Name: the colon-26 carcinoma tumor-bearing mouse as a model for the study of cancer cachexia
Uploaded: 2016-11-30T14:00:00
Description: Watch this Scientific Journal Video about The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia at JoVE.com

We thank Richard Lieber and Shannon Bremner for their ImageJ macro and instructions. While at Thomas Jefferson University, this work was supported by the Pennsylvania Department of Health CURE Grant TJU No. 080-37038-AI0801. Subsequently, this study was supported by a grant to AB from the National Institutes of Health (R21CA190028), and by grants to TAZ from the National Institutes of Health (R01CA122596, R01CA194593), the IU Simon Cancer Center, the Lustgarten Foundation, the Lilly Foundation, Inc., and the IUPUI Pancreas Signature Center.

Declaración de Ética: Todos los estudios descritos fueron aprobados por los Comités de Cuidado y Uso de Animales Institucional de la Universidad Thomas Jefferson y la Escuela de Medicina de la Universidad de Indiana. 1. C26 crecimiento celular y Preparación <ol><li> Obtener células de cáncer colorrectal C26 (Universidad Estatal de Ohio Medical Center (OSUMC)) y preparar el medio de crecimiento completo (es decir, alta glucosa de Dulbecco Modificado de Eagle Medium (DMEM) que...

<ol>
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Especialmente en sus últimas etapas, el cáncer colorrectal se asocia con el desarrollo de la caquexia, que es responsable de los resultados y las reducciones en la calidad de vida del paciente más pobres. Muchos estudios se han centrado en el tratamiento de condiciones secundarias al cáncer; Sin embargo, a pesar de muchos esfuerzos en este sentido, aún no existe una terapia aprobada para la caquexia por cáncer 21. Por lo tanto, es imperativo que los modelos animales se asemejan a la patología humana ta...

La atrofia muscular es una complicación grave de diversas condiciones clínicas tales como el cáncer, sepsis, hígado, cirrosis, insuficiencia cardíaca y renal, enfermedad pulmonar obstructiva crónica, y el SIDA. En particular, el desgaste muscular es evidente en al menos 50% de los pacientes con cáncer de 1. La pérdida de músculo esquelético en el cáncer resulta de una mayor degradación de las proteínas debido a la sobre-activación de los sistemas proteolíticos del músculo esquelético y / o de disminución de la síntesis de proteínas 6. La lipólisis es también evidente, lo que lleva a la disminución de tejido adiposo. Clínicamente, la caquexia está asociada con una reducción de la calidad y la duración de la vida y se estima que es la causa de la muerte en 20 - 30% de los pacientes de cáncer de 7. El uso de modelos experimentales que se asemejan a la enfermedad humana tan estrechamente como sea posible sería beneficioso. Un modelo animal óptima se caracteriza por una alta reproducibilidad, así como por la interferencia limitada de diferentes terapias y los factores impredecibles dela dieta, el sexo y los antecedentes genéticos que se asocian generalmente con la condición clínica 8. Hasta el momento, la caquexia del cáncer se ha estudiado principalmente en modelos animales que se caracterizan por el trasplante de células de cáncer o de inyección de carcinógenos, aunque un nuevo método es el uso de ratones modificados genéticamente susceptibles al desarrollo de cáncer. Los ratones que llevan el carcinoma C26 (también referidos como de colon-26 y adenocarcinoma) representar un modelo bien caracterizado y utilizado ampliamente de la caquexia del cáncer de 2,5. El crecimiento de los resultados C26 tumorales en el cuerpo y la pérdida de peso muscular, principalmente mediante el aumento de la grasa y el catabolismo proteico 9. Generalmente, un peso del tumor 10% versus el peso corporal total se asoció con una reducción de 20-25% en peso del músculo esquelético y un mayor agotamiento de la grasa de 3,10. Hepatomegalia y esplenomegalia también se observan con el crecimiento tumoral, junto con la activación de la respuesta de fase aguda y la elevación de pro-inflalos niveles de citoquinas mmatory 3,11. Entre estos, se sabe que la IL-6 juega un papel fundamental en la mediación de pérdida de masa muscular en el modelo C26, a pesar de que esta citocina no es probablemente el único inductor de caquexia 12. Elevada IL-6 causa atrofia muscular a través de la activación de la vía de JAK / STAT3, y la inhibición de este factor de transcripción puede prevenir atrofia muscular 3,4. Durante pérdida de masa muscular inducida por C26, como en muchas condiciones de atrofia muscular, la masa muscular se pierde en gran medida a través de reducciones en el contenido de proteína del músculo a través de las fibras musculares, no a través de la muerte celular o pérdida de fibras 13. En C26 caquexia, un cambio hacia áreas de sección transversal más pequeñas se observa tanto en glucolíticas y oxidativas fibras 2. Esto también es coherente con una reducción de la fuerza muscular 5. Muchos grupos de todo el mundo han aprovechado el modelo C26 con el fin de descubrir nuevos mediadores de la pérdida de masa muscular o fármacos clínicamente relevantes para el cáncer cacHexia. Sin embargo, se ha informado de muchos procedimientos diferentes para el uso de este modelo, aumentando las preocupaciones acerca de la consistencia de los datos obtenidos y que presenta obstáculos al reproducibilidad en diferentes condiciones experimentales. Aquí mostramos un uso típico de este modelo para el estudio de la caquexia por cáncer que produce datos estandarizados y reproducibles.

