Agradecemos a los Dres. Diane Palmieri y Patricia Steeg del NCI para que nos proporciona las células MDA-MB231Br. Agradecemos al Dr. Ralph Mason, el Sr. Jason Reneau y la Sra. Ramona Lopes para el apoyo técnico y colegial. Este trabajo fue apoyado en parte por el IDEA DOD Premios W81XWH-08-1-0.583 y W81XWH-12-1-0.317. Experimentos de resonancia magnética se realizaron en el Centro de Investigación de Imagen Avanzados, un centro de NIH BTRP # P41-RR02584, y la inyección intracardiaca guiada por ecografía se realizó con VisualSonics Vevo 770 bajo 1S10RR02564801.

Todos los animales procedimientos fueron aprobados por el Comité de la Universidad de Texas Southwestern Medical Center Institucional Cuidado de Animales y el uso. 1. Preparación de las células MDA-MB231/Br-GFP <ol><li> Recuperar y cultivar las células MDA-MB231/Br-GFP (amablemente proporcionados por los Dres. Palmieri y Steeg, NCI) en medio DMEM que contiene 10% de SFB, 1% de glutamina y 1% de penicilina / estreptomicina. </li><li> Observar la condición de las células y de color del medio, y reemplazar el medio cada 2-3 días. </li><li> Tripsinizar y recoger las células cuando se alcanza el 80% de confluencia como se indica en los pasos 1.3.1-1.3.5: <ol><li> Retire el medio viejo por completo y añadir 5 ml de PBS (1x) para lavar las células suavemente. Retire el PBS del plato. </li><li> Añadir 1,5 ml de tripsina en el plato; inclinar el plato suavemente para asegurar que todas las células están cubiertas por la tripsina. Coloque el plato de nuevo a la incubadora de células y la mantendrá a 37 ° C durante 1 min. </li><li> Sacar la fuente de la incubadora y observarlas células bajo microscopio óptico para asegurar las células desprenden de la placa. </li><li> Añadir 3 ml de medio para detener el efecto de la tripsina. Recoger la mezcla de células en un tubo de centrífuga. Centrifugar la mezcla a 2000 rpm durante 5 min. </li><li> Retire el medio con cuidado y volver a suspender las células en 5 ml de medio libre de suero hasta que sea homogénea. </li></ol></li><li> Contar el número apropiado de células y resuspender en medio DMEM libre de suero con una concentración final de 1,75 x 10 5 células en 100 l de volumen. <ol><li> Tomar 50 l mezcla de células y añadir 50 l de azul de tripano. Después de mezclar, se toma 10 l mezcla y con cuidado añadir a la diapositiva recuento celular. Utilice varias muestras para asegurar el número de células estimación precisa. </li><li> Pegue en la diapositiva recuento celular, uno de los extremos a la vez y el contador de células empieza a contar automáticamente. Tome promedio de todos los resultados del recuento y calcular el número total de células. </li><li> Centrifugar las células de nuevo y resuspender enel volumen apropiado de medio libre de suero para dar lugar a la concentración final de 1,75 x 10 5 células/100 l de volumen. </li></ol></li><li> Coloque las células en hielo antes de la inyección intracardiaca. </li></ol> 2. Guiada por imagen de ultrasonido de inyección intracardiaca <ol><li> Utilice ratones desnudos hembra (BALB / c nu / nu) entre 6-8 semanas de edad. </li><li> Entrar en el sistema de imágenes. Inicialice el transductor 704 (40 MHz). </li><li> Iniciar un nuevo estudio y complete la información. </li><li> Anestesie (3% isoflurane/100% O 2 en una cámara de inducción) y mantener los animales con isoflurano (2%) en el 100% de O 2 (1 dm 3 / min) durante todo el procedimiento a través de un cono de la nariz. La anestesia se confirma cuando se observa ningún reflejo de retirada con pizca dedo del pie. </li><li> Ajuste la temperatura de la tabla de imagen a 37 ° C. Pegue el ratón anestesiado en la mesa de formación de imágenes se calienta en posición supina. </li><li> Mantener el gel de ultrasonido a 37 ° C antes de imenvejecimiento. Aplique el gel en el pecho del ratón. </li><li> Monte el transductor en el soporte. Baje el transductor hasta que se alcanza la profundidad de imagen deseada. Mueva la etapa hasta el ventrículo izquierdo se identifica con la aorta ascendente como punto de referencia (Figura 1 A). Bloquee la etapa en la que se visualiza una vista clara