La investigación reportada en esta publicación fue apoyada por el Centro Nacional para el Avance de las Ciencias Traslacionales bajo el número de premio UL1TR003096 (MDE), National Heart, Lung, and Blood Institute Predoctoral Fellowship in Lung Diseases Training Program 5T32HL134640 (MLD).

Todos los métodos descritos aquí han sido aprobados por el Comité Institucional de Cuidado y Uso de Animales (IACUC) de la Universidad de Alabama en Birmingham.
1. Protocolo experimental
<ol><li>Sacrifique el ratón utilizando un método IACUC aprobado. Aquí, usamos la dislocación cervical de un ratón anestesiado con un 5% de isoflurano. Utilice un ratón adecuado para el estudio; aquí, usamos un ratón FVB de 8 semanas de edad</li>
	<li>Con tijeras quirúrgicas, realice una incisión horizontal de 3,5-5 mm en el centro del abdomen inferior. A continuación, inserte tijeras quirúrgicas en el pequeño agujero creado a partir de la incisión y corte verticalmente por la línea media central hasta justo debajo del cuello del ratón.</li>
	<li>Tire de la piel hacia atrás con los dedos e inspeccione los ganglios linfáticos axilares.</li>
	<li>Con tijeras quirúrgicas, realice una incisión lateral de 3,5 mm para abrir la cavidad abdominal y, a continuación, corte en la dirección anterior hasta la parte inferior del tórax. Inspeccione los órganos de la cavidad abdominal: hígado, bazo, riñones, etc.</li>
	<li>Con el plano de las tijeras quirúrgicas, o usando fórceps, mueva el hígado para exponer el diafragma. Inspeccione el diafragma en busca de crecimiento tumoral o metástasis. A continuación, recorte suavemente el diafragma en el lado derecho del operador, lo que le permite expandirse. Corte suavemente el diafragma de derecha a izquierda para exponer la cavidad torácica y los pulmones. Tenga cuidado de no cortar los pulmones.</li>
	<li>Corte a través del extremo lateral de la caja torácica izquierda (a la derecha del operador) para inspeccionar el lóbulo izquierdo de los pulmones.</li>
	<li>Mueva suavemente los lóbulos derecho del pulmón fuera del camino y corte el extremo lateral de la caja torácica derecha y retire la caja torácica. NOTA: La extracción de la caja torácica es opcional, aunque la eliminación permite una visión más clara de la inflación pulmonar posterior.<ol>
<li>Si se requiere tejido pulmonar fresco o congelado, utilice fórceps hemostat para sujetar el bronco del lóbulo izquierdo y resecar el pulmón izquierdo con tijeras quirúrgicas antes de la perfusión.</li>
	</ol>
</li>
	<li>Usando los fórceps para levantar el tejido que cubre la tráquea, cortar cualquier exceso de tejido. A continuación, corte suavemente el tejido delgado que recubre la tráquea para exponer las vías respiratorias.</li>
	<li>Corte la arteria renal con tijeras quirúrgicas.</li>
	<li>Para perfundir los pulmones, utilice una jeringa de 3 ml con una aguja de 22 G para inyectar 1 pbs con 10 U/ml de heparina en el ventrículo derecho del corazón. Perfundir lentamente los pulmones a aproximadamente 300 μL/s con PBS/heparina. Los pulmones con frecuencia se vuelven blancos. 2.5 mL de PBS se utilizan generalmente en este paso.</li>
	<li>Para la inflación pulmonar, utilice una jeringa de 3 ml con una aguja de 22 G, esta vez mantenida paralela a la tráquea. Inserte la aguja en la tráquea e inyecte un 10% de formalina con una velocidad de flujo no superior a ~200 μL/s hasta que los pulmones se hayan inflado por completo. Una vez inflados los pulmones, la formalina se reflujo fuera de la tráquea. Sujete la aguja en su lugar durante unos segundos más y luego retíriga.<ol>
<li>(Opcional) Antes de retirar la inflación de agujas y pulmones, utilice hilo de sutura para atar la tráquea. Para lograr esto, utilice 4 pulgadas de hilo de sutura que sostenga el punto de la rosca con un pequeño par de fórceps. Coloque el hilo en el lado dorsal de la tráquea y tire a través para hacer un bucle alrededor de la aguja. A continuación, haga un nudo de mano alrededor de la aguja. Tire del nudo apretado, retire la aguja de la tráquea, cierre el nudo.</li>
	</ol>
</li>
	<li>Utilice fórceps para levantar el corazón, inserte tijeras quirúrgicas directamente detrás de los pulmones y corte el tejido conectivo mientras levanta el corazón para resecar los pulmones.</li>
	<li>Corta el corazón para extraerlo de los pulmones.</li>
	<li>Coloque los pulmones un casete etiquetado con el ID del ratón o la identificación del estudio. Coloque el cassette en formalina tamponada al 10% y fije para 24-48 h. Los pulmones se pueden dejar en fijación durante más de un año si se desea.</li>
	<li>Transfiera el casete que contiene los pulmones al 70% de etanol y proceda al procesamiento para la histología.</li>
</ol>

