Abstract 3858: Validation of digital spatial profiling of key immuno-oncology targets for mouse FFPE preclinical models
Authors: Warren Sarah , Metz Heather , Barker Kristi , Gong JingJing , VanSchoiack Alison , Huynh Quoc , Merritt Chris , Dennis Lucas , Liang Yan , Beechem Joseph M. 
Published: August 2018

Abstract
Mouse models of cancer are an invaluable biomedical research tool in which characterization of the abundance, distribution and co-localization of immuno-oncology targets is critical for the advancement of disease understanding. Nanostring Technologies' Digital Spatial Profiling (DSP) technology allows for the simultaneous analysis of 10s to 100s of proteins from discrete regions of interest (ROI), providing a spatial context to multiplexed data. The purpose of this study is to validate an antibody panel designed to characterize key tumor and immunology markers on the DSP platform. DSP technology utilizes barcoded oligo tags that are covalently linked to detection reagents, in this case, antibodies. The workflow for a typical DSP experiment consists of incubating a formalin-fixed, paraffin-embedded (FFPE) tissue with a cocktail of oligo-labeled primary antibodies and three fluorescently labeled visualization markers. The visualization markers and tissue morphology guide the selection of ROIs. UV light then illuminates each ROI, releasing the oligo tags through a UV photocleavable linker. Tags are collected through microcapillary aspiration and then deposited into a microtiter plate for further analysis on the standard nCounter® platform. In order to validate antibodies for this technology, the specificity and sensitivity of each antibody was evaluated for proper immunohistochemistry (IHC) staining pattern on appropriate tissues as well as signal-to-noise ratios of positive counts above background. Panels will also undergo an evaluation of single vs. multiplex interactions to ensure there are no deleterious effects of multiplexing antibodies. Immunohistochemical analysis of antibodies chosen for panel displays indistinguishable staining patterns on mouse control tissues for unconjugated primary antibodies and oligo-conjugated primary antibodies. Visualization markers allow for an ROI selection strategy that utilizes a range of circular ROIs (e.g., 50-650um) as well as custom masking of areas of interest. Key immuno-oncology targets including B7-H3, beta-catenin, CD19, CD25, CD3, CD4, histone H3, Ly-6G, pan-cytokeratin, PD-1, PD-L1, F4/80, Ki-67, STAT3, etc., have been validated thus far. Ongoing validation efforts will continue to expand this antibody panel. Nanostring Technologies' DSP platform is applicable not only to human tissue but also to mouse preclinical samples. Antibody specificity, sensitivity and interaction within a cocktail have been validated for this application. DSP technology allows for the multiplexing of important mouse immuno-oncology markers so that researchers can evaluate the abundance as well as the spatial distribution of these key targets simultaneously.
Citation Format: Sarah Warren, Heather Metz, Kristi Barker, JingJing Gong, Alison VanSchoiack, Quoc Huynh, Chris Merritt, Lucas Dennis, Yan Liang, Joseph M. Beechem. Validation of digital spatial profiling of key immuno-oncology targets for mouse FFPE preclinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3858.