The deregulation of microRNAs (miRNAs) plays an important role in human hepatocarcinogenesis. In this study, we highlight exosomes as mediators involved in modulating miRNA profiles in hepatocellular carcinoma (HCC) cells. First, we examined the different miRNA expression profiles in HCC cells and HCC cell–derived exosomes. Next, coculture experiments indicated that HCC cell–derived exosomes promoted the cell growth, migration, and invasion of HCC cells and had the ability to shuttle miRNAs to recipient cells. Further, our data showed that Vps4A, a key regulator of exosome biogenesis, was frequently down‐regulated in HCC tissues. The reduction of Vps4A in HCC tissues was associated with tumor progression and metastasis. In vitro studies revealed that Vps4A repressed the growth, colony formation, migration, and invasion of HCC cells. We further investigated the role and involvement of Vps4A in suppressing the bioactivity of exosomes and characterized its ability to weaken the cell response to exosomes. By small RNA sequencing, we demonstrated that Vps4A facilitated the secretion of oncogenic miRNAs in exosomes as well as accumulation and uptake of tumor suppressor miRNAs in cells. A subset of Vps4A‐associated miRNAs was identified. Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that the phosphatidylinositol‐3‐kinase/Akt signaling pathway was the most likely candidate pathway for modulation by these miRNAs. Indeed, we proved that the phosphatidylinositol‐3‐kinase/Akt pathway was inactivated by Vps4A overexpression. Conclusion: Exosome‐mediated miRNA transfer is an important mechanism of self‐modulation of the miRNA expression profiles in HCC cells, and Vps4A may function as a tumor suppressor, which utilizes exosomes as mediators to regulate the secretion and uptake of miRNAs in hepatoma cells; these observations provide new insights into the development of HCC. (Hepatology 2015;61:1284–1294)

Immune checkpoint inhibitors (ICIs) are widely used for treating hepatocellular carcinoma (HCC), yet their efficacy remains limited, with suboptimal response rates. The predictive power of the current biomarker, programmed death ligand-1 (PD-L1), is limited by detection variability and glycosylation, underscoring the need for complementary biomarkers to enhance predictive accuracy. In this study, mass spectrometry was employed to identify proteomic alterations in HCC tissues from responders and nonresponders to anti-programmed cell death-1 (PD-1) therapy. Survival analysis established the role of Yin Yang 1 (YY1) in determining ICI efficacy. Coculture models of hepatoma and CD8+ T cells revealed the immunosuppressive function of YY1. Transcriptome sequencing identified polypeptide N-acetylgalactosaminyltransferase 16 (GALNT16) as a transcriptional target of YY1, and subsequent Western blot and coimmunoprecipitation assays demonstrated that GALNT16 augments PD-L1 expression. Furthermore, in vivo mouse models demonstrated that YY1 knockdown potentiated the efficacy of anti-PD-1 therapy, an effect that was partially reversed by GALNT16 overexpression. Specifically, YY1 upregulates GALNT16, which in turn promotes PD-L1 glycosylation and stability, leading to diminished CD8+ T cell activity. Thus, GALNT16 knockdown rescued the compromised CD8+ T cell cytotoxicity induced by YY1. Collectively, these results elucidate the YY1/GALNT16/PD-L1 axis as a pivotal mechanism underlying HCC resistance to ICI therapy. This highlights the therapeutic potential of targeting PD-L1 glycosylation pathways.

