The authors would like to acknowledge the Netherlands Organization for Scientific Research (NWO, grant 17.106.329)

Los experimentos con animales fueron aprobados por el comité de bienestar de los animales, del Centro Médico de la Universidad de Leiden (LUMC). 1. Preparación de células, Instrumentos y Ratones <ol><li> Un día antes de la operación, se afeitan el / gamma SCID (NSG) ratones NOD a partir del cuarto pezón a la línea media y pesan los ratones para comprobar que todos los ratones tienen pesos más o menos comparables. </li><li> Autoclave una tijera, dos pinzas y dos pinzas de mosquitos rectas (Figura 1A). </li><li> En el día de la operación, lavar el adenocarcinoma de mama humano (MDA-MB-231) celdas que se inyecta una vez con tampón fosfato salino (PBS), pH 7,4 y trypsinize las células. Se inactiva la tripsina mediante la adición de 10 ml que contiene suero DMEM los medios de comunicación en la parte superior de las células. Centrifugar las células a 1200 rpm durante 7 min a TA para eliminar el suero resuspendiendo las células, ya sea en PBS o en medios sin suero. Centrifugar de nuevo para eliminar trazas de suero completo. Resuspender las células en PBS omedios de comunicación. </li><li> Contar las células con un hemocitómetro y calcular la cantidad de células. Utilice 500.000 células / ratón en no más de 150 l. Facultativo de PBS o medio, resuspender las células en matrigel. NOTA: Determinar la cantidad de células en función del tipo de célula utilizado. </li><li> Coloque las células en unos tubos de microcentrífuga estériles y mantenerlos en hielo. </li><li> Llenar tubo de centrífuga cónico uno 50 ml con 35 ml de PBS pH 7,4 y otro tubo de centrífuga cónico de 50 ml con etanol al 70%. Remoje las esponjas de algodón en cada tubo. </li></ol> 2. Inyección ortotópico <ol><li> Realizar la cirugía en una campana estéril para mantener un ambiente estéril. Anestesiar el ratón por inyección subcutánea de la mezcla de xilazina / ketamina a una dosis de 10 mg / kg, 100 mg / kg de peso corporal, respectivamente. Fijar el ratón sobre una almohadilla térmica. Confirme el éxito de la anestesia por la falta de reacción ante pizca dedo del pie. NOTA: Si uno no está muy familiarizado con los procedimientos quirúrgicos, la cirugía puede tomar más tiempo. Arreglarlas dosis de anestesia en consecuencia. </li><li> Aplique un ungüento oftálmico en los ojos de ratones, para evitar que los ojos se sequen. </li><li> Limpie el área afeitada utilizando el bastoncillo de algodón humedecido en etanol preparado en el paso 1.6. Alternando matorrales utilizando betadine y alcohol tres veces en forma circular que gira lejos del lugar de la incisión planificada debe ser realizada. Alternativamente, utilizar ionóforo como desinfectante. </li><li> Hacer una pequeña incisión entre el cuarto pezón y la línea media con una tijera y hacer un bolsillo insertando el hisopo de algodón humedecido con PBS pH 7,4. </li><li> Use una pinza para exponer la almohadilla de grasa mamaria. Observe la almohadilla de grasa por su color blanco. </li><li> Apriete la almohadilla de grasa con la otra pinza de su base; al hacer esto, exponer completamente la almohadilla de grasa (Figura 1B) para realizar inyecciones fácilmente. </li><li> Homogeneizar la mezcla de células pipeteando arriba y abajo. Se aspira suavemente 50 l de suspensión celular en una jeringa de insulina y inyectar enla almohadilla de grasa mamaria mediante la celebración de la aguja en posición horizontal. Confirme la inyección correcta comprobando si hay inflamación de la almohadilla de grasa. </li><li> Suelte