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Abstract Checkpoint kinase 1 (CHK1) is critical for cell survival under replication stress (RS). CHK1 inhibitors (CHK1i’s) in combination with chemotherapy have shown promising results in preclinical studies but have displayed minimal efficacy with substantial toxicity in clinical trials. To explore combinatorial strategies that can overcome these limitations, we perform an unbiased high-throughput screen in a non-small cell lung cancer (NSCLC) cell line and identify thioredoxin1 (Trx1), a major component of the mammalian antioxidant-system, as a determinant of CHK1i sensitivity. We establish a role for redox recycling of RRM1, the larger subunit of ribonucleotide reductase (RNR), and a depletion of the deoxynucleotide pool in this Trx1-mediated CHK1i sensitivity. Further, the TrxR inhibitor auranofin, an approved anti-rheumatoid arthritis drug, shows a synergistic interaction with CHK1i via interruption of the deoxynucleotide pool. Together, we show a pharmacological combination to treat NSCLC that relies on a redox regulatory link between the Trx system and mammalian RNR activity.

Summary: Antipsychotic drugs have been shown to have antitumor effects but have had limited potency in the clinic. Here, we unveil that pimozide inhibits lysosome hydrolytic function to suppress fatty acid and cholesterol release in glioblastoma (GBM), the most lethal brain tumor. Unexpectedly, GBM develops resistance to pimozide by boosting glutamine consumption and lipogenesis. These elevations are driven by SREBP-1, which we find upregulates the expression of ASCT2, a key glutamine transporter. Glutamine, in turn, intensifies SREBP-1 activation through the release of ammonia, creating a feedforward loop that amplifies both glutamine metabolism and lipid synthesis, leading to drug resistance. Disrupting this loop via pharmacological targeting of ASCT2 or glutaminase, in combination with pimozide, induces remarkable mitochondrial damage and oxidative stress, leading to GBM cell death in vitro and in vivo. Our findings underscore the promising therapeutic potential of effectively targeting GBM by combining glutamine metabolism inhibition with lysosome suppression.

Summary: Cholesterol is a structural component of cell membranes. How rapidly growing tumor cells maintain membrane cholesterol homeostasis is poorly understood. Here, we found that glioblastoma (GBM), the most lethal brain tumor, maintains normal levels of membrane cholesterol but with an abundant presence of cholesteryl esters (CEs) in its lipid droplets (LDs). Mechanistically, SREBP-1 (sterol regulatory element-binding protein 1), a master transcription factor that is activated upon cholesterol depletion, upregulates critical autophagic genes, including ATG9B, ATG4A, and LC3B, as well as lysosome cholesterol transporter NPC2. This upregulation promotes LD lipophagy, resulting in the hydrolysis of CEs and the liberation of cholesterol from the lysosomes, thus maintaining plasma membrane cholesterol homeostasis. When this pathway is blocked, GBM cells become quite sensitive to cholesterol deficiency with poor growth in vitro. Our study unravels an SREBP-1-autophagy-LD-CE hydrolysis pathway that plays an important role in maintaining membrane cholesterol homeostasis while providing a potential therapeutic avenue for GBM.

Preclinical in vitro models play an important role in studying cancer cell biology and facilitating translational research, especially in the identification of drug targets and drug discovery studies. This is particularly relevant in breast cancer, where the global burden of disease is quite high based on prevalence and a relatively high rate of lethality. Predictive tools to select patients who will be responsive to invasive or morbid therapies (radiotherapy, chemotherapy, immunotherapy, and/or surgery) are relatively lacking. To be clinically relevant, a model must accurately replicate the biology and cellular heterogeneity of the primary tumor. Addressing these requirements and overcoming the limitations of most existing cancer cell lines, which are typically derived from a single clone, we have recently developed conditional reprogramming (CR) technology. The CR technology refers to a co-culture system of primary human normal or tumor cells with irradiated murine fibroblasts in the presence of a Rho-associated kinase inhibitor to allow the primary cells to acquire stem cell properties and the ability to proliferate indefinitely in vitro without any exogenous gene or viral transfection. This innovative approach fulfills many of these needs and offers an alternative that surpasses the deficiencies associated with traditional cancer cell lines. These CR cells (CRCs) can be reprogrammed to maintain a highly proliferative state and reproduce the genomic and histological characteristics of the parental tissue. Therefore, CR technology may be a clinically relevant model to test and predict drug sensitivity, conduct gene profile analysis and xenograft research, and undertake personalized medicine. This review discusses studies that have applied CR technology to conduct breast cancer research.

