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.

<ol>
	<li>Tan, B., Fearon, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cachexia:+prevalence+and+impact+in+medicine.">Cachexia: prevalence and impact in medicine.</a> Curr Opin Clin Nutr Metab Care. 11, 400-407 (2008).</li><li>Aulino, P., 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=Molecular+cellular+and+physiological+characterization+of+the+cancer+cachexia-inducing+C26+colon+carcinoma+in+mouse.">Molecular cellular and physiological characterization of the cancer cachexia-inducing C26 colon carcinoma in mouse.</a> BMC cancer. 10, 363 (2010).</li><li>Bonetto, A., 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=STAT3+activation+in+skeletal+muscle+links+muscle+wasting+and+the+acute+phase+response+in+cancer+cachexia.">STAT3 activation in skeletal muscle links muscle wasting and the acute phase response in cancer cachexia.</a> PloS One. 6, e22538 (2011).</li><li>Bonetto, A., 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=JAK/STAT3+pathway+inhibition+blocks+skeletal+muscle+wasting+downstream+of+IL-6+and+in+experimental+cancer+cachexia.">JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia.</a> Am J Physiol Endocrinol Metab. 303, E410-E421 (2012).</li><li>Bonetto, A., 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=Deacetylase+inhibitors+modulate+the+myostatin/follistatin+axis+without+improving+cachexia+in+tumor-bearing+mice.">Deacetylase inhibitors modulate the myostatin/follistatin axis without improving cachexia in tumor-bearing mice.</a> Current Cancer Drug Targets. 9, 608-616 (2009).</li><li>Acharyya, S., 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=Cancer+cachexia+is+regulated+by+selective+targeting+of+skeletal+muscle+gene+products.">Cancer cachexia is regulated by selective targeting of skeletal muscle gene products.</a> The Journal of Clinical Investigation. 114, 370-378 (2004).</li><li>Fearon, 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=Definition+and+classification+of+cancer+cachexia:+an+international+consensus.">Definition and classification of cancer cachexia: an international consensus.</a> The Lancet Oncology. 12, 489-495 (2011).</li><li>Holecek, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle+wasting+in+animal+models+of+severe+illness.">Muscle wasting in animal models of severe illness.</a> Int J Exp Pathol. 93, 157-171 (2012).</li><li>Acharyya, S., 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=Dystrophin+glycoprotein+complex+dysfunction:+a+regulatory+link+between+muscular+dystrophy+and+cancer+cachexia.">Dystrophin glycoprotein complex dysfunction: a regulatory link between muscular dystrophy and cancer cachexia.</a> Cancer Cell. 8, 421-432 (2005).</li><li>Benny Klimek, ., E, M., 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=Acute+inhibition+of+myostatin-family+proteins+preserves+skeletal+muscle+in+mouse+models+of+cancer+cachexia.">Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia.</a> Biochemical and Biophysical Research Communications. 391, 1548-1554 (2010).</li><li>Pedroso, F. 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B., 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=Increased+efficacy+and+decreased+systemic-effects+of+botulinum+toxin+A+injection+after+active+or+passive+muscle+manipulation.">Increased efficacy and decreased systemic-effects of botulinum toxin A injection after active or passive muscle manipulation.</a> Dev Med Child Neurol. 49, 907-914 (2007).</li><li>Murphy, K., Lynch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Update+on+emerging+drugs+for+cancer+cachexia.">Update on emerging drugs for cancer cachexia.</a> Expert Opin Emerg Drugs. 14, 619-632 (2009).</li><li>Seto, D. N., Kandarian, S. C., Jackman, R. 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The authors have nothing to disclose.