<ol>
	<li>Hsia, C. C., Hyde, D. M., Ochs, M., Weibel, E. R., Structure, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+official+research+policy+statement+of+the+American+Thoracic+Society/European+Respiratory+Society:+standards+for+quantitative+assessment+of+lung+structure.">An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure.</a> American Journal of Respiratory and Critical Care Medicine. 181 (4), 394-418 (2010).</li><li>Vasilescu, D. M., Knudsen, L., Ochs, M., Weibel, E. R., Hoffman, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+murine+lung+preparation+for+detailed+structural+evaluation+via+micro-computed+tomography.">Optimized murine lung preparation for detailed structural evaluation via micro-computed tomography.</a> Journal of Applied Physiology. 112 (1), 159-166 (2012).</li><li>Blumler, P., Acosta, R. H., Thomas-Semm, A., Reuss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lung+fixation+for+the+preservation+of+air+spaces.">Lung fixation for the preservation of air spaces.</a> Experimental Lung Research. 30 (1), 73-82 (2004).</li><li>Oldmixon, E. H., Suzuki, S., Butler, J. P., Hoppin, F. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perfusion+dehydration+fixes+elastin+and+preserves+lung+air-space+dimensions.">Perfusion dehydration fixes elastin and preserves lung air-space dimensions.</a> Journal of Applied Physiology. 58 (1), 105-113 (1985).</li><li>Braber, S., Verheijden, K. A., Henricks, P. A., Kraneveld, A. D., Folkerts, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+fixation+methods+on+lung+morphology+in+a+murine+model+of+emphysema.">A comparison of fixation methods on lung morphology in a murine model of emphysema.</a> American Journal of Physiology-Lung Cellular and Molecular Physiology. 299 (6), 843-851 (2010).</li><li>Renne, R., 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=Recommendation+of+optimal+method+for+formalin+fixation+of+rodent+lungs+in+routine+toxicology+studies.">Recommendation of optimal method for formalin fixation of rodent lungs in routine toxicology studies.</a> Toxicologic Pathology. 29 (5), 587-589 (2001).</li><li>Limjunyawong, N., Mock, J., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instillation+and+Fixation+Methods+Useful+in+Mouse+Lung+Cancer+Research.">Instillation and Fixation Methods Useful in Mouse Lung Cancer Research.</a> Journal of Visualized Experiments. (102), e52964 (2015).</li><li>Lum, H., Mitzner, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+10%+formalin+fixation+on+fixed+lung+volume+and+lung+tissue+shrinkage.+A+comparison+of+eleven+laboratory+species.">Effects of 10% formalin fixation on fixed lung volume and lung tissue shrinkage. A comparison of eleven laboratory species.</a> American Review of Respiratory Disease. 132 (5), 1078-1083 (1985).</li><li>Edmonds, M. D., 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=MicroRNA-31+initiates+lung+tumorigenesis+and+promotes+mutant+KRAS-driven+lung+cancer.">MicroRNA-31 initiates lung tumorigenesis and promotes mutant KRAS-driven lung cancer.</a> Journal of Clinical Investigation. 126 (1), 349-364 (2016).</li><li>Zhao, K., 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=Wogonin+suppresses+melanoma+cell+B16-F10+invasion+and+migration+by+inhibiting+Ras-medicated+pathways.">Wogonin suppresses melanoma cell B16-F10 invasion and migration by inhibiting Ras-medicated pathways.</a> PLoS One. 9 (9), 106458 (2014).</li></ol>

Los autores no tienen nada que revelar.

El procedimiento descrito anteriormente para la perfusión, inflación y fijación de los pulmones de ratón es ideal para una preparación rápida y eficiente de los pulmones de ratón para la histología y el análisis patológico de tumores pulmonares. El procedimiento no requiere ningún equipo especial y se puede realizar en menos de 6 minutos por ratón. El procedimiento no requiere un volumen fijo para la inflación ni una presión fluida constante. Debido a que este procedimiento no está estandarizado, no se rec...

