Heij, L. R.; Hayat, S.; Reichel, K.; Maryam, S.; Rourke, C.; Tan, X.; van den Braber, M.; Verhoeff, J.; Halder, M.; Peisker, F.; Wiltberger, G.; Bednarsch, J.; Heise, D.; Campello Deierl, J.; Lang, S.; Ulmer, F.; Luedde, T.; Dahl, E.; Jonigk, D.; Nolting, J.; Sivakumar, S.; Siveke, J.; Rocha, F.; Baba, H.; Andersen, J.; Garcia Vallejo, J.; Kramann, R.; Neumann, U.

Objective: Cholangiocarcinoma (CCA) is a deadly disease, and treatment options besides surgery are limited. This cancer entity is characterized by abundant stroma and the tumor microenvironment (TME) plays an important role in the poor response rate to therapeutics; however, underlying pathways are unknown. Design: To fill this gap, we used high-dimensional cytometry to analyze 16 human CCA samples and identified cell cluster crosstalk in seven human CCA samples using single-cell transcriptomics. Results: We showed an increased immune checkpoint expression on various key T cell subsets in the tumor niche. PD-1 (p<0.001), ICOS (p=0.002) and TIGIT (p=0.03) were elevated on intratumoral residential memory CD8 T cells (TRM) and PD-1 (p=0.03) and ICOS (p=0.02) were elevated on intratumoral (EM) CD8 cells. We also identified regulatory T cells to be more abundant in the tumor (p=0.002), while CD8 EM cells fail to reach the tumor (p=0.02). To further explore the immunosuppressive TME, we used snRNA sequencing on 7 pCCA samples. We identified a shift in crosstalk between the stromal and hepatic stellate cells (SCHSC) to a specific cholangiocytes cluster, mainly present in the poor outcome group. In addition, we identified new cellular crosstalk with macrophage-myeloid cells. The effector memory cells are sparse in the cancer tissue and spatial distribution indicates that the peripheral liver tissue can be used for stratification for personalized care. Conclusions: This study sheds light on the etiology of the immunosuppressive TME in CCA and uncovers pathways that are promising for further examination.