CAM Assay: raw image data, predicted vessels and a visualised version for human interpretation.

The multifaceted applications of angiogenesis quantification software

Updated in January 2022 by:

Fanny Dobrenova, Siegfried Schwarz and Elisa Opriessnig

Using the latest angiogenesis quantification software products available on the market researchers are now able to accurately study the nature of human blood vessel formation and easily characterize the morphology features of vascular structures. Find out how using automated angiogenesis analysis software can help scholars efficiently quantify blood vessel growth processes. 

In this article we compare five angiogenesis quantification software solutions to help you untap the potential of AI-driven image analysis in vasculogenesis research. With the help of novel angiogenic assay software applications researchers can easily evaluate vascular development and angiogenesis inhibition based on different quantitative parameters.

The angiogenic blood vessel growth process

Angiogenesis, or the formation of new blood vessels, involves the strict regulation of multiple signaling pathways by which newly formed blood vessels emerge from the endothelial cells of pre-existing ones such as arteries, veins, and capillaries. Angiogenesis primarily occurs during embryogenesis and vessel reproduction in the form of vasculogenesis, but it can also be viewed as a salient process in different pathologic conditions, including cancer and inflammation, throughout the lifespan of an organism. 

angiogenesis vs vasculogenesis infographic

Fig 1: how does angiogenesis differ from vasculogenesis

angiogenesis vs vasculogenesis

Fig 1: how does angiogenesis differ from vasculogenesis

Ongoing angiogenesis is even considered an indication of cancer. In fact, the vascular endothelial growth factor pathway plays a pivotal role in tumor angiogenesis. Many cancers exploit this angiogenic activity to stimulate angiogenic tumor growth and supply nutrients to the tumor. 

Further, tumor angiogenesis results in cancer cell invasion and metastasis. For this reason, tumor angiogenesis plays an important role in the regulation of cancer progression, although not completely understood at this point (Zhao and Adjei, 2015). 

tumor angiogenesis

Fig 2: the tumor angiogenesis process. Attribution: Mjeltsch, CC BY-SA 4.0, via Wikimedia Commons

tumor angiogenesis

Fig 2: the tumor angiogenesis process. Attribution: Mjeltsch, CC BY-SA 4.0, via Wikimedia Commons

The study of angiogenesis is a crucial part of tumor research, because it can help reduce both morbidity and mortality from carcinomas. The discovery of angiogenic inhibitors in particular can help prevent neogenic blood vessel formation and tumor cell proliferation (Nishida et al., 2006).  

Angiogenic growth factors

While angiogenesis takes place during embryo development above all, the major physical causes which stimulate angiogenic processes in fully-developed organisms, are tissue ischemia, hypoxia, inflammation and stress. There are a number of specific factors released by tumor cells known to stimulate or inhibit angiogenesis over time, including vascular growth factors, tumor angiogenesis growth factors, inflammatory cytokines etc.. 

VEGF - vascular endothelial growth factors - and VEGF receptors are part of the major angiogenesis signaling pathways. There are five VEGF glycoproteins, which can be distinguished, namely VEGF-A, VEGF-B, VEGF-C, VEGF-D and VEGF-E (Lee et al., 2015). 

The placental growth factors PLGF 1 and 2 are also a part of the VEGF family. VEGF-A and its receptors KLT/VEGFR1 and VEGFR-2 (a tumor angiogenesis receptor) are considered to be the main target areas of current antiangiogenic agents. VEGF-A, for example, can be targeted by applying a specific therapeutic agent to inhibit microvessel growth.    

 

Recent articles suggest that VEGF may also have an additional effect in cancer progression due to the autocrine stimulation of VEGF-receptors in tumor cells. There is increasing evidence of the presence of VEGFRs in liquid and solid tumor cells, e.g. in melanoma, prostate cancer, breast cancer, as well as in leukemia (Lee et al., 2015).

However, the relevance of this expression pattern is still subject to further studies. Tumor growth might not only occur due to angiogenesis induced by VEGF, but can also be the result of direct stimulation via VEGFRs. Thus, endothelial cell-independent pathways may serve as the basis for useful future targets of cancer therapy methods that reach far beyond vascular endothelial growth factors (Lee et al., 2015).  

Angiogenic markers and angiogenesis quantification

Several endothelial cell markers (e.g. PECAM-1/CD31, CD34, vWF) and angiogenesis protein markers are commonly used in immunohistochemistry (IHC) stains of human FFPE tumor sections. Quantitative data obtained from angiogenesis models can include the endothelial cell count or the expression levels of RNAs encoding proteins associated with neovascularization. 

