The uses of CAM Assay angiogenesis analysis software
In this article we highlight the uses of CAM assay angiogenesis analysis software in the field of pathophysiology. 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 chick chorioallantoic membrane (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.
To apply this analysis technique a small cut is made on the shell of a fertilized egg. Tumor cells and tumor growth agents can be implanted on the CAM membrane of the chicken embryo or angiogenesis inhibitors can be injected (Figure 1).
As the chick chorioallantoic membrane 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 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.
Angiogenesis 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
Table 1: Overview of angiogenic growth factors. An angiogenic growth factor is a complex protein compound that serves as regulators of vasculogenesis. It is essential to measure the expression level of these factors during angiogenesis assays.
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 (Table 1 and Table 2). 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
Table 2: 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 (Figure 2).
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 (Figure 3) 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.
The CAM assay method proves to be very efficient in terms of time resources, too. Dr. Ghaffary stated that “within 72 hours, we identify whether these factors were inhibiting or stimulating growth.”
Researcher Lorenz Faihs also acknowledges the potential of the CAM model. “The CAM Assay can be used as a bioreactor. For example, bone grafts or even tumors can be cultured and studied on the CAM,” explains Lorenz.
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,” shares Dr. Ghaffari.
Further, she explains how the IKOSA software has helped her not only count the number of new vessels, but also measure the thickness of the vascular branches. The manual method (Figure 4) to do this 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.
“With IKOSA, we can avoid bias, because it is not affected by the “human” factors,” shares Lorenz Faihs.
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 1pm. And this is only part of the experiment.”
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. 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
Using our CAM Assay angiogenesis analysis software 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.
If you are interested in testing our CAM assay application, simply log into your IKOSA account and accelerate your CAM analysis workflows.
Full interview provided in the PDF file below. No email required.
As, M. N., Deshpande, R., Kale, V. P., Bhonde, R. R., & Datar, S. P. (2018). Establishment of an in ovo chick embryo yolk sac membrane (YSM) assay for pilot screening of potential angiogenic and anti‐angiogenic agents. Cell biology international, 42(11), 1474-1483.
Deryugina, E. I., & Quigley, J. P. (2008). Chick embryo chorioallantoic membrane models to quantify angiogenesis induced by inflammatory and tumor cells or purified effector molecules. Methods in enzymology, 444, 21-41.
Ghaffari-Tabrizi-Wizsy, N., Passegger, C. A., Nebel, L., Krismer, F., Herzer-Schneidhofer, G., Schwach, G., & Pfragner, R. (2019). The avian chorioallantoic membrane as an alternative tool to study medullary thyroid cancer. Endocrine connections, 8(5), 462-467.
Winter, R., Dungel, P., Reischies, F. M. J., Rohringer, S., Slezak, P., Smolle, C., ... & Schicho, K. (2018). Photobiomodulation (PBM) promotes angiogenesis in-vitro and in chick embryo chorioallantoic membrane model. Scientific reports, 8(1), 1-9.