Drug Sensitivity of Circulating Tumor Cells as a Game-Changer in Subsequent-Line Therapy for Biliary Tract Carcinoma

Drug Sensitivity of Circulating Tumor Cells as a Game-Changer in Subsequent-Line Therapy for Biliary Tract Carcinoma | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Drug Sensitivity of Circulating Tumor Cells as a Game-Changer in Subsequent-Line Therapy for Biliary Tract Carcinoma Huafei Li, Shuangqun Chen, Xiaoxia Kou, Yuan Tian, Cong Wu, Huiying Liu, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7201269/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 02 Feb, 2026 Read the published version in Cancer Cell International → Version 1 posted 9 You are reading this latest preprint version Abstract Biliary tract carcinoma (BTC) is an aggressive cancer with a poor prognosis, and chemotherapy’s effectiveness is limited, especially after first-line therapy failure. Circulating tumor cells (CTCs) offer a platform for in vitro drug-sensitivity testing to optimize subsequent-line chemotherapy, but the clinical efficacy and prognostic value remains well-established. In this study, we retrospectively analyzed 85 advanced BTC patients, with 25 receiving CTC-based drug-sensitivity-guided chemotherapy (CSBT), 15 receiving FOLFOX, and 45 receiving empirical therapy. CTCs were enriched and tested for drug sensitivity using a glucose uptake assay. Therapeutic efficacy, including patient response, progression-free survival (PFS), overall survival (OS), and toxicity profiles, was evaluated. The results indicated that the objective response rate (ORR) was 16% in CSBT, 6.7% in FOLFOX, and 4.4% in the empirical group. The disease control rate (DCR) was significantly higher in CSBT group (56%) compared to the FOLFOX (20%) and empirical therapy (22.2%; p < 0.05) groups. Median PFS was significantly prolonged in the CSBT group (5.4 months) versus the FOLFOX (1.9 months) and empirical therapy (2.7 months; p < 0.05) groups. Median OS was extended in the CSBT group (12 months), with a significant improvement in OS during the first year of treatment ( p < 0.05). Toxicity profiles were similar across all groups. In conclusion, this study for the first time proved that CTC-based drug-sensitivity testing offers a potential approach to guide chemotherapy for advanced BTC. Furthermore, this approach does not increase the risk of severe adverse events, highlighting its potential as a safe and effective strategy for improving patient prognosis in advanced BTC. Circulating tumor cells In vitro drug screening Anti-cancer drug sensitivity testing Biliary tract cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Background Biliary tract cancer (BTC) ranks as the second most prevalent primary liver malignancy following hepatocellular carcinoma (HCC), accounting for approximately 3% of all gastrointestinal cancers [ 1 , 2 ]. Among BTC types, adenocarcinoma is the most common pathological subtype, which is notorious for its aggressive nature, difficulty in early diagnosis, and exceedingly poor prognosis, with a global five-year survival rate of less than 5% [ 3 , 4 ]. While surgical resection remains the only potential curative treatment for BTC, nearly two-thirds of patients are diagnosed at a stage where the cancer is locally advanced, unresectable, or metastatic, eliminating surgery as a viable option [ 4 , 5 ]. For these patients, systematic chemotherapy remains the major therapeutics, with the combination of gemcitabine plus cisplatin (GC) serving as the current frontline standard regimen [ 6 ]. Despite initial responses to first-line chemotherapy, most patients eventually experience disease progression, facing with the issue of receiving subsequent-line treatments [ 7 ]. The selection of an appropriate subsequent-line treatment is challenging due to limited standardized options and variable patient responses [ 8 , 9 ]. One regimen that has been investigated is FOLFOX, which combines folinic acid, fluorouracil, and oxaliplatin. The ABC-06 trial, a phase III study, evaluated FOLFOX plus active symptom control (ASC) versus ASC alone in patients with advanced BTC who had progressed after first-line GC therapy. The results indicated a modest improvement in overall survival (OS) for the FOLFOX plus ASC group, suggesting its potential as a second-line option [ 6 ]. However, the efficacy of subsequent-line therapies of BTC remains limited, with significant challenges still present. A key challenge is cancer heterogeneity, which varies both within individual tumors and across patients. This heterogeneity complicates treatment by contributing to varied therapeutic responses, thereby diminishing the overall efficacy of current treatment strategies [ 10 , 11 ]. Additionally, the rarity of BTC has limited the availability of large-scale randomized controlled trials, which hinders the establishment of standardized second-line or subsequent-line therapeutic regimens. To address these challenges, ongoing research is focusing on personalized medicine, which holds the potential to revolutionize cancer treatment and improve patient outcomes [ 12 ]. Traditional approaches to personalized medicine involve identifying predictive biomarkers of treatment sensitivity or resistance in tumor cells [ 13 ]. For example, in colorectal cancer, panitumumab is effective only in patients without KRAS mutations. Thus, to ensure optimal outcomes, patients are screened for KRAS mutations prior to receiving panitumumab in the clinic [ 14 ]. Similarly, patients with non-small cell lung cancer (NSCLC) are tested for specific epidermal growth factor receptor (EGFR) mutations prior to receiving treatments like gefitinib or erlotinib [ 15 ]. Although advancements in cancer genomics over the past two decades have broadened our understanding, there remain many instances in which genomic analysis does not yield effective or sensitive therapeutic targets [ 16 ]. Even when actionable genomic alterations are identified, patient responses to the targeted therapy are not guaranteed. Furthermore, for most standard chemotherapeutic agents, no reliable genetic markers have been identified to predict patient response accurately. The issues of intrinsic and acquired chemoresistance significantly impact treatment outcomes, contributing to treatment failure in over 90% of patients with metastatic BTC [ 17 , 18 ]. For these patients, in vitro drug screening using patient-derived tumor cells offers a valuable approach. This method enables the confirmation of drug sensitivity, prediction of therapeutic efficacy, and identification of additional treatment options [ 19 ]. Currently, enrolling such heavily pretreated patients—with multi-drug resistance (MDR) after progression under second or third-line treatments— in clinical trials is challenging, because they often do not meet the stringent entry criteria. Pursuing compassionate use of non-FDA-approved drugs or off-label use of FDA-approved drugs through individualized “N-of-1” studies can be time-consuming, costly, and frequently impractical. In contrast, in vitro drug testing provides a feasible solution for screening potential drug combinations, benefiting both physicians and patients [ 16 ]. In recent years, circulating tumor cells (CTCs) have garnered increasing attention in cancer research [ 20 ]. Multiple studies have revealed that CTCs appear early in the disease course, with their presence in the bloodstream correlating strongly with disease severity [ 21 , 22 ]. Clinicians are now considering CTCs as a form of “fluid biopsy”, offering real-time insights into the tumor’s current status. CTCs are thought to reflect, in part, the extensive heterogeneity of the tumor [ 22 , 23 ]. CTCs have become a focal point to target for therapy due to the fact they are the primary route for metastasis to occur [ 24 ]. Consequently, they are extensively studied at the individual patient level for various characterizations, such as assessing their epithelial-to-mesenchymal transition (EMT) state and identifying surface markers associated with specific drug responses [ 17 , 25 ]. Despite their potential significance, clinical studies exploring the predictive impact of CTCs on chemotherapeutic response and drug sensitivity remain limited. In this retrospective study, we delved into the transformative potential of drug sensitivity testing on CTCs in shaping subsequent-line chemotherapy strategies for patients with advanced BTC who have experienced relapse post-initial treatment. Our analysis underscores the pivotal role of CTC-based drug sensitivity assessments in enabling the selection of more efficacious chemotherapy protocols. This personalized approach not only holds the promise of promoting patient survival but also significantly enhances the clinical outcomes for individuals battling advanced BTC (Fig. 1 ). This study positions CTC drug sensitivity testing as a game-changer in the realm of subsequent-line chemotherapy for advanced BTC, offering a tailored and potentially more successful treatment trajectory. 2. Materials and methods 2.1 Patients and study design. A retrospective analysis was conducted on a total of 85 patients with advanced BTC at the Department of Oncology Biotherapy, the Third Affiliated Hospital of Navy Medical University (Shanghai, China) between February 2018 and September 2023. Among them, 26 patients received subsequent chemotherapy regimens based on the results of CTC drug sensitivity testing (CTC drug sensitivity-based therapy, CSBT), 15 patients were treated with the standard FOLFOX regimen, and the chemotherapy regimens of the other 45 patients were based on physician experience (experience-based therapy, EBT) (Fig. 1 ). All patients had a definitive diagnosis confirmed by pathology from surgery or liver biopsy, and their medical records were complete. The inclusion criteria for cases are as follows: (1) patients aged between 18 and 80 years, (2) an Eastern Cooperative Oncology Group (ECOG) performance status of 3 or lower, (3) the presence of clearly measurable cancer lesions, (4) patients with locally advanced disease without distant metastasis were deemed unresectable following a multidisciplinary assessment, (5) baseline blood tests and biochemical indicators within specified ranges: neutrophil count >1.5×10 9 /L, hemoglobin > 90 g/L, platelet count > 75×10 9 /L, serum total bilirubin < 33.5 µmol/L, alanine aminotransferase (ALT) < 100U/L, aspartate aminotransferase (AST) < 100U/L, and serum creatinine (SCR) < 160 µmol/L, (6) conventional empirical therapy had been administered as the first-line treatment for patients experiencing disease progression. Exclusion criteria: (1) Presence of other malignancies; (2) women who are pregnant or breastfeeding; (3) history of severe allergy to one or more drugs; (4) patients with severe infections requiring treatment; (5) presence of active bleeding or intestinal obstruction; (6) individuals with severe liver disease, kidney disease, respiratory diseases, or uncontrolled diabetes, hypertension, coronary heart disease, and other chronic conditions. 2.2 Samples collection and CTC enrichment Patient-derived CTCs were efficiently isolated and enriched through microfluidic methods. Briefly, fresh blood samples, totaling 30 ml, were collected from the patient's peripheral vein and collected in BD Vacutainer EDTA tubes (Becton, Dickinson and Company, NJ, USA). The samples were meticulously mixed with anticoagulants by gently inverting the tubes several times. Following centrifugation, the plasma layer was discarded, and the remaining sample underwent erythrocyte lysis to eliminate red blood cells (RBCs). The nucleated cells were then resuspended in PBS containing 1% BSA and processed through the FlowCell™ CTC enrichment system (Polaris Biology, Shanghai, China), which effectively removed residual RBCs and white blood cells (WBCs). This sophisticated method yielded approximately 100–400 CTCs per patient. 