Spatial profiling of immune cell markers in FFPE tumor tissues using the RNAscope&[trade] HiPlex v2 in situ hybridization assay

Abstract

Abstract The tumor microenvironment (TME) is highly complex, comprised of tumor cells, immune cells, stromal cells, and extracellular matrix. This study demonstrates a highly sensitive and specific multiplexed technique, the RNAscope HiPlex v2 in situ hybridization (ISH) assay for spatial and transcriptomic profiling of target genes to assess immune regulation in FFPE tumor tissues. The RNAscope HiPlex v2 assay iteratively multiplexes up to 12 targets in frozen and FFPE tissues. The novel FFPE reagent effectively reduces background autofluorescence, improving the signal to noise ratio. Here we investigate spatial expression of 12 immuno-oncology target genes, including tumor markers, immune checkpoint markers, immunosuppression markers, immune cell markers and secreted chemokines The targets were simultaneously registered using HiPlex image registration software v2 that enables background subtraction. We visualized T cell infiltration and activation within tumors using CD3, CD8 and IFNG expression. Subsets of pro- and anti-inflammatory macrophages were detected by CD68 and CD163 expression Hypoxia markers HIF1A and VEGF indicated immunosuppressive state of tumor cells. Preliminary analysis and quantification of the HIF1A expression using HALO® image analysis showed higher copy numbers in the lung tumor as compared to the other tumors. We additionally showed the differential expression of immune checkpoint markers PDCD1, and CD274 within the TME. Using a highly sensitive multiplexed RNAscope HiPlex v2 ISH assay, 12 key targets were spatially resolved in four different tumor types. The assay can provide valuable understanding of the biological crosstalk among various cell types in complex and heterogeneous tissues.