Abstract 1183: High-plex spatial profiling of whole FFPE tissue sections using InSituPlex® technology for discovery applications
Authors: Mohammed Abdul Majeed , Xia Zhixin , Chatterjee Gourab , Hwang Kevin , Manesse Mael 
Published: August 2019

Abstract
Background: Comprehensive spatial phenotyping is an increasingly important pathology research tool for investigating the highly complex biology of tumors and their micro-environment. High multiplex immunohistochemical (IHC) strategies enable spatial profiling of tumor samples and expand the depth of information obtained from a single tissue section. Conventional IHC technologies overcome the limitations of low multiplexing by testing serial sections; however, this method does not provide evidence of true marker colocalization and results in inaccurate phenotyping. More recent multiplex methods involving iterative staining and stripping steps have been developed but suffer from low-throughput workflow and can potentially damage samples. Alternative strategies using localized release of mass- or oligonucleotide-tags are not only low-throughput but are also high-cost and require specialized instruments. In this study, we developed a highly streamlined 15-plex fluorescence IHC assay to identify and subtype lymphocytes, macrophages, dendritic cells, antigen presenting cells, and tumor cells on single FFPE tissue sections in a single work day.
Methods: InSituPlex® technology was used to perform high-multiplex immune profiling (15 targets) of different FFPE tumor sections. Slides were stained with a Leica® Bond®RX autostainer using a cocktail of 15 primary antibodies. Whole slide images were obtained using commercially available multi-color fluorescence slide scanners. The resulting images were then segmented and analyzed for different phenotypes and their spatial relationships using the Indica Labs HALOTM software. Tissue sections processed with single-antibody staining were used as references to characterize the performance of the 15-plex assay.
Results: We demonstrate a 15-plex immuno-fluorescence assay on whole FFPE tumor tissue sections in a single work day using the InSituPlex technology. Equivalence of assay performance between the 15-plex and individually stained reference samples confirmed assay specificity and sensitivity in the high multiplexed format. High-dimensional data obtained from the 15-plex assay allowed for a detailed sub-classification of various cell types and their spatial relationship on each single section.
Conclusions: InSituPlex technology empowers immuno-oncology research through the staining of 15 different immune and tumor markers on single FFPE tumor sections, with a fast, automated workflow and reproducible assay. The resulting high-dimensional images are then used to spatially profile various sub-populations of lymphocytes, macrophages, and tumor cells. This study demonstrates the promises of the InSituPlex technology for translational research and discovery applications where rapid imaging of a large number of biomarkers over the entire tissue is highly desired.
Citation Format: Abdul Majeed Mohammed, Zhixin Xia, Gourab Chatterjee, Kevin Hwang, Mael Manesse. High-plex spatial profiling of whole FFPE tissue sections using InSituPlex® technology for discovery applications [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 1183.