Abstract 490: Highly multiplexed single-cell spatial analysis of tissue specimens using CODEX

Characterizing the complexities of the tumor microenvironment is elemental to understanding disease mechanisms. The spatial relationships between infiltrating immune cells and the remodeling of the cellular matrix is widely recognized as a key component to defining tumor heterogeneity. Current methodologies for analyzing the spatial dimension in tissues, like traditional immunofluorescence (IF) and immunohistochemistry (IHC), are limited to a few parameters at a time, restricting the scope of identifiable cells. Conversely, single-cell technologies like mass cytometry and NGS-based tools provide multiplexing capabilities, but at the expense of the associated spatial information. Here, we present a novel multiplexed imaging technology, termed CODEX, (CO-Detection by indEXing) that combines the high parameter capabilities of single-cell methodologies with the associated spatial dimension. The CODEX technology involves labeling antibodies with oligonucleotide-based Barcodes followed by a single staining step. Over 50 parameters are measured within a single tissue through fully-automated, iterative cycles of adding and removing corresponding dye-conjugated Reporters. Unlike other cyclic IF approaches involving multiple antibody staining and stripping steps, the CODEX platform involves a single initial staining step and subsequent gentle and relatively fast manipulation of the tissue thereafter. This provides a faster workflow and prevents tissue degradation. Other multiplexed imaging technologies, including imaging cytometry and MIBI, require expensive equipment precluding their routine use across various labs. The CODEX technology, developed by Akoya, is comprised of a fluidics instrument that interfaces with existing microscope hardware, as suite of reagents and associated control and analysis software. Over 100 antibody clones have been validated for this platform with more than ten tissue types analyzed, including both FFPE and fresh-frozen from human and mouse samples. The CODEX technology can be used to ascertain complex cellular niches and spatial associations between multiple cell types based on the staining pattern of more than 50 parameters. The CODEX viewer software package enables users to interact with both raw data and cell annotated tissue maps to determine the underlying spatial relationships within each dataset. CODEX data from various normal and cancer tissue types is shown here with corresponding single-cell analysis of key tissue features.