We would like to thank Pascal DuBois for proof-reading the article. The authors would also like to thank the personnel of the Animal Facility at Hunter College of The City University of New York. This work was supported by a NIH RCMI grant to Hunter College.

Declaración de Ética Todos los estudios con animales fueron aprobados por el Cuidado y Uso de Animales Comité Institucional (IACUC) de Hunter College de la City University de Nueva York. Nota: ocho ratones BALB / c fueron utilizados en este procedimiento experimental. Cinco ratones BALB / c fueron implantados con 5 x 10 6 células de carcinoma hepatocelular BNL 1ME A.7R.1 ratón para producir los tumores primarios. Tres ratones BALB / c sin implantaciones se utilizaron como controles. BNL 1ME A.7R.1 es una línea celular de carcinoma hepatocelular murino que se derivó por transformación química de la no tumorigénicos línea celular de hepatocitos murinos BNL CL.2. BNL 1ME A.7R.1 se obtuvo de la American Type Culture Collection (ATCC). 1. Pre-cirugía <ol><li> Asegúrese de que los ratones de todas las cirugías de supervivencia son saludables. Para asegurar que los ratones están sanos, inspeccionar signos de deshidratación por abombamiento de la piel. No utilice los ratones con cualquier evidencia de enfermedad o distress en estos experimentos. </li><li> Pesar ratones para asegurarse de que no son de bajo peso. Asegúrese de que el peso es al menos 16-20 gramos. </li><li> Preparar 5 × células HCC ratón 10 6 BNL 1ME A.7R.1 para la implantación por ratón: <ol><li> Aspirar los medios de comunicación a partir de 10 ml de 60% - placa de cultivo de células confluentes 70% de las células de HCC A.7R.1 ratón BNL 1me. Lavar dos veces con 5 ml de PBS. Añadir 2 ml de tripsina al plato y se incuba a 37 ° C en un incubador humidificado con aire y 5% de CO 2 durante 5 min. </li><li> Posteriormente, pipeta de 10 l tripsinizaron células en un hemocitómetro manual para el recuento de células bajo un microscopio con una ampliación de 10X. Contar el número total de células que se encuentran en los cuatro grandes cuadrados de las esquinas y multiplicar el factor de dilución por el número total de células. Luego, divida por el número de plazas contadas y multiplíquelo por 10 6 para la concentración de células (células por ml). </li><li> Usando el cálculo, recolectar 5 millones de células por ratón en estéril1,5 ml tubos de microcentrífuga. Etiqueta de 1,5 ml tubos de microcentrífuga. </li></ol></li><li> Resuspender las 5 × 10 6 células BNL 1ME A.7R.1 ratón HCC en 100 l de tampón fosfato salino (PBS). Mantenga estas células en hielo hasta la implantación en cirugía. </li><li> Realizar las actividades necesarias para la preparación de los ratones para la cirugía, tales como la inducción de la anestesia y el pelo de recorte de distancia de los equipos y materiales estériles para evitar la contaminación. </li><li> Inducir la anestesia general en los ratones por inhalación de isoflurano usando un vaporizador de isoflurano junto con oxígeno a una velocidad de flujo constante de 1 concentración de L por minuto (aprox. 2,1%). Nota: El sistema es autosuficiente, e incluye un vaporizador de precisión, un suministro de oxígeno y un filtro de carbón activado que está vinculada a un circuito de respiración necesaria para la recogida de gases / anestésico. </li><li> Asegúrese de que la anestesia es la adecuada para evitar que el animal sometido a dolor durante la cirugía. Realizar toe-pinch y observar animales por falta de responsiveness para confirmar la anestesia. </li><li> En ese momento, cambiar la línea de la válvula, el sello de la caja y abierto al cono de la nariz. </li><li> Tan pronto como el ratón se anestesia, aplique una pequeña cantidad de una pomada oftálmica para las córneas como una medida preventiva de secado y daños. </li><li> Posteriormente, mover el animal a la zona de preparación, con el cono de la nariz correctamente colocado y conectado (para la anestesia de mantenimiento). Preparar el sitio de la incisión quirúrgica para la cirugía por la depilación en esta zona de preparación. </li><li> Asegúrese de que el área de la cirugía es en un lugar libre de toda otra actividad. </li><li> Asegúrese de que la superficie de trabajo es robusto y debidamente desinfectados, inmaculado y libre de desorden antes de la cirugía. Coloque un pedazo estéril de lino o bandeja quirúrgica sobre ella. </li><li> Asegúrese de que el campo operatorio es permitida sólo a las personas que llevan a cabo la cirugía. Asegúrese de que la sala de cirugía se limpia y se barrió con un limpiador apropiado y rocía con un disinfe apropiadactant. </li><li> Asegúrese de que también se coloca lejos de puertas y ventanas como corrientes de aire puede esparcir el polvo que conduce a la contaminación del sitio. </li><li> Realizar la cirugía de supervivencia