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.

Tous les protocoles de recherche décrits ont été examinés par l&#39;Université de Chicago Institutional Review Board (IRB). Le consentement éclairé a été obtenu à partir de chaque patient avant la chirurgie et l&#39;étude a été approuvé par l&#39;Université de Chicago CISR. Une sécurité biologique type armoire 2 et des gants devraient être utilisés lors de la manipulation des tissus humains pour la protection et de réduire le risque de contamination des cellules. 1. Isolement et culture de cellules stromales primaires non transformés <ol><li> Collection de tissus humains et de préparation. <ol><li> Obtenir des échantillons de omentum humain, 2 cm 3, enlevé au cours d&#39;une intervention chirurgicale abdominale, et immerger le tissu immédiatement en RT-solution saline tamponnée au phosphate (PBS) (figure 2A). Un morceau de épiploon 3 cm x 2 cm donne généralement 1 million de cellules primaires humaines mésothéliales (HPMC) et 200.000 fibroblastes humains primaires ou des fibroblastes normaux omentales (NOF). </li><li> Isoler la PBS le tissu a été immergé dans de 0,5 g pendant 3min dès que possible après la collecte (&lt;2 h dans du PBS) et transférer le tissu à frais 20 ml de PBS dans un tube conique de 50 ml. Coupez le tissu en 5mm 3 pièces en hachant avec scalpels dans un diamètre de 15 cm, boîte de culture stérile (figure 2B). </li></ol></li><li> Isolement de cellules mésothéliales humaines primaires 8 <ol><li> Pour isoler HPMC, transférer le tissu haché dans 50 ml tubes coniques et attendre 1 min pour les morceaux solides de flotter vers le haut (figure 2C &amp; D). HPMC et les globules rouges (RBC) sont présents dans le liquide sur le fond. Utiliser une pipette pour éliminer le liquide de la partie inférieure du tube et dans un nouveau tube conique. Spin le liquide vers le bas à 0,5 g pendant 3 min et aspirer le surnageant du culot HPMC / RBC. <ol><li> Répétez le processus à trois reprises: ajouter 20 ml de PBS au tissu hachée, laisser le tissu se lever, enlever le liquide du fond avec une pipette et dans le tube HPMC / RBC, spin-down, et retirer lesurnageant. Après que tous les tours sont terminés et le surnageant est aspiré, le culot contiendra HPMC et RBC. </li></ol></li><li> Plaque de toutes les cellules de la pastille dans un 2 flacon de 75 cm dans 15 ml de milieu de croissance complet (DMEM avec 10% de sérum bovin fœtal [FBS], 1% de vitamines MEM [93 mg / L], 1% MEM acides aminés non essentiels [81,4 mg / L], 1% de pénicilline streptomycine [Pen-Strep, 100 U / ml de pénicilline et 100 ug / ml de streptomycine]). </li><li> Effectuer un lavage PBS secondaire pour isoler HPMC supplémentaires. <ol><li> Pour le secondaire PBS laver, secouez le tissu solide restant à 200 rpm, 37 ° C pendant 10 min dans 20 ml de PBS, puis attendre 1 min pour que le tissu flottent à la surface. Retirer le liquide depuis le fond du tube dans un nouveau tube, centrifuger à 0,5 g pendant 3 min, et aspirer le surnageant. </li><li> Plate toute la HPMC / RBC de la PBS secondaire laver dans un 75 cm 2 ballon séparé dans 15 ml de milieu de croissance. </li></ol></li><li> Pour isoler toute remaining HPMC, secouez le tissu haché à 200 rpm, 37 ° degrés C pendant 10 min dans 20 ml d&#39;un 1: 1 0,25% mM EDTA trypsine 25: solution de PBS en volume. Laisser le tissu à la hausse, de recueillir le liquide au fond du tube dans un nouveau tube de 50 ml, tourner vers le bas et à 0,5 gx 3 min. <ol><li> Aspirer le surnageant et de la plaque toute la HPMC / RBC dans un 75 cm 2 ballon séparé dans 15 ml de milieu de croissance. </li></ol></li><li> Culture des cellules étalées à 37 ° C et 5% de CO2 dans un environnement humidifié. Flux cellules étalées en ajoutant 15 ml de milieu de croissance complète les jours 3 et 5 sans enlever les médias dépensé. HPMC peut être cultivé pendant 5-7 jours avant de se séparer. <ol><li> Utilisez faible passage HPMC (jusqu&#39;à passage 2) dans toutes les expériences de minimiser dédifférenciation et la modification du phénotype d&#39;origine. Utiliser un microscope à champ large de préférence avec un objectif 20x avec différentiel plastique capacités de contraste d&#39;interférence ou objectif 4-20x avec contraste de phase capabilities (filtres de phase de contraste sur microscope) pour prendre toutes les images de cellules, et un objectif 10x () dans le champ lumineux d&#39;analyser immunohistochimique 3,3&#39;-diaminobenzidine (DAB) coloration. </li></ol></li><li> Vérifiez que le HPMC sont cubiques et expriment la cytokératine 8 et vimentine en effectuant immunohistochimie 8 (figure 3A, C, E). </li></ol></li><li> NOF isolement 8 <ol><li> Préparer 10 ml d&#39;un stock de III solution de type collagénase (10x) en combinant un mélange 1: 1 de 100x Pen-Strep: 1 1.500 unités activité / ml de collagénase de type III: activité 714 unités / ml de hyaluronidase