We thank all residents and attending physicians, notably Dr. A.F. Haney (the University of Chicago, Department of Obstetrics and Gynecology) for collecting omental biopsies. Also, we thank Stacey Tobin and Gail Isenberg for carefully editing this manuscript. This work was supported by Bears Care, the charitable beneficiary of the Chicago Bears Football Club, the National Institute of Neurological Disorders and Stroke (NINDS) R21 NS075702, and the National Cancer Institute grant R01 CA111882 to E.L.

Tutti i protocolli di ricerca descritti sono stati esaminati dalla University of Chicago Institutional Review Board (IRB). Il consenso informato è stato ottenuto da ogni paziente prima dell&#39;intervento chirurgico e lo studio è stato approvato dalla University of Chicago IRB. Un tipo cappa di sicurezza biologica 2 e guanti devono essere utilizzate per la manipolazione dei tessuti umani per la protezione e per ridurre il rischio di contaminazione delle cellule. 1. Isolamento e la coltura di cellule stromali primarie non trasformati <ol><li> Collezione tessuti umani e di preparazione. <ol><li> Ottenere esemplari di omento umana, 2 cm 3, rimossi durante un intervento chirurgico addominale, e immergere subito tessuto RT tampone fosfato salino (PBS) (Figura 2A). Un pezzo di omento 3 cm x 2 cm produce tipicamente 1 milione di cellule primarie umane mesoteliali (HPMC) e 200.000 fibroblasti umani primari o fibroblasti omentali normali (NOF). </li><li> Centrifugare il PBS il tessuto è stato immerso in a 0,5 xg per 3min più presto possibile dopo la raccolta (&lt;2 ore in PBS) e trasferire il tessuto fresco 20 ml PBS in una provetta conica da 50 ml. Tagliare il tessuto in 5mm 3 pezzi da tritare con bisturi in un diametro di 15 centimetri, piatto di coltura sterile (Figura 2B). </li></ol></li><li> Primaria mesothelial umano isolamento delle cellule 8 <ol><li> Per isolare HPMC, trasferire il tessuto tritato in 50 ml provette coniche e attendere 1 minuto per i pezzi solidi di galleggiare verso l&#39;alto (Figura 2C &amp; S). HPMC e globuli rossi (RBC) saranno presenti nel liquido sul fondo. Utilizzare una pipetta per rimuovere il liquido dal fondo della provetta e in un nuovo tubo conico. Spin il liquido verso il basso a 0,5 xg per 3 min e aspirare il surnatante dal pellet HPMC / RBC. <ol><li> Ripetere il processo per tre volte: aggiungere 20 ml di PBS al tessuto tritato, permettono il tessuto a salire, rimuovere il liquido dal basso con una pipetta e nella provetta HPMC / RBC, spin down, e rimuovere ilsurnatante. Dopo che tutti i giri sono stati completati e il surnatante viene aspirato, il pellet conterrà HPMC e RBC. </li></ol></li><li> Piatto tutte le cellule del pellet in un pallone 75 cm 2 in 15 ml di terreni di crescita completo (DMEM con 10% di siero fetale bovino [FBS], 1% vitamine MEM [93 mg / L], 1% di acidi MEM amminoacidi non essenziali [81.4 mg / L], 1% di penicillina streptomicina [Pen-Strep, 100 U / ml di penicillina e 100 mg / ml di streptomicina]). </li><li> Eseguire un lavaggio secondario PBS per isolare HPMCs supplementari. <ol><li> Per la PBS secondario lavare, scuotere il tessuto solido residuo a 200 rpm, 37 ° C per 10 minuti in 20 ml di PBS, quindi attendere 1 minuto per il tessuto di galleggiare verso l&#39;alto. Rimuovere il liquido dal fondo della provetta in un nuovo tubo, centrifugare a 0,5 xg per 3 min, e aspirare il surnatante. </li><li> Piatto tutto il HPMC / RBC dal PBS secondario lavare in un pallone di 75 cm separata 2 in 15 ml di terreni di crescita completa. </li></ol></li><li> Per isolare ogni rNumero residua HPMC, agitare il tessuto macinata a 200 rpm, 37 ° C ° per 10 min