Den Colon-26 Carcinoma Tumor-bærende mus som en model for Studiet af Cancer Kakeksi
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
Muskelsvind er en alvorlig komplikation af forskellige kliniske tilstande, såsom cancer, sepsis, lever, skrumpelever, hjerte og nyresvigt, kronisk obstruktiv lungesygdom, og AIDS. Især muskelsvind er tydeligt i mindst 50% af patienter med cancer 1. Tab af skeletmuskulatur i kræft resultater fra øget proteinnedbrydning på grund af over-aktivering af skelet muskler proteolytiske systemer og / eller fra nedsat proteinsyntese 6. Lipolyse er også tydeligt, hvilket fører til nedbrydning af fedtvæv. Klinisk kakeksi er forbundet med nedsat kvalitet og længden af livet og skønnes at være årsag til dødsfald i 20 – 30% af kræftpatienter 7. Anvendelse af eksperimentelle modeller, der ligner den humane sygdom så tæt som muligt ville være gavnligt. En optimal dyremodel er kendetegnet ved høj reproducerbarhed, samt ved begrænset interferens fra forskellige terapier og uforudsigelige faktorerkost, køn, og genetisk baggrund, der normalt er forbundet med den kliniske tilstand 8. Hidtil har cancer kakeksi undersøgt hovedsagelig i dyremodeller karakteriseret ved transplantation af cancerceller eller injektion af kræftfremkaldende stoffer, selv om en ny metode er at bruge genetisk modificerede mus er modtagelige for udvikling af kræft.
Mus med C26 carcinom (også omtalt som kolon-26 og adenocarcinom) repræsenterer en velkarakteriseret og ekstensivt anvendte model af cancer kakeksi 2,5. Væksten af C26 tumor resultater i krop og muskler vægttab, primært gennem øget fedt og protein katabolisme 9. Generelt er en 10% tumor vægt versus samlede kropsvægt forbundet med en reduktion på 20-25% i skeletmuskulatur vægt og en større udtømning af fedt 3,10. Hepatomegali og splenomegali også observeret med tumorvækst, sammen med aktiveringen af den akutte fase respons og højden af pro-inflationsmåletmmatory cytokinniveauer 3,11. Blandt disse er det velkendt, at IL-6 spiller en central rolle i mediering muskelsvind i C26-modellen, selv om dette cytokin er sandsynligvis ikke den eneste inducer af kakeksi 12. Forhøjet IL-6 forårsager muskelatrofi gennem aktivering af JAK / STAT3-vejen, og inhibering denne transskriptionsfaktor kan forhindre muskelsvind 3,4.
Under C26-induceret muskelsvind, som i mange betingelser for muskelatrofi, er muskelmasse tabt hovedsagelig gennem reduktioner i muskel protein indhold på tværs muskelfibre, ikke gennem celledød eller tab af fibre 13. I C26 kakeksi, er et skift i retning af mindre tværsnitsareal observeret i både glycolytiske og oxidative fibre 2. Dette er også i overensstemmelse med reduceret muskelstyrke 5. Mange grupper verden over har draget fordel af C26 model for at opdage nye mediatorer af muskelsvind eller klinisk relevante lægemidler til kræft cacHexia. Imidlertid er der rapporteret mange forskellige procedurer for brugen af denne model, at hæve bekymringer om konsistensen af de opnåede data og udgør hindringer for reproducerbarhed i forskellige eksperimentelle betingelser. Her rapporterer vi en typisk brug af denne model for studiet af kræft kakeksi, der giver standardiserede og reproducerbare data.
Protocol
Representative Results
Discussion
Især i sine seneste stadier, er kolorektal cancer forbundet med udviklingen af kakeksi, som er ansvarlig for fattigere resultater og reduktioner i patientens livskvalitet. Mange undersøgelser har fokuseret på behandling af tilstande sekundært til cancer; Men trods mange bestræbelser i denne retning, er der stadig ingen godkendt behandling for cancer kakeksi 21. Således er det bydende nødvendigt, at dyremodeller ligne den menneskelige patologi så tæt som muligt for at maksimere oversættelse af …
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 |
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