EVs were isolated from the bile of patients with extrahepatic cholangiocarcinoma (eCCA) or benign hepatobiliary diseases (4 patients with choledocholithiasis, 1 patient with bile duct injury) to determine the differentially expressed circRNAs. Transmission electron microscopy (TEM) confirmed the exosomal morphology of the membrane‐encapsulated bile extracts (Fig. 1A). One hundred microliters of EVs (2.9‐3.1 × 1011 EVs/mL) was isolated from 50 mL bile. For nanoparticle tracking analysis assay, the experiments were performed at 1:6,000 dilution, leading to particle concentrations around 5.0 × 107 EVs/mL with a 92.1 ± 34.7 nm in size (Fig. 1A). The particles were positive for the exosomal markers Alix, CD63, and tumor susceptibility 101 relative to the groups of unextracted bile and total cholangiocyte lysates (Fig. 1A). CircRNA microarray analysis revealed 85 circRNAs (fold‐change > 2; P < 0.05) that were differentially expressed between the BEVs of eCCA and healthy controls. Fig. 1B shows the hierarchical clustering of the top 30 up‐regulated and down‐regulated circRNAs. We used eCCA/adjacent normal tissues from the same 5 patients to study circRNA expression profiles using ribosomal RNA–depleted RNA sequencing (RNA‐seq). Among the 140 differentially expressed circRNAs (fold‐change > 2; P < 0.05), the top 30 up‐regulated and down‐regulated circRNAs are shown in Fig. 1B. Analysis of differentially expressed circRNAs in EVs and tissues revealed three commonly dysregulated circRNAs (Supporting Fig. S1A). Quantitative real‐time PCR (qRT‐PCR) showed that, of these circRNAs (hsa_circRNA_102064, hsa_circRNA_100364, and hsa_circRNA_101721), hsa_circRNA_102064 showed the most dysregulated expression in 20 pairs of CCA and normal tissues (5 eCCA and 15 iCCA; Supporting Fig. S1B). Hsa_circRNA_102064 is synthesized from exons 23‐26 of ERBB2 (Supporting Fig. S1C). To help distinguish this from the known circ‐ERBB2, we named this circRNA “circ‐CCAC1”. Sanger sequencing validated the back‐spliced junction of circ‐CCAC1 (Supporting Fig. S1D). Moreover, it harbored a loop structure resistant to ribonuclease R (Supporting Fig. S1E). Total RNA was extracted to detect the expression of circ‐CCAC1 and linear ERBB2 after treatment with actinomycin D at different time points. Linear ERBB2 showed a shorter half‐life compared with circ‐CCAC1, highlighting the stability of circ‐CCAC1 (Supporting Fig. S1F).

EVs were isolated from the bile of patients with extrahepatic cholangiocarcinoma (eCCA) or benign hepatobiliary diseases (4 patients with choledocholithiasis, 1 patient with bile duct injury) to determine the differentially expressed circRNAs. Transmission electron microscopy (TEM) confirmed the exosomal morphology of the membrane‐encapsulated bile extracts (Fig. 1A). One hundred microliters of EVs (2.9‐3.1 × 1011 EVs/mL) was isolated from 50 mL bile. For nanoparticle tracking analysis assay, the experiments were performed at 1:6,000 dilution, leading to particle concentrations around 5.0 × 107 EVs/mL with a 92.1 ± 34.7 nm in size (Fig. 1A). The particles were positive for the exosomal markers Alix, CD63, and tumor susceptibility 101 relative to the groups of unextracted bile and total cholangiocyte lysates (Fig. 1A). CircRNA microarray analysis revealed 85 circRNAs (fold‐change > 2; P < 0.05) that were differentially expressed between the BEVs of eCCA and healthy controls. Fig. 1B shows the hierarchical clustering of the top 30 up‐regulated and down‐regulated circRNAs. We used eCCA/adjacent normal tissues from the same 5 patients to study circRNA expression profiles using ribosomal RNA–depleted RNA sequencing (RNA‐seq). Among the 140 differentially expressed circRNAs (fold‐change > 2; P < 0.05), the top 30 up‐regulated and down‐regulated circRNAs are shown in Fig. 1B. Analysis of differentially expressed circRNAs in EVs and tissues revealed three commonly dysregulated circRNAs (Supporting Fig. S1A). Quantitative real‐time PCR (qRT‐PCR) showed that, of these circRNAs (hsa_circRNA_102064, hsa_circRNA_100364, and hsa_circRNA_101721), hsa_circRNA_102064 showed the most dysregulated expression in 20 pairs of CCA and normal tissues (5 eCCA and 15 iCCA; Supporting Fig. S1B). Hsa_circRNA_102064 is synthesized from exons 23‐26 of ERBB2 (Supporting Fig. S1C). To help distinguish this from the known circ‐ERBB2, we named this circRNA “circ‐CCAC1”. Sanger sequencing validated the back‐spliced junction of circ‐CCAC1 (Supporting Fig. S1D). Moreover, it harbored a loop structure resistant to ribonuclease R (Supporting Fig. S1E). Total RNA was extracted to detect the expression of circ‐CCAC1 and linear ERBB2 after treatment with actinomycin D at different time points. Linear ERBB2 showed a shorter half‐life compared with circ‐CCAC1, highlighting the stability of circ‐CCAC1 (Supporting Fig. S1F).