Visualizing immune cells markers and cytokines to characterize tumor infiltrating immune cells using the spatial RNAscope™ HiPlex v2 in situ hybridization assay

Understanding spatial interactions between various cell types and their activation states in the TME is crucial for implementing successful immunotherapy strategies against various types of cancer. Here we demonstrate a newly developed HiPlex v2 assay with capability of iteratively multiplexing up to 12-48 targets in FFPE, fixed and fresh frozen samples. Using a novel FFPE reagent that effectively reduces background autofluorescence and improves the signal to noise ratio, we investigated spatial expression of 12 oncology and immuno-oncology target genes, including tumor markers, immune checkpoint markers, immunosuppression markers, immune cell markers and secreted chemokine RNA expression profile within the TME. We show that RNAscope HiPlex v2 image analysis and quantification provides a highly resolved spatial multiplexing solution to interrogate complex tissues and investigate biologically meaningful questions.