a 20 ml di una miscela 1: 1 0,25% tripsina mM EDTA 25: soluzione PBS in volume. Lasciare il tessuto a salire, raccogliere il liquido sul fondo della provetta in un nuovo tubo 50 ml, e spin giù a 0.5 gx 3 min. <ol><li> Aspirare il surnatante e piastra tutto il HPMC / RBC in un pallone di 75 cm 2 separata in 15 ml di terreni di crescita completa. </li></ol></li><li> Cultura le cellule piastrate a 37 ° C e 5% CO 2 in un ambiente umidificato. Mangimi cellule placcato con l&#39;aggiunta di 15 ml di media piena crescita nei giorni 3 e 5 senza rimuovere i media spesi. HPMC può essere coltivato per 5-7 giorni prima della scissione. <ol><li> Utilizzare a basso passaggio HPMC (fino a passaggio 2) in tutti gli esperimenti per ridurre al minimo dedifferenziazione e la modifica di fenotipo originale. Utilizzare un microscopio a grande campo, preferibilmente con un obiettivo 20x con funzionalità di contrasto interferenza differenziali plastica o obiettivo 4-20x con capabilitie contrasto di fases (filtri di contrasto di fase sul microscopio) per l&#39;adozione di tutte le immagini delle cellule, e un obiettivo 10x () in campo chiaro per analizzare immunoistochimica 3,3&#39;-diaminobenzidina (DAB) colorazione. </li></ol></li><li> Verificare che il HPMC sono cubica ed esprimere citocheratina 8 e vimentina eseguendo immunoistochimica 8 (figura 3A, C, E). </li></ol></li><li> NOF isolamento 8 <ol><li> Preparare 10 ml di uno stock di soluzione di tipo collagenasi III (10x) mediante la combinazione di un 1: 1: 1 miscela di 100x Pen-Strep: 1.500 unità di attività / ml di collagenasi di tipo III: attività 714 unità / ml di ialuronidasi in PBS. </li><li> Agitare il tessuto omento macinata, che rimane dopo l&#39;isolamento HPMC a 200 rpm, 37 ° C per 6 ore in 10 ml di soluzione di 10x collagenasi tipo III diluito nei media senza siero (DMEM con 1% vitamine MEM [93 mg / L], 1% MEM aminoacidi non essenziali [81,4 mg / L], 1% di penicillina streptomicina [Pen-Strep, 100 U / ml di penicillina e 100 mg / ml di streptomicina]). Tessuto digerito sarà opaco e potrebbe avere alcuni detriti fibroso (Figura 2E). </li><li> Centrifugare le cellule NOF soluzione contenente in sospensione a 0,5 xg per 3 min a RT, aspirare il supernatante e piastra il pellet in un pallone 75 cm 2 in 13 ml di terreni di crescita completa. </li><li> Rimuovere vecchi media e sostituirlo con 15 ml di fresco terreni di crescita completo dopo 24 ore. NOF può essere coltivato per 1-3 giorni prima della scissione. Nota proliferazione del NOF cesserà quando le cellule raggiungono confluenza. </li><li> Confermare che i fibroblasti umani primari sono, cellule allungate piatte ed esprimono vimentina, ma non citocheratina 8 eseguendo immunoistochimica 8 (figura 3B, D, F). Utilizzare a basso passaggio NOF per esperimenti (idealmente prima del passaggio 3) per ridurre al minimo dedifferentiation e modifica di fenotipo originale. Utilizzare un microscopio a grande campo con un obiettivo 20x con funzionalità di plastica DIC dell&#39;adozione di tutte le immagini in fase di cellule,e un obiettivo 10x in campo chiaro per analizzare immunoistochimica DAB colorazione. </li></ol></li></ol> 2. Placcatura Cultura organotipica 8 <ol><li> NOF uscita dal pallone di coltura 75 cm da risciacquo con 10 ml di PBS seguita da 3 ml di 0,25% / 25 mM EDTA tripsina per non più di 5 min. Neutralizzare tripsina con almeno 3 volte il volume del terreno di coltura completo. </li><li> Trasferire le cellule trypsinized in una provetta da 50 ml e spin down a 0.5 gx 3 min. Rimuovere il surnatante e portare le cellule back up in 5 ml di media piena crescita. Utilizzare un contatore di cellule