大腸-26癌腫瘍を有するがん悪液質の研究のためのモデルとしてマウスを
Summary
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.
Abstract
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.
Introduction
筋萎縮は、癌、敗血症、肝臓、肝硬変、心臓や腎不全、慢性閉塞性肺疾患、およびAIDSなどの様々な臨床症状の重篤な合併症です。特に、筋消耗、癌1患者の少なくとも50％において明らかです。原因および/またはからの骨格筋タンパク質分解システムの過剰活性化に増加したタンパク質分解からの癌の結果で骨格筋の損失は、タンパク質合成6を減少させました 。脂肪分解は、脂肪組織の枯渇をもたらすも明らかです。臨床的には、悪液質を低減し、品質及び寿命の長さと関連し、20における死亡の原因であると推定されている-癌患者7の30％。できるだけヒトの疾患に似ている実験モデルの使用は有益であろう。最適な動物モデルは、異なる治療法との予測不可能な要因から高い再現性により、同様に制限された干渉によって特徴づけられます通常、臨床状態8に関連付けられている食事、性別、遺伝的背景。新しい方法は、癌の発生に影響を受けやすい遺伝的に改変されたマウスを使用することであるが、これまで、癌性悪液質は、癌細胞または発癌物質の注入の移植によって特徴付けられる動物モデルにおいて主に研究されてきました。
C26癌を有するマウスは、癌性悪液質2,5のよく特徴付けられたと広範囲に使用されるモデルを表す（結腸-26および腺癌と呼ばれます）。主に強化脂肪及びタンパク質異化9を介して身体および筋肉重量損失C26腫瘍結果の成長。一般に、全体重に対して10％の腫瘍重量は、骨格筋重量の20％〜25％の減少および脂肪3,10の高い枯渇と関連しています。肝腫および脾腫も、急性期反応の活性化及びプロinflaの上昇に伴って、腫瘍の成長で観察されていますmmatoryサイトカインレベル3,11。これらの中でも、同様にこのサイトカインはおそらく、悪液質12の唯一の誘導因子ではないにもかかわらず、IL-6は、C26モデルにおいて筋肉消耗を媒介する重要な役割を果たしていることが知られています。上昇したIL-6は、JAK / STAT3経路の活性化を介して筋萎縮を引き起こし、この転写因子を阻害すること3,4筋肉消耗を防ぐことができます。
C26誘発性筋萎縮時には、筋萎縮の多くの条件のように、筋肉量がない細胞死または繊維13の損失を介して、主に筋線維全体の筋肉のタンパク質含有量の減少によって失われます。 C26の悪液質では、より小さな断面積へのシフトは、解糖および酸化繊維2の両方で観察されました。これはまた、減少筋力5と一致しています。多くのグループが、世界中のがんCACのための筋萎縮の新しいメディエーターまたは臨床的に関連する薬物を発見するために、C26モデルの利点をとっていますhexia。しかし、このモデルの使用のために多くの異なる手順は、得られたデータの整合性についての懸念を高め、異なる実験条件で再現性の障壁を装った、報告されています。ここでは、標準化と再現性のあるデータが得られ、癌性悪液質の研究のためのこのモデルの典型的な使用を報告しています。
Protocol
Representative Results
Discussion
特にその最新の段階で、結腸直腸癌は、貧しい転帰と患者の生活の質の低下の原因である悪液質の開発と関連しています。多くの研究は、癌の二次的症状の治療に焦点を当てています。しかし、この方向に多くの努力にもかかわらず、癌性悪液質21には承認された治療法はまだありません。従って、動物モデルは、所見の変換を最大にするためにできるだけ近いヒト病理に似ているこ…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| Cell culture Flasks | Falcon – Becton Dickinson | 35-5001 | |
| DMEM | Cellgro | 10-017-CV | |
| FBS | Gibco | 26140 | |
| Streptomycin-Penicillin | Cellgro | 30-002-CI | |
| CD2F1 mice | Harlan | 060 | |
| Anesthesia apparatus | EZ-Anesthesia | EZ-7000 | |
| 2-Methyl Butane | Sigma-Aldrich | M32631 | |
| OCT | Tissue-Tek | 4583 | |
| Cryostat | Leica | CM1850 | |
| Cork disks | Electron Microscopy Sciences | 63305 | |
| Superfrost plus glass slides | VWR | 48311-703 | |
| Anti-Laminin Rabbit polyclonal antibody | Sigma-Aldrich | L9393 | |
| Anti-Dystrophin Mouse Monoclonal antibody | Vector Laboratories | VP-D508 | |
| Alexa Flour 594 anti-mouse IgG | Life Technologies | A11062 | |
| Alexa Flour 594 anti-rabbit IgG | Life Technologies | A21211 | |
| Hematoxylin | Sigma-Aldrich | GHS216 | |
| Eosin | Sigma-Aldrich | HT110332 | |
| Xylene | Acros Organics | 422680025 | |
| Cytoseal-XYL | Thermo | 8312-4 | |
| Microscope | Zeiss | Observer.Z1 | |
| Bamboo Tablet | Wacom | CTH-661 | |
| Prism 7.0 for Mac OS X | GraphPad Software, Inc. | ||
| Excel for Mac 2011 | Microsoft Corp. | ||
| Image J | US National Institutes of Health | IJ1.46 | http://rsbweb.nih.gov/ij/download.html |
| Microtainer | BD | 365873 |
Riferimenti
- Tan, B., Fearon, K. Cachexia: prevalence and impact in medicine. Curr Opin Clin Nutr Metab Care. 11, 400-407 (2008).
- Aulino, P., et al. Molecular cellular and physiological characterization of the cancer cachexia-inducing C26 colon carcinoma in mouse. BMC cancer. 10, 363 (2010).
