Este estudio es financiado en parte por el Departamento de Defensa premio IDEA de Cáncer de Mama W81XWH-08-1-0583 y la NIH / NCI CA141348-01A1 (DZ) y FAMRI premio científico clínico (DS). Infraestructura de imagen se proporciona por el Programa de Pequeños Animales del Suroeste de Investigación de imagen apoyado en parte por U24 CA126608 y Simmons Cancer Center (P30 CA142543) y NIH 1S10RR024757-01.

Todos los animales procedimientos fueron aprobados por el Cuidado de Animales institucional y el empleo de la Universidad de Texas, Centro Médico del Sudoeste. 1. En el ensayo in vitro de HIF-1α bioluminiscencia <ol><li> Material y métodos: <ul><li> Humanas de cáncer de mama metastásico línea celular MDA-MB231 transfectadas con un gen novedoso HIF-1-dependiente, 5HRE-ODD-luc fue generada por el Dr. Harada. </li><li> En condiciones de hipoxia, la mayor expresión de dominio degradación dependiente de oxígeno (ODD)-luciferasa proteína de fusión se debe a cinco copias del elemento de respuesta a la hipoxia (5HRE). La presencia de ODD provoca la degradación del ODD-Luc proteína resultante en muy baja actividad de la luciferasa de fondo en condiciones normoxic. Por lo tanto, este novedoso sistema se puede utilizar para detectar las regiones de hipoxia en un tumor de imagen en tiempo real. La construcción de este vector de expresión 5HRE-ODD-luc ha sido reportado por Harada et al 1,2. </li></ul></li><li> Las células de cultivo en normoxia o hipoxia: <ul><li> Mantener la recombinante MDA-MB231 células en suero fetal bovino al 10% (FBS)-DMEM medio que contiene una glutamina%, los antibióticos de 400 mg / ml de G418 y el 1% penicilina / estreptomicina. </li><li> Para el análisis in vitro de HIF-1α bioluminiscencia, la placa de 3 x 10 5 MDA-MB231 células que expresan 5HRE-ODD-luc vector en cada pocillo de dos períodos de seis platos así. </li><li> Permiten a las células para fijar el plato de la pared después de incubación durante la noche y luego transferir un plato en una cámara de hipoxia (Billups-Rothenberg, Inc. Del Mar, CA) para los estudios de hipoxia, mientras que mantener el plato otra en condiciones normoxic (21% O 2). </li><li> Volver a montar la cámara y la cámara de gas con un 0,1% de O 2 mediante la conexión de la tubería de conexión de entrada con un cilindro de gas. Nota: Los dos de entrada y salida de las abrazaderas puerto debe estar abierto durante este procedimiento. </li><li> Desconecte la fuente de gas después de la cámara se ha purgado y la cámara del sello por el cierre de las abrazaderas de plástico. </li><li> Coloque la cámara en una incubadora a 37 ° C con 5% de CO 2. Nota: La cámara debe estar húmeda para evitar la evaporación excesiva de las culturas. Esto se puede lograr mediante la colocación de 10 a 20 ml de agua estéril en la cámara. </li></ul></li><li> Bioluminiscencia ensayo: <ul><li> Después de la incubación de 24 horas, se elimina el medio y lavar las células rápidamente con PBS enfriado con hielo (2X). </li><li> Añadir 1 ml de PBS frío con 100 l de luciferina (Biotecnología de Oro, St Louis, MO). </li><li> BL adquirir imágenes (sistema IVIS Spectrum, Caliper Life Sciences, Hopkinton, MA) con tiempos de exposición diferentes (1, 30, 60, 180 s). </li><li> Medir la intensidad de la luz en cada pozo usando el software Image Vivir (Caliper Life Sciences). </li></ul></li></ol> 2. Establecimiento de un modelo de cáncer de mama