per contare le cellule recuperate dal pallone di coltura. </li><li> Diluire le cellule nel volume appropriato di supporto piena crescita al piatto 2.000-4.000 NOF per 100 microlitri su un piatto nero, chiaro a fondo, 96 pozzetti. Aggiungere 0,5 mg / 100 ml di coda di topo collagene I alla miscela cella prima placcatura. Let cellule siedono a 37 ° C in 5% CO 2 in un ambiente umidificato per almeno 4 ore, o fino a quando le cellulead aderire alla superficie della piastra. </li><li> Rilasciare HPMC dal pallone di coltura usando gli stessi metodi descritti in fasi 2.1 e 2.2. Diluire le cellule nella quantità appropriata di terreni di crescita completo alla tavola 10.000-20.000 HPMC per 50 microlitri in cima al NOF già placcato con collagene I in piastre da 96 pozzetti. Lasciare le cellule siedono a 37 ° C in 5% CO 2 in un ambiente umidificato per almeno 18 ore prima di iniziare esperimenti. </li><li> La proteina fluorescente verde Cultura (GFP) esprimente le cellule tumorali dell&#39;ovaio HeyA8 nei media piena crescita. Le cellule tumorali dell&#39;ovaio HeyA8 sono fluorescente da espressione di un vettore lentivirale che esprime copepodi cGFP (pCDH-CMV-MCS-EF1). Le cellule tumorali dell&#39;ovaio HeyA8 dovrebbe essere 80-90% confluenti il ​​giorno di utilizzo (fase di crescita logaritmica). Rilasciate le cellule dalla piastra di coltura e contare con le stesse modalità descritte nei passaggi 2.1-2.2 per le cellule primarie. </li><li> Consentire cellule di cancro ovarico di recuperare nei media piena crescita per 15-20 minuti a 37 ° C in untubo conico con coperchio sigillato liberamente. </li></ol> 3. Adesione saggio 8 <ol><li> Dopo il recupero, agglomerare le cellule di cancro ovarico, facendo girare per 3 min a 0,5 xg e poi rimuovere il surnatante e diluire le cellule nel volume adeguato di mezzi privi di siero per ottenere una concentrazione di 50.000 cellule / 100 microlitri. </li><li> Capovolgere le piastre a 96 pozzetti con / NOF colture organotipiche HPMC per rimuovere il supporto spesi, e aggiungere 100 ml di sospensione cellulare di cancro nei media senza siero in ciascun pozzetto. Incubare la piastra a 37 ° C in 5% CO 2 in un ambiente umidificato per 30 minuti a 4 ore. </li><li> Capovolgere la piastra a 96 pozzetti per rimuovere i media e le cellule non aderenti. Utilizzare una pipetta multicanale a bassa potenza da Dispensare attenzione e delicatezza 100 ml di soluzione in ciascuno bene, e invertire di nuovo piatto. Ripetere la PBS lavare una volta di più. </li><li> Inverti per rimuovere PBS e aggiungere 100 microlitri paraformaldeide 4% in ogni pozzetto. Lasciare la piastra a sedere per 20 min per fissare le cellule. Dopo 20 minuti, capovolgere la piastra e aggiungere 100 microlitri di PBS. </li><li> Si noti che la fluorescenza totale di pozzetti possono essere segnalati o il numero di cellule può essere quantificata. Per la quantificazione, prima piastra una curva standard in duplicato utilizzando cellule HeyA8 ad una concentrazione di 1 milione di cellule / ml. <ol><li> Rendere la curva standard riducendo la velocità 1 milione di cellule, rimuovendo i media, e consentendo loro di sedersi in 1 ml di paraformaldeide 4% per 20 min. </li><li> Spin cellule di nuovo e portare fino in 1 ml di PBS (cellule raccontano per confermare 1.000.000 / concentrazione ml). Piatto 50.000, 40.000, 30.000, 20.000, 10.000, 5000, 1000, e 0 cellule in ciascun pozzetto in duplicato, e aggiungere PBS per un volume totale finale di 50 microlitri in ciascun pozzetto. </li></ol></li><li> Fondo leggere la piastra di coltura su un lettore di fluorescenza piatto (eccitazione = 488 nm, emissione = 528 nm), il