con instrumentos estériles. De cualquier remojo instrumentos en un desinfectante de acuerdo con la recomendación o autoclave del fabricante. Utilice suministros estériles básicos que están listos para su uso. </li><li> Instrumentos separados según su función para ayudar a prevenir la contaminación de las zonas estériles. </li><li> Afeitado aproximadamente 1 pulgada alrededor del sitio de la incisión (incisión de línea media) para acceder al hígado del animal. Quitar el pelo del animal lejos del sitio de la cirugía. </li><li> Después del afeitado, frote el área con un desinfectante y jabón (betadine quirúrgica matorral) y lavar el exceso de desinfectante de distancia con una solución de PBS estéril. Realizar esto varias veces, con todo el proceso de borrado de una duración aproximada de 5 minutos. Retire y descartados los guantes usados. </li><li> Después, cambiar a la ropa de protección, como Surgical vestido, top matorrales y la máscara de la cara. A continuación, desinfectar las manos con solución exfoliante adecuada antes de ponerse los guantes quirúrgicos. Nota: Este es un paso necesario para evitar que la excreción y la contaminación de la zona quirúrgica con contaminantes potenciales. </li></ol> 2. Cirugía <ol><li> Proporcionar una fuente de calor externa para el calor durante todo el período de la anestesia. El animal puede experimentar algo de hipotermia debido a la anestesia provoca alteraciones de la termorregulación; por lo tanto, mantener la temperatura corporal. </li><li> Coloque el ratón sobre la espalda durante toda la cirugía. </li><li> Fijar las extremidades delanteras, patas traseras y la cola del ratón a la mesa de cirugía con trozos de cintas quirúrgicas. </li><li> Haga una incisión recta inmediatamente inferior a la xyphisternum. </li><li> Usar un retractor quirúrgico auto-retención estéril para exponer el hígado. </li><li> Con una jeringa que tiene una aguja 20G, lenta y cuidadosamente inyectar 100 l de PBS que contenía 5 × 10 6 BNL 1ME A.7R.1células de ratón HCC en el lóbulo derecho del hígado. </li><li> Después de retirar la aguja, aplique un hisopo de algodón estéril durante al menos 1 minuto para detener cualquier sangrado potencial debido a la alta vascularización del hígado. </li><li> Cierre el tejido subcutáneo con una sutura absorbible seguido de cierre de la piel utilizando grapas quirúrgicas. Posteriormente, limpiar la herida una vez más con matorrales betadine. Nota: Esto asegurará que el cierre de la herida quirúrgica. </li><li> Subcutánea dar 100 l de solución salina normal. </li></ol> 3. Después de la cirugía <ol><li> Inmediatamente después de la cirugía, transferir el ratón a una jaula limpia con una tarjeta de identificación después de la cirugía para notificar a los cuidadores de la condición del ratón. Proporcionar alimentos y agua. Nota: En nuestra experiencia, un ratón que acaba sometido a cirugía le irá mejor si alojados solo con comida húmeda y agua durante el período de 24 horas después de la cirugía inicial. </li><li> Mantenga el animal caliente con una lámpara de calor que genera calor no supera 38 ºC. Use una termómetro para medir la temperatura hasta que el ratón está totalmente recuperado. </li><li> Casa del animal en una jaula de preferencia con ropa de cama de papel para garantizar la comodidad. </li><li> Después de la recuperación inicial, el seguimiento del animal para detectar signos de incomodidad, sangrado, dolor y angustia. Dar buprenorfina (0.05 -0.1 mg / kg) para aliviar el dolor. Supervisar de cerca la actividad, el comportamiento y apariencia, así como el agua y el consumo de alimentos durante aproximadamente 2 h. Si ambulatoria, mover el animal de nuevo a la zona de una vivienda normal regular de los ratones y seguir vigilando a los 6-12 hr intervalos de 24 horas después de la cirugía. Realizar una comprobación de peso si se desea. </li><li> Sin embargo, si el animal está teniendo problemas de recuperación, tomar las medidas adecuadas para el cuidado de apoyo. Si los ratones no pueden recuperarse, con humanidad eutanasia a los ratones a través de asfixia con CO2 y dislocación cervical. Siga las pautas institucionales aprobados para la eutanasia. </li><li> Después de la cirugía, compruebe si hay evidencia clínica de HCC que suele ser observable reducción tan significativa en la condición corporal. Nota: En este punto, un tumor primario ha desarrollado probablemente en el hígado y depósitos metastásicos probable que se han desarrollado en los pulmones. </li><li> En este punto, sacrificar todos los ratones (experimental y control) que participan en el experimento por eutanasia humanitaria. Someter los ratones a la asfixia con dióxido de carbono: liberar muy lentamente el dióxido de carbono en la cámara durante un período de 5 -10 min. Esto provocará un paro respiratorio. </li></ol>