<table border="0" cellpadding="0" cellspacing="0" width="661">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Cell culture Flasks</td>
			<td style="width:205px;">Falcon - Becton Dickinson</td>
			<td style="width:104px;">35-5001</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">DMEM</td>
			<td style="width:205px;">Cellgro</td>
			<td style="width:104px;">10-017-CV</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">FBS</td>
			<td style="width:205px;">Gibco</td>
			<td style="width:104px;">26140</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Streptomycin-Penicillin&#160;</td>
			<td style="width:205px;">Cellgro</td>
			<td style="width:104px;">30-002-CI</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">CD2F1 mice</td>
			<td>Harlan</td>
			<td>060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Anesthesia apparatus</td>
			<td style="width:205px;">EZ-Anesthesia</td>
			<td style="width:104px;">EZ-7000</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">2-Methyl Butane</td>
			<td style="width:205px;">Sigma-Aldrich</td>
			<td style="width:104px;">M32631</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">OCT</td>
			<td style="width:205px;">Tissue-Tek</td>
			<td style="width:104px;">4583</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Cryostat</td>
			<td style="width:205px;">Leica</td>
			<td style="width:104px;">CM1850</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Cork disks</td>
			<td>Electron Microscopy Sciences</td>
			<td>63305</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">Superfrost plus glass slides</td>
			<td>VWR</td>
			<td>48311-703</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">Anti-Laminin Rabbit polyclonal antibody</td>
			<td>Sigma-Aldrich</td>
			<td>L9393</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">Anti-Dystrophin Mouse Monoclonal antibody</td>
			<td>Vector Laboratories</td>
			<td>VP-D508</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">Alexa Flour 594 anti-mouse IgG</td>
			<td>Life Technologies</td>
			<td>A11062</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">Alexa Flour 594 anti-rabbit IgG</td>
			<td>Life Technologies</td>
			<td>A21211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hematoxylin</td>
			<td>Sigma-Aldrich</td>
			<td>GHS216</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eosin</td>
			<td>Sigma-Aldrich</td>
			<td>HT110332</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xylene</td>
			<td>Acros Organics</td>
			<td>422680025</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Cytoseal-XYL</td>
			<td style="width:205px;">Thermo</td>
			<td style="width:104px;">8312-4</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Microscope</td>
			<td style="width:205px;">Zeiss</td>
			<td style="width:104px;">Observer.Z1&#160;</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Bamboo Tablet</td>
			<td style="width:205px;">Wacom</td>
			<td style="width:104px;">CTH-661</td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Prism 7.0 for Mac OS X</td>
			<td style="width:205px;">GraphPad Software, Inc.</td>
			<td style="width:104px;"></td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:167px;">Excel for Mac 2011</td>
			<td style="width:205px;">Microsoft Corp.</td>
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			<td height="42" style="height:42px;width:167px;">Image J</td>
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			<td style="width:104px;">IJ1.46</td>
			<td style="width:185px;">http://rsbweb.nih.gov/ij/download.html</td>
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			<td height="22" style="height:22px;width:167px;">Microtainer</td>
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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.

C26 cinética de crecimiento de tumores muestran una fase de latencia para la primera 7-8 d después de la inyección, seguido por un crecimiento celular exponencial (4-5 d). La masa tumoral llega finalmente ~ 10% del peso corporal (alrededor de 2 g; Figura 1A-B). Durante la primera fase, el tumor puede ser localizado por palpación solamente y aparece como una pequeña protuberancia de la piel. En la segunda fase, el tumor se observa como una masa bajo ...

Watch this Scientific Journal Video about The Colon-26 Carcinoma Tumor-bearing Mouse as a Model for the Study of Cancer Cachexia at JoVE.com

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

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 ...