2.3 Assessment of drug susceptibility As the number of CTCs in patient peripheral blood is relatively low (ranging from a few to several dozen per milliliter), the Glucose uptake assay was employed for drug sensitivity assessment. Generally, the patient CTCs (approximately 100–400 based on the patient’s condition) were evenly distributed and inoculated into 384-well plates (with about 5–10 tumor cells per well). Cells were cultured in RPMI1640 cell culture medium without glucose supplemented with 20% Fetal Bovine Serum (FBS, Thermo Fisher Scientific, Massachusetts, U.S.A.). To each well, 0.4 mmol/L of the fluorescently labeled glucose analog, 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG, Thermo Fisher Scientific, Massachusetts, U.S.A.), was added. The chemotherapeutic agents to be tested were also added for a 24-hour incubation. The concentration of the chemotherapeutic drugs is set as the average IC50 value of multiple BTC cell lines (1.0 IC50) and half of IC50 values (0.5 IC50), with three replicate wells for each concentration. After 24 hours, a fluorescently labeled CD45 antibody is added to each well and further incubated for 30 minutes. The EVOS™ FL M7000 imaging system (Thermo Fisher Scientific, Massachusetts, U.S.A.) is used to photograph each well, and a neural network-based algorithm is utilized to analyze cell morphology, CD45 expression, and cellular glycometabolism levels. By comparing the average glycometabolism rate (GMR) of CTCs in drug-treated wells with that of the control group (tumor cells untreated with chemotherapy drugs), the drug sensitivity (activity inhibition rate of different chemotherapy regimens at various concentrations on CTCs) can be calculated according to the following equations: Drug sensitivity where average GMR in experimental group represented the relative glucose uptake rates of CTCs at the concentrations of 0.5 IC50 and 1.0 IC50. The sensitivity of CTCs was categorized into four distinct levels: highly sensitive (less than 0.70), moderately sensitive (ranging from 0.70 to 0.90), and resistant (more than 0.90). The objective is to identify the most effective chemotherapeutic regimen based on the established sensitivity levels. The alternative chemotherapeutic regimens to be tested mainly include: Gemcitabine plus Cisplatin (GC regimen), S-1 plus Oxaliplatin (SOX regimen), Gemcitabine plus Capecitabine (GX regimen), Nab-paclitaxel plus Gemcitabine (AG regimen), Oxaliplatin plus Leucovorin plus Fluorouracil (FOLFOX regimen), Irinotecan plus Leucovorin plus Fluorouracil (FOLFIRI regimen). 2.4 Therapeutic efficacy evaluation All patients underwent serum tumor markers, blood routine examination, blood biochemistry test, electrocardiogram, CT or MRI examination before and after each cycle of chemotherapy. Efficacy was evaluated every two cycles of chemotherapy according to the Response Evaluation Criteria in Solid Tumors (RECIST), which categorizes responses into complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). Objective response rate (ORR) and disease control rate (DCR) were calculated as follows: ORR = CR rate + PR rate, DCR = CR rate + PR rate + SD rate. Progression-free survival (PFS) is defined as the time from the start of chemotherapy to tumor progression, and overall survival (OS) is defined as the time from the start of chemotherapy to the patient’s death, loss to follow-up, or the last follow-up. Follow-ups were conducted through hospital visits or telephone calls. Adverse reactions were evaluated according to the National Cancer Institute-common toxicity criteria (NCI-CTC) 4.0, graded from 0 to 4. 2.5 Statistical analysis Data analysis was conducted utilizing SPSS software (version 23.0). For continuous variables, data are presented as the mean ± standard deviation (x ± s), with group comparisons performed using independent samples t-tests. Categorical data are depicted as frequencies and percentages, with group comparisons carried out via chi-square tests or Fisher’s exact test when appropriate. Survival analysis was executed employing the Kaplan-Meier estimation to generate survival curves, and survival time comparisons were assessed using the log-rank test. The significance level (α) was set at 0.05. 3. Results 3.1 Patient characteristics A total of 85 patients were enrolled in this study, categorized into three groups: 25 patients in the CSBT group, where the therapeutic regimens were determined based on CTC drug susceptibility testing results; 15 patients treated with FOLFOX-based chemotherapy; and 45 patients receiving experience-based therapy (EBT) (Fig. 1 ). Detailed patient characteristics are presented in Table 1 . Analysis revealed that general condition, tumor staging, and the incidence of distant metastasis were largely comparable across the three groups, suggesting a balanced patient profile. Table 1 Baseline demographic and clinical characteristics of patients in different groups. CSBT ( N = 25) FOLFOX ( N = 15) EBT ( N = 45) P value Age, years Median 57.7 55.8 55.978 0.78 Range 33–80 37–74 37–75 Gender Male 14 6 25 0.5418 Female 11 9 20 Primary tumor site Intrahepatic bile duct 14 8 19 0.8151 Extrahepatic bile duct 4 3 9 Gallbladder cancer 7 4 17 TNM stage ⅢB 4 1 2 0.3894 Ⅳ 16 10 27 IVB 5 4 16 Abbreviation: TNM, tumor-node-metastasis 3.2 Improved treatment efficacy of patients receiving CTC-based therapy Figure 2 presents a detailed comparison of the efficacy outcomes across three chemotherapy regimens. Patients in the CSBT group achieved the highest ORR at 16%, with 1 out of 25 achieving CR and 3 out of 25 PR. In comparison, the ORR was 6.7% for the FOLFOX-based therapy group, with no CR and 1 out of 15 patients achieving PR, while the EBT group had the lowest ORR at 4.4%, with no CR and 2 out of 45 patients achieving PR (Figs. 2 A and 2 C). However, although the CSBT group demonstrated a notably higher ORR, the differences among groups were not statistically significant (p = 0.235). In contrast, the DCR showed significant differences among groups. The CSBT group achieved the highest DCR at 56%, significantly outperforming the FOLFOX group (20%) (p = 0.046) and the EBT group (22.2%) (p = 0.008) (Figs. 2 B and 2 C). These findings indicate that the therapeutic regimens based on CTC drug sensitivity testing are more effective in controlling disease progression. The lack of statistical significance in ORR differences suggests that a larger cohort might be needed to fully capture the potential efficacy disparities between the regimens. However, the significant difference in DCR highlights the clear advantage of CTC-based therapy in achieving disease control, underscoring its potential for disease control. 3.3 Prolonged PFS of patients in CSBT group. Subsequently, we compared the progression-free survival of BTC patients in different groups. As depicted in Fig. 3 A, drug sensitivity testing significantly improved the progression-free survival of patients. Generally, the median progression-free survival (mPFS) for patients treated with FOLFOX regimen and EBT was 1.9 ± 0.20 months and 2.6 ± 0.27 months, respectively, with no statistically significant difference observed between these two groups. In contrast, patients treated with CSBT demonstrated a markedly prolonged mPFS of 5.4 ± 1.17 months (*p < 0.05). These findings are further supported by the risk function shown in Fig. 3 B, which highlights a consistently lower initial and sustained risk of disease progression in patients receiving CSBT. This underscores the potential benefit of tailoring treatment based on drug susceptibility to achieve improved outcomes. 3.4 Prolonged OS of patients in CSBT group. In our comprehensive analysis, we observed a modest yet noteworthy extension in the OS of patients who were administered therapeutics based on CTC drug sensitivity testing. Specifically, these patients exhibited a median OS (mOS) of 12 ± 1.58 months, as depicted in Fig. 4 A and Table 2 . This finding is particularly significant when juxtaposed with the mOS of 5.1 ± 1.10 months for those treated with FOLFOX regimen, and 7.8 ± 1.88 months for patients receiving empiric therapy. However, the statistical analysis did not yield a significant difference among groups. The risk function graph presented in Fig. 4 B offers a visual representation of these survival outcomes. The green line, representing the CSBT group, begins at a lower point on the graph, signifying a reduced initial risk of mortality. Over the course of the study period, this line consistently remains slightly below the others, suggesting a sustained lower risk of death. However, as time progresses, the risk function curves for all groups tend to converge, a trend that underscores the notion that, in the long term, the risk of death among patients may become more homogeneous across different groups. Table 2 Survival analysis among groups. CSBT FOLFOX EBT mPFS (months) 5.4 ± 1.17 1.9 ± 0.20 2.6 ± 0.27 mOS (months) 12 ± 1.58 5.1 ± 1.10 7.8 ± 1.88 Given the above-mentioned trends of OS, and significant extension in PFS observed in the CSBT group, we did survival analysis of patients during the initial 12 months of treatment, elucidating the short-term impact of drug sensitivity assessment on patient outcomes. The initial year of treatment is often marked by the most rapid changes in disease status, the survival analysis during which will provide a more nuanced understanding of the treatment effects during the critical initial phase of patient management. The survival curves in Fig. 4 C demonstrate that, the green line (CSBR group) likely demonstrates a statistically higher survival rate compared to the blue (FOLFOX group) and orange (EBT group) lines, respectively. This suggests that patients receiving CSBT have a better chance of surviving the first year. The curves may show a gradual convergence as the 12-month mark approaches, indicating that while the initial survival benefit is evident, it may not be as pronounced towards the end of the first year. Accordingly, the risk functions illustrated in Fig. 4 D reflects a reduced initial risk of death for patients treated with CSBT. Over the first year, this green line remains below the other two, suggesting a consistently lower risk of death. These results suggest that drug sensitivity-based therapeutics offer an initial survival advantage. Patients treated with these regimens exhibit better short-term survival rates and a lower risk of death early in the treatment course. However, the convergence of survival curves and risk functions towards the end of the first year implies that this advantage may not be as significant or may diminish over the short term. This analysis underscores the need for continued monitoring and further research to assess the long-term implications of these initial survival benefits and to determine if they translate into sustained improvements in overall survival outcomes. 