Mitochondrial dysfunction and glycolysis activation are improtant hallmarks of hepatocellular carcinoma (HCC). NOP2 is an S-adenosyl-L-methionine-dependent methyltransferase that regulates the cell cycle and proliferation activities. In this study, found that NOP2 contributes to HCC progression by promoting aerobic glycolysis. Our results revealed that NOP2 was highly expressed in HCC and that it was associated with unfavorable prognosis. NOP2 knockout in combination with sorafenib enhanced sorafenib sensitivity, which, in turn, led to marked tumor growth inhibition. Mechanistically, we identified that NOP2 regulates the c-Myc expression in an m5C-modification manner to promote glycolysis. Moreover, our results revealed that m5C methylation induced c-Myc mRNA degradation in an eukaryotic translation initiation factor 3 subunit A (EIF3A)-dependent manner. In addition, NOP2 was found to increase the expression of the glycolytic genes LDHA, TPI1, PKM2, and ENO1. Furthermore, MYC associated zinc finger protein (MAZ) was identified as the major transcription factor that directly controlled the expression of NOP2 in HCC. Notably, in a patient-derived tumor xenograft (PDX) model, adenovirus-mediated knockout of NOP2 maximized the antitumor effect and prolonged the survival of PDX-bearing mice. Our cumulative findings revealed the novel signaling pathway MAZ/NOP2/c-Myc in HCC and uncovered the important roles of NOP2 and m5C modifications in metabolic reprogramming. Therefore, targeting the MAZ/NOP2/c-Myc signaling pathway is suggested to be a potential therapeutic strategy for the treatment of HCC.

Abstract Hepatocellular carcinoma (HCC) is one of the most common public health challenges, worldwide. Because of molecular complexity and tumor heterogeneity, there are no effective predictive models for prognosis of HCC. This underlines the unmet need for accurate prognostic models for HCC. Analysis of GSE14520 data from gene omnibus (GEO) database identified multiple differentially expressed mRNAs (DEMs) between HCC and normal tissues. After randomly stratifying the patients into the training and testing groups, we performed univariate, lasso, and multivariable Cox regression analyses to delineate the prognostic gene signature in training set. We then used Kaplan–Meier plot, time‐dependent receiver operating characteristic (ROC), multivariable Cox regression analysis of clinical information, nomogram, and decision curve analysis (DCA) to evaluate the predictive and overall survival value of a novel five‐gene signature (CNIH4, SOX4, SPP1, SORBS2, and CCL19) within and across sets, separately and combined. We also validated the prognostic value of the five‐gene signature using The Cancer Genome Atlas—Liver Hepatocellular Carcinoma (TCGA‐LIHC), GSE54236 and International Cancer Genome Consortium (ICGC) sets. Multivariable Cox regression analysis revealed that the five‐gene signature and tumor node metastasis (TNM) stage were independent prognostic factors for overall survival of HCC patients in GSE14520 and TCGA‐LIHC. Combining TNM stage clinical pathological parameters and nomogram greatly improved the prognosis prediction of HCC. Further gene set enrichment analysis (GSEA) revealed enrichment of KEGG pathways related to cell cycle in the high‐risk group and histidine metabolism in the low‐risk group. Finally, all these five mRNAs are overexpressed between 12 pairs of HCC and adjacent normal tissues by quantitative real‐time PCR validation. In brief, a five‐gene prognostic signature and a nomogram were identified and constructed, respectively, and further validated for their HCC prognostic value. The five‐gene risk score together with TNM stage models could aid in rationalizing customized therapies in HCC patients.