Angiogenesis markers are measured with standardized angiogenic protein assays on the basis of particular clinical parameters like VEGF levels (Rykala et al., 2011). Specialized image analysis software can play a central role in angiogenesis quantification based on these IHC protein markers. Such tools are commonly used for molecular tumor profiling, monitoring of tumor progression and estimating tumor malignancy. In addition to its clinical uses, IHC quantification software has proven to be an invaluable tool in a variety of experimental models for the study of pathological angiogenesis (Shih et al., 2002).  

endothelial cell origin angiogenesis

Fig 3: The generation of endothelial cells plays a central role in the angiogenic process.

endothelial cells origin angiogenesis

Fig 3: The generation of endothelial cells plays a central role in the angiogenic process.

Angiogenesis inhibition in cancer treatment   

Angiogenesis inhibition plays a vital role in cancer treatment. Angiogenesis inhibitory factors serve as cancer-fighting agents by interfering with various steps in the blood vessel growth. For example, they block the formation and growth of new blood vessels that support tumor progression.

Angiogenesis inhibitors may be used as monotherapy or in combination with other anti-cancer drugs. However, preclinical and clinical studies have shown higher therapeutic efficiency using a combined treatment regime in contrast with individual treatments (El-Kenawi & El-Remessy, 2013). 

How do angiogenesis inhibitors work

Different angiogenesis inhibitors are currently applied in the treatment of many cancers (Petrovic et al., 2016; Goel and Mercurio, 2014). Angiogenesis inhibitors can be classified into direct inhibitors, which target endothelial cells in the growing vasculature or indirect inhibitors, which block the activity and expression of angiogenesis inducers. Indirect inhibitors include targeted therapy concepts against oncogenes, conventional chemotherapeutic agents or other drugs aiming at other cells of the tumor microenvironment (El-Kanawi & El-Remessy, 2013). 

direct vs indirect angiogenesis inhibitors

Fig 4: Angiogenesis inhibitors can be grouped into two major categories: direct and indirect ones.

direct vs indirect angiogenesis inhibitors

Fig 4: Angiogenesis inhibitors can be grouped into two major categories: direct and indirect ones.

The suppression of vascular endothelial growth factors (VEGF) is often described in literature. This approach includes not only direct anti-VEGF treatments, either alone or in combination with chemotherapy, but also immunomodulatory drugs with antiangiogenic properties and receptor tyrosine kinase inhibitors, targeting VEGF receptors and their signaling. 

Other approaches aim at the inhibition of VEGF receptor tyrosine kinase activity. Treatment with receptor tyrosine kinase inhibitors may appear relatively non-specific to angiogenesis as many other growth factor receptors share structural similarities in their tyrosine kinase domain. 

The prevailing idea of all of these concepts is to specifically target angiogenesis and other endothelial cell functions. This aspect of VEGF-targeted therapy has been extensively studied (Goel and Mercurio, 2014).

Angiogenesis inhibitor factor examples 

Among the most commonly used VEGF-targeting inhibitory agents are Avastin (Bevacizumab), Aflibercept (Zaltrap) and Ramucirumab (Cyramza). Current research gives insights into the antiangiogenic effects of novel angiogenic inhibitors. For example, promising preclinical studies revealed that Cilengitide, a selective integrin inhibitor, reduces vascular density, vascular permeability and increases survival rates in a model of orthotopically-implanted glioblastoma in rats. Inhibition of FGFR-1–4, PDGFRβ, and VEGFR-1–3 with Dovitinib demonstrated anti-tumor activity in xenografts models of renal cell carcinoma (Ramjiawan et al., 2017). 

angiogenesis inhibition

Fig 5: angiogenesis inhibition with anti-angiogenic agents. Attribution: Mjeltsch, CC BY-SA 4.0, via Wikimedia Commons

angiogenesis inhibition

Fig 5: angiogenesis inhibition with anti-angiogenic agents. Attribution: Mjeltsch, CC BY-SA 4.0, via Wikimedia Commons

The applications of quantification software in angiogenesis stimulation and inhibition research

Using an elaborate angiogenesis analysis model allows researchers to examine the effects of stimulatory and inhibitory agents on vascular formation and growth.  

In vitro angiogenic assays are performed on cell culture and are used to examine specific functions and processes. In vitro angiogenesis assays can be classified into categories such as endothelial proliferation models, endothelial migration models and endothelial cell differentiation models.  

In vivo assays provide a more thorough assessment of essential angiogenesis quantification parameters than in vitro and ex vivo assays, since they allow researchers to study angiogenesis dynamics in a living organism.    

Ex vivo assays make use of organ or embryo culture to examine elaborate angiogenic processes. These models are more complex than in vitro assays, since they involve the interaction of vascular structures with different organ cells and surrounding tissue besides endothelial cells.     

Table 1 displays an overview of the most common types of in vitro, in vivo and ex vivo angiogenic assays discussed in recent research literature.      

