Este trabajo fue financiado en parte por DK089016 y RR030244 L30 (MAC), CA153036 (AS), y P30 DK52574-(to the Core Washington University Digestive Diseases Research). AIT era un Howard Hughes Medical Institute Medical Research Fellow de Formación.

1. Colitis asociada al cáncer de inducción <ol><li> Ponga a un lado las jaulas de sexo y edad, los ratones 6-8 semanas de edad que se utilizarán para los grupos experimental y control. Los ratones pueden ser etiquetados individualmente con marcas de cola o clips de oído. </li><li> En el día 0, los pesos iniciales de registro y se inyecta por vía intraperitoneal a cada ratón (IP) con 10 mg / kg de solución AOM de trabajo (1 mg / ml en solución salina isotónica, diluido a partir de 10 mg / ml de solución madre en dH2O mantuvo a -20 ° C) . Basado en la experiencia, esta dosis puede ajustarse entre 7-14 mg / kg y / o repetido temprano en el experimento. </li></ol> Precaución: OMA es un agente genotóxico volátil y debe ser manejado con cuidado de acuerdo con el acompañamiento MSDS. Las diluciones se prepararon en una campana química, mantenido en hielo, y se desecha siguiendo protocolos institución específica. <ol start="3"><li> Hacer un% 2,5 (2,5 g/100 ml) solución de DSS en agua destilada y se pasan a través de un f acetato de celulosa 0,22 micrasilter por vacío. Esta dosis se puede ajustar entre 1-3.5% dependiendo de la cepa del ratón y el medio ambiente. Una vez preparada, la solución de DSS puede mantenerse refrigerado hasta por 1 semana. </li><li> En el día 7, de suministro de solución de DSS en ratones como su agua potable. Aproximadamente 250 ml / jaula será necesario cada vez que se proporcionan DSS nueva para un máximo de 5 ratones / jaula, sin embargo, estos son sólo estimaciones y pueden variar en función del tipo de botellas de agua utilizados en su instalación de animales. </li><li> Para proporcionar un suministro continuo de DSS durante siete días, la solución de DSS debe ser sustituido en botellas limpias tres veces (cada 2-3 días) durante este período. Algunos investigadores medir la cantidad de DSS consumidos antes de reemplazar con nueva solución como una medida de la exposición. </li><li> El día 14, cambia de nuevo a las jaulas de agua potable estándar durante dos semanas. </li><li> Repetir los pasos (1,4) - (1,6) en los días 28 y 49 para proporcionar un segundo y tercer ciclo de DSS. Un DSS &quot;ciclo&quot; se compone de una semana de DSS en el agua de bebidaseguido de 2 semanas de regular (autoclave) de agua. </li></ol> 2. Colitis Evaluación clínica y la progresión tumoral <ol><li> Los ratones se pesaron / observado 2-3 veces por semana. Pérdida de porcentaje en peso respecto a la línea base se utiliza como una medida sustituta de la gravedad de la colitis. Las evaluaciones regulares de sangrado rectal, diarrea o prolapso puede hacerse en función de objetivos experimentales e indicará con arreglo a los sistemas de puntuación diferentes. 9, 10 </li><li> Una pérdida de peso de hasta el 10% a lo largo de 2-3 días de diarrea y sangrado rectal se puede esperar en la semana después de un ciclo completo de DSS (observación diaria puede ser útil durante este inmediato post-DSS período). </li></ol> Es posible que algunos ratones no pueden recuperarse; ratones que pierden más de 20% de su peso son menos probabilidades de sobrevivir y puede requerir la eutanasia precoz. Una sola inyección IP de 0,5 a 1,0 ml de solución salina en estos ratones puede ser una medida útil de apoyo a corregir FLUI<html> d volumen perdido debido a la diarrea. 3. Murino Endoscopy (Opcional) <ol><li> Endoscopia siguiendo el ciclo de segundo o tercero de DSS se puede realizar para confirmar el crecimiento del tumor in vivo antes de su sacrificio. Como la OMA / DSS modelo es bastante fiable en la producción de tumores, este paso no es necesario en general. Sólo unos pocos ratones se evaluó usando este método, ya que tiene el potencial de interrumpir los tumores de ratón. 11 </li><li> Se anestesia el ratón con sedación inhalada o inyectable. </li><li> Después de ratón es anestesiado, asegure el ratón a un tablero plano con cinta en la cola y el pecho. </li><li> Haga avanzar el endoscopio murino en el recto ratón suavemente con solución salina para inflar y riego para mejorar la vista intraluminal y eliminar contenido fecal. (Tenga en cuenta que la endoscopia también se puede realizar usando la insuflación de aire como una alternativa a la solución salina y la técnica esto puede ofrecer una mejor visualización del tumor. 