Existen una variedad de modelos de ratón de oncogenesis pulmonar y metástasis del cáncer hasta el pulmón que van desde GEMMs complejos hasta modelos inducidos por carcinógenos hasta modelos singénicos y xenógrafos, donde las células cancerosas se inyectan a través de intracardiaco, intratorácico, la vena de la cola u otros métodos para establecer tumores dentro del pulmón. Todos estos modelos comparten la necesidad común de evaluación histológica de histología pulmonar y patología. Por lo tanto, es necesario tener un método robusto pero rápido para realizar necropsias de ratones mientras se perfunden los pulmones para eliminar el exceso de sangre, e inflar y fijar los pulmones para visualizar claramente la arquitectura pulmonar. La velocidad es un componente crítico de este procedimiento, ya que puede ser necesario recoger los pulmones de docenas de ratones en un solo punto de tiempo. Este procedimiento se puede realizar en menos de 6 minutos por ratón.
Si bien este procedimiento es más que suficiente para evaluar la histología tumoral, no se recomienda para aquellos que deseen realizar estereología o mediciones morfométricas de los pulmones. Estas mediciones requieren que la inflación pulmonar se normalice, al igual que el cálculo de la superficie absoluta del pulmón, el volumen absoluto y el tamaño y el número1del alveolar. Este método tampoco es óptimo para algunos enfoques de imágenes. Por ejemplo, la toma de imágenes de los pulmones a través de μCT para el análisis morfométrico ex vivo requiere que los pulmones permanezcan llenos de aire2. Cuando la preservación de los espacios y dimensiones del aire son la principal preocupación, se recomienda fijar los pulmones mediante técnicas de deshidratación por perfusión3,4. Una de las mayores preocupaciones de este modelo es el potencial de rupturing de las paredes alveolares, disminuyendo su uso en estudios de enfisema; sin embargo, el procedimiento recomendado para la fijación de pulmones para el estudio del enfisema sigue siendo bastante similar, ya que se recomienda fijar los pulmones ya sea por instilación intratraqueal de 10% de formalina (similar al protocolo descrito aquí) bajo presión constante del líquido o por fijación in situ5.
La ventaja del procedimiento descrito aquí es que no requiere presión constante del líquido, sino que infla los pulmones hasta que se han expandido por completo, disminuyendo así el tiempo necesario para el procedimiento. El procedimiento aquí descrito se asemeja mucho a los métodos recomendados por un armamentoarium de la Sociedad de Patología Toxicológica, donde se formó un subcomité para recomendar los mejores métodos de fijación pulmonar para estudios toxicológicos. La mayoría de los científicos de este subcomité recomendaron arreglar los pulmones mediante instilación intratraqueal con una jeringa, aunque había diferentes recomendaciones sobre el momento en que el pulmón quedó en el fijador6. Por lo tanto, mientras que existe una variedad de métodos de inflación pulmonar y fijación, el método descrito aquí se propone para ser el método óptimo para inflar rápidamente y fijar los pulmones para la evaluación histológica tumoral aguas abajo.

<table>
	<tbody>
		<tr>
			<td>10% buffered formalin</td>
			<td>Fisher</td>
			<td>23-245685</td>
			<td></td>
		</tr>
		<tr>
			<td>22 G Needle</td>
			<td>BD</td>
			<td>305155</td>
			<td></td>
		</tr>
		<tr>
			<td>3 mL syringe</td>
			<td>BD</td>
			<td>309656</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Decon</td>
			<td>2405</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Harvard Apparatus</td>
			<td>72-8595</td>
			<td></td>
		</tr>
		<tr>
			<td>Heparin</td>
			<td>Fisher</td>
			<td>H19</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline (PBS)</td>
			<td>Corning</td>
			<td>21-030-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical scissors</td>
			<td>Harvard Apparatus</td>
			<td>72-8428</td>
			<td></td>
		</tr>
	</tbody>
</table>

perfusion and inflation of the mouse lung for tumor histology

La capacidad de evaluar la histología pulmonar es fundamental para los campos de la investigación del cáncer de pulmón y la metástasis del cáncer. Es igualmente importante realizar necropsias rápida y eficientemente a partir de estudios sin sacrificar la calidad de los tejidos adquiridos. El objetivo de este protocolo es presentar un método para perfundir, inflar y fijar rápidamente los pulmones del ratón para el análisis histológico posterior. Este método no estandariza la inflación pulmonar; por lo tanto, no requiere ningún procedimiento o equipo especial y en su lugar simplemente inculca fijación directamente a través de la tráquea después de perfusión a través del corazón. Esto permite una estimación suficiente del tamaño del tumor, histología y puntuación. Esto también permite la recolección de tejido congelado antes de la fijación del tejido pulmonar. Este método es limitado en el sentido de que no permite la cuantificación morfométrica posterior del pulmón; sin embargo, es más que suficiente para el análisis de tumores pulmonares a partir de modelos de ratón genéticamente modificados (GEMMs), modelos singénicos, así como estudios de tumores y metástasis xenógrafos.