la almohadilla de grasa suavemente. </li><li> Suturar la incisión utilizando un controlador de aguja. Hacer tres tiros girando la sutura alrededor del conductor de la aguja en sentido horario y anti-horario y hacia la derecha. Deben utilizarse dos nudos por sutura. NOTA: Asegúrese de que los nudos están apretados de lo contrario los ratones pueden abrir las suturas. </li><li> Después de la cirugía, se inyecta un analgésico, tal como Temgesic en 0,05-0,1 mg / kg de peso corporal, por vía subcutánea con el fin de aliviar el dolor. </li><li> No deje a los animales sin vigilancia hasta que adquieran conciencia y mantener decúbito esternal. Coloque todos los animales en diferentes jaulas hasta que estén completamente recuperados. </li><li> Para mantener la esterilidad, utilizar jaulas autoclave y agua. Cambie las jaulas cada tres días para mantener el ambiente limpio. </li></ol> 3. Recolección de Órganos para el Análisis <ol><li> Aldía de la cosecha, 8 semanas después de la implantación de las células, llenar 15 ml tubos de centrífuga cónicos con 3 ml de solución de Bouin para cada ratón. Además, utilizar dos 15 ml tubos llenos con 5 ml de solución de formalina por ratón. </li><li> Anestesiar a los animales mediante la inyección de xilazina / ketamina, a una dosis de 10 mg / kg 100 mg / kg de peso corporal por vía subcutánea. Espere hasta que el animal pierde toe retirada pizca reflejo. </li><li> Fijar el animal con agujas sobre una base. Haga una larga incisión en la línea media vertical con tijeras. Haga dos incisiones horizontales justo debajo de la pata delantera y por encima de la pata trasera. Exponer el tumor fijando la piel a la base. Medir el volumen del tumor usando un calibrador. </li><li> Disociar el tumor de la piel usando tijeras. Snap congelar una parte del tumor en nitrógeno líquido para el aislamiento de ARN. Coloque la otra parte, en el tubo de centrífuga cónico lleno de formol para realizar inmunohistoquímica tras la inclusión en parafina. </li><li> Tomar suavemente los pulmones. Coloque elpulmón izquierdo en solución de Bouin. Mantenga el pulmón en solución durante 3 días. Observe focos metastásicos superficial claramente a simple vista. Opcionalmente, coloque el pulmón derecho en formalina para verificar micrometástasis mediante hematoxilina y eosina (H &amp; E) tinción. NOTA: Si bien, la metástasis pulmonar observado con frecuencia en el cáncer de mama, también puede ser que desee para recoger los huesos, el hígado, el cerebro y el bazo para analizar la metástasis. Las células en la zona metastásico son más densas y morfológicamente diferente y por lo tanto se pueden distinguir fácilmente de tejido pulmonar. </li><li> Un día después de la cosecha, aspirar la solución de formalina de tubos de 15 ml y reemplazar con etanol al 70%. Insertar los tejidos en parafina y realizar estudios de inmunohistoquímica. </li><li> Para el análisis de sangre, abre la cavidad torácica con unas tijeras. Retirar 450 l de sangre por punción cardiaca. Coloque la muestra de sangre en un tubo de microcentrífuga que contiene 50 l de 3,2% de citrato de sodio. Después de recoger la sangre, girar a 2.000 xg durante 10 millasn. Almacenar las muestras de plasma a -20 ° C hasta su uso posterior. NOTA: Hay varias otras técnicas de recogida de sangre de uso común en la práctica 20 y la técnica que se debe utilizar depende de la configuración experimental y elección del científico. Si no se requiere muestra de sangre, la eutanasia del animal de acuerdo con el protocolo de animal o de acuerdo con las directrices de la institución. </li></ol>