In vitro assays

In vivo assays

Ex vivo assays

Boyden chamber assay

Martigel plug assay

Rat aortic ring assay

Endothelial tube formation assay (EFTA)

Corneal micropocket assay

Chick aortic arch assay

Phagokinetic track assay

Chick chorioallantoic membrane (CAM) assay

Choroid sprouting assay

MTT assay

Hindlimb ischemia assay

Retina model assay

Matrix invasion assay

Zebrafish assay

Human placental vessels assay

Fibrin bead assay

Disc assay (DAS) 

Skeletal muscle explant assay

Matrix Metalloproteinase (MMP) assay

Sponge implantation method

Bovine/murine retinal explant assay

Table 1: Angiogenesis assays at a glance

How to choose the angiogenesis assay that fits your needs

There is not a single all-round angiogenesis assay applicable to all scenarios as the specifics of each method prevent the development of one standard procedure. Due to the heterogeneity and diversity of tissues and the complexities of angiogenic reactions, it seems to be an uphill task to develop a single assay for all experimental designs (Shahid et al., 2017). In line with the purpose of your research and depending on which aspect of angiogenesis you want to look at and which cells need to be included, different factors should be considered. 

A number of angiogenesis assays enable an evaluation of pro- or anti-angiogenic activity of stimulating  or inhibitory agents on the basis of their pro- or anti-proliferative, migratory and/or tube formation effects on ECs (Stryker et al., 2019). For this reason, in vitro and in vivo assays are used. In vivo assays allow early stage evaluations, while in vivo methods offer a living microenvironment. Here are some tips on choosing the right assay according to the current state of research. 

choosing an angiogenesis assay

Fig. 6: choosing the right angiogenesis assay

choosing the right angiogenesis assay

Fig. 6: choosing the right angiogenesis assay

First of all, the release rate [R] and the spatial and temporal concentration distribution [C] of a tested compound need to be estimated with the help of the chosen assay in order to evaluate dose-response curves. The method has to yield information on oncogene expression and angiogenic growth factor levels. 

Next, the assays must be designed in a way that quantitative measuring parameters of the newly formed vessels can be defined. This means the chosen methodology must enable you to obtain quantitative data on parameters such as surface area [A], volume [V], vascular length [L], number of vessels in the network [N], fractal dimensions of the network [Df], and extent of basement membrane [BM]. 

In addition, the design of the assay should allow for weighing quantitative measures of morphological characteristics of new vessels such as endothelial cell migration [MR], proliferation rate [PR], canalization rate [CR], blood flow rate [F], and vascular permeability [P]. It is also vital that a clear demarcation between a newly formed vessel and the parent vessels can be detected with the help of the assay.  

When doing the assessment, in vitro methods must always be verified by in vivo methods and an angiogenesis assay for long-term and non-invasive monitoring should be preferred. When choosing an assay, economic, ethical, robustness, and reliability aspects need to be considered as well in order to ensure a smooth workflow (Shahid et al., 2017; Norrby, 2006). 

With the help of advanced deep learning solutions researchers are able to fully automate complete angiogenic assay workflows and obtain quantitative data on vascular formation processes and markers. Such software products largely increase throughput and reproducibility in angiogenesis image analysis. Below we provide an overview of five IKOSA angiogenesis analysis software solutions based on popular types of angiogenic assays.         

CAM assay analysis software  

The chorioallantoic membrane (CAM) method is widely used in ex ovo research to quantify neovascularization. Moreover, the CAM Assay is applied in in vivo cancer and wound healing research for the quantitative analysis of the angiogenic and anti-angiogenic processes.    

The CAM Assay model is commonly performed to study vascular growth patterns in the membrane lining developed around a chicken embryo on the inner surface of an egg shell. Current CAM Assay software enables researchers to automatically extract information on morphological and spatial parameters of the vascular area on the chorioallantoic membrane. Using such applications, parameters such as vascular density, blood vessel diameter, surface roughness, vessel length and lacunarity can be easily quantified.   

​CAM assay IKOSA Prisma microscopy image analysis software

CAM Assay

​CAM Grid Assay IKOSA Prisma image analysis software for microscopy

CAM Grid Assay

Fig 7: The IKOSA Prisma software portfolio features two easy-to-use application modules for the quantification of blood vessels on an avian chorioallantoic membrane (CAM).

If you are currently studying angiogenic growth using the chorioallantoic membrane (CAM) model, the IKOSA CAM Assay application can provide you with an automated state-of-the-art solution to quantify the neovascularization process. The IKOSA CAM Assay application is future-proof in this regard as it allows flexibility. By employing the CAM Assay software you are now able to automatically gain valuable quantitative insights into the vessel area, length, branching points and thickness of blood vessels.   

Alternatively, the IKOSA CAM Grid Assay has been designed for the segmentation of new blood vessels on a chorioallantoic membrane placed on a polymerized grid. This method allows you to collect quantitative data on parameters such as vessel area and number of vessels.       

