Development and utility of precision FISH/IF multiomic panel to elucidate T cell functionality in the tumor microenvironment

Immunohistochemistry (IHC) is a widely utilized tool for visualizing biomarker expression in tissues of interest. However, not all biomarkers are amenable to investigation by IHC, including secreted factors, such as cytokines. In these cases, in situ hybridization (ISH) may be a suitable alternative. The combination of IHC and ISH approaches allows for investigation of protein and RNA targets in the same multiomic assay, offering greater utility and flexibility than traditional assays. Furthermore, image and spatial analyses can be applied to elucidate the colocalization, proximity, and spatial relationship of RNA and protein biomarkers in the same sample. Flagship Biosciences, in collaboration with Advanced Cell Diagnostics (ACD), is developing a multiplex IHC/ISH fluorescent, which aims to identify and characterize subsets of T lymphocytes in the context of cancer. Using the RNAscope™ Multiomic LS assay we detected CD8, CD3, and Ki67 proteins alongside IL4 , IFNG , and GZMB RNA expression to assess the activation and functional state of different T cell subsets based on biomarker expression profiles. Here, we demonstrate the capacity of this analysis on a set of FFPE lung samples from healthy and non-small cell lung cancer subjects. Our approach and analysis highlights the potential of spatial multiomics assays to bridge immunobiology with cancer research. Data generated could prove useful for phenotyping tumors, monitoring therapy response, or better understanding immune cell dynamics within the tumor microenvironment.