ridimensionamento alti pozzi (50.000 in curva standard). Utilizzare la curva standard per calcolare il numero di cellule di cancro ovarico aderito alla organotypcultura ic in ciascun pozzetto (Figura 4A-C). </li></ol> 4. Proliferazione Assay 10 <ol><li> Dopo che le cellule tumorali dell&#39;ovaio recuperare (vedere i passi 2.5 e sopra 2.6), agglomerare le cellule facendo girare per 3 min a 0,5 xg e quindi diluire le cellule nel volume appropriato di 1% FBS supporti (DMEM con 1% FBS, 1% MEM vitamine [93 mg / L], 1% MEM aminoacidi essenziali [81,4 mg / L], 1% di penicillina streptomicina [Pen-Strep, 100 U / ml di penicillina e 100 mg / ml di streptomicina]) per ottenere una concentrazione di 4.000 cellule / 150 ml. </li><li> Capovolgere la piastra a 96 pozzetti con i HPMC / NOF colture organotipiche di estrarre il supporto spesi, e aggiungere 150 ml di sospensione cellulare cancro nel 1% dei media FBS in ciascun pozzetto. Incubare la piastra a 37 ° C in 5% CO 2 in un ambiente umidificato per 72 hr. </li><li> Fondo leggere la piastra di coltura su un lettore di piastre a fluorescenza una volta al giorno (di eccitazione = 488 nm, emissione = 528 nm) per valutare la relativa proliferazionedi cellule. Continuare il test per 96 ore, cambiando i media a 48 ore (Figura 5A-C). Fare attenzione a non disturbare la cultura quando si cambia supporto (invertendo il piatto è preferibile). </li></ol> 5. Invasione Assay 8 <ol><li> Prima di placcatura la cultura 3D, aggiungere 7,5 mg coda di topo collagene I in 200 l di PBS a 24 pozzetti inserto piastra di coltura (8 micron dimensione dei pori). Lasciate che la piastra di incubare O / N, quindi rimuovere con cautela la PBS con una pipetta immediatamente prima placcatura cultura organotipica HPMC / NOF sugli inserti collagene rivestite (step 2, sopra). </li><li> Preparare le cellule tumorali ovariche come al punto 3.1. Al piatto le cellule tumorali ovariche sulle colture organotipiche sugli inserti, utilizzare con cautela una pipetta per rimuovere l&#39;eccesso supporto dalla parte superiore degli inserti. Aggiungere 100 ml di sospensione cellulare tumore ovarico in mezzi privi di siero in ciascun pozzetto. </li><li> Aggiungere 750 ml di media piena crescita nel pozzo sotto l&#39;inserto di indurre cellule del cancro invasion nelle culture organotipiche. Incubare la piastra a 37 ° C in 5% CO 2 in un ambiente umidificato per 24 ore. Nota: le cellule tumorali ovariche che invadono la cultura organotipica attraverserà l&#39;inserto e rimanere sul lato inferiore dell&#39;inserto. </li><li> Dopo 24 ore, cogliere l&#39;inserto con le pinze e brevemente sciacquarlo in un bicchiere di PBS, quindi spostare l&#39;inserto in un pozzo vuoto. Scrub parte superiore di ogni inserto con entrambi i lati di un batuffolo di cotone per un totale di 1 min. Introdurre rapidamente l&#39;inserto in un piatto contenente 750 ml 4% paraformaldeide in ogni pozzetto. Consentire ad ogni inserto di sedersi in paraformaldeide per 10 minuti prima di trasferire gli inserti nei pozzetti riempiti con 750 l di PBS. </li><li> Vedi le cellule invase (aderito e fissati al fondo di inserti) con un microscopio a 2,5x. Quando l&#39;intero pozzo è all&#39;interno del campo di vista, regolare l&#39;ingrandimento di 10x e prendere 5 immagini, uno vicino al centro e 1 in ciascun quadrante del pozzo, evitandoprendendo immagini troppo vicino ai bordi. Seguire lo stesso schema per ogni bene. </li><li> Utilizzare il programma del produttore per contare il numero di cellule in ogni immagine per ottenere un numero medio di cellule invase per campo in ogni pozzetto (Figura 6A-C) in base al loro protocollo. </li></ol>