As one of the most common internal modifications in eukaryotic mRNA, N6-methyladenosine (m6A) modification is involved in the pathogenesis of many diseases, including hepatocellular carcinoma (HCC). In this study, we explored the prognostic significance of the expression of RNA binding motif protein 15B (RBM15B) in HCC, by studying specimens collected from clinical subjects. RBM15B is highly expressed in HCC patients and indicates a poor prognosis. Functionally, overexpression of RBM15B promotes HCC cell proliferation and invasion and induces sorafenib resistance in HCC cells. Mechanistically, we confirmed that RBM15B is transcriptionally activated by YY1 and regulates the stability of TRAM2 mRNA in an m6A-dependent manner. Overall, our results reveal a YY1-RBM15B-TRAM2 regulatory axis and highlight the critical role of RBM15B and m6A modifications in HCC. These findings may provide a novel mechanism and therapeutic targets for the treatment of HCC.

PurposeWe aimed to investigate the restoration of CD8+PD-1+ T cells through adoptive T-cell therapy (ACT) in relation to the prognosis and the therapeutic response to anti-PD-1 in patients with advanced pancreatic cancer (APC).MethodsA total of 177 adult patients who underwent tumor resection as initial treatment for pancreatic ductal adenocarcinoma (PDAC) from February 2013 to July 2019 at Zhongnan Hospital of Wuhan University were enrolled in this study. Another cohort of 32 patients with APC was prospectively enrolled from Capital Medical University Cancer Center between June 1, 2013, and May 30, 2019.ResultsOf the 177 patients who received tumor resection, 67 tumor samples showed overexpression of PD-L1 and 110 patients with low expression of PD-L1. We found that overexpressed PD-L1 was a significant prognostic factor related to overall survival (OS). Furthermore, we tested the percentage of peripheral CD8+PD-1+ T cells in all patients and found that it was significantly correlated with the PD-L1 expression and the prognosis of patients with PDAC. The peripheral blood T lymphocyte subtypes were tracked for 30 months, and CD8+PD-1+ cells were shown to decrease. After that, we performed ACT for patients with APC in another cancer center. We found that the ratios of posttreatment of ACT/pre-ACT CD8+PD-1+ T cells were significantly related to the prognosis of patients with APC. Moreover, patients with combined treatment of ACT with anti-PD-1 had significantly favorable OS.ConclusionsThis study showed that the CD8+PD-1+ T-cell level was related to the expression of PD-L1. Restoring CD8+PD-1+ T cells in patients with APC by treatment of ACT significantly benefits the prognosis and facilitates the response to anti-PD-1.

ObjectiveTo explore the independent predictive factors of spontaneous tumor rupture (STR) in patients undergoing curative resection of hepatocellular carcinoma (HCC), and to evaluate the impact of STRHCC on long-term survival after hepatectomy.MethodsThe clinicopathological parameters of 106 patients with STRHCC and 201 patients with non-ruptured HCC who underwent hepatectomy from January 2007 to November 2011 at the Eastern Hepatobiliary Surgery Hospital and Zhongnan Hospital of Wuhan University were analyzed using propensity score matching (PSM) and a logistic regression model.ResultsFactors including hypertension, cirrhosis, total bilirubin (TB), tumor size, and ascites were independent predictors of STR. For all 307 HCC patients, the 1-, 3- and 5-year overall survival (OS) rates were 54.0%, 37.3% and 33.8%, respectively. After PSM, the 1-, 3-, and 5-year OS rates in the ruptured group remained significantly lower at 41.5%, 23.5%, and 17.5% when compared with the non-ruptured group at 70.8%, 47.1%, and 37.6%, respectively, while the 1-, 3-, and 5-year disease-free survival (DFS) rates between the groups did not differ significantly (50.4%, 35.1%, 27.1% vs 55.4%, 38.2%, 27.4%). STRHCC was significantly associated with increased risk of OS, but not of shorter DFS. No significant difference in postoperative morbidity or hospital death was observed between the groups.ConclusionFactors including hypertension, liver cirrhosis, higher TB levels, tumor size > 5cm, and ascites are significant predictors of STR. The recurrence rate of patients in the ruptured group was significantly higher than that of patients in the non-ruptured group. STR results in poorer OS but not DFS in patients undergoing curative resection for HCC. STRHCC has no impact on postoperative morbidity and mortality after hepatectomy.

