Abstract
Cholangiocarcinoma (CCA) is a deadly cancer, characterized by abundant stroma. The tumor microenvironment (TME) plays an important role in its aggressive behavior and poor response to therapeutics; however, the underlying pathways are unknown. To fill this gap, we used multiplexed immunohistochemistry, high-dimensional cytometry, and single cell transcriptomics. Our findings confirm an abundance of regulatory T cells (Tregs) and a lack of effector memory T cells within the tumor. Tumor-infiltrating T cells show signs of exhaustion. Using our transcriptomic data, we revealed cellular crosstalk in poor prognosis patients. This crosstalk is driven by stromal cells and macrophages. Among the responsible receptor-ligand pairs are GAS6-AXL, VCAN-TLR2, and EGFR-TGF-β. The multiple mechanisms leading to the exclusion of relevant immune cells needed for an anti-cancer response and mechanisms leading to active immune suppression are part of complex cell-cell crosstalk. This study provides a deeper insight into the immune exhausted phenotype in CCA.
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Data availability
The code used in analyzing this data is available at https://github.com/hayatlab/cholangiocarcinoma_ici. The raw data generated in this study using single nuclei RNA-sequencing will be made available via the European Nucleotide Archive (ENA) under the accession code PRJEB97203. Bulk transcriptomic data was retrieved from the Gene Expression Omnibus (GEO) dataset GSE132305.
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Acknowledgements
This study was funded by Deutsche Forschungsgesellschaft (DFG, German Research Foundation) – Project ID 403224013 – SFB 1382 (UN and TL). This research project was supported by the START Program of the Faculty of Medicine of the RWTH Aachen University, Project ID 19/23 (LH).
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L.H., Si.H., K.R., J.J.G.V., R.K., and U.N. designed this study. S.L., F.U., X.T., G.W., J.B., M.B., F.P., D.H., J.N., J.V., Z.H.,. A.G., and M.H. contributed to sample collection and processing. L.H., Si.H,. K.R., S.M., C.R., Z.H., A.G., J.H., and J.J.G.V. were responsible for data analysis and interpretation. L.H., D.J., and H.B. provided histological expertise. L.H., Si.H., S.M., R.K., J.A., J.D., and C.R. performed statistical analysis and interpretation. L.H., Si.H., S.S., F.R., J.J.G.V., J.A., C.R., and K.R. contributed to the first draft of this manuscript. U.N., R.K., S.S., T.L., D.J., E.D., J.S., H.B., J.H., Sy.H., and J.J.G.V. provided the infrastructure and supervised the study. All authors contributed to the data analysis and manuscript writing.
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Heij, L., Hayat, S., Reichel, K. et al. Multimodal single-cell profiling reveals crosstalk between macrophages and stromal cells in poor prognostic cholangiocarcinoma patients. npj Precis. Onc. (2026). https://doi.org/10.1038/s41698-026-01292-6
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DOI: https://doi.org/10.1038/s41698-026-01292-6
