Abstract LB-101: Quantitative imaging of protein expression using multiplex quantum dot immunohistochemistry

Abstract

Abstract Introduction: Immunohistochemistry (IHC) is an important tool for studying protein expression in the tumor microenvironment. Advances in imaging hardware now permit an entire slide to be digitized at high magnification so that software can analyze protein expression within individual cells across the entire tissue section. The chromaphores and organic fluorescent probes used in traditional IHC limit the utility of quantitative IHC, since only relatively few proteins can be measured simultaneously. A new class of fluorescent labels made of semiconductor nanocrystals, known as quantum dots (QD) has unique optical properties that have the potential to overcome limitations of traditional IHC techniques and expand the usefulness of quantitative image analysis approaches. The narrowband emission spectra of these novel probes enable them to be highly multiplexed without significant spectral overlap, providing independent measurements of protein expression with minimal crosstalk. Methods: We have developed a collection of software algorithms around the QD-IHC protocol to analyze protein expression, co-expression, and subcellular localization in whole-slide QD-IHC images. This system segments the digitized tissue into individual cells, their subcellular compartments and multicellular structures to generate comprehensive digital descriptions. Features are calculated for each cell to represent the morphology, protein expression, co-expression, and subcellular localization. Each cell is mapped to their closest multicellular structure, enabling protein expression features to be analyzed as a function of distance to vessels, necrosis, etc. We evaluated the use of QD-IHC for analysis of formalin-fixed, paraffin-embedded glioblastoma tissues against known pathways using streptavidin-biotin detection and applied our software system to analyze expression and co-expression patterns. Features of protein expression were calculated for individual cells along with distance-to-vessel measurements. Results: A five-plex staining was achieved and sections were stained and digitized on a whole-slide scanner (Pannoramic from 3D Histech). The digitized tissues were analyzed using our software system to generate measurements of protein expression in millions of cells. These measurements were used to generate visualizations, including analyses of the variations of expression with respect to distance-to-nearest-vessel. We are continuing to enhance our software capabilities to include statistical tests and updated imaging and analysis capabilities will be presented. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-101. doi:1538-7445.AM2012-LB-101