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The authors declare that they have no competing financial interests.

Inyección ortotópico de células de cáncer de mama es un poderoso modelo para estudiar todos los aspectos del crecimiento del cáncer. La implantación de estas células en la almohadilla de grasa mamaria de los ratones se debe realizar cuidadosamente para evitar la variación en el crecimiento del tumor. Lo más importante, la inyección de la misma cantidad de células a cada ratón es crucial. Para ello, se debe trypsinize las células rigurosamente sin afectar la viabilidad de las células. Las células no viable...

El cáncer es una enfermedad muy compleja que ha sido objeto de estudios por más siglos. El cáncer de mama es el tipo de cáncer más común; se produce predominantemente en las mujeres, pero puede también ocurrir esporádicamente en los hombres 27. La enfermedad es causada principalmente por la pérdida de mecanismo de control de la división celular de gobierno que a su vez conduce a un crecimiento infinito de las células en el cuerpo. Estas disfunciones pueden ser causadas por varios mecanismos: primero, las células sanas necesitan señales de crecimiento de las células circundantes con el fin de proliferar mientras que las células de cáncer hacen sus propios factores de crecimiento y aumentan la expresión de los receptores del factor de crecimiento obteniendo así una tasa de proliferación superior 1; segundo, las células cancerosas son menos susceptibles a las señales anti-proliferativos 8; tercero, para equilibrar el número de células en también es necesaria la muerte de las células del cuerpo; Sin embargo, las células de cáncer de escape de la muerte celular programada, denominada apoptosis 14; cuarto, las células se adhieren a matrices extracelularescon el fin de sobrevivir, pero las células tumorales pueden crecer sin la necesidad de unión y muestran resistencia a la anoikis 19; quinto, la activación de la telomerasa evita el acortamiento de los telómeros y evita que la senescencia replicativa 21; por último pero no menos importante, la omisión del control de calidad del ADN tras los resultados de la mitosis en el contenido genético alterado 15,16. Con el fin de identificar los oncogenes o supresores tumorales que juegan un papel en esta proliferación desregulada, experimentos de crecimiento de tumor en ratones son cruciales. El crecimiento primario del tumor generalmente no es la principal razón de la muerte. La migración de las células cancerosas desde el sitio primario a un sitio secundario, denominado metástasis, es la causa principal de muerte en la mayoría de los pacientes de cáncer 22. Metástasis implica invasión de células tumorales, intravasación, viajando a través de la circulación, evitando inmune ataque, la extravasación y el crecimiento en el sitio secundario. Epitelial a mesenquimal (EMT) es un proceso clave enmetástasis e implica un cambio en los perfiles de expresión génica produciendo células con mayor movilidad y capacidad de invasión, que son requisitos previos para la célula de metástasis 12. Como el proceso canceroso es la resultante de una combinación de diversas acciones, incluyendo las interacciones recíprocas entre las células cancerosas, las células del estroma y las células pro- y anti-inflamatorias, un enfoque in vitro de cáncer a menudo no proporciona completa información sobre el proceso canceroso. De manera similar, los tratamientos contra el cáncer que afectan a la vasculatura del tumor a menudo pueden no ser estudiados in vitro, por lo tanto el uso de enfoques in vivo es inevitable. Para estudiar la progresión del cáncer de mama, se han desarrollado diferentes métodos experimentales. El modelo más ampliamente utilizado es la inyección subcutánea de células de cáncer de mama en ratones 5. En esta configuración experimental, el investigador puede introducir una amplia gama de alteraciones a una línea celular de elección in vitro (es decir,upregulation, regulación a la baja de las proteínas) e inyectar las células debajo de la piel. Aunque este método es sencillo y el proceso de inyección es simple, sin necesidad de realizar la cirugía en los ratones, el sitio en el que se inyecta el tumor no representa el ambiente del tumor mamario local y la ausencia de este entorno puede resultar en el desarrollo del cáncer de mama que se difiere de la observada en patología humana. En segundo lugar, los ratones genéticamente modificados se utilizan frecuentemente como una herramienta in vivo para estudiar la progresión del cáncer de mama. En este modelo, el oncogén (es decir PYMT, Neu) la expresión es conducida por un promotor específico de tejido mamario que conduce a la formación espontánea de s tumor de mama. Esta configuración experimental es útil para estudiar el aspecto de tratamiento de la enfermedad mediante la inyección de drogas o anticuerpos mientras comprueba el tamaño del tumor en el tiempo 3. Sin embargo, la cría de estos ratones con otras cepas de ratones deficientes o mutados en un gen de interés también podría dar insERECHOS en el papel de diferentes proteínas en el crecimiento del tumor de mama 24. La desventaja de este modelo es que es propenso a la variación en el tamaño del tumor y el número. Por otra parte, el nivel de expresión del transgen depende del sitio de integración en el genoma y se puede cambiar de una cepa de ratón a otro 4. En este modelo, la expresión del transgén se puede lograr por todas las células con origen epitelial mientras que en la enfermedad humana, sólo una subpoblación de células expresan el oncogén o regular a la baja los niveles supresores de tumores 26. Para estudiar la metástasis, las células del cáncer de mama también se pueden inyectar por vía intravenosa (un modelo denomina metástasis experimental) 25. Sin embargo, este enfoque sólo recapitula el proceso metastásico parcialmente; elude el requisito de las células tumorales para invadir y intravasate, y se inicia desde el punto en el que las células tumorales son fácilmente presente en la circulación. En nuestro trabajo, utilizamos una inyección ortotópicoion modelo para estudiar la implicación de los genes de interés en la progresión del cáncer de mama 13. Nos sobreexpresan la proteína en las células del cáncer de mama humano e inyectarlos en la almohadilla de grasa mamaria de ratones NOD / SCID gamma (NSG) ratones. Este método es ventajoso en muchos aspectos: permite cambios genéticos muy rápidos y diversos en la línea celular inyectada, que abarca todo el proceso de la progresión del cáncer de mama de crecimiento del tumor primario a la metástasis en los sitios patológicamente relevantes, y también proporciona un buen modelo experimental para estudiar el impacto de los tratamientos terapéuticos en etapas tempranas o tardías de la enfermedad. Además, el uso de este modelo se puede investigar el papel de las proteínas del estroma frente derivadas de células de cáncer en progresión de la enfermedad mediante el uso de ratones o células modificadas genéticamente. Aunque las inyecciones subcutáneas son más fáciles de realizar, modelos ortotópicos dan lugar a una población de células de cáncer más tumorigénico y más metastásico. Por lo tanto, los resultados obtenidos por medio de la inyección subcutáneas podría ser falsos negativos o falsos positivos 6,17 fomentar el uso de modelos ortotópico para estudiar el crecimiento del tumor.

