta causa di decessi per cancro correlati negli Stati Uniti 1. Nonostante un iniziale risposta positiva alle terapie, da 70 a 90 per cento delle donne con tumore ovarico sviluppare nuove metastasi, e la ricorrenza è spesso fatale 2. Si tratta, quindi, necessario capire come metastasi secondarie sorgono al fine di sviluppare migliori trattamenti per il cancro fase intermedia e tardiva dell&#39;ovaio. Metastasi del cancro ovarico si verifica quando le cellule maligne si staccano dal tumore primario e diffondere in tutta la cavità peritoneale. Le cellule diffusi possono formare aggregati multicellulari, o sferoidi, che uno rimanga unattached, o impianto sul organi all&#39;interno della cavità peritoneale 3 (figura 1, film 1). Tutti gli organi all&#39;interno della cavità peritoneale sono rivestite con un unico strato continuo di cellule mesoteliali 4-6 (Figura 2). Tuttavia, le cellule mesoteliali sono assenti da sottomasse tumorali peritoneali, come rivelato dagli studi al microscopio elettronico di sezioni umani asportati 3,5-7 tessuto tumorale (Figura 2). Questo suggerisce che le cellule mesoteliali sono esclusi da sotto la massa tumorale da un processo sconosciuto. Precedente esperimenti in vitro hanno dimostrato che cellule primarie di cancro ovarico collegare in modo più efficace matrice extracellulare che di cellule mesoteliali 8, e studi più recenti hanno dimostrato che le cellule primarie peritoneali mesoteliali in realtà costituiscono una barriera per l&#39;adesione delle cellule del cancro ovarico e l&#39;invasione (rispetto alla adesione e l&#39;invasione su supporti che non sono stati coperti con le cellule mesoteliali) 9,10. Questo suggerisce che le cellule mesoteliali fungere da barriera contro il cancro ovarico metastasi. I meccanismi cellulari e molecolari con cui le cellule del cancro ovarico violare questa barriera, ed escludere il mesotelio hanno, fino a poco tempo, è rimasto sconosciuto. Qui si descrive °metodologia e di un test in vitro che modella l&#39;interazione tra sferoidi di cellule di cancro ovarico e cellule mesoteliali in vivo (figura 3, Movie 2). Il nostro protocollo è stato adattato da metodi descritti in precedenza per analizzare le interazioni delle cellule tumorali ovariche con monostrati mesoteliali 8-16, ed è stata descritta in un rapporto che mostra che le cellule tumorali ovariche utilizzano un integrina-dipendente l&#39;attivazione della miosina e la forza di trazione per promuovere l&#39;esclusione del cellule mesoteliali da sotto un tumore 17 sferoide. Questo modello si avvale di time-lapse microscopia a fluorescenza per monitorare le due popolazioni cellulari in tempo reale, fornendo informazioni spaziale e temporale sull&#39;interazione. Le cellule del carcinoma ovarico esprimere la proteina fluorescente rossa (RFP), mentre le cellule mesoteliali esprimere la proteina fluorescente verde (GFP). RFP che esprimono sferoidi di cellule di cancro ovarico allegare alla GFP-esprimere monostrato mesoteliali. La diffusione sferoidi, invadere, eforzare le cellule mesoteliali parte creando un foro nel monostrato. Questo foro è visualizzato come lo spazio negativo (nero) nell&#39;immagine GFP. L&#39;area del foro può essere misurata per analizzare quantitativamente differenze di attività di gioco tra le popolazioni di controllo e sperimentali di cancro ovarico e / o cellule mesoteliali. Questo saggio necessita soltanto un piccolo numero di cellule di cancro ovarico (100 cellule per sferoidali X 20-30 sferoidi per condizione), quindi è possibile eseguire questo saggio con preziosi campioni di cellule del tumore primario. Inoltre, questa analisi può essere facilmente adattato per lo screening ad alto rendimento.

Watch this Scientific Journal Video about In vitro Mesothelial Clearance Assay that Models the Early Steps of Ovarian Cancer Metastasis at JoVE.com

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

Il saggio di liquidazione mesoteliali qui descritto si avvale di cellule fluorescente e microscopia time-lapse video visualizzare e misurare quantitativamente le interazioni di sferoidi multicellulari di cancro ovarico e monostrati di cellule mesoteliali. Questo saggio modella i primi passi di metastasi del cancro ovarico.

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 ...

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

14.4K Views.  Harvard Medical School. Il saggio di liquidazione mesoteliali qui descritto si avvale di cellule fluorescente e microscopia time-lapse video visualizzare e misurare quantitativamente le interazioni di sferoidi multicellulari di cancro ovarico e monostrati di cellule mesoteliali. Questo saggio modella i primi passi di metastasi del cancro ovarico.

Video: In vitro Mesothelial Clearance Assay that Models the Early Steps of 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 ...