metástasis cerebral <ol><li> Preparación de las células MDA-MB231/5HRE-ODD-luc <ul><li> Recuperar y cultivar las células en un medio de MDA-MB231/5HRE-ODD-luc DEME que contiene 10% de SFB, 1% y glutamina 1% de penicilina / estreptomicina. </li><li> Vuelva a colocar el medio cada 2-3 días. Tripsinize y lavar las células cuando llegan a 80% de confluencia. </li><li> Contar el número apropiado de células y resuspender en medio sin suero DEME libres con un 25% Matrigel (BD Biosciences, San Jose, CA) con una concentración final de 10 5 células en 4 l de volumen. </li><li> Células de lugar en el hielo antes de la inyección intracraneal. </li></ul></li><li> Implantación quirúrgica <ul><li> Mujer ratones desnudos (BALB / c nu / nu; Instituto Nacional del Cáncer, Bethesda, MD) a las 4-6 semanas de edad son utilizados en este estudio. </li><li> Anestesiar (3% isoflurano / O2 en una cámara de inducción; isoflurano de Baxter International Inc., Deerfield, IL, EE.UU.) y mantener a los animales con isoflurano (1%) en oxígeno (1 dm 3 / min) durante el procedimiento quirúrgico 3. </li><li> La piel parietal derecho debe ser preparado con betadine y alcohol al 70% antes de la incisión. </li><li> El uso de un taladro de alta velocidad, una fresa agujero de 1 mm en el hemisferio derecho del cráneo, 1 mm por delante de la sutura coronal y 2 mm lateral a la sutura sagital. </li><li> Dibuja 4 l mezcla de células (10 5 células), utilizando una jeringa de 10 l Hamilton (Hamilton Company, Reno, NV). Inyectar las células directamente en el derecho diencéfalo caudal, 1,5 mm por debajo de la duramadre con una medida 32G aguja Hamilton. Mantenga la aguja en su lugar durante unos 30 s antes de la retirada. El uso de una aguja fina 32G minimiza el daño tisular. </li><li> Llenar el agujero de trépano con cera de hueso y cerca del cuero cabelludo con suturas absorbibles. </li><li> Preparar a la región de la incisión con alcohol al 70%. </li><li> Aplicar la buprenorfina analgesia cada 12 horas durante dos días. </li></ul></li></ol> 3. En imágenes de bioluminiscencia in vivo de tumores dinámica de la hipoxia en las metástasis cerebrales del cáncer de mama <ul><li> Iniciar imágenes de bioluminiscencia longitudinal (sistema IVIS Spectrum) dos semanas después de la implantación intracraneal y repetir una vez por semana durante 8-10 Semanas. </li><li> Anestesiar a tres ratones en un momento (3% isoflurano / O 2 en una cámara de inducción) </li><li> Administrar una solución de D-luciferina (120 mg / kg en PBS en un volumen total de 80 l; Biotecnología de oro) por vía subcutánea en la región del cuello de cada ratón como se describe en detalle anteriormente 4. </li></ul> D-luciferina no es tóxico y se ha demostrado que es capaz de penetrar intacta la barrera hematoencefálica (BBB) ​​y las membranas celulares 3,5. <ul><li> Lugar el 3 ratones en la cámara de imágenes y mantenida con isoflurano (1%) en oxígeno (1 dm 3 / min) durante la exploración. </li><li> Cinco minutos después de la inyección luciferina, BL adquirir imágenes con una serie de tiempo de exposición diferentes (1, 30, 60, 180 s). </li></ul> Nuestras observaciones muestran que el tiempo máximo de emisión de luz es cerca de 5 minutos tras la administración subcutánea de la luciferina en la región del cuello 3,4. <ul><li> Analizar los datos con el software de imágenes de vida (Caliper Life Sciences) con el recuento absoluto de fotones (fotones / s) en una región de interés (ROI), dibujado manualmente para delinear la señal de BLI del cerebro. </li><li> Tiempo de trama curva curso de cuenta de fotones para indicar la dinámica de tumor hipoxia. </li><li> Inmediatamente después de la última BLI, administrar pimonidazole, el marcador de hipoxia y una hora más tarde, el sacrificio de los ratones y diseccionar el cerebro. Insertar el cerebro entero en la temperatura óptima de corte (OCT) de mediano y congelación de -80 ° C congelador. Posterior tinción histológica violeta de cresilo y tinción inmunohistoquímica contra la luciferasa, marcador de hipoxia pimonidazole, y HIF-1α-realizado para validar las observaciones de imágenes. </li></ul> 4. Los resultados representativos: Como se muestra en la Figura 1, significativamente mayor señal de BLI se observó después de las células transfectadas se incubaron en la cámara de hipoxia (1% O 2) durante 24 horas. La emisión de luz débil se observó en las células de control en normoxia. Esto puede deberse a la población de células hacinamiento después de la cultura de 24 horas de 3 x 10 5 células, lo que indujo una señal de tensión a las células de HIF-1α sobreexpresan. Sin embargo, los resultados in vivo BLI fueron validados por tinción inmunohistoquímica mostrando colocalización entre la hipoxia tumoral y la expresión de la luciferasa. Una serie de tiempos de exposición automático permite la adquisición continua de una serie de imágenes, lo que facilita la captura de una señal débil con mayor tiempo de exposición, y las señales de fuerte uso de un tiempo más corto sin saturar el CCD. <img alt="Figura 1" src="/files/ftp_upload/3175/3175fig1.jpg" /> Figura 1 Ensayo in vitro de HIF-1α bioluminiscencia Fila superior:. 3 x 10 5 células se incubaron en MDA-MB231/5HRE-ODD-luc cada pocillo de una 6-y-placa en una cámara de hipoxia (0,1% O 2) por 24 horas antes de que se retiró el medio, se lavan y se sustituye por 1 ml de PBS. Inmediatamente después de 100 l luciferina se añadió en cada pozo, BLI fue adquirido con una serie de tiempos de exposición (1, 30, 60, 180 s). Luminiscencia fuerte se observó en los pozos representante Fila inferior:. Como control, 3 x 10 5 células se incubaron en normoxia (21% O 2) emite luz débil. <img alt="Figura 2" src="/files/ftp_upload/3175/3175fig2.jpg" /> En la figura 2 imágenes de bioluminiscencia in vivo de la dinámica de la hipoxia tumoral. A) una señal de luz débil del lado derecho del cerebro de los ratones fue el primero en visualizar cinco semanas después de la implantación intracraneal MDA-MB231/5HRE-ODD-luc de las células de cáncer de mama. Aumento de la señal óptica se observó durante otras 6 semanas, lo que indica un aumento hipoxia tumoral. B) La figura muestra la curva de evolución en el tiempo de recuento de fotones cuantitativo de la señal de la luz. <img alt="Figura 3" src="/files/ftp_upload/3175/3175fig3.jpg" /> Figura 3 Colocaliztion de la luciferasa y la hipoxia detectada por tinción inmunohistoquímica. Un cerebro de un ratón congelado que lleva una metástasis de cáncer de mama MDA-MB231/5HRE-ODD-luc incrustado en octubre se seccionó. Una sección de 10 m immunostained con un marcador de hipoxia, pimonidazole, reveló la heterogeneidad intratumoral de la hipoxia, que se correlaciona espacialmente con expresión de la luciferasa detectados por los anti-manchas luciferasa. Barra de escala, 100 micras.

