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.

Natural selection shapes traits during evolution including animal coloration known to be important for concealment and communication and color has been particularly salient in the explosive radiation of cichlid fish species in the rift valley lakes of East Africa. Though selection can produce variation in color via genetic substrates during early development, plasticity in coloration can occur through endocrine, neural, and transcriptional cues in response to various environmental stimuli. It is well known that some animals often change color to match their visual ecology. Adult male cichlid fish (Astatotilapia burtoni, Lake Tanganyika) can switch between blue and yellow body colors. Different colors result from the expression of pigment-bearing cells, which differ in density and function between these two color morphs. We show that A. burtoni switches from yellow to blue depending on their visual environment by downregulating endothelin receptor B (EdnRB) mRNA via DNA hypermethylation at a single cytosine residue within its promoter. EdnRB functions in yellow chromatophores to signal the aggregation of yellow pigments, making yellow less visible. Taken together, the regulation of EdnRB through DNA methylation in yellow chromatophores, in part, contribute to pigmentation changes from blue to yellow, depending on the visual environment.

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BackgroundMultiplexed immunofluorescence (mIF) allows the visualization of multiple biomarkers in a single tumor tissue section, while at the same time preserving the spatial biology of the tumor microenvironment (TMI). CO-Detection by indEXing (CODEX®) and Phenoptics™ platforms are complementary mIF technologies that span the full spectrum of cancer research, from discovery to translational and clinical research. CODEX® is ultra-high plex and allows imaging of up to 40 antigens on a single tissue section with single-cell resolution. Phenoptics™ is an established mIF platform that enables high-throughput whole slide multispectral image acquisition and tissue interrogation with up to 8 markers plus DAPI. Here we present a study that compares shared sets of immune and tumor markers between the CODEX® and Phenoptics™ platforms. This cross-platform comparison provides a conceptual framework for researchers to translate biomarker signatures from discovery to high-throughput translational studies.Materials and MethodsSerial sections of human formalin-fixed paraffin embedded non-small cell lung cancer (NSCLC) and tonsils were analyzed. An initial screen with a 28-plex CODEX® antibody panel revealed multiple biomarkers of interest, including CK, CD8, Ki67, PD-L1 and PD-1; all of these biomarkers showed abundant expression in the TMI. Building on this result, we next developed a 6-plex Opal™ Phenotpics™ panel. This panel was screened and analyzed via high-throughput whole slide scanning of sample tissues. Image processing and data analysis were conducted similarly for both datasets so that repeatability and consistency of measurements could be established.ResultsBoth CODEX® and Phenoptics™ detected the same cell phenotypes and displayed similar frequencies of cells expressing CK, CD8, Ki67, PD-L1 and PD-1 in serial sections of tonsil and NSCLC tissues. These observations were consistent and cross-validated in data from CODEX® and Phenoptics™ platforms. Crucially, this means that the two approaches can be made analytically equivalent, and hence, that they can be used in conjunction with each other as research progresses along the continuum from discovery to translational and clinical research.ConclusionsOur cross-platform comparison provides a conceptual framework for biomarkers discovered on the CODEX® platform to be translated to the Phenoptics™ platform for high-throughput translational studies. The resulting comprehensive phenotyping and quantification data retain spatial context and provide unprecedented insight into tumor biology.Abstract P01.04 Figure 1Disclosure InformationO. Braubach: A. Employment (full or part-time); Significant; Akoya Biosciences. M. Gallina: A. Employment (full or part-time); Significant; Akoya Biosciences. B. Remeniuk: A. Employment (full or part-time); Significant; Akoya Biosciences. C. Wang: A. Employment (full or part-time); Significant; Akoya Biosciences. N. Nikulina: A. Employment (full or part-time); Significant; Akoya Biosciences. R. Bashier: A. Employment (full or part-time); Significant; Akoya Biosciences. J. Kennedy-Darling: A. Employment (full or part-time); Significant; Akoya Biosciences. C. Hoyt: A. Employment (full or part-time); Significant; Akoya Biosciences.

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. 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. Over 50 parameters are measured within a single tissue through fully-automated, iterative cycles of adding and removing dye-conjugated Reporters to corresponding nucleic acid based Barcodes. The CODEX technology, developed by Akoya, is comprised of a fluidics instrument that interfaces with existing microscope hardware, a 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 their staining pattern. CODEX data from various normal and cancer tissue types are 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.