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.