12) </li></ol>e &quot;&gt; 4. Ratón Sacrificio y cosecha Colon <ol><li> El día 70, cada ratón OMA / DSS tratada debe albergar múltiples tumores de colon y estar listo para la evaluación. Esta fecha puede retrasarse semanas o meses si los tumores más grandes son deseados. (Es de destacar que los ratones con prolapso rectal severa pueden necesitar ser sacrificados temprano para evitar excesivas molestias a los animales como por los Comités Institucionales de cuidado de los animales.) </li><li> Antes de la necropsia, individualmente eutanasia a los ratones con dislocación cervical y el isoflurano (u otro método aprobado institucionalmente). El peso final y otras mediciones se pueden hacer en este momento. </li><li> Coloque el ratón con su lado ventral expuesta en una tabla de cortar. Cubrir el abdomen con 70% de etanol para evitar que los pelos de la contaminación de los contenidos del abdomen durante la extracción del colon. </li><li> Use pinzas para agarrar la línea media del abdomen y hacer una pequeña incisión para exponer el peritoneo. </li><li> Extienda la incisión en cada lado del abdomen a lo largo de la margi costaln. </li><li> Utilice unas tijeras para cortar a través de la pelvis para que el colon se pueden cosechar hasta la unión anorrectal. Esto es importante porque la lesión colitis DSS es mayor en el recto distal y, correspondientemente, esta es la región de mayor desarrollo del tumor. </li><li> Tejidos adicionales, tales como los ganglios linfáticos mesentéricos también pueden cosecharse en este momento. </li></ol> 5. Preparación del colon para el análisis macroscópico <ol><li> Usando una aguja de 18G sonda unida a una jeringa de 5 ml, lavar el colon con helado de buffer fosfato salino (PBS) de tal manera que las salidas de fluido en la dirección fisiológica. Si el tejido del colon se utilizan para ARN o proteína basada en el análisis, puede mantenerse en frío mediante el empleo de pre-refrigerado bandejas de trabajo. </li><li> Abra el colon enrojecida longitudinalmente a lo largo de su mesenterio y la pintura en la bandeja de frío. Los tumores son más fácilmente visualizados cuando el colon se coloca sobre una superficie oscura. Alternativamente, 1% de tinte azul Alcian se puede aplicar para resaltar tumors. Evaluar el número y tamaño del tumor con una regla o calibrador digital. </li><li> Pequeñas secciones de tejido normal y tumoral adyacente se puede escindir en este punto para el análisis futuro por el ARN, proteína, o métodos de inmunohistoquímica (IHC), dejando una parte del colon restante para su evaluación histológica. </li></ol> Fotografía digital de las muestras de colon brutos inmediatamente después de la etapa 5,2 puede ser útil para el análisis preciso de la carga tumoral, especialmente si partes significativas de los dos puntos se escindió para otras formas de análisis, como se describe anteriormente. La carga tumoral (%) se calcula como el área del tumor / área total del colon usando software libre como ImageJ. 6. Preparación del colon para su evaluación histológica <ol><li> Aplicar 10% de formalina para el colon restante en la bandeja de frío mediante una jeringa o un dedo. Permitiendo que el tejido a fijar durante al menos 30 segundos de esta manera facilita la transferencia a la cuenca de fijación. Asegúrese residual formalina es wiped apagado antes de pintar dos puntos de nueva construcción inaugurado en la misma bandeja. </li><li> Transferir dos puntos a un recipiente lleno de formol al 10% y precisar en los bordes. </li><li> Después de 3 horas de la fijación, transferir dos puntos a 70% de etanol. Los dos puntos pueden ser conservados en etanol al 70% indefinidamente. </li><li> Tres secciones longitudinales de dos puntos (proximal, medio y distal / recto) puede ser estabilizado en 2% de agar y presentado para su inclusión en parafina. Asegurarse de que el colon proximal y distal del colon / recto están orientados de manera uniforme para cada ratón. </li><li> Displasia, daño cripta, y la inflamación puede ser descrito y evaluado por un individuo experimentado de acuerdo con protocolos publicados previamente. 