Introduction

Cholangiocarcinoma (CCA) is an aggressive malignancy of the bile ducts, associated with poor prognosis and limited therapeutic options1. Early detection and precise molecular characterization are critical for improving clinical outcomes. However, conventional diagnostic approaches, such as tissue biopsies, are invasive and often fail to capture the molecular heterogeneity of the tumor. As a result, there is a growing need for less invasive and more comprehensive diagnostic methods.

Liquid biopsy has emerged as a promising alternative for molecular analysis in various cancers, including CCA2. Bile fluid, which directly interacts with tumor cells, represents a unique and valuable resource for liquid biopsy in CCA. It contains tumor-derived components, such as proteins, RNA, and circulating tumor DNA (ctDNA), which serve as biomarkers for disease detection and monitoring. Several studies have highlighted the potential of bile in elucidating the molecular landscape of CCA3,4,5. For example, ctDNA analysis from bile has been shown to detect tumor-specific mutations, offering insights into potential therapeutic targets and prognostic factors5. Additionally, bile is rich in proteins, messenger RNA (mRNA), and microRNA (miRNA), providing a deeper understanding of the underlying cancer biology6. These attributes position bile as a powerful tool for liquid biopsy in the context of CCA.

Despite its potential, the absence of standardized protocols for bile sample preparation has hindered its integration into routine clinical practice. Variables such as storage conditions, processing methods, and storage duration significantly impact the stability of bile-derived biomarkers, potentially compromising the reliability of molecular testing. Although previous studies have explored the utility of bile in molecular analysis, there remains a lack of clear.

guidelines for bile handling in the context of liquid biopsy. There is no clear guideline on bile sample handling and storage conditions. This study directly addresses this gap by determining how long bile samples can be stored at room temperature without significant degradation, thereby providing practical insights for clinical implementation. This study seeks to address this gap by systematically evaluating the optimal conditions for bile sample preparation and storage for protein and genetic analyses in CCA. By establishing best practices for handling bile samples, this work aims to improve the reliability and reproducibility of bile-based molecular diagnostics, enhancing their diagnostic and prognostic applications in CCA7.

Materials and methods

Patients

This prospective study was approved by the Institutional Review Board of Keimyung University Dongsan Medical Center (DSMC 2022-07-034). Bile fluid was collected from six patients diagnosed with extrahepatic cholangiocarcinoma (eCCA) between December 2021 and June 2022. To minimize potential confounding factors, strict inclusion criteria were applied. None of the patients included in this study had received chemotherapy, warfarin, or other medications known to affect hepatic metabolism prior to bile collection. Additionally, patients with pre-existing liver diseases such as viral hepatitis, alcoholic liver disease, or cirrhosis were excluded. Diagnoses were confirmed through a combination of surgical resection, needle biopsy, or cytology, and percutaneous transhepatic biliary drainage (PTBD). Bile was collected at least 3 days after PTBD, ensuring that samples were obtained after resolution of cholangitis, reducing the risk of inflammatory or infection-related alterations in bile composition. Patients either underwent curative resection or received palliative therapies based on their metastatic status8.

Bile collection and preparation

Bile samples were divided into four aliquots and processed at 1, 3, 5, and 7 h after sample collection. During this period, the samples were stored at room temperature, maintained at approximately 25 °C (range: 20–25 °C). The samples were centrifuged at 16,000 × g for 10 min at 4 °C to collect the supernatant. The pellets were resuspended in a two-fold volume of phosphate-buffered saline and re-centrifuged under the same conditions to obtain bile pellets9. All samples were stored for the analysis of circulating tumor DNA (ctDNA) at room temperature and analyzed at four-time intervals: 1, 3, 5, and 7 h after collection at − 80 °C for further analysis.

Chemicals and reagents

The following kits and reagents were used: NucleoSpin® cfDNA XS Kit (Macherey-Nagel), ddPCR™ KRAS Screening Multiplex Kit (Bio-Rad), E- and N-Cadherin assay kits (Biomatik), and antibodies (Abcam). Protein concentrations were determined using the Bradford assay kit (Thermo Scientific), and all other chemicals were sourced from Sigma (St. Louis, USA).

DNA extraction from bile samples

Circulating tumor DNA (ctDNA) was extracted from bile fluid supernatants following centrifugation at 16,000 × g for 10 min at 4 °C, as described by Abi Zabron et al.8. DNA extraction was performed on 270 µL aliquots using the NucleoSpin® cfDNA XS Kit, and ctDNA quality and quantity were analyzed using the Agilent 2100 Bioanalyzer. Extracted ctDNA samples were stored at − 20 °C for subsequent use.