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The authors declare that they have no competing financial interests.

En este artículo se da una descripción detallada del método que se informó recientemente por Ogunwobi y colegas del éxito del establecimiento de un modelo de ratón singénico ortotópico de HCC metástasis (Figura 2) 3. La tasa de absorción de los tumores para este procedimiento es generalmente alto. Hemos observado previamente una tasa de absorción del tumor de 100% 3. Sin embargo, la tasa de absorción puede ser variable en función de la competencia del investigador. En ...

Hepatocellular carcinoma (HCC) is one of the most lethal cancers, with poor prognosis and low life expectancy. Nearly all cancer-related deaths are due to the metastatic spread of the disease from the originating organ to additional distant organs2-6.HCC progression is a complex process. Therefore, being able to model the tumor microenvironment that is naturally found in metastatic HCC in animal models can prove to be a successful and useful way to reveal relevant mechanisms in humans.7Unfortunately, the mechanisms of cancer metastasis are still largely unclear. Therefore, there is a need to establish animal models that will enable us to elucidate the underlying molecular mechanisms of metastases of cancers such as HCC3,5.
Mouse model systems are a very useful approach for delineating mechanisms and evaluating novel strategies for treatment of metastatic human cancers5,8. The various mouse model systems that presently exist are a testament to efforts of researchers to correctly depict the complexity of the disease5,8,9.
Ogunwobi and colleagues recently used a survival surgical approach to demonstrate establishment of a novel orthotopic syngeneic mouse model for the study of metastasis in HCC3. Their work established a &#8220;patient-like&#8221; mouse model that recapitulates features of aggressive and metastatic HCCs3. They further demonstrated that this mouse model system can be used to study the biology of circulating tumor cells, and that this holds potential for gaining novel insights into the mechanisms of cancer metastasis3.
The aim of this paper is to describe in detail the methodology used in establishing this &#8220;patient-like&#8221; orthotopic syngeneic mouse model of HCC metastasis3. The methodology of how to implant BNL 1ME A.7R.1 mouse HCC cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c3 using survival surgery will be described. Unlike other mouse xenograft tumor models where human tumor cells are implanted into immune deficient mice, this system is syngeneic and is, therefore, suitable for studying the role of the immune system in tumor metastasis3,5,8. This approach will likely gain widespread use for studying the mechanisms of metastasis in solid organ cancers.

