Background The canonical WNT-β-catenin signaling pathway is vital for development and tissue homeostasis but becomes strongly tumorigenic when dysregulated. and alter the transcriptional signature of a cell to promote malignant transformation. However, thorough characterization of these transcriptomic signatures has been challenging because traditional methods lack either spatial information, multiplexing, or sensitivity/specificity. To overcome these challenges, we 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™ Digital Spatial Profiler (DSP) RNA assays. Using these methods, we sought to spatially profile and compare gene expression signatures of tumor niches with high and low CTNNB1 expression.

Methods After screening 120 tumor cores from multiple tumors for CTNNB1 expression by the RNAscope assay, we identified melanoma as the tumor type with the highest CTNNB1 expression while prostate tumors had the lowest expression. Using the RNAscope Multiplex Fluorescence assay we selected regions of high CTNNB1 expression within 3 melanoma tumors as well as regions with low CTNNB1 expression within 3 prostate tumors. These selected regions of interest (ROIs) were then transcriptionally profiled using the GeoMx DSP RNA assay for a set of 78 genes relevant in immuno-oncology. Target genes that were differentially expressed were further visualized and spatially assessed using the RNAscope Multiplex Fluorescence assay to confirm GeoMx DSP data with single cell resolution.

Results The GeoMx DSP analysis comparing the melanoma and prostate tumors revealed that they had significantly different gene expression profiles and many of these genes showed concordance with CTNNB1 expression. Furthermore, immunoregulatory targets such as ICOSLG, CTLA4, PDCD1 and ARG1, also demonstrated significant correlation with CTNNB1 expression. On validating selected targets using the RNAscope assay, we could distinctly visualize that they were not only highly expressed in melanoma compared to the prostate tumor, but their expression levels changed proportionally to that of CTNNB1 within the same tumors suggesting that these differentially expressed genes may be regulated by the WNT-β-catenin pathway.

Conclusions In summary, by combining the RNAscope ISH assay and the GeoMx DSP RNA assay into one joint workflow we transcriptionally profiled regions of high and low CTNNB1 expression within melanoma and prostate tumors and identified genes potentially regulated by the WNT- β-catenin pathway. This novel workflow can be fully automated and is well suited for interrogating the tumor and stroma and their interactions.GeoMx Assays are for RESEARCH ONLY, not for diagnostics.