ARCAS: A Tool to Identify Enriched Spatial Co-localization in Biomedical Images
Creators: name: Corliss, Bruce A.
name: Ray, H. Clifton
name: Cronk, Stephen M.
name: Yates, Paul A.
name: Peirce, Shayn M.
Publisher: name: Zenodo
Year: 2015
Subjects: colocalization
vascular biology

description: Poster presentation from 2015 Biomedical Engineering Society at Tampa, Florida. <strong>Title of Abstract: </strong>ARCAS: A Tool to Identify Enriched Spatial Co-localization in Biomedical Images Bruce A. Corliss<sup>1</sup>, Howard Ray<sup>1</sup>, Stephen M. Cronk<sup>1</sup>, Paul A. Yates<sup>1,2</sup>, Shayn M. Peirce<sup>1</sup>. 1. Biomedical Engineering Department, University of Virginia 2. Department of Ophthalmology, University of Virginia <strong>Introduction:</strong> Spatial co-localization of cells in images of tissues, such as the localization of a perivascular cell adjacent to an endothelial cell, a stem cell next to a differentiated cell, or a macrophage next to a tumor cell, may be indicative of important cell behaviors, such as cellular homing, chemotaxis, or cell-cell communication. There is a lack of quantitative tools and metrics to determine whether the observed fraction of cells colocalizing with other cells in the image is statistically significant over random behavior. The fraction of randomly colocalizing cells changes with each image based on vascular density, cell size, and number of cells observed. We present a new MATLAB program, the Automated Random Cell Association Simulator (ARCAS), that can be used to determine if the co-localization of two different cell populations in an image is greater than random chance. We validated the tool by using it to evaluate whether mouse adipose derived stem cells (mASCs) injected into the vitreous, previously shown by our group to prevent pathological vessel dropout in the retina, exhibit greater than random co-localization with retinal vessels. <strong>Materials and Methods:</strong> Murine ASCs (mASCs) were isolated from epididymal adipose tissue from C57Bl/6J mice using published methods<sup>1</sup>. All mASCs were labeled with DiI. A portion of mASCs were fixed with 4% PFA, which served as the “dead cell” control. Viable mASCs were intravitreally injected into the eyes of Akimba mice, an established transgenic murine model of diabetic retinopathy<sup>2</sup> in which we have previously shown that injected mASCs confer therapeutic benefit by preserving vascular density<sup>1</sup>.Dead mASCs were intravitreally injected into the contralateral eye. Retinas were isolated, wholemounted, and stained with lectin to visualize the vasculature. Images were acquired with a confocal microscope. The lectin+ vasculature was thresholded in ImageJ, and mASCs were quantified with ImageJ’s Cell Counter plug-in. Images were imported into the MATLAB program, and the random colocalization fraction was calculated with a binomial distribution based on vascular area fraction, cell size, and number of cells. The extent of co-localization of mASCs with blood vessels was validated with a Monte Carlo model and hypergeometric distribution. <strong>Results and Discussion:</strong> While all viable injected mASCs showed greater than random co-localization with the vasculature (Figure 1), they were not consistently significantly greater than cells randomly placed- only 58% of images had a significantly enriched colocalization (binomial test, p&lt;0.05, n=8 mice). A similar trend is seen with co-localization from the dead cell control group, with only 37% of images obtaining attaining statistical significance (binomial test, p&lt;0.05, n=7 mice). If injected cells were preferentially migrating to the vasculature, all images for each treatment group would obtain significance compared to random behavior. <strong>Figure 1.</strong> <strong>Images of both viable and dead injected mASCs exhibited greater than random colocalization with vessels as predicted by a binomial model. </strong>Images from each retina are grouped together and the binomial association fraction of cells is calculated based on the vascular density and observed number of injected cells in the field of view. Images were acquired at 20x magnification, with 3-5 zstacks acquired per retina at random stage locations. <strong>Conclusions: </strong>Previously, we hypothesized that co-localization of mASCs with the vasculature was necessary for the vascular protection observed with this treatment. Our present results suggest that co-localization of viable injected mASCs with the vasculature may be no more frequent than co-localization of dead mASCs with the vasculature, implicating paracrine mechanisms of action over cell-cell interactions. Tools, such as these, that provide the statistics to analyze images of multi-cell tissues enable quantitative analyses that shed light on possible biological mechanisms. <strong>Acknowledgements</strong>: NIH EY022063 (to P.A.Y., and S.M.P.),and NIH T32GM008715-16 (to B.C.) <strong>References: 1. </strong>Cronk, Stephen M. <em>et al. </em>Stem Cells Trans. Med. 2015, Online Only. <strong>2.</strong> Wisniewska-Kruk, Joanna <em>et al. Exp. Eye Research. 2014, 122:123-131.</em>