Spatial profiling of NUT carcinoma reveals an immunosuppressive myeloid-dominant microenvironment

Background: NUT carcinoma (NC) is an extremely rare and fatal fusion-driven malignancy, that can arise anywhere in the body. Because of its rarity, NC remains poorly understood and treatment relies on chemotherapy and radiotherapy regimens with limited response. Here, we aimed to generate the first comprehensive spatial single-cell profiling of NC to characterize the disease’s tumor immune microenvironment and uncover clinically relevant features. Methods: We performed multiplexed spatial proteomics using MACSima imaging cyclic staining on formalin-fixed paraffin-embedded tumors from the largest pediatric and young adult patients NC cohort to date. Therefore, we generated a comprehensive antibody panel which enabled the single-cell phenotyping of NC and established a downstream spatial analysis workflow to associate spatial features with clinical outcomes. Results: Spatial profiling of 553,331 cells revealed an immunosuppressive tumor immune microenvironment dominated by myeloid populations, including myeloid-derived suppressor cells, M2-like macrophages, and granulocytes. We identified novel, recurrent cellular neighborhoods in NC, including granulocytic suppressive regions and tertiary lymphoid structure–like organizations in a subset of patients. Notably, we describe that myeloid-derived suppressor cells-to-CD8+ T cell neighbor preferences are associated with lower survival in our cohort. Conclusions: In summary, we generated the first spatial single-cell resolved atlas of NUT carcinoma and identified myeloid cell states and spatial preferences that suggest potential therapeutic opportunities for myeloid-targeting strategy. This work established a blueprint for the spatial analysis of other rare and understudied malignancies, where small cohort sizes have historically limited the tumor immune microenvironment’s characterization.