The Multifaceted Applications of Angiogenesis Quantification Software

The Angiogenic Blood Vessel Growth Process

Angiogenesis, or the formation of new blood vessels, involves the strict regulation of multiple signaling pathways through 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.

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

Furthermore, tumor angiogenesis can result in cancer cell invasion and metastasis and it plays an important role in the regulation of cancer progression, although not completely understood yet (Lugano et al., 2020).

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 (Jiang et al., 2020). Several successful angiogenesis inhibitors have been developed for various medical purposes. One notable example is Bevacizumab, which is an anti-angiogenic drug used in the treatment of various cancers, including colorectal, lung, and kidney cancer, among others. Bevacizumab works by inhibiting the activity of vascular endothelial growth factor (VEGF), thereby reducing the formation of new blood vessels in tumors (Haibe et al., 2020). There are also several ongoing clinical trials investigating and evaluating anti-angiogenic agents in combination with immune checkpoint inhibitors (ICIs) in solid tumors (Lopes-Coelho et al., 2021). 

Angiogenic Growth Factors

The major physical causes that stimulate angiogenic processes in fully-developed organisms are tissue ischemia, hypoxia, inflammation, and stress. There are several 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 a 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 antiangiogenic agents. VEGF-A, for example, can be targeted by applying a specific therapeutic agent to inhibit microvessel growth. 

Some articles suggest that VEGF may also have an additional effect on 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 proteins associated with neovascularization.

Angiogenesis markers are measured with standardized angiogenic protein assays based on particular clinical parameters like VEGF levels (Rykala et al., 2011). Specialized image analysis software also plays a central role in the quantification of angiogenic protein markers. Such tools are commonly used for molecular tumor profiling, monitoring 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 (Kuri et al., 2022).

Angiogenesis Inhibition in Cancer Treatment

Angiogenesis inhibitory factors serve as cancer-fighting agents by interfering with various steps in 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

Numerous angiogenesis inhibitors are presently utilized in the management of various cancer types (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 therapy concepts against oncogenes, conventional chemotherapeutic agents, or other drugs targeting other cells of the tumor microenvironment (El-Kanawi & El-Remessy, 2013).

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

Angiogenesis Inhibitor Factor Examples

Among the most commonly used VEGF-targeting inhibitory agents are Avastin (Bevacizumab), Aflibercept (Zaltrap), and Ramucirumab (Cyramza) (Ramjiawan et al., 2017). Past 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, and 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 antitumor activity in xenograft models of renal cell carcinoma (Ramjiawan et al., 2017). Current clinical studies target multiple elements within angiogenic pathways, which could potentially offer a solution for anti-angiogenic treatments. For example, Lenvatinib, a promising multi-kinase inhibitor, has demonstrated effectiveness in treating renal cell carcinoma, differentiated thyroid cancer, and hepatocellular carcinoma, primarily due to its anti-angiogenic properties. It targets key receptors including VEGFR, FGFR, PDGFRα, KIT, and RET. Lenvatinib went on a journey from research to clinical application. (Capozzi et al., 2019).

The Use of Image Analysis 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 used to examine specific functions and processes. These assays can be classified such as: 

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 are closer to the physiological environment and 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.

How to Choose The Angiogenesis Assay That Fits Your Needs?

There is not a single all-around angiogenesis assay applicable to every research design 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). 

The factors to be considered in your research will vary depending on the purpose and specific aspect of angiogenesis you wish to investigate, as well as the types of cells that need to be included.

Several angiogenesis tests allow us to assess whether certain substances promote or inhibit blood vessel growth by looking at their impact on the growth, movement, and tube formation of endothelial cells  (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.

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 morphological 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 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).

Such software products largely increase throughput and reproducibility in angiogenesis image analysis. Below, we provide an overview of five IKOSA image analysis solutions, which will help you obtain the optimal results while conducting the angiogenic assay of your choice. Each of these image analysis applications relies on a powerful deep learning algorithm that is able to quickly and reliably process huge amounts of image data. 

