The uses of CAM Assay angiogenesis analysis software

14 Jan, 2021 | Blog posts, Interviews, Prisma

As the chick chorioallantoic membrane (CAM) model raises in popularity in scientific literature, many researchers express interest in using automated bioimage analysis software tailored to the specifics of the CAM method.

Our team contacted Dr. Nassim Ghaffari Tabrizi-Wizsy and researcher Lorenz Faihs, experts in the field of angiogenesis studies, to shed light on the applications of microscopy image analysis software alongside the CAM technique. Dr. Nassim Ghaffari Tabrizi-Wizsy is a researcher at the Institute of Pathophysiology and Immunology at the Medical University of Graz. She has a long list of publications on novel avian chorioallantoic membrane methods for the study of tumor angiogenesis and angiogenesis inhibitor screening (Winter et al., 2018; Ghaffari-Tabrizi-Wizsy et al., 2019). Our article throws light on the experiences of Dr. Ghaffari and her team with computational methods for the evaluation of angiogenic processes.

The CAM Assay Angiogenesis Analysis Method explained

The CAM assay angiogenesis analysis method is an in vivo animal model widely used in the study of cancer cell proliferation and wound healing as well as in tissue engineering. The CAM model enables the evaluation of changes in the blood vessel network of the vascularized membrane of a developing chicken embryo during incubation.

Ex ovo angiogenesis assays provide in vivo culture experimental environments in which the nature of vascular network development can be observed and quantified. Alongside with a number of other ex ovo models like the chick yolk sac membrane (YSM) assay (As et al., 2018) and the early chick embryo blood island assay (Zhou et al., 2018), the CAM model (Deryugina & Quigley, 2018) is widely used for the study of tumor formation, tumor angiogenesis and metastasis

Tips and tricks

To apply this analysis technique a small cut has to be made on the shell of a fertilized egg. Tumor cells and tumor growth agents can then be implanted on the CAM membrane of the chicken embryo or angiogenesis inhibitors can be injected.

The chick chorioallantoic membrane (CAM) model uses ex ovo environments to study angiogenesis.
The chick chorioallantoic membrane (CAM) model uses ex ovo environments to study angiogenesis. Image kindly provided by Dr. Nassim Ghaffari Tabrizi-Wzsy

The role of in vivo models in the study of neovascularization

Angiogenesis is a dynamic process taking place throughout the entire lifecycle of an organism, starting during embryo development. Although angiogenesis is a part of physiological neovascularization activity, it can be stimulated in adult organisms through pathogenic processes in the body such as tumor growth, cell migration and proliferation or inflammation. Angiogenic growth factors are thought to be important in the regulation of these mechanisms. 

Did you know?

Angiogenic growth factors are complex protein compounds that serve as regulators of vasculogenesis. It is essential to measure the expression level of these factors during angiogenesis assays.

Below you can find an overview of common angiogenic growth factors: 

  • Angiogenin (ANG)
  • Vascular Endothelial Growth Factors (VEGF)
  • Transforming Growth Factors (TGF)
  • Epidermal Growth Factors (EGF)
  • Fibroblast Growth Factors (FGF)
  • Platelet-derived Growth Factors (PDGF)

According to Dr. Ghaffari, in vivo angiogenesis assays offer valuable insights into the induction and suppression of angiogenesis, as they allow researchers to observe tissue response and complex host organism interactions to angiogenic and antiangiogenic agents. Such factors enable the control and evaluation of angiogenesis-related changes. Dr. Ghaffari and her colleagues are currently working on a platform for ex ovo research projects on angiogenesis stimulation and inhibition.

Direct Angiogenesis Inhibitors Indirect Angiogenesis Inhibitors
AngiostatinIressa
EndostatinSunitinib
ArrestinAvastin
CanstatinTarceva
TumstatinAMG-706
Table: Overview of angiogenesis inhibition agents. These active agents are used in the treatment of cancer by inhibiting the proliferation of cancer and endothelial cells. Direct inhibitors target endothelial cells and prevent them from reacting to angiogenic growth factors. Indirect inhibitors act by inhibiting the expression of angiogenic growth factors in the tumor.

Experts on the CAM assay principle

We wanted to know how exactly the chorioallantoic membrane assay is performed in experimental lab settings. Dr. Ghaffary explains that the underlying principles involved in blood vessel formation are similar across different animal species. Angiogenesis research makes use of this to observe vascular network formation during chick embryo development and subsequently apply the findings to human therapy. 

