Background The immune system plays a critical role in combating several types of malignancies. Immunotherapies including checkpoint blockade therapies have utilized the innate ability of the immune system to identify and eliminate diseased cells. It has also been established that the profile of tumor-infiltrating lymphocytes (TILs) can to some extent predict patient response and overall survival. Traditionally, standard techniques such as flow cytometry, immunofluorescence have enabled successful assessment of tumor immune profile but identifying information about their activation states and cytotoxic effects has been challenging. Spatially visualizing the expression of soluble factors such as cytokines and chemokines along with immune cell markers can provide information about the immune cell composition and enable a comprehensive understanding of mechanisms underlying immune recruitment, infiltration and exclusion.

Methods To address this, we have developed a fully automated spatial multiomic protocol on the COMET™ that enables RNA detection using the RNAscope™ HiPlexPro assay combined with protein detection using sequential immunofluorescence (seqIF™, PMID: 37813886) to integrate same-section sequential detection of up to 12 RNAs followed by up to 24 proteins. This workflow allows the user to detect any RNA and protein target of interest by utilizing the vast catalog menu of RNAscope probes or generate a custom design for RNA targets and the use of standard, non-conjugated primary antibody for protein detection.

Results Here, we have demonstrated the precise spatial profiling of FFPE solid tumors through the detection of key cytokines indicative of activated T cells and macrophages by using cytokine RNA probes such as IFNG, IL-1B, TNFA, TGFB, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12 and IL-17 in combination with cell marker antibodies such as CD3,CD8,CD4, FOXP3 and CD68 .In addition, we were also able to visualize T cell recruiting chemokines and receptors such as CXCL10, CXCL9, CXCL13, CXCL12, CXCR3 and CXCR4, and spatially map tumor infiltrating T cells within tissue context and with subcellular resolution.

Conclusions The use of RNAscope HiPlexPro on COMET™ along with seqIF allows true multiomic analysis with simultaneous visualization of RNA and protein targets for immune cell profiling. Detecting cytokine and chemokine expression provides vital information about immune cell activation and recruitment, thereby increasing our understanding of phenomenon such as immune exclusion which is key in establishing predictive signatures for immunotherapies.