Aberrant DNA methylation is a hallmark of nasopharyngeal carcinoma (NPC) pathogenesis. The aberrant DNA methylation patterns in NPC, particularly in its cancer stem cells (CSCs), and their underlying significance require further elucidation. We integratively performed DNA methylome and transcriptome combined with single-nucleus RNA sequencing to investigate DNA methylation and gene expression patterns of NPC and CSCs. Unlike Epstein-Barr virus (EBV)-negative cells, NPC and CSCs harboring EBV displayed global DNA hypermethylation and they were more oncogenic and immunosuppressive. By correlating DNA methylation and gene expression profiles, we disclosed potential relationships between aberrant DNA methylation, tumorigenesis, metastasis, immunotherapy response, and radiotherapy resistance of NPC. After validating with datasets from GEO and TCGA, we identified aberrant DNA methylation-associated biomarkers including 9 NPC-specific diagnostic markers that had significantly higher DNA methylation levels in NPC than in normal tissues and 8 types of cancers, and 12 potential prognostic markers that were highly correlated to cell cycle dysregulation. Notably, 2 of these potential biomarkers highly expressed in CSCs were validated at the single-cell level. Our study not only identified new potential diagnostic and prognostic biomarkers but also provided new insight into aberrant DNA methylation-associated pathogenesis of NPC, which is beneficial for the development of precision diagnosis and treatment schemes.

BACKGROUND:Fusobacterium nucleatum (F. nucleatum) harbors multiple virulence-associated factors, notably Fap2, FomA, and FadA, which collectively mediate its pathogenic potential through diverse mechanisms including host cell adhesion, tissue invasion, and immunomodulatory interactions. OBJECTIVE:This study was designed to construct and characterize recombinant vaccine candidates expressing multi-epitope antigens derived from these three virulence factors, followed by evaluation of their capacity to induce specific immune responses against F. nucleatum. METHODS:Computational prediction and screening of antigenic epitopes from Fap2, FomA, and FadA using the SVMTriP algorithm; Individual ligation of three synthesized multi-epitope-encoding sequences into the pGEX-1λT expression vector (with empty vector as negative control); Successful transformation of recombinant plasmids into Escherichia coli BL21 (DE3) competent cells; Expression analysis of recombinant multi-epitope proteins; Quantitative assessment of serum antigen-specific IgA and IgG levels in immunized C57BL/6 mice. RESULTS:Molecular confirmation of recombinant strains through comprehensive validation including PCR amplification, restriction enzyme digestion, and DNA sequencing. Differential immune responses observed: Fap2-and FadA-based constructs preferentially stimulated IgA production, FomA-derived antigen predominantly elicited IgG responses. CONCLUSION:We have successfully developed and verified recombinant vaccine strains expressing multi-epitope antigens from F. nucleatum virulence factors Fap2, FomA, and FadA. These constructs demonstrated the capacity to provoke distinct antigen-specific antibody responses in mice, highlighting their promising potential as vaccine candidates against F. nucleatum infections.

