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

Abstract

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. Overall, the CODEX platform is an accessible and versatile technology for high parameter, spatial profiling of tissue specimens. Citation Format: Gajalakshmi Dakshinamoorthy, Jaskirat Singh, Joseph Kim, Nadya Nikulina, Roya Bashier, Sejal Mistry, Maria E. Gallina, Atri Choksi, Meenu Perera, Ashley Wilson, Julia Kennedy-Darling. Highly multiplexed single-cell spatial analysis of tissue specimens using CODEX [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 490.

Overview of multiplex immunohistochemistry/immunofluorescence techniques in the era of cancer immunotherapy

“…Unlike conventional IF and IHC which are limited to measuring few parameters simultaneously and single‐cell technologies like mass cytometry that have limited spatial information, CODEX uses high‐throughput technology to detect more than 50 biomarkers simultaneously in a single tissue sample. In addition, it provides information about the relative abundance and expression of these biomarkers at a spatial level [22].…”

“…Although a study reported minimal steric hindrance when large panels of CODEX antibodies are used for staining, it was found that there are some cases where the signal might be lower [22]. Hence, there is still a need for the users to perform proper controls for specific antibodies and combinations of interest.…”

“…Several other studies[20‐22] have utilized different types of mIHC/IF to assess their tumor samples and obtained the data required for analysis. However, these methods were not without their limitations.…”

ME-VAE: Multi-Encoder Variational AutoEncoder for Controlling Multiple Transformational Features in Single Cell Image Analysis

“…1,2,3 Until recently, analyzing single cell imaging data in a similar fashion was limited to extracting mean intensity profiles, predefined shape, textural and morphological features, and images stained with only a few markers. Emerging multiplexed imaging technologies such as cyclic immunofluorence (CYCIF), 4 multiplexed immunohistochemistry (mIHC), 5 CO-Detection by indEXing (CODEX), 6 and Multiplexed ion beam imaging (MIBI) 7 can create images stained with a large number of markers, expanding the depth of features. Robust analysis methods for high dimensional multiplexed imaging data, however, are not as well developed as single cell RNAseq (scRNAseq) analysis tools.…”

CGAT: Cell Graph ATtention Network for Grading of Pancreatic Disease Histology Images

“…This visually driven approach has been marred with subjectivity, with very low inter-observer agreements in many cases ( 8 , 9 ). Moreover, though Hematoxylin and Eosin (H&E) is the most widely used staining paradigm, the emergence of molecular and antigenic-based staining paradigms, such as multiplexed immuno-florescence and CODEX had allowed for the identification of more than 20 antigens on the cell’s surface, enabling richer tissue information to be available ( 10 , 11 ). It is possible to now characterize multiple sub types of different cell phenotypes, of which immune cells are of great interest.…”