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.
C26 tumor-bearing mice are a commonly used experimental model of cancer cachexia22-24. This model closely resembles the human disease, showing reductions in body, muscle, and fat mass, as well as muscle fiber atrophy and increased expression of inflammatory genes and ubiquitin ligases consistent with marked protein hypercatabolism2,5. Despite this, a few disparities with data obtained in cancer patients have been reported25,26. Indeed, some features of the model, including the non-physiologic growth environment (e.g., an ectopic tumor grown subcutaneously instead of orthotopically implanted in the GI tract), the relatively short experimental period compared to other models (e.g., genetic or orthotopic injection of tumors), the dependency on IL-6 action, and the necessary use of CD2F1 or Balb/c mice may represent severe limitations and could constrain the interpretation of results.
The current protocol has proven to be highly reproducible across many experiments performed in our laboratory, maintaining the same characteristics and allowing comparisons between results obtained at different times, in different geographic locations, and by different researchers3,10. However, in order to promote the reproducibility of data, it is important to establish clear and common guidelines.
As gathered from the literature, several caveats might prevent the reproduction of the same data in two different laboratories. For the same reasons, the use of different protocols may generate different phenotypes and outcomes, as well as lead to conflicting and questionable results. Indeed, differences in performing this animal model have been reported, mainly resulting from the strain (Balb/c or CD2F1)2,5,27-29 or sex of mice used17, the type of tumor that was implanted (a cell suspension3,29 rather than a solid tumor in a graft2,30), the tumor source (NCI, ATCC, or OSUMC), the number of C26 cells that were injected, and the site of injection or implantation (the flanks6,17,31 or the dorsal region3,10,32). No direct comparison of the effects associated with the implantation of C26 cell lines obtained from different sources (mainly OSUMC versus NCI) has ever been performed, thus preventing us from drawing any definitive conclusions. However, it is likely that the choice of the source of cells may also significantly influence the expected outcomes. When considering the choice of males versus females, investigators should be reminded of significant differences in the outcomes. Indeed, as reported by Cosper and Leinwand17, male tumor-bearing mice, due to the absence of estrogens, may show a more severe phenotype than females, including greater cardiac mass loss and mortality, a more robust pro-inflammatory response to the tumor, and greater cardiac autophagy.
Especially for those investigators that are not familiar with this model and may initially face problems, based on both a power analysis and our previous experience, the use of at least 6 animals per experimental condition (n = 6) is advisable in order to detect statistically significant differences. It is also critical that randomization is performed carefully, so that initial body weights in the experimental animals do not differ significantly among groups. Similarly, it is advised that, in order to avoid inter-operator variability, the same investigator perform the tissue and organ collection on every animal. Further, it is imperative that tissues (particularly muscle) are frozen as fast as possible in order to preserve the RNAs and enzymatic structure and activity, especially if the goal of the study is to assess gene expression or to evaluate enzymatic activities. Moreover, in the attempt to determine muscle CSA, we showed that reporting the fiber area is generally accepted and representative of muscle fiber size. Here, we present two distinct methods to assess muscle CSA. As shown in Figures 4-5, both methods were able to detect a significant reduction in myofiber size between controls and tumor hosts, although the degree of wasting appeared different.
This discrepancy may result from the fact that, although both methods are acceptable ways to assess muscle fiber size, quantification of muscle characteristics from H&#38;E-stained slides is still a manual or semi-automatic process, most often labor-intensive, time-consuming, and affected by limited accuracy. Based on our experience, we believe that the IF-based method is a more accurate technique to report muscle CSA. Indeed, the number of fibers that can be measured automatically by taking advantage of this technique is significantly larger, thus increasing the accuracy of the measurement. Of note, for both techniques, an alternative method of reporting muscle size is assessing the Feret&#39;s diameter. Interestingly, this is considered a very reliable tool due to the fact that this parameter is largely independent of the angle of sectioning. Other parameters, such as the &#34;minimal inner diameter&#34; and the &#34;minimal outer diameter&#34; are also insensitive to the plane of sectioning and can be used instead of the Feret&#39;s diameter as alternative indications of the fiber size.
In conclusion, although the cachexia community clearly needs to establish more physiological models for the study of tumor-associated muscle wasting, we believe that mice bearing the C26 colon carcinoma represent a well-standardized and easy-to-use model to investigate molecular alterations and physiological abnormalities usually detected after the occurrence of a tumor. Future applications will involve the investigation of whether orthotopic implantation of C26 cells into the colon might represent a proper and more physiological model of colorectal cancer cachexia.

