Abstract 2092: Multiomic fluorescent co-detection of biomarkers in the tumor microenvironment using a new RNAscope™ assay on Roche DISCOVERY™ ULTRA

Abstract Understanding the interactions between different cell types in the tumor microenvironment (TME) is crucial for advancing cancer and immuno-oncology research. New spatial transcriptomic and proteomic technologies enable a context-based study of the TME. Automated RNAscope™ assays allow high throughput RNA in situ hybridization (ISH) and can be coupled with immunolocalization of proteins within the same tissue section. However, visualization of multiple biomarkers with standard chromogenic detection becomes challenging as the number of targets increases, due to the lack of color contrast for resolving different signals. Using fluorescent dyes enables imaging of each target in an independent channel and overlaying of each channel for the combined interpretation. Fluorescent analysis allows for greater confidence of signal identity when analyzing and quantifying complex cellular interactions. This is especially relevant in cancer research, where ISH can be used to visualize expression of multiple tumor biomarkers and combined with immunofluorescence (IF) to provide comprehensive information about the interaction between a tumor, infiltrating cells, and the surrounding environment. We developed a new assay enabling fluorescent detection of multiple RNAs that expands the current RNAscope™ assay capabilities on the Roche DISCOVERY™ ULTRA System. Here, we introduce a new, fully automated protease-free workflow to enable the fluorescent co-detection of multiple RNAs and proteins. This workflow includes harmonized cell conditioning for effective combined RNA and antigen retrieval, and a new pretreatment reagent, the protease-free VS PretreatPro, allowing for RNA detection without disruption of protease-sensitive epitopes. Following tissue pretreatment, new RNAscopeTM Duplex Fluorescent reagents were used to detect two target RNA species with fluorophore-conjugated tyramide dyes, followed by sequential detection of multiple proteins by IF in the same sample. To demonstrate the capabilities of this new assay, we analyzed the interaction of tumors with their microenvironment by co-detecting tumor expression of hypoxia markers as HIF1A and VEGFA, and chemokines as CXCL9, CXCL10 by ISH, followed by immunofluorescent detection of cell-surface markers to identify cells within the TME, including CD163+ macrophages, CD3+ T cells, as well as protease-sensitive targets, including CD45 (pan-hematopoietic) and CD20 for identification of B cells. Further, we analyzed the anti-tumor response and immune checkpoints identifying the expression of IFNG, GZMB, LAG3, and CTLA4 by ISH, together with PD-1 and PD-L1 by IF. The combination of multiple dyes produced high-intensity signals and negligible background. This new automated multiomic assay demonstrated high sensitivity and specificity for the detection of diverse targets in tumor tissue. Citation Format: Renzo Adilardi, Rose Delvillar, Wendy French, Qi Shen, Emerald Doolittle, Anushka Dikshit, Henry Lamparski, Maithreyan Srinivasan. Multiomic fluorescent co-detection of biomarkers in the tumor microenvironment using a new RNAscope™ assay on Roche DISCOVERY™ ULTRA [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 2092.