del ventrículo izquierdo. </li><li> Dibujar 100 l de mezcla de células en una jeringuilla de 1 ml con una aguja de calibre 22. Colocar y fijar la jeringa en el montaje de jeringa. </li><li> Mueva la jeringa hacia adelante hacia el pecho del ratón y mover con cuidado de lado a lado hasta que la punta de la aguja está en el campo de la imagen de la vista (antes de entrar en el ratón). </li><li> Ajuste la altura de la aguja y el ángulo para apuntar hacia abajo el ventrículo izquierdo. </li><li> Penetrar la aguja de la jeringa en el espacio intercostal con prontitud a través de capas de piel y músculo en el ventrículo izquierdo, bajo la guía de la ecografía. Una indicación de la inserción con éxito de la aguja en el ventrículo izquierdoCLE es el reflujo de la sangre arterial fresca (de color rosa en contraste con la sangre venosa de color rojo oscuro) en la jeringa </li><li> Inyectar la mezcla de células lentamente (Figura 1B). </li><li> Al terminar, retire la aguja, levante el transductor, limpiar el gel de ultrasonido con una gasa humedecida y retire la cinta. </li><li> Prepare una jaula limpia con una almohadilla precalentado. </li><li> Colocar el animal en el teclado y observe al animal hasta la recuperación completa. </li><li> Monitorear el comportamiento del animal cada 24 horas durante dos días. </li><li> Condiciones generales y signos neurológicos de complicaciones en ratones experimentales se monitorean rutinariamente. </li></ol> 3. MRI Seguimiento del desarrollo del tumor intracraneal <ol><li> Utilice un Tesla imán 9.4 para supervisar el desarrollo de las metástasis cerebrales intracraneales. </li><li> Iniciar MRI dos semanas después de la implantación del tumor y repetir una vez por semana durante un máximo de tres semanas. </li><li> Sedar a los animales con un 3% isoflurano y mantenerlos bajo genanestesia ral (1,5% de isoflurano). </li><li> Controlar y mantener la temperatura corporal del animal y la respiración constante durante todo el experimento. </li><li> Alta resolución multicorte (14 cortes con 1 mm de espesor, sin espacio) T 1 - y en T2 imágenes coronales, que abarca la región del lóbulo frontal en la fosa posterior, se adquieren con los siguientes parámetros: T imágenes potenciadas en 1: girar eco rebanada múltiple (SEMS), TR / TE = 400 msec/20 ms, matriz: 256 x 256, FOV 20 x 20 mm, de resolución del plano: 78 x 78 m 2. T imágenes ponderadas 2: giro rápido eco de slice múltiple (FSEMS) secuencias, TR / TE = 2500 msec/48 ms, 8 trenes de eco, matriz: 256 x 256, FOV 20 x 20 mm, de resolución del plano: 78 x 78 micras 28,9. Lesiones tumorales aparecen más brillantes que los tejidos cerebrales normales en las imágenes potenciadas T-2. </li><li> El tamaño del tumor se determina en T-2 imágenes ponderadas por esbozar manualmente el accesporción de la masa cing en cada imagen mediante el uso de programas de MATLAB escritas por nosotros 8. Habida cuenta de la mayor parte del tumor diámetros son menores que el espesor de corte (1 mm), el tamaño tumoral se presenta como en la zona de avión y no el volumen. </li></ol> 4. H &amp; E tinción Confirmando las metástasis <ol><li> Sacrificar los ratones inmediatamente después de escanear el último MR, diseccionar los cerebros con tumores e incrustarlo en el medio de octubre y congelado en -80 ° C. </li><li> Sección de una serie de 10 micras de espesor cerebro coronal especímenes con criostato. </li><li> Realizar tinción H &amp; E en las secciones del cerebro. </li></ol>

<ol>
	<li>Schouten, L. J., 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=Incidence+of+brain+metastases+in+a+cohort+of+patients+with+carcinoma+of+the+breast,+colon,+kidney,+and+lung+and+melanoma.">Incidence of brain metastases in a cohort of patients with carcinoma of the breast, colon, kidney, and lung and melanoma.</a> Cancer. 94, 2698-2705 (2002).</li><li>Lin, N. U., 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=CNS+metastases+in+breast+cancer.">CNS metastases in breast cancer.</a> J. Clin. Oncol. 22, 3608-3617 (2004).</li><li>Eichler, A. 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No hay conflictos de interés declarado.

