ApplIcAtIon note

Definiens Developer and Definiens Architect

Analysis of Blood Vessel Formation from Full Image Sections Using Definiens Developer and Definiens Architect
Introduction
The search of drugs for an effective, non-toxic antiangiogenesis therapy to control the growth of blood vessels remains an important contemporary theme in cancer drug development. The discovery of a biological control for angiogenesis is critical to the clinical treatment of solid tumor growth. Translating an angiogenesis lead compound from the laboratory to the clinic can involve assays to: (1) define the molecular target1, (2) determine whether endothelial cell migration and proliferation are inhibited1, (3) test for an angiogenesis activator or inhibitor using the normally a-vascular cornea, which enables quantification of the number of vessels and the rate of vascular in-growth1, or (4) rigorously test an angiogenesis inhibitor to evaluate it in a vascularized organ such as the brain2. In migration and proliferation assays, one of the biggest challenges is the precise determination of endothelial cells lining real blood vessels, which includes the exact differentiation between real and false lumens and the staining of tissue distortion and background. This application note describes the use of Definiens Developer and Definiens Architect for the novel automated analysis of blood vessel formation in full section images.

Description of the Experiment

The application is developed for recognizing blood vessels using immunostaining to define endothelial cells lining real blood vessels. The application aims to count the number of blood vessels. In addition, blood vessel characteristics can be collected, such as data about the size, shape, staining intensity amongst others. The application has been primarily developed for images acquired with 10x magnification, since this magnification was assumed to be the most desired/commonly used image acquisition setting. Modification of the application for other magnifications is relatively simple.

Method
A major challenge in angiogenesis image analysis is to extract the blood vessels as structures of interest in an automated and reliable manner. Differences between treatment groups are automatically detected and potentially subtle patterns of activity can be found by using the Definiens Enterprise Image Intelligence Suite.

Key words
Angiogenesis Image Analysis Definiens Architect Definiens Developer

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Angiogenesis Analysis Protocol
The overall goal of the analysis protocol is the precise identification, extraction, and quantification of the blood vessel formation during different treatments with potential drug candidates. The main steps of the application consist of the following subroutines: Background detection Nuclei detection Detection of stain Lumen detection Identification of blood vessels Export of customized measures During first steps, background and cell nuclei are identified, followed by finding the components of the blood vessels (i.e. stained epithelial cells, lumens). In a subsequent step, the application proceeds with the recognition of complete vessels, and, finally with the segmentation of the blood vessel boarders.

Figure 1 Original image

Angiogenesis Analysis Result

With a successful angiogenesis image analysis different result displays can be visualized as shown in the following figures. In the image (see figure 2) cell nuclei and also blood vessel walls are detected. In the image (see figure 3) complete blood vessels, including the lumen as part of the vessel structure, are successfully identified. After a successful detection of blood vessels and other regions of interest the next steps required are to export a number of meaningful parameters that best describe any detected differences between treatment groups. A number of customized measures could be created such as blood vessel area, vessel length and total vessel length. These measures can be exported as CSV files and the data can be plotted to compare differences in drug treatment. This analysis represents a challenging task due to the relatively low intensity of the endothelial cell immuno staining in the blood vessels, high background level due to necrosis and background emerging from stroma-tissue.
Figure 2 Analysis results showing the identified blood vessel structures
(green) and eliminated background staining (grayed out)

Figure 3 Analysis results showing stained blood vessel walls (red),

possible lumen structures (aquamarine blue), cell nuclei (blue) and
background (grayed out).

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Conclusions
During this study, blood vessels with lumens were identified, tissue distortions were not classified as lumens, and background was excluded. In addition, stained blood vessel walls, possible lumen structures, cell nuclei and background were analyzed. Of perhaps greater significance is the ease with which a complex analysis was reduced to straightforward practice using Definiens software. Creating a custom application within Definiens Developer and then converting the components into individual actions for use in Definiens Architect allows complex custom made analysis to be deployed and reused by non-expert users. This application note demonstrates the capabilities of the Definiens Enterprise Image Intelligence Suite for the analysis of complex heterogeneous image information in full image sections. Subtle structures such as blood vessels can be reliably detected and quantified. Differences in the intensity of immunostained endothelial cells in the blood vessels and high background levels do not represent a challenge for this analysis approach. Acknowledgements Images courtesy of Juha Kononen MD PhD, Beecher Instruments Inc., Sun Prairie, WI 53590, USA www.beecherinstuments.com

References
1 2

Cancer Res. 1994; 54:2654 -2660 Am J Pathol. 1990; 137:1121-1142

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