The clinical backdrop makes the finding urgent. Surgical resection remains the only curative option for cholangiocarcinoma, yet because early-stage disease produces few symptoms, only 30 to 40 percent of patients are candidates for an operation. Even among those who undergo complete tumor removal, recurrence is common and the five-year overall survival rate falls below 40 percent. Current guidelines recommend six months of adjuvant capecitabine after curative surgery, while for advanced disease the combination of cisplatin and gemcitabine, known as CisGem, stands as the first-line standard of care. But chemotherapy resistance is high, and patients receiving CisGem experience progression-free survival and overall survival of only about 8.0 and 11.7 months, respectively. Targeted agents and immunotherapies have begun to reshape the landscape, with the IDH-1 inhibitor ivosidenib demonstrating activity in the ClarIDHy phase III trial for patients with IDH-1-mutant tumors, and pembrolizumab indicated for tumors deficient in mismatch repair proteins or showing high microsatellite instability. Still, the field suffers from a shortage of specific biomarkers and effective therapeutic targets.

CD39, also known as ectonucleoside triphosphate diphosphohydrolase 1, or ENTPD1, is a membrane-bound enzyme that hydrolyzes extracellular ATP and ADP into anti-inflammatory adenosine. It is normally expressed in organs such as the bladder, brain, lung, spleen, thymus, and placenta, primarily on endothelial and immune cells. Its abundance varies dramatically across immune populations: more than 90 percent of B lymphocytes, monocytes, and neutrophils in peripheral blood carry it, roughly 20 to 30 percent of CD4-positive T cells including memory T cells and regulatory T cells express it, fewer than 5 percent of CD8-positive T cells do, and only 2 to 5 percent of natural killer cells display it on their surface. Crucially, CD39 has been found overexpressed in a range of solid tumors, including colorectal cancer, lung cancer, pancreatic cancer, head and neck cancer, and chronic lymphocytic leukemia, and high expression has been linked to poor outcomes in clear cell renal cell carcinoma, non-small-cell lung cancers, hepatocellular carcinoma, ovarian carcinoma, and gastric cancers. Its role in cholangiocarcinoma, however, had remained unexplored until now.

To fill that gap, the researchers began with a systematic search for genes that behave abnormally in bile duct cancer. RNA sequencing of 12 paired tumor and adjacent non-tumor samples from their institution identified 3,383 upregulated and 3,258 downregulated genes. They then cross-referenced these results with RNA-seq data from the TCGA dataset, comprising 9 pairs, and a GEO dataset, also 9 pairs, and found 21 genes abnormally expressed across all three cohorts. Ten upregulated candidates were selected for validation by quantitative reverse transcription PCR in 24 paired tumor and normal tissue samples. Only one survived the scrutiny: CD39 was significantly upregulated in tumor tissue, and both RT-qPCR and western blotting confirmed that its levels were especially elevated in tumors from patients with lymph node metastasis.

The clinical significance became even clearer when the team turned to immunohistochemistry in a much larger cohort of 180 cholangiocarcinoma patients. Consistent with the molecular data, CD39 was highly expressed in tumor tissue compared with adjacent non-tumor tissue, with a P value below 0.001. Aberrant expression was also observed in patients with lymph node metastasis compared with those without, again at P < 0.001. Survival analysis revealed that patients with high CD39 expression had significantly worse overall survival, with P < 0.001. High expression correlated with a higher rate of lymph node metastasis, P = 0.007, and with more advanced TNM stage, P = 0.004. Most importantly, multivariable Cox proportional hazards regression established CD39 expression as an independent prognostic factor for overall survival, meaning its predictive power held even after accounting for other clinical variables.

Having established the correlation, the researchers moved to causation. They first profiled CD39 expression across human cholangiocarcinoma cell lines, finding it low in the RBE line and high in TFK-1, and used these two lines for gain- and loss-of-function experiments. Cell Counting Kit-8 assays showed that knocking down CD39 inhibited proliferation, while overexpressing it significantly boosted proliferative capacity. Transwell assays told a parallel story for motility: silencing CD39 suppressed the migratory and invasive abilities of TFK-1 cells, whereas forcing its expression enhanced migration and invasion in RBE cells. The pattern extended into living organisms. In cell-line-derived xenograft models using immunodeficient B-NDG mice, tumor volume and weight rose significantly when CD39 was overexpressed and fell when it was knocked down. The number of metastatic nodules in the liver increased in the overexpression group and decreased in the knockdown group, directly implicating CD39 in the metastatic spread of the disease.