El protocolo anterior permite una perfusión rápida, inflación y fijación de los pulmones del ratón. Las cifras que se muestran a continuación representan la importancia de cada paso. La Figura 1 representa los pulmones manchados de H&E que han sido perfundidos con PBS y pulmones en los que se ha saltado el paso de perfusión o los pulmones no se perfundieron correctamente. Como se muestra, el exceso de sangre en los pulmones mal impregnados crea una histología menos que ideal y puede ...

Watch this Scientific Journal Video about Perfusion and Inflation of the Mouse Lung for Tumor Histology at JoVE.com

Perfusion and Inflation of the Mouse Lung for Tumor Histology

El propósito de este método es presentar un método simple y eficiente para la perfusión, inflación y fijación de los pulmones del ratón para el examen de la patología pulmonar y la evaluación de metástasis en el pulmón.

Perfusión e inflación del pulmón del ratón para la histología tumoral

The ability to evaluate lung histology is critical for the fields of lung cancer research and cancer metastasis. It is equally important to perform ...

perfusion-and-inflation-of-the-mouse-lung-for-tumor-histology

Research

JoVE Journal

Cancer Research

17.5K vistas.  University of Alabama at Birmingham. El propósito de este método es presentar un método simple y eficiente para la perfusión, inflación y fijación de los pulmones del ratón para el examen de la patología pulmonar y la evaluación de metástasis en el pulmón.

Video: Perfusion and Inflation of the Mouse Lung for Tumor Histology

Lung Tumor Cell Recruitment Assay

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we demonstrate a simple assay to evaluate tumor cell extravasation or micrometastasis. In this assay, tumor cells were injected through the tail vein, and after a short period, the lungs were dissected and digested to count the accumulated labeled tumor cells. This assay skips the initial step of primary tumor invasion into the blood vessel and facilitates the study of events in the distant organ where tumor metastasis occurs. The number of cells injected into the blood vessel can be optimized to observe a limited number of metastases. It has been reported that stromal cells in the distant organ contribute to metastasis. Thus, this assay could be a useful tool to explore potential therapeutic drugs or devices for prevention of tumor metastasis.

This manuscript describes a method to quantify tumor cell accumulation in the lungs in an animal model of tumor metastasis.

Ensayo de reclutamiento de células de pulmón Tumor

To investigate the molecular mechanisms governing tumor metastasis, various assays using the mouse as a model animal have been proposed. Here, we ...

Investigación

Investigación sobre el cáncer

The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and activation of lipolysis and proteolysis systems. Cancer patients with cachexia benefit less from anti-neoplastic therapies and show increased mortality1. Several animal models have been established in order to investigate the molecular causes responsible for body and muscle wasting as a result of tumor growth. Here, we describe methodologies pertaining to a well-characterized model of cancer cachexia: mice bearing the C26 carcinoma2-4. Although this model is heavily used in cachexia research, different approaches make reproducibility a potential issue. The growth of the C26 tumor causes a marked and progressive loss of body and skeletal muscle mass, accompanied by reduced muscle cross-sectional area and muscle strength3-5. Adipose tissue is also lost. Wasting is coincident with elevated circulating levels of pro-inflammatory cytokines, particularly Interleukin-6 (IL-6)3, which is directly, although not entirely, responsible for C26 cachexia. It is well-accepted that a primary mechanism by which the C26 tumor induces muscle tissue depletion is the activation of skeletal muscle proteolytic systems. Thus, expression of muscle-specific ubiquitin ligases, such as atrogin-1/MAFbx and MuRF-1, represent an accepted method for the evaluation of the ongoing muscle catabolism2. Here, we present how to execute this model in a reproducible manner and how to excise several tissues and organs (the liver, spleen, and heart), as well as fat and skeletal muscles (the gastrocnemius, tibialis anterior, and quadriceps). We also provide useful protocols that describe how to perform muscle freezing, sectioning, and fiber size quantification.