3.5 No statistically significant differences among groups. As presented in Table 3 , a comparative analysis of chemotherapy-related adverse events was conducted across different groups, with particular emphasis on grade 3–4 toxicities, which are indicative of greater severity. In terms of hematological adverse events, anemia was the most frequently observed, impacting 48%, 40%, and 28.9% of patients in the CTC-based, FOLFOX-based, and experience-based therapeutic groups, respectively. Notably, there was no significant variation in the incidence across all grades or specifically in grade 3–4 cases. Neutropenia and thrombocytopenia were also documented. Table 3 Chemotherapy-related adverse events in different treatment groups. CSBT ( N = 25) FOLFOX ( N = 15) EBT ( N = 45) Adverse events Overall Grade 3–4 Overall Grade 3–4 Overall Grade 3–4 Hematological Anemia 12 1 6 0 13 0 Neutropenia 5 2 2 1 7 1 Thrombocytopenia 10 2 5 1 14 4 Nonhematological Fatigue 13 3 8 1 20 1 Anorexia 14 2 7 0 12 1 Vomiting 5 1 3 1 5 2 Diarrhea 2 0 1 1 3 1 Sensory neuropathy 2 0 2 0 2 0 Hand-foot syndrome 2 1 0 6 0 Allergy 0 0 0 0 0 0 Liver dysfunction 2 0 1 0 2 0 When examining nonhematological adverse events, fatigue stood out as the most common, impacting 52% of patients in the CSTB group, 53.3% in the FOLFOX-based therapy group, and 44.4% in the EBT group. Anorexia was another frequently reported side effect, affecting 56% of patients in the CTC-based therapy group, 46.7% in the FOLFOX-based therapy group, and 26.7% in the experience-based therapy group. Additional side effects such as vomiting and diarrhea were also reported throughout the chemotherapy period. Although the frequencies of these side effects vary, there are no statistically significant differences among groups. This suggests that while each regimen may have a distinct profile of adverse events, the severity of these events does not significantly differ, allowing for a balanced consideration of efficacy and safety in treatment decisions. 4. Discussion This study provides valuable insights into the potential of CTCs in guiding chemotherapy for advanced BTC. Our findings demonstrate that CTC-based drug sensitivity testing can significantly influence treatment outcomes. Patients in the CSTB achieved notably improved ORRs, significantly improved DCRs and prolonged PFS, compared to those receiving FOLFOX or empiric therapy. To the best of our knowledge, this is the first study to systematically investigate the impact of CTC drug sensitivity-guided clinical decision-making on patient outcomes. Over the past decades, personalized medicine has played an increasingly critical role in cancer treatment, driven by the need to address the molecular heterogeneity of neoplastic diseases [ 26 ]. While tissue biopsy remains the gold standard for profiling tumor molecular characteristics and informing treatment decisions, it is invasive and provides limited insight into the clonal evolution of tumors during disease progression [ 27 ]. In this context, liquid biopsies have emerged as a promising alternative. Non-invasive, easily repeatable, and accessible, liquid biopsies have been extensively studied for their potential to revolutionize clinical decision-making by enabling real-time tumor monitoring [ 28 , 29 ]. In vitro drug screening using patient-derived tumor cells provides a valuable approach for confirming drug sensitivity, predicting therapeutic efficacy, and identifying alternative treatment strategies [ 11 ]. CTCs, as a major component of liquid biopsy, are ideal candidates for such analyses. They offer prognostic and predictive information and dynamically reflect the evolving characteristics of tumor cells, particularly their drug sensitivities [ 30 , 31 ]. However, the rarity of CTCs in the bloodstream has historically limited their clinical application. In this study, we successfully isolated 100–400 CTCs per patient using a microfluidic method, overcoming a key technical barrier. Although this number is insufficient for traditional viability assays like CCK-8 or MTT, we employed the innovative 2-NBDG method, which enabled metabolic activity assessments with minimal cell input. The 2-NBDG method leverages the Warburg effect, whereby cancer cells exhibit increased glycolysis [ 32 ]. By monitoring glucose metabolism via the fluorescent 2-NBDG analog, this method provides a label-free, real-time assessment of drug sensitivity [ 33 ]. Previous studies have validated its utility in some cancer types, and our findings demonstrate its applicability in CTC-based drug sensitivity testing [ 33 , 34 ]. Using this novel methodology, 25 patients in the CSBT group were treated based on CTC drug sensitivity testing results. Statistical analyses revealed that the CSBT group achieved an ORR of 16% and a DCR of 56%, both remarkably higher than those observed in the two control groups. Besides, CTC drug sensitivity testing significantly improved the mPFS of patients to 5.4 ± 1.17 months. It is noteworthy that these clinical outcomes appear to be prior to those of the clinically recommended second-line regimen as reported previously. In a randomized Phase 3 ABC-06 trial, the ASC + mFOLFOX regimen showed significant clinical efficacy, with an ORR of 5.0%, a DCR of 33%, a median PFS of 4.0 months, and a median OS of 6.2 months [ 6 , 35 ]. The observed improvements in ORR and DCR emphasize the potential of personalized, CTC-guided chemotherapy to address challenges posed by tumor heterogeneity and interpatient variability in treatment response. These findings align with growing evidence supporting the use of liquid biopsy-based approaches for dynamic tumor monitoring and treatment optimization. The significant extension of PFS in the CTC group suggests that CTC-guided chemotherapy may effectively delay disease progression, providing patients with valuable additional time. This is particularly critical given the limited treatment options and poor prognosis associated with advanced BTC. As for the OS of patients, patients in CTC drug sensitivity testing group exhibited a median OS (mOS) of 12 ± 1.58 months, although noteworthy extended, the statistical analysis did not yield a significant difference among groups. However, survival curves indicated an early survival benefit for patients receiving CTC-guided therapy, with improved survival rates during the first year of treatment. The convergence of survival curves beyond the 12-month mark suggests that while the initial benefits are clear, their long-term impact warrants further investigation. This analysis underscores the need for continued monitoring and further research to assess the long-term implications of these initial survival benefits and to determine if they translate into sustained improvements in overall survival outcomes. Importantly, the toxicity profiles of the treatments were comparable across all groups, with no significant differences in the incidence of severe adverse events. This demonstrates that the efficacy advantages of CTC-guided therapy do not come at the cost of increased toxicity, a critical consideration in the management of advanced cancer. However, this study has several limitations. The relatively small sample size reduces the generalizability of the findings and increases the risk of bias. Additionally, potential confounding factors, such as variations in treatment timing and prior therapies, were not fully accounted for. Future research should focus on larger, prospective, randomized controlled trials to validate these results. Further studies should also explore the underlying mechanisms of CTCs in predicting drug response, identify novel therapeutic targets, and evaluate the cost-effectiveness of integrating CTC-based approaches into standard clinical practice. CTC-based drug sensitivity testing emerges as a transformative approach in guiding subsequent-line chemotherapy for advanced BTC. CTC-guided therapy notably enhances ORRs, significantly improves DCRs and PFS, and provides a short-term OS advantage during the first year of treatment. Although no statistically significant differences in OS were observed across the entire study period, the trends and early survival benefits suggest that personalized chemotherapy informed by CTC drug sensitivity assessments can address tumor heterogeneity and optimize therapeutic outcomes. Intriguingly, the results imply that to maximize patient benefit, increasing the frequency of CTC drug sensitivity testing during the treatment course, such as annually, could potentially and significantly improve patient prognosis and survival. However, this hypothesis necessitates validation through further clinical research. 5. Conclusion CTC-based drug sensitivity testing has emerged as a transformative approach in guiding subsequent-line chemotherapy for advanced BTC. CTC-guided therapy significantly improves ORRs, DCRs, and PFS, while offering a short-term OS advantage during the first year of treatment. Although no statistically significant differences in OS were observed over the entire study period, the trends and early survival benefits suggest that personalized chemotherapy based on CTC drug sensitivity assessments can address tumor heterogeneity and optimize therapeutic outcomes. Notably, the results imply that increasing the frequency of CTC drug sensitivity testing during the treatment course - for example, to annual testing - could potentially improve patient prognosis and survival. However, this hypothesis requires validation through further clinical research. Abbreviations BTC: Biliary Tract Carcinoma CTC: Circulating Tumor Cells CSBT: CTC-Based Drug Sensitivity-Guided Therapy FOLFOX: Folinic Acid, Fluorouracil, and Oxaliplatin EBT: Experience-Based Therapy ORR: Objective Response Rate DCR: Disease Control Rate PFS: Progression-Free Survival OS: Overall Survival NCI-CTC: National Cancer Institute-Common Toxicity Criteria ECOG: Eastern Cooperative Oncology Group IC50: Half Maximal Inhibitory Concentration ALT: Alanine Aminotransferase AST: Aspartate Aminotransferase SCR: Serum Creatinine GC: Gemcitabine and Cisplatin MDR: Multi-Drug Resistance 2-NBDG: 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose BSA: Bovine Serum Albumin RPMI1640: Roswell Park Memorial Institute Medium 1640 FBS: Fetal Bovine Serum CD45: Cluster of Differentiation 45 RECIST: Response Evaluation Criteria in Solid Tumors Declarations Authors’ contribution H Li and J Qiu: Conceptualization, methodology, validation, formal analysis, data curation, investigation, manuscript revision, and project administration; S Chen, X Kou, and H Liu: Methodology, data collection, validation, formal analysis, writing—original draft; C Wu, and Y Tian: Formal analysis, data curation, visualization, and methodology. All authors read and approved the final manuscript Funding This work was supported by National Key R&D Program of China, MOST (2023YFC2510000), Natural Science Foundation of Shanghai (Grant No. 21ZR1422800). Availability of data and materials Not applicable. Ethical approval: This in a retrospective study. The Ethics Committee of Shanghai University (Approval No. ECSHU 2024-275) provided institutional ethical approval for this study. Informed consent was obtained from all patients for the use of their medical records in the study prior to the inclusion of their case details. The study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Acknowledgements Shanghai ChemAn Biotech Co., Ltd. should be deeply acknowledged for their technical support in the enrichment and drug sensitivity testing of CTCs. References Rizvi S, Khan SA, Hallemeier CL, Kelley RK, Gores GJ. Cholangiocarcinoma - evolving concepts and therapeutic strategies. Nat Rev Clin Oncol. 