Background and aims: Despite growing evidence linking pretransplant exposure to immune checkpoint inhibitors (ICIs) to increased allograft rejection risk after liver transplantation (LT), a lack of comparative studies to definitively establish the correlation between ICI exposure and adverse short-term outcomes after LT exists. This study aimed to analyze the impact of preoperative ICI exposure on short-term post-LT prognosis and allograft rejection risk. Methods: This retrospective cohort study included 121 recipients who underwent LT for hepatocellular carcinoma (HCC) between June 2019 and March 2023. The recipients were categorized into ICI (n = 35) and non-ICI (n = 86) exposure groups based on pretransplant ICI exposure. Demographics, clinical characteristics, and short-term outcomes were compared between the cohorts. Kaplan-Meier analysis evaluated the impact of ICI exposure on graft survival. Univariate and multivariate logistic regression models assessed the impact of patient characteristics on allograft rejection. Results: Recipients with or without ICI exposure exhibited comparable demographic baseline characteristics. The incidences of early allograft dysfunction and biliary and vascular complications were similar between both groups. Post-transplant infection incidence was 37.1% and 20.9% in the ICI and non-ICI groups, respectively (P = 0.064). Allograft rejection rates were significantly higher in the ICI group than in the non-ICI group (22.9% vs. 5.8%, P = 0.015). The ICI group exhibited a higher 90-day post-transplant mortality rate than that of the non-ICI group (14.3% vs. 2.3%, P = 0.034). Logistic regression analyses demonstrated that allograft rejection independently correlated with 90-day post-transplant mortality, with ICI exposure being an independent risk factor for allograft rejection. In recipients with ICI exposure, a shorter interval between ICIs and LT (washout period) was significantly associated with a higher allograft rejection risk, with the optimal washout period identified as 21 days for predicting 90-day rejection-free survival (P = 0.0001). Moreover, in recipients with allograft rejection, the peripheral CD4+/CD8+ T cell ratio was much lower in the ICI group than in the non-ICI group. Conclusions: Pretransplant ICI exposure was an independent risk factor for allograft rejection and was significantly associated with 90-day post-transplant mortality after LT for HCC. A ≤21-day washout period was significantly associated with allograft rejection. Future multicenter studies with larger cohorts and prospective designs are essential to validate these findings, confirm causality, and establish standardized clinical guidelines for ICI use before transplantation. Trail registration: ClinicalTrials.gov NCT05913583.

BACKGROUND:Pre-transplant exposure to immune checkpoint inhibitors (ICIs) significantly increases the risk of allograft rejection after liver transplantation (LT); however, whether ICI-related rejection leads to increased graft loss remains controversial. Therefore, this study aimed to investigate the association between ICI-related allograft rejection and perioperative graft loss. METHODS:This was a retrospective analysis of adult liver transplant recipients with early biopsy-proven T-cell-mediated rejection (TCMR) at Liver Transplantation Center of Sun Yat-sen Memorial Hospital from June 2019 to September 2024. The pathological features, clinical characteristics, and perioperative graft survival were analyzed. RESULTS:Twenty-eight patients who underwent early TCMR between June 2019 and September 2024 were included. Based on pre-LT ICI exposure, recipients were categorized into ICI-related TCMR (irTCMR, n = 12) and conventional TCMR (cTCMR, n = 16) groups. Recipients with irTCMR had a higher median Banff rejection activity index (RAI) (6 vs . 5, P = 0.012) and more aggressive tissue damage and inflammation. Recipients with irTCMR showed higher proportion of treatment resistance, achieving a complete resolution rate of only 8/12 compared to 16/16 for cTCMR. Graft loss occurred in 5/12 of irTCMR recipients within 90 days after LT, with no graft loss in cTCMRs recipients. Cox analysis demonstrated that irTCMR with an ICI washout period of <30 days was an independent risk factor for perioperative graft loss (hazard ratio [HR], 6.540; 95% confidence interval [CI], 1.067-40.067, P = 0.042). CONCLUSION:IrTCMR is associated with severe pathological features, increased resistance to treatment, and higher graft loss in adult liver transplant recipients.

Cancer is a significant challenge to society and public health in the 21st century. According to GLOBOCAN 2020, there were 19.3 million new cancer cases with approximately 10.0 million deaths in 2020 globally. By 2040, 28 million new cases and 16.2 million deaths are estimated. With the escalating challenges of cancer and limitations of conventional therapies like surgery, chemotherapy, and radiotherapy, the development of novel therapies such as immunotherapy and targeted therapy is required. Immunotherapy, especially immune checkpoint inhibitors (ICIs), has become a significant advancement in cancer treatment, combating tumors by activating the immune system. This review offers a thorough overview of ICIs, including their classification, mechanisms of action, and adverse events. It also examines the application of ICIs across various cancer types especially on advanced or unresectable malignancies, such as head and neck squamous cell carcinoma, esophageal cancer, non-small cell lung cancer, breast cancer, hepatocellular carcinoma, and bladder cancer, highlighting their therapeutic potential and the challenges they face. By providing a comprehensive analysis, this review aims to construct a reference system for clinicians to better understand and utilize ICIs in treating cancer.

