Molecularly guided digital spatial profiling for multiplexed analysis of gene expression with spatial and single cell resolution

The tumor microenvironment (TME) is a network of complex interactions between the tumor and surrounding immune cells. To identify new predictive biomarkers to better stratify patients, it is essential to comprehensively characterize the immune cells within the TME at the molecular level. We have developed a novel workflow combining the single molecule and single cell visualization capabilities of the RNAscope in situ hybridization (ISH) assay with the highly multiplexed spatial profiling capabilities of the GeoMx(TM) Digital Spatial Profiler (DSP) RNA assays (Research Use Only). The fully automated RNAscope Multiplex Fluorescent assay was used to visually identify CD3E (T-cell)-enriched regions and CD19 and CD20 (B-cell)-enriched regions within FFPE human lung cancer tissues. Using the GeoMx DSP, 10 CD3E-enriched regions of interest (ROI) and 10 CD19-enriched ROI were spatially profiled for 78 genes related to immune-oncology research. The RNAscope Multiplex Fluorescence assay was used again to visually confirm the differentially expressed genes between the T and B-cell-enriched regions with single cell resolution. To show a workflow combining RNAscope molecularly guided visualization and GeoMx DSP profiling is feasible, we confirmed that both assay protocols are compatible and tested the fully automated workflow. We compared the differentially expressed genes within the T cell and B cell-enriched ROI. The RNAscope assay confirmed that the CD3E (T-cell)-enriched ROI demonstrated significantly higher expression of the checkpoint markers (PD-1, PD-L1, CTLA4, ICOSLG) as well as inflammatory markers (CCL5, CXCL9, IFNG) compared to the CD19-enriched ROI. We present a robust workflow that overcomes the historical limitations of ISH and IHC by combining high resolution imaging with high plex profiling. With this workflow, the RNAscope ISH technology can molecularly guide the GeoMx DSP to precisely profile ROI while retaining the morphological context.