Spatiotemporal Mapping of Tertiary Lymphoid Structure Heterogeneity Shapes Immune Niches and Clinical Outcomes in Intrahepatic Cholangiocarcinoma

Abstract Intrahepatic cholangiocarcinoma (iCCA) is a highly lethal malignancy with limited therapeutic options. The spatial architecture and functional diversity of tertiary lymphoid structures (TLSs) in iCCA remain unclear. Here, we present a multimodal spatial atlas of TLSs and identified Intra-tumoral TLSs (iTLSs) as independent prognostic markers. Bulk proteomic profiling of 214 discovery and 155 validation cases identified a four-tier TLS-based TME classification system and supported development of a TLS-predictive random forest classifier. Imaging mass cytometry revealed that iTLS⁺ tumors harbor structured immune architectures, where M1-like tissue-resident macrophages (RTMs), dendritic cells, and CXCL13⁺ CD4⁺ T cells co-localize to form antigen-presenting neighborhoods (apc-CNs) spatially coupled to TLS core regions (TLScore-CNs). Single-cell spatial transcriptomics further resolved 61 TLSs into 14 spatial niches and defined a pseudo-temporal maturation continuum: aggregated, activated, and post-activated. Intra-niche communication, primarily mediated by ifnCAFs, iCAFs, and CXCL12⁺ macrophages, evolved dynamically with maturation. Single-nucleus RNA-sequencing combined with Tangram-based spatial mapping revealed CXCL12⁺ macrophages and iCAFs forming a peripheral band in aggregated TLSs, while ifnCAFs infiltrated TLS interiors during activation. These findings define TLS heterogeneity and provide insights for stroma-directed immunotherapy. Teaser An atlas of tertiary lymphoid structures reveals immune-stroma interactions in Intrahepatic cholangiocarcinoma.