En el presente estudio, hemos demostrado que el ultrasonido de inyección ventricular izquierda guiada por imagen garantiza la precisión de modo que se observó cada animal en este estudio desarrollado metástasis cerebrales y no la muerte del animal. La belleza de la inyección guiada por imagen es que el camino de la penetración de la aguja de la piel y, finalmente, en el ventrículo izquierdo puede ser controlada y ajustada debajo de la imagen, que es distinto del procedimiento manual que requiere estrictos puntos ...

Metástasis cerebral es la neoplasia maligna intracraneal más común en los adultos. El pronóstico es muy malo, con una supervivencia media de 4-6 meses, incluso con tratamiento agresivo. El cáncer de mama es una de las tres principales cánceres primarios con una elevada morbilidad de metástasis cerebral 1-3. Varias líneas de cáncer de mama cerebro-trópicos son capaces de desarrollar metástasis cerebrales después intracardiaca o inyección intracarotídea 4. La inyección directa de las células tumorales en el ventrículo izquierdo puede pasar por alto el lecho capilar pulmonar y por lo tanto aumentar la incidencia de la formación de metástasis cerebrales y reducir al mínimo las metástasis viscerales. La línea MDA-MB231Br es una de las líneas humanas de cáncer de mama más utilizados para el desarrollo de metástasis cerebral en modelos de roedores 5,6. Al igual que muchos otros estudios 4,7, hemos llevado a cabo un procedimiento manual de la inyección intracardiaca en nuestros estudios anteriores. Sin embargo, la tasa de éxito sólo 50% se obtuvo con el manual deinyección y una fracción de los ratones murieron a causa de los procedimientos invasivos repetidos si los ensayos anteriores fracasaron. Aquí, estamos introduciendo el uso de un procedimiento de imagen guiada para asegurar la inyección de células de cáncer de mama en busca del cerebro hacia el ventrículo izquierdo de los ratones atímicos. Longitudinal alta resolución MRI se aplica a seguir el desarrollo de las metástasis cerebrales intracraneales.

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			<td style="width:147px;"> </td>
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			<td height="21" style="height:21px;width:413px;">Trypsin</td>
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			<td height="21" style="height:21px;width:413px;">Automatic cell counter</td>
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			<td height="21" style="height:21px;width:413px;">Isoflurane</td>
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			<td height="21" style="height:21px;width:413px;">VisualSonics Vevo 770 High-Resolution Imaging System </td>
			<td>Visual Sonics Inc.</td>
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			<td height="21" style="height:21px;">9.4T horizontal bore magnet with a Varian INOVA Unity system </td>
			<td>Agilent</td>
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ultrasound imaging-guided intracardiac injection to develop a mouse model of breast cancer brain metastases followed by longitudinal mri

Metástasis cerebrales del cáncer de mama, que se producen en el 30% de los pacientes con cáncer de mama en estadio IV, se asocia con una elevada mortalidad. La mediana de supervivencia es de sólo 6 meses. Es fundamental contar con modelos animales adecuados para imitar la propagación hemodinámico de las células metastásicas en el escenario clínico. Aquí, estamos introduciendo el uso de la ecografía de pequeños animales para guiar una inyección precisa de las células de cáncer de mama tropical cerebrales en el ventrículo izquierdo de los ratones atímicos. RM longitudinal se utiliza para evaluar la iniciación intracraneal y el crecimiento de las metástasis cerebrales. Inyección intracardiaca guiada por ultrasonido garantiza no sólo una inyección precisa y por la presente una mayor tasa de éxito, pero también disminuyó significativamente la tasa de mortalidad, en comparación con nuestro procedimiento manual anterior. In vivo de alta resolución MRI permite la visualización de lesiones multifocales hiperintensas, tan pequeñas como 310 micras de diámetro en T 2 imágenes con ponderación a las 3 semanas después de la inyección. Follow de la RM revela el crecimiento del tumor intracraneal y aumento del número de metástasis que distribuyen a lo largo de todo el cerebro.

Con la alta resolución espacial de la RM (78 micras de resolución del plano), lesiones hiperintensas pueden ser identificados tan pequeñas como 310 micras de diámetro (Figura 2). Dado que las metástasis en este estudio son muy pequeñas y el desarrollo de necrosis y edema es mínima, la lesión hiperintensa en las imágenes potenciadas T 2-realmente representaba la masa tumoral. Estudios de resonancia magnética longitudinales permiten en la evaluaci...