dans du PBS. </li><li> Agiter le tissu haché épiploon qui reste après HPMC isolement à 200 rpm, 37 ° C pendant 6 heures dans 10 ml de la III solution de type 10x collagénase dilué dans un milieu sans sérum (DMEM avec 1% MEM vitamines [93 mg / l], 1% MEM acides aminés non essentiels [81,4 mg / L], 1% de pénicilline streptomycine [Pen-Strep, 100 U / ml de pénicilline et 100 ug / ml de streptomycine]). Tissu digéré examinera opaque et peut avoir quelques débris fibreux (figure 2E). </li><li> Centrifuger les cellules NOF de solution contenant en suspension à 0,5 x g pendant 3 minutes à température ambiante, aspirer le surnageant et le culot de plaque dans une fiole 75 de 2 cm dans 13 ml de milieu de croissance complet. </li><li> Retirer les anciens médias et les remplacer par 15 ml de milieu de croissance complet frais après 24 h. NOF peut être cultivé pendant 1-3 jours avant de se séparer. Remarque prolifération de la NOF cessera lorsque les cellules atteignent la confluence. </li><li> Vérifiez que les fibroblastes humains primaires sont des cellules plates, allongées et expriment la vimentine mais ne cytokératine 8 en effectuant immunohistochimie 8 (figure 3B, D, F). Utilisez faible passage NOF pour les expériences (idéalement avant le passage 3) pour minimiser dédifférenciation et la modification du phénotype d&#39;origine. Utiliser un microscope à champ large avec un objectif 20x avec des capacités DIC en plastique de prendre toutes les images de phase de cellules,et un objectif 10x dans le champ lumineux d&#39;analyser coloration immunohistochimique de DAB. </li></ol></li></ol> 2. Placage la Culture organotypique 8 <ol><li> NOF de sortie du flacon de culture de 75 cm par rinçage avec 10 ml de PBS suivis par 3 ml de trypsine à 0,25% / EDTA 25 mM à pas plus de 5 min. Neutraliser la trypsine avec au moins 3 fois le volume de milieu de croissance complet. </li><li> Transfert cellules trypsinisées dans un tube conique de 50 ml et centrifuger à 0,5 gx 3 min. Retirer le surnageant et amener les cellules de retour dans 5 ml de milieu de croissance. Utiliser un compteur de cellules pour compter les cellules récupérées à partir du flacon de culture. </li><li> Diluer cellules dans le volume approprié de milieu de croissance complet à l&#39;assiette 2,000-4,000 NOF par 100 pi sur une plaque noire, fond transparent, 96 puits. Ajouter 0,5 ug / 100 ul de collagène de queue de rat I du mélange de cellules avant l&#39;étalement. Cellules laisser reposer à 37 ° C dans 5% de CO 2 dans un environnement humide pendant au moins 4 heures, ou jusqu&#39;à ce que les cellulesà adhérer à la surface de la plaque. </li><li> HPMC de sortie du flacon de culture en utilisant les mêmes procédés décrits dans les étapes 2.1 et 2.2. Diluer les cellules de la quantité appropriée de milieu de croissance complet à l&#39;assiette 10.000-20.000 HPMC par 50 pi au-dessus du NOF déjà plaqué avec du collagène I dans des plaques à 96 puits. Laissez les cellules sont assis à 37 ° C dans 5% de CO 2 dans un environnement humide pendant au moins 18 heures avant le début des expériences. </li><li> Culture protéine verte fluorescente (GFP) exprimant les cellules cancéreuses de l&#39;ovaire HeyA8 dans les médias de croissance. Les cellules cancéreuses de l&#39;ovaire HeyA8 sont marquées par fluorescence par l&#39;expression d&#39;un vecteur lentiviral exprimant copépode CGFP (pCDH-CMV-MCS-EF1). Les cellules cancéreuses de l&#39;ovaire HeyA8 devrait être de 80 à 90% de confluence le jour de l&#39;utilisation (en phase de croissance logarithmique). Libérer les cellules de la plaque de la culture et de compter en utilisant les mêmes méthodes décrites dans les étapes 2.1-2.2 pour des cellules primaires. </li><li> Laisser les cellules cancéreuses d&#39;ovaire de récupérer dans un milieu de croissance complet pendant 15-20 min à 37 ° C dans untube conique avec le couvercle lâche scellé. </li></ol> 3. Essai d&#39;adhésion 8 <ol><li> Après la récupération, sédimenter les cellules de cancer de l&#39;ovaire en faisant tourner pendant 3 min à 0,5 XG, puis éliminer le surnageant et diluer les cellules dans le volume approprié de milieu sans sérum pour obtenir une concentration de 50.000 cellules / 100 pi. </li><li> Inverser les plaques à 96 puits avec les cultures organotypiques / NOF HPMC pour enlever milieu usé, et ajouter 100 pi de la suspension de cellules de cancer en milieu sans sérum à chaque puits. Incuber la plaque à 37 ° C dans 5% de CO2 dans un environnement humidifié pendant 30 min pour 4 heures. </li><li> Inversez la plaque de 96 puits pour enlever les médias et les cellules non-adhérentes. Utilisez une pipette multicanaux à faible puissance à pipette soigneusement et doucement 100 pi de PBS dans chaque puits, et inverser à nouveau la plaque. Répétez la PBS laver une fois de plus. </li><li> Inversez pour enlever PBS et ajouter 100 ul paraformaldéhyde 4% à chaque