Characterization of immune cell phenotypes through quantification of the 12-plex spatial RNAscope™ HiPlex v2 assay using the HALO® image analysis platform

Comprehensive spatial analysis is ideally accomplished by combining multiplexing technologies that allow simultaneous visualization of multiple target genes within the same sample with image analysis tools that can provide quantitative, single cell level data to accurately interpret gene expression within the context of the tissue.

Having been optimized for detection of target RNA in FFPE samples, the RNAscope™ HiPlex v2 assay provides a multiplexing approach ideally suited for investigating target genes in tumor tissues. We recently demonstrated the capabilities of the HiPlex v2 assay for multiplex fluorescent detection of up to 12 targets in fresh/fixed frozen and FFPE tissues. When combined with quantitative analysis, multiplexed in situ RNA detection provides the ability to explore complex interactions between immune cells, tumor cells, endothelial cells, and stromal cells, thus providing an important tool for cancer research.

In this application note, we demonstrate how the HALO® image analysis platform can be employed with HiPlex v2 to

• quantitatively assess differential expression of 12 RNA targets,
• quantify six distinct cell phenotypes based on gene expression, and
• analyze spatial relationships between cell phenotypes within the Tumor Microenvironment (TME).