Watch this Scientific Journal Video about Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI at JoVE.com

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

Un cáncer de cerebro modelo murino de metástasis de mama se establece con ultrasonido intracardiaco inyección guiada por imágenes de células MDA-MB231/Br-GFP. Desarrollo de las metástasis intracraneales multifocales ha sido supervisado longitudinalmente utilizando alta resolución 9.4 T MRI.

Guiada por imagen de ultrasonido de inyección intracardiaca de desarrollar un modelo de ratón de cáncer de mama metástasis cerebrales Seguido por Longitudinal MRI

Breast cancer brain metastasis, occurring in 30% of breast cancer patients at stage IV, is associated with high mortality. The median survival is only 6 ...

ultrasound-imaging-guided-intracardiac-injection-to-develop-mouse

Research

JoVE Journal

Medicine

18.6K vistas.  University of Texas Southwestern Medical Center.  Un cáncer de cerebro modelo murino de metástasis de mama se establece con ultrasonido intracardiaco inyección guiada por imágenes de células MDA-MB231/Br-GFP. Desarrollo de las metástasis intracraneales multifocales ha sido supervisado longitudinalmente utilizando alta resolución 9.4 T MRI. 

Video: Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by Longitudinal MRI

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

Imágenes in vivo de tumores bioluminiscencia Dinámica hipoxia de la metástasis cerebral cáncer de mama en un modelo de ratón

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Investigación

Medicina

MRI-guided Disruption of the Blood-brain Barrier using Transcranial Focused Ultrasound in a Rat Model

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a significant obstacle to pharmaceutical treatments of brain disorders as it limits the passage of molecules from the vasculature into the brain tissue to molecules less than approximately 500 Da in size6. FUS induced BBB disruption (BBBD) is temporary and reversible4 and has an advantage over chemical means of inducing BBBD by being highly localized. FUS induced BBBD provides a means for investigating the effects of a wide range of therapeutic agents on the brain, which would not otherwise be deliverable to the tissue in sufficient concentration. While a wide range of ultrasound parameters have proven successful at disrupting the BBB2,5,7, there are several critical steps in the experimental procedure to ensure successful disruption with accurate targeting. This protocol outlines how to achieve MRI-guided FUS induced BBBD in a rat model, with a focus on the critical animal preparation and microbubble handling steps of the experiment.

Microbubble-mediated focused ultrasound disruption of the blood-brain barrier (BBB) is a promising technique for non-invasive targeted drug delivery in the brain1-3. This protocol outlines the experimental procedure for MRI-guided transcranial BBB disruption in a rat model.

Resonancia magnética guiada por disrupción de la barrera sangre-cerebro con ultrasonido transcraneal Enfocado en un modelo de rata

Focused ultrasound (FUS) disruption of the blood-brain barrier (BBB) is an increasingly investigated technique for circumventing the BBB1-5. The BBB is a ...

Multi-modal Imaging of Angiogenesis in a Nude Rat Model of Breast Cancer Bone Metastasis Using Magnetic Resonance Imaging, Volumetric Computed Tomography and Ultrasound

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell proliferation in the bone marrow cavity as well as for interaction of tumor and bone cells resulting in local bone destruction. Our aim was to develop a model of experimental bone metastasis that allows in vivo assessment of angiogenesis in skeletal lesions using non-invasive imaging techniques.
For this purpose, we injected 105 MDA-MB-231 human breast cancer cells into the superficial epigastric artery, which precludes the growth of metastases in body areas other than the respective hind leg1. Following 25-30 days after tumor cell inoculation, site-specific bone metastases develop, restricted to the distal femur, proximal tibia and proximal fibula1. Morphological and functional aspects of angiogenesis can be investigated longitudinally in bone metastases using magnetic resonance imaging (MRI), volumetric computed tomography (VCT) and ultrasound (US).
MRI displays morphologic information on the soft tissue part of bone metastases that is initially confined to the bone marrow cavity and subsequently exceeds cortical bone while progressing. Using dynamic contrast-enhanced MRI (DCE-MRI) functional data including regional blood volume, perfusion and vessel permeability can be obtained and quantified2-4. Bone destruction is captured in high resolution using morphological VCT imaging. Complementary to MRI findings, osteolytic lesions can be located adjacent to sites of intramedullary tumor growth. After contrast agent application, VCT angiography reveals the macrovessel architecture in bone metastases in high resolution, and DCE-VCT enables insight in the microcirculation of these lesions5,6. US is applicable to assess morphological and functional features from skeletal lesions due to local osteolysis of cortical bone. Using B-mode and Doppler techniques, structure and perfusion of the soft tissue metastases can be evaluated, respectively. DCE-US allows for real-time imaging of vascularization in bone metastases after injection of microbubbles7.
In conclusion, in a model of site-specific breast cancer bone metastases multi-modal imaging techniques including MRI, VCT and US offer complementary information on morphology and functional parameters of angiogenesis in these skeletal lesions.

