Abstract 2681: Co-detection of a tumor-infiltrating lymphocyte immunofluorescence (IF) panel and cytokine RNA in-situ hybridization (ISH) markers in non-small cell lung cancer (NSCLC) tumor microenvironment using combined MultiOmyx and RNAscope platforms
Authors: Todorov Courtney , Gozo Maricel , Nunns Harry , Parnell Erinn , Kuo Judy , Leones Eric , Nagy Mate , Au Qingyan 
Published: July 2021

Abstract
Co-detection of RNA and protein can greatly expand the data output from a single specimen, providing critical information such as the source of secreted proteins (e.g. cytokines) or cell type specific transcript levels. MultiOmyx is a proprietary immunofluorescence (IF) platform for the visualization and characterization of up to 60 protein biomarkers in a single formalin-fixed paraffin-embedded (FFPE) section. RNAscope Multiplex is a highly sensitive fluorescent in-situ hybridization (ISH) assay that can detect up to 3 RNA markers in a single FFPE section. Combination of MultiOmyx IF with RNAscope Multiplex ISH would therefore provide a novel and powerful platform to co-detect multiple RNA and protein markers in a single slide/sample. However, the RNAscope Multiplex assay includes a protease pretreatment step, which may compromise downstream antibody-antigen interaction and thereby the IF signal. This study is a validation of the sensitivity, specificity, reproducibility, and repeatability of an integrated MultiOmyx IF and RNAscope ISH assay. Depending on the context, cytokines interleukin-10 (IL10) and interferon gamma (IFNγ) have both been shown to either induce immunosuppression and favor tumor growth or promote an anti-tumor response. The mechanisms and cues determining the pro- or anti-tumor activity of IL10 and IFNγ are still poorly understood. Spatiotemporal characterization of IFNG and IL10 is therefore critical to help define the dynamic relationship of cytokines and the immune system within the tumor microenvironment. Therefore, for validation of the integrated MultiOmyx-RNAscope platform, RNA ISH markers for IL10 and IFNγ were combined with the MultiOmyx 12 marker tumor infiltrating lymphocyte (TIL) panel (CD3, CD4, CD8, CD20, CD68, CD56, CD45RO, PD-1, PD-L1, CTLA4, FOXP3, and tumor marker PanCK) on FFPE human NSCLC samples. This combined MultiOmyx-RNAscope workflow was performed for three individual runs using triplicate NSCLC samples for each run. Intensity and cell classification for each ISH and TIL marker was quantified using the proprietary MultiOmyx Analytics pipeline. The results demonstrate that the integrated assay maintains sensitivity and specificity of the TIL IF markers in the integrated workflow when benchmarked to an IF alone workflow. Furthermore, these results can be used to characterize expression of IL10 and IFNγ within immune cell subsets represented by the TIL IF panel (e.g. T cells, NK cells, macrophages). This integrated approach can be used to spatially correlate the distribution of cytokine and immune cell expression within the tumor microenvironment. Therefore, the RNAscope-MultiOmyx IF assay provides a robust and powerful platform to simultaneously co-detect RNA with protein IF in a single specimen.
Citation Format: Courtney Todorov, Maricel Gozo, Harry Nunns, Erinn Parnell, Judy Kuo, Eric Leones, Mate Nagy, Qingyan Au. Co-detection of a tumor-infiltrating lymphocyte immunofluorescence (IF) panel and cytokine RNA in-situ hybridization (ISH) markers in non-small cell lung cancer (NSCLC) tumor microenvironment using combined MultiOmyx and RNAscope platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2681.