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Angiogenesis is the process of formation of new blood vessels from pre-existing vessels or endothelial cell progenitors. It plays a crucial role in tumor growth and metastasis. Tumor angiogenesis have been widely studied as an important target for suppressing tumor growth and metastasis. Here, we describe an in vivo chick embryo chorioallantoic membrane (CAM) model. The chick embryo chorioallantoic membrane is an extraembryonic and is rich of blood vessels. After exposing the vascular zone of the CAM, a sterilized filter-paper disk is employed, which is used as a carrier for being loaded with various chemicals, drugs or virus. Finally, the CAM was fixed and spread on glass slide, and the blood vessels were quantified by counting the number of blood vessel branch points. Compared with the matrigel plug angiogenesis assay, in which tumor cells are mixed with the matrigel gel (expensive) and injected into the mice, subsequently using immunohistochemistry (IHC) staining (time consuming) with the endothelial marker to indicate the presence of the newly formed capillaries, the main advantages of CAM model are its low cost, simplicity, reproducibility, and reliability. Thus, the CAM can be widely used in vivo to study both angiogenesis and anti-angiogenesis.

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In vivo Chick Chorioallantoic Membrane (CAM) Angiogenesis Assays

Cancer Biology > Angiogenesis > Drug discovery and analysis > Metabolism
Authors: Zhenguo Chen
Zhenguo ChenAffiliation: Department of Cell Biology, Southern Medical University, Guangzhou, China
Bio-protocol author page: a850
Zhihua Wen
Zhihua WenAffiliation: Department of Cell Biology, Southern Medical University, Guangzhou, China
Bio-protocol author page: a851
 and Xiaochun Bai
Xiaochun BaiAffiliation: Department of Cell Biology, Southern Medical University, Guangzhou, China
For correspondence: baixc15@smu.edu.cn
Bio-protocol author page: a852
Vol 3, Iss 18, 9/20/2013, 9538 views, 3 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.913

[Abstract] Angiogenesis is the process of formation of new blood vessels from pre-existing vessels or endothelial cell progenitors. It plays a crucial role in tumor growth and metastasis. Tumor angiogenesis have been widely studied as an important target for suppressing tumor growth and metastasis. Here, we describe an in vivo chick embryo chorioallantoic membrane (CAM) model. The chick embryo chorioallantoic membrane is an extraembryonic and is rich of blood vessels. After exposing the vascular zone of the CAM, a sterilized filter-paper disk is employed, which is used as a carrier for being loaded with various chemicals, drugs or virus. Finally, the CAM was fixed and spread on glass slide, and the blood vessels were quantified by counting the number of blood vessel branch points. Compared with the matrigel plug angiogenesis assay, in which tumor cells are mixed with the matrigel gel (expensive) and injected into the mice, subsequently using immunohistochemistry (IHC) staining (time consuming) with the endothelial marker to indicate the presence of the newly formed capillaries, the main advantages of CAM model are its low cost, simplicity, reproducibility, and reliability. Thus, the CAM can be widely used in vivo to study both angiogenesis and anti-angiogenesis.

Keywords: Chick chorioallantoic membrane, Angiogenesis, Blood vessel branch

Materials and Reagents

  1. Fertilized E6 chicken embryos (from Poultry Center of South China Agricultural University)
  2. 0.1% Benzalkonium Bromide (from Guangzhou Chemical Reagent Factory, diluted in sterile water before use)
  3. Methanol and acetone (1:1 in volume)
  4. Filter-paper disk (Whatman, catalog number: 1441150)
  5. Packing film (Parafilm)

Equipment

  1. Incubator
  2. Glass slide
  3. Ophthalmic forceps

Procedure

  1. Fertilized E6 chicken embryos (48 ± 5 g) were cleaned with 0.1% Benzalkonium Bromide and preincubated at 37.5 °C in 85% humidity for 2 days.
  2. Egg morphology appears like a meta-ellipse, with a relatively larger side and a smaller one, and the air sac is usually located on the larger side right behind the shell. After disinfection of the shell center outside the air sac with 0.1% Benzalkonium Bromide, a hole highlighted with marker pen was buffed and drilled gently over the air sac with a nipper not to break the shell, and the vascular zone was easy to be identified on the CAM (Figure 1).