The mechanistic breakthrough came from co-immunoprecipitation followed by mass spectrometry. Applying stringent filters, including confidence scores of at least 100 in the mass spectrometric assays and absence from corresponding anti-sense control groups, the team identified nine candidate proteins that physically interact with CD39. Western blotting confirmed that one of them, Annexin A2, or ANXA2, specifically binds CD39. ANXA2 is a calcium-mediated phospholipid-binding protein known to regulate cell survival and communication with the extracellular environment, and it has been implicated in the metastasis of gastric, colorectal, prostate, and breast cancers. Notably, the interaction with CD39 did not change the total amount of ANXA2 in the cells; instead, it altered its phosphorylation state. Knocking down CD39 suppressed phosphorylation of ANXA2 at the tyrosine 24 residue, a modification previously linked to malignant transformation, epithelial-mesenchymal transition, tumor invasion, and metastasis.

Downstream of ANXA2, the team traced the signal into two well-known cancer-promoting programs. Gene Set Enrichment Analysis of RNA-seq data from both tumor tissue and cell lines showed that the PI3K/AKT pathway and the epithelial-mesenchymal transition, or EMT, process were significantly influenced in cases with high CD39 expression. Western blotting confirmed the bioinformatics: knocking down CD39 reduced the phosphorylation of PI3K, AKT, and mTOR at the S2448 site without changing their total protein levels, while overexpression enhanced their phosphorylation. CD39 levels also reshaped the EMT machinery, with knockdown lowering the expression of Claudin-1, Twist-1, Vimentin, and N-cadherin while raising E-cadherin, and overexpression producing the opposite pattern. The decisive proof came from a rescue experiment in which the researchers mutated the Tyr24 site of ANXA2, substituting alanine for tyrosine. This single amino acid change reversed nearly everything CD39 had done: the proliferative boost measured by CCK-8, the migratory and invasive gains seen in transwell assays, the EMT marker shifts, and the PI3K/AKT phosphorylation all collapsed once the phosphorylation site was eliminated.

The authors are candid about the limitations of their work. The study is retrospective, and the prognostic role of CD39 will need validation in a prospective multi-center cohort. The number of samples and cell lines was modest, and larger cohorts would strengthen the statistical power and generalizability of the findings. It also remains unclear exactly how the CD39/ANXA2 axis influences cholangiocarcinoma progression, and how CD39 modulates ANXA2 phosphorylation and the downstream signaling cascades requires further investigation. Even so, the convergence of evidence from human tissue, cell culture, animal models, and mechanistic biochemistry makes a compelling case. CD39 stands out as both a biomarker that could identify patients at highest risk and a therapeutic target whose blockade might starve bile duct tumors of a key growth and spread pathway. For a cancer with so few options, that dual promise is a rare and welcome development.

Subject of Research: The role of CD39 expression in the progression, metastasis, and prognosis of cholangiocarcinoma

Article Title: The aberrant expression of CD39 is associated with the dismal outcome of patients with cholangiocarcinoma

Article References: Zheng, B., Shen, S., Wang, J., Sun, W., Wan, W., Liu, J., Zhang, D., Ni, X., Suo, T., Liu, H., Ni, X., & Liu, H. (2025). The aberrant expression of CD39 is associated with the dismal outcome of patients with cholangiocarcinoma. Clinical Cancer Bulletin, 4(1), Article 1. https://doi.org/10.1007/s44272-025-00028-0

Image Credits: AI Generated

DOI: 10.1007/s44272-025-00028-0

Keywords: CD39, cholangiocarcinoma, ANXA2, PI3K/AKT signaling, EMT, phosphorylation, prognostic biomarker, biliary tract cancer, ENTPD1, metastasis, immunohistochemistry, therapeutic target