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bioRxiv · 10.64898/2026.01.02.697439

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

Gao, L.·Mei, J.·Hong, L.·Jin, Y.·Cheng, J.·Sun, X.·Liu, C.·Li, B.·Meng, X.·Lin, B.·Sun, Y.·Zhao, P.·Chen, M.·Guo, R.·Xin, S.·Yun, J.·Martinez-Reyes, I.·Wei, W.·Fang, W.·Bao, X.
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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. TeaserAn atlas of tertiary lymphoid structures reveals immune-stroma interactions in Intrahepatic cholangiocarcinoma.

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Gao, L., Mei, J., Hong, L., Jin, Y., Cheng, J., Sun, X., Liu, C., Li, B., Meng, X., Lin, B., Sun, Y., Zhao, P., Chen, M., Guo, R., Xin, S., Yun, J., Martinez-Reyes, I., Wei, W., Fang, W., Bao, X.. 2026-01-06. Spatiotemporal Mapping of Tertiary Lymphoid Structure Heterogeneity Shapes Immune Niches and Clinical Outcomes in Intrahepatic Cholangiocarcinoma. https://doi.org/10.64898/2026.01.02.697439

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