    Figure 1. The vascular zone of the CAM

  3. Two drops of normal saline were then added to moisten the inner shell membrane adjacent to the CAM so that the membrane was easy to be separated from CAM.
  4. After being clamped and raised by ophthalmic forceps, the membrane and the CAM separated unforcedly, and then a 1 x 1 cm window on the membrane was sectioned to expose the vascular zone.
  5. A 5 mm x 5 mm sterilized filter-paper disks, which were used as a carrier for being directly loaded with indicated concentrations of chemicals or virus, were then directly applied and adhere to the vascular zone with right density of vascular.
  6. Upon sealing the openings with sterile flexible packing film, the eggs were further incubated for indicated periods.
  7. Finally, a mix of methanol and acetone (1:1 in volume) was directly added to immerse and fix the blood vessels of the experiment zone.
  8. After being clamped and raised by ophthalmic forceps, the CAM was easy to be separated from the embryo, and it was cut and spread on glass slide, and the blood vessels were viewed, photographed and quantified by counting the number of blood vessel branch points (Figure 2).


    Figure 2. Blood vessel branch points on CAM. Arrow indicates new-formed blood vessel branches.

Acknowledgments

This protocol was adapted from the following published papers: Wen et al. (2013); Chen et al. (2014). This work was supported by The State Key Development Program for Basic Research of China (2009CB 918904, 2013CB945203), National Natural Sciences Foundation of China (30870955, 91029727, 30900555) and Program for New Century Excellent Talents in University (NCET-08-0646).

References

  1. Chen, Z., Zhang, Y., Jia, C., Wang, Y., Lai, P., Zhou, X., Wang, Y., Song, Q., Lin, J., Ren, Z., Gao, Q., Zhao, Z., Zheng, H., Wan, Z., Gao, T., Zhao, A., Dai, Y. and Bai, X. (2014). mTORC1/2 targeted by n-3 polyunsaturated fatty acids in the prevention of mammary tumorigenesis and tumor progression. Oncogene 33(37): 4548-4557.
  2. Wen, Z. H., Su, Y. C., Lai, P. L., Zhang, Y., Xu, Y. F., Zhao, A., Yao, G. Y., Jia, C. H., Lin, J., Xu, S., Wang, L., Wang, X. K., Liu, A. L., Jiang, Y., Dai, Y. F. and Bai, X. C. (2013). Critical role of arachidonic acid-activated mTOR signaling in breast carcinogenesis and angiogenesis. Oncogene 32(2): 160-170.


How to cite this protocol: Chen, Z., Wen, Z. and Bai, X. (2013). In vivo Chick Chorioallantoic Membrane (CAM) Angiogenesis Assays. Bio-protocol 3(18): e913. DOI: 10.21769/BioProtoc.913; Full Text



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7/6/2016 12:34:03 AM  

surendra reddy
yogivemanauniversity

sir . please tell me the controls(positve and negetive) which you are used and what concentrations are used

7/10/2016 7:40:45 PM  

Zhenguo Chen (Author)
Department of Cell Biology,Southern Medical University

Thanks for your interest. Could you please specify for what positive and negative controls you mentioned.

7/13/2016 10:53:39 PM  

surendra reddy
yogivemanauniversity

for CAM i have taken suramin disodium salt as controller . please tell me the other controllers

7/13/2016 11:16:09 PM  

Zhenguo Chen (Author)
Department of Cell Biology,Southern Medical University

You use suramin disodium as a control for what chemicals?

7/13/2016 11:38:27 PM  

surendra reddy
yogivemanauniversity

phycocyanin

7/14/2016 12:08:51 AM  

Zhenguo Chen (Author)
Department of Cell Biology,Southern Medical University

But we did not mention phycocyanin in the protocol.

Reply

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2/9/2016 9:47:56 AM  

Hruaia Pautu
Entomology Research Institute

Firstly, I've been looking for CAM assay, most of them did not mention details protocol.
Thanks for your article.
Secondly, For cleaning purpose besides 0.1% Benzalkonium Bromide, what else can we use?

7/10/2016 7:31:59 PM  

Zhenguo Chen (Author)
Department of Cell Biology,Southern Medical University

To our knownledge, neither 75%alcohol nor iodophor was suitable, so 0.1% Benzalkonium Bromide is the best choice. Thanks for your inquiry。

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4/8/2015 9:13:22 AM  

glad mohesh
SSSMCRI

dear author.your article is informative, however as a new aspirant to learn this technique kindly help me with your pdf or video files.thankyou

4/15/2015 7:36:15 PM  

Zhenguo Chen (Author)
Department of Cell Biology,Southern Medical University

A PDF can be downloaded directly from the website.Thank you for your interest.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
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