10, 13-15 Los ratones pueden ser sacrificado antes de 70 días si el experimentador está interesado en la detección de los eventos tempranos de displásicas. </li></ol> 7. Los resultados representativos El modelo de OMA / DSS se describe en este documento permite al investigador generar fiable tumores de colon en ratones.El crecimiento tumoral en este modelo está directamente influenciada por el proceso inflamatorio asociado. Gravedad de la colitis deben ser controlados clínicamente siguiendo la pérdida de peso y la presencia de diarrea / hematoquecia (Figura 2). Estos signos de actividad de la enfermedad tienden a comenzar por día 5 del ciclo de DSS y de cuatro o más días después de DSS se retira. En raras ocasiones, los ratones con una carga tumoral rectal significativa puede desarrollar prolapso rectal. Después de la diarrea DSS segundo o tercer ciclo puede llegar a ser persistente. Típicamente los tumores están presentes e identificables por colonoscopia murino antes del tercer ciclo de DSS (Figura 3). El tiempo adicional y un tercer curso de los resultados DSS en tumores más grandes en el momento de la cosecha (Figura 4). El uso de la aplicación tópica de Alcian mancha azul puede ser utilizado para poner de relieve los tumores (Figura 5). Las fotografías de los tumores de colon ayudará en la generación de las mediciones del tumor que pueden ser utilizados para comparar cuantitativamente la producción de tumores y el tamañoentre los grupos experimentales (Figura 6). Fijos y embebidos en parafina las muestras de colon se puede entonces evaluar para histología o con el uso de la tinción inmunohistoquímica (Figura 7 y 8). <img src="/files/ftp_upload/4100/4100fig1.jpg" alt="Figura 1" /> Figura 1. Esquema de la OMA y la administración de DSS. AOM (10 mg / kg) se inyecta en el día 0. Al comienzo de la segunda semana (día 7), 2,5% de solución de DSS se administra a ratones en su agua potable. Siete días de DSS es seguido por dos semanas de agua tratada en autoclave. Un adicional de dos ciclos de DSS se administran antes de su sacrificio. <img src="/files/ftp_upload/4100/4100fig2.jpg" alt="Figura 2" /> Figura 2. Ratón respecto al valor basal durante AOM y administración DSS peso. Tenga en cuenta que en la semana siguiente a cada ciclo de DSS, los ratones<html> e 5-10% de su peso corporal. La pérdida de peso en este experimento es un marcador sustituto de la gravedad de la colitis. <img src="/files/ftp_upload/4100/4100fig3.jpg" alt="Figura 3" /> Figura 3. Vista de tumores en el colon distal mediante endoscopia murino en el día 50 de OMA / DSS tratamiento. Tenga en cuenta las múltiples masas polipoides que obstruyen el lumen del colon distal (b, c) en comparación con el colon normal (a). <img src="/files/ftp_upload/4100/4100fig4.jpg" alt="Figura 4" /> Figura 4. Longitudinalmente abierto colon de ratón que ilustra aspecto macroscópico de los tumores. Tenga en cuenta la mayor carga del tumor en el distal del colon / recto (imagen superior izquierda), y la textura característica rugated del colon proximal (imagen superior derecha) con el crecimiento tumoral poco. Una vista de cerca del colon distal muestra numerosos tumores de diferentes tamaños (abajo). ove_content &quot;fo: keep-together.within-page =&quot; always &quot;&gt; <img src="/files/ftp_upload/4100/4100fig5.jpg" alt="Figura 5" /> Los tumores Figura 5. Resaltados por la aplicación de Alcian azul mancha. Nótese cómo el colorante enfatiza la textura normal del colon, así como los bordes de cada tumor individual. Tinción de este tipo puede ser útil en la medición precisa de las zonas tumorales por regla o medida digital. <img src="/files/ftp_upload/4100/4100fig6.jpg" alt="La figura 6" /> Figura 6. Distribución representativa del número medio de tumores por ratón tratados con OMA / DSS. Tenga en cuenta que la mayoría de los tumores se localizan en el colon distal y son &lt;2 mm de tamaño. <img src="/files/ftp_upload/4100/4100fig7.jpg" alt="Figura 8" /> Figura 7. Parafina embebido en secciones longitudinales del colon en cassette (arriba) unad en la diapositiva después de tinción con HE (abajo). Tenga en cuenta que la mancha azul Alcian residual no interfiere con H &amp; E tinción. Un tumor grande es un círculo en la imagen de la diapositiva (en el círculo). Las denominaciones &quot;distal&quot;, &quot;medio&quot; y &quot;proximal&quot; resultan de seccionar el colon en tercios entre el ciego y el ano. <img src="/files/ftp_upload/4100/4100fig8.jpg" alt="Figura 8" /> Figura 8. Histología Representante de un tumor resultante de la OMA / DSS administración en el colon distal. H &amp; E, BrdU, y β-catenina portaobjetos teñidos a 50x (panel superior) y 400X (panel inferior), respectivamente, demuestran cambios displásicos similares a los adenocarcinomas de colon humano.