<ol>
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Oncol. 93, 170-181 (2004).</li><li>Suzuki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HMOCC-1,+a+human+monoclonal+antibody+that+inhibits+adhesion+of+ovarian+cancer+cells+to+human+mesothelial+cells.">HMOCC-1, a human monoclonal antibody that inhibits adhesion of ovarian cancer cells to human mesothelial cells.</a> Gynecol. Oncol. 95, 290-298 (2004).</li><li>Kishikawa, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+distinct+pattern+of+peritoneal+involvement+shown+by+in+vitro+and+in+vivo+ovarian+cancer+dissemination+models.">Two distinct pattern of peritoneal involvement shown by in vitro and in vivo ovarian cancer dissemination models.</a> Invas. Metast. 15, 11-21 (1995).</li><li>Casey, R. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+an+in+vitro+assay+to+measure+the+invasion+of+ovarian+carcinoma+cells+through+mesothelial+cell+monolayers.">Establishment of an in vitro assay to measure the invasion of ovarian carcinoma cells through mesothelial cell monolayers.</a> Clin. Exp. Metastasis. 20, 343-356 (2003).</li><li>Niedbala, M. J., Crickard, K., Bernacki, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interactions+of+human+ovarian+tumor+cells+with+human+mesothelial+cells+grown+on+extracellular+matrix.">Interactions of human ovarian tumor cells with human mesothelial cells grown on extracellular matrix.</a> Exp. Cell Res. 160, 499-513 (1985).</li><li>White, E. A., Kenny, H. A., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-Dimensional+modeling+of+ovarian+cancer.">Three-Dimensional modeling of ovarian cancer.</a> Adv. Drug Deliv. Rev. 79-80, 184-192 (2014).</li><li>Kenny, H. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+high+throughput+screening+using+a+primary+human+three-dimensional+organotypic+culture+predicts+in+vivo+efficacy.">Quantitative high throughput screening using a primary human three-dimensional organotypic culture predicts in vivo efficacy.</a> Nature Comm. , (2015).</li><li>Sawada, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=c-Met+overexpression+is+a+prognostic+factor+in+ovarian+cancer+and+an+effective+target+for+inhibition+of+peritoneal+dissemination+and+invasion.">c-Met overexpression is a prognostic factor in ovarian cancer and an effective target for inhibition of peritoneal dissemination and invasion.</a> Cancer Res. 67, 1670-1680 (2007).</li><li>Kenny, H. A., Lengyel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MMP-2+functions+as+an+early+response+protein+in+ovarian+cancer+metastasis.">MMP-2 functions as an early response protein in ovarian cancer metastasis.</a> Cell cycle. 8, 683-688 (2009).</li><li>Sawada, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loss+of+E-cadherin+promotes+ovarian+cancer+metastasis+via+alpha+5-integrin,+which+is+a+therapeutic+target.">Loss of E-cadherin promotes ovarian cancer metastasis via alpha 5-integrin, which is a therapeutic target.</a> Cancer Res. 68, 2329-2339 (2008).</li><li>Mitra, A. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-independent+activation+of+c-Met+by+fibronectin+and+&#945;(5)&#946;(1)-integrin+regulates+ovarian+cancer+invasion+and+metastasis.">Ligand-independent activation of c-Met by fibronectin and &#945;(5)&#946;(1)-integrin regulates ovarian cancer invasion and metastasis.</a> Oncogene. 30, 1566-1576 (2011).</li></ol>