<table border="0">
	<tbody>
		<tr>
			<td>Micro Dissecting Tweezer&#160;</td>
			<td>Roboz</td>
			<td>(RS-5040)</td>
			<td>Tip .20 x .12mm</td>
		</tr>
		<tr>
			<td>Graefe Micro Dissecting Forceps, serrated curved tip</td>
			<td>Roboz</td>
			<td>(RS-5111)</td>
			<td>1 X 2 teeth, curved tip width 0.6mm</td>
		</tr>
		<tr>
			<td>Micro Dissecting Retractor-Agricola (3 by 3 prongs)</td>
			<td>Roboz</td>
			<td>(RS-6501)</td>
			<td>Blunt 3 X 3 prongs, depth 4mm, spread 25mm</td>
		</tr>
		<tr>
			<td>Micro Dissecting Retractor-Goldstein (3 by 3 prongs)</td>
			<td>Roboz</td>
			<td>(RS-6503)</td>
			<td>Blunt, 3 X 3 prongs, depth 4mm, spread 19mm</td>
		</tr>
		<tr>
			<td>Jameson Caliper (Measures tumors)</td>
			<td>Roboz</td>
			<td>(RS-6466)</td>
			<td>80mm/3 inch scale, chrome plated&#160;</td>
		</tr>
		<tr>
			<td>Micro Dissecting Scissors, large ring sissors, straight and sharp</td>
			<td>Roboz</td>
			<td>(RS-5852)</td>
			<td>23mm blades, length 4 inches, flat shanks and large rings</td>
		</tr>
		<tr>
			<td>Scalpel with blades, for delicate dissecting procedures</td>
			<td>Roboz</td>
			<td>(RS-9861-36)</td>
			<td></td>
		</tr>
		<tr>
			<td>Scalpel handle&#160;</td>
			<td>Roboz</td>
			<td>(RS-9884)</td>
			<td>Solid</td>
		</tr>
		<tr>
			<td>Littauer Stitch Sissors</td>
			<td>Roboz</td>
			<td>(RS-7074)</td>
			<td>Length 4.5 inches</td>
		</tr>
		<tr>
			<td>Brown needle holder, for easy suture tying</td>
			<td>Roboz</td>
			<td>(RS-7960)</td>
			<td>Convex jaw, fine serrations</td>
		</tr>
		<tr>
			<td>Reflex 7MM wound clips with reflex 7 clip applier</td>
			<td>Roboz</td>
			<td>(RS-9262)</td>
			<td>safe, secure alternative method of wound closure</td>
		</tr>
		<tr>
			<td>Instrument tray and lid</td>
			<td>Roboz</td>
			<td>RT-1350S</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-clipper with detachable blade</td>
			<td>Roboz</td>
			<td>(RS-5903)</td>
			<td></td>
		</tr>
		<tr>
			<td>Germinator 500 (the Germ Terminator) Dry Sterilizer</td>
			<td>Roboz</td>
			<td>Ds-400, Ds-401, DS-501</td>
			<td>For fast decontamination of micro-dissecting instruments.&#160;&#160; Instruments decontaminate within 15 seconds.&#160;</td>
		</tr>
		<tr>
			<td>Microinjection needles</td>
			<td>VWR</td>
			<td>BD305125</td>
			<td>25G Needle</td>
		</tr>
	</tbody>
</table>

a "patient-like" orthotopic syngeneic mouse model of hepatocellular carcinoma metastasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of cancer metastasis are still unclear. Animal models are essential for elucidating the mechanisms and for evaluating novel strategies for the treatment of metastatic cancers. Here, an in-depth description of a &#8220;patient-like&#8221; orthotopic syngeneic mouse model for exploring the mechanisms of metastasis of solid organ tumors is provided. The survival surgical implantation of BNL 1ME A.7R.1 mouse hepatocellular carcinoma cells directly into the liver (the organ of origin) of the inbred wild-type immune competent laboratory mouse strain, BALB/c is described. The success and reproducibility of this methodology recommends it for widespread use in elucidating the biological mechanisms of solid organ cancer metastasis.

Los hígados de ratones Balb / c se implantaron con 5 × 10 6 células de HCC ratón BNL 1ME A.7R.1. La evidencia clínica de desarrollo de HCC fue observable de los 63 días posteriores a la cirugía. En consecuencia, los ratones fueron sacrificados humanitariamente. La necropsia se realizó en los ratones sacrificados. Los pulmones y el hígado fueron resecados y un examen cuidadoso bruto se realizó para identificar tumores macroscópicos. En un ratón, se observó un tumor superficial en la superficie del...

Watch this Scientific Journal Video about A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis at JoVE.com

A &quot;Patient-Like&quot; Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

La diseminación metastásica del cáncer es la principal causa de muerte por cáncer. Proporcionamos una descripción en profundidad de nuestra metodología de la cirugía de supervivencia para el establecimiento de un sistema de modelo de ratón singénico &quot;-paciente como&quot; ortotópico para el estudio de los mecanismos de la metástasis en tumores de órganos sólidos.

