Fig 1: TRAIL-R+immune cells in the TIME are essential in facilitating tumor progression. (A and B) Uniform manifold approximation and projection (UMAP) plots of single cells from 34 human CCA tumors in public scRNA-seq data sets (GSE210066, GSE189903, GSE138709, GSE125449, and HRA000863). (A) CCA cells (7426) were colored (red) by TRAIL (TNFSF10) expression and expression of epithelial cell markers (violet) in these tumor cells. (B) Immune cells (156,063) were colored by TRAIL-R1 (TNFRSF10A) expression (left) and TRAIL-R2 (TNFRSF10B) expression (right). Positive cells (in red) were defined as those with a unique molecular identifier of the specific gene larger than 1.7. (C) Graph depicts Kaplan–Meier survival curve of 117 iCCA patients from the International Cancer Genome Consortium cohort (GSE89747), stratified according to expression of the TRAIL-R1 gene (TRAIL-R1 high and TRAIL-R1 low). The Gehan–Breslow–Wilcoxon test was performed to assess the overall survival of the 2 groups. Wilcoxon P value is provided. (D) Immunoblot analysis of TRAIL expression in SB, KPPC, and FAC mouse CCA cell lysates. β-actin (ACTIN) was used as a loading control. (E) Schematic of SB syngeneic CCA model in WT and Trail-r-/- mice. (F) Average tumor weights of WT and Trail-r-/- mice (n > 15). (G) Representative photographs of livers from panel F. Scale bar: 0.5 cm. (H) Schematic of KPPC syngeneic CCA model in WT and Trail-r-/- mice. (I) Average tumor weights of WT and Trail-r-/- mice (n > 10). (J) Representative photographs of livers from panel I. Scale bar: 0.5 cm. (K) Schematic of FAC syngeneic CCA model in WT and Trail-r-/- mice. (L) Average tumor weights of WT and Trail-r-/- mice (n = 4). (M) Representative photographs of livers from panel L. Scale bar: 0.5 cm. Data are represented as means ± SD. Unpaired Student t test was used. ∗P < .05, ∗∗∗P < .001. ANXA, Annexin; EPCAM, Epithelial Cell Adhesion Molecule.