- Bonetto, A., et al. STAT3 activation in skeletal muscle links muscle wasting and the acute phase response in cancer cachexia. PloS One. 6, e22538 (2011).
- Bonetto, A., et al. JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia. Am J Physiol Endocrinol Metab. 303, E410-E421 (2012).
- Bonetto, A., et al. Deacetylase inhibitors modulate the myostatin/follistatin axis without improving cachexia in tumor-bearing mice. Current Cancer Drug Targets. 9, 608-616 (2009).
- Acharyya, S., et al. Cancer cachexia is regulated by selective targeting of skeletal muscle gene products. The Journal of Clinical Investigation. 114, 370-378 (2004).
- Fearon, K., et al. Definition and classification of cancer cachexia: an international consensus. The Lancet Oncology. 12, 489-495 (2011).
- Holecek, M. Muscle wasting in animal models of severe illness. Int J Exp Pathol. 93, 157-171 (2012).
- Acharyya, S., et al. Dystrophin glycoprotein complex dysfunction: a regulatory link between muscular dystrophy and cancer cachexia. Cancer Cell. 8, 421-432 (2005).
- Benny Klimek, ., E, M., et al. Acute inhibition of myostatin-family proteins preserves skeletal muscle in mouse models of cancer cachexia. Biochemical and Biophysical Research Communications. 391, 1548-1554 (2010).
- Pedroso, F. E., et al. Inflammation, organomegaly, and muscle wasting despite hyperphagia in a mouse model of burn cachexia. Journal of Cachexia, Sarcopenia and Muscle. 3, 199-211 (2012).
- Soda, K., Kawakami, M., Kashii, A., Miyata, M. Manifestations of cancer cachexia induced by colon 26 adenocarcinoma are not fully ascribable to interleukin-6. International journal of cancer. 62, 332-336 (1995).
- Costelli, P., et al. IGF-1 is downregulated in experimental cancer cachexia. American journal of physiology. Regulatory, Integrative and Comparative Physiology. 291, R674-R683 (2006).
- Palus, S., Akashi, Y., von Haehling, S., Anker, S. D., Springer, J. The influence of age and sex on disease development in a novel animal model of cardiac cachexia. International Journal of Cardiology. 133, 388-393 (2009).
- Norman, K., et al. Effect of sexual dimorphism on muscle strength in cachexia. Journal of Cachexia, Sarcopenia and Muscle. 3, 111-116 (2012).
- Stephens, N. A., et al. Sexual dimorphism modulates the impact of cancer cachexia on lower limb muscle mass and function. Clinical Nutrition. 31, 499-505 (2012).
- Cosper, P. F., Leinwand, L. A. Cancer causes cardiac atrophy and autophagy in a sexually dimorphic manner. Ricerca sul cancro. 71, 1710-1720 (2011).
- Ullman-Cullere, M. H., Foltz, C. J. Body condition scoring: a rapid and accurate method for assessing health status in mice. Laboratory Animal Science. 49, 319-323 (1999).
- Bonetto, A., Andersson, D. C., Waning, D. L. Assessment of muscle mass and strength in mice. Bonekey Rep. 4, 732 (2015).
- Minamoto, V. B., et al. Increased efficacy and decreased systemic-effects of botulinum toxin A injection after active or passive muscle manipulation. Dev Med Child Neurol. 49, 907-914 (2007).
- Murphy, K., Lynch, G. Update on emerging drugs for cancer cachexia. Expert Opin Emerg Drugs. 14, 619-632 (2009).
- Seto, D. N., Kandarian, S. C., Jackman, R. W. A Key Role for Leukemia Inhibitory Factor in C26 Cancer Cachexia. The Journal of Biological Chemistry. 290, 19976-19986 (2015).
- Judge, S. M., et al. Genome-wide identification of FoxO-dependent gene networks in skeletal muscle during C26 cancer cachexia. BMC Cancer. 14, 997 (2014).
- Kliewer, K. L., et al. Adipose tissue lipolysis and energy metabolism in early cancer cachexia in mice. Cancer Biol Ther. 16, 886-897 (2015).
- Aversa, Z., et al. Changes in myostatin signaling in non-weight-losing cancer patients. Ann Surg Oncol. 19, 1350-1356 (2012).
- Bonetto, A., et al. Early changes of muscle insulin-like growth factor-1 and myostatin gene expression in gastric cancer patients. Muscle Nerve. 48, 387-392 (2013).
- Lazarus, D. D., et al. A new model of cancer cachexia: contribution of the ubiquitin-proteasome pathway. The American Journal of Physiology. 277, E332-E341 (1999).
- al-Majid, S., McCarthy, D. O. Resistance exercise training attenuates wasting of the extensor digitorum longus muscle in mice bearing the colon-26 adenocarcinoma. Biol Res Nurs. 2, 155-166 (2001).
- Bonetto, A., et al. JAK/STAT3 pathway inhibition blocks skeletal muscle wasting downstream of IL-6 and in experimental cancer cachexia. American journal of physiology. Endocrinology and metabolism 303. 303, E410-E421 (2012).
- Samuels, S. E., et al. Liver protein synthesis stays elevated after chemotherapy in tumour-bearing mice. Cancer Lett. 239, 78-83 (2006).
- Cornwell, E. W., Mirbod, A., Wu, C. L., Kandarian, S. C., Jackman, R. W. C26 cancer-induced muscle wasting is IKKbeta-dependent and NF-kappaB-independent. PloS One. 9, e87776 (2014).
- Penna, F., et al. Muscle wasting and impaired myogenesis in tumor bearing mice are prevented by ERK inhibition. PloS One. 5, e13604 (2010).
