Abstract 5304: Profiling the tumor- immune landscape with a same-section multiomics assay

Abstract Checkpoint therapies and engineered immune cell therapies are increasingly becoming the mainstay when it comes to cancer treatments. Continued and improved success of these therapies relies on our deep understanding of the checkpoint marker and immune cell biology, cell-cell interaction in the tumor microenvironment (TME), and immune cell activation. 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 cell recruitment, infiltration and exclusion. To address this, we have developed a fully automated spatial multiomics protocol on the COMET™ platform that enables RNA detection using the RNAscope™ HiPlex Pro assay combined with protein detection using sequential immunofluorescence (seqIF™, (Rivest et al, 2023) ) to integrate same-section sequential detection of up to 12 RNAs followed by up to 24 proteins. This workflow allows the detection of any RNA and protein target of interest by utilizing existing RNAscope probes or generating a custom design for any RNA target and use standard, non-conjugated primary antibodies for protein detection. We used Lunaphore’s HORIZON™ software to analyze this multiomics dataset. 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 targeting IFNG, IL-1B, TNFA, TGFB1, IL-4, IL-6, IL-8, IL-10 among others in combination with cell marker antibodies detecting 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 and CXCR3, and spatially map tumor-infiltrating T cells within tissue context and with subcellular resolution. To that end, we successfully demonstrated same-slide RNA-protein detection for profiling the TME. Using single-cell spatial multiomics we were able to identify unique phenotypes within the tumors that might directly correlate to immunosuppressive or immunostimulatory microenvironments. Detailed mapping of the cellular components of the TME has the potential to better predict therapeutic response for checkpoint and immune-based therapies. Citation Format: Anushka Dikshit, Cansaran Saygili Demir, Rose Delvillar, Emerald Doolittle, Alice Comberlato, Arec Manoukian, Maria-Giuseppina Procopio, Pino Bordignon, Saska Brajkovic, Maithreyan Srinivasan. Profiling the tumor- immune landscape with a same-section multiomics assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5304.