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El tratamiento de ratones con OMA y DSS rápida y efectivamente los modelos humanos cáncer de colitis asociada. Hipótesis con respecto a factores hereditarios contribuyen a la colitis cánceres asociados pueden ser fácilmente estudiado con ratones genéticamente modificados. 13, 16 Alternativamente, el efecto de objetivos farmacológicos en colitis asociada a cáncer puede ser estudiada mediante el empleo de ratones de tipo salvaje. Si bien este modelo es muy valorado por los i...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Ratones C57BL/6J </td><td> Jackson Laboratory </td><td> 000664 </td><td></td></tr><tr><td> Azoximetano (AOM) </td><td> Sigma Aldrich </td><td> A5486-100MG </td><td> Solución stock: diluido a 10 mg / ml en agua destilada que se mantuvo a -20 ° C como 0,5 a 1 ml de alícuotas. Solución de trabajo: diluir stock a 1 mg / ml en solución salina isotónica (0,9%) de solución salina </td></tr><tr><td> El dextrano sulfato de sodio (DSS) </td><td> TDB Consultoría </td><td> DB001 </td><td> MW 40 kDa (36-50 kDa preparaciones procedentes de otras fuentes son aceptables; El mismo lote se debe utilizar para un único experimento) 6 </td></tr><tr><td> Coloviewsistema miniendoscopic </td><td> Karl Storz </td><td> Múltiple </td><td> Ver Becker et al. Para la explicación detallada de los equipos y la configuración. 11 </td></tr><tr><td> TPP Rapid FilterMax 500 ml Botella-Filter, 0,22 m PES </td><td> Midwest Scientific </td><td> TP99500 </td><td> Cualquier filtro de cultivo de tejidos estándar es aceptable </td></tr><tr><td> Alcohol etílico 200 Proof ASC / USP </td><td> Fármaco-AAPER (u otro) </td><td> 11ACS200 </td><td> Diluir hasta 70% en agua destilada </td></tr><tr><td> USP isoflurano, </td><td> Mayordomo Animal Health Supply </td><td> 4029405 </td><td> Coloque el ratón en el tarro de cristal con una gasa o un paño empapado en anestésico pequeño </td></tr><tr><td> 18G Aguja Recta Gavage </td><td> Braintree Scientific </td><td> N-008 </td><td></td></tr><tr><td> Tampón fosfato salino (PBS) </td><td> SigmaAldrich </td><td> P5493 </td><td> Diluir a 1X (0,01 M) en agua destilada </td></tr><tr><td> Bandeja fría (Tissue Tek II Placa Fría) </td><td> Fisher Scientific </td><td> NC9491941 </td><td> Almacenar a -20 ° C </td></tr><tr><td> ImageJ software </td><td> NIH (descarga gratuita) </td><td></td><td> <a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> </td></tr><tr><td> Formaldehído (37%) </td><td> Fisher Scientific </td><td> F79-500 </td><td> Diluir a 10% en PBS </td></tr><tr><td> BD Bacto Agar </td><td> Fisher Scientific </td><td> DF0140-01-0 </td><td> Use placa calefactora para crear 2% de solución en agua destilada </td></tr><tr><td> Miltex Pinza Dressing Eye </td><td> MedPlus Inc. </td><td> 18-780 </td><td></td></tr><tr><td> Miltex Tijeras oculares </td><td> MedPlus Inc. </td><td> 18-1430 </td> &lt;td&gt; puntos curvos evitar daños en dos puntos durante la apertura. </td></tr><tr><td> Azul de Alcian 8GX (polvo) </td><td> Sigma Aldrich </td><td> A5268 </td><td> Añadir 1 g de polvo a 100 ml de 3% de ácido acético (3 ml de ácido acético glacial + 97 ml de agua destilada) </td></tr><tr><td> 1 ml jeringa de tuberculina con adjunto x 26 G 3/8 de aguja intradérmica bisel </td><td> BD </td><td> 305946 </td><td> Para la inyección de AOM </td></tr></tbody></table>

