MiCASA is a new method for quantifying cellular organization

ARTICLE Received 14 Sep 2016 | Accepted 7 Apr 2017 | Published 30 May 2017 DOI: 10.1038/ncomms15619 OPEN MiCASA is a new method for quantifying cellular organization Andrew Sornborger1, Jie Li2, Cullen Timmons2,w, Floria Lupu2, Jonathan Eggenschwiler2, Yousuke Takahama3 & Nancy R. Manley2 While many tools exist for identifying and quantifying individual cell types, few methods are available to assess the relationships between cell types in organs and tissues and how these relationships change during aging or disease states. We present a quantitative method for evaluating cellular organization, using the mouse thymus as a test organ. The thymus is the primary lymphoid organ responsible for generating T cells in vertebrates, and its proper structure and organization is essential for optimal function. Our method, Multitaper Circularly Averaged Spectral Analysis (MiCASA), identifies differences in the tissue-level organization with high sensitivity, including defining a novel type of phenotype by measuring variability as a specific parameter. MiCASA provides a novel and easily implemented quantitative tool for assessing cellular organization. 1 Department of Mathematics, University of California, Davis, California 95616, USA. 2 Department of Genetics, Paul D. Coverdell Center, University of Georgia, 500 DW Brooks Drive, Athens, Georgia 30602, USA. 3 Division of Experimental Immunology, Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan. w Present address: Department of Emergency Medicine, Vanderbilt University Hospital, Nashville, Tennessee 37232, USA. Correspondence and requests for materials should be addressed to A.S. (email: ) or to N.R.M. (email: ). NATURE COMMUNICATIONS | 8:15619 | DOI: 10.1038/ncomms15619 | www.nature.com/naturecommunications 1 ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms15619 T he lack of quantitative methods to assess and describe the organization, as opposed to composition, of tissues and organs is a significant technical and theoretical barrier in the study of many organs and tissues. Organs are more than the sum of their component parts—functional competence requires that these parts not only be present in the appropriate proportions, but also be arranged in specific ways. However, there are few quantitative tools for evaluating and comparing tissue organization. As a result, organization is usually assessed by qualitative and subjective methods (whether it ‘looks organized’). This lack of tools constitutes a critical problem; without a quantitative framework to characterize the functional organization of organs and tissues, we currently do not have a language to describe its disintegration during disease or involution, or benchmarks to evaluate regenerative therapies. The thymus is an excellent example of the connection between organization and function. The thymus consists of developing T cells, or thymocytes, supported by a complex cellular environment containing a variety of resident cell types, including thymic epithelial cells (TEC), dendritic cells, vasculature and mesenchymal cells1. These cell types comprise multiple microenvironments that direct and support thymocytes to develop from immature progenitors into mature T cells that are both self-tolerant (will not attack the body’s own cells) and self-restricted (only recognize antigens in a specific ‘self’ context). T-cell development in the thymus is not a cell autonomous process, but requires interactions with the thymic microenvironments that provide signals for their survival, proliferation and differentiation2–5. Failure of these events results in immunodeficiency or autoimmunity. Thymic output is quantitatively and qualitatively correlated with peripheral immune function. Loss of thymic output occurs during aging and because of a wide variety of conditions including genetic disorders, disease and cancer therapies such as irradiation and chemotherapy6,7. Transient or permanent thymic rejuvenation thus has major consequences for human health. Establishing quantitative, predictive models of thymic structure and function could have significant implications for understanding the process of immunosenescence and for evaluating the effectiveness of clinical interventions. In spite of its critical role in the generation of cellular immunity, the composition and organization of thymic microenvironments and the mechanisms that promote proper development and function are not fully understood. To date, no quantitative models of thymus organ structure and function exist in the literature. In this report, we have addressed this need by developing a statistical framework for measuring cellular associations that quantify cellular organization in an organ or tissue, in this case the mouse postnatal thymus. We then use this tool to evaluate organ structure in previously published mutant strains with defects in TEC differentiation and organ structure. These analyses show that Multitaper Circularly Averaged Spectrum Analysis (MiCASA) can detect statistically robust phenotypic differences at earlier stages than can be identified by the eye, and can identify novel types of phenotypic differences including changes in the variability (Variance) of cellular organization. We also show MiCASA analysis of two different sets of markers in wild-type mouse fetal spinal cord, demonstrating that the resulting methodology may be used, more generally, for characterizing cellular organization elsewhere, including tissues outside of the immune system. Results Calculating relative spatial distributions of cell types. To develop both a rapid screening method for assessing thymic 2 organization and a quantitative method to assess specific cellular associations within the thymus, we developed a statistical framework for measuring cellular associations based on cellular correlation functions. We calculate these correlation functions in the frequency domain and average them over all angles, providing a summary of the distribution of individual cell types and associations between cell types. We call our method MiCASA. Similar analytical methods are commonly used in cosmology to describe and quantify structure in the distribution of galaxies throughout the universe8–10. In the resulting graphs, the ordinate measures structure within a cellular distribution (log-spectra) or correspondences between two distinct cell distributions (atanh-coherence). The abscissa represents the logarithm of spatial frequency, which, in turn, is inversely proportional to the intercellular distance. Thus, this axis essentially measures characteristic cellular separations, with distances decreasing to the right of the graph. Each parameter can be evaluated for statistical significance, giving a quantitative and sensitive measure of cellular organization. Because of the ability of each parameter’s distribution to be analysed for statistical significance, our approach can yield a great deal of detailed informati (...truncated)