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.

Todos los protocolos de investigación descritos han sido revisados ​​por la Universidad de Chicago Junta de Revisión Institucional (IRB). El consentimiento informado se obtuvo de cada paciente antes de la cirugía y el estudio fue aprobado por la Universidad de Chicago IRB. Un gabinete de seguridad biológica de tipo 2 y los guantes se deben utilizar para la manipulación de tejidos humanos para su protección y para reducir el riesgo de contaminación de las células. 1. Aislamiento y cultivo de células estromales no transformadas primarias <ol><li> Recogida de tejido humano y preparación. <ol><li> Obtener muestras de epiplón humana, 2 cm 3, eliminado durante una cirugía abdominal, y sumergirse de inmediato el tejido en solución salina tamponada con fosfato de RT (PBS) (Figura 2). Un pedazo de epiplón 3 cm x 2 cm típicamente produce 1 millón de células mesoteliales primarios humanos (HPMC) y 200.000 fibroblastos humanos primarios o fibroblastos normales omental (NOF). </li><li> Gira por la PBS el tejido se sumerge en 0,5 g durante 3min tan pronto como sea posible después de la recogida (&lt;2 h en PBS) y transferir el tejido fresco a 20 ml de PBS en un tubo cónico de 50 ml. Cortar el tejido dentro de 5 mm 3 piezas mediante el picado con escalpelos en un diámetro de 15 cm, placa de cultivo estéril (Figura 2B). </li></ol></li><li> Mesotelial humana primaria aislamiento de células 8 <ol><li> Para aislar HPMC, transferir el tejido picado en 50 ml tubos cónicos y esperar 1 min para los trozos sólidos floten a la parte superior (Figura 2 C + D). HPMC y los glóbulos rojos (RBC) estarán presentes en el líquido en la parte inferior. Usar una pipeta para eliminar el líquido de la parte inferior del tubo y en un nuevo tubo cónico. Girar el líquido hacia abajo a 0,5 xg durante 3 min y aspirar el sobrenadante del sedimento de HPMC / RBC. <ol><li> Repita el proceso tres veces: añadir 20 ml de PBS al tejido picada, deje el tejido se eleve, eliminar el líquido de la parte inferior con una pipeta y en el tubo de HPMC / RBC, girar hacia abajo, y extraiga laflotante. Después de todos los giros se han completado y se aspira el sobrenadante, el sedimento contiene HPMC y RBC. </li></ol></li><li> Plate todas las células del pellet en un matraz 75 cm 2 en 15 ml de medio de crecimiento completo (DMEM con 10% de suero bovino fetal [FBS], 1% de vitaminas MEM [93 mg / L], 1% de ácidos MEM aminoácidos no esenciales [81.4 mg / L], 1% de penicilina estreptomicina [Pen-Strep, 100 U / ml de penicilina y 100 mg / ml de estreptomicina]). </li><li> Realizar un lavado PBS secundaria para aislar HPMCs adicionales. <ol><li> Para lavar el PBS secundaria, agitar el tejido sólido que queda a 200 rpm, 37 ° C durante 10 minutos en 20 ml de PBS, a continuación, espere 1 minuto para el tejido flote a la cima. Retirar el líquido de la parte inferior del tubo en un nuevo tubo, centrifugar a 0,5 xg durante 3 min, y aspirar el sobrenadante. </li><li> Placa todo el HPMC / RBC desde el PBS secundaria de lavado en un matraz separado 75 cm 2 en 15 ml de medio de crecimiento completo. </li></ol></li><li> Para aislar cualquier remaining HPMC, agitar el tejido picado a 200 rpm, 37 ° C grados durante 10 min en 20 ml de una mezcla 1: 1 0,25% de EDTA mM de tripsina 25: solución de PBS en volumen. Permitir que el tejido se eleve, recoger el líquido en la parte inferior del tubo en un nuevo tubo de 50 ml, y girar hacia abajo en 0,5 gx 3 min. <ol><li> Aspirar el sobrenadante y la placa todo el HPMC / RBC en un matraz separado 75 cm 2 en 15 ml de medio de crecimiento completo. </li></ol></li><li> Cultivar las células en placas a 37 ° C y 5% de CO 2 en un ambiente humidificado. Alimentación células sembradas por la adición de 15 ml de medio de cultivo completo en los días 3 y 5 sin necesidad de retirar los medios gastados. HPMC puede ser cultivadas durante 5-7 días antes de la división. <ol><li> Utilice HPMC de bajo paso (hasta el paso 2) en todos los experimentos para minimizar desdiferenciación y la modificación del fenotipo inicial. Utilice un microscopio de campo amplio de preferencia con un objetivo de 20x con capacidad de contraste de interferencia diferencial de plástico o objetivo 4-20x con contraste de fase capabilities (filtros de fase de contraste en el microscopio) para tomar todas las imágenes de las células, y un objetivo de 10x () en campo claro para analizar inmunohistoquímica 3,3&#39;-diaminobencidina (DAB) tinción. </li></ol></li><li> Confirme que la HPMC son cúbicas y expresar citoqueratina 8 y vimentina realizando inmunohistoquímica 8 (Figura 3 A, C, E). </li></ol></li><li> NOF aislamiento 8 <ol><li> Preparar 10 ml de un stock de solución de colagenasa de tipo III (10x) mediante la combinación de una mezcla 1: 1 de Pen-Strep 100x:: 1 1.500 unidades de actividad / ml de colagenasa de tipo III: actividad 714 unidades / ml de hialuronidasa en PBS. </li><li> Agitar el tejido epiplón picada que permanece después del aislamiento HPMC en 200 rpm, 37 ° C durante 6 horas en 10 ml de el tipo de solución 10x colagenasa III diluido en medio libre de suero (DMEM con 1% de MEM vitaminas [93 mg / l], 1% aminoácidos no esenciales MEM [81.4 mg / L], 1% penicilina estreptomicina [Pen-Strep, 100 U / ml de penicilina y 100 mg / ml de estreptomicina]). Tejido digerido se verá opaca y puede tener algunos escombros fibrosa (Figura 2E). </li><li> Centrifugar las células NOF solución que contiene en suspensión a 0,5 × g durante 3 min a RT, aspirar el sobrenadante y el sedimento de la placa en un matraz de 75 cm 2 en 13 ml de medio de crecimiento completo. </li><li> Retire los viejos medios y reemplazar con 15 ml de medio de cultivo completo fresco después de 24 horas. NOF puede cultivarse durante 1-3 días antes de la división. Nota proliferación de la NOF cesará cuando las células alcanzan la confluencia. </li><li> Confirme que los fibroblastos humanos primarios son células planas, alargadas y expresan vimentina pero no citoqueratina 8 realizando inmunohistoquímica 8 (Figura 3B, D, F). Utilice NOF bajo paso para experimentos (idealmente antes paso 3) para reducir al mínimo desdiferenciación y la modificación del fenotipo inicial. Utilice un microscopio de campo amplio con un objetivo de 20x con plástico capacidades DIC para la toma de todas las imágenes de fase de las células,y un objetivo de 10x en campo claro para analizar tinción inmunohistoquímica DAB. </li></ol></li></ol> 2. Revestimiento del organotípicos Cultura 8 <ol><li> NOF de lanzamiento del matraz de cultivo 75 cm de un enjuague con 10 ml de PBS, seguido de 3 ml de 0,25% de tripsina / EDTA 25 mM durante no más de 5 min. Neutralizar la tripsina con al menos 3 veces el volumen de medio de crecimiento completo. </li><li> Transferencia de las células tratadas con tripsina en un tubo cónico de 50 ml y centrifugar a 0,5 gx 3 min. Eliminar el sobrenadante y llevar a las células una copia de seguridad en 5 ml de medio de cultivo completo. Utilice un contador de células para contar las células recuperadas del matraz de cultivo. </li><li> Diluir las células en el volumen apropiado de medio de crecimiento completo a la placa 2.000-4.000 NOF por 100 l en una placa de negro, de fondo transparente, de 96 pocillos. Añadir 0,5 g / 100 l de colágeno de cola de rata I de la mezcla de células antes de la siembra. Deje que las células se sientan a 37 ° C en 5% de CO 2 en un ambiente humidificado durante al menos 4 horas, o hasta que las célulasa adherirse a la superficie de la placa. </li><li> Soltar HPMC del matraz de cultivo usando los mismos métodos descritos en los pasos 2.1 y 2.2. Diluir las células en la cantidad apropiada de medio de crecimiento completo a la placa de 10.000-20.000 HPMC por cada 50 l en la parte superior de la NOF ya chapada con colágeno I en placas de 96 pocillos. Deje que las células se sientan a 37 ° C en 5% de CO 2 en un ambiente humidificado durante al menos 18 horas antes de comenzar los experimentos. </li><li> Cultura proteína fluorescente verde (GFP) expresando las células del cáncer de ovario HeyA8 en medio de crecimiento completo. Células de cáncer ovárico HeyA8 están etiquetados con fluorescencia mediante la expresión de un vector lentiviral que expresa copépodos CGFP (Pcdh-CMV-MCS-EF1). Células de cáncer ovárico HeyA8 deben ser 80-90% de confluencia en el día de uso (fase de crecimiento logarítmica). Liberar células de la placa de cultivo y contar usando los mismos métodos descritos en los pasos 2.1-2.2 para las células primarias. </li><li> Permitir que las células de cáncer de ovario se recuperen en medio de crecimiento completo para 15-20 minutos a 37 ° C en untubo cónico con la tapa sellada débilmente. </li></ol> 3. Ensayo de adhesión 8 <ol><li> Después de la recuperación, sedimentar las células de cáncer de ovario haciendo girar durante 3 min a 0,5 × g y luego eliminar el sobrenadante y diluir las células en el volumen apropiado de medio libre de suero para lograr una concentración de 50.000 células / 100 l. </li><li> Invertir las placas de 96 pocillos con los cultivos organotípicos / NOF HPMC para eliminar medio gastado, y añadir 100 l de la suspensión de células de cáncer en medio libre de suero a cada pocillo. Incubar la placa a 37 ° C en 5% de CO 2 en un ambiente humidificado durante 30 min a 4 h. </li><li> Invierta la placa de 96 pocillos para eliminar los medios de comunicación y las células no adherentes. Utilice una pipeta multicanal a baja potencia para pipetear con cuidado y suavidad 100 l de PBS en cada pocillo, e invertir la placa de nuevo. Repita el PBS lavar una vez más. </li><li> Invertir para eliminar PBS y añadir 100 l de paraformaldehído al 4% a cada