Abstract 1371: Spatially-resolved, multiplexed digital characterization of protein distribution and abundance in FFPE tissue sections
Authors: Cesano Alessandra , Beechem Joseph , Webster Philippa , Merritt Chris , Jung Jaemyeong , Dunaway Dwayne , Geiss Gary , Warren Sarah , Mills Gordon 
Published: July 2016

Abstract
Intratumoral heterogeneity has emerged as a critical challenge to the implementation of targeted therapeutics. Historically, immunohistochemistry (IHC) has been used to assess spatial heterogeneity of proteins; however, it has been difficult to quantify protein abundance at high multiplex and wide dynamic range. Here, we report the development and validation of a spatially-resolved, antibody-based proteomic approach with a “barcoding-potential” to quantify up to 800 targets with 5.5 logs (base 10) of dynamic range in a single formalin-fixed paraffin-embedded (FFPE) slide. By labeling antibodies with photocleavable oligos which are recognized by NanoString® nCounter® fluorescent barcodes and subsequently exposing them to focused UV light, we have developed an nCounter assay capable of quantifying protein abundance in a predefined spatial region of a tissue section.
Methods: A slide-mounted FFPE tissue section is bound with a multiplexed cocktail of primary antibody-oligo conjugates, and a microfluidic flow cell is attached to the slide. Using a simple modification of a standard microscope, regions of interest are identified by light or fluorescence microscopy and are sequentially illuminated with UV light to release the oligos. Following each illumination cycle, an eluent is collected and analyzed, resulting in digital counts that correspond to the abundance of each targeted protein in sequentially illuminated areas. We demonstrate a high degree of linearity (0.97 &lt; R2 &lt; 0.99) for the number of observed counts versus area of UV illumination, with current detection spatial resolution down to 100 μm x 100 μm, or approximately 100 cells.
Application: FFPE slides from resected breast cancers are bound with an antibody cocktail (10+ plex, including HER2, EGFR, PR and others) and visualized by light microscopy. Regions of interest are identified, and oligo barcodes from those regions are released by UV illumination and digitally quantified by nCounter analysis. This enables multiplexed detection and comparison of proteins of interest from discrete regions within the tumor and adjacent normal tissue, enabling systematic interrogation of the heterogeneous tumor microenvironment.
Conclusion: Application of this NanoString barcoded antibody platform to ongoing clinical studies is intended to elucidate novel responses to immunotherapy and other targeted therapies. Further development of this technology will enable the multiplexed analysis of up to 800 protein targets from a single FFPE section and facilitate detailed interrogation of spatial interactions within a tissue. The ability to measure DNA, RNA, and protein from FFPE tissue may enable the discovery of immune biomarkers in tumors and the development of companion diagnostics.
Citation Format: Alessandra Cesano, Joseph Beechem, Philippa Webster, Chris Merritt, Jaemyeong Jung, Dwayne Dunaway, Gary Geiss, Sarah Warren, Gordon Mills. Spatially-resolved, multiplexed digital characterization of protein distribution and abundance in FFPE tissue sections. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1371.