Abstract 3955: Spatially resolved, multiplexed digital characterization of protein and mRNA distribution and abundance in formalin-fixed, paraffin-embedded (FFPE) tissue sections based on NanoString’s Digital Spatial Profiling (DSP) technology: applications to immuno-oncology (IO) and tumor heterogeneity
Authors: Merritt Chris , Jung Jaemyeong , Ong Giang , Liang Yan , Pakiam Fiona , Dunaway Dwayne , Sprague Isaac , Warren Sarah , Mills Gordon B. , Beechem Joseph 
Published: July 2017

Abstract
Introduction: As intra-tumoral heterogeneity has emerged as a challenge in development of targeted cancer therapeutics, the tissue context of biomarker levels and colocalization of key immunoregulatory proteins has become an increasingly important aspect for understanding tumor immune responses, patient classification, and stratification. Historically, immunohistochemistry and in situ hybridization have been used to assess spatial heterogeneity of targets in clinical samples. These approaches, however, have limited multiplexing capacity and dynamic range. Here, we use DSP technology, a spatially resolved approach for quantifying up to 800 protein or RNA targets with over 5 logs of dynamic range in a single FFPE slide to overcome these limits.
Methods: The technology uses DNA oligo tags for either protein or RNA detection. For protein detection, a cocktail of 30+ primary antibodies (Abs), each with a unique, photocleavable oligo tag, and 1-3 fluorescently (FL) labeled Abs was applied to a slide-mounted FFPE tissue section. Regions of interest (ROI), selected based on a FL imaging scan of the entire tissue, were illuminated sequentially with focused UV light to release the oligos. Following each UV cycle, eluent was collected from the local ROI, moved to a microtiter plate, hybridized to NanoString® barcodes, and then analyzed with an nCounter® Analysis System. The resulting digital counts corresponded to the abundance of each targeted protein in the ROI. For RNA detection, a cocktail of multiple UV-cleavable in situ hybridization probes were used in a similar manner.
Results: We demonstrate multiplexed detection from discrete ROIs within a tumor and adjacent normal tissue, enabling systematic interrogation of a heterogeneous tumor microenvironment. In control samples, we found expected levels of protein and RNA targets. We further demonstrate that this approach enables analysis of target abundance from individually selected cells, both contiguous and non-contiguous with the same phenotype, and enables multiplexed detection of key IO targets. Finally, we demonstrate detection of key IO RNA targets using direct hybridization of oligo-labeled probes.
Conclusions: With further development, our novel DSP approach to capture the spatial context of protein and RNA levels will have many applications in biomarker and translational research. The ability to digitally measure RNA and protein at up to 800-plex from FFPE tissues could facilitate drug mechanism-of-action and response studies within the tumor microenvironment. Quantitative, high-plex data should also greatly accelerate the discovery of IO biomarkers in tumors and the development of companion diagnostics for targeted therapies.
Citation Format: Chris Merritt, Jaemyeong Jung, Giang Ong, Yan Liang, Fiona Pakiam, Dwayne Dunaway, Isaac Sprague, Sarah Warren, Gordon B. Mills, Joseph Beechem. Spatially resolved, multiplexed digital characterization of protein and mRNA distribution and abundance in formalin-fixed, paraffin-embedded (FFPE) tissue sections based on NanoString’s Digital Spatial Profiling (DSP) technology: applications to immuno-oncology (IO) and tumor heterogeneity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3955. doi:10.1158/1538-7445.AM2017-3955