Droplet digital PCR (ddPCR)

Droplet digital PCR was performed using the ddPCR™ KRAS Screening Multiplex Kit on a Bio-Rad QX200 system. Two-dimensional fluorescence amplitude plots were generated using QuantaSoft™ software (version 1.7, https://www.bio-rad.com/en-kr/life-science/digital-pcr/qx200-droplet-digital-pcr-system/quantasoft-software-regulatory-edition) to differentiate between mutant and wild-type DNA. Amplification was conducted on a C1000 Touch Thermal Cycler (Bio-Rad). The fragment size distribution of bile ctDNA was analyzed using the Agilent 2100 Bioanalyzer. Mutant KRAS DNA concentration was estimated using the Poisson distribution, with a cut-off value of 1.7 copies/µL for KRAS mutations.

Protein assay

Protein concentrations in bile were determined using the Bradford method, with bovine serum albumin as the standard. Absorbance was measured at 593 nm using an Infinite M 200 Pro microplate reader (TECAN, USA).

Enzyme-Linked immunosorbent assay (ELISA)

The activities of E- and N-Cadherin in bile were quantified using enzyme-linked immunosorbent assay (ELISA) kits. Diluted bile samples (100 µL) and standards were added to 96-well plates pre-coated with capture antibodies. After incubation and washing, avidin-HRP conjugation was added. The reaction was developed using tetramethylbenzidine (TMB) substrate, and absorbance was measured at 450 nm using an Infinite M 200 Pro microplate reader after the addition of stop solution.

Statistical analysis

Statistical analyses were conducted using R software (Version 4.4.2, https://web-r.org) and Sigma plot software (Version14.0, https://sigmaplot.softnic.kr). Continuous variables were presented as mean ± standard deviation (SD) and compared using independent t-tests. Categorical variables were analyzed using χ² tests. Time-dependent changes in variables were assessed using repeated measures ANOVA. A p-value of < 0.05 was considered statistically significant. To evaluate the clinical significance of the findings, effect sizes were calculated alongside P-values. Eta-squared (η²) was used to quantify the proportion of variance explained by the independent variable in repeated measures ANOVA. Effect sizes were interpreted as small (η² ≤ 0.01), medium (η² ≈ 0.06), and large (η² ≥ 0.14) based on Cohen’s guidelines.

Results

Bile preparation

For each patient, at least 10 mL of bile was collected, and 0.5 mL of each sample was processed within 7 h at room temperature (20–25 °C). First, the sample was centrifuged at 12,000 rpm for 10 min, and the supernatant (bile fraction) was saved. The pellet was resuspended in 1 mL of chilled PBS and centrifuged at 12,000 rpm for 5 min. This washing step was repeated once. After the final centrifugation, the supernatant was discarded, and the air-dried pellet was saved as the bile pellet fraction (Fig. 1).

Fig. 1
figure 1

A schematic diagram illustrating the preparation process of bile fluid samples collected from patients with cholangiocarcinoma (CCA). The samples were stored for the analysis of circulating tumor DNA (ctDNA) at room temperature and analyzed at four-time intervals: 1, 3, 5, and 7 h after collection.

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Protein concentration in bile

Protein concentrations in bile from six patients with cholangiocarcinoma (CCA) were analyzed at 1, 3, 5, and 7 h after initial collection using the Bradford method. Minor fluctuations in protein concentration were observed; however, no significant changes were detected over the 7-hour period (repeated measures ANOVA; P = 0.828, η² = 0.007). These results indicate that the effect size was negligible, suggesting minimal biological variability in protein concentrations across the observed time points. Thus, protein concentrations in bile remain stable at room temperature for up to 7 h, supporting its suitability for extended storage in clinical and research settings without significant degradation (Fig. 2). No significant changes in ctDNA concentration were observed over the 7-hour period in either the wild-type or mutant KRAS groups (repeated measures ANOVA; P = 0.926, η² = 0.059 and P = 0.399, η² = 0.059, respectively). The small effect sizes indicate minimal biological variability in ctDNA levels over time, suggesting that bile-derived ctDNA remains stable at room temperature for up to 7 h, ensuring its reliability for molecular analysis and biomarker assessment.

Fig. 2
figure 2

Protein concentration in bile samples from six CCA patients measured at 1, 3, 5, and 7 h after collection using the Bradford method. No significant changes in protein concentration were observed over time (Repeated measures ANOVA; P = 0.828).

