Spatial biology methods have shed light on the complexity of the tumor microenvironment (TME) at the single-cell level, revealing common and rare cell types and their intercellular interactions in a spatial context. Hyperplex immunofluorescence allows the identification of multiple biomarkers on the same sample, enabling immune cell profiling within the TME. However, revealing in-depth TME activation status requires mapping of cytokines/chemokines, currently achievable by RNA in situ hybridization techniques on a separate serial section. The ability to spatially profile both RNA and protein markers on the same section is needed to facilitate investigations of specific cell populations, such as immune cells and their activation states.

Here, we show a novel multiomics approach that integrates RNAscope™ and sequential immunofluorescence (seqIF™) to simultaneously identify RNA and protein targets on the same tissue slide. The analysis is fully automated on COMET™, an advanced tissue staining and imaging platform with precise temperature control and full workflow automation, ensuring optimal efficiency and reproducibility. Our integrated protocol combines up to three cycles of RNA detection with seqIF™, where two markers are identified in each cycle for a final dataset including 12-plex RNA and 20-plex protein multiomics panel.

The panel is designed to identify key components of the TME, such as macrophages, dendritic cells, fibroblasts, and tumor-infiltrating lymphocytes (TILs). RNA probes are selected to provide further information about the activation state of TILs by targeting key biomarkers like co-inhibitory receptors and transcription factors, together with secreted molecules such as cytokines and proteases. The simultaneous detection of RNA and proteins provides extensive insights into the TME molecular landscape, revealing co-expression patterns and relationships between RNA and proteins within individual cells. The signature of an immunosuppressive microenvironment is also studied, focusing on TIL activation states and immune checkpoint expression.

Our results affirm the successful implementation of the combined RNAscope™ and seqIF™ protocols on the COMET™ platform to allow same-section spatial multiomics analysis. Preserving spatial context and intercellular relationships, this technology will help unravel complex cellular interactions among distinct cell populations, thus opening new avenues for personalized medicine and identifying therapeutic targets.