2018;15(2):95-111. Xie C, McGrath NA, Monge BC, Fu J. Systemic treatment options for advanced biliary tract carcinoma. J Gastroenterol. 2020;55(10):944-957. Brindley PJ, Bachini M, Ilyas SI, Khan SA, Loukas A et al . Cholangiocarcinoma. Nat Rev Dis Primers. 2021;7(1):65. Vithayathil M, Khan SA. Current epidemiology of cholangiocarcinoma in Western countries. J Hepatol. 2022;77(6):1690-1698. Banales JM, Marin J, Lamarca A, Rodrigues PM, Khan SA et al . Cholangiocarcinoma 2020: the next horizon in mechanisms and management. Nat Rev Gastroenterol Hepatol. 2020;17(9):557-588. Rizzo A, Cusmai A, Giovannelli F, Acquafredda S, Rinaldi L et al . 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Trastuzumab plus FOLFOX for HER2-positive biliary tract cancer refractory to gemcitabine and cisplatin: a multi-institutional phase 2 trial of the Korean Cancer Study Group (KCSG-HB19-14). Lancet Gastroenterol Hepatol. 2023;8(1):56-65. Barthel S, Falcomata C, Rad R, Theis FJ, Saur D. Single-cell profiling to explore pancreatic cancer heterogeneity, plasticity and response to therapy. Nat Cancer. 2023;4(4):454-467. Tian Y, Wang X, Wu C, Qiao J, Jin H et al . A protracted war against cancer drug resistance. Cancer Cell Int. 2024;24(1):326. Ma G, Yang X, Liang Y, Wang L, Li D et al . Precision medicine and bladder cancer heterogeneity. Bull Cancer. 2018;105(10):925-931. Guo L, Kong D, Liu J, Zhan L, Luo L et al . Breast cancer heterogeneity and its implication in personalized precision therapy. Exp Hematol Oncol. 2023;12(1):3. Fakih MG, Salvatore L, Esaki T, Modest DP, Lopez-Bravo DP et al . Sotorasib plus Panitumumab in Refractory Colorectal Cancer with Mutated KRAS G12C. N Engl J Med. 2023;389(23):2125-2139. Passaro A, Mok T, Attili I, Wu YL, Tsuboi M et al . Adjuvant Treatments for Surgically Resected Non-Small Cell Lung Cancer Harboring EGFR Mutations: A Review. JAMA Oncol. 2023;9(8):1124-1131. Pich O, Bailey C, Watkins TBK, Zaccaria S, Jamal-Hanjani M et al . The translational challenges of precision oncology. Cancer Cell. 2022;40(5):458-478. Hughes AD, Marshall JR, Keller E, Powderly JD, Greene BT et al . Differential drug responses of circulating tumor cells within patient blood. Cancer Lett. 2014;352(1):28-35. Mlynarczyk-Bonikowska B, Kowalewski C, Krolak-Ulinska A, Marusza W. Molecular Mechanisms of Drug Resistance in Staphylococcus aureus. Int J Mol Sci. 2022;23(15). van de Wetering M, Francies HE, Francis JM, Bounova G, Iorio F et al . Prospective derivation of a living organoid biobank of colorectal cancer patients. Cell. 2015;161(4):933-945. van de Donk N. How We Manage Newly Diagnosed Multiple Myeloma With Circulating Tumor Cells. J Clin Oncol. 2023;41(7):1342-1349. Liu X, Song J, Zhang H, Liu X, Zuo F et al . Immune checkpoint HLA-E:CD94-NKG2A mediates evasion of circulating tumor cells from NK cell surveillance. Cancer Cell. 2023;41(2):272-287. Zhang W, Xu F, Yao J, Mao C, Zhu M et al . Single-cell metabolic fingerprints discover a cluster of circulating tumor cells with distinct metastatic potential. Nat Commun. 2023;14(1):2485. He S, Li P, Chen X, Yu Z. Fluid biopsy for circulating tumor cells in an occult ovarian cancer patient exhibiting bilateral supraclavicular lymph node metastases: A case report. Oncol Lett. 2013;6(2):448-452. Cortes-Hernandez LE, Eslami-S Z, Pantel K, Alix-Panabieres C. Circulating Tumor Cells: From Basic to Translational Research. Clin Chem. 2024;70(1):81-89. Rupp B, Ball H, Wuchu F, Nagrath D, Nagrath S. Circulating tumor cells in precision medicine: challenges and opportunities. Trends Pharmacol Sci. 2022;43(5):378-391. Longo DL. Tumor heterogeneity and personalized medicine. N Engl J Med. 2012;366(10):956-957. Martins I, Ribeiro IP, Jorge J, Goncalves AC, Sarmento-Ribeiro AB et al . Liquid Biopsies: Applications for Cancer Diagnosis and Monitoring. Genes (Basel). 2021;12(3). Heitzer E, Haque IS, Roberts C, Speicher MR. Current and future perspectives of liquid biopsies in genomics-driven oncology. Nat Rev Genet. 2019;20(2):71-88. Martin-Alonso C, Tabrizi S, Xiong K, Blewett T, Sridhar S et al . Priming agents transiently reduce the clearance of cell-free DNA to improve liquid biopsies. Science. 2024;383(6680):eadf2341. Grzybowska EA. Circulating Tumor Cells: Pathological, Molecular and Functional Characteristics. Int J Mol Sci. 2024;25(15). Lin D, Shen L, Luo M, Zhang K, Li J et al . Circulating tumor cells: biology and clinical significance. Signal Transduct Target Ther. 2021;6(1):404. Shamshoum M, Kuperman OA, Shadmi SK, Itkin M, Malitsky S et al . 2-NBDG Uptake in Gossypium hirsutum in vitro ovules: exploring tissue-specific accumulation and its impact on hexokinase-mediated glycolysis regulation. Front Plant Sci. 2023;14(1242150. Tang Y, Wang Z, Li Z, Kim J, Deng Y et al . High-throughput screening of rare metabolically active tumor cells in pleural effusion and peripheral blood of lung cancer patients. Proc Natl Acad Sci U S A. 2017;114(10):2544-2549. Millon SR, Ostrander JH, Brown JQ, Raheja A, Seewaldt VL et al . Uptake of 2-NBDG as a method to monitor therapy response in breast cancer cell lines. Breast Cancer Res Treat. 2011;126(1):55-62. Lamarca A, Palmer DH, Wasan HS, Ross PJ, Ma YT et al . Second-line FOLFOX chemotherapy versus active symptom control for advanced biliary tract cancer (ABC-06): a phase 3, open-label, randomised, controlled trial. Lancet Oncol. 2021;22(5):690-701. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 02 Feb, 2026 Read the published version in Cancer Cell International → Version 1 posted Editorial decision: Revision requested 28 Aug, 2025 Reviews received at journal 17 Aug, 2025 Reviews received at journal 08 Aug, 2025 Reviewers agreed at journal 07 Aug, 2025 Reviewers agreed at journal 07 Aug, 2025 Reviewers invited by journal 07 Aug, 2025 Editor assigned by journal 25 Jul, 2025 Submission checks completed at journal 24 Jul, 2025 First submitted to journal 24 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7201269","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":498007047,"identity":"4ef30048-b863-47b2-b2c5-cc279ce4c9cf","order_by":0,"name":"Huafei Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYBACPmYeIGnDwMPPAGIwMBPWwgbWksbAI9lAtBYGiBYGgwNEa2HnPSbxI8FGxvj82WMSDBXWiQ3sZw8QcBhfmmRPQhqP2Y28NAmGM+mJDTx5CYT8YibB++MwUAuQwdh2OLFBgseAoBbJPwn/eYz7zwC1/CNSizRPwgGgshyglgbitBhbyyQk80jcyDG2SDiWbtzGk4NfCz//GcObbxLs7Pn7zxje+FBjLdvPfga/FlSQALKXBPWjYBSMglEwCnAAABA2NU8LA2N9AAAAAElFTkSuQmCC","orcid":"","institution":"Shanghai University","correspondingAuthor":true,"prefix":"","firstName":"Huafei","middleName":"","lastName":"Li","suffix":""},{"id":498007049,"identity":"45380f80-d736-4037-9860-6b68f10ce16b","order_by":1,"name":"Shuangqun Chen","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Shuangqun","middleName":"","lastName":"Chen","suffix":""},{"id":498007051,"identity":"f4bc5527-e074-4e0e-a021-f8c794592d88","order_by":2,"name":"Xiaoxia Kou","email":"","orcid":"","institution":"the Third Affiliated Hospital of Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xiaoxia","middleName":"","lastName":"Kou","suffix":""},{"id":498007052,"identity":"3842f9de-8524-472d-8881-7be495fe8aa4","order_by":3,"name":"Yuan Tian","email":"","orcid":"","institution":"Shanghai University","correspondingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Tian","suffix":""},{"id":498007055,"identity":"58442524-8b8d-4664-81a3-705892398fb0","order_by":4,"name":"Cong Wu","email":"","orcid":"","institution":"the First Affiliated Hospital of Naval Medical University, Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cong","middleName":"","lastName":"Wu","suffix":""},{"id":498007056,"identity":"15604d95-55dc-48be-9b5d-c5441cf1c324","order_by":5,"name":"Huiying Liu","email":"","orcid":"","institution":"the Third Affiliated Hospital of Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Huiying","middleName":"","lastName":"Liu","suffix":""},{"id":498007059,"identity":"3a33eff5-d563-4e3b-a08e-3b1f80486207","order_by":6,"name":"Jinrong Qiu","email":"","orcid":"","institution":"the Third Affiliated Hospital of Naval Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jinrong","middleName":"","lastName":"Qiu","suffix":""}],"badges":[],"createdAt":"2025-07-24 04:23:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7201269/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7201269/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12935-026-04198-2","type":"published","date":"2026-02-02T15:56:57+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88949940,"identity":"f9f8cde5-7a63-4469-8fa4-5da6ed3f8c1b","added_by":"auto","created_at":"2025-08-13 05:42:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":231612,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSchematic diagram of patient grouping and study design.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7201269/v1/efae0967a0b3e58a4a5efa41.png"},{"id":88949956,"identity":"01ecab82-1a71-457a-bf19-7aaf6b3d770a","added_by":"auto","created_at":"2025-08-13 05:42:14","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":257828,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eComparison of treatment efficacy among groups. \u003c/strong\u003e(A) Composition of treatment outcomes among different groups after therapy. CR: complete remission, PR: partial remission, SD: stable disease, PD: progressive disease. (B) Comparison of ORR and DCR of patients receiving different therapeutic regimens among groups. (C) Summary of patient numbers with different treatment responses across groups. ns: not significant (p\u0026gt;0.05), *: p\u0026lt;0.05, **: p\u0026lt;0.01.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7201269/v1/90beab8ecdc107b52ff9ab2f.png"},{"id":88949942,"identity":"cf92de71-2e23-4939-af62-5c22a398543d","added_by":"auto","created_at":"2025-08-13 05:42:13","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":123007,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eProgression-free survival analysis among different groups.\u003c/strong\u003e(A) Kaplan-Meier survival curves showing the PFS of patients in different groups (*p\u0026lt;0.05). (B) Risk function analysis indicating a consistently lower initial and sustained risk of disease progression in the CSBT group, highlighting the benefit of tailoring treatment based on CTC drug sensitivity.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7201269/v1/0da87cd321c9c3d722196065.png"},{"id":88950827,"identity":"76bdae3d-2bee-4511-a229-ea4b0d437956","added_by":"auto","created_at":"2025-08-13 05:50:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":271516,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eOverall survival analysis of patients among groups. \u003c/strong\u003e(A) Kaplan-Meier survival curves showing the OS of patients in three groups. (B) Risk function analysis indicating a reduced initial risk of mortality in the CSBT group compared to the FOLFOX and EBT groups, with the risk curves converging over time. (C) Short-term survival analysis during the first 12 months, highlighting a higher survival rate in the CSBT group, particularly during the critical early treatment phase. (D) Risk function analysis during the first 12 months, showing consistently lower mortality risk in the CSBT group, emphasizing the short-term survival advantage of CTC-based therapy. ns: not significant (p\u0026gt;0.05), *: p\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7201269/v1/b5254ad38c82d24aaa6c64b6.png"},{"id":102233956,"identity":"c9cbd8ae-e3f3-4ac3-a09c-8fb496e3fb23","added_by":"auto","created_at":"2026-02-09 16:00:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1795906,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7201269/v1/cae9911d-e7c1-43ec-9773-dcc381a27fcf.