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CtDNA concentrations

Mutant KRAS DNA concentrations in the bile of six CCA patients were assessed over time. Four patients had values below the cut-off of 1.5 copies/µL, indicating wild-type KRAS (Fig. 3A), while two patients exhibited values exceeding the cut-off, consistent with the presence of a KRAS mutation (Fig. 3B). No significant changes in ctDNA concentration were observed over the 7-hour period in either the wild-type or mutant KRAS groups (repeated measures ANOVA; P = 0.926, η² = 0.059 and P = 0.399, η² = 0.059, respectively). The small effect sizes indicate minimal biological variability in ctDNA levels over time, suggesting that bile-derived ctDNA remains stable at room temperature for up to 7 h, ensuring its reliability for molecular analysis and biomarker assessment.

Fig. 3
figure 3

Concentrations of ctDNA in bile samples from six patients with BTC. (A) Four patients with wild-type KRAS had ctDNA concentrations below the cut-off value of 1.5 copies/µL. (B) Two patients with KRAS mutations exhibited ctDNA concentrations above the cut-off value. No significant changes in ctDNA concentrations were observed over time for either group (Repeated measures ANOVA; P = 0.926 and P = 0.399, respectively).

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E- and N-Cadherin enzyme activity

E-Cadherin and N-Cadherin enzyme activities, key markers of epithelial-mesenchymal transition (EMT), were evaluated in bile from one CCA patient. No significant changes in enzyme activity were observed for either marker over the 7-hour period (Fig. 4, P = 0.650). These findings suggest that no significant metabolic alterations occurred in the bile within 7 h following collection.

Fig. 4
figure 4

Enzyme activities of E-Cadherin (A) and N-Cadherin (B) in bile from one BTC patient, measured at 1, 3, 5, and 7 h after collection using ELISA. No significant changes in enzyme activity were observed over time (Repeated measures ANOVA; P = 0.650).

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Discussion

One of the key findings of this study is the absence of significant changes in protein concentration, ctDNA levels, and EMT marker activity over the 7-hour period. While these results may be considered negative findings, their implications are highly significant for the practical use of bile-based liquid biopsy. The stability of ctDNA and protein biomarkers over time suggests that bile can serve as a reliable sample source for molecular diagnostics, even in real-world clinical settings where immediate sample processing may not always be feasible. This offers practical advantages for sample collection and transportation, particularly in settings where immediate freezing or processing is not possible.

This result aligns with previous studies suggesting that bile proteins are relatively robust under similar conditions10. Protein stability is essential for downstream applications, including proteomic profiling and biomarker discovery, which are crucial for understanding CCA pathogenesis and prognosis. These findings validate the practical utility of bile for protein-based assays in clinical and research settings.

Mutant KRAS DNA concentrations were analyzed in six patients, among whom four exhibited wild-type KRAS (below the cut-off of 1.5 copies/µL) and two showed mutant KRAS levels exceeding the cut-off (Fig. 3). Importantly, no significant changes in ctDNA concentration were observed over the 7-hour period in either group (P = 0.926 and P = 0.399, respectively). These findings are consistent with previous reports indicating that ctDNA remains stable under controlled conditions11,12. KRAS mutations are well-documented drivers of CCA pathogenesis, and their detection in bile may offer a non-invasive approach to molecular diagnostics13. The stability of ctDNA over several hours provides flexibility in sample handling, facilitating its application in diagnostic and prognostic workflows.

E-Cadherin and N-Cadherin activities, markers of EMT, also showed no significant changes over the 7-hour observation period (P = 0.650, Fig. 4). We observed no significant changes in E-Cadherin and N-Cadherin enzyme activities in bile over a 7-hour period. This stability suggests that these cadherin-associated enzymatic activities remain largely unaffected by short-term ex vivo conditions. One possible explanation is that bile, a relatively stable biofluid, may provide a protective environment that minimizes enzymatic degradation. Additionally, the absence of external stimuli, such as cellular interactions or inflammatory mediators, could contribute to the preservation of enzyme activity. These findings indicate that short-term storage does not significantly impact cadherin enzyme activity in bile, supporting its feasibility for biomarker analysis within this timeframe.

EMT is a critical process in cancer progression and metastasis, with these markers being strongly associated with poor prognosis in CCA14. The stability of EMT markers in bile further supports its use as a medium for studying tumor biology and developing targeted therapies.