pocillo. Permita que la plancha reposar por 20 millasn para fijar las células. Después de 20 minutos, invierta la placa y añadir 100 l de PBS. </li><li> Tenga en cuenta que la fluorescencia total de pozos puede ser reportada o el número de células puede ser cuantificado. Para la cuantificación, primera placa de una curva estándar por duplicado utilizando células HeyA8 a una concentración de 1 millón de células / ml. <ol><li> Hacer la curva estándar desacelerándose 1 millón de células, la eliminación de los medios de comunicación, y que les permite sentarse en 1 ml de paraformaldehído al 4% durante 20 min. </li><li> Células girar de nuevo y traer en 1 ml de PBS (células de recuento para confirmar 1.000.000 / ml de concentración). Plate 50000, 40000, 30000, 20000, 10000, 5000, 1000, y 0 células en cada pocillo en duplicado, y añadir PBS para un volumen total final de 50 l en cada pocillo. </li></ol></li><li> Bottom leer la placa de cultivo en un lector de placa fluorescente (excitación = 488 nm, emisión = 528 nm), escalando altos pozos (50.000 en la curva estándar). Utilice la curva estándar para calcular el número de células de cáncer de ovario se adhirieron a la organotypcultura ic en cada pocillo (Figura 4A-C). </li></ol> 4. Ensayo de proliferación de 10 <ol><li> Después de que las células de cáncer de ovario se recuperan (consulte los pasos 2.5 y 2.6 arriba), sedimentar las células por centrifugación durante 3 min a 0,5 xg y luego diluir las células en el volumen apropiado de 1% de FBS medios (DMEM con 1% de FBS, 1% MEM vitaminas [93 mg / l], 1% MEM no esenciales aminoácidos [81,4 mg / l], 1% de penicilina estreptomicina [Pen-Strep, 100 U / ml de penicilina y 100 mg / ml de estreptomicina]) para lograr una concentración de 4.000 células / 150 l. </li><li> Invierta la placa de 96 pocillos con los / cultivos organotípicos NOF HPMC para eliminar medio gastado, y añadir 150 l de la suspensión de células de cáncer en el 1% de medios FBS a cada pocillo. Incubar la placa a 37 ° C en 5% de CO 2 en un ambiente humidificado durante 72 hr. </li><li> Bottom leer la placa de cultivo en un lector de placa fluorescente una vez al día (excitación = 488 nm, emisión = 528 nm) para evaluar la proliferación relativade las células. Continuar el ensayo durante 96 horas, cambiando los medios de comunicación a las 48 h (Figura 5A-C). Tenga cuidado de no molestar a la cultura al cambiar los medios de comunicación (invirtiendo la placa es preferible). </li></ol> 5. Invasión Ensayo 8 <ol><li> Antes plateando la cultura 3D, agregue el colágeno de cola de rata 7,5 g I en 200 l de PBS a un 24 pocillos de inserción placa de cultivo (8 micras de tamaño de poro). Deje que la placa incubado O / N, a continuación, retire con cuidado la PBS con una pipeta inmediatamente antes plateando la cultura organotípico HPMC / NOF en los insertos recubiertos con colágeno (paso 2 anterior). </li><li> Preparar las células de cáncer de ovario que en el paso 3.1. Para la placa de las células de cáncer de ovario en los cultivos organotípicos en los insertos, utilice cuidadosamente una pipeta para eliminar el exceso de medios de comunicación de la parte superior de los insertos. Añadir 100 l de la suspensión de células cáncer de ovario en medio libre de suero a cada pocillo. </li><li> Añadir 750 l de medio de cultivo completo en el pozo por debajo de la inserción para inducir las células de cáncer eninvasión en los cultivos organotípicos. Incubar la placa a 37 ° C en 5% de CO 2 en un ambiente humidificado durante 24 hr. Nota: células de cáncer ovárico que invaden el cultivo organotípico cruzará el inserto y permanecer en el lado inferior del inserto. </li><li> Después de 24 horas, sujete el inserto con pinzas y brevemente enjuague en un vaso de precipitados de PBS, a continuación, mueva el inserto en un pozo vacío. Frote la parte superior de cada inserto con ambos lados de un hisopo de algodón para un total de 1 min. Colocar rápidamente el inserto en una placa que contiene 750 l de paraformaldehído al 4% en cada pocillo. Permitir que cada inserto para sentarse en paraformaldehído durante 10 min antes de transferir los insertos en los pozos llenos con 750 l de PBS. </li><li> Ver las células invadidas (adheridas y se fija a la parte inferior de inserciones) con un microscopio con un aumento de 2,5 veces. Cuando todo el bien está dentro del campo de visión, ajustar la ampliación de 10x y tomar 5 fotos, uno cerca del centro y 1 en cada cuadrante del pozo, evitandotomando imágenes que están demasiado cerca de los bordes. Siga el mismo patrón para cada pozo. </li><li> Utilice el programa del fabricante para contar el número de células en cada imagen para obtener un número medio de células invadidas por campo en cada pocillo (Figura 6A-C) de acuerdo con su protocolo. </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.