Mice bearing the Colon-26 (C26) carcinoma represent a classical model of cancer cachexia. Progressive muscle wasting occurs in association with tumor growth, over-expression of muscle-specific ubiquitin ligases, and reductions in muscle cross-sectional area. Fat loss is also observed. Cachexia is studied in a time-dependent manner with increasing severity of wasting.

El Colon-26 Carcinoma portadores de tumores de ratón como modelo para el estudio del cáncer de caquexia

Cancer cachexia is the progressive loss of skeletal muscle mass and adipose tissue, negative nitrogen balance, anorexia, fatigue, inflammation, and ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Destino celular genoma pre-enriquecimiento y toda amplificación sola célula caracterización aguas abajo

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

The Establishment of a Lung Colonization Assay for Circulating Tumor Cell Visualization in Lung Tissues

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by CTCs critically contributes to early lung metastatic processes, has been vigorously investigated. As such, animal models are the only approach that captures the full systemic process of metastasis. Given that problems occur in previous experimental designs for examining the contributions of CTCs to blood vessel extravasation, we established an in vivo lung colonization assay in which a long-term-fluorescence cell-tracer, carboxyfluorescein succinimidyl ester (CFSE), was used to label suspended tumor cells and lung perfusion was performed to clear non-specifically trapped CTCs prior to lung removal, confocal imaging, and quantification. Polymeric fibronectin (polyFN) assembled on CTC surfaces has been found to mediate lung colonization in the final establishment of metastatic tumor tissues. Here, to specifically test the requirement of polyFN assembly on CTCs for lung colonization and extravasation, we performed short term lung colonization assays in which suspended Lewis lung carcinoma cells (LLCs) stably expressing FN-shRNA (shFN) or scramble-shRNA (shScr) and pre-labeled with 20 &#956;M of CFSE were intravenously inoculated into C57BL/6 mice. We successfully demonstrated that the abilities of shFN LLC cells to colonize the mouse lungs were significantly diminished in comparison to shScr LLC cells. Therefore, this short-term methodology may be widely applied to specifically demonstrate the ability of CTCs within the circulation to colonize the lungs.

An animal model is needed to decipher the role of circulating tumor cells (CTCs) in promoting lung colonization during cancer metastasis. Here, we established and successfully performed an in vivo assay to specifically test the requirement of polymeric fibronectin (polyFN) assembly on CTCs for lung colonization.

El establecimiento de un ensayo de colonización pulmonar para la visualización de células tumorales en tejidos pulmonares de la circulación

Metastasis is the major cause of cancer death. The role of circulating tumor cells (CTCs) in promoting cancer metastasis, in which lung colonization by ...

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography (18F-FDG PET) imaging. Co-mutations in the KRAS proto-oncogene and the LKB1 tumor suppressor gene are frequent events in lung cancer that drive hypermetabolic, glycolytic tumor growth. A critical pathway regulating the growth and metabolism of these tumors is the mechanistic target of the rapamycin (mTOR) pathway, which can be effectively targeted using selective catalytic mTOR kinase inhibitors. The mTOR inhibitor MLN0128 suppresses glycolysis in mice bearing tumors with Kras and Lkb1 co-mutations, referred to as KL mice. The therapy response in KL mice is first measured by 18F-FDG PET and computed tomography (CT) imaging before and after the delivery of MLN0128. By utilizing 18F-FDG PET/CT, researchers are able to measure dynamic changes in the glucose metabolism in genetically engineered mouse models (GEMMs) of lung cancer following a therapeutic intervention with targeted therapies. This is followed by ex vivo autoradiography and a quantitative immunohistochemical (qIHC) analysis using morphometric software. The use of qIHC enables the detection and quantification of distinct changes in the biomarker profiles following treatment as well as the characterization of distinct tumor pathologies. The coupling of PET imaging to quantitative histology is an effective strategy to identify metabolic and therapeutic responses in vivo in mouse models of disease.

Utilizing 18F-FDG PET/CT Imaging and Quantitative Histology to Measure Dynamic Changes in the Glucose Metabolism in Mouse Models of Lung Cancer

In this protocol, we describe how to utilize [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography (18F-FDG PET/CT) imaging to measure the tumor metabolic response to the targeted therapy MLN0128 in a Kras/Lkb1 mutant mouse model of lung cancer and coupled imaging with high resolution ex vivo autoradiography and quantitative histology.

