Background
Spatial biology techniques have revolutionized our approach toward the study of the tumor microenvironment (TME) and its complex cellular interplay. By preserving the spatial information and allowing analyses at single-cell resolution, an unprecedented understanding of the cellular composition of cancer tissues has been made possible. On the one hand, multiplex immunofluorescence (mIF) methods have enabled precise profiling of immune cells and other key cellular players of the TME while uncovering their spatial distribution and interactions. On the other hand, in situ hybridization (ISH) technologies have been shown to provide complementary information to protein profiling, such as the mapping of cytokine- and chemokine-expressing cells, which are essential to comprehend signaling networks and immune activation statuses.
Methods
We made use of a novel multiomics approach that combines these two biological inputs by integrating RNAscope™ HiPlex Pro Assay (1) and sequential immunofluorescence (seqIF™) (2) protocols to achieve same-section co-detection of RNA and protein targets. The combined workflow is fully automated on COMET™, an advanced tissue staining and imaging platform (Figure 1). Through precise control over temperature and reagent distribution, the instrument ensures each assay’s maximum efficiency and reproducibility. The integrated multiomics protocol presented here allows for up to three RNAscope™ detection cycles combined with twelve seqIF™ cycles for a total of 12-plex RNA and 24-plex protein panel in less than a day.