Abstract 170: A novel protease-free workflow for spatial multiomics analysis of the tumor microenvironment in breast cancer

Abstract Introduction: Spatial multiomics is revolutionizing our understanding of the tumor microenvironment (TME) by enabling simultaneous analysis of multiple analytes within their tissue context. Combining in situ hybridization with immunofluorescence enables detection of key RNA and protein markers, together, on the same section. However, traditional ISH protocols that use enzyme-based pretreatment were incompatible with certain IHC/IF antibodies. Here, we present a novel protease-free workflow for a spatial multiomics panel, applied to human breast cancer samples, to investigate the interplay between immune cell infiltration and tumor cell signaling. Methods: Formalin-fixed paraffin-embedded (FFPE) breast cancer tissue sections underwent a protease-free staining protocol. The panel comprised three protein markers: CD3 (all T cells), CD8 (cytotoxic T cells), and PanCK (tumor cells). The RNAscope™ Multiomic LS in situ hybridization (ISH) assay was employed to detect IFNG (T cell activation), CXCL9 (chemokine produced by tumor cells), and CXCR3 (receptor for CXCL9 on CD8+ T cells). Spatial analysis algorithms were used to quantify co-expression and proximity of these markers. Results: Our protease-free workflow enabled simultaneous visualization of protein and RNA targets with high sensitivity and specificity. Preliminary data demonstrate distinct spatial patterns of immune cell infiltration and activation within the breast TME. We observed correlations between IFNG expression in CD8+ T cells and CXCL9 expression in PanCK+ tumor cells, suggesting a potential mechanism of T cell recruitment and activation. Conclusions: This novel protease-free spatial multiomics panel provides a powerful tool for dissecting the complex interactions between tumor cells and the immune system in breast cancer. By preserving both protein and RNA integrity, this approach offers valuable insights into the spatial organization of the TME and may identify novel therapeutic targets and biomarkers. Citation Format: Patrick Savickas. A novel protease-free workflow for spatial multiomics analysis of the tumor microenvironment in breast cancer [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 170.