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10084 - Mouse, Breast Cancer, Pos MRI Contrast Ratio

INTRODUCTION
In magnetic resonance imaging (MRI) a contrast agent is used to enhance soft tissue of interest. In a study [See REFERENCES: 1] a method was developed to detect a nanoparticle based contrast agent, specifically designed to target tumor tissue. Polyethylene glycol (PEG) coats the nanoparticles and can be targeted by antibodies through immunohistochemistry (IHC) staining, using diaminobenzidine (DAB).

This APP automatically detects the PEG positive areas in the tissue as well as the ratio compared to the total tissue area, which should give an indication of the tumor growth. 

KEYWORDS
Polyethylene glycol, nanoparticles, immunohistochemistry

METHODS
Hematoxylin was used to stain nuclei (blue) and the brown chromogen diaminobenzidine (DAB) was used to visualize PEG-positive tissue areas as described in the staining protocol [See EXAMPLES: Figure 1 and 2]. VISIOmorph was used to calculate the amount of PEG in the tissue expressed as % of tissue area. The preprocessing consisted of DAB and Hematoxylin color de-convolution. The blue color channel was used to detect the remaining tissue. Three levels of DAB chromogen intensity (high, medium and low) was classified using the Bayesian classification method [See EXAMPLES: Figure 2]. This was done to allow for the possibility to fine tune the configuration and/or to analyze the PEG intensities separately. However, no significant differences were observed when using separate PEG intensities when comparing tumor tissue. Therefore, in the final settings the PEG intensities were added together and the total amount of PEG in the tissue was calculated [See EXAMPLES: Figure 3]. 

QUANTITATIVE OUTPUT VARIABLES
PEGtot: Total PEG stained tissue area 
Total Tissue area: Total tissue area 
PEG (% of Tissue area): Percentage of tissue stained with PEG


AUXILIARY APPS (included)
Auxiliary APPs are used for additional process steps, e.g. finding Region of Interest (ROI).

There are no Auxiliary APPs available.


STAINING PROTOCOL
As described in Eriksson et al [See REFERENCES: 1]:

Paraformaldehyde fixed muscle and tumor tissues collected before injection and at 30 minutes and 2-4 h post-dose were paraffin embedded and thereafter cut (4 µm) and positioned on glass tissue slides. Prior to staining, the slides were placed in an antigen-retrieval solution using an automated pre-treatment module (PT-Link; Dako, Glostrup, Denmark). Slides were stained for PEG in an automated immunohistochemistry robot (Autostainer; Dako) using a primary rabbit anti-PEG-B-47 antibody (Abcam, Cambride, MA) at a concentration of 0.6 µg/ml and a secondary goat-anti rabbit antibody (Dako) at 1:200 dilution. Slides were counterstained with hematoxylin, dehydrated and mounted. The stained slides were digitalized using an ultra-high-resolution slide scanner (Aperio Scan Scope, Aperio Technologies, CA USA) and total PEG-staining was quantified with the VisomorphDP software (Visiopharm, Hoersholm, Denmark) and total PEG-staining was quantified with the Visomorph software (Visiopharm, Hoersholm, Denmark).


ADDITIONAL INFORMATION
This APP was developed by Caroline Sandén at Medetect AB in collaboration with Dept. Airway Inflammation at Lund University, for Spago Nanomedical AB.

Spago Nanomedical is developing a nanoparticle based contrast agent, SpagoPix, for diagnosis of tumors with MRI. The proprietary nanoparticles provide exceptional MR signal enhancement and selectively target tumor tissue via the passive EPR effect (Enhanced Permeability and Retention), which results in improved tumor detection over current contrast agents. The tumor targeting properties were illustrated in a study published in PlosOne (see references) where MRI was used to locate tumors in a clinically relevant mouse breast cancer model (MMTV-PyMT) injected with SpagoPix. The imaging results were supplemented and confirmed with quantification of SpagoPix by chemical analysis (ICP) and with immunohistochemistry using antibodies directed against the polyethylene glycol (PEG) surface coating on the nanoparticles. 

The APP utilizes the EngineTM and Viewer software modules, where EngineTM offers an execution platform to expand processing capability and speed of image analysis. Viewer gives a fast review together with several types of image adjustment properties ex. outlining of regions, annotations and direct measures of distance, curve length, radius, etc. 
By adding the AuthorTM module the APP can be customized to fit other purposes. AuthorTM offers a comprehensive and dedicated set of tools for creating new fit-for-purpose analysis APPs, and no programming experience is required.


REFERENCES
1.Novel Nano-Sized MR Contrast Agent Mediates Strong Tumor Contrast Enhancement in an Oncogene-Driven Breast Cancer Model. Eriksson P-O et al., PLoS ONE 9(10): e107762. doi:10.1371/journal.pone.0107762, 2014. 

RUO
FIGURE 1
FIGURE 1
Close up mouse breast tissue displaying negative nuclei (blue) and PEG positive areas (brown).
FIGURE 2
FIGURE 2
The original image was classified into tissue or PEG positive areas with high, medium and low DAB chromogen intensity.
FIGURE 3
FIGURE 3
A post processing step was used to change the low, medium and high PEG intensities into one, which was used in the calculation step.