Gallbladder carcinoma (GBC) is an extremely aggressive tumor of the biliary tract with a bleak prognosis, and the evidence supporting the benefit of available systemic therapy for advanced GBC is scarce. Herein, this study intended to investigate the real-world outcome of chemotherapy combined with programmed death-1 (PD-1) inhibitor for the management of unresectable or recurrent GBC. From January 2018 to December 2023, consecutive patients who were treated with systematic treatment, including chemotherapy or the combination of chemotherapy plus PD-1 inhibitor, for unresectable or recurrent GBC were retrospectively identified. Clinical data regarding baseline characteristics, therapeutic response, adverse events (AEs), and oncological outcomes were collected. The eligible patients were allocated to combination therapy arm (n = 46) and mono-chemotherapy arm (n = 19). After propensity score matching (PSM), 16 patients were allocated in each arm. The overall survival (OS) and progression-free survival (PFS) of combination therapy were marginally superior to mono-chemotherapy both before and after PSM. The combination therapy exhibited advantage over mono-chemotherapy in regards to partial response (PR) (before PSM: P = 0.009; after PSM: P = 0.037) and objective response rate (ORR) (before PSM: P = 0.006; after PSM: P = 0.015). In combined therapy cohort, 1 patient achieve a complete response, and 13 patients were assessed as appropriate for surgical excision, among which 1 patient refused further surgical intervention. In patients with unresectable or recurrent GBC, the combination of chemotherapy and PD-1 inhibitor as first-line therapy exhibited prolonged OS and PFS, and increased PR and ORR over those receiving chemotherapy alone, with an acceptable toxicity profile. The combination therapy may be a potential conversion therapy in unresectable GBC patients.

Perturb-Seq combines CRISPR (clustered regularly interspaced short palindromic repeats)-based genetic screens with single-cell RNA sequencing readouts for high-content phenotypic screens. Despite the rapid accumulation of Perturb-Seq datasets, there remains a lack of a user-friendly platform for their efficient reuse. Here, we developed PerturbDB (http://research.gzsys.org.cn/perturbdb), a platform to help users unveil gene functions using Perturb-Seq datasets. PerturbDB hosts 66 Perturb-Seq datasets, which encompass 4 518 521 single-cell transcriptomes derived from the knockdown of 10 194 genes across 19 different cell lines. All datasets were uniformly processed using the Mixscape algorithm. Genes were clustered by their perturbed transcriptomic phenotypes derived from Perturb-Seq data, resulting in 421 gene clusters, 157 of which were stable across different cellular contexts. Through integrating chemically perturbed transcriptomes with Perturb-Seq data, we identified 552 potential inhibitors targeting 1409 genes, including an mammalian target of rapamycin (mTOR) signaling inhibitor, retinol, which was experimentally verified. Moreover, we developed a ‘Cancer’ module to facilitate the understanding of the regulatory role of genes in cancer using Perturb-Seq data. An interactive web interface has also been developed, enabling users to visualize, analyze and download all the comprehensive datasets available in PerturbDB. PerturbDB will greatly drive gene functional studies and enhance our understanding of the regulatory roles of genes in diseases such as cancer.

Harnessing the cGAS-STING DNA sensing pathway in dendritic cells (DCs) to enhance anti-tumor immunity in immunosuppressive pancreatic tumors remains a significant challenge. While manganese (Mn2+) enhances cGAS sensitivity to DNA, the precise mechanisms and potential of Mn-DNA complexes in this process are unclear. Here, this work introduces a strategy to encapsulate Mn-DNA complexes into DC-derived immunogenic extracellular vesicle (EVDC@Mn-DNA) to trigger robust anti-tumor immunity. This work shows that Mn2+ induces tumor cell-DNA transition to Z-DNA, strengthening cGAS binding and promoting its phase condensation for optimal activation in DCs. Using this newly developed Raft-Ultra method, this work engineers immunogenic EVs derived from tumor cell lysate-pulsed DCs, loaded with Mn-DNA complexes (EVDC@Mn-DNA). These EVs efficiently deliver Mn-DNA complexes to DCs, activating the cGAS-STING pathway both in vitro and in vivo. In animal models, EVDC@Mn-DNA administration enhances vascular function, as evidenced by increased blood flow and perfusion, improved anti-PD-L1 delivery, reduced hypoxia, and elevated endothelial cell-ICAM1 expression, which facilitates T cell adhesion. This approach expands the intratumoral population of activated DCs and T cells and promotes the formation of larger tertiary lymphoid structures, ultimately suppressing orthotopic pancreatic tumor growth. Overall, this EVDC@Mn-DNA strategy reprograms intratumoral DCs, restores vascular-immune homeostasis, and potentiates anti-tumor immunity in pancreatic cancer.