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Medicina

MAME Models for 4D Live-cell Imaging of Tumor: Microenvironment Interactions that Impact Malignant Progression

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in real-time of cell:cell interactions. Our overall goal was to develop models that recapitulate the architecture of preinvasive breast lesions to study their progression to an invasive phenotype. Specifically, we developed models to analyze interactions among pre-malignant breast epithelial cell variants and other cell types of the tumor microenvironment that have been implicated in enhancing or reducing the progression of preinvasive breast epithelial cells to invasive ductal carcinomas. Other cell types studied to date are myoepithelial cells, fibroblasts, macrophages and blood and lymphatic microvascular endothelial cells. In addition to the MAME models, which are designed to recapitulate the cellular interactions within the breast during cancer progression, we have developed comparable models for the progression of prostate cancers. 
Here we illustrate the procedures for establishing the 3D cocultures along with the use of live-cell imaging and a functional proteolysis assay to follow the transition of cocultures of breast ductal carcinoma in situ (DCIS) cells and fibroblasts to an invasive phenotype over time, in this case over twenty-three days in culture. The MAME cocultures consist of multiple layers. Fibroblasts are embedded in the bottom layer of type I collagen. On that is placed a layer of reconstituted basement membrane (rBM) on which DCIS cells are seeded. A final top layer of 2% rBM is included and replenished with every change of media. To image proteolysis associated with the progression to an invasive phenotype, we use dye-quenched (DQ) fluorescent matrix proteins (DQ-collagen I mixed with the layer of collagen I and DQ-collagen IV mixed with the middle layer of rBM) and observe live cultures using confocal microscopy. Optical sections are captured, processed and reconstructed in 3D with Volocity visualization software. Over the course of 23 days in MAME cocultures, the DCIS cells proliferate and coalesce into large invasive structures. Fibroblasts migrate and become incorporated into these invasive structures. Fluorescent proteolytic fragments of the collagens are found in association with the surface of DCIS structures, intracellularly, and also dispersed throughout the surrounding matrix. Drugs that target proteolytic, chemokine/cytokine and kinase pathways or modifications in the cellular composition of the cocultures can reduce the invasiveness, suggesting that MAME models can be used as preclinical screens for novel therapeutic approaches.

We have developed 3D coculture models for live-cell imaging in real-time of interactions among breast tumor cells and other cells in their microenvironment that impact progression to an invasive phenotype. These models can serve as preclinical screens for drugs to target paracrine-induced proteolytic, chemokine/cytokine and kinase pathways implicated in invasiveness.

Modelli per MAME 4D Live-Imaging cellule di tumore: Interazioni microambiente che Impact progressione maligna

We have developed 3D coculture models, which we term MAME (mammary architecture and microenvironment engineering), and used them for live-cell imaging in ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multi-modal Imaging di angiogenesi in un modello di ratto Nudo di metastasi ossee cancro al seno utilizzando la risonanza magnetica, tomografia computerizzata volumetrica e Ultrasuoni

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical treatments and improved platinum-based chemotherapies, there has been little improvement in EOC survival rates for more than four decades 1,2. Whilst stage I tumors have 5-year survival rates &gt;85%, survival rates for stage III/IV disease are &lt;40%. Thus, the high rates of mortality for EOC could be significantly decreased if tumors were detected at earlier, more treatable, stages 3-5. At present, the molecular genetic and biological basis of early stage disease development is poorly understood. More specifically, little is known about the role of the microenvironment during tumor initiation; but known risk factors for EOCs (e.g. age and parity) suggest that the microenvironment plays a key role in the early genesis of EOCs. We therefore developed three-dimensional heterotypic models of both the normal ovary and of early stage ovarian cancers. For the normal ovary, we co-cultured normal ovarian surface epithelial (IOSE) and normal stromal fibroblast (INOF)&nbsp;cells, immortalized by retrovrial transduction of the catalytic subunit of human telomerase holoenzyme (hTERT) to extend the lifespan of these cells in culture. To model the earliest stages of ovarian epithelial cell transformation, overexpression of the CMYC oncogene in IOSE cells, again co-cultured with INOF cells. These heterotypic models were used to investigate the effects of aging and senescence on the transformation and invasion of epithelial cells. Here we describe the methodological steps in development of these three-dimensional model; these methodologies aren't specific to the development of normal ovary and ovarian cancer tissues, and could be used to study other tissue types where stromal and epithelial cell interactions are a fundamental aspect of the tissue maintenance and disease development.

Heterotypic Three-dimensional In Vitro Modeling of Stromal-Epithelial Interactions During Ovarian Cancer Initiation and Progression

We describe methodologies for establishing in vitro heterotypic three-dimensional models comprising ovarian fibroblasts and normal ovarian surface or ovarian cancer epithelial cells. We discuss the use of these models to study stromal-epithelial interactions that occur during ovarian cancer development.

Eterotipica tridimensionale<em&gt; In Vitro</emModellazione&gt; di stromale-epiteliali interazioni Durante iniziazione e la progressione del cancro ovarico

Epithelial ovarian cancers (EOCs) are the leading cause of death from gynecological malignancy in Western societies. Despite advances in surgical ...

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 ...

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 ...

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 ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

Tridimensionale (3D) Tumore Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

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

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

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

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

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

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

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.

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 ...

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

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