Our study underscores the stability of key biomarkers in bile at room temperature, supporting its potential as a diagnostic and prognostic medium in CCA. The 7-hour stability window was selected in the present study based on real-world clinical workflow. Unlike blood sample, it requires several hours (typically 3–5 h) for sufficient bile accumulation before sample collection via procedures such as ERCP, ENBD or PTBD. Given these practical constraints, it is often impossible to process bile samples immediately after collection. To ensure a clinically relevant assessment, we designed our study to extend beyond the typical 3–5-hour collection period and evaluated bile stability up to 7 h. It may ensure that biomarker integrity is preserved even in cases where sample processing is delayed due to logistical challenges in hospital settings. While our findings demonstrate that bile retains molecular integrity for up to 7 h, prolonged storage and varying storage conditions (e.g., refrigeration or freezing) must be further investigated to ensure reproducibility and reliability in clinical applications. When comparing our findings with previous studies, it has been reported that storage temperature and duration can influence the stability of bile components. Reichen et al. examined bile from pigs stored at -15 °C for 17 days and found that most components remained stable, except for unconjugated bilirubin, which increased over time. Additionally, total bilirubin levels decreased by approximately 25% after 17 days at 4 °C and by 70% at 37 °C. These findings suggest that certain bile components are temperature-sensitive15. Another study investigated the stability of cell-free DNA (cfDNA) in bile. When bile samples were collected in cfDNA-preserving tubes and stored at room temperature for 10 days, cfDNA integrity was remained intact16. However, in non-preservative tubes, cfDNA degraded over time. Similarly, when bile samples were stored at 4 °C for two months in cfDNA-preserving tubes, cfDNA stability was maintained, whereas slight degradation was observed in standard tubes.

The demonstrated stability of proteins, ctDNA, and EMT markers for up to 7 h offers flexibility in sample handling and processing. These findings contrast with recommendations for immediate centrifugation and storage at -80 °C to prevent degradation3,5,9,17. Biofluids such as bile are inherently more vulnerable to enzymatic degradation compared to tissue samples17. The composition of bile, including bile acids, cholesterol, and proteins, increases its susceptibility to molecular breakdown1,8,9,18. The observed stability of ctDNA and protein levels in bile over the 7-hour period suggests that bile may provide a protective environment for these biomarkers. Several biological and chemical factors may contribute to this stability. Unlike plasma, where ctDNA is rapidly degraded by nucleases, bile has a distinct composition that may protect ctDNA from enzymatic degradation. Bile contains bile salts, phospholipids, and mucins, which may form micelles or vesicle-like structures that encapsulate ctDNA and prevent nuclease-mediated degradation. Similarly, proteins in bile may be stabilized by hydrophobic interactions with bile acids or binding to other molecular carriers, such as albumin or exosome membranes, reducing susceptibility to proteolytic degradation. While the exact mechanism remains unclear, this study provides initial evidence that bile can serve as a stable medium for biomarker preservation. While overall KRAS mutant ctDNA levels remained stable, inter-patient variability was observed. This is presumed to be related to various factors, including tumor burden, tumor heterogeneity, and mutation clonality; however, as the variations observed were all within the cut-off value range, they did not have a significant impact on the overall results.

This study has a few limitations, including the small sample size (n = 6) and the lack of validation in larger, multi-center cohorts. Only one patient’s bile was analyzed for EMT markers, and this restricts the generalizability of our findings. Therefore, the generalizability of these results may be limited by inter-individual variability in bile composition, and it may introduce selection bias, as the patient’s individual disease characteristics could influence enzyme activity measurements. Larger cohorts are needed to validate our results and better understand the influence of patient-specific factors, such as liver conditions, infections, or medications, on bile composition and biomarker stability. Although the primary aim of this study was to evaluate how long protein and ctDNA remain stable in bile at room temperature, rather than to compare absolute biomarker levels between different patient groups, the lack of a control group could be another limitation. The application of bile biomarkers in the context of CCA diagnosis and prognosis holds significant promise, and ongoing efforts should aim to expand the utility of bile in precision oncology.

In conclusion, this study demonstrates that ctDNA and protein biomarkers in bile remain stable for up to 7 h at room temperature, supporting the feasibility of bile-based liquid biopsy in cholangiocarcinoma. These findings suggest that bile could be a viable diagnostic medium, offering practical advantages for clinical workflows where immediate sample processing is not always feasible. Further studies with larger patient populations and long-term validation are needed to confirm these results. Despite limitations, our study provides a foundation for developing standardized protocols for bile sample handling and storage, facilitating the integration of bile-based liquid biopsy into clinical practice.