该<em>卵内</em>鸡胚尿囊膜（CAM）含量为肝癌的有效异种移植模型
Summary
The chick chorioallantoic membrane (CAM) is immunodeficient and highly vascularized, making it a natural in vivo model of tumor growth and angiogenesis. In this protocol, we describe a reliable method of growing three-dimensional, vascularized hepatocellular carcinoma (HCC) tumors using the CAM assay.
Abstract
鸡胚绒毛尿囊膜（CAM）开始7天受精后制定和成熟通过12天的CAM是自然免疫缺陷和高度血管，使其成为肿瘤植入的理想系统。此外，所述CAM包含细胞外基质蛋白，如纤连蛋白，层粘连蛋白，胶原蛋白，整合素（v）的β3，和MMP-2，使其成为有吸引力的模型来研究肿瘤侵袭和转移。科学家长期以来采取所述CAM的生理学优点通过使用它作为血管生成的模型。最近，CAM分析已被修改，使其成为对各种癌症的体内异种移植物模型体系桥接基本体外工作和更复杂的动物癌症模型之间的差距。 CAM分析允许肿瘤生长，抗肿瘤疗法，和亲肿瘤分子途径以生物相关的系统，既具有成本和时间有效的研究。在这里，我们描述了CAM的Xen的发展肝细胞癌（HCC）的高达93％的胚胎存活率和可靠的肿瘤ograft模型上导致立体，血管瘤的生长。
Introduction
肝细胞癌（HCC）是癌症死亡率在世界1中的第三大原因。目前，只有30％的肝癌患者有资格有可能治愈手术治疗2，和全身化疗不灵验3。因此，有迫切未满足的临床需要新的肝癌的治疗，并且适用于新的代理程序有效的测试模型系统的发展。鸡胚绒毛尿囊膜（CAM）试验提供了在体内测试潜在的抗肿瘤药物的可重复的，具有成本效益，并快速介质通量方法。
CAM分析已被广泛用于研究血管生成4。还已经成功地发展成癌症的肿瘤异种移植物模型，包括成胶质细胞瘤5，胰腺癌6，黑素瘤7-9和骨肉瘤10-11 无论是在OVO 12 和前OVO13技术已被用在文献中，以改变从协议到协议的细节。一个主要的挑战CAM移植瘤模型是胚胎死亡的操控卵子后的发病率比较高，与已发表 的鸡胚死亡率从25 – 50％的11-14。
在这篇文章中，我们描述了一个卵内肝癌移植瘤模型，可靠地产生三维，血管瘤的病理类似未分化肝癌生长的发展。我们已经适应首先由Ossowski 等人 14所述的协议，并已实现了高达93％鸡胚存活率具有极高的肿瘤植入。
Protocol
Representative Results
Discussion
在这个协议中几个关键步骤最有可能占了改进的胚胎存活以及肿瘤生长的增加的可靠性。通过施加抽吸到空气囊丢弃所述CAM远离壳是侵入性比其它现有的方法（使用针从卵，钝性分离等除去白蛋白）以下。使用无菌图钉创造这种方法需要使用两个小孔是协议中最具挑战性的部分。使用过大的力可能会损坏到CAM及其脉管通过过度穿透的推针或者甚至可能导致破裂或破坏的蛋。使用图钉动手实践，使?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
A.S. was partially supported by grants from the National Institutes of Health (NIH) National Institute of Dental and Craniofacial Research (5R03DE021741-02) and the National Cancer Institute (1K08CA154963-01A1).
The Howard Hughes Medical Institute provided funding for M.L. through the HHMI Medical Research Fellows Program.
Materials
| Name of Reagent/ Equipment | Company | Catalog Number | Comments/Description |
| Premium incubated eggs | Charles River | N/A | http://www.criver.com/files/pdfs/avian/av_c_spf_egg_price_list.aspx |
| Egg incubators | GQF | Hova-Bator 2362N | |
| Rotating egg trays | GQF | 1611 Automatic Egg Turner | |
| Egg candler | Lyon | Hi-Power 950-070 | |
| Dremel 100 rotary tool with 15/16 cut-off wheel | Dremel | 100-N/7 | |
| Sterile forceps, push pin, dissection scissors, Scotch tape | |||
| Matrigel | BD Biosciences | 356234 | |
| Cryogenic vials, external thread with silicone washer | Corning | 430659 | |
| Collagenase from Clostridium histolyticum | Sigma | C9891 |
References
- Sangiovanni, A., et al. Increased survival of cirrhotic patients with a hepatocellular carcinoma detected during surveillance. Gastroenterology. 126 (4), 1005-1014 (2004).
- Lopez, P. M., Villanueva, A., Llovet, J. M. Systematic review: evidence-based management of hepatocellular carcinoma–an updated analysis of randomized controlled trials. Aliment Pharmacol Ther. 23 (11), 1535-1547 (2006).
- Llovet, J. M., Burroughs, A., Bruix, J. Hepatocellular carcinoma. Lancet. 362 (9399), 1907-1917 (2003).
- Folkman, J. What is the evidence that tumors are angiogenesis dependent. J Natl Cancer Inst. 82, 4-6 (1990).
- Hagedorn, M., et al. Accessing key steps of human tumor progression in vivo by using an avian embryo model. Proc Natl Acad Sci U S A. 102 (5), 1643-1648 (2005).
- Dumartin, L., et al. Netrin-1 mediates early events in pancreatic adenocarcinoma progression, acting on tumor and endothelial cells. Gastroenterology. 138 (4), 1595-1606 (2010).
- Aguirre-Ghiso, J. A., Estrada, Y., Liu, D., Ossowski, L. ERK(MAPK) activity as a determinant of tumor growth and dormancy; regulation by p38(SAPK). Cancer Res. 63 (7), 1684-1695 (2003).
- Baroni, T. E., et al. Ribonomic and short hairpin RNA gene silencing methods to explore functional gene programs associated with tumor growth arrest. Methods Mol Biol. 383, 227-244 (2007).
- Lopez-Rivera, E., et al. Inducible nitric oxide synthase drives mTOR pathway activation and proliferation of human melanoma by reversible nitrosylation of TSC2. Cancer Res. 74 (4), 1067-1078 (2014).
- Balke, M., et al. A short-term in vivo model for giant cell tumor of bone. BMC Cancer. 11, 241 (2011).
- Balke, M., et al. Morphologic characterization of osteosarcoma growth on the chick chorioallantoic membrane. BMC Res Notes. 3, 58 (2010).
- Sys, G. M., et al. The in ovo CAM-assay as a xenograft model for sarcoma. J Vis Exp. (77), e50522 (2013).
- Dohle, D. S., et al. Chick ex ovo culture and ex ovo CAM assay: how it really works. J Vis Exp. (33), (2009).
- Ossowski, L., Reich, E. Experimental model for quantitative study of metastasis. Cancer Res. 40 (7), 2300-2309 (1980).
- Ghanekar, A., Ahmed, S., Chen, K., Adeyi, O. Endothelial cells do not arise from tumor-initiating cells in human hepatocellular carcinoma. BMC Cancer. 13, 485 (2013).
- Ho, J. W., et al. Effects of a novel immunomodulating agent, FTY720, on tumor growth and angiogenesis in hepatocellular carcinoma. Mol Cancer Ther. 4 (9), 1430-1438 (2005).
- Hagedorn, M., et al. VEGF coordinates interaction of pericytes and endothelial cells during vasculogenesis and experimental angiogenesis. Dev Dyn. 230 (1), 23-33 (2004).
