Abstract 1187: Toward high-throughput, high-multiplex FFPE tumor tissue assays for translational research: 7-color, whole slide imaging

Abstract

Background: Immuno-oncology research and predictive biomarkers discovery requires the ability to rapidly identify, quantify, and spatially map many cell sub-types of the tumor environment in FFPE tissue sections. Multiplexed immunofluorescence (IF) enables identification of multiple targets within separate spectral, or color, channels, which in turn enables more in-depth analysis of rare tissue material relative to traditional methods. Although many methods for multiplexed IF exist, they are often costly and require time-consuming assay development and long imaging times. The Ultivue® InSituPlex® technology enables rapid, optimized staining of multiple targets utilizing widely-used immunohistochemistry (IHC) staining and fluorescence imaging instrumentation. Here, we demonstrate the application of InSituPlex technology to image 6 targets and a nuclear counterstain in a 7-color single whole-slide scan, without linear unmixing-based image reconstruction. This technique expands the subtyping depth that can be achieved in a single imaging round and on a single section, and also increases sample-to-answer throughput with a streamlined, single-day workflow. Methods: InSituPlex technology was used to perform 6-plex immune profiling of different FFPE tumor sections. Slides were stained with a cocktail of primary antibodies using a Leica® Bond® RX autostainer and imaged on commercially available fluorescent slide scanners. Subsequent image analysis of the whole slide scans was carried out using Indica Labs HALO® software. Results: In this poster, we present an assay for pathology research that enables fast immuno-profiling in tumor tissues, using a streamlined staining workflow and 7-color imaging. Newly selected fluorophores were used with corresponding optical filter pairs, which increased the number of spectral channels available on commercially available slide scanners while limiting channel cross-talk. Analysis led to the identification of key immune cell phenotypes through marker colocalization without significant ambiguity between targets tagged with spectrally adjacent fluorescent labels. Conclusion: The newly-expanded fluorophore configuration of the InSituPlex technology enables high throughput identification of 6 targets and a nuclear counterstain on whole tumor sections, empowering translational and immuno-oncology research. Citation Format: Amanda J. Bares, Eloise M. Wheeler, Maël Manesse. Toward high-throughput, high-multiplex FFPE tumor tissue assays for translational research: 7-color, whole slide imaging [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1187.