Combining Immunohistochemistry and In Situ Hybridization to Characterize Immunosuppression in the Tumor Microenvironment

Abstract While immune-based cancer treatments have been successfully deployed in the clinic for hematological malignancies, treatment of solid tumors has proven more challenging. Comprehensive understanding of the dynamic landscape of the tumor microenvironment (TME) will lead to new therapies and improved efficacy of current therapies. Immunohistochemistry (IHC) and in situ hybridization (ISH) are the two techniques at the forefront of TME characterization. Combining RNA and protein detection with ISH and IHC reveals cell-type specific gene expression and the cellular sources of secreted proteins. Traditional ISH-IHC workflows require harsh pre-treatment of tissues prior to detection with primary antibodies, destroying the target epitope for some antibody clones. Bio-Techne’s RNAscopeTM RNA protein Codetection Assay improves the likelihood of successful antibody staining by incubating with primary antibody first, before initiating the RNAScope pretreatment step. With minimal optimization time, we were able to visualize protein expression using IHC validated antibodies for important immunosuppression biomarkers such as HIF-1 alpha, FoxP3, PD-L1, and CD11b. Using a cancer tissue microarray (TMA), we show this workflow is compatible with many tissue types, including pancreas, uterus, liver, breast, lung, prostate, and colon tissue. In combination with RNAScope probes CD8A, CD4, PD-1, TGFB1, and VEGF we were able to identify immunosuppressive cells in the TME, including Regulatory T cells (Tregs) and tumor associated macrophages (TAMs). Adoption of this workflow will streamline profiling of the TME, facilitating a more comprehensive evaluation of solid tumors for biomarker discovery and immunotherapy development.