puits. Laisser la plaque reposer pendant 20 min pour fixer les cellules. Après 20 minutes, retourner la plaque et ajouter 100 ul de PBS. </li><li> Notez que la fluorescence totale de puits peut être rapporté ou le nombre de cellules peut être quantifiée. Pour la quantification, première plaque une courbe standard en double exemplaire, en utilisant des cellules HeyA8 à une concentration de 1 millions de cellules / ml. <ol><li> Faire de la courbe standard par filage soit 1 million de cellules, de retirer le média, et en leur permettant de siéger dans 1 ml de paraformaldéhyde 4% pendant 20 min. </li><li> Cellules Spin ramènerai et dans 1 ml de PBS (cellules de recomptage confirment 1 million / ml de concentration). Plate 50.000, 40.000, 30.000, 20.000, 10.000, 5000, 1000, et 0 cellules dans chaque puits en double, et d&#39;ajouter PBS pour un volume total final de 50 ul dans chaque puits. </li></ol></li><li> Bottom lire la plaque de culture sur un lecteur de plaque fluorescente (excitation = 488 nm, émission = 528 nm), mise à l&#39;échelle haute puits (50.000 sur la courbe standard). Utilisez la courbe standard pour calculer le nombre de cellules de cancer de l&#39;ovaire ont adhéré à la organotypic culture dans chaque puits (figure 4A-C). </li></ol> 4. Essai de prolifération 10 <ol><li> Après que les cellules de cancer de l&#39;ovaire récupérer (voir les étapes 2.5 et 2.6 ci-dessus), d&#39;une pastille les cellules par centrifugation pendant 3 minutes à 0,5 x g et ensuite dilué les cellules dans le volume approprié de 1% de FBS médias (DMEM avec 1% de FBS, 1% de MEM vitamines [93 mg / L], 1% MEM non essentiels acides aminés [81,4 mg / l], 1% de pénicilline streptomycine [Pen-Strep, 100 U / ml de pénicilline et 100 pg / ml de streptomycine]) pour obtenir une concentration de 4000 cellules / 150 ul. </li><li> Inversez la plaque de 96 puits avec les HPMC / cultures organotypiques NOF pour enlever milieu usé, et ajouter 150 pi de la suspension de cellules de cancer de 1% de FBS à chaque puits. Incuber la plaque à 37 ° C dans 5% de CO2 dans un environnement humidifié pendant 72 heures. </li><li> Bottom lire la plaque de culture sur un lecteur de plaque fluorescente une fois par jour (excitation = 488 nm, émission = 528 nm) pour évaluer la prolifération rapportdes cellules. Continuer l&#39;essai pendant 96 heures, en changeant les médias à 48 h (figure 5A-C). Soyez prudent de ne pas perturber la culture lors du changement de média (inversant la plaque est préférable). </li></ol> 5. Invasion Assay 8 <ol><li> Avant la culture 3D de placage, ajouter 7,5 ug de collagène de queue de rat I dans 200 ul de PBS dans une plaque de culture de 24 insert de puits (8 um de taille de pore). Laissez la plaque d&#39;incubation O / N, puis retirez soigneusement la PBS avec une pipette immédiatement avant l&#39;ensemencement de la culture organotypique HPMC / NOF sur les inserts revêtus de collagène (étape 2, ci-dessus). </li><li> Préparer les cellules de cancer ovarien comme à l&#39;étape 3.1. Pour plaquer les cellules cancéreuses de l&#39;ovaire sur les cultures organotypiques sur les inserts, utiliser avec précaution une pipette pour enlever l&#39;excès des médias à partir du haut des inserts. Ajouter 100 ul de la suspension de cellules de cancer ovarien dans du milieu sans sérum à chaque puits. </li><li> Ajouter 750 ul de milieu de pleine croissance dans le bien en dessous de l&#39;insert pour induire des cellules cancéreuses dansinvasion dans les cultures organotypiques. Incuber la plaque à 37 ° C dans 5% de CO2 dans un environnement humidifié pendant 24 heures. Nota: Les cellules de cancer ovarien qui envahissent la culture organotypique traversera l&#39;insert et rester sur le côté inférieur de l&#39;insert. </li><li> Après 24 h, saisir l&#39;insert avec des pinces et brièvement rincer dans un bécher de PBS, puis déplacer l&#39;insert dans un puits vide. Frotter la partie supérieure de chaque insert avec deux côtés d&#39;un coton-tige pour un total de 1 min. Placer rapidement l&#39;insert dans une plaque contenant 750 pi 4% de paraformaldehyde dans chaque puits. Permettre à chaque insert de siéger dans paraformaldéhyde pendant 10 minutes avant de transférer les inserts dans les puits remplis de 750 pi de PBS. </li><li> Afficher les cellules envahies (collées et fixées sur le fond du inserts) avec un microscope à grossissement 2,5x. Lorsque l&#39;ensemble du bien est dans le champ de vision, ajuster l&#39;agrandissement de 10x et de prendre 5 photos, l&#39;un près du centre et 1 dans chaque quadrant du puits, en évitantprendre des images qui sont trop près des bords. Suivez la même pour chaque puits. </li><li> Utilisez le programme du fabricant à compter le nombre de cellules dans chaque image pour obtenir un nombre moyen de cellules envahies par champ dans chaque puits (figure 6A-C) en fonction de leur protocole. </li></ol>