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><tbody><tr><td>Cell culture Flasks</td><td>Falcon - Becton Dickinson</td><td>35-5001</td><td></td></tr><tr><td>DMEM</td><td>Cellgro</td><td>10-017-CV</td><td></td></tr><tr><td>FBS</td><td>Gibco</td><td>26140</td><td></td></tr><tr><td>Streptomycin-Penicillin&amp;nbsp;</td><td>Cellgro</td><td>30-002-CI</td><td></td></tr><tr><td>CD2F1 mice</td><td>Harlan</td><td>060</td><td></td></tr><tr><td>Anesthesia apparatus</td><td>EZ-Anesthesia</td><td>EZ-7000</td><td></td></tr><tr><td>2-Methyl Butane</td><td>Sigma-Aldrich</td><td>M32631</td><td></td></tr><tr><td>OCT</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Cryostat</td><td>Leica</td><td>CM1850</td><td></td></tr><tr><td>Cork disks</td><td>Electron Microscopy Sciences</td><td>63305</td><td></td></tr><tr><td>Superfrost plus glass slides</td><td>VWR</td><td>48311-703</td><td></td></tr><tr><td>Anti-Laminin Rabbit polyclonal antibody</td><td>Sigma-Aldrich</td><td>L9393</td><td></td></tr><tr><td>Anti-Dystrophin Mouse Monoclonal antibody</td><td>Vector Laboratories</td><td>VP-D508</td><td></td></tr><tr><td>Alexa Flour 594 anti-mouse IgG</td><td>Life Technologies</td><td>A11062</td><td></td></tr><tr><td>Alexa Flour 594 anti-rabbit IgG</td><td>Life Technologies</td><td>A21211</td><td></td></tr><tr><td>Hematoxylin</td><td>Sigma-Aldrich</td><td>GHS216</td><td></td></tr><tr><td>Eosin</td><td>Sigma-Aldrich</td><td>HT110332</td><td></td></tr><tr><td>Xylene</td><td>Acros Organics</td><td>422680025</td><td></td></tr><tr><td>Cytoseal-XYL</td><td>Thermo</td><td>8312-4</td><td></td></tr><tr><td>Microscope</td><td>Zeiss</td><td>Observer.Z1&amp;nbsp;</td><td></td></tr><tr><td>Bamboo Tablet</td><td>Wacom</td><td>CTH-661</td><td></td></tr><tr><td>Prism 7.0 for Mac OS X</td><td>GraphPad Software, Inc.</td><td></td><td></td></tr><tr><td>Excel for Mac 2011</td><td>Microsoft Corp.</td><td></td><td></td></tr><tr><td>ImageJ</td><td>US National Institutes of Health</td><td>IJ1.46</td><td>http://rsbweb.nih.gov/ij/download.html</td></tr><tr><td>Microtainer</td><td>BD</td><td>365873</td><td></td></tr></tbody></table>