Se estableció un modelo organotípico del microambiente peritoneal para evaluar la función individual y colectiva (s) de ambos los componentes celulares y innatas del microambiente en la diseminación del cáncer de ovario. Se proporcionan los protocolos específicos para el chapado y personalizar el cultivo organotípico 3D para investigar la adhesión de células de ovario cáncer, la proliferación y la invasión. Células epiplón humanos primarios mesoteliales y fibroblastos fueron aisladas de los pacientes y se ...

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.

El cultivo organotípico se ensambló mezclando primero los fibroblastos humanos primarios con colágeno de tipo I y luego superponiendo esta cultura con 5 veces el número de células mesoteliales. El cultivo se incubó durante al menos 18 horas antes de que se añadieron células de cáncer de ovario para estudiar la adhesión, invasión o proliferación. Cada ensayo se repitió con múltiples (n = 3-5) culturas 3D obtenidos de diferentes pacientes y numerosos pozos fueron probados en cada condición para la adhesión...

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.

Modelado de los primeros pasos de cáncer de ovario Difusión en una cultura organotípicos del Peritoneal Cavity Humano

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

Research

JoVE Journal

Medicine

26.5K vistas.  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.

Modelado y de imagen de 3 dimensiones invasión celular colectiva

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

Investigación

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.

Ensayo in vitro Liquidación mesoteliales que modela los pasos iniciales de la metástasis del cáncer ovárico

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.

Heterotípicos tridimensional<em&gt; In Vitro</em&gt; Modelado de estromal-epitelial interacciones durante la iniciación y progresión del cáncer de ovario

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.

Excitotóxica Estimulación del cerebro Microslices como<em&gt; In vitro</em&gt; Modelo de Carrera

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.

Una<em&gt; Ex vivo</em&gt; Modelo para estudiar la acción hormonal en el pecho humano

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.

En vivo y ex vivo para estudiar Cáncer de ovario metastásico colonización de Estructuras Vía lugar en Peritoneal 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 organotípicos de alto rendimiento para la Caracterización de la sensibilidad a los fármacos de las células del mieloma múltiple primaria

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 cuantificación de la intra-peritoneal del cáncer ovárico Metástasis

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 citología tubárica del tubo de Falopio como herramienta prometedora para la detección temprana del cáncer de ovario

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.

Pasos de preparación para la medición de la reactividad en ratón arteriolas retinianas Ex Vivo

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