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Drug Sensitivity of Circulating Tumor Cells as a Game-Changer in Subsequent-Line Therapy for Biliary Tract Carcinoma","fulltext":[{"header":"1. Background","content":"\u003cp\u003eBiliary tract cancer (BTC) ranks as the second most prevalent primary liver malignancy following hepatocellular carcinoma (HCC), accounting for approximately 3% of all gastrointestinal cancers [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Among BTC types, adenocarcinoma is the most common pathological subtype, which is notorious for its aggressive nature, difficulty in early diagnosis, and exceedingly poor prognosis, with a global five-year survival rate of less than 5% [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile surgical resection remains the only potential curative treatment for BTC, nearly two-thirds of patients are diagnosed at a stage where the cancer is locally advanced, unresectable, or metastatic, eliminating surgery as a viable option [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. For these patients, systematic chemotherapy remains the major therapeutics, with the combination of gemcitabine plus cisplatin (GC) serving as the current frontline standard regimen [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Despite initial responses to first-line chemotherapy, most patients eventually experience disease progression, facing with the issue of receiving subsequent-line treatments [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The selection of an appropriate subsequent-line treatment is challenging due to limited standardized options and variable patient responses [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. One regimen that has been investigated is FOLFOX, which combines folinic acid, fluorouracil, and oxaliplatin. The ABC-06 trial, a phase III study, evaluated FOLFOX plus active symptom control (ASC) versus ASC alone in patients with advanced BTC who had progressed after first-line GC therapy. The results indicated a modest improvement in overall survival (OS) for the FOLFOX plus ASC group, suggesting its potential as a second-line option [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eHowever, the efficacy of subsequent-line therapies of BTC remains limited, with significant challenges still present. A key challenge is cancer heterogeneity, which varies both within individual tumors and across patients. This heterogeneity complicates treatment by contributing to varied therapeutic responses, thereby diminishing the overall efficacy of current treatment strategies [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Additionally, the rarity of BTC has limited the availability of large-scale randomized controlled trials, which hinders the establishment of standardized second-line or subsequent-line therapeutic regimens.\u003c/p\u003e\u003cp\u003eTo address these challenges, ongoing research is focusing on personalized medicine, which holds the potential to revolutionize cancer treatment and improve patient outcomes [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Traditional approaches to personalized medicine involve identifying predictive biomarkers of treatment sensitivity or resistance in tumor cells [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. For example, in colorectal cancer, panitumumab is effective only in patients without KRAS mutations. Thus, to ensure optimal outcomes, patients are screened for KRAS mutations prior to receiving panitumumab in the clinic [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Similarly, patients with non-small cell lung cancer (NSCLC) are tested for specific epidermal growth factor receptor (EGFR) mutations prior to receiving treatments like gefitinib or erlotinib [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAlthough advancements in cancer genomics over the past two decades have broadened our understanding, there remain many instances in which genomic analysis does not yield effective or sensitive therapeutic targets [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Even when actionable genomic alterations are identified, patient responses to the targeted therapy are not guaranteed. Furthermore, for most standard chemotherapeutic agents, no reliable genetic markers have been identified to predict patient response accurately. The issues of intrinsic and acquired chemoresistance significantly impact treatment outcomes, contributing to treatment failure in over 90% of patients with metastatic BTC [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eFor these patients, \u003cem\u003ein vitro\u003c/em\u003e drug screening using patient-derived tumor cells offers a valuable approach. This method enables the confirmation of drug sensitivity, prediction of therapeutic efficacy, and identification of additional treatment options [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Currently, enrolling such heavily pretreated patients\u0026mdash;with multi-drug resistance (MDR) after progression under second or third-line treatments\u0026mdash; in clinical trials is challenging, because they often do not meet the stringent entry criteria. Pursuing compassionate use of non-FDA-approved drugs or off-label use of FDA-approved drugs through individualized \u0026ldquo;N-of-1\u0026rdquo; studies can be time-consuming, costly, and frequently impractical. In contrast, \u003cem\u003ein vitro\u003c/em\u003e drug testing provides a feasible solution for screening potential drug combinations, benefiting both physicians and patients [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eIn recent years, circulating tumor cells (CTCs) have garnered increasing attention in cancer research [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Multiple studies have revealed that CTCs appear early in the disease course, with their presence in the bloodstream correlating strongly with disease severity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Clinicians are now considering CTCs as a form of \u0026ldquo;fluid biopsy\u0026rdquo;, offering real-time insights into the tumor\u0026rsquo;s current status. CTCs are thought to reflect, in part, the extensive heterogeneity of the tumor [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eCTCs have become a focal point to target for therapy due to the fact they are the primary route for metastasis to occur [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Consequently, they are extensively studied at the individual patient level for various characterizations, such as assessing their epithelial-to-mesenchymal transition (EMT) state and identifying surface markers associated with specific drug responses [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Despite their potential significance, clinical studies exploring the predictive impact of CTCs on chemotherapeutic response and drug sensitivity remain limited.\u003c/p\u003e\u003cp\u003eIn this retrospective study, we delved into the transformative potential of drug sensitivity testing on CTCs in shaping subsequent-line chemotherapy strategies for patients with advanced BTC who have experienced relapse post-initial treatment. Our analysis underscores the pivotal role of CTC-based drug sensitivity assessments in enabling the selection of more efficacious chemotherapy protocols. This personalized approach not only holds the promise of promoting patient survival but also significantly enhances the clinical outcomes for individuals battling advanced BTC (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This study positions CTC drug sensitivity testing as a game-changer in the realm of subsequent-line chemotherapy for advanced BTC, offering a tailored and potentially more successful treatment trajectory.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003e2.1 Patients and study design.\u003c/h2\u003e\n \u003cp\u003eA retrospective analysis was conducted on a total of 85 patients with advanced BTC at the Department of Oncology Biotherapy, the Third Affiliated Hospital of Navy Medical University (Shanghai, China) between February 2018 and September 2023. Among them, 26 patients received subsequent chemotherapy regimens based on the results of CTC drug sensitivity testing (CTC drug sensitivity-based therapy, CSBT), 15 patients were treated with the standard FOLFOX regimen, and the chemotherapy regimens of the other 45 patients were based on physician experience (experience-based therapy, EBT) (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). All patients had a definitive diagnosis confirmed by pathology from surgery or liver biopsy, and their medical records were complete. The inclusion criteria for cases are as follows: (1) patients aged between 18 and 80 years, (2) an Eastern Cooperative Oncology Group (ECOG) performance status of 3 or lower, (3) the presence of clearly measurable cancer lesions, (4) patients with locally advanced disease without distant metastasis were deemed unresectable following a multidisciplinary assessment, (5) baseline blood tests and biochemical indicators within specified ranges: neutrophil count \u0026gt;1.5\u0026times;10\u003csup\u003e9\u003c/sup\u003e/L, hemoglobin\u0026thinsp;\u0026gt;\u0026thinsp;90 g/L, platelet count\u0026thinsp;\u0026gt;\u0026thinsp;75\u0026times;10\u003csup\u003e9\u003c/sup\u003e /L, serum total bilirubin\u0026thinsp;\u0026lt;\u0026thinsp;33.5 \u0026micro;mol/L, alanine aminotransferase (ALT)\u0026thinsp;\u0026lt;\u0026thinsp;100U/L, aspartate aminotransferase (AST)\u0026thinsp;\u0026lt;\u0026thinsp;100U/L, and serum creatinine (SCR)\u0026thinsp;\u0026lt;\u0026thinsp;160 \u0026micro;mol/L, (6) conventional empirical therapy had been administered as the first-line treatment for patients experiencing disease progression. Exclusion criteria: (1) Presence of other malignancies; (2) women who are pregnant or breastfeeding; (3) history of severe allergy to one or more drugs; (4) patients with severe infections requiring treatment; (5) presence of active bleeding or intestinal obstruction; (6) individuals with severe liver disease, kidney disease, respiratory diseases, or uncontrolled diabetes, hypertension, coronary heart disease, and other chronic conditions.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e2.2 Samples collection and CTC enrichment\u003c/h2\u003e\n \u003cp\u003ePatient-derived CTCs were efficiently isolated and enriched through microfluidic methods. Briefly, fresh blood samples, totaling 30 ml, were collected from the patient\u0026apos;s peripheral vein and collected in BD Vacutainer EDTA tubes (Becton, Dickinson and Company, NJ, USA). The samples were meticulously mixed with anticoagulants by gently inverting the tubes several times. Following centrifugation, the plasma layer was discarded, and the remaining sample underwent erythrocyte lysis to eliminate red blood cells (RBCs). The nucleated cells were then resuspended in PBS containing 1% BSA and processed through the FlowCell\u0026trade; CTC enrichment system (Polaris Biology, Shanghai, China), which effectively removed residual RBCs and white blood cells (WBCs). This sophisticated method yielded approximately 100\u0026ndash;400 CTCs per patient.