>Laparoscopic hepatectomy is now a widely accepted surgical technique in hepatobiliary surgery and is comparably safe and efficient to open hepatectomy [ 1, 2 ]. In liver transplantation, studies have underscored the safety of laparoscopic or robot-assisted procedures in donor hepatectomy [ 3-5 ]. Donors undergoing laparoscopic or robot-assisted hepatectomy experience reduced postoperative complications and shorter recovery periods [ 6-8 ]. Recently, surgeons in Seoul National University Hospital reported several consecutive living donor liver transplantation(LDLT) with pure laparoscope, hybrid laparoscopic with robotic-assistance, and total robot-assistance [ 9-11 ]. Following closely, surgeons in King Faisal Specialist Hospital at Saudi Arabia reported 3 fully robotic donor hepatectomy and robotic recipient liver graft implantation [ 12 ]. However, to the best of our knowledge, the laparoscopic implantation for a full-size liver graft has not been reported.

Immune checkpoint inhibitor (ICI)-based immunotherapy has emerged as the most promising strategy for hepatocellular carcinoma (HCC) downstaging prior to liver transplantation (LT). However, further evidence is required to assess the feasibility and safety of pretransplant ICI exposure. We retrospective analyzed 159 HCC patients who underwent LT at our institution from June 2019 to December 2023, and 39 recipients (39/159, 24.5

Patients with cholangiocarcinoma (CCA) with lymph node metastasis (LNM) have the worst prognosis, even after complete resection; however, the underlying mechanism remains unclear.Here, we established CAF-derived PDGF-BB as a regulator of LMN in CCA.Proteomics analysis revealed upregulation of PDGF-BB in CAFs derived from patients with CCA with LMN (LN + CAFs).Clinically, the expression of CAF-PDGF-BB correlated with poor prognosis and increased LMN in patients with CCA, while CAF-secreted PDGF-BB enhanced lymphatic endothelial cell (LEC)-mediated lymphangiogenesis and promoted the trans-LEC migration ability of tumor cells.Co-injection of LN + CAFs and cancer cells increased tumor growth and LMN in vivo.Mechanistically, CAF-derived PDGF-BB activated its receptor PDGFR-β and its downstream ERK1/2-JNK signaling pathways in LECs to promote lymphoangiogenesis, while it also upregulated the PDGFR-β-GSK-P65mediated tumor cell migration.Finally, targeting PDGF-BB/PDGFR-β or the GSK-P65 signaling axis prohibited CAF-mediated popliteal lymphatic metastasis (PLM) in vivo.Overall, our findings revealed that CAFs promote tumor growth and LMN via a paracrine network, identifying a promising therapeutic target for patients with advanced CCA.