Fibrin Tube Formation assay IKOSA Prisma microscopy image analysis software

Fibrin Tube Formation Assay

Spheroid Sprouting Assay IKOSA Prisma image analysis software for microscopy

Spheroid Sprouting Assay

Network Formation assay IKOSA Prisma microscopy image analysis software

Network Formation Assay

Fig 8: The IKOSA Prisma portfolio includes a list of versatile angiogenesis quantification software solutions. Find the right one for your research project.

Software solutions for the analysis of angiogenic sprouting

Angiogenic sprouting refers to the morphogenesis of hierarchical networks of vascular sprouts such as arterioles, venules and highly branched capillaries providing efficient blood flow to body organs. Angiogenic sprouting models are widely applied by researchers to examine the dynamics of cancer cell invasion in blood vessel sprouting in vitro. The spheroid sprouting assay method makes use of endothelial cell spheroids or tumor organoids to study this process. 

The spheroid sprouting assay is one of the methods used to quantify the migration of cells as an indicator of angiogenic response. For this purpose spheroids are embedded on a collagen, matrigel of fibrin medium matrix. The migration of cells into the medium involves either the formation of single-cell sprouts or that of complex capillary-like structures. 

Using a specialized image quantification algorithm complex data on different parameters describing the cell invasion process can be collected. Measures mentioned in literature are spheroid core area, edging cell area, detached cell area and envelope radius i.e. the distance between the spheroid border and the maximum point reached by migrating cells (Blacher et al. 2014).     

Using advanced AI-backed software researchers are currently able to perform elaborate time-series analyses on images of angiogenic sprouting models. Such applications allow users to extract spatial and temporal features describing angiogenesis sprouting mechanisms. 

The IKOSA Spheroid Sprouting Assay application allows you to study crucial sprouting parameters of endothelial cell spheroids based on time-lapse images. This particular image analysis software product is perfectly suited for the quantification of features such as the length, number, area and circularity of sprouts.

Tube formation quantification software

Endothelial cell biology has found its rightful place in recent studies. Endothelial cell culture methods are widely used in the study of vascular network formation over time. In vitro vascular network formation research often makes use of endothelial cell samples to examine the development of vessel-like structures or tubes.      

The endothelial tube formation assay (ETFA) is a widely used method to examine the capillary-like growth of endothelial cells on a fibrin matrix. EFTA is a commonly used in vitro method, applied in experimental wound healing and angiogenesis research to study the induction or inhibition of tube formation. 

Matrigel is a cell culture medium widely-used in the context of the tube formation assay method. For this purpose endothelial cells are plated on the matrigel medium in an extracellular matrix. The formation of tube-like structures together with the differentiation of endothelial cells can be easily observed and quantified in this manner.  

AI-driven microscopy image analysis software allows researchers to examine the pseudo-vascular structure of a 3-dimensional fibrin network. This specialized software enables scholars to automatically and precisely detect and quantify extremities, branch structures, segments and junctions of an endothelial cells tubular network. 

The IKOSA Fibrin Tube Formation Assay application helps you gain valuable insights into vital parameters such as number, area, length and branching points of endothelial fibrin tubes in 3D image data.

Angiogenesis analysis software and vascular network formation

Angiogenesis network formation research can greatly benefit from automated deep learning applications. For instance, these applications could support scientific studies on how to block the formation of new blood vessels in order to suppress tumor growth. In other words, researchers are seeking strategies to cut the adequate nutrient supply in the vascular network of cultured endothelial tissue.

The IKOSA Network Formation Assay application allows users to automatically collect relevant information on multiple quantitative parameters such as area, length, branching points and number of tubes from the 2D microscopy image of vascular networks.

Network Formation assay IKOSA Prisma microscopy image analysis software

Network Formation Assay

Fig. 9: Quantify angiogenic processes with the IKOSA Network Formation Assay.

Taking all this into account, it is now time for you to start your own angiogenesis assays. KML offers full flexibility using one of our IKOSA products. If you have recently developed new angiogenesis image analysis assays in your lab and you would like to have them automated, reach out to us at +43 680 156 7596 or drop us a brief message at  ikosa@kmlvision.com.

Acknowledgements

We would like to thank the following project team for the opportunity to use CAM Assay images in this article:  

Dr. Nassim Ghaffari Tabrizi-Wizsy (Otto Loewi Research Center, Immunology and Pathophysiology, Medical University of Graz) provided the expertise of working with the CAM Assay. Lorenz Faihs performed the experiments and imaging/analysis, as well as DI Dr. Peter Dungel and A.o. Univ.-Prof. Mag. DDr. Kurt Schicho (Ludwig Boltzmann Institute for Experimental and Clinical Traumatology and University Clinic for Cranio-, Maxillofacial and Oral Surgery, Medical University of Vienna), who planned the project.

See references