<table border="0" cellpadding="0" cellspacing="0" width="1093">
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:185px;">Name of material/equipment</td>
			<td style="width:172px;">Company</td>
			<td style="width:95px;">Catalog number</td>
			<td style="width:641px;">Comments/Description</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Bouin&#39;s solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT10132</td>
			<td>Used for investigating the metastasis on lungs</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Formalin solution</td>
			<td>Sigma-Aldrich</td>
			<td>HT501128</td>
			<td>Used to fix the tissues</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Matrigel, growth factor reduced</td>
			<td>Corning</td>
			<td>356230</td>
			<td>Cells can be resuspended in matrigel for injection</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Mosquito forceps</td>
			<td>Fine Science Tools</td>
			<td>13008-12</td>
			<td>Used for stiching</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Angled forceps</td>
			<td>Electron microscopy sciences</td>
			<td>72991-4c</td>
			<td>These make the exposure of mammary fat pad easier</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Scissors</td>
			<td>B Braun Medicals</td>
			<td>BC056R</td>
			<td>Used to cut open the mice</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Straight forceps</td>
			<td>B Braun Medicals</td>
			<td>BD025R</td>
			<td>This is used to open up the skin to expose mammary fat pad</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">NOD scid gamma mice</td>
			<td>Charles River</td>
			<td>005557</td>
			<td>Experimental animal used for experiment</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">MDA-MB-231</td>
			<td>Sigma-Aldrich</td>
			<td>92020424</td>
			<td>Experimental cells used for injections</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Oculentum simplex</td>
			<td>Teva Pharmachemie</td>
			<td></td>
			<td>Opthalmic ointment used to prevent drying out of eyes</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Betadine</td>
			<td>Fischer Scientific</td>
			<td>19-898-859</td>
			<td>Ionophore, used to disinfect the surgical area</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylazin/Ketamine</td>
			<td>Sigma-Aldrich</td>
			<td>X1251, K2753</td>
			<td>Use injected anesthesia as 10mg/kg and 100mg/kg body weight respectively</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Temgesic</td>
			<td>Schering-Plough</td>
			<td></td>
			<td>Use the painkiller as 0,05-0,1mg/kg body weight</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">DMEM</td>
			<td>Life sciences</td>
			<td>11995</td>
			<td>For trypsin neutralization,use media with serum(FBS:media 1:10 volume); for injection, use media with no serum</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Buffered sodium citrate</td>
			<td>Aniara</td>
			<td>A12-8480-10</td>
			<td>Use the volume ratio as citrate:blood; 1:9</td>
		</tr>
	</tbody>
</table>