The use of the CAM model in a lab environment
This image demonstrates the use of the  CAM model in a lab environment. Image kindly provided by Dr. Nassim Ghaffari Tabrizi-Wzsy.

In addition, researchers can control the vascularization process of the chick chorioallantoic membrane by adding an active agent such as an angiogenesis inhibitor to its tissue. Thus, Dr. Ghaffary and her colleagues can easily assess changes in the number of blood vessels and the vascular network structure.

For this purpose, researchers place an area of the CAM tissue on a collagen matrix to obtain quantitative information on the parameters of the newly formed vascular network. Finally, the quantitative outcomes are compared to the results from control group experiments, where no inhibition agent has been added.

CAM Assay angiogenesis collagen matrix
Avian chorioallantoic membrane tissue on a collagen matrix. Image kindly provided by Dr. Nassim Ghaffari Tabrizi-Wzsy.

The CAM assay method proves to be very efficient in terms of time resources, too. 

Within 72 hours, we identify whether these factors were inhibiting or stimulating growth.

Dr. Nassim Ghaffari Tabrizi-Wzsy, Researcher, Medical University of Graz

Researcher Lorenz Faihs also acknowledges the potential of the CAM model and has made it the focus of his PhD work.

The CAM Assay can be used as a bioreactor. For example, bone grafts or even tumors can be cultured and studied on the CAM.

Lorenz Faihs, Researcher, Medical University of Vienna

Yet, in order to obtain consistent results a standardized methodology to quantify the observed changes is needed. This is where targeted angiogenesis analysis software solutions come into play.

Quantifying the CAM assay method with specialized angiogenesis analysis software

Automated angiogenesis analysis software allows researchers to easily quantify the results of the CAM assay method.

I’m very happy to automate the counting of new blood vessels process with IKOSA and get reliable results without wasting too many resources and being able to focus on our priorities.

Dr. Nassim Ghaffari Tabrizi-Wzsy, Researcher, Medical University of Graz

Further, she explains how the IKOSA Prisma CAM Assay application has helped her not only count the number of new vessels, but also measure the thickness of the vascular branches. The manual method has proven to be cumbersome and time-consuming.

Moreover, the results of manual protocol can be highly subjective. There is always an element of human bias involved, since each researcher evaluates changes in the vascular network differently, as the scholars explain.

Manual CAM assay angiogenesis analysis
A manual counting protocol of the branching points on CAM images.Image kindly provided by Dr. Nassim Ghaffari Tabrizi-Wzsy.

With IKOSA, we can avoid bias, because it is not affected by the “human” factors

Lorenz Faihs, Researcher, Medical University of Vienna

Dr. Ghaffary, when asked how much time it took her to complete the blood vessel quantification protocol manually states:

I remember that we could start counting at 8 am and finish by 1 pm. And this is only part of the experiment.

Dr. Nassim Ghaffari Tabrizi-Wzsy, Researcher, Medical University of Graz

Lorenz Faihs reports trying to use image analysis software developed for other purposes to quantify the results of the CAM assay. But this has also proven ineffective, since the tools have not been targeted to the specifics of the CAM method.

That is why Dr. Graffary and her team reached out to DDr. Michael Mayrhofer at KML Vision to develop a specialized CAM assay application targeted to their research needs. On being asked why they chose our service from a number of alternative software products the researchers shared that they had been looking for a company that understands their field of work, their particular problems and is open to dialogue with science.

Performing automated cell image segmentation with the IKOSA CAM Assay Application

The specialized CAM Assay app included in the IKOSA Prisma portfolio offers valuable insights into a number of reporting parameters central to the CAM methodology.

CAM Assay Angiogenesis Analysis Software
Automated CAM image analysis with the IKOSA software. Image kindly provided by Dr. Nassim Ghaffari Tabrizi-Wzsy.

Using our CAM Assay angiogenesis analysis app allows you to count the number of blood vessels, vessel paths and branching points on a chick chorioallantoic membrane as well as to obtain quantitative information on vessel total length and thickness. This unique image analysis application supports different imaging modalities including multichannel, time series and z-stack image data.

Full interview provided in the PDF file below. No email required.

Written by Fanny Dobrenova and Elisa Opriessnig

Updated in October 2022

If you are interested in testing our CAM assay application and you already are signed up, simply log into your IKOSA account and accelerate your CAM analysis workflows.

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