Un &quot;paciente-Like&quot; ortotópico Singénico modelo de ratón de carcinoma hepatocelular Metástasis

The majority of cancer-related deaths are caused by the metastasis of the cancer rather than the primary tumor itself. Yet, the underlying mechanisms of ...

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A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

12.1K vistas.  Hunter College of the City University of New York.  La diseminación metastásica del cáncer es la principal causa de muerte por cáncer. Proporcionamos una descripción en profundidad de nuestra metodología de la cirugía de supervivencia para el establecimiento de un sistema de modelo de ratón singénico "-paciente como" ortotópico para el estudio de los mecanismos de la metástasis en tumores de órganos sólidos. 

Video: A "Patient-Like" Orthotopic Syngeneic Mouse Model of Hepatocellular Carcinoma Metastasis

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

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 ...

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 ...

An Orthotopic Mouse Model of Anaplastic Thyroid Carcinoma

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer cells into immunodeficient mice. Subcutaneous xenograft models have been valuable for preclinical screening and evaluation of new therapeutic treatments. There are a number of advantages to using a subcutaneous model; 1) rapid, 2) reproducible, and 3) tumor establishment, growth, and response to therapeutic agents may be monitored by visual inspection. However, substantial evidence has shed light on the short-comings of subcutaneous xenograft models1-3. For instance, medicinal treatments demonstrating curative properties in subcutaneous xenograft models often have no notable impact on the human disease. The microenvironment of the site of xenographic transplantation or injection lies at the heart of this dissimilarity.
Orthotopic tumor xenograft models provide a more biologically relevant context in which to study the disease. The advantages of implanting diseased cells or tissue into their anatomical origin equivalent within a host animal includes a suitable site for tumor-host interactions, development of disease-related metastases and the ability to examine site-specific influence on investigational therapeutic remedies. Therefore, orthotopic xenograft models harbor far more clinical value because they closely reproduce human disease. For these reasons, a number of groups have taken advantage of an orthotopic thyroid cancer model as a research tool4-7.
Here, we describe an approach that establishes an orthotopic model for the study of anaplastic thyroid carcinoma (ATC), which is highly invasive, resists treatment, and is virtually fatal in all diagnosed patients. Cultured ATC cells are prepared as a dissociated cellular suspension in a solution containing a basement membrane matrix. A small volume is slowly injected into the right thyroid gland. Overall appearance and health of the mice are monitored to ensure minimal post-operative complications and to gauge pathological penetrance of the cancer. Mice are sacrificed at 4 weeks, and tissue is collected for histological analysis. Animals may be taken at later time-points to examine more advance progression of the disease. Production of this orthotopic mouse model establishes a platform that accomplishes two objectives: 1) further our understanding of ATC pathology, and 2) screen current and future therapeutic agents for efficacy in combating ATC.

Generation of an orthotopic mouse model of anaplastic thyroid carcinoma is described here. This technique employs surgical placement of human anaplastic thyroid cancer cells into the thyroid of immunodeficient mice, thus creating a more clinically relevant setting to study disease progression as well as screen innovative therapeutic interventions.

Un modelo de ratón ortotópico de carcinoma de tiroides anaplásico

Several types of animal models of human thyroid carcinomas have been established, including subcutaneous xenograft and orthotopic implantation of cancer ...

Generation of Subcutaneous and Intrahepatic Human Hepatocellular Carcinoma Xenografts in Immunodeficient Mice

In vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate the human disease provide a valuable platform for research into disease pathophysiology and for the preclinical evaluation of novel therapies. We present a variety of methods to generate subcutaneous or orthotopic human HCC xenografts in immunodeficient mice that could be utilized in a variety of research applications. With a focus on the use of primary tumor tissue from patients undergoing surgical resection as a starting point, we describe the preparation of cell suspensions or tumor fragments for xenografting. We describe specific techniques to xenograft these tissues i) subcutaneously; or ii) intrahepatically, either by direct implantation of tumor cells or fragments into the liver, or indirectly by injection of cells into the mouse spleen. We also describe the use of partial resection of the native mouse liver at the time of xenografting as a strategy to induce a state of active liver regeneration in the recipient mouse that may facilitate the intrahepatic engraftment of primary human tumor cells. The expected results of these techniques are illustrated. The protocols described have been validated using primary human HCC samples and xenografts, which typically perform less robustly than the well-established human HCC cell lines that are widely used and frequently cited in the literature. In comparison with cell lines, we discuss factors which may contribute to the relatively low chance of primary HCC engraftment in xenotransplantation models and comment on technical issues that may influence the kinetics of xenograft growth. We also suggest methods that should be applied to ensure that xenografts obtained accurately resemble parent HCC tissues.