modeling colitis-associated cancer with azoxymethane (aom) and dextran sulfate sodium (dss)

Los individuos con enfermedad inflamatoria intestinal (EII), tales como la enfermedad de Crohn (CD) o colitis ulcerativa (UC) están en mayor riesgo de desarrollar cáncer colorrectal (CRC) sobre los individuos sanos. Este riesgo es proporcional a la duración y extensión de la enfermedad, con una incidencia acumulada de hasta un 30% en los individuos con larga UC con afectación colónica generalizada. 1 displasia de colon en la EII y la colitis asociada a cáncer (CAC) se cree que se desarrollan como resultado de ciclos repetidos de lesión de la célula epitelial y la reparación, mientras que estas células se bañan en un medio de citoquinas inflamatoria crónica. 2 Mientras que los cánceres espontáneos y la colitis asociada a compartir la calidad de los adenocarcinomas siendo, la secuencia de eventos moleculares subyacentes se cree que es diferente. 3 Esta distinción argumenta la necesidad de modelos animales específicos del CAC. Varios modelos de ratón existen en la actualidad para el estudio de CAC. Sulfato de dextrano sodium (DSS), un agente con efectos tóxicos directos sobre el epitelio del colon, puede ser administrado en el agua potable a los ratones en varios ciclos para crear un estado inflamatorio crónico. Con una duración suficiente, algunos de estos ratones desarrollan tumores. 4 desarrollo tumoral se aceleró en este modelo si se administra en un entorno pro-cancerígenos. Estos incluyen ratones con mutaciones genéticas en las vías de tumorigénesis (APC, p53, MSH2), así como ratones tratados previamente con agentes genotóxicos (azoximetano [OMA], 1,2-dimetilhidracina [DMH]). 5 La combinación de DSS con OMA como un modelo de colitis asociada a cáncer ha ganado popularidad por su reproducibilidad, potencia, bajo precio y facilidad de uso. Aunque tienen un mecanismo compartido, AOM se ha encontrado para ser más potentes y estables en solución que la DMH. Mientras que el desarrollo de tumores en otros modelos generalmente requiere varios meses, los ratones inyectados con AOM y posteriormente tratado con DSS desarrollar tumores adecuados en uns poco como 7-10 semanas. 6, 7 Finalmente, AOM y DSS se puede administrar a ratones de cualquier fondo genético (knock out, transgénicos, etc) sin cruzamiento para una cepa específica tumorigénico. Aquí, demostramos un protocolo para la inflamación impulsada por la tumorigénesis de colon en ratones utilizando una única inyección de AOM seguido de tres de siete días ciclos de DSS durante un período de semanas 10. Este modelo induce tumores con cambios histológicos y moleculares muy parecidas a las que ocurren en humanos CAC y proporciona un modelo muy valioso para el estudio de la oncogénesis y la quimioprevención en esta enfermedad. 8

Watch this Scientific Journal Video about Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS at JoVE.com

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

Se demuestra un protocolo en el que la administración del agente genotóxico azoximetano (AOM), seguido por tres ciclos de la sódico pro-inflamatoria agente sulfato de dextrano (DSS) rápida y consistente genera tumores de colon en ratones con similitudes morfológicas y moleculares a las que se observan en la colitis humana asociada a cáncer.