orthotopic injection of breast cancer cells into the mammary fat pad of mice to study tumor growth.

<html> El crecimiento del cáncer de mama puede ser estudiado en ratones utilizando una gran cantidad de modelos. La manipulación genética de las células de cáncer de mama puede ayudar a comprender las funciones de las proteínas implicadas en la progresión oncogénica o ayuda a descubrir nuevos supresores tumorales. Además, la inyección de células de cáncer en ratones con diferentes genotipos podría proporcionar una mejor comprensión de la importancia del compartimiento del estroma. Muchos modelos pueden ser útiles para investigar ciertos aspectos de la progresión de la enfermedad, pero no recapitular todo el proceso canceroso. En contraste, las células de cáncer de mama injerto a la almohadilla de grasa mamaria de ratones mejores recapitula la localización de la enfermedad y la presencia del compartimiento del estroma apropiada y por lo tanto mejor imita enfermedad cancerosa humana. En este artículo, describimos cómo implantar las células del cáncer de mama en ratones ortotópicamente y explicar cómo recolectar tejidos para analizar la: 14px; line-height:. 28px; &quot;&gt; medio del tumor y la metástasis a órganos distantes uso de este modelo, muchos aspectos (crecimiento, la angiogénesis y la metástasis) de cáncer pueden ser investigados simplemente proporcionando un ambiente adecuado para las células tumorales crezcan.

La aplicación exitosa del &quot;modelo ortotópico de cáncer de mama&quot; se basa en la inyección adecuada de las células en la almohadilla de grasa mamaria. Errores experimentales tales como la inoculación de las células imprecisa o fugas pueden conducir a variaciones en el tamaño del tumor o incluso la ausencia de un tumor que conduce a la formación de una estructura similar a la que mira una almohadilla de grasa mamaria inyectado con un tampón de control (Figura 2A). La tasa de crecimiento ...

Watch this Scientific Journal Video about Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth. at JoVE.com

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

El cáncer es una enfermedad compleja que se ve influenciada por el tejido que rodea al tumor, así como pro- local y anti-inflamatorias mediadores. Por lo tanto, los modelos de inyección de ortotrópicos, en lugar de los modelos subcutáneos pueden ser útiles para estudiar la progresión del cáncer de una manera que imita mejor la patología humana.

Inyección ortotópico de células de cáncer de mama en el Mamaria Fat Pad de ratones para estudiar el crecimiento del tumor.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

orthotopic-injection-breast-cancer-cells-into-mammary-fat-pad-mice-to

Research

JoVE Journal

Medicine

78.3K vistas.  Leiden University Medical Center.  El cáncer es una enfermedad compleja que se ve influenciada por el tejido que rodea al tumor, así como pro- local y anti-inflamatorias mediadores. Por lo tanto, los modelos de inyección de ortotrópicos, en lugar de los modelos subcutáneos pueden ser útiles para estudiar la progresión del cáncer de una manera que imita mejor la patología humana. 