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e2.3 Assessment of drug susceptibility\u003c/h2\u003e\n \u003cp\u003eAs the number of CTCs in patient peripheral blood is relatively low (ranging from a few to several dozen per milliliter), the Glucose uptake assay was employed for drug sensitivity assessment. Generally, the patient CTCs (approximately 100\u0026ndash;400 based on the patient\u0026rsquo;s condition) were evenly distributed and inoculated into 384-well plates (with about 5\u0026ndash;10 tumor cells per well). Cells were cultured in RPMI1640 cell culture medium without glucose supplemented with 20% Fetal Bovine Serum (FBS, Thermo Fisher Scientific, Massachusetts, U.S.A.). To each well, 0.4 mmol/L of the fluorescently labeled glucose analog, 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG, Thermo Fisher Scientific, Massachusetts, U.S.A.), was added. The chemotherapeutic agents to be tested were also added for a 24-hour incubation. The concentration of the chemotherapeutic drugs is set as the average IC50 value of multiple BTC cell lines (1.0 IC50) and half of IC50 values (0.5 IC50), with three replicate wells for each concentration. After 24 hours, a fluorescently labeled CD45 antibody is added to each well and further incubated for 30 minutes. The EVOS\u0026trade; FL M7000 imaging system (Thermo Fisher Scientific, Massachusetts, U.S.A.) is used to photograph each well, and a neural network-based algorithm is utilized to analyze cell morphology, CD45 expression, and cellular glycometabolism levels. By comparing the average glycometabolism rate (GMR) of CTCs in drug-treated wells with that of the control group (tumor cells untreated with chemotherapy drugs), the drug sensitivity (activity inhibition rate of different chemotherapy regimens at various concentrations on CTCs) can be calculated according to the following equations:\u003c/p\u003e\n \u003cp\u003eDrug sensitivity \u003cimg src=\"data:image/png;base64,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\"\u003e\u0026nbsp;where average GMR in experimental group represented the relative glucose uptake rates of CTCs at the concentrations of 0.5 IC50 and 1.0 IC50. The sensitivity of CTCs was categorized into four distinct levels: highly sensitive (less than 0.70), moderately sensitive (ranging from 0.70 to 0.90), and resistant (more than 0.90). The objective is to identify the most effective chemotherapeutic regimen based on the established sensitivity levels.\u003c/p\u003e\n \u003cp\u003eThe alternative chemotherapeutic regimens to be tested mainly include: Gemcitabine plus Cisplatin (GC regimen), S-1 plus Oxaliplatin (SOX regimen), Gemcitabine plus Capecitabine (GX regimen), Nab-paclitaxel plus Gemcitabine (AG regimen), Oxaliplatin plus Leucovorin plus Fluorouracil (FOLFOX regimen), Irinotecan plus Leucovorin plus Fluorouracil (FOLFIRI regimen).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e2.4 Therapeutic efficacy evaluation\u003c/h2\u003e\n \u003cp\u003eAll patients underwent serum tumor markers, blood routine examination, blood biochemistry test, electrocardiogram, CT or MRI examination before and after each cycle of chemotherapy. Efficacy was evaluated every two cycles of chemotherapy according to the Response Evaluation Criteria in Solid Tumors (RECIST), which categorizes responses into complete response (CR), partial response (PR), stable disease (SD), and progressive disease (PD). Objective response rate (ORR) and disease control rate (DCR) were calculated as follows: ORR\u0026thinsp;=\u0026thinsp;CR rate\u0026thinsp;+\u0026thinsp;PR rate, DCR\u0026thinsp;=\u0026thinsp;CR rate\u0026thinsp;+\u0026thinsp;PR rate\u0026thinsp;+\u0026thinsp;SD rate. Progression-free survival (PFS) is defined as the time from the start of chemotherapy to tumor progression, and overall survival (OS) is defined as the time from the start of chemotherapy to the patient\u0026rsquo;s death, loss to follow-up, or the last follow-up. Follow-ups were conducted through hospital visits or telephone calls. Adverse reactions were evaluated according to the National Cancer Institute-common toxicity criteria (NCI-CTC) 4.0, graded from 0 to 4.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\n \u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e\n \u003cp\u003eData analysis was conducted utilizing SPSS software (version 23.0). For continuous variables, data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (x\u0026thinsp;\u0026plusmn;\u0026thinsp;s), with group comparisons performed using independent samples t-tests. Categorical data are depicted as frequencies and percentages, with group comparisons carried out via chi-square tests or Fisher\u0026rsquo;s exact test when appropriate. Survival analysis was executed employing the Kaplan-Meier estimation to generate survival curves, and survival time comparisons were assessed using the log-rank test. The significance level (\u0026alpha;) was set at 0.05.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Patient characteristics\u003c/h2\u003e\u003cp\u003eA total of 85 patients were enrolled in this study, categorized into three groups: 25 patients in the CSBT group, where the therapeutic regimens were determined based on CTC drug susceptibility testing results; 15 patients treated with FOLFOX-based chemotherapy; and 45 patients receiving experience-based therapy (EBT) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Detailed patient characteristics are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Analysis revealed that general condition, tumor staging, and the incidence of distant metastasis were largely comparable across the three groups, suggesting a balanced patient profile.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBaseline demographic and clinical characteristics of patients in different groups.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCSBT (\u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;25)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFOLFOX (\u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEBT (\u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;45)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eP\u003c/em\u003e value\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eAge, years\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMedian\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e57.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e55.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e55.978\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.78\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRange\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e33\u0026ndash;80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e37\u0026ndash;74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e37\u0026ndash;75\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGender\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eMale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003e0.5418\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFemale\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e11\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePrimary tumor site\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIntrahepatic bile duct\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.8151\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eExtrahepatic bile duct\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGallbladder cancer\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e17\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTNM stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u0026nbsp;\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eⅢB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e0.3894\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eⅣ\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eIVB\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eAbbreviation: TNM, tumor-node-metastasis\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Improved treatment efficacy of patients receiving CTC-based therapy\u003c/h2\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e presents a detailed comparison of the efficacy outcomes across three chemotherapy regimens. Patients in the CSBT group achieved the highest ORR at 16%, with 1 out of 25 achieving CR and 3 out of 25 PR. In comparison, the ORR was 6.7% for the FOLFOX-based therapy group, with no CR and 1 out of 15 patients achieving PR, while the EBT group had the lowest ORR at 4.4%, with no CR and 2 out of 45 patients achieving PR (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). However, although the CSBT group demonstrated a notably higher ORR, the differences among groups were not statistically significant (p\u0026thinsp;=\u0026thinsp;0.235).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn contrast, the DCR showed significant differences among groups. The CSBT group achieved the highest DCR at 56%, significantly outperforming the FOLFOX group (20%) (p\u0026thinsp;=\u0026thinsp;0.046) and the EBT group (22.2%) (p\u0026thinsp;=\u0026thinsp;0.008) (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). These findings indicate that the therapeutic regimens based on CTC drug sensitivity testing are more effective in controlling disease progression.\u003c/p\u003e\u003cp\u003eThe lack of statistical significance in ORR differences suggests that a larger cohort might be needed to fully capture the potential efficacy disparities between the regimens. However, the significant difference in DCR highlights the clear advantage of CTC-based therapy in achieving disease control, underscoring its potential for disease control.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Prolonged PFS of patients in CSBT group.\u003c/h2\u003e\u003cp\u003eSubsequently, we compared the progression-free survival of BTC patients in different groups. As depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, drug sensitivity testing significantly improved the progression-free survival of patients. Generally, the median progression-free survival (mPFS) for patients treated with FOLFOX regimen and EBT was 1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20 months and 2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27 months, respectively, with no statistically significant difference observed between these two groups. In contrast, patients treated with CSBT demonstrated a markedly prolonged mPFS of 5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17 months (*p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). These findings are further supported by the risk function shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, which highlights a consistently lower initial and sustained risk of disease progression in patients receiving CSBT. This underscores the potential benefit of tailoring treatment based on drug susceptibility to achieve improved outcomes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Prolonged OS of patients in CSBT group.