71 Integration of RNA in situ hybridization and sequential immunofluorescence for same-slide fully automated multi-omics analysis of the tumor microenvironment

BackgroundSpatial biology has transformed our understanding of the tumor microenvironment (TME) by enabling the study of tissue composition and intercellular interactions at a single-cell level while preserving spatial context.1–3 Hyperplex immunofluorescence (IF) techniques allow the simultaneous detection of multiple protein biomarkers, enabling immune cell profiling in the TME.4 Similarly, RNA in situ hybridization (ISH) techniques have enabled the detection of RNA biomarkers, such as soluble factors with high sensitivity and specificity.5 Combining the detection of key RNA and protein targets can provide valuable insights into unique infiltrating immune cell populations and their activation states.In this study, we propose a novel approach that combines RNAscope™ and sequential immunofluorescence (seqIF™) protocols for the simultaneous detection of RNA and protein targets. The integrated same-slide multi-omics protocol is automated on the COMET™ platform, an advanced platform for tissue staining that uses precise temperature control and automation capabilities, ensuring reproducibility and efficiency in the workflow.MethodsThe RNAscope HiPlex assay was automated on COMET™ for RNA detection and combined with the seqIF™ protocol for the detection of protein biomarkers. By integrating the two, we could sequentially detect multiple RNAs and proteins in the same tissue sections, preserving the spatial relationship between different molecular species.ResultsWe developed an integrated protocol for RNA and protein detection with three cycles of RNA detection (four fluorescent channels per cycle), enabling a total of 12-plex RNA panel detection, followed by consecutive cycles of seqIF™, with two protein markers detected per cycle. We included antibodies to detect infiltration of T cells, B cells, macrophages, and other immune cells in combination with RNA probes for key biomarkers such as chemokines and cytokines. The automated process on COMET™ seamlessly synchronized all protocol steps, including imaging, and allowed multi-omics analysis without any user intervention. By combining RNA and protein codetection, we gained extensive insights into the TME molecular landscape, uncovering co-expression patterns and relationships between RNA and proteins within individual cells.ConclusionsOur results demonstrate the successful implementation of the combined RNAscope and seqIF™ protocols on COMET™. Preserving spatial context and intercellular relationships, this appro