The overall goals of this protocol are to execute the Colon 26 In Vivo model in a reproducible manor, and to properly investigate the progressive muscle wasting and weakness that occur in association with tumor growth. This model can help answer key questions in a cancer cachexia field about tumor induced muscle loss, muscle weakness, increased muscle catabolism, and hyper inflammatory cytokine levels. The main advantage of this experimental model is that change is consistent with a cachectic phenotype can be easily started in a time dependent manor as the wasting severity increases.Demonstrating these procedures will be Rafael Barreto, a technician from my lab, and Joseph Rupert, a PhD student from Teresa Zimmer's group. To implant the C 26 tumors use a sterile, one millimeter insulin syringe, equipped with a 26 gauge needle to quickly inject one times 10 to the sixth cells subcutaneously and interscapularly into the fat pads of individual, adult, CD two F one mice. Return the animals to their cage after tumor injection, with monitoring.Checking the mice, and recording their body weights daily. When the body weight loss in the tumor host, versus the control animals, reaches five, 10, or 15%compared to their initial body weights, draw one to one point five milliliters of blood by cardiac puncture, and transfer the individual blood samples into empty 18 milligram potassium EDTA tubes. Place the tubes on ice until all of the blood has been collected.Then, centrifuge the samples, collecting the plasma and serum at the end of the centrifugation. To avoid further contamination, spray 70%ethanol over the entire body. Then place the animal on a dissection bed in the supine position, and attach one hind limb to an elevated burette clamp to extend the limb vertically.Using Dumont forceps, grasp the superficial skin of the hind limb and use curved fine scissors to gently remove the skin and fascia, exposing the underlying muscle bellies of the lower limb. Identify the gastrocnemius muscle on the posterior lower limb by its origin at the lateral and medial condyles on the posterior femur, and its insertion at the calcaneus, vial the calcaneal tendon. Use the forceps to grasp the distal end of the gastrocnemius and pull the muscle belly toward it's origin.Then, use scissors to cut the muscle at its origin, as close as possible to the femur and transfer the muscle to a pre-weighed dish. Weigh the dish immediately, and transfer the muscle to a pre-labeled cryo tube. Next, identify the tibialis anterior muscle from its origin at the anterolateral surface of the tibia and its insertion at the medial cuneiform via its distal tendon.Then, grasp the foot by the digits with the index finger and thumb, and insert the tip of the Dumont forceps immediately under the superficial distal tendon of the tibialis. Move the forceps such that the blunt side can be used to detach the tibialis muscle belly from the underlying connective tissue, and use the fine curved scissors to cut the distal tendon. Then, cut the muscle at the origin, as close as possible to the tibia.When all of the muscles have been collected, immerse the bottom half of a plastic, 50 milliliter beaker containing isopentane into liquid nitrogen. When the isopentane becomes slightly viscous, with a solid white laminate lining visible inside the beaker, pour a few drops of embedding medium on a chuck. Then, pick up a muscle by the tendon, and carefully position the end of the fresh muscle on top of the embedding medium.Holding it vertically, relative to the cork. Then, keeping the muscle vertical, dip the chuck and attached muscle into the isopentane bath for seven to 15 seconds until the tissue is completely frozen. A well frozen specimen will appear chalky white.Then, place the muscle in a specimen tube for immediate storage at negative 80 degrees Celsius. To preserve the RNA and enzymatic structure and tibialis samples, it is required to freeze the muscle tissues as soon as possible after their extraction. To section the frozen muscle tissue, first set the working temperature of the Cryostat inner chamber to negative 23 degrees Celsius.Allow the negative 80 degrees Celsius specimen to acclimate to the working temperature for a few hours. Then, obtain multiple eight micron thick sections of the specimen at the mid belly region of the muscle, perpendicular to the mounting axis. Collecting the sections on room temperature glass slides as they are cut.Keep the samples inside the Cryostat chamber until all of the sections have been collected. Then, store the samples at negative 80 degrees Celsius until further analysis. C 26 tumor growth kinetics demonstrate a lag phase for the first seven to eight days after injection, followed by exponential cell growth for four to five days.With the tumor mass eventually reaching approximately 10%of the animal's body weight. Tumor bearing mice appear wasted and exhibit disheveled fur at the end of the experimental period. With 20 to 30%reduction in skeletal muscle weight.The cardiac muscle is also significantly reduced in weight. Although to a lesser extent compared to the other muscles. Interestingly, hepatomegaly and splenomagly are also generally detected in tumor hosts.While the fat mass, similar to the skeletal muscle mass, is severely depleted. Skeletal muscle weight loss is also consistent with, and proportional to, the reduction in muscle fiber size. As observed by immunofluorescent morphometric evaluation of muscle fiber cross-sectional areas.In particular, the frequency distribution analysis demonstrates a shift towards smaller sized fibers in C 26 bearing mice. Suggesting that the whole muscle undergoes atrophy in the presence of a C 26 tumor. Similar results can also be observed by HNE analysis.Although the magnitude of change in the muscle cross-sectional area associated with the cancer growth is slightly different. Proper use of a C 26 model allows for the investigation of tumor derived cachexia. The model closely resembles a human disease showing an overall reduction in mass as well as muscle fiber atrophy, inflammation, and hyper catabolism.When performing this experiment, the strain of six of the mice, the source, and number of tumor cells, and the site of implantation can be modified. Such modifications can lead to differences in the expected outcomes. Despite a few known limitations, such as the non-physiologic growth environment, the dependency on IL six action, and the necessary use of CD two F one or Valve C mice, the C 26 model has greatly contributed to understanding of the molecular mechanisms of cancer induced cachexia.After watching this video, you should have a good understanding of how to replicate the C 26 cancer cachexia model to study the progressive muscle wasting and weakness that typically occur, concurrent with tumor development. Be aware that working with the C 26 model requires familiarity with proper animal handling techniques. Best practices should always be used to execute this protocol as humanely as possible.

Research

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Cancer Research

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