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Background NUT carcinoma (NC) is a rare, aggressive cancer defined by NUTM1 gene rearrangements, often arising in midline structures such as the head, neck, and thorax. NC is associated with rapid progression, poor clinical outcomes, and resistance to standard treatments. Although the complexity of NC has been studied extensively, the disease remains incompletely understood. To address this critical scientific and clinical gap, we aimed to uncover key characteristics of immune and tumor cells within the tumor immune microenvironment (TIME) of NC, identifying prognostic factors and potential therapeutic targets to substantially improve patient outcomes.Methods We analyzed 14 NC tumor samples from children, adolescents, and young adults using classical immunohistochemistry, whole-genome DNA sequencing (WGS), RNA sequencing, and multiplexed tissue imaging. We employed MACSima imaging cyclic staining (MICS) to attain a detailed spatial profile of the TIME in NC. Our ultra-high content imaging pipeline 1 was optimized for formalin-fixed, paraffin-embedded (FFPE) samples, utilizing a custom immunophenotyping panel targeting 72 markers. This enabled deep profiling of cellular heterogeneity in tumor and immune cell populations, sub-phenotyping of immune cells, and mapping of cellular neighborhoods using sophisticated bioinformatic workflows to uncover interactions and niches in NC.Results Our comprehensive characterization of the immune landscape in NC revealed novel infiltration and interaction signatures. In particular, high proportions of intratumoral granulocytes, myeloid-derived suppressor cells (MDSCs), and M2-like macrophages could be associated with worse outcomes. Furthermore, we identified previously unreported immunologically ‘hot’ NC tumors that may benefit from immune checkpoint blockade and other immunotherapies. To uncover novel actionable targets in NC, we screened for the expression of immune checkpoint molecules and target antigens. B7-H3 emerged as particularly promising due to its uniform expression across all NC samples. We evaluated the therapeutic potential by utilizing B7-H3-targeted chimeric antigen receptor (CAR)-T cells, demonstrating specific elimination of NC both in vitro and in NC xenograft models.Conclusions This study provides the first large-scale, spatially resolved immunophenotyping of NC, offering a valuable framework for identifying and validating new diagnostic and therapeutic options. By comprehensively mapping the TIME in NC, we aim to advance precision immunotherapy approaches tailored to this challenging malignancy. Our findings highlight prognostic immune signatures and identify B7-H3 as a promising target for CAR-T cell therapy in NC, paving the way for novel, more effective treatments for patients with NC.Acknowledgements We thank the patients and families for their willingness to contribute to scientific research by participating in the study. We thank the NGS Core Facility, and the Omics IT and Data Management Core Facility (ODCF) of the German Cancer Research Center (DKFZ) for providing excellent WGS, RNA-seq, and data management services.Reference Scheuermann S, Kristmann B, Engelmann F, et al. Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging. Front Immunol. 2024;15:1383932. Published 2024 Mar 19. doi:10.3389/fimmu.2024.1383932Ethics Approval All patients or theri legal representatives gave their written informed consent for biobanking and the use of biomaterials and clinical data for scientific assessment, as approved by the ethics committee of the University Hospital Tübingen (ethics approval No. 879/2020B02).