Abstract 1179: Deep spatial immuno-profiling through high biomarker colocalization in FFPE tumor tissue sections
Authors: Mohammed Abdul , Wood Douglas , Manesse Mael 
Published: August 2019

Abstract
Background: The recent emergence of highly multiplexed immunohistochemistry (IHC) has the potential to revolutionize immuno-oncology and pathology research as it enables the identification of complex cell subtypes and their potential interactions in the tumor environment. Comprehensive classification of the different cell types in immuno-oncology presents a unique challenge as many of the relevant biomarkers are common to large subsets of cell types within a particular cell class. Therefore, a key feature for accurate phenotypic classification is the ability to detect a high number of colocalized biomarkers. Current IHC technologies offer a low subtyping level due to steric constraints, spectral overlap between dyes, or depletion of deposition sites. Alternative strategies using mass- or cleavable oligonucleotide- tags can potentially achieve high colocalization but are hindered by their low-throughput, high-cost, and elaborate post-acquisition image reconstruction. In this study, we demonstrate how the Ultivue® InSituPlex® technology enables researchers to reliably label and detect at least 4 markers in the same compartment (cytoplasmic or nuclear) on single cells in tumor tissue samples.
Methods: Different FFPE tumor sections were labeled, imaged, and profiled for 15 targets using InSituPlex technology. Staining was carried out in a single workday on a Leica BOND RX autostainer using a cocktail of the 15 primary antibodies relevant to immuno-oncology. Commercially available multi-color fluorescence slide scanners were used to obtain whole slide images. The resulting images were then segmented and analyzed for different phenotypes and their spatial relationships using Indica Labs HALO® software. Additional dimensionality reduction algorithms were used to visualize the high-dimensional phenotypic data of cells with many overlapping biomarkers.
Results: In this poster, we demonstrate the detection of at least 4 co-localized biomarkers on the same cell in FFPE tissue sections. Analysis of the high dimensional, spatially resolved data obtained from a 15-plex assay provided phenotypic information of different lymphocytes, macrophages, dendritic cells, antigen presenting cells, and tumor cell populations with multiple colocalized biomarkers.
Conclusions: InSituPlex technology empowers pathology research through colocalization analysis of many markers on single cells over entire sections of tissue for accurate differentiation and subtyping of cell populations in the tumor microenvironment. As exploratory research in immuno-oncology expands, advancements in multiplex spatial profiling technologies promise to offer greater insights by providing a systems-level view of the biology.
Citation Format: Abdul Mohammed, Douglas Wood, Mael Manesse. Deep spatial immuno-profiling through high biomarker colocalization in FFPE tumor tissue sections [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 1179.