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No hay conflictos de interés declarado.

El cáncer de mama metástasis cerebral se produce en el 30% de los pacientes con cáncer de mama en etapa IV. Se asocia con una elevada morbilidad y mortalidad y tiene una supervivencia media de 13 meses 6. Es necesario disponer de modelos animales apropiados para imitar esta enfermedad clínicamente devastadoras para facilitar la comprensión de su inicio y la progresión intracraneal, así como los perfiles de fisiopatológicas. En este caso, hemos desarrollado un cáncer de mama ortotópico modelo de met?...

<table border="1"><tbody><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> D-luciferina </td><td> Oro Biotecnología </td><td> L-123 </td><td> 120 mg / kg en PBS en un volumen total de 80 l para el estudio in vivo </td></tr><tr><td> Isoflurano </td><td> Baxter International Inc. </td><td> 1001936060 </td><td></td></tr><tr><td> Matrigel </td><td> BD Biosciences </td><td> 354234 </td><td></td></tr><tr><td> Hamilton jeringa </td><td> Hamilton Company </td><td> 1701 </td><td></td></tr><tr><td> 32G Hamilton aguja </td><td> Hamilton Company </td><td> 7803-04 </td><td></td></tr><tr><td> Cámara de hipoxia </td><td> Billups-Rothenberg, Inc. </td><td> MIC-101 </td><td></td></tr><tr><td> Sistema de imágenes de bioluminiscencia </td><td> Caliper Life Sciences </td><td> IVIS sistema Spectrum </td><td></td></tr><tr><td> G418 </td><td> Fisher Scientific </td><td> SV3006901 </td><td></td></tr></tbody></table>

in vivo bioluminescence imaging of tumor hypoxia dynamics of breast cancer brain metastasis in a mouse model

Es bien sabido que la hipoxia tumoral juega un papel importante en la promoción de la progresión maligna y que afectan negativamente a la respuesta terapéutica. Existe poco conocimiento sobre el terreno, en la hipoxia in vivo, durante el desarrollo del tumor intracraneal de tumores cerebrales malignos debido a la falta de medios eficaces para su seguimiento en estos tumores ortotópico profunda. Imágenes de bioluminiscencia (BLI), basado en la detección de la luz emitida por las células vivas que expresan un gen de la luciferasa, ha sido adoptado rápidamente por la investigación del cáncer, en particular, para evaluar el crecimiento del tumor o cambio el tamaño del tumor en respuesta al tratamiento en estudios preclínicos en animales. Por otra parte, al expresar un gen bajo el control de una secuencia promotora, la expresión de genes específicos pueden ser monitoreados de forma no invasiva por BLI. En situaciones de estrés hipóxico, las respuestas de señalización están mediados principalmente por el factor inducible por hipoxia 1α (HIF-1α) para conducir la transcripción de varios genes. Por lo tanto, hemos utilizado un reportero construir HIF-1α, 5HRE-ODD-luc, transfectadas establemente en el cáncer de mama humano MDA-MB231 (MDA-MB231/5HRE-ODD-luc). Ensayo in vitro de HIF-1α bioluminiscencia es realizada por incubación de las células transfectadas en una cámara de hipoxia (0,1% O 2) durante 24 horas antes de BLI, mientras que las células en normoxia (21% O 2) servir como un control. Flujo de fotones observados significativamente mayor para las células en condiciones de hipoxia sugiere un aumento de HIF-1α unirse a su promotor (elementos HRE), en comparación con los de normoxia. Las células se inyectan directamente en el cerebro del ratón para establecer un cáncer de mama cerebro modelo de metástasis. En imágenes de bioluminiscencia in vivo de la dinámica de la hipoxia tumoral se inicia dos semanas después de la implantación y repitió una vez por semana. BLI revela aumento de las señales de luz desde el cerebro a medida que avanza tumor, lo que indica la hipoxia aumentó tumor intracraneal. Estudio histológico e inmunohistoquímico se utilizan para confirmar los resultados de las imágenes en vivo. Aquí, vamos a introducir los enfoques de análisis in vitro de HIF-1α bioluminiscencia, la creación quirúrgica de una metástasis de mama cáncer cerebral en un ratón desnudo y la aplicación de imágenes de bioluminiscencia en vivo para controlar la hipoxia tumoral intracraneal.

Watch this Scientific Journal Video about In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model at JoVE.com

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

Imágenes de bioluminiscencia de inducible por hipoxia-1α factor de actividad se aplica al seguimiento del desarrollo de la hipoxia tumoral intracraneal en un modelo de ratón de cáncer de mama metástasis cerebrales.

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 ...

in-vivo-bioluminescence-imaging-tumor-hypoxia-dynamics-breast-cancer

Research

JoVE Journal

Medicine

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

19.5K vistas.  University of Texas Southwestern Medical Center. Imágenes de bioluminiscencia de inducible por hipoxia-1α factor de actividad se aplica al seguimiento del desarrollo de la hipoxia tumoral intracraneal en un modelo de ratón de cáncer de mama metástasis cerebrales.