Utilizando 18F-FDG PET/CT la proyección de imagen e histología cuantitativa para medir cambios dinámicos en el metabolismo de la glucosa en modelos de ratón de cáncer de pulmón

A hallmark of advanced tumors is a switch to aerobic glycolysis that is readily measured by [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography ...

Acellular and Cellular Lung Model to Study Tumor Metastasis

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor cells with a natural matrix and continual flow of nutrients, as well as a model that shows the interaction of tumor cells with normal cellular components and a natural matrix. The acellular ex vivo lung model is created by isolating a rat heart-lung block and removing all the cells using the decellularization process. The right main bronchus is tied off and tumor cells are placed in the trachea by a syringe. The cells move and populate the left lung. The lung is then placed in a bioreactor where the pulmonary artery receives a continual flow of media in a closed circuit. The tumor grown on the left lung is the primary tumor. The tumor cells that are isolated in the circulating media are circulating tumor cells and the tumor cells in the right lung are metastatic lesions. The cellular ex vivo lung model is created by skipping the decellularization process. Each model can be used to answer different research questions.

Here, we present a protocol for an ex vivo lung cancer model that mimics the steps of tumor progression and helps to isolate a primary tumor, circulating tumor cells, and metastatic lesions.

Modelo de pulmón acelular y celular para el estudio de metástasis de Tumor

It is difficult to isolate tumor cells at different points of tumor progression. We created an ex vivo lung model that can show the interaction of tumor ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

Digital de la reacción en cadena de polimerasa para la variación genética en un paciente de la poliposis adenomatosa familiar esporádico con el formato de Chip en tubo

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

Detección de la progresión del tumor pulmonar en ratones por imágenes por ultrasonido

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

Pathological Analysis of Lung Metastasis Following Lateral Tail-Vein Injection of Tumor Cells

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous metastasis models are available. Thus, the experimental metastasis model involving tail-vein injection of suitable cell lines is a mainstay of metastasis research. When cancer cells are injected into the lateral tail-vein, the lung is their preferred site of colonization. A potential limitation of this technique is the accurate quantification of the metastatic lung tumor burden. While some investigators count macrometastases of a pre-defined size and/or include micrometastases following sectioning of tissue, others determine the area of metastatic lesions relative to normal tissue area. Both of these quantification methods can be exceedingly difficult when the metastatic burden is high. Herein, we demonstrate an intravenous injection model of lung metastasis followed by an advanced method for quantifying metastatic tumor burden using image analysis software. This process allows for investigation of multiple end-point parameters, including average metastasis size, total number of metastases, and total metastasis area, to provide a comprehensive analysis. Furthermore, this method has been reviewed by a veterinary pathologist board-certified by the American College of Veterinary Pathologists (SEK) to ensure accuracy.

Intravenous injection of cancer cells is often used in metastasis research, but the metastatic tumor burden can be difficult to analyze. Herein, we demonstrate a tail-vein injection model of metastasis and include a novel approach to analyze the resulting metastatic lung tumor burden.

Análisis patológico de metástasis pulmonares después de la inyección lateral de células tumorales en la vena de la cola

Metastasis, the primary cause of morbidity and mortality for most cancer patients, can be challenging to model preclinically in mice. Few spontaneous ...

Generation of Zebrafish Larval Xenografts and Tumor Behavior Analysis

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly visualizing the response to anti-cancer therapies (chemo, radiotherapy, and biologicals), angiogenesis and metastasis with single cell resolution, places the zebrafish xenograft model as a top choice to develop preclinical studies.

The zebrafish larval xenograft assay presents several experimental advantages compared to other models, but probably the most striking is the reduction of size scale and consequently time. This reduction of scale allows single cell imaging, the use of a relatively low number of human cells (compatible with biopsies), medium-high-throughput drug screenings, but most importantly enables a significant reduction of the time of the assay. All these advantages make the zebrafish xenograft assay extremely attractive for future personalized medicine applications.

Many zebrafish xenograft protocols have been developed with a wide diversity of human tumors; however, a general and standardized protocol to efficiently generate zebrafish larval xenografts is still lacking. Here we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Here, we provide a step-by-step protocol, with tips to generate xenografts and guidelines for tumor behavior analysis, whole-mount immunofluorescence, and confocal imaging quantification.

Generación de Xenoinjertos Larvales de Pez Cebra y Análisis de Comportamiento Tumoral

Zebrafish larval xenografts are being widely used for cancer research to perform in vivo and real-time studies of human cancer. The possibility of rapidly ...