The authors have nothing to disclose.

Un modello organotipica del microambiente peritoneale è stato istituito per valutare la funzione individuale e collettiva (s) di entrambi i componenti cellulari e innate del microambiente in ovarico diffusione del cancro. Sono disponibili i protocolli specifici per la placcatura e personalizzare la cultura organotipica 3D per indagare ovarico adesione delle cellule tumorali, la proliferazione e l&#39;invasione. Cellule primarie umane omentali mesothelial e fibroblasti sono stati isolati da pazienti e utilizzati in un p...

Ovarian cancer is the deadliest gynecologic malignancy1. The majority of patients are diagnosed after the cancer has disseminated throughout the peritoneal cavity. Once the cancer has spread throughout the peritoneal cavity, cytoreductive surgery and chemotherapy are often not sufficient treatment to prevent cancer recurrence and chemoresistance, resulting in a less than 30% 5-year survival rate. Ovarian cancer metastasis is predominantly limited to the peritoneal cavity, and several other cancer types, including gastric, pancreatic, and colon cancers, metastasize to the same anatomic sites in the peritoneal cavity. In general, ovarian cancer cells detach from the in situ carcinoma in the fallopian tube or the primary ovarian tumor, travel in peritoneal fluid as single cells or spheroids, and attach to mesothelium-lined surfaces of the omentum, bowel, and abdominal wall2. 
The tumor microenvironment plays an important role in disease progression and chemoresistance in many cancers3-6. The peritoneal cavity is a unique microenvironment, with a mesothelial cell monolayer covering the majority of surfaces (Figure 1A)7. The mesothelial lining acts as a barrier that creates a low-friction surface, which tends to be protective against cancer cell adhesion8. Immediately underneath this mesothelial-lined surface is a layer made predominantly of fibroblasts and extracellular matrix (ECM), which promote cancer cell adhesion and invasion8. Ovarian cancer cells secrete factors that induce changes in the mesothelial cell lining that enhance ovarian cancer cell adhesion, invasion, and metastasis9,10. Ovarian cancer cells adhere to the mesothelial surface via integrin and CD44-mediated mechanisms (Figure 1B)11-16. 
Historically, several 3D models have been developed to investigate ovarian cancer interactions with the microenvironment. Some of the first models studied ovarian cancer-ECM interface17-21, ovarian cancer-mesothelial cell communication13,14,21-24, or both25 (reviewed by us 26). Niedbala et al. discovered that ovarian cancer cells display a quicker and firmer adhesion to ECM than to mesothelial cells or to plastic alone25. However, these models did not histologically resemble the peritoneal microenvironment. Therefore, we established a 3D organotypic model to more thoroughly replicate the ovarian cancer microenvironment. In order to better understand the role of the microenvironment and the interaction between cancer and peritoneal cells in the peritoneal dissemination of ovarian cancer, we have developed a 3D organotypic in vitro culture model of the peritoneal cavity lining (schematic in Figure 1C). The proposed model is composed of primary human fibroblasts and ECM, covered with a layer of primary human mesothelial cells-each cell type is isolated from human omentum. Histologically, this model resembles the normal peritoneal or omental lining, and provides a surface on which we can study the tumor microenvironment, the interaction between cancer cells and normal tissue, and the processes of cancer cell adhesion, invasion, and proliferation8.