Video: Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Performing and Processing FNA of Anterior Fat Pad for Amyloid

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis. Since the clinical presentation of this disease is so variable and non-specific, the associated risks of these biopsies are too great for the diagnostic yield. Other sites that have a lower biopsy risk, such as skin or gingival, are also relatively invasive and expensive. In addition, these biopsies may not always have sufficient amyloid deposits to establish a diagnosis. Fat pad aspiration has demonstrated good clinical correlation with low cost and minimal morbidity. However, there are no standardized protocols for performing this procedure or processing the aspirated specimen, which leads to variable and nonreproducible results. The most frequently utilized modality for detecting amyloid in tissue is an apple-green birefringence on Congo red stained sections using a polarizing microscope. This technique requires cell block preparation of aspirated material. Unfortunately, patients presenting in early stage of amyloidosis have minimal amounts of amyloid which greatly reduces the sensitivity of Congo red stained cell block sections of fat pad aspirates. Therefore, ultrastructural evaluation of fat pad aspirates by electron microscopy should be utilized, given its increased sensitivity for amyloid detection. This article demonstrates a simple and reproducible procedure for performing anterior fat pad aspiration for the detection of amyloid utilizing both Congo red staining of cell block sections and electron microscopy for ultrastructural identification.

Fat pad aspiration is a preferred, minimally invasive, and low cost approach as compared to other methods to detect amyloid for diagnosis of systemic amyloidosis. This video article demonstrates a procedural outline for performing fat pad aspiration with appropriate processing of the specimen for the optimal diagnostic outcome.

Realización y procesamiento de la PAAF de almohadilla grasa anterior de amiloide

Historically, heart, liver, and kidney biopsies were performed to demonstrate amyloid deposits in amyloidosis.  Since the clinical presentation of this ...

Investigación

Medicina

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent until many years after removal of the primary tumor and adjuvant therapy. A likely explanation of this phenomenon is that tumor cells have seeded metastatic sites, are resistant to conventional therapies, and remain dormant for long periods of time 1-4.
The existence of dormant cancer cells at secondary sites has been described previously as quiescent solitary cells that neither proliferate nor undergo apoptosis 5-7. Moreover, these solitary cells has been shown to disseminate from the primary tumor at an early stage of disease progression 8-10 and reside growth-arrested in the patients' bone marrow, blood and lymph nodes 1,4,11. Therefore, understanding mechanisms that regulate dormancy or the switch to a proliferative state is critical for discovering novel targets and interventions to prevent disease recurrence. However, unraveling the mechanisms regulating the switch from tumor dormancy to metastatic growth has been hampered by the lack of available model systems.
in vivo and ex vivo model systems to study metastatic progression of tumor cells have been described previously 1,12-14. However these model systems have not provided in real time and in a high throughput manner mechanistic insights into what triggers the emergence of solitary dormant tumor cells to proliferate as metastatic disease. We have recently developed a 3D in vitro system to model the in vivo growth characteristics of cells that exhibit either dormant (D2.OR, MCF7, K7M2-AS.46) or proliferative (D2A1, MDA-MB-231, K7M2) metastatic behavior in vivo . We demonstrated that tumor cells that exhibit dormancy in vivo at a metastatic site remain quiescent when cultured in a 3-dimension (3D) basement membrane extract (BME), whereas cells highly metastatic in vivo readily proliferate in 3D culture after variable, but relatively short periods of quiescence. Importantly by utilizing the 3D in vitro model system we demonstrated for the first time that the ECM composition plays an important role in regulating whether dormant tumor cells will switch to a proliferative state and have confirmed this in in vivo studies15-17. Hence, the model system described in this report provides an in vitro method to model tumor dormancy and study the transition to proliferative growth induced by the microenvironment.

