Bex-Plex Enables Simultaneous Visualization of Protein Markers and Ribonucleic Acid Transcripts for Deep Spatial...

Here, we present a protocol to enable simultaneous spatial profiling of protein and ribonucleic acid (RNA) markers at single-cell resolution in intact tissues. One of the core issues surrounding solid tumor cancers is the inability to predict patient responsiveness to therapeutic intervention. Deciphering these complexities requires quantitative measurement of cellular behaviors, interactions, and phenotypes within intact tissues. Specifically, the proteome and transcriptome provide overlapping but nonredundant information that can be critical to designing rational immunotherapeutics. However, there is not currently a straightforward and cost-effective way to deeply profile both the transcriptional and functional state of all cells in the tumor microenvironment while retaining their critical spatial localization information. To this end, we have successfully combined the IBEX (iterative bleaching extends multiplexity) platform with HiPlex RNAscope to simultaneously visualize high-plex protein markers and high-plex ribonucleic acid (RNA) transcripts with single-cell resolution. This approach incorporates a modified RNA target retrieval step to preserve nuclear morphology and enable accurate image alignment across cycles. "Bex-Plex" enables us to perform an unmodified IBEX protocol on fixed frozen tissues followed by the HiPlex RNAscope protocol, in which we use a modified RNA target retrieval to ensure nuclear-level alignment with IBEX cycles. This protocol is optimized for fixed frozen tissues and enables integrated spatial analysis of transcriptional and functional cellular states. The power to profile at this single-cell level not only provides a deep well of information about the tissue itself but can also empower predictions about the nuanced cellular interactions that drive therapeutic responsiveness, providing a framework for studying spatial biology in tumor microenvironments and related systems.