Objective:To investigate surgical strategies and the corresponding benefits for patients with perihilar cholangiocarcinoma(pCCA).Methods:A total of 81 patients with pCCA who underwent radical excision in the Department of Biliary and Pancreatic Surgery of Sun Yat-Sen Memorial Hospital between January 2014 and December 2021 were retrospectively collected.The cohort consisted of 50 male and 31 female patients,with an age of (62.5±11.5)years(range:26 to 83 years).Seventy-five cases were diagnosed with jaundice,60 of whom received preoperative biliary drainage,while 20 patients received portal vein embolization.Their serum bilirubin level within one week before the operation( M(IQR)) was 44.3 (41.9) μmol/L(range:8.0 to 344.2 μmol/L).Preoperative imaging examinations were performed to evaluate the Bismuth-Corlette type of pCCA,showing 3,6,21,27,and 24 cases of Bismuth-Corlette type Ⅰ,Ⅱ,Ⅲa,Ⅲb,and Ⅳ,respectively.The primary outcome was overall survival (OS),and the secondary outcomes were relapse-free survival (RFS),90-day postoperative morbidity and 90-day postoperative mortality.OS and RFS were estimated using the Kaplan-Meier method and compared by the Log-rank test.Significant prognostic factors were determined using univariate and multivariable Cox proportional hazard regression analyses. Results:In the cohort of 81 pCCA patients,67 cases(82.7%) underwent major hepatectomy while 3 cases received major hepatectomy combined with pancreaticoduodenectomy.Thirty-four patients underwent hepatectomy combined with vascular resection and reconstruction(18 cases of portal vein resection and reconstruction alone;9 cases of hepatic artery resection and reconstruction alone;7 cases of combination of portal vein and hepatic artery resection and reconstruction).Margin negative(R0 excision) were achieved in 53.1%(43/81) of these patients.The operation duration was (627±136)minutes(range:565 to 940 minutes),and the intraoperative blood loss was 400(455)ml(range:200 to 2 800 ml).The 90-day postoperative mortality was 3.7%(3/81).Grade 3-4 postoperative morbidity was 23.4% (19/81) according to the Clavien-Dindo classification of surgical complications.Up to the last follow-up at September 2022,the follow-up time was 34.0(24.2)months (range:0.4 to 103.6 months).Three patients who died within 90 days after surgery were excluded from the survival analysis.The median OS was 36.10 months (95% CI:18.23 to 42.97 months) and the 1-,3-and 5-year OS rates were 85.3%,46.8% and 27.3%,respectively.The median OS of 41 patients with negative margins was 47.83 months(95% CI:36.90 to 58.80 months) and that of 37 patients with positive margins was 20.47 months(95% CI:10.52 to 30.58 months).The median RFS of 70 patients with R0 and R1 resection was 24.50 months(95% CI:12.15 to 31.85 months)and the 1-,3-and 5-year RFS rates were 65.2%,45.7% and 29.9%,respectively.The median RFS of 41 patients with R0 resection was 38.57 months(95% CI:21.50 to 55.63 months) and that of 29 patients with R1 resection was 10.83 months(95% CI:2.82 to 19.86 months). Conclusions:The primary therapy for pCCA is radical surgical resection.A precise preoperative evaluation and sufficient preparation can reduce postoperative morbidity.Surgical treatment can achieve a better survival outcome by increasing the radical resection rate.

BACKGROUND:Intrahepatic cholangiocarcinoma (ICC) is a malignant disease characterized by onset occult, rapid progression, high relapse rate, and high mortality. However, data on how the tumor microenvironment (TME) regulates ICC metastasis at the transcriptomic level remains unclear. This study aimed to explore the mechanisms and interactions between hepatocytes and ICC cells.METHODS:We analyzed the interplay between ICC and liver microenvironment through cytokine antibody array analysis. Then we investigated the role of N6-methyladenosine (m6A) modification and the downstream target in vitro, in vivo experiments, and in clinical specimens.RESULTS:Our study demonstrated that cytokine CCL3, which is secreted by hepatocytes, promotes tumor metastasis by regulating m6A modification via vir-like m6A methyltransferase associated (VIRMA) in ICC cells. Moreover, immunohistochemical analyses showed that VIRMA correlated with poor outcomes in ICC patients. Finally, we confirmed both in vitro and in vivo that CCL3 could activate VIRMA and its critical downstream target SIRT1, which fuels tumor metastasis in ICC.CONCLUSIONS:In conclusion, our results enhanced our understanding of the interaction between hepatocytes and ICC cells, and revealed the molecular mechanism of the CCL3/VIRMA/SIRT1 pathway via m6A-mediated regulation in ICC metastasis. These studies highlight potential targets for the diagnosis, treatment, and prognosis of ICC.

