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Background Triple Negative Breast Cancer (TNBC) is a severely aggressive form of breast cancer, spreads rapidly, is highly heterogeneous in nature, and remains a subtype with few treatment options. Defined by the absence of ER/PR and HER2 expression, development of targeted therapy options for TNBC is challenging given the lack of disease-specific biomarkers. 1 Spatial biology has advanced significantly in recent years and the techniques that now exist to investigate the tumor microenvironment (TME) are easier and more versatile than ever before. This study utilized a fully automated multiplexed immunofluorescence (mIF) and RNA in situ hybridization (ISH) system to interrogate the immune profile of TNBC from a multiomic approach.Methods Using the COMET™ integrated staining and imaging platform, RNAscope™ and sequential immunofluorescence (seqIF) assays were performed on FFPE triple-negative breast cancer and normal breast tissue sections. A high-plex multiomic panel of biomarkers featuring fourteen protein markers (CD3, CD4, CD8, FoxP3, CD56, CD16, CD20, CD68, CD11c, aSMA, PD-L1, F4/80, CD45, and Ki67), two directly-conjugated tumor markers (panCK and CK18), and four RNA targets (PPIB, GAPDH, SERPINA1, and ACE2) was designed to explore and better understand the tumor landscape of this complex cancer subtype. Sample pre-processing steps were done utilizing the Epredia© PT Module.Results This study demonstrates the COMET’s capability of merging RNA and protein staining in a user-friendly and fully automated manner. The ability to visualize 4 RNA and 16 protein markers on the same slide of FFPE cancerous and normal breast tissues drastically reduced the number of samples needed without sacrificing data points. This multiomic approach ( figure 1) also allows for the comprehensive spatial profiling of numerous cell types (T-cells, proliferating cells, macrophages, B-cells, etc.) with possible RNA co-detection for the same target on the same cells. Finally, usage of direct and/or indirect primary antibodies and the capacity for four fluorescent channels increases the user’s choice in antibodies.Conclusions Our results highlight the compatibility of combined RNAscope™ and seqIF high-plex protocols with same-section FFPE slides and variable antibody formats. The automation of the instrument, high-plex nature of the assay, and protease-free protocol significantly reduces consumption of tissue, and the gentle staining procedure allows for additional downstream tissue use. Further examination of multiomic biomarker panels in patient tumor microenvironments could provide additional information to enhance treatment or immunotherapy options.Acknowledgements We would like to acknowledge the ACDBio team for advisement throughout the RNA probe design process and the Lunaphore team for their ongoing technical support.Reference Zhou H, Chen DD. Prognosis of patients with triple-negative breast cancer: A population-based study from Seer Database. Clinical Breast Cancer. 2023;23:85-94.Abstract 76 Figure 1Multiomic staining of breast tumor tissue. Breast tumor tissue stained with panCK, SERPINA1, ACE2, and DAPI showing SERPINA1 secretion by cancerous cells into stroma