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.

Ethics Statement: All studies described were approved by the Institutional Animal Care and Use Committees of the Thomas Jefferson University and Indiana University School of Medicine.
1. C26 Cell Growth and Preparation
<ol>
	<li>Obtain C26 colorectal cancer cells (Ohio State University Medical Center (OSUMC)) and prepare complete growth medium (i.e., high-glucose Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) containing 10% Fetal Bovine Serum (FBS), 1 mM sodium pyruvate, 1% gluta...

<ol>
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Especially in its latest stages, colorectal cancer is associated with the development of cachexia, which is responsible for poorer outcomes and reductions in patient quality of life. Many studies have focused on the treatment of conditions secondary to cancer; however, despite many efforts in this direction, there is still no approved therapy for cancer cachexia21. Thus, it is imperative that animal models resemble the human pathology as closely as possible in order to maximize the translation of findings....

Muscle wasting is a serious complication of various clinical conditions such as cancer, sepsis, liver, cirrhosis, heart and kidney failure, chronic obstructive pulmonary disease, and AIDS. In particular, muscle wasting is evident in at least 50% of patients with cancer1. Loss of skeletal muscle in cancer results from increased protein degradation due to the over-activation of the skeletal muscle proteolytic systems and/or from decreased protein synthesis6. Lipolysis is also evident, leading to the depletion of adipose tissue. Clinically, cachexia is associated with reduced quality and length of life and is estimated to be the cause of death in 20 - 30% of cancer patients7. Use of experimental models that resemble the human disease as closely as possible would be beneficial. An optimal animal model is characterized by high reproducibility, as well as by limited interference from different therapies and the unpredictable factors of diet, sex, and genetic background that are usually associated with the clinical condition8. So far, cancer cachexia has been studied mainly in animal models characterized by transplantation of cancer cells or injection of carcinogens, although a new method is to use genetically modified mice susceptible to the development of cancer.
Mice bearing the C26 carcinoma (also referred to as colon-26 and adenocarcinoma) represent a well-characterized and extensively used model of cancer cachexia2,5. The growth of the C26 tumor results in body and muscle weight loss, mainly through enhanced fat and protein catabolism9. Generally, a 10% tumor weight versus total body weight is associated with a reduction of 20-25% in skeletal muscle weight and a greater depletion of fat3,10. Hepatomegaly and splenomegaly are also observed with tumor growth, along with the activation of the acute phase response and the elevation of pro-inflammatory cytokine levels3,11. Among these, it is well known that IL-6 plays a pivotal role in mediating muscle wasting in the C26 model, even though this cytokine is probably not the only inducer of cachexia12. Elevated IL-6 causes muscle atrophy through activation of the JAK/STAT3 pathway, and inhibiting this transcription factor can prevent muscle wasting3,4.
During C26-induced muscle wasting, as in many conditions of muscle atrophy, muscle mass is lost largely through reductions in muscle protein content across muscle fibers, not through cell death or loss of fibers13. In C26 cachexia, a shift towards smaller cross-sectional areas is observed in both glycolytic and oxidative fibers2. This is also consistent with reduced muscle strength5. Many groups worldwide have taken advantage of the C26 model in order to discover new mediators of muscle wasting or clinically relevant drugs for cancer cachexia. However, many different procedures for the use of this model have been reported, raising concerns about the consistency of the obtained data and posing barriers to reproducibility in different experimental conditions. Here we report a typical use of this model for the study of cancer cachexia that yields standardized and reproducible data.

<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>
			<td style="width:104px;"></td>
			<td style="width:185px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:167px;">Image J</td>
			<td style="width:205px;">US National Institutes of Health</td>
			<td style="width:104px;">IJ1.46</td>
			<td style="width:185px;">http://rsbweb.nih.gov/ij/download.html</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:167px;">Microtainer</td>
			<td style="width:205px;">BD</td>
			<td style="width:104px;">365873</td>
			<td style="width:185px;"></td>
		</tr>
	</tbody>
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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 tumor growth kinetics show a lag phase for the first 7 - 8 d after injection, followed by exponential cell growth (4 - 5 d). The tumor mass eventually reaches ~10% of the body weight (about 2 g; Figure 1A-B). During the first phase, the tumor can be located by palpation only and appears as a small protrusion of the skin. In the second phase, the tumor is observed as a mass under the skin. Rarely, the tumor becomes ulcerated, resulting in an open wound...

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.

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.

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