An In Vitro System to Study Tumor Dormancy and the Switch to Metastatic Growth

A modified 3-D in vitro system is presented in which growth characteristics of several tumor cell lines in reconstituted basement membrane correlate with the dormant or proliferative behavior of the tumor cells at a metastatic secondary site in vivo.

Un In Vitro Sistema para el Estudio de latencia del tumor y el salto hacia el crecimiento metastásico

Recurrence of breast cancer often follows a long latent period in which there are no signs of cancer, and metastases may not become clinically apparent ...

An Orthotopic Bladder Tumor Model and the Evaluation of Intravesical saRNA Treatment

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder tumor model. The mouse model is established by urethral catheterization under inhaled general anesthetic. Chemical burn is then introduced to the bladder mucosa using intravesical silver nitrate solution to disrupt the bladder glycosaminoglycan layer and allows cells to attach. Following several washes with sterile water, human bladder cancer KU-7-luc2-GFP cells are instilled through the catheter into the bladder to dwell for 2 hours. Subsequent growth of bladder tumors is confirmed and monitored by in vivo bladder ultrasound and bioluminescent imaging. The tumors are then treated intravesically with saRNA formulated in lipid nanoparticles (LNPs). Tumor growth is monitored with ultrasound and bioluminescence. All steps of this procedure are demonstrated in the accompanying video.

Establishing an orthotopic bladder tumor model to evaluate antitumor effects of intravesically delivered saRNA and monitoring tumor growth by ultrasound and bioluminescent imaging.

Un modelo de tumor vesical ortotópica y la Evaluación de tratamiento intravesical SARNA

We present a novel method for treating bladder cancer with intravesically delivered small activating RNA (saRNA) in an orthotopic xenograft mouse bladder ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos neuroendocrinos líneas de células tumorales como un modelo tridimensional para el estudio de la terapia humana tumor neuroendocrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

Orthotopic Aortic Transplantation in Mice for the Study of Vascular Disease

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. This is largely true, but there are a number of important principles that can reduce the difficulty of these procedures and enhance reproducibility. Orthotopic aortic transplantation is an excellent procedure in which to learn these principles because it involves only two end-to-end anastomoses, but requires good suturing technique and handling of the vessels for consistent success. This procedure begins with the procurement of a length of abdominal aorta from a donor animal, followed by division of the native aorta in the recipient. The procured aorta is then placed between the divided ends of the recipient aorta and sutured into place using end-to-end anastomoses. To accomplish this objective successfully requires a high degree of concentration, good tools, a steady hand, and an appreciation of how easily the vasculature of a mouse can be damaged, resulting in thrombosis. Learning these important principles is what occupies most of the beginner's time when learning microsurgery in small rodents. Throughout this protocol, we refer to these important points. This model can be used to study vascular disease in a variety of different experimental systems1-8. In the context shown here, it is most often used for the study of post-transplant vascular disease, a common long-term complication of solid organ transplantation in which intimal hyperplasia occurs within the allograft. The primary advantage of the model is that it facilitates quantitative morphometric analyses and the transplanted vessel lies contiguous to the endogenous vessel, which can serve as an additional control9. The technique shown here is most often used for mice weighing 18-25 grams. We have accumulated most of our experience using the C57BL/6J, BALB/cJ, and C3H/HeJ strains.

We describe a technique in which a section of the abdominal aorta from a mouse is transplanted orthotopically to just below the renal arteries in an allogeneic or syngeneic recipient. This technique can be useful in studies in which transplantation of large arteries of uniform size is deemed advantageous.