In the pathogenesis of bone metastasis, angiogenesis is a crucial process and therefore represents a target for imaging and therapy. Here, we present a rat model of site-specific breast cancer bone metastasis and describe strategies to non-invasively image angiogenesis in vivo using magnetic resonance imaging, volumetric computed tomography and ultrasound.

Multimodal de imágenes de la angiogénesis en un modelo de rata desnuda de las metástasis óseas del cáncer de pecho utilizando imágenes de resonancia magnética, tomografía computarizada y la ecografía volumétrica

Angiogenesis is an essential feature of cancer growth and metastasis formation. In bone metastasis, angiogenic factors are pivotal for tumor cell ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

Imágenes en directo de las respuestas de drogas en el microambiente tumoral en modelos de ratón de cáncer de mama

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Initiation of Metastatic Breast Carcinoma by Targeting of the Ductal Epithelium with Adenovirus-Cre: A Novel Transgenic Mouse Model of Breast Cancer

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in exponential tumor growth and metastasis to distal tissues and the collapse of anti-tumor immunity. Many useful animal models exist to study breast cancer, but none completely recapitulate the disease progression that occurs in humans. In order to gain a better understanding of the cellular interactions that result in the formation of latent metastasis and decreased survival, we have generated an inducible transgenic mouse model of YFP-expressing ductal carcinoma that develops after sexual maturity in immune-competent mice and is driven by consistent, endocrine-independent oncogene expression. Activation of YFP, ablation of p53, and expression of an oncogenic form of K-ras was achieved by the delivery of an adenovirus expressing Cre-recombinase into the mammary duct of sexually mature, virgin female mice. Tumors begin to appear 6 weeks after the initiation of oncogenic events. After tumors become apparent, they progress slowly for approximately two weeks before they begin to grow exponentially. After 7-8 weeks post-adenovirus injection, vasculature is observed connecting the tumor mass to distal lymph nodes, with eventual lymphovascular invasion of YFP+ tumor cells to the distal axillary lymph nodes. Infiltrating leukocyte populations are similar to those found in human breast carcinomas, including the presence of &#945;&#946; and &#947;&#948; T cells, macrophages and MDSCs. This unique model will facilitate the study of cellular and immunological mechanisms involved in latent metastasis and dormancy in addition to being useful for designing novel immunotherapeutic interventions to treat invasive breast cancer.

Activation of latent mutations with adenovirus-Cre into the mammary ductal system results in a clinically relevant metastatic breast cancer. Incorporation of a YFP promoter allows tracking of distal metastatic tumor cells. This model is useful to study latent metastasis, anti-tumor immunity, and for designing novel immunotherapies to treat breast cancer.

Inicio de la metástasis de carcinoma de mama mediante la Focalización del epitelio ductal con adenovirus Cre: Un modelo de ratón transgénico de Novela del Cáncer de Mama

Breast cancer is a heterogeneous disease involving complex cellular interactions between the developing tumor and immune system, eventually resulting in ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

La cuantificación de la invasividad de las células del cáncer de mama El uso de un modelo (3D) Tres Dimensiones

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Trauma cerebral aguda en ratones seguido de Longitudinal dos fotones de imágenes

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

A Preclinical Murine Model of Hepatic Metastases

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, murine pancreatic tumor model of hepatic metastases via a hemispleen injection of syngeneic murine pancreatic tumor cells is described. This model mimics many of the clinical conditions in patients with metastatic disease to the liver. Mice consistently develop metastases in the liver allowing for investigation of the metastatic process, experimental therapy testing, and tumor immunology research.

A preclinical, murine model of hepatic metastases performed via a hemispleen injection technique.

A preclínicos modelo murino de metástasis hepática

Numerous murine models have been developed to study human cancers and advance the understanding of cancer treatment and development. Here, a preclinical, ...

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

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

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 ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans biopelícula Desarrollo de Cuerpos Extraños médicamente relevantes en un modelo de ratón subcutánea Seguido por bioluminiscencia Imaging