Multiplexed RNA-protein co-detection to explore tumor-immune landscape using a novel automated RNAscope&[trade] assay

Abstract

Abstract Development of next-generation immunotherapies need a highly sensitive multiomics platform to evaluate diagnostic and therapeutic biomarkers. Tissue heterogeneity poses immense challenges to understanding underlying molecular mechanisms using techniques such as qRT-PCR or bulk sequencing. Single-cell RNA sequencing can provide information about precise cellular composition of tissues, data analysis can be cumbersome and spatial context is lost. With single-cell spatial platforms such as RNAscope, target gene and protein expression can be visualized to characterize cell types and tissue neighborhoods. Here, we demonstrate a novel method for the simultaneous detection of RNA and protein using a modified co-detection assay. This novel co-detection assay enables visualization of a combination of up to 12 RNA and/or protein targets on the same sample. We used a set of antibodies targeting key immune and tumor cell markers-PD1, CD3, CD4, CD8, CD68, FOXP3 and KRT17, along with RNA biomarkers to interrogate the tumor microenvironment (TME) in human FFPE tumor samples. Using a combination of RNA and protein targets, we characterized different cell types such as T cells, macrophages, and tumor cells in the TME. We also developed a novel method for visualizing intercellular interactions between PD1 and PD-L1, offering insights into popular checkpoint mechanisms that are targeted for therapeutic intervention in cancer treatment. The assay offers a powerful technique for visualizing target RNA biomarkers in specific cell-types identified by cell-marker protein expression. This is a valuable tool for multiomic analysis and accurate interrogation of complex tissues to obtain insights into novel biomarkers and therapeutic targets.