Unveiling the IKOSA Angiogenesis Analysis Portfolio

CAM Assay Analysis Software

The Chorioallantoic Membrane (CAM) method is widely used in ex ovo research to quantify neovascularization and to study vascular growth patterns in the membrane lining developed around a chicken embryo on the inner surface of an eggshell. Moreover, the CAM Assay is applied to in vivo cancer and wound healing research for the quantitative analysis of the angiogenic and anti-angiogenic processes.

The IKOSA CAM Assay App enables researchers to automatically extract information on morphological and spatial parameters of the vascular area on the chorioallantoic membrane, such as:

The IKOSA CAM Assay application is future-proof in this regard as it allows flexibility. 

We developed this state-of-the-art algorithm for the analysis of CAM images in close cooperation with leading scientists from the Institute for Molecular and Cellular Anatomy at the University of Regensburg, the Otto Loewi Research Center at the Medical University of Graz, and the Ludwig Boltzmann Institute for Experimental and Clinical Traumatology in Vienna. 

We’d like to express a special thanks to researchers Dr. Silke Härteis and Dr. Nassim Ghaffari Tabrizi-Wizsy for their valuable input. 

Learn how using the IKOSA CAM Application algorithm was put to practice in an article on the quantification of tumor-induced angiogenesis in a 3D in vivo tutor model. One further publication of the research group showcases the use of our CAM Assay Application for quantifying renal cyst growth in kidney tissue. 

Our cooperation with the team of Dr. Nassim Ghaffari Tabrizi-Wizsy at the Medical University of Graz gave rise to another automated tool for quantifying changes in vasculature in CAM images. The IKOSA CAM Grid Assay Application has been developed 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: 

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 during blood vessel sprouting in vitro studies. The spheroid sprouting assay makes use of endothelial cell spheroids or tumor organoids to study this process. 

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

The IKOSA Spheroid Sprouting Assay Application enables the investigation of critical sprouting parameters of endothelial cell spheroids using time-lapse images. This allows you to extract spatial and temporal information on angiogenesis sprouting mechanisms. The application is perfectly suited for the quantification of features such as:

This distinctive spheroid sprouts analysis solution is the result of our collaboration with the Department of Obstetrics and Gynecology at the Medical University of Graz and the Ocular Angiogenesis Group at the Department of Medical Biology of Amsterdam UMC. The development of this application wouldn’t have been possible without the assistance of researchers Dr. Ursula Hiden, Jasmin Strutz MSc, and Dr. Ingeborg Klaassen. 

Our cooperation partners have a proven record of publications on endothelial cell response during angiogenesis, including studies on:  

Fibrin Tube Formation Quantification Software

Endothelial cell culture techniques are extensively employed for investigating the progression of vessel-like structures or tubes as part of vascular network formation studies over time. 

The Endothelial Tube Formation Assay (ETFA) is a widely accepted approach for assessing the capillary-like expansion of endothelial cells on a fibrin matrix. 

Therefore, EFTA is a popular in vitro method applied in experimental wound healing and angiogenesis research to study the induction or inhibition of tube formation.

Matrigel is a solid basement membrane matrix typically used in the Tube Formation Assay, helping endothelial cells with the formation of tube-like structures and differentiation. This method is typically used to study the effects of various compounds on tube formation and for morphological characterization.

AI-driven image analysis software allows researchers to automatically detect and quantify extremities, branch structures, segments, and junctions of an endothelial cell tubular network.

Specifically designed for that purpose, the IKOSA Fibrin Tube Formation Assay application will help researchers gain valuable insights into vital parameters such as:

We’d like to thank Dr. Ursula Hiden and Jasmin Strutz MSc from the Department of Obstetrics and Gynecology at the Medical University of Graz for their support on this project. Find out how their team applied the Fibrin Tube Formation Assay in an article examining outgrowth, proliferation, viability, angiogenesis, and phenotype of primary human endothelial cells. 

Analysis of 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.

Developed in collaboration with the Department of Obstetrics and Gynecology at the Medical University of Graz, the IKOSA Network Formation Assay Application allows users to automatically collect relevant information on multiple quantitative parameters such as: 

Find out how the research team at the Medical University of Graz applies the Network Formation Assay for studying placental angiogenesis.