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Background Glioblastoma (GBM), known as one of the most aggressive primary brain tumors, presents significant challenges in diagnosis and treatment. 1 Traditional immunohistochemistry often falls short in capturing the complexity of the tumor microenvironment (TME) within GBM. With an increasing emphasis on biomarker discovery and targeting complex diseases like GBM, spatial biology has emerged as a fundamental approach, revealing multiple biomarkers while preserving spatial tissue information. The pivotal task of transforming information-rich multiplex images into crucial quantitative data remains a significant hurdle. In this study, we present an innovative workflow for generating and analyzing hyperplex images.Methods We used the COMET™ platform, which performs fully automated spatial multiomics on tissue sections of human GBM samples. On COMET™, we integrated the RNAscope™ HiPlex Pro technology 2 with a sequential immunofluorescence (seqIF™) approach3 to detect RNA and proteins on a single tissue section simultaneously. A multiomics panel of 12 RNAs and 24 proteins was used to study immune and cancer cell populations and their cellular activity within the TME. Multiplex immunofluorescence images were analyzed with the HORIZON™ software.Results Deep-cell phenotyping was performed on the multiomics datasets using HORIZON™ to identify multi-dimensional insights about the sample. A batch analysis was performed on multiple regions of interest to apply the same analysis workflow, encompassing AI-driven cell segmentation, followed by RNA signal detection, feature extraction, and supervised classification. Additionally, we performed spatial analysis of cellular phenotypes of interest that unveiled organizational relationships within the TME.Conclusions This work demonstrates how spatial multiomics offers a comprehensive view of the TME, advancing our understanding of GBM pathology. Leveraging the single-cell resolution of spatial multiomics and an intuitive image analysis approach will pave the way for researchers to seamlessly navigate the complex immune and cancer networks within the TME.Acknowledgements This work was funded by the Research Foundation Flanders (FWO), Kom op tegen kanker (KOTK) and KULeuven.References Schaff LR, Mellinghoff IK. Glioblastoma and Other primary brain malignancies in adults: a review. JAMA. 2023 Feb 21;329(7):574–587.Wang F, et al., RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues. J Mol Diagn. 2012 Jan;14(1):22–9.Rivest F, et al, Fully automated sequential immunofluorescence (seqIF) for hyperplex spatial proteomics. Sci Rep. 2023 Oct 9;13(1):16994.Ethics Approval The samples were collected under protocol S59804 and S61081 as approved by the ethics board of KULeuven/UZLeuven