<table>
	<tbody>
		<tr>
			<td>1. Isolation and culture of primary cells</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Scientific</td>
			<td>SH3001304</td>
			<td></td>
		</tr>
		<tr>
			<td>Single-edged razor blades</td>
			<td>Fisher Scientific</td>
			<td>12-640</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm culture dishes</td>
			<td>BD Biosciences</td>
			<td>353025</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass flask</td>
			<td>?</td>
			<td>?</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Life Technologies</td>
			<td>16000044_3616914956</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with L-Glutamine</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Vitamins</td>
			<td>Corning</td>
			<td>25-020-Cl</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM Nonessential amino acids</td>
			<td>Corning</td>
			<td>25-025-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Shaker&#160;</td>
			<td>Thermo-Fisher</td>
			<td>MaxQ 4450</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5702</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo-Fisher</td>
			<td>Forma Series II Water Jacketed CO2 Incubator Model 3100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA, 1x (0.25%)</td>
			<td>Corning</td>
			<td>25-053-CI</td>
			<td></td>
		</tr>
		<tr>
			<td>Hyaluronidase</td>
			<td>Worthington Biochemical</td>
			<td>LS002592</td>
			<td></td>
		</tr>
		<tr>
			<td>T-75 Flasks</td>
			<td>BD Biosciences</td>
			<td>353136</td>
			<td></td>
		</tr>
		<tr>
			<td>T-175 Flasks</td>
			<td>BD Biosciences</td>
			<td>353112</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>Rainin</td>
			<td>P2, P10, P20, P200 and P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet tips</td>
			<td>Corning</td>
			<td>Filtered tips P2, P10, P20, P200 and P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>2. Plating 3D culture</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Counter</td>
			<td>Invitrogen</td>
			<td>Countess</td>
			<td></td>
		</tr>
		<tr>
			<td>Countess Cell Counting Chamber Slides</td>
			<td>Invitrogen</td>
			<td>C10313</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue Stain (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen Type I (Rat Tail)</td>
			<td>BD Biosciences</td>
			<td>354236</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well plate, clear bottom, black</td>
			<td>BD Biosciences</td>
			<td>353219</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>3. Adhesion assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Multichannel pipet</td>
			<td>Eppendorf</td>
			<td>Xplorer 300</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde solution 4% in PBS</td>
			<td>Santa Cruz Biotechnology</td>
			<td>sc-281692</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Molecular Devices</td>
			<td>Minimax</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>5. Invasion assay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Inserts (8um, 24-well)</td>
			<td>BD Biosciences</td>
			<td>353097</td>
			<td></td>
		</tr>
		<tr>
			<td>Cotton swabs</td>
			<td>Q-tips</td>
			<td>cotton swabs</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>Axiovert 200m</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Profiler</td>
			<td>public domain</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>BD Biosciences</td>
			<td>353047</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Reagent/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>6. Antibodies</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Integrin &#945;V&#946;3 Antibody, clone LM609</td>
			<td>EMD Millipore</td>
			<td>MAB1976</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta 1&#160;</td>
			<td>Oncosynergy</td>
			<td>OS2966</td>
			<td></td>
		</tr>
		<tr>
			<td>Alpha 5 [CD49e]</td>
			<td>ID Pharmingen</td>
			<td>555615</td>
			<td></td>
		</tr>
		<tr>
			<td>Beta 4 [CD104]</td>
			<td>EMD Millipore</td>
			<td>MAB 2058</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling the early steps of ovarian cancer dissemination in an organotypic culture of the human peritoneal cavity

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. Instead, ovarian cancer cells exfoliated from the primary tumor are carried by peritoneal fluid to metastatic sites within the peritoneal cavity. These sites, most notably the abdominal peritoneum and omentum, are organs covered by a mesothelium-lined surface. To investigate the processes of ovarian cancer dissemination, we assembled a complex three-dimensional culture system that reconstructs the lining of the peritoneal cavity in vitro. Primary human fibroblasts and mesothelial cells were isolated from human omentum. The fibroblasts were then mixed with extracellular matrix and covered with a layer of the primary human mesothelial cells to mimic the peritoneal and omental surfaces encountered by metastasizing ovarian cancer cells. The resulting organotypic model is, as shown, used to examine the early steps of ovarian cancer dissemination, including cancer cell adhesion, invasion, and proliferation. This model has been used in a number of studies to investigate the role of the microenvironment (cellular and acellular) in early ovarian cancer dissemination. It has also been successfully adapted to high throughput screening and used to identify and test inhibitors of ovarian cancer metastasis.

La coltura organotipica stato assemblato mescolando dapprima fibroblasti umani primari con collagene di tipo I e quindi sovrapponendo questa cultura con 5 volte il numero di cellule mesoteliali. La cultura è stata incubata per almeno 18 ore prima di cellule di cancro ovarico sono stati aggiunti per studiare l&#39;adesione, invasione o la proliferazione. Ciascun test è stato ripetuto con multiplo (n = 3-5) culture 3D ottenuti da pazienti diversi e numerosi pozzi sono stati testati in ciascuna condizione per l&#39;adesi...

Watch this Scientific Journal Video about Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity at JoVE.com

Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation.

