Abstract 2713: Characterization of GM-CSF and G-CSF expressing cell subtypes in the tumor microenvironment using the Integrated MultiOmyx-RNAscope assay

Abstract Granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF) are cytokines known to stimulate differentiation and activation of granulocytes, monocytes, macrophages, and dendritic cells (DCs) as well as non-immune cells including endothelial, epithelial and fibroblasts. GM-CSF and G-CSF can be produced by many different cell types, including immune cells, fibroblasts, endothelial cells and have also been shown to be upregulated in multiple human cancers. Studies have further demonstrated the immune-modulatory effects of GM-CSF/G-CSF can promote both anti-tumorigenic or pro-tumorigenic activity through regulation of neutrophils/M1 macrophages/T-effectors or myeloid-derived suppressor cells (MDSCs)/M2 macrophages/Tregs, respectively. Therefore, modulators to promote or reduce the activity of GM-CSF/G-CSF have been explored for tumor therapeutic intervention, often in combination with other therapies such as chemotherapy. However, to further dissect and target anti or pro-tumor GM-CSF/G-CSF modulation requires a better understanding of the distribution and identity of GM-CSF/G-CSF secreting cells within the tumor micro-environment (TME). To characterize GM-CSF/G-CSF expression in the TME, we use the Integrated MultiOmyx-RNAscope platform. MultiOmyxTM (NeoGenomics Laboratoies, Inc) is a proprietary multiplex immunofluorescence (mIF) platform for the visualization and characterization of up to 60 protein biomarkers in a single formalin-fixed paraffin-embedded (FFPE) section and offers high-resolution spatial and quantitative analysis of protein expression in tissue samples. RNAscopeTM (Bio-Techne) Multiplex is a highly sensitive fluorescent in-situ hybridization (ISH) assay that can detect up to 3 RNA markers in a single FFPE section. The Integrated MultiOmyx-RNAscope assay allows for simultaneous detection of both protein and RNA markers in a single sample. Herein we report the design and use of a novel panel of commercially-available RNAscope ISH probes and IF antibodies broad enough to characterize GM-CSF and G-CSF subpopulations in the TME of a variety of tumor indications including lung, prostate, melanoma, and colon cancer. Using this panel in combination with proprietary deep-learning based image analysis, GM-CSF and G-CSF expressing cells can be characterized into different immune and TME subtypes. Understanding of the variety and phenotype of GM-CSF and G-CSF expressing cells in the TME will help improve the understanding and development of targeted therapies for cancer treatment. Citation Format: Courtney Todorov, Kevin Gallagher, Victoria Huang, Harry Nunns, Marianne Thio, Eric Leones, Flora Sahafi, Erinn A. Parnell, Qingyan Au. Characterization of GM-CSF and G-CSF expressing cell subtypes in the tumor microenvironment using the Integrated MultiOmyx-RNAscope assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2713.