Modelado Colitis asociada a cáncer con azoximetano (AOM) y Sodio sulfato de dextrano (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

modeling-colitis-associated-cancer-with-azoxymethane-aom-dextran

Research

JoVE Journal

Medicine

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

46.5K vistas.  Washington University School of Medicine. Se demuestra un protocolo en el que la administración del agente genotóxico azoximetano (AOM), seguido por tres ciclos de la sódico pro-inflamatoria agente sulfato de dextrano (DSS) rápida y consistente genera tumores de colon en ratones con similitudes morfológicas y moleculares a las que se observan en la colitis humana asociada a cáncer.

Video: Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

Modeling Stroke in Mice - Middle Cerebral Artery Occlusion with the Filament Model

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are very limited. To meet the need for novel therapeutic approaches, experimental stroke research frequently employs rodent models of focal cerebral ischaemia. Most researchers use permanent or transient occlusion of the middle cerebral artery (MCA) in mice or rats.
Proximal occlusion of the middle cerebral artery (MCA) via the intraluminal suture technique (so called filament or suture model) is probably the most frequently used model in experimental stroke research. The intraluminal MCAO model offers the advantage of inducing reproducible transient or permanent ischaemia of the MCA territory in a relatively non-invasive manner. Intraluminal approaches interrupt the blood flow of the entire territory of this artery. Filament occlusion thus arrests flow proximal to the lenticulo-striate arteries, which supply the basal ganglia. Filament occlusion of the MCA results in reproducible lesions in the cortex and striatum and can be either permanent or transient. In contrast, models inducing distal (to the branching of the lenticulo-striate arteries) MCA occlusion typically spare the striatum and primarily involve the neocortex. In addition these models do require craniectomy. In the model demonstrated in this article, a silicon coated filament is introduced into the common carotid artery and advanced along the internal carotid artery into the Circle of Willis, where it blocks the origin of the middle cerebral artery. In patients, occlusions of the middle cerebral artery are among the most common causes of ischaemic stroke. Since varying ischemic intervals can be chosen freely in this model depending on the time point of reperfusion, ischaemic lesions with varying degrees of severity can be produced. Reperfusion by removal of the occluding filament at least partially models the restoration of blood flow after spontaneous or therapeutic (tPA) lysis of a thromboembolic clot in humans.
In this video we will present the basic technique as well as the major pitfalls and confounders which may limit the predictive value of this model.

Filamentous occlusion of the Middle cerebral artery is a common model for studying ischemic stroke in mice.

Modelado de accidente cerebrovascular en ratones - Oclusión arteria cerebral media con el modelo de filamento

Stroke is among the most frequent causes of death and adult disability, especially in highly developed countries. However, treatment options to date are ...

Investigación

Medicina

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 ...

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 ...

Retinal Detachment Model in Rodents by Subretinal Injection of Sodium Hyaluronate

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the occurrence of subretinal hemorrhage can affect photoreceptor cell death in the detached retina. Hence, it is advantageous to create reproducible RDs without subretinal hemorrhage for evaluating photoreceptor cell death. We modified a previously reported method to create bullous and persistent RDs in a reproducible location with rare occurrence of subretinal hemorrhage. The critical step of this modified method is the creation of a self-sealing scleral incision, which can prevent leakage of sodium hyaluronate after injection into the subretinal space. To make the self-sealing scleral incision, a scleral tunnel is created, followed by scleral penetration into the choroid with a 30 G needle. Although choroidal hemorrhage may occur during this step, astriction with a surgical spear reduces the rate of choroidal hemorrhage. This method allows a more reproducible and reliable model of photoreceptor death in diseases that involve RD such as rhegmatogenous RD, retinopathy of prematurity, diabetic retinopathy, central serous chorioretinopathy, and age-related macular degeneration (AMD).

Creating experimental retinal detachments with a reproducible and sustained height of detachment, and without subretinal hemorrhage, is important for studying the pathophysiology of photoreceptor cell loss in retinal disease and evaluating potential therapeutic interventions. Here, we report such a method in detail.