<ol>
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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 modèle organotypique du microenvironnement péritonéale a été créé pour évaluer la fonction (s) individuelle et collective à la fois des composants cellulaires et innées du microenvironnement dans la diffusion de cancer de l&#39;ovaire. Les protocoles spécifiques pour le placage et la personnalisation de la culture organotypique 3D pour étudier l&#39;ovaire adhérence des cellules cancéreuses, la prolifération et l&#39;invasion sont fournis. Cellules humaines omentales mésothéliales et des fibroblastes...

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 culture organotypique a été assemblé en mélangeant d&#39;abord des fibroblastes humains primaires avec collagène de type I et recouvrant cette culture avec 5 fois le nombre de cellules mésothéliales. La culture a été incubée pendant au moins 18 heures avant que les cellules cancéreuses de l&#39;ovaire ont été ajoutés pour étudier l&#39;adhérence, l&#39;invasion ou la prolifération. Chaque essai a été répété avec de multiples (n = 3-5) cultures 3D obtenues à partir de différents patients et de ...

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.

Modélisation des étapes précoces de cancer de l&#39;ovaire Diffusion dans une culture organotypique de la cavité péritonéale humain

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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26.5K vues.  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. 

Vidéo: 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.

Modélisation et Imagerie en 3 dimensions l&#39;invasion des cellules collectives

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

Recherche

Médecine

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.

Test in vitro avec dégagement mésothéliales que les modèles les étapes précoces de métastases du cancer de l&#39;ovaire

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.

Hétérotypique tridimensionnelle<em&gt; In Vitro</em&gt; Modélisation des interactions stroma-épithéliales lors de l&#39;initiation et la progression du cancer des ovaires

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.

Excitotoxiques stimulation de Microslices cerveau comme un<em&gt; In vitro</em&gt; Modèle de l&#39;AVC

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.

Une<em&gt; Ex vivo</em&gt; Modèle pour étudier action hormone dans le sein humain

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.

Dans vivo et ex vivo approches pour étudier Cancer de l&#39;ovaire métastatique colonisation de Lactée spot Structures en péritonéale adipeux

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 système organotypiques à haut débit pour la caractérisation de la sensibilité des médicaments cellules du myélome multiple primaire

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.

Quantification du commerce intra-péritonéale Cancer de l&#39;ovaire Métastase

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.

La cytologie tubaire de la trompe de Fallope est un outil prometteur pour la détection précoce du cancer de l&#39;ovaire

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.

Étapes de préparation pour la mesure de la réactivité chez les souris des artérioles rétiniennes Ex Vivo

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