Spatial multi-omics analyses to reveal KRT5 ⁺ tumor cell orchestration of an immune-excluded microenvironment in thymic carcinoma.

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Background: Thymic carcinoma is a rare and aggressive malignancy with limited therapeutic strategies and poor clinical outcomes. The spatial organization of its tumor immune microenvironment (TIME) remains poorly defined, hindering biomarker discovery and therapeutic development. Methods: We integrated 10× Genomics Visium HD spatial transcriptomics, multiplex immunofluorescence, and bulk RNA-seq of resected thymic carcinoma to characterize tumor-immune spatial interactions.Subsequent functional validation was conducted using in vitro co-culture assays and in vivo subcutaneous tumor models. Results: Spatial multi-omics analyses revealed that thymic carcinoma exhibited a KRT5-driven immune-excluded microenvironment characterized by CD8 ⁺ T cell exhaustion, M2-macrophage polarization, and extracellular matrix (ECM) remodeling-associated fibroblasts accumulation. Functionally, KRT5 knockout in TC-1889 cells (KRT5-KO) significantly reduced proliferative and invasive abilities in vitro and in vivo. Proteomic analyses demonstrated downregulation of SOX2 and S100P following KRT5 depletion, indicating attenuation of stemness-associated and tumor-promoting programs. Moreover, the co-culture assay further demonstrated that KRT5-KO attenuated PD-1 and TIM-3 expression on CD8 ⁺ T cells, decreased CD163 and CD206 on M2 macrophage, and reduced ECM-remodeling phenotypes (POSTN and CTHRC1) in fibroblasts. Conclusions: KRT5 ⁺ tumor cells define a spatially restricted immunosuppressive niche in thymic carcinoma and promote tumor progression through stemness-associated and microenvironmental remodeling programs. Targeting KRT5-associated pathways may represent a potential strategy to overcome immune exclusion and enhance immunotherapeutic efficacy in this rare malignancy.