\u003c/h2\u003e\u003cp\u003eIn our comprehensive analysis, we observed a modest yet noteworthy extension in the OS of patients who were administered therapeutics based on CTC drug sensitivity testing. Specifically, these patients exhibited a median OS (mOS) of 12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58 months, as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. This finding is particularly significant when juxtaposed with the mOS of 5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10 months for those treated with FOLFOX regimen, and 7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88 months for patients receiving empiric therapy. However, the statistical analysis did not yield a significant difference among groups. The risk function graph presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB offers a visual representation of these survival outcomes. The green line, representing the CSBT group, begins at a lower point on the graph, signifying a reduced initial risk of mortality. Over the course of the study period, this line consistently remains slightly below the others, suggesting a sustained lower risk of death. However, as time progresses, the risk function curves for all groups tend to converge, a trend that underscores the notion that, in the long term, the risk of death among patients may become more homogeneous across different groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eSurvival analysis among groups.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eCSBT\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFOLFOX\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eEBT\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emPFS (months)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003emOS (months)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e7.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.88\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eGiven the above-mentioned trends of OS, and significant extension in PFS observed in the CSBT group, we did survival analysis of patients during the initial 12 months of treatment, elucidating the short-term impact of drug sensitivity assessment on patient outcomes. The initial year of treatment is often marked by the most rapid changes in disease status, the survival analysis during which will provide a more nuanced understanding of the treatment effects during the critical initial phase of patient management. The survival curves in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC demonstrate that, the green line (CSBR group) likely demonstrates a statistically higher survival rate compared to the blue (FOLFOX group) and orange (EBT group) lines, respectively. This suggests that patients receiving CSBT have a better chance of surviving the first year. The curves may show a gradual convergence as the 12-month mark approaches, indicating that while the initial survival benefit is evident, it may not be as pronounced towards the end of the first year. Accordingly, the risk functions illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD reflects a reduced initial risk of death for patients treated with CSBT. Over the first year, this green line remains below the other two, suggesting a consistently lower risk of death.\u003c/p\u003e\u003cp\u003eThese results suggest that drug sensitivity-based therapeutics offer an initial survival advantage. Patients treated with these regimens exhibit better short-term survival rates and a lower risk of death early in the treatment course. However, the convergence of survival curves and risk functions towards the end of the first year implies that this advantage may not be as significant or may diminish over the short term. This analysis underscores the need for continued monitoring and further research to assess the long-term implications of these initial survival benefits and to determine if they translate into sustained improvements in overall survival outcomes.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.5 No statistically significant differences among groups.\u003c/h2\u003e\u003cp\u003eAs presented in Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, a comparative analysis of chemotherapy-related adverse events was conducted across different groups, with particular emphasis on grade 3\u0026ndash;4 toxicities, which are indicative of greater severity. In terms of hematological adverse events, anemia was the most frequently observed, impacting 48%, 40%, and 28.9% of patients in the CTC-based, FOLFOX-based, and experience-based therapeutic groups, respectively. Notably, there was no significant variation in the incidence across all grades or specifically in grade 3\u0026ndash;4 cases. Neutropenia and thrombocytopenia were also documented.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eChemotherapy-related adverse events in different treatment groups.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"8\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c4\" namest=\"c3\"\u003e\u003cp\u003eCSBT (\u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;25)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e\u003cp\u003eFOLFOX (\u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e\u003cp\u003eEBT (\u003cem\u003eN\u003c/em\u003e\u0026thinsp;=\u0026thinsp;45)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eAdverse events\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOverall\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eGrade 3\u0026ndash;4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOverall\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eGrade 3\u0026ndash;4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eOverall\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003eGrade 3\u0026ndash;4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003eHematological\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAnemia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eNeutropenia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eThrombocytopenia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e10\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"7\" rowspan=\"8\"\u003e\u003cp\u003eNonhematological\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eFatigue\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAnorexia\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e14\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e12\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eVomiting\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDiarrhea\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eSensory neuropathy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eHand-foot syndrome\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eAllergy\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLiver dysfunction\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c8\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eWhen examining nonhematological adverse events, fatigue stood out as the most common, impacting 52% of patients in the CSTB group, 53.3% in the FOLFOX-based therapy group, and 44.4% in the EBT group. Anorexia was another frequently reported side effect, affecting 56% of patients in the CTC-based therapy group, 46.7% in the FOLFOX-based therapy group, and 26.7% in the experience-based therapy group. Additional side effects such as vomiting and diarrhea were also reported throughout the chemotherapy period.\u003c/p\u003e\u003cp\u003eAlthough the frequencies of these side effects vary, there are no statistically significant differences among groups. This suggests that while each regimen may have a distinct profile of adverse events, the severity of these events does not significantly differ, allowing for a balanced consideration of efficacy and safety in treatment decisions.\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study provides valuable insights into the potential of CTCs in guiding chemotherapy for advanced BTC. Our findings demonstrate that CTC-based drug sensitivity testing can significantly influence treatment outcomes. Patients in the CSTB achieved notably improved ORRs, significantly improved DCRs and prolonged PFS, compared to those receiving FOLFOX or empiric therapy. To the best of our knowledge, this is the first study to systematically investigate the impact of CTC drug sensitivity-guided clinical decision-making on patient outcomes.\u003c/p\u003e\u003cp\u003eOver the past decades, personalized medicine has played an increasingly critical role in cancer treatment, driven by the need to address the molecular heterogeneity of neoplastic diseases [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. While tissue biopsy remains the gold standard for profiling tumor molecular characteristics and informing treatment decisions, it is invasive and provides limited insight into the clonal evolution of tumors during disease progression [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In this context, liquid biopsies have emerged as a promising alternative. Non-invasive, easily repeatable, and accessible, liquid biopsies have been extensively studied for their potential to revolutionize clinical decision-making by enabling real-time tumor monitoring [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003cem\u003eIn vitro\u003c/em\u003e drug screening using patient-derived tumor cells provides a valuable approach for confirming drug sensitivity, predicting therapeutic efficacy, and identifying alternative treatment strategies [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. CTCs, as a major component of liquid biopsy, are ideal candidates for such analyses. They offer prognostic and predictive information and dynamically reflect the evolving characteristics of tumor cells, particularly their drug sensitivities [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. However, the rarity of CTCs in the bloodstream has historically limited their clinical application. In this study, we successfully isolated 100\u0026ndash;400 CTCs per patient using a microfluidic method, overcoming a key technical barrier. Although this number is insufficient for traditional viability assays like CCK-8 or MTT, we employed the innovative 2-NBDG method, which enabled metabolic activity assessments with minimal cell input.\u003c/p\u003e\u003cp\u003eThe 2-NBDG method leverages the Warburg effect, whereby cancer cells exhibit increased glycolysis [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. By monitoring glucose metabolism via the fluorescent 2-NBDG analog, this method provides a label-free, real-time assessment of drug sensitivity [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Previous studies have validated its utility in some cancer types, and our findings demonstrate its applicability in CTC-based drug sensitivity testing [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eUsing this novel methodology, 25 patients in the CSBT group were treated based on CTC drug sensitivity testing results. Statistical analyses revealed that the CSBT group achieved an ORR of 16% and a DCR of 56%, both remarkably higher than those observed in the two control groups. Besides, CTC drug sensitivity testing significantly improved the mPFS of patients to 5.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.17 months. It is noteworthy that these clinical outcomes appear to be prior to those of the clinically recommended second-line regimen as reported previously. In a randomized Phase 3 ABC-06 trial, the ASC\u0026thinsp;+\u0026thinsp;mFOLFOX regimen showed significant clinical efficacy, with an ORR of 5.0%, a DCR of 33%, a median PFS of 4.0 months, and a median OS of 6.2 months [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThe observed improvements in ORR and DCR emphasize the potential of personalized, CTC-guided chemotherapy to address challenges posed by tumor heterogeneity and interpatient variability in treatment response. These findings align with growing evidence supporting the use of liquid biopsy-based approaches for dynamic tumor monitoring and treatment optimization.\u003c/p\u003e\u003cp\u003eThe significant extension of PFS in the CTC group suggests that CTC-guided chemotherapy may effectively delay disease progression, providing patients with valuable additional time. This is particularly critical given the limited treatment options and poor prognosis associated with advanced BTC.\u003c/p\u003e\u003cp\u003eAs for the OS of patients, patients in CTC drug sensitivity testing group exhibited a median OS (mOS) of 12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58 months, although noteworthy extended, the statistical analysis did not yield a significant difference among groups. However, survival curves indicated an early survival benefit for patients receiving CTC-guided therapy, with improved survival rates during the first year of treatment. The convergence of survival curves beyond the 12-month mark suggests that while the initial benefits are clear, their long-term impact warrants further investigation. This analysis underscores the need for continued monitoring and further research to assess the long-term implications of these initial survival benefits and to determine if they translate into sustained improvements in overall survival outcomes.\u003c/p\u003e\u003cp\u003eImportantly, the toxicity profiles of the treatments were comparable across all groups, with no significant differences in the incidence of severe adverse events. This demonstrates that the efficacy advantages of CTC-guided therapy do not come at the cost of increased toxicity, a critical consideration in the management of advanced cancer.\u003c/p\u003e\u003cp\u003eHowever, this study has several limitations. The relatively small sample size reduces the generalizability of the findings and increases the risk of bias. Additionally, potential confounding factors, such as variations in treatment timing and prior therapies, were not fully accounted for. Future research should focus on larger, prospective, randomized controlled trials to validate these results. Further studies should also explore the underlying mechanisms of CTCs in predicting drug response, identify novel therapeutic targets, and evaluate the cost-effectiveness of integrating CTC-based approaches into standard clinical practice.\u003c/p\u003e\u003cp\u003eCTC-based drug sensitivity testing emerges as a transformative approach in guiding subsequent-line chemotherapy for advanced BTC. CTC-guided therapy notably enhances ORRs, significantly improves DCRs and PFS, and provides a short-term OS advantage during the first year of treatment. Although no statistically significant differences in OS were observed across the entire study period, the trends and early survival benefits suggest that personalized chemotherapy informed by CTC drug sensitivity assessments can address tumor heterogeneity and optimize therapeutic outcomes. Intriguingly, the results imply that to maximize patient benefit, increasing the frequency of CTC drug sensitivity testing during the treatment course, such as annually, could potentially and significantly improve patient prognosis and survival. However, this hypothesis necessitates validation through further clinical research.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eCTC-based drug sensitivity testing has emerged as a transformative approach in guiding subsequent-line chemotherapy for advanced BTC. CTC-guided therapy significantly improves ORRs, DCRs, and PFS, while offering a short-term OS advantage during the first year of treatment. Although no statistically significant differences in OS were observed over the entire study period, the trends and early survival benefits suggest that personalized chemotherapy based on CTC drug sensitivity assessments can address tumor heterogeneity and optimize therapeutic outcomes. Notably, the results imply that increasing the frequency of CTC drug sensitivity testing during the treatment course - for example, to annual testing - could potentially improve patient prognosis and survival. However, this hypothesis requires validation through further clinical research.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBTC: Biliary Tract Carcinoma\u003c/p\u003e\n\u003cp\u003eCTC: Circulating Tumor Cells\u003c/p\u003e\n\u003cp\u003eCSBT: CTC-Based Drug Sensitivity-Guided Therapy\u003c/p\u003e\n\u003cp\u003eFOLFOX: Folinic Acid, Fluorouracil, and Oxaliplatin\u003c/p\u003e\n\u003cp\u003eEBT: Experience-Based Therapy\u003c/p\u003e\n\u003cp\u003eORR: Objective Response Rate\u003c/p\u003e\n\u003cp\u003eDCR: Disease Control Rate\u003c/p\u003e\n\u003cp\u003ePFS: Progression-Free Survival\u003c/p\u003e\n\u003cp\u003eOS: Overall Survival\u003c/p\u003e\n\u003cp\u003eNCI-CTC: National Cancer Institute-Common Toxicity Criteria\u003c/p\u003e\n\u003cp\u003eECOG: Eastern Cooperative Oncology Group\u003c/p\u003e\n\u003cp\u003eIC50: Half Maximal Inhibitory Concentration\u003c/p\u003e\n\u003cp\u003eALT: Alanine Aminotransferase\u003c/p\u003e\n\u003cp\u003eAST: Aspartate Aminotransferase\u003c/p\u003e\n\u003cp\u003eSCR: Serum Creatinine\u003c/p\u003e\n\u003cp\u003eGC: Gemcitabine and Cisplatin\u003c/p\u003e\n\u003cp\u003eMDR: Multi-Drug Resistance\u003c/p\u003e\n\u003cp\u003e2-NBDG: 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose\u003c/p\u003e\n\u003cp\u003eBSA: Bovine Serum Albumin\u003c/p\u003e\n\u003cp\u003eRPMI1640: Roswell Park Memorial Institute Medium 1640\u003c/p\u003e\n\u003cp\u003eFBS: Fetal Bovine Serum\u003c/p\u003e\n\u003cp\u003eCD45: Cluster of Differentiation 45\u003c/p\u003e\n\u003cp\u003eRECIST: Response Evaluation Criteria in Solid Tumors\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eH Li and J Qiu: Conceptualization, methodology, validation, formal analysis, data curation, investigation, manuscript revision, and project administration; S Chen, X Kou, and H Liu: Methodology, data collection, validation, formal analysis, writing\u0026mdash;original draft; C Wu, and Y Tian: Formal analysis, data curation, visualization, and methodology. All authors read and approved the final manuscript\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Key R\u0026amp;D Program of China, MOST (2023YFC2510000), Natural Science Foundation of Shanghai (Grant No. 21ZR1422800).\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEthical approval:\u0026nbsp;\u003c/strong\u003eThis in a retrospective study. The Ethics Committee of Shanghai University (Approval No. ECSHU 2024-275) provided institutional ethical approval for this study. Informed consent was obtained from all patients for the use of their medical records in the study prior to the inclusion of their case details. The study was performed in accordance with the ethical standards as laid down in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShanghai ChemAn Biotech Co., Ltd. should be deeply acknowledged for their technical support in the enrichment and drug sensitivity testing of CTCs.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRizvi S, Khan SA, Hallemeier CL, Kelley RK, Gores GJ. Cholangiocarcinoma - evolving concepts and therapeutic strategies. 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Lancet Oncol. 2021;22(5):690-701.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Circulating tumor cells, In vitro drug screening, Anti-cancer drug sensitivity testing, Biliary tract cancer","lastPublishedDoi":"10.21203/rs.3.rs-7201269/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7201269/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBiliary tract carcinoma (BTC) is an aggressive cancer with a poor prognosis, and chemotherapy\u0026rsquo;s effectiveness is limited, especially after first-line therapy failure. Circulating tumor cells (CTCs) offer a platform for \u003cem\u003ein vitro\u003c/em\u003e drug-sensitivity testing to optimize subsequent-line chemotherapy, but the clinical efficacy and prognostic value remains well-established. In this study, we retrospectively analyzed 85 advanced BTC patients, with 25 receiving CTC-based drug-sensitivity-guided chemotherapy (CSBT), 15 receiving FOLFOX, and 45 receiving empirical therapy. CTCs were enriched and tested for drug sensitivity using a glucose uptake assay. Therapeutic efficacy, including patient response, progression-free survival (PFS), overall survival (OS), and toxicity profiles, was evaluated. The results indicated that the objective response rate (ORR) was 16% in CSBT, 6.7% in FOLFOX, and 4.4% in the empirical group. The disease control rate (DCR) was significantly higher in CSBT group (56%) compared to the FOLFOX (20%) and empirical therapy (22.2%; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) groups. Median PFS was significantly prolonged in the CSBT group (5.4 months) versus the FOLFOX (1.9 months) and empirical therapy (2.7 months; \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) groups. Median OS was extended in the CSBT group (12 months), with a significant improvement in OS during the first year of treatment (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Toxicity profiles were similar across all groups. In conclusion, this study for the first time proved that CTC-based drug-sensitivity testing offers a potential approach to guide chemotherapy for advanced BTC. Furthermore, this approach does not increase the risk of severe adverse events, highlighting its potential as a safe and effective strategy for improving patient prognosis in advanced BTC.\u003c/p\u003e","manuscriptTitle":"Drug Sensitivity of Circulating Tumor Cells as a Game-Changer in Subsequent-Line Therapy for Biliary Tract Carcinoma","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-13 05:42:08","doi":"10.21203/rs.3.rs-7201269/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-28T12:45:55+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-17T11:34:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-08T04:53:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"95495228309121542836151263042162684675","date":"2025-08-08T03:22:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148413774205441638660700637247856069511","date":"2025-08-07T17:46:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-07T08:22:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-25T09:49:57+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-24T09:26:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cancer Cell International","date":"2025-07-24T04:22:02+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cancer-cell-international","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ccin","sideBox":"Learn more about [Cancer Cell International](http://cancerci.biomedcentral.com/)","snPcode":"12935","submissionUrl":"https://submission.nature.com/new-submission/12935/3","title":"Cancer Cell International","twitterHandle":"@OncoBioMed","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"93afe91d-3628-43df-9ae8-142c3c49e756","owner":[],"postedDate":"August 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-09T16:00:02+00:00","versionOfRecord":{"articleIdentity":"rs-7201269","link":"https://doi.org/10.1186/s12935-026-04198-2","journal":{"identity":"cancer-cell-international","isVorOnly":false,"title":"Cancer Cell International"},"publishedOn":"2026-02-02 15:56:57","publishedOnDateReadable":"February 2nd, 2026"},"versionCreatedAt":"2025-08-13 05:42:08","video":"","vorDoi":"10.1186/s12935-026-04198-2","vorDoiUrl":"https://doi.org/10.1186/s12935-026-04198-2","workflowStages":[]},"version":"v1","identity":"rs-7201269","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7201269","identity":"rs-7201269","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}