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; in this case, the mouse is excluded from the experimental group and is humanely euthanized.
Body weight is unchanged in the first phase, but it is significantly reduced in the second phase, when it reaches 10 - 15% of the initial body weight (30% in the case of tumor-free weight; Figure 1A). Tumor-bearing mice appear wasted and showed disheveled fur at the end of the experimental period, with a body condition (BC) score equal to 118 (Figure 1C). BC1 represents a severely emaciated mouse, where skeletal structures are evident and the vertebrae are distinctly segmented. Body weight loss is mainly accounted for by both skeletal muscle and fat tissue wasting (Figure 1B). Body weight loss is consistent with a reduction of about 20 - 30% in skeletal muscle weight, in particular in the gastrocnemius, tibialis anterior, and quadriceps (Figure 2). The cardiac muscle is also significantly reduced in weight, although to a lesser extent when compared to the other skeletal muscles (Figure 2). Interestingly, hepatomegaly (+16%, p &#60; 0.01) and splenomegaly (+110%, p &#60; 0.01) are generally detected in tumor hosts, while fat mass, similar to skeletal muscle, is severely depleted (-70%, p &#60; 0.001; Figure 3).
Skeletal muscle weight loss is also consistent with and proportional to the reduction in muscle fiber size, as observed after morphometric evaluation of muscle fiber CSA by means of an IF method (Figure 4A-B). In particular, the frequency distribution analysis showed a shift towards smaller-size fibers in C26-bearing mice, thus suggesting that the whole muscle undergoes atrophy in the presence of a C26 tumor (Figure 4C). Similar results can be observed by taking advantage of a traditional H&#38;E-based methodology for the quantification of fiber size, although the magnitude of the change in muscle CSA associated with cancer growth is slightly different (38% versus control, p &#60; 0.01 for the IF-based method; -18% versus control, p &#60; 0.01 for the H&#38;E-based method; Figure 5).
<img alt="Figure 1" src="/files/ftp_upload/54893/54893fig1.jpg" /> 
Figure 1: Body Weights in Control and C26-bearing Mice. A) Body weight curve in control and C26 hosts over the entire experimental period (14 days after tumor injection). B) Initial body weight (IBW), final tumor-free body weight (FBW), and tumor weight in control and C26-bearing animals. C) Representative pictures of controls and C26-bearers at the time of sacrifice (day 14 after tumor inoculation). Data are expressed as the mean SD. n = 6. Significance of the differences: *p &#60; 0.05, **p &#60; 0.01, ***p &#60; 0.001 versus the control group using Student&#39;s t-test. <a href="http://cloudflare.jove.com/files/ftp_upload/54893/54893fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54893/54893fig2.jpg" /> 
Figure 2: Muscle Weights in Control and C26-bearing Mice. The tibialis, gastrocnemius, quadriceps, and heart weights in control and C26 tumor-bearers are reported as the weight/100 mg IBW. The data are expressed as the mean SD. n = 6. Significance of the differences: **p &#60; 0.01 and ***p &#60; 0.001 versus the control group using Student&#39;s t-test.
<img alt="Figure 3" src="/files/ftp_upload/54893/54893fig3.jpg" /> 
Figure 3: Organ Weights in Control and C26-bearing Mice. The liver, spleen, and epididymal fat pad weights in control and C26 tumor-bearers are reported as the weight/100 mg IBW. The data are expressed as the mean SD. n = 6. Significance of the differences: **p &#60; 0.01 versus the control group.
<img alt="Figure 4" src="/files/ftp_upload/54893/54893fig4.jpg" /> 
Figure 4: Morphometry for Myofiber Size by Immunofluorescence. A) Fluorescent immunohistochemical reaction of tibialis sections using an anti-dystrophin antibody (Vector Laboratories) from control and C26 hosts. Magnification: 10X. Size bar: 100 &#181;m. B) Quantification of CSA in the tibialis muscle measured with the ImageJ macro. C) Frequency distribution of CSA in the tibialis muscle of control and C26-bearing mice. The data are expressed as the mean &#177; SD. n = 6 for each group. Significance of the differences: **p &#60; 0.01 versus the control group using Student&#39;s t-test.
<img alt="Figure 5" src="/files/ftp_upload/54893/54893fig5.jpg" /> 
Figure 5: Morphometry for Myofiber Size by H&#38;E. A) Hematoxylin &#38; eosin staining of tibialis sections from control and C26 hosts. Magnification: 20X. Size bar: 100 &#181;m. B) Quantification and frequency distribution of CSA in the tibialis muscle. The data are expressed as the mean SD. n = 6. Significance of the differences: ***p &#60; 0.001 versus the control group using Student&#39;s t-test.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Authors</td>
			<td>Mouse strain</td>
			<td>Mouse gender</td>
			<td>Cell number</td>
			<td>Origin</td>
			<td>Implanted as solid tumor?</td>
			<td>Site of injection</td>
			<td>Days</td>
		</tr>
		<tr>
			<td>Lazarus et al., 1999</td>
			<td>CD2F1</td>
			<td>M</td>
			<td>5 x 105</td>
			<td>NCI</td>
			<td>NO</td>
			<td>Dorsal region</td>
			<td>17</td>
		</tr>
		<tr>
			<td>Al-Majid and McCarthy, 2001</td>
			<td>CD2F1</td>
			<td>F</td>
			<td>2.5 x 106</td>
			<td>OSUMC</td>
			<td>NO</td>
			<td>Dorsal region</td>
			<td>17</td>
		</tr>
		<tr>
			<td>Samuels et al., 2001</td>
			<td>Balb/c</td>
			<td>M</td>
			<td>0.5 g/ml of tumor suspension</td>
			<td>ns</td>
			<td>YES</td>
			<td>Dorsal region</td>
			<td>Up to 11</td>
		</tr>
		<tr>
			<td>Acharyya et al., 2004</td>
			<td>CD2F1</td>
			<td>M</td>
			<td>1 x 107</td>
			<td>OSUMC</td>
			<td>NO</td>
			<td>Right flank</td>
			<td>Up to 24</td>
		</tr>
		<tr>
			<td>Aulino et al., 2010</td>
			<td>Balb/c</td>
			<td>ns</td>
			<td>0.5 mm3</td>
			<td>NCI</td>
			<td>YES</td>
			<td>Dorsal region</td>
			<td>21</td>
		</tr>
		<tr>
			<td>Benny-Klimek et al., 2010</td>
			<td>CD2F1</td>
			<td>F</td>
			<td>1 x 106</td>
			<td>OSUMC</td>
			<td>NO</td>
			<td>Dorsal region</td>
			<td>14</td>
		</tr>
		<tr>
			<td>Bonetto et al., 2011; 2012</td>
			<td>CD2F1</td>
			<td>F</td>
			<td>1 x 106</td>
			<td>OSUMC</td>
			<td>NO</td>
			<td>Dorsal region</td>
			<td>14</td>
		</tr>
		<tr>
			<td>Cosper and Leinwand, 2010</td>
			<td>CD2F1</td>
			<td>M+F</td>
			<td>5 x 105</td>
			<td>ns</td>
			<td>NO</td>
			<td>Right flank</td>
			<td>Up to 27</td>
		</tr>
		<tr>
			<td>Murphy et al., 2012</td>
			<td>CD2F1</td>
			<td>ns</td>
			<td>5 x 105</td>
			<td>NCI/OSUMC</td>
			<td>NO</td>
			<td>Dorsal region</td>
			<td>14</td>
		</tr>
		<tr>
			<td>Cornwell et al., 2014</td>
			<td>CD2F1</td>
			<td>M</td>
			<td>5 x 105</td>
			<td>NCI</td>
			<td>NO</td>
			<td>Right&#38;Left flank</td>
			<td>Up to 26</td>
		</tr>
		<tr>
			<td>Assi et al., 2016</td>
			<td>Balb/c</td>
			<td>ns</td>
			<td>1 x 106</td>
			<td>Cell Line Service</td>
			<td>NO</td>
			<td>Dorsal region</td>
			<td>Up to 22</td>
		</tr>
	</tbody>
</table>
Table 1: Comparison of the Different Protocols used for C26 Implantation. Different protocols for C26 implantation are provided, with particular reference to the mouse strain used, the cell number and type of tumor inoculated, the cell origin, the site of injection, and the length of the experimental period. OSUMC: Ohio State University Medical Center. NCI: National Cancer Institute. Commercial cell line service NS: not specified.