Desprendimiento de Retina modelo en roedores por inyección subretiniana de Hialuronato de Sodio

Subretinal injection of sodium hyaluronate is a widely accepted method of inducing retinal detachment (RD). However, the height and duration of RD or the ...

Modeling Spontaneous Metastatic Renal Cell Carcinoma (mRCC) in Mice Following Nephrectomy

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully recapitulate early- and late-stage metastatic disease progression. Typical tumor implantation models utilize ectopic or orthotopic primary tumor implantation, but few include systemic spontaneous metastatic disease that mimics the clinical setting. This protocol describes the key steps to develop RCC disease progression stages similar to patients. First, it uses a highly metastatic mouse tumor cell line in a syngeneic model to show orthotopic tumor cell implantation. Methods include superficial and internal implantation into the sub-capsular space with cells combined with matrigel to prevent leakage and early spread. Next it describes the procedures for excision of tumor-bearing kidney (nephrectomy), with critical pre- and post- surgical mouse care. Finally, it outlines the steps necessary to monitor and assess micro-and macro-metastatic disease progression, including bioluminescent imaging as well provides a detailed visual necropsy guide to score systemic disease distribution. The goal of this protocol description is to facilitate the widespread use of clinically relevant metastatic RCC models to improve the predictive value of future therapeutic testing.&#160;

Modeling Spontaneous Metastatic Renal Cell Carcinoma mRCC in Mice Following Nephrectomy

Models of spontaneous metastatic renal cell carcinoma (RCC) disease progression can be used for evaluating treatments in a clinically relevant setting. This protocol demonstrates different procedures for orthotopic kidney tumor cell implantation, proper nephrectomy, and finally outlines a necropsy guide for visual and bioluminescent scoring of metastatic burden and localization.

Modelado espontánea metastásico Carcinoma de células renales (CCR metastásico) en ratones después de la nefrectomía

One of the key challenges to improved testing of new experimental therapeutics in renal cell carcinoma (RCC) is the development of models that faithfully ...

Working with Human Tissues for Translational Cancer Research

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human specimens, precious samples must be handled with the greatest care in order to ensure integrity of organs, tissues, and cells to the highest degree. Unfortunately, tissue processing by definition requires extraction of tissues from the host, a change which can cause great cellular stress and have major repercussions on subsequent analyses. These stresses could result in the specimen being no longer representative of the site from which it was retrieved. Therefore, a strict protocol must be adhered to while processing these specimens to ensure representativeness. The desired assay(s) must also be taken into consideration in order to ensure that an optimal technique is used for sample processing. Outlined here is a protocol for tissue retrieval, processing and various analyses which may be performed on processed tissue in order to maximize downstream production from limited tissue samples.

Translational cancer research is dependent on extraction of human tissues. Much work has gone into optimizing extraction methods for ex vivo analysis. Here, we describe tissue processing methods allowing for maximal data output from limited samples.

Trabajar con Tejidos Humanos para la Investigación del Cáncer Traslacional

Medical research for human benefit is greatly impeded by the necessity for human tissues and subjects. However, upon obtaining consent for human ...

Modeling Encephalopathy of Prematurity Using Prenatal Hypoxia-ischemia with Intra-amniotic Lipopolysaccharide in Rats

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best advance translational objectives and uncover new therapeutic strategies for brain injury associated with preterm birth, preclinical models of EoP must include similar mechanisms of prenatal global injury observed in humans and involve multiple components of the maternal-placental-fetal system. Ideally, models should produce a similar spectrum of functional deficits in the mature animal and recapitulate multiple aspects of the pathophysiology. To mimic human systemic placental perfusion defects, placental underperfusion and/or chorioamnionitis associated with pathogen-induced inflammation in early preterm birth, we developed a model of prenatal transient systemic hypoxia-ischemia (TSHI) combined with intra-amniotic lipopolysaccharide (LPS). In pregnant Sprague Dawley rats, TSHI via uterine artery occlusion on embryonic day 18 (E18) induces a graded placental underperfusion defect associated with increasing CNS damage in the fetus. When combined with intra-amniotic LPS injections, placental inflammation is increased and CNS damage is compounded with associated white matter, gait and imaging abnormalities. Prenatal TSHI and TSHI+LPS prenatal insults meet several of the criteria of an EoP model including recapitulating the intrauterine insult, causing loss of neurons, oligodendrocytes and axons, loss of subplate, and functional deficits in adult animals that mimic those observed in children born extremely preterm. Moreover, this model allows for the dissection of inflammation induced by divergent injury types.

