206 A new automated RNAscope^TM assay for the fluorescent co-detection of multiple RNA and protein biomarkers on Roche DISCOVERY ULTRA™

<h3>Background</h3> Spatial transcriptomic and proteomic technologies allow for context-based study of the tumor microenvironment (TME). Understanding interactions between different cell types in the TME is crucial for advancing immunotherapy strategies against various types of cancer. Automated RNAscope^TM assays allow high throughput RNA in situ hybridization (ISH) and can be coupled with immunolocalization of proteins within the same tissue section. However, some epitopes are sensitive to the ISH protease pretreatment, highlighting the need for a protease-free solution. Additionally, 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 punctate RNA signals from distinct targets. 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 and the colocalization of biomarkers. This is especially relevant in cancer immunotherapy, where ISH can be used to visualize expression of multiple cytokines and combined with immunofluorescence (IF) to provide comprehensive information about the activation states of immune cells across the TME. <h3>Methods</h3> Expanding on current RNAscope^TM assay capabilities on the Roche DISCOVERY ULTRA™ System, we developed a new assay enabling fluorescent detection of multiple RNAs. Additionally, we also developed a new, fully automated ISH-IF 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 RNAscope^TM Multiplex Fluorescent reagents were used to detect two target RNA species with TSA dyes. Finally, IF was combined sequentially to detect multiple proteins. <h3>Results</h3> For proof-of-concept, we used this new automated multiplex, multiomic assay to view the complex interaction of tumors with their microenvironment by co-detecting tumor expression of hypoxia markers <i>HIF1A</i> and <i>VEGFA</i> by ISH while simultaneously detecting immune cells within the TME using the protease-sensitive hematopoietic cell surface marker CD45, as well as CD163+ macrophages by IF. The combination of multiple dyes produced high-intensity signals for each target and negligible background. <h3>Conclusions</h3> This new ISH assay demonstrated high sensitivity and specificity for targets with dissimilar expression levels, and together with the new VS PretreatPro enabled robust detection of protease-sensitive epitopes. This novel assay opens new possibilities for automated multiomic analysis of multiple biomarkers.