BackgroundThis study aims to (1) identify preoperative testing-based characteristics associated with enhanced prognosis and survival for cholangiocarcinoma patients, and (2)create a distinctive nomogram to anticipate each patient's cancer-specific survival (CSS).MethodsRetrospective analysis was performed on 197 CCA patients who underwent radical surgery at Sun Yat-sen Memorial Hospital; they were divided into a 131-person "training cohort" and a 66-person "internal validation cohort." The prognostic nomogram was created following a preliminary Cox proportional hazard regression search for independent factors influencing the patients' CSS. Its applicable domain was examined via an external validation cohort, which included 235 patients from the Sun Yat-sen University Cancer Center.ResultsThe median follow-up period for the 131 patients in the training group was 49.3 months (range, 9.3 to 133.9 months). One-, three-, and five-year CSS rates were 68.7%, 24.5%, and 9.2%, respectively, with the median CSS length being 27.4 months (range: 1.4 to 125.2 months). PLT, CEA, AFP, tumor location, differentiation, lymph node metastasis, chemotherapy, and TNM stage were determined to be independent risk factors for CCA patients by univariate and multivariate Cox proportional hazard regression analysis. We were able to accurately predict postoperative CSS after incorporating all of these characteristics into a nomogram. The AJCC's 8th edition staging method's C-indices were statistically substantially (P < 0.001) lower than the nomogram's C-indices (0.84, 0.77, and 0.74 in the training, internal and external validation cohorts respectively).ConclusionsA realistic and useful model for clinical decision-making and the optimization of therapy is presented as a nomogram that includes serum markers and clinicopathologic features for predicting postoperative survival in cholangiocarcinoma.

This study aimed to assess whether postoperative adjuvant chemoimmunotherapy could lead to better clinical outcomes for high-risk patients with perihilar cholangiocarcinoma (pCCA). In the cohort study, we retrospectively reviewed patients who received surgical resection for pCCA with curative intent from January 2018 to December 2021 at the Sun Yat-sen Memorial Hospital. The patients at high risk for relapse were further analyzed. Among them, 20 patients received adjuvant chemoimmunotherapy, 28 patients received adjuvant chemotherapy, and 33 patients received surgery alone. The oncological outcomes and drug-associated adverse events were evaluated. The 2-year overall survival (OS) rates in patients treated with adjuvant chemoimmunotherapy, adjuvant chemotherapy, and surgery alone were 80.0

Background and Aims: Patients with persistent positive hepatitis B surface antigen (HBsAg), even with a low HBV-DNA load, have a higher risk of hepatocellular carcinoma (HCC) than those without HBV infection. Given that tumor stemness has a critical role in the occurrence and maintenance of neoplasms, this study aimed to explore whether HBsAg affects biological function and stemness of HCC by regulating microRNA, and to explore underlying mechanisms. Methods: We screened out miR-203a, the most significant down-regulated microRNA in the microarray analysis of HBsAg-positive samples and focused on that miRNA in the ensuing study. In vitro and in vivo functional experiments were performed to assess its regulatory function. The effect of miR-203a on stemness and the possible correlation with BMI1 were analyzed in this study. Results: MiR-203a was significantly down-regulated in HBsAg-positive HCC with the sharpest decrease shown in microarray analysis. The negative correlation between miR-203a and HBsAg expression was confirmed by quantitative real-time PCR after stimulation or overexpression/knockdown of HBsAg in cells. We demonstrated the function of miR-203a in inhibiting HCC cell proliferation, migration, clonogenic capacity, and tumor development in vivo. Furthermore, the overexpression of miR-203a remarkably increases the sensitivity of tumor cells to 5-FU treatment and decreases the proportion of HCC cells with stem markers. In concordance with our study, the survival analysis of both The Cancer Genome Atlas database and samples in our center indicated a worse prognosis in patients with low level of miR-203a. We also found that BMI1, a gene maintains the self-renewal capacity of stem cells, showed a significant negative correlation with miR-203a in HCC specimen (p<0.001). Similarly, opposite BMI1 changes after overexpression/knockdown of miR-203a were also confirmed in vitro. Dual luciferase reporting assay suggested that miR-203a may regulate BMI1 expression by direct binding. Conclusions: HBsAg may promote the development of HCC and tumor stemness by inhibiting miR-203a, resulting in poor prognosis. miR-203a may serve as a crucial treatment target in HBsAg-positive HCC. More explicit mechanistic studies and animal experiments need to be conducted as a next step.