Modellazione Piazza precoci di cancro ovarico Diffusione in una cultura Organotipica della Cavità peritoneale umano

The pattern of ovarian cancer metastasis is markedly different from that of most other epithelial tumors, because it rarely spreads hematogenously. ...

modeling-early-steps-ovarian-cancer-dissemination-an-organotypic

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JoVE Journal

Medicine

26.5K Views.  University of Chicago. Here, we present a protocol to construct a three-dimensional in vitro model of the lining of the peritoneal cavity, composed of primary human mesothelial cells and fibroblasts layered with extracellular matrix, as a tool to investigate ovarian cancer cell adhesion, invasion, and proliferation. 

Video: Modeling the Early Steps of Ovarian Cancer Dissemination in an Organotypic Culture of the Human Peritoneal Cavity

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

Modellazione e imaging 3-Dimensional Invasion cella Collettiva

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

Ricerca

Medicina

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 ...

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 ...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and heterogeneity of patient-derived multiple myeloma (MM) cells. This approach consists in seeding primary MM cells freshly extracted from bone marrow aspirates into microfluidic chambers implemented in multi-well plates, each consisting of a reconstruction of the bone marrow microenvironment, including extracellular matrix (collagen or basement membrane matrix) and stroma (patient-derived mesenchymal stem cells) or human-derived endothelial cells (HUVECs). The chambers are drugged with different agents and concentrations, and are imaged sequentially for 96 hr through bright field microscopy, in a motorized microscope equipped with a digital camera. Digital image analysis software detects live and dead cells from presence or absence of membrane motion, and generates curves of change in viability as a function of drug concentration and exposure time. We use a computational model to determine the parameters of chemosensitivity of the tumor population to each drug, as well as the number of sub-populations present as a measure of tumor heterogeneity. These patient-tailored models can then be used to simulate therapeutic regimens and estimate clinical response.

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Un sistema organotipica High Throughput per la caratterizzazione di droga sensibilità di primaria multicampo cellule del mieloma

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

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 ...

Tubal Cytology of the Fallopian Tube as a Promising Tool for Ovarian Cancer Early Detection

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to the lack of markers or tools for the ovarian cancer identification, the early detection of HGSC remains challenging. Direct sampling of the fallopian tube can enhance sensitivity for detection of neoplastic cells when the tumor is not grossly visible. We developed a procedure to collect fallopian tube cells directly from freshly received surgical specimens, which has shown excellent correlation with histological findings. This approach lays a foundation for the future utility of minimally invasive laparoscopic screening in high-risk patient populations.

We explored a tubal cytologic method by sampling the fallopian tube directly post-surgical excision as a tool of ovarian cancer early detection. Here, we present a protocol to collect fallopian tube cells from freshly received surgical specimens.

Tubal Cytologia del Tubo Fallopiano come strumento promettente per il rilevamento precoce del cancro ovarico

Currently, it is widely accepted that the vast majority of ovarian high-grade serous carcinoma (HGSC) originate from the fallopian tube. However, due to ...

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular diseases, such as diabetic retinopathy, hypertensive retinopathy, and possibly glaucoma. Therefore, retinal microvascular preparations are pivotal tools for physiological and pharmacological studies to delineate the underlying pathophysiological mechanisms and to design therapies for the diseases. Despite the wide use of mouse models in ophthalmic research, studies on retinal vascular reactivity are scarce in this species. A major reason for this discrepancy is the challenging isolation procedures owing to the small size of these retinal blood vessels, which is ~ &#8804; 30 &#181;m in luminal diameter. To circumvent the problem of direct isolation of these retinal microvessels for functional studies, we established an isolation and preparation technique that enables ex vivo studies of mouse retinal vasoactivity under near-physiological conditions. Although the present experimental preparations will specifically refer to the mouse retinal arterioles, this methodology can readily be employed to microvessels from rats.

Preparation Steps for Measurement of Reactivity in Mouse Retinal Arterioles Ex Vivo

Many vision-threatening ocular diseases are associated with dysfunctional retinal microvessels. Therefore, the measurement of retinal arteriole responses is important to investigate the underlying pathophysiological mechanisms. This article describes a detailed protocol for mouse retinal arteriole isolation and preparation to assess the effects of vasoactive substances on vascular diameter.

Procedura di preparazione per la misura della reattività in Mouse arteriole retiniche Ex Vivo

Vascular insufficiency and alterations in normal retinal perfusion are among the major factors for the pathogenesis of various sight-threatening ocular ...