El trasplante ortotópico aórtica en los ratones para el Estudio de la Enfermedad Vascular

Vascular procedures involving anastomoses in the mouse are generally thought to be difficult and highly dependent on the skill of the individual surgeon. ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

Una novela de alta resolución<em&gt; En vivo</em&gt; Técnica de escaneo para el Estudio de la respuesta dinámica de estructuras intracraneales al crecimiento del tumor y Terapéutica

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

Tres Culturas dimensionales: una herramienta para estudiar normal Acinar Arquitectura vs transformación maligna de las células de la mama

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Tracking the Mammary Architectural Features and Detecting Breast Cancer with Magnetic Resonance Diffusion Tensor Imaging

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of life and survival of breast cancer patients. In this paper a new approach for the detection of breast cancer is described, based on tracking the mammary architectural elements using diffusion tensor imaging (DTI). 
The paper focuses on the scanning protocols and image processing algorithms and software that were designed to fit the diffusion properties of the mammary fibroglandular tissue and its changes during malignant transformation. The final output yields pixel by pixel vector maps that track the architecture of the entire mammary ductal glandular trees and parametric maps of the diffusion tensor coefficients and anisotropy indices. 
The efficiency of the method to detect breast cancer was tested by scanning women volunteers including 68 patients with breast cancer confirmed by histopathology findings. Regions with cancer cells exhibited a marked reduction in the diffusion coefficients and in the maximal anisotropy index as compared to the normal breast tissue, providing an intrinsic contrast for delineating the boundaries of malignant growth. Overall, the sensitivity of the DTI parameters to detect breast cancer was found to be high, particularly in dense breasts, and comparable to the current standard breast MRI method that requires injection of a contrast agent. Thus, this method offers a completely non-invasive, safe and sensitive tool for breast cancer detection.

We describe how to obtain parametric and vector maps of the diffusion tensor of the breast using magnetic resonance imaging. The protocol and final output following imaging processing are tailored for tracking breast architectural features and detecting breast malignancy.

Seguimiento de las mamarias características de la arquitectura y la detección de cáncer de mama con resonancia magnética con tensor de difusión de imágenes

Breast cancer is the most common cause of cancer among women worldwide. Early detection of breast cancer has a critical role in improving the quality of ...

Assessment of Viability of Human Fat Injection into Nude Mice with Micro-Computed Tomography

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft tissue filler as it is readily available, easily obtained, inexpensive, and inherently biocompatible.1 However, despite its burgeoning popularity, fat grafting is hampered by unpredictable results and variable graft survival, with published retention rates ranging anywhere from 10-80%. 1-3
To facilitate investigations on fat grafting, we have therefore developed an animal model that allows for real-time analysis of injected fat volume retention. Briefly, a small cut is made in the scalp of a CD-1 nude mouse and 200-400 &#181;l&#160;of processed lipoaspirate is placed over the skull. The scalp is chosen as the recipient site because of its absence of native subcutaneous fat, and because of the excellent background contrast provided by the calvarium, which aids in the analysis process. Micro-computed tomography (micro-CT) is used to scan the graft at baseline and every two weeks thereafter. The CT images are reconstructed, and an imaging software is used to quantify graft volumes.
Traditionally, techniques to assess fat graft volume have necessitated euthanizing the study animal to provide just a single assessment of graft weight and volume by physical measurement ex vivo. Biochemical and histological comparisons have likewise required the study animal to be euthanized. This described imaging technique offers the advantage of visualizing and objectively quantifying volume at multiple time points after initial grafting without having to sacrifice the study animal. The technique is limited by the size of the graft able to be injected as larger grafts risk skin and fat necrosis. This method has utility for all studies evaluating fat graft viability and volume retention. It is particularly well-suited to providing a visual representation of fat grafts and following changes in volume over time.

Fat grafting is an essential technique for reconstructing soft tissue deficits. However, it remains an unpredictable procedure characterized by variable graft survival. Our goal was to devise a mouse model that utilizes a novel imaging method to compare volume retention between differing techniques of fat graft preparation and delivery.

Evaluación de la viabilidad de la inyección de grasa humana en ratones desnudos con micro-tomografía computarizada

Lipotransfer is a vital tool in the surgeon&#8217;s armamentarium for the treatment of soft tissue deficits of throughout the body. Fat is the ideal soft ...

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 ...