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 ...

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Cancer Research

16.1K Views.  Indiana University School of Medicine. 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. 

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

Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo&#160;Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches1,2. ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Orthotopic Mouse Model of Colorectal Cancer

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature cancer cells growing in their natural location, replicate human disease with high fidelity. Two techniques can be used to establish this model. Both techniques are similar and require mouse anesthesia and laparotomy for exposure of the cecum. One technique involves injection of a colorectal cancer cell suspension into the cecal wall. Cancer cells are first grown in culture, harvested when subconfluent and prepared as a single cell suspension. A small volume of cells is injected slowly to avoid leakage. The other technique involves transplantation of a piece of subcutaneous tumor onto the cecum. A mouse with a previously established subcutaneous colorectal tumor is euthanized and the tumor is removed using sterile technique. The tumor piece is divided into small pieces for transplantation to another mouse. Prior to transplantation, the cecal wall is lightly damaged to facilitate tumor cell infiltration. The time to developing primary tumors and liver metastases will vary depending on the technique, cell line, and mouse species used. This orthotopic mouse model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

Two techniques can be used to establish this model: injection of a cancer cell suspension into the cecal wall or transplantation of a piece of subcutaneous tumor onto the cecum. This model is useful for studying the natural progression of colorectal cancer and testing new therapeutic agents against colorectal cancer.

The traditional subcutaneous tumor model is less than ideal for studying colorectal cancer. Orthotopic mouse models of colorectal cancer, which feature ...