Disclosure: P. Li: None. J. Jiang: None. Z. Fan: None. L. Feng: None. F. Yang: None. Objective This study aimed to investigate the correlation between gut microbiota composition, virulence factor (VF) gene alterations, and hepatic metabolic inflammation in overweight/obese children, providing novel insights for the prevention and management of obesity-related metabolic disorders in pediatric populations.Methods: A nested case-control study was conducted. Thirty-one overweight/obese children [aged 6.1 (3.9–8.6) years; 17 males] were recruited from the "Southwest China Childhood Obesity Prevention Study" at West China Second University Hospital of Sichuan University (August 2021–April 2022). Twenty-two healthy children [aged 5.3 (5.1–5.4) years; 10 males] served as controls. Body mass index (BMI), fasting blood glucose (FBG), liver enzymes, uric acid, and other metabolic-inflammatory biomarkers were measured. Fecal samples were analyzed via metagenomic sequencing to characterize gut microbiota composition, VF gene abundance, and KEGG signaling pathways. Group comparisons were performed using t-tests or Mann-Whitney U tests; Spearman’s rank correlation was applied for association analysis.Results: The overweight/obese group exhibited significantly higher BMI [20.44 (17.45–22.80) kg/m² vs. 13.45 (12.25–14.28) kg/m²; P = 0.000] and elevated metabolic-inflammatory markers, including alanine aminotransferase (ALT) [17.00 (10.00–21.00) U/L vs. 15.00 (13.00–17.00) U/L; P = 0.011] and γ-glutamyl transferase (γ-GT) [15.00 (10.00–18.00) U/L vs. 11.00 (9.00–12.00) U/L; P < 0.001]. Metagenomic analysis revealed higher abundances of Coprococcus_A [0.76 (0.00–3.11) vs. 0.00 (0.00–0.00); FDR < 0.05] and Parasutterella [0.89 (0.08–1.79) vs. 0.00 (0.00–0.08); FDR < 0.05] in the overweight/obese group. Overweight/obese children demonstrated greater VF gene diversity, with higher Simpson [0.9919 (0.9915–0.9928) vs. 0.9905 (0.9898–0.9912); P < 0.05] and Shannon indices [5.51 (5.42–5.56) vs. 5.37 (5.30–5.43); P < 0.05], and a higher number of annotated VF types [941.00 (886.00–977.00) vs. 890.00 (806.75–919.50); P = 0.028]. FBG, γ-GT, and ALT levels positively correlated with VF abundance (r > 0.3, FDR < 0.05), with γ-GT showing the strongest association with lipopolysaccharide biosynthesis-related VFs (LpxH, LpxB, LpxK).Conclusion: Overweight/obese children display elevated hepatic metabolic-inflammatory markers, which may be linked to gut microbiota dysbiosis and enrichment of VF genes. These findings highlight the potential role of gut microbial virulence factors in modulating liver immune-metabolic pathways, offering a novel perspective for targeting gut-liver axis interactions in childhood obesity management. Presentation: Monday, July 14, 2025

Background Epstein-Barr virus (EBV) is a significant global public health concern because of its association with various malignancies and autoimmune diseases. Over 90% of the global population is chronically infected with EBV, impacting numerous cancer-related cases annually. However, none of the effective prophylactic vaccines against EBV is approved at present.Methods In this study, we developed a novel vaccine candidate based on epitope peptides from the receptor-binding domain of EBV-encoded gp350 glycoprotein to prevent EBV infection. These epitope peptides detected a binding capability with host cells were then fused by flexibility linkers and expressed in Escherichia coli to reduce the unnecessary glycan modifications to simulate their free-glycan status. The fused recombinant protein (L350) was displayed on the surface of ferritin-based nanoparticle. The immunogenicity of the L350-ferritin nanoparticle was evaluated in Balb/c mice, and the neutralizing titers of sera from immunized mice were detected by means of an infection blocking assay in an in vitro cell model.Results All the five epitope peptides could bind to AKATA cells, and their fused recombinant protein (L350) was successfully presented on the surface of self-assembled ferritin nanoparticles. Sera from the L350-ferritin nanoparticle-immunized mice showed high titers of both L350 protein-specific and gp350D123 protein-specific antibodies, and sera from gp350D123 protein-immunized mice could also recognize L350 protein well. Most importantly, the L350-ferritin nanoparticle induced efficient neutralizing antibodies to block EBV-GFP infection in AKATA cells and also constructed a strong antigen-specific B-cell memory in immunized mice. Moreover, histopathological changes of main tissues from all vaccinated mice were not observed.Conclusion These data indicate that the L350-ferritin nanoparticle vaccine candidate has considerable potential application in preventing EBV infection and provides a promising basis for developing prophylactic EBV vaccines.