Objective: This study aimed to determine the association between prognosis of COVID-19 patients with and without cancer. Moreover, we compared the prognosis of cancer patients subjected to anti-tumor therapy with those who have not undergone anti-tumor therapy in the past 6 months.Methods and Results: A total of 7,926 adult patients with COVID-19 were retrospectively enrolled in Hubei Province,China between December 31, 2019 and February 20, 2020. Two hundred and seventy seven cancer patients (cancer group, median age 64 [IQR 56–70] years; 50.90% male) and 7,649 non-cancer patients were identified (non-cancer group, median age 55 [IQR 42–64] years; 48.19% male). The mortality rate was lower in the non-cancer group compared to the cancer group (4.50 vs. 9.03%; P < 0.001). The duration between onset and admission shorter in the cancer group (Days, 9 [IQR 5–18]) compared to the non-cancer group (Days, 10; [IQR 6–19]; P = 0.036). ICU occupancy was higher in the cancer group (n[%], 30[10.83%]) than in the non-cancer group (n[%], 314[4.11%]). In reviewing the anti-tumor therapy, data from 277 selected cancer patients were obtained out of which 74 patients had undergone anti-tumor therapy (mean age 65 [IQR 51–67] years; 45.95% male), 203 had not undergone anti-tumor therapy (non-anti-tumor therapy group, mean age 63 [IQR 53–75] years; 49.75% male) in the past 6 months. The mortality rate for the anti-tumor therapy group and the non-anti-tumor therapy group was similar (9.46 vs. 8.87%; P = 0.879).Conclusion: The mortality rate was higher in COVID-19 patients with cancer compared to those without cancer. Moreover, anti-tumor therapy in the past 6 months did not worsen the prognosis of cancer patients with COVID-19.

Background: An iatrogenic bile duct injury (IBDI) is a severe complication that has a great impact on the physical and mental quality of life of the patients, especially for patients with postoperative benign biliary stricture. The effective measures for end-to-end biliary-to-biliary anastomosis intraoperative are essential to prevent the postoperative bile duct stricture, but also a challenge even to the most skilled biliary tract surgeon.Objective: A postoperative benign biliary stricture is an extremely intractable complication that occurs following IBDI. This study aimed to introduce a novel end-to-end biliary-to-biliary anastomosis technique named fish-mouth-shaped (FMS) end-to-end biliary-to-biliary reconstruction and determine the safety and effectiveness for preventing the postoperative benign biliary stricture in both rats and humans.Methods: In this study, 18 patients with biliary injury who underwent an FMS reconstruction procedure were retrospectively analyzed. Their general information, disease of the first hospitalization, operation method, and classification of bile duct injury (BDI) were collected. The postoperative complications were evaluated immediately perioperatively and the long-term complications were followed up at the later period of at least 5 years. An IBDI animal model using 18 male rats was developed for animal-based evaluations. A bile duct diathermy injury model was used to mimic BDI. The FMS group underwent an FMS reconstruction procedure while the control group underwent common end-to-end biliary-to-biliary anastomosis, a sham operation group was also established. The blood samples, liver, spleen, and common bile duct tissues were harvested for further assessments.Results: In the retrospective study, there was no postoperative mortality and no patient developed cholangitis during the 5-years postoperation follow-up. In the study of IBDI animal models, compared with the control group, the FMS reconstruction procedure reduced the occurrence of benign biliary stenosis, liver function damage, and jaundice. The blood tests as well as morphological and pathological observations revealed that rats in the FMS reconstruction group had a better recovery than those in the control group.Conclusions: An FMS reconstruction procedure is a safe and efficient BDI treatment method.

Glioblastoma (GBM) is the most lethal primary brain tumor. With limited therapeutic options, novel therapies are desperately needed. Recent studies have shown that GBM acquires large amounts of lipids for rapid growth through activation of sterol regulatory element-binding protein 1 (SREBP-1), a master transcription factor that regulates fatty acid and cholesterol synthesis, and cholesterol uptake. Interestingly, GBM cells divert substantial quantities of lipids into lipid droplets (LDs), a specific storage organelle for neutral lipids, to prevent lipotoxicity by increasing the expression of diacylglycerol acyltransferase 1 (DGAT1) and sterol-O-acyltransferase 1 (SOAT1), which convert excess fatty acids and cholesterol to triacylglycerol and cholesteryl esters, respectively. In this review, we will summarize recent progress on our understanding of lipid metabolism regulation in GBM to promote tumor growth and discuss novel strategies to specifically induce lipotoxicity to tumor cells through disrupting lipid storage, a promising new avenue for treating GBM.

Background: Neuroendocrine tumors are heterogeneous malignancies that originate from the neuroendocrine system. Previous studies show that this cancer type mainly localizes in the gastrointestinal tract and often metastasizes to the liver. Primary liver neuroendocrine tumors are very rare and primary hepatic neuroendocrine tumors (PHNET) with concurrent hepatocellular carcinoma (HCC) are extremely rare. To the best of our knowledge, only few PHNET cases have been identified, making their diagnosis difficult. Here, we report the biggest ever reported and “deceiving” lesion of a mixed neuroendocrine-non-neuroendocrine neoplasm in the liver, aiming to raise awareness and improve treatment of the disease.Case Presentation: Here, we report a preoperative misdiagnosed case that presented with hepatocellular carcinoma clinical features and no extrahepatic tumors. Postoperative pathology confirmed that it was a mixed neuroendocrine-non-neuroendocrine neoplasm. The patient was then referred for etoposide and cisplatin-based chemotherapy. No disease recurrence was observed at the 6-month follow-up.Conclusion: We report a very rare and easily misdiagnosed case and we speculate that there were “undifferentiated cells” undergoing neuroendocrine and hepatocellular carcinoma differentiation, during which some hepatocellular carcinoma cells express neuroendocrine features. We recommend proper surgery and postoperative platinum-based chemotherapy in the management of this disease.

We recently demonstrated that glioblastoma, the most lethal brain cancer, upregulates diacylglycerol O-acyltransferase 1 (DGAT1) to store excess fatty acids into triglycerides to prevent lipotoxicity and promote tumor growth. Targeting DGAT1 resulted in marked tumor cell death by triggering extensive oxidative stress, indicating that DGAT1 could be a promising target for cancer therapy.

Summary: Recently, lipid metabolism reprogramming has been further evidenced in malignancies via the observation of large amounts of lipid droplets (LDs) in human tumors, including in glioblastoma (GBM), the most lethal primary brain tumor. However, the role played by LDs in tumor cells remains unknown. Here, we show that triglycerides (TG), the major components of LDs, serve as a critical energy reservoir to support GBM cell survival. TG/LDs rapidly diminished in GBM cells upon glucose reduction, whereas inhibiting fatty acid oxidation or autophagy resulted in the accumulation of TG/LDs and strongly potentiated GBM cell death. Immunofluorescence imaging and time-lapse videos showed that LDs are hydrolyzed by autophagy to release free fatty acids that mobilize into mitochondria for energy production. Our study demonstrates that autophagy-mediated hydrolysis of TG/LDs maintains energy homeostasis and GBM survival upon glucose reduction, suggesting that limiting TG/LDs utilization might be necessary upon treating GBM.

Hepatocellular carcinoma (HCC) remains hard to diagnose early and cure due to a lack of accurate biomarkers and effective treatments. Hence, it is necessary to explore the tumorigenesis and tumor progression of HCC to discover new biomarkers for clinical treatment. We performed weighted gene co-expression network analysis (WGCNA) to explore hub genes that have high correlation with clinical information. In this study, we found 13 hub genes (GTSE1, PLK1, NCAPH, SKA3, LMNB2, SPC25, HJURP, DEPDC1B, CDCA4, UBE2C, LMNB1, PRR11, and SNRPD2) that have high correlation with histologic grade in HCC by analyzing TCGA LIHC dataset. All of these 13 hub genes could be used to effectively distinguish high histologic grade from low histologic grade of HCC through analysis of the ROC curve. The overall survival and disease-free survival information showed that high expression of these 13 hub genes led to poor prognosis. Meanwhile, these 13 hub genes had significantly different expression in HCC tumor and non-tumor tissues. We downloaded GSE6764, which contains corresponding clinical information, to validate the expression of these 13 hub genes. At the same time, we performed quantitative real-time PCR to validate the differences in the expression tendencies of these 13 hub genes between HCC tumor tissues and non-tumor tissues and high histologic grade and low histologic grade. We also explored mutation and methylation information of these 13 hub genes for further study. In summary, 13 hub genes correlated with the progression and prognosis of HCC were discovered by WGCNA in our study, and these hub genes may contribute to the tumorigenesis and tumor progression of HCC.

Abstract Reprogramming of lipid metabolism is a newly recognized hallmark of malignancy. Increased lipid uptake, storage and lipogenesis occur in a variety of cancers and contribute to rapid tumor growth. Lipids constitute the basic structure of membranes and also function as signaling molecules and energy sources. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors in the endoplasmic reticulum, play a central role in the regulation of lipid metabolism. Recent studies have revealed that SREBPs are highly up-regulated in various cancers and promote tumor growth. SREBP cleavage-activating protein is a key transporter in the trafficking and activation of SREBPs as well as a critical glucose sensor, thus linking glucose metabolism and de novo lipid synthesis. Targeting altered lipid metabolic pathways has become a promising anti-cancer strategy. This review summarizes recent progress in our understanding of lipid metabolism regulation in malignancy, and highlights potential molecular targets and their inhibitors for cancer treatment.

Dysregulated lipid metabolism is a characteristic of malignancies. Sterol regulatory element binding protein 1 (SREBP-1), a transcription factor playing a central role in lipid metabolism, is highly activated in malignancies. Here, we unraveled a link between miR-29 and the SCAP (SREBP cleavage-activating protein)/SREBP-1 pathway in glioblastoma (GBM) growth. Epidermal growth factor receptor (EGFR) signaling enhances miR-29 expression in GBM cells via upregulation of SCAP/SREBP-1, and SREBP-1 activates miR-29 expression via binding to specific sites in its promoter. In turn, miR-29 inhibits SCAP and SREBP-1 expression by interacting with their 3′ UTRs. miR-29 transfection suppressed lipid synthesis and GBM cell growth, which were rescued by the addition of fatty acids or N-terminal SREBP-1 expression. Xenograft studies showed that miR-29 mimics significantly inhibit GBM growth and prolong the survival of GBM-bearing mice. Our study reveals a previously unrecognized negative feedback loop in SCAP/SREBP-1 signaling mediated by miR-29 and suggests that miR-29 treatment may represent an effective means to target GBM.

Constitutively activated oncogenic signaling via genetic mutations such as in the EGFR/PI3K/Akt and Ras/RAF/MEK pathways has been recognized as a major driver for tumorigenesis in most cancers. Recent insights into tumor metabolism have further revealed that oncogenic signaling pathways directly promote metabolic reprogramming to upregulate biosynthesis of lipids, carbohydrates, protein, DNA and RNA, leading to enhanced growth of human tumors. Therefore, targeting cell metabolism has become a novel direction for drug development in oncology. In malignant gliomas, metabolism pathways of glucose, glutamine and lipid are significantly reprogrammed. Moreover, molecular mechanisms causing these metabolic changes are just starting to be unraveled. In this review, we will summarize recent studies revealing critical gene alterations that lead to metabolic changes in malignant gliomas, and also discuss promising therapeutic strategies via targeting the key players in metabolic regulation.

The sustained clinical activity of the BRAF inhibitor vemurafenib (PLX4032/RG7204) in patients with BRAF(V600) mutant melanoma is limited primarily by the development of acquired resistance leading to tumor progression. Clinical trials are in progress using MEK inhibitors following disease progression in patients receiving BRAF inhibitors. However, the PI3K/AKT pathway can also induce resistance to the inhibitors of MAPK pathway.The sensitivity to vemurafenib or the MEK inhibitor AZD6244 was tested in sensitive and resistant human melanoma cell lines exploring differences in activation-associated phosphorylation levels of major signaling molecules, leading to the testing of co-inhibition of the AKT/mTOR pathway genetically and pharmacologically. There was a high degree of cross-resistance to vemurafenib and AZD6244, except in two vemurafenib-resistant cell lines that acquired a secondary mutation in NRAS. In other cell lines, acquired resistance to both drugs was associated with persistence or increase in activity of AKT pathway. siRNA-mediated gene silencing and combination therapy with an AKT inhibitor or rapamycin partially or completely reversed the resistance.Primary and acquired resistance to vemurafenib in these in vitro models results in frequent cross resistance to MEK inhibitors, except when the resistance is the result of a secondary NRAS mutation. Resistance to BRAF or MEK inhibitors is associated with the induction or persistence of activity within the AKT pathway in the presence of these drugs. This resistance can be potentially reversed by the combination of a RAF or MEK inhibitor with an AKT or mTOR inhibitor. These combinations should be available for clinical testing in patients progressing on BRAF inhibitors.

Purpose: This study is to investigate the [68Ga]Ga-DOTA-FAPI PET/CT diagnosis performance in biliary tract carcinoma (BTC) and analyze the association between [68Ga]Ga-DOTA-FAPI PET/CT and clinical indexes. Methods: A prospective study (NCT 05264688) was performed between January 2022 and July 2022. Fifty participants were scanned using [68Ga]Ga-DOTA-FAPI and [18F]FDG PET/CT and acquired pathological tissue. We employed the Wilcoxon signed-rank test to compare the uptake of [68Ga]Ga-DOTA-FAPI and [18F]FDG, and the McNemar test was used to compare the diagnostic efficacy between the two tracers. Spearman or Pearson correlation was used to assess the association between [68 Ga]Ga-DOTA-FAPI PET/CT and clinical indexes. Results: In total, 47 participants (mean age 59.09 ± 10.98 [range 33–80 years]) were evaluated. The [68Ga]Ga-DOTA-FAPI detection rate was greater than [18F]FDG in primary tumors (97.62% vs. 85.71%), nodal metastases (90.05% vs. 87.06%), and distant metastases (100% vs. 83.67%). The uptake of [68Ga]Ga-DOTA-FAPI was higher than [18F]FDG in primary lesions (intrahepatic cholangiocarcinoma, 18.95 ± 7.47 vs. 11.86 ± 0.70, p = 0.001; extrahepatic cholangiocarcinoma, 14.57 ± 6.16 vs. 8.80 ± 4.74, p = 0.004), abdomen and pelvic cavity nodal metastases (6.91 ± 6.56 vs. 3.94 ± 2.83, p < 0.001), and distant metastases (pleural, peritoneum, omentum, and mesentery, 6.37 ± 4.21 vs. 4.50 ± 1.96, p = 0.01; bone, 12.15 ± 6.43 vs. 7.51 ± 4.54, p = 0.008). There was a significant correlation between [68Ga]Ga-DOTA-FAPI uptake and fibroblast-activation protein (FAP) expression (Spearman r = 0.432, p = 0.009), carcinoembryonic antigen (CEA) (Pearson r = 0.364, p = 0.012), and platelet (PLT) (Pearson r = 0.35, p = 0.016). Meanwhile, a significant relationship between [68Ga]Ga-DOTA-FAPI metabolic tumor volume and carbohydrate antigen199 (CA199) (Pearson r = 0.436, p = 0.002) was confirmed. Conclusion: [68Ga]Ga-DOTA-FAPI had a higher uptake and sensitivity than [18F]FDG in the diagnosis of BTC primary and metastatic lesions. The correlation between [68Ga]Ga-DOTA-FAPI PET/CT indexes and FAP expression, CEA, PLT, and CA199 were confirmed. Trial registration: clinicaltrials.gov: NCT 05,264,688. [ABSTRACT FROM AUTHOR]