Biology

Modeling Colitis-Associated Cancer with Azoxymethane (AOM) and Dextran Sulfate Sodium (DSS)

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal cancer (CRC) over healthy individuals. This risk is proportional to the duration and extent of disease, with a cumulative incidence as high as 30% in individuals with longstanding UC with widespread colonic involvement.1 Colonic dysplasia in IBD and colitis associated cancer (CAC) are believed to develop as a result of repeated cycles of epithelial cell injury and repair while these cells are bathed in a chronic inflammatory cytokine milieu.2 While spontaneous and colitis-associated cancers share the quality of being adenocarcinomas, the sequence of underlying molecular events is believed to be different.3 This distinction argues the need for specific animal models of CAC.
Several mouse models currently exist for the study of CAC. Dextran sulfate sodium (DSS), an agent with direct toxic effects on the colonic epithelium, can be administered in drinking water to mice in multiple cycles to create a chronic inflammatory state. With sufficient duration, some of these mice will develop tumors.4 Tumor development is hastened in this model if administered in a pro-carcinogenic setting. These include mice with genetic mutations in tumorigenesis pathways (APC, p53, Msh2), as well as mice pre-treated with genotoxic agents (azoxymethane [AOM], 1,2-dimethylhydrazine [DMH]).5 
The combination of DSS with AOM as a model for colitis associated cancer has gained popularity for its reproducibility, potency, low price, and ease of use. Though they have a shared mechanism, AOM has been found to be more potent and stable in solution than DMH. While tumor development in other models generally requires several months, mice injected with AOM and subsequently treated with DSS develop adequate tumors in as little as 7-10 weeks.6, 7 Finally, AOM and DSS can be administered to mice of any genetic background (knock out, transgenic, etc.) without cross-breeding to a specific tumorigenic strain. Here, we demonstrate a protocol for inflammation-driven colonic tumorigenesis in mice utilizing a single injection of AOM followed by three seven-day cycles of DSS over a 10 week period. This model induces tumors with histological and molecular changes closely resembling those occurring in human CAC and provides a highly valuable model for the study of oncogenesis and chemoprevention in this disease.8

Modeling Colitis-Associated Cancer with Azoxymethane AOM and Dextran Sulfate Sodium DSS

We demonstrate a protocol in which administration of the genotoxic agent azoxymethane (AOM) followed by three cycles of the pro-inflammatory agent dextran sulfate sodium (DSS) rapidly and consistently generates colon tumors in mice with morphologic and molecular similarities to those seen in human colitis-associated cancer.

Individuals with inflammatory bowel disease (IBD), such as Crohn's disease (CD) or ulcerative colitis (UC) are at increased risk of developing colorectal ...

Medicine

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the tumor microenvironment, play a crucial role in metastatic CRC progression and predict poor patient prognosis. However, there is a lack of satisfactory mouse models to study the crosstalk between metastatic cancer cells and CAFs. Here, we present a method to investigate how liver metastasis progression is regulated by the metastatic niche and possibly could be restrained by stroma-directed therapy. Portal vein injection of CRC organoids generated a desmoplastic reaction, which faithfully recapitulated the fibroblast-rich histology of human CRC liver metastases. This model was tissue-specific with a higher tumor burden in the liver when compared to an intra-splenic injection model, simplifying mouse survival analyses. By injecting luciferase-expressing tumor organoids, tumor growth kinetics could be monitored by in vivo imaging. Moreover, this preclinical model provides a useful platform to assess the efficacy of therapeutics targeting the tumor mesenchyme. We describe methods to examine whether adeno-associated virus-mediated delivery of a tumor-inhibiting stromal gene to hepatocytes could remodel the tumor microenvironment and improve mouse survival. This approach enables the development and assessment of novel therapeutic strategies to inhibit hepatic metastasis of CRC.

Portal vein injection of colorectal cancer (CRC) organoids generates stroma-rich liver metastasis. This mouse model of CRC hepatic metastasis represents a useful tool to study tumor-stroma interactions and develop novel stroma-directed therapeutics such as adeno-associated virus-mediated gene therapies.

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (&#181;CT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.

This protocol describes the orthotopic implantation of patient-derived cancer cells in the cecum wall of immunodeficient mice. The model recapitulates advanced colorectal cancer metastatic disease and allows for the evaluation of new therapeutic drugs in a clinically relevant scenario of lung and liver metastases.