Human tumor xenografts in immunodeficient mice are valuable tools to study cancer biology. Specific protocols to generate subcutaneous and intrahepatic xenografts from human hepatocellular carcinoma cells or tumor fragments are described. Liver regeneration induced by partial hepatectomy in recipient mice is presented as a strategy to facilitate intrahepatic engraftment.

Generación de subcutánea e intrahepática hepatocelular carcinoma humano xenoinjertos en ratones inmunodeficientes

In  vivo experimental models of hepatocellular carcinoma (HCC) that recapitulate  the human disease provide a valuable platform for research into disease  ...

An Orthotopic Murine Model of Human Prostate Cancer Metastasis

Our laboratory has developed a novel orthotopic implantation model of human prostate cancer (PCa). As PCa death is not due to the primary tumor, but rather the formation of distinct metastasis, the ability to effectively model this progression pre-clinically is of high value. In this model, cells are directly implanted into the ventral lobe of the prostate in Balb/c athymic mice, and allowed to progress for 4-6 weeks. At experiment termination, several distinct endpoints can be measured, such as size and molecular characterization of the primary tumor, the presence and quantification of circulating tumor cells in the blood and bone marrow, and formation of metastasis to the lung. In addition to a variety of endpoints, this model provides a picture of a cells ability to invade and escape the primary organ, enter and survive in the circulatory system, and implant and grow in a secondary site. This model has been used effectively to measure metastatic response to both changes in protein expression as well as to response to small molecule therapeutics, in a short turnaround time.

This orthotopic model of human prostate cancer allows for quantification of tumor size, circulating tumor cells, and formation of distinct metastasis to the lung. As cells must escape the primary organ, enter the blood stream, and implant into a secondary site, this model effectively recapitulates the scenario in humans.

Un modelo murino de cáncer de próstata humano Metástasis ortotópico

Our laboratory has developed a novel orthotopic implantation model of  human prostate cancer (PCa). As PCa death is not due to the primary tumor, but  ...

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 ...

The In Ovo Chick Chorioallantoic Membrane (CAM) Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and highly vascularized, making it an ideal system for tumor implantation. Furthermore, the CAM contains extracellular matrix proteins such as fibronectin, laminin, collagen, integrin alpha(v)beta3, and MMP-2, making it an attractive model to study tumor invasion and metastasis. Scientists have long taken advantage of the physiology of the CAM by using it as a model of angiogenesis. More recently, the CAM assay has been modified to work as an in vivo xenograft model system for various cancers that bridges the gap between basic in vitro work and more complex animal cancer models. The CAM assay allows for the study of tumor growth, anti-tumor therapies, and pro-tumor molecular pathways in a biologically relevant system that is both cost- and time-effective. Here, we describe the development of CAM xenograft model of hepatocellular carcinoma (HCC) with embryonic survival rates of up to 93% and reliable tumor take leading to growth of three-dimensional, vascularized tumors.

The In Ovo Chick Chorioallantoic Membrane CAM Assay as an Efficient Xenograft Model of Hepatocellular Carcinoma

The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.

los<em&gt; En Ovo</em&gt; Chick corioalantoidea de membrana (CAM) Ensayo como un modelo de xenoinjerto eficiente de carcinoma hepatocelular

The chick chorioallantoic membrane (CAM) begins to develop by day 7 after fertilization and matures by day 12. The CAM is naturally immunodeficient and ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.

Dynamic Contrast mejorada de imagen de resonancia magnética de un modelo ortotópico de cáncer pancreático del ratón

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

Ortotópico Implantación y Seguimiento de la célula inmune periférica en el modelo II-45 Singénico Rata Mesotelioma

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...