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

Multi-photon Imaging of Tumor Cell Invasion in an Orthotopic Mouse Model of Oral Squamous Cell Carcinoma

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient management strategies. Orthotopic mouse models of oral cancer have been developed to facilitate the study of factors that impact invasion and serve as model system for evaluating anti-tumor therapeutics. In these systems, visualization of disseminated tumor cells within oral cavity tissues has typically been conducted by either conventional histology or with in vivo bioluminescent methods. A primary drawback of these techniques is the inherent inability to accurately visualize and quantify early tumor cell invasion arising from the primary site in three dimensions. Here we describe a protocol that combines an established model for squamous cell carcinoma of the tongue (SCOT) with two-photon imaging to allow multi-vectorial visualization of lingual tumor spread. The OSC-19 head and neck tumor cell line was stably engineered to express the F-actin binding peptide LifeAct fused to the mCherry fluorescent protein (LifeAct-mCherry). Fox1nu/nu mice injected with these cells reliably form tumors that allow the tongue to be visualized by ex-vivo application of two-photon microscopy. This technique allows for the orthotopic visualization of the tumor mass and locally invading cells in excised tongues without disruption of the regional tumor microenvironment. In addition, this system allows for the quantification of tumor cell invasion by calculating distances that invaded cells move from the primary tumor site. Overall this procedure provides an enhanced model system for analyzing factors that contribute to SCOT invasion and therapeutic treatments tailored to prevent local invasion and distant metastatic spread. This method also has the potential to be ultimately combined with other imaging modalities in an in vivo setting.

A comprehensive overview of the techniques involved in generating a mouse model of oral cancer and quantitative monitoring of tumor invasion within the tongue through multi-photon microscopy of labeled cells is presented. This system can serve as a useful platform for the molecular assessment and drug efficacy of anti-invasive compounds.

Multi-fotón imágenes de invasión de células tumorales en un modelo de ratón ortotópico de carcinoma oral de células escamosas

Loco-regional invasion of head and neck cancer is linked to metastatic risk and presents a difficult challenge in designing and implementing patient ...

Investigación

Medicina

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Combinación de radioterapia en un modelo ortotópico de tumor cerebral del ratón

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

La implantación estereotáctica intracraneal y<em&gt; En vivo</em&gt; Radiología bioluminiscente de xenoinjertos de tumores en un sistema modelo de ratón de glioblastoma multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

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 ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse de imágenes de Primaria preneoplásicas células epiteliales mamarias derivadas de los modelos de ratones genéticamente modificados del cáncer de mama

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

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 ...

Ultrasound Imaging-guided Intracardiac Injection to Develop a Mouse Model of Breast Cancer Brain Metastases Followed by 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 months. It is critical to have suitable animal models to mimic the hemodynamic spread of the metastatic cells in the clinical scenario. Here, we are introducing the use of small animal ultrasound imaging to guide an accurate injection of brain tropical breast cancer cells into the left ventricle of athymic nude mice. Longitudinal MRI is used to assessing intracranial initiation and growth of brain metastases. Ultrasound-guided intracardiac injection ensures not only an accurate injection and hereby a higher successful rate but also significantly decreased mortality rate, as compared to our previous manual procedure. In vivo high resolution MRI allows the visualization of hyperintense multifocal lesions, as small as 310 &#181;m in diameter on T2-weighted images at 3 weeks post injection. Follow-up MRI reveals intracranial tumor growth and increased number of metastases that distribute throughout the whole brain.

A breast cancer brain metastasis mouse model is established with ultrasound imaging-guided intracardiac injection of MDA-MB231/Br-GFP cells. Development of multifocal intracranial metastases has been monitored longitudinally using high-resolution 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 ...

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 ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

En vivo de imágenes óptico de tumores del cerebro y la artritis Usando fluorescente SAPC-DOPS nanovesículas

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...