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Intrapulmonary Inoculation of Multicellular Spheroids to Model Orthotopic NSCLC

Construction of An Orthotopic Xenograft Model of Non-Small Cell Lung Cancer Mimicking Disease Progression and Predicting Drug Activities

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models based on subcutaneous inoculation of cancer cell suspensions do not recapitulate the tumor microenvironment in NSCLC. Herein we describe a murine orthotopic lung cancer xenograft model that employs the intrapulmonary inoculation of three-dimensional multicellular spheroids (MCS). Specifically, fluorescent human NSCLC cells (A549-iRFP) were cultured in low-attachment 96-well microplates with collagen for 3 weeks to form MCS, which were then inoculated intercostally into the left lung of athymic nude mice to establish the orthotopic lung cancer model.
Compared with the original A549 cell line, MCS of the A549-iRFP cell line responded similarly to anticancer drugs. The long-wavelength fluorescent signal of the A549-iRFP cells correlated strongly with common markers of cancer cell growth, including spheroid volume, cell viability, and cellular protein level, thus allowing dynamic monitoring of the cancer growth in vivo by fluorescent imaging. After inoculation into mice, the A549-iRFP MCS xenograft reliably progressed through phases closely resembling the clinical stages of NSCLC, including the expansion of the primary tumor, the emergence of neighboring secondary tumors, and the metastases of cancer cells to the contralateral right lung and remote organs. Moreover, the model responded to the benchmark antilung cancer drug, cisplatin with the anticipated toxicity and slower cancer progression. Therefore, this murine orthotopic xenograft model of NSCLC would serve as a platform to recapitulate the disease&#39;s progression and facilitate the development of potential anticancer drugs.

Author Spotlight: Establishing a Murine Non-Small Cell Lung Cancer Model for Developing Nanoformulations of Anticancer Drugs

This study presents an orthotopic non-small cell lung cancer (NSCLC) model based on intrapulmonary inoculation of multicellular spheroids of fluorescent A549-iRFP cells. The model recapitulates clinical NSCLC stages and responds to cisplatin, according to dynamic in vivo monitoring of long-wavelength fluorescence.

Non-small cell lung cancer (NSCLC) is a highly lethal disease with a complex and heterogeneous tumor microenvironment. Currently, common animal models ...

Murine Distal Colostomy, A Novel Model of Diversion Colitis in C57BL/6 Mice

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a diverting enterostomy. The etiology of this disease remains ill-defined but appears to differ from that of classical inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. Research aimed to decipher the pathophysiological mechanisms leading to the development of this disease has been severely hampered by the lack of an appropriate murine model. This protocol generates a murine model of DC that facilitates the study of the immune system's role and its interaction with the microbiome in the development of DC. In this model using C57BL/6 animals, distal parts of the colon are excluded from the fecal stream by creating a distal colostomy, triggering the development of mild to moderate inflammation in the excluded bowel segments and reproducing the hallmark lesions of human DC with a moderate systemic inflammatory response. In contrast to the rat model, a large number of genetically-modified murine models on the C57BL/6 background are available. The combination of these animals with our model allows the potential roles of individual cytokines, chemokines, or receptors of bioactive molecules (e.g., interleukin (IL)-17; IL-10, chemokine CXCL13, chemokine receptors CXCR5 and CCR7, and the sphingosine-1-phosphate receptor 4) to be assessed in the pathogenesis of DC. The availability of congenic mouse strains on the C57BL/6 background largely facilitates transfer experiments to establish the roles of distinct cell types involved in the etiology of DC. Finally, the model offers the opportunity to assess the influences of local interventions (e.g., modification of the local microbiome or local anti-inflammatory therapy) on mucosal immunity in affected and non-affected bowel segments and the on systemic immune homeostasis.

Murine distal colostomy provides a murine model for human diversion colitis, a predominantly lymphocytic colitis in colon segment excluded from the fecal stream.

Diversion colitis (DC) is a frequent clinical condition occurring in patients with bowel segments excluded from the fecal stream as a result of a ...

Spontaneous Metastasis Mouse Models: A Platform to Study the Metastatic Potential of Orthotopically Injected Colorectal Cancer Cells

Source: Okazawa, Y., et al. <a href="https://www.jove.com/v/57374"> High-sensitivity Detection of Micrometastases Generated by GFP Lentivirus-transduced Organoids Cultured from a Patient-derived Colon Tumor.</a>&#160;J. Vis. Exp.&#160;(2018).
This video describes the protocol to establish a spontaneous metastasis mouse model of colorectal cancer patient-derived xenografts or PDXs. These xenograft models partially mimic the features of human carcinomas and understand the critical aspects of human malignancies, including invasion and metastasis.&#160;

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

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