Encephalopathy of prematurity encompasses the central nervous system abnormalities associated with injury from preterm birth. This report describes a clinically relevant rat model of in utero transient systemic hypoxia-ischemia and intra-amniotic lipopolysaccharide administration (LPS) that mimics chorioamnionitis, and the related impact of infectious stimuli and placental underperfusion on CNS development.

Modelado Encefalopatía del Prematuro El uso prenatal hipoxia-isquemia con lipopolisacárido intraamniótica en ratas

Encephalopathy of prematurity (EoP) is a term that encompasses the central nervous system (CNS) abnormalities associated with preterm birth. To best ...

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.

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 Chemotherapy Resistant Leukemia In Vitro

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and progress in this site. The model presented in the current report utilizes human primary bone marrow stromal cells and osteoblasts as two representative cell types from the marrow niche that influence tumor cell phenotype. The in vitro co-culture conditions described for human leukemic cells with these primary niche components support the generation of a chemoresistant subpopulation of tumor cells that can be efficiently recovered from culture for analysis by diverse techniques. A strict feeding schedule to prevent nutrient fluxes followed by gel type 10 cross-linked dextran (G10) particles recovery of the population of tumor cells that have migrated beneath the adherent bone marrow stromal cells (BMSC) or osteoblasts (OB) generating a "phase dim" (PD) population of tumor cells, provides a consistent source of purified therapy resistant leukemic cells. This clinically relevant population of tumor cells can be evaluated by standard methods to investigate apoptotic, metabolic, and cell cycle regulatory pathways as well as providing a more rigorous target in which to test novel therapeutic strategies prior to pre-clinical investigations targeted at minimal residual disease.

Modeling Chemotherapy Resistant Leukemia In Vitro

The current report summarizes a protocol that can be utilized to model the influence of the bone marrow microenvironment niche on leukemic cells with emphasis placed on enrichment of the most chemoresistant subpopulation.

Modelado Quimioterapia Leucemia resistente<em&gt; In Vitro</em

It is well established that the bone marrow microenvironment provides a unique site of sanctuary for hematopoietic diseases that both initiate and ...

Modeling and Simulations of Olfactory Drug Delivery with Passive and Active Controls of Nasally Inhaled Pharmaceutical Aerosols

There are many advantages of direct nose-to-brain drug delivery in the treatment of neurological disorders. However, its application is limited by the extremely low delivery efficiency (&#60; 1%) to the olfactory mucosa that directly connects the brain. It is crucial to develop novel techniques to deliver neurological medications more effectively to the olfactory region. The objective of this study is to develop a numerical platform to simulate and improve intranasal olfactory drug delivery. A coupled image-CFD method was presented that synthetized the image-based model development, quality meshing, fluid simulation, and magnetic particle tracking. With this method, performances of three intranasal delivery protocols were numerically assessed and compared. Influences of breathing maneuvers, magnet layout, magnetic field strength, drug release position, and particle size on the olfactory dosage were also numerically studied.
From the simulations, we found that clinically significant olfactory dosage (up to 45%) were feasible using the combination of magnet layout and selective drug release. A 64 -fold higher delivery of dosage was predicted in the case with magnetophoretic guidance compared to the case without it. However, precise guidance of nasally inhaled aerosols to the olfactory region remains challenging due to the unstable nature of magnetophoresis, as well as the high sensitivity of olfactory dosage to patient-, device-, and particle-related factors.

This manuscript reviews the modeling and simulations of different protocols to deliver medications to the olfactory region in image-based nasal airway models. Multiple software modules are used to develop the anatomically accurate nose model, generate computational mesh, simulate nasal airflows, and predict particle deposition at the olfactory region.

Modelado y Simulación de olfativa Drug Delivery con controles pasivos y activos por vía nasal de aerosoles farmacéuticos inhalados

There are many advantages of direct nose-to-brain drug delivery in the treatment of neurological disorders. However, its application is limited by the ...