BACKGROUND:Intrauterine growth restriction (IUGR) with rapid postnatal catch-up growth has been associated with adipose tissue inflammation and metabolic dysfunction. The long-term persistence of these abnormalities and their relationship with different catch-up growth patterns remain unclear. METHODS:To investigate the long-term metabolic consequences of IUGR in relation to different catch-up growth patterns. An experimental animal study using a rat model of IUGR induced by maternal protein restriction during gestation. Abdominal adipose tissue transcriptome profiles in male rats were analyzed at 3 and 9 months of age, considering variations in catch-up growth patterns. The primary outcomes included markers of adipose tissue inflammation and metabolic function. RESULTS:Among IUGR offspring, approximately 50% demonstrated slow catch-up growth and remained undernourished at 3 months of age. Transcriptome analysis revealed persistent adipose tissue inflammation and metabolic alterations that progressed with age. These abnormalities were present in both rapid and slow catch-up growth groups, although offspring with rapid catch-up growth exhibited more adverse manifestations. CONCLUSION:IUGR was associated with long-term adipose tissue inflammation and metabolic dysfunction, independent of catch-up growth pattern. These findings suggest that IUGR may have lasting metabolic consequences regardless of postnatal growth trajectory.

Background: Epstein-Barr virus (EBV) infects approximately 95% of the global population, causing numerous malignancy-related cases annually and some autoimmune diseases. EBV-encoded gp350, gH, gL, gp42 and gB glycoproteins are identified as antigen candidates for their key role in viral entry, and nanoparticle vaccines displaying them were developed for the advantage of inducing cross-reactive B cell responses. Methods: To develop liposomes displaying nanoparticle vaccine, we synthesized liposomes to present the well-identified EBV-encoded gp350D123 glycoprotein on their surface to imitate the viral structure, through the conjugation between N-hydroxysuccinimide (NHS) groups on the liposomes and primary amine of antigens to form stable amide bond. Then we assessed the immunogenicity of the biomimetic Lipo-gp350D123 nanoparticle vaccine in Balb/c mice immunized experiments. Results: The results showed that the sera samples from Lipo-gp350D123 nanoparticle vaccine immunized mice collected at weeks 8, 10 and 12 had higher titers of gp350D123 protein-specific antibodies, compared to monomer gp350D123 protein control, and higher titers of neutralizing antibodies to block EBV-GFP infection in AKATA cells. Meanwhile, the Lipo-gp350D123 nanoparticle vaccine also induced higher percentage of CD8+ IFN-γ+ T cells in the spleen, but without significance in CD4+ IFN-γ+ T cells, and these isolated splenocytes showed a higher level of secreted IFN-γ. Moreover, no significant histopathological changes were observed in all vaccinated mice. Conclusions: Altogether these data demonstrated that the liposome displaying promoted the immunogenicity of antigens, and the Lipo-gp350D123 nanoparticle vaccine candidate had potential application in blocking EBV infection. The liposome nanoparticle was a useful vector for antigen displaying to elicit effective immunity.

We aimed to investigate the efficacy and safety of transarterial chemoembolization (TACE) or hepatic arterial infusion chemotherapy (HAIC) combined with immune checkpoint inhibitors (ICIs) and angiogenesis inhibitors in unresectable hepatocellular carcinoma (uHCC). The endpoints were the objective response rate (ORR), disease control rate (DCR), conversion rate, progression-free survival (PFS), overall survival (OS), and the incidence of adverse events (AEs). Stratified analyses were accomplished based on local treatment and evaluation criteria. Totally, 4930 individuals from 76 studies were recruited. For initial uHCC treated with the triple therapy, the pooled pathologic complete response (pCR) rate, major pathologic response (MPR) rate, and conversion resection rate were 29.91%, 44.81%, and 30.98%; the ORR and DCR were 38.52% and 84.42% according to RECIST 1.1, 57.82% and 85.82% by mRECIST 1.1. Furthermore, PFS rates at 6-months, 12-months, 18-months, 24-months, and 30-months were 74.77%, 44.30%, 30.97%, 22.71%, and 15.35%; while OS rates at 6-months, 12-months, 18-months, 24-months, 30-months, and 36-months were 94.94%, 76.95%, 58.17%, 45.19%, 27.38%, and 17.79%, respectively. The pooled results showed that the pooled PFS of triple therapy was superior to that of the control group (HR=0.74, 95% CI: 0.71-0.77), so was OS (HR=0.68, 95% CI: 0.65-0.72). The pooled rate of any grade AEs was 91.93%, and grade 3 or higher AEs was 34.50%. There were no fatal AEs reported in any of the included studies. The triple therapy of TACE/HAIC combined with ICIs and angiogenesis inhibitors was promising in uHCC with good efficacy and tolerated toxicity; however, the potential influence of confounding factors cannot be entirely excluded.

In order to understand the laboratory biosafety status of Jiaxing from 2021 to 2023, before and after the end of the COVID-19 strict control strategy, this study used Zhejiang local standard DB33/T 2540 to conduct biosafety quality control inspection and risk identification, and used Chinese industry standard RB/T 040 for risk analysis and assessment. The results showed that the major problems in biosafety management were from organization management, laboratory housekeeping, material and label management, and facilities and equipment, accounting for 39.76%, 28.97% and 14.69% respectively. Statistical analysis showed that the significant improvement of laboratory filing requirements (χ 2 = 5.84, P = 0.016) was the main reason for the decrease of organization management problems (χ 2 = 5.007, P = 0.025). The problems of laboratory housekeeping, experimental material and safety label management had become increasingly prominent (χ 2 = 6.192, P = 0.013), especially the nonstandard use of biosafety label (χ 2 = 5.218, P = 0.022). Assessment results showed that all found problems as identified risk factors in the past 3 years were determined at the level of medium or low. These suggested that in 2021-2023, the overall laboratory biosafety risk level in Jiaxing was controllable and acceptable, and the organization management had been improved greatly. At the same time, the management of laboratory housekeeping, materials and labels, especially the use of biosafety labels, had become increasingly prominent and need to be standardized and strengthened.

INTRODUCTION AND OBJECTIVES:Transient elastography (TE) has been widely used in clinical practice, but noninvasive prediction models based on TE for the assessment of liver fibrosis in primary biliary cholangitis (PBC) has not been reported before. The aim of this study is to develop the simple, accurate and noninvasive prediction models for the histologic staging of PBC based on TE. PATIENTS AND METHODS:Serologic testing, liver stiffness measurement (LSM), and histological assessment of 144 patients with PBC were collected retrospectively. In model group consisted of 96 patients, LSM and alkaline phosphatase (ALP) were identified as two independent predictors of PBC histological stage, which were used to establish two noninvasive models, Model A and Model B, to predict significant fibrosis (Ludwig stage ≥ 2) and advanced fibrosis (Ludwig stage ≥ 3), respectively. The performance of noninvasive models was then validated in the validation group (48 patients). RESULTS:The area under the ROC curve (AUROC) of Model A was 0.870 (95 % CI, 0.802-0.939) in the model group and 0.914 (95 % CI, 0.803-1.000) in the validation group. The AUROC of Model B was 0.948 (95 % CI, 0.888-0.998) in the model group and 0.906 (95 % CI, 0.824-0.987) in validation group. The above results were higher than the other five serologic markers. CONCLUSIONS:The Model A and Model B were the efficient noninvasive models with high accuracy in predicting the histological stages of PBC.

Orthobunyaviruses, including La Crosse virus (LACV), Oropouche virus (OROV), Schmallenberg virus (SBV), and Akabane virus (AKAV), pose substantial threats to global public health and livestock industries. This review focuses on the interplay between these viruses and the host immune systems, highlighting key mechanisms of viral entry and immune evasion. The viruses exploit vulnerabilities in host innate immunity, particularly through nonstructural protein NSs, which disrupts type I interferon signaling and transcriptional machinery. Additionally, this review delineates how host restriction factors counteract viral proliferation through compartmentalized defense mechanisms including BST-2 and MxA. The review also discusses antiviral strategies, including vaccines and inhibitors. Hence, this review synthesizes current knowledge on host recognition, immune evasion, and therapies for Orthobunyaviruses infections (focusing on LACV, OROV, AKAV, SBV) to guide targeted antiviral and broad-spectrum countermeasure development against emerging Orthobunyaviruses threats.

The COVID-19 pandemic has made assessing vaccine efficacy more challenging. Besides neutralizing antibody assays, systems vaccinology studies use omics technology to reveal immune response mechanisms and identify gene signatures in human peripheral blood mononuclear cells (PBMCs). However, due to their low proportion in PBMCs, profiling the immune response signatures of dendritic cells (DCs) is difficult. Here, we develop a predictive model for evaluating early immune responses in dendritic cells. We establish a THP-1-derived dendritic cell (TDDC) model and stimulate their maturation in vitro with an optimal dose of attenuated yellow fever 17D (YF-17D). Transcriptomic analysis reveals that type I interferon (IFN-I)-induced immunity plays a key role in dendritic cells. IFN-I regulatory biomarkers (IRF7, SIGLEC1) and IFN-I-inducible biomarkers (IFI27, IFI44, IFIT1, IFIT3, ISG15, MX1, OAS2, OAS3) are identified and validated in vitro and in vivo. Furthermore, we apply this TDDC approach to various types of vaccines, providing novel insights into their early immune response signatures and their heterogeneity in vaccine recipients. Our findings suggest that a standardizable TDDC model is a promising predictive approach to assessing early immunity in DCs. Further research into vaccine efficacy assessment approaches on various types of immune cells could lead to a systemic regimen for vaccine development in the future.

Engineered bacteria are widely used in cancer treatment because live facultative/obligate anaerobes can selectively proliferate at tumor sites and reach hypoxic regions, thereby causing nutritional competition, enhancing immune responses, and producing anticancer microbial agents in situ to suppress tumor growth. Despite the unique advantages of bacteria-based cancer biotherapy, the insufficient treatment efficiency limits its application in the complete ablation of malignant tumors. The combination of nanomedicine and engineered bacteria has attracted increasing attention owing to their striking synergistic effects in cancer treatment. Engineered bacteria that function as natural vehicles can effectively deliver nanomedicines to tumor sites. Moreover, bacteria provide an opportunity to enhance nanomedicines by modulating the TME and producing substrates to support nanomedicine-mediated anticancer reactions. Nanomedicine exhibits excellent optical, magnetic, acoustic, and catalytic properties, and plays an important role in promoting bacteria-mediated biotherapies. The synergistic anticancer effects of engineered bacteria and nanomedicines in cancer therapy are comprehensively summarized in this review. Attention is paid not only to the fabrication of nanobiohybrid composites, but also to the interpromotion mechanism between engineered bacteria and nanomedicine in cancer therapy. Additionally, recent advances in engineered bacteria-synergized multimodal cancer therapies are highlighted.

Although liver transplantation is the gold-standard therapy for end-stage liver disease, the shortage of suitable organs results in only 25% of waitlisted patients undergoing transplants. Three-dimensional (3D) bioprinting is an emerging technology and a potential solution for personalized medicine applications. This review highlights existing 3D bioprinting technologies of liver tissues, current anatomical and physiological limitations to 3D bioprinting of a whole liver, and recent progress bringing this innovation closer to clinical use. We reviewed updated literature across multiple facets in 3D bioprinting, comparing laser, inkjet, and extrusion-based printing modalities, scaffolded versus scaffold-free systems, development of an oxygenated bioreactor, and challenges in establishing long-term viability of hepatic parenchyma and incorporating structurally and functionally robust vasculature and biliary systems. Advancements in liver organoid models have also increased their complexity and utility for liver disease modeling, pharmacologic testing, and regenerative medicine. Recent developments in 3D bioprinting techniques have improved the speed, anatomical, and physiological accuracy, and viability of 3D-bioprinted liver tissues. Optimization focusing on 3D bioprinting of the vascular system and bile duct has improved both the structural and functional accuracy of these models, which will be critical in the successful expansion of 3D-bioprinted liver tissues toward transplantable organs. With further dedicated research, patients with end-stage liver disease may soon be recipients of customized 3D-bioprinted livers, reducing or eliminating the need for immunosuppressive regimens.

Studies with genetically modified mice have implicated the transcriptional regulator STAT3 as a key modulator of bone development. STAT3-OKO knockout mouse lines were generated in two genetic backgrounds, pure C57BL/6 (STAT3-OKO-BL) and mixed C57BL/6, CD1 (STAT3-OKO-M). Both lines exhibited defective postnatal bone development resulting in reduced body weight and shortened femurs that displayed low bone mineral density as well as cortical widening and thinning in the diaphyseal region. Remarkably, each of these defects displayed sexual dimorphism that was dependent on genetic background: the phenotype was entirely male-specific in STAT3-OKO-M but not in STAT3-OKO-BL, in which defects were similar in both sexes. However, both lines exhibited a male-specific bone defect in mineralization, and also in bone mechanical properties related to bone quality, such as yield stress and ultimate stress. On the other hand, bone mechanical properties such as ultimate force, that may reflect density and macrostructure rather than bone quality, showed male-specific defects only in STAT3-OKO-M. These findings suggest that STAT3 may regulate multiple sex-dependent mechanisms in bone development that control either mineralization or bone accrual, and that the sex-dependence of at least some of these mechanisms is affected by genetic background. Finally, we used CRISPR/Cas9 to generate STAT3-deficient preosteoblastic cells from immortalized wild-type bone marrow stem cells and showed that the defective osteoblastic differentiation of STAT3-ablated cells was associated with reduced gene expression of Wnt3a and Wnt5a, consistent with other studies that identify Wnt signaling pathways as potential effector mechanisms for STAT3-mediated regulation of bone development.

Natural killer (NK) cells play an important role in immune rejection in solid organ transplantation. To mitigate human NK cell activation in xenotransplantation, introducing inhibitory ligands on xenografts via genetic engineering of pigs may protect the graft from human NK cell-mediated cytotoxicity and ultimately improve xenograft survival. In this study, non-classical HLA class I molecules HLA-E and HLA-G were introduced in an immortalized porcine liver endothelial cell line with disruption of five genes (GGTA1, CMAH, β4galNT2, SLA-I α chain, and β-2 microglobulin) encoding three major carbohydrate xenoantigens (αGal, Neu5Gc, and Sda) and swine leukocyte antigen class I (SLA-I) molecules. Expression of HLA-E and/or HLA-G on pig cells were confirmed by flow cytometry. Endogenous HLA-G molecules as well as exogenous HLA-G VL9 peptide could dramatically enhance HLA-E expression on transfected pig cells. We found that co-expression of HLA-E and HLA-G on porcine cells led to a significant reduction in human NK cell activation compared to the cells expressing HLA-E or HLA-G alone and the parental cell line. NK cell activation was assessed by analysis of CD107a expression in CD3-CD56+ population gated from human peripheral blood mononuclear cells. CD107a is a sensitive marker of NK cell activation and correlates with NK cell degranulation and cytotoxicity. HLA-E and/or HLA-G on pig cells did not show reactivity to human sera IgG and IgM antibodies. This in vitro study demonstrated that co-expression of HLA-E and HLA-G on genetically modified porcine endothelial cells provided a superior inhibition in human xenoreactive NK cells, which may guide further genetic engineering of pigs to prevent human NK cell mediated rejection.

Background and Aims: Collagen beta(1-O) galactosyltransferase 25 domain 1 (GLT25D1) is associated with collagen production and glycosylation, and its knockout in mice results in embryonic death. However, its role in liver fibrosis remains elusive, particularly in hepatic stellate cells (HSCs), the primary collagen-producing cells associated with liver fibrogenesis. Herein, we aimed to elucidate the role of GLT25D1 in HSCs. Methods: Bile duct ligation (BDL)-induced mouse liver fibrosis models, primary mouse HSCs (mHSCs), and transforming growth factor beta 1 (TGF-beta 1)-stimulated LX-2 human hepatic stellate cells were used in in vivo and in vitro studies. Stable LX-2 cell lines with either GLT25D1 overexpression or knockdown were established using lentiviral transfection. RNA-seq was performed to investigate the genomic differences. HPLCMS/MS were used to identify glycosylation sites. Scanning electronic microscopy (SEM) and second-harmonic generation/two-photon excited fluorescence (SHG/TPEF) were used to image collagen fibril morphology. Results: GLT25D1 expression was upregulated in nonparenchymal cells in human cirrhotic liver tissues. Meanwhile, its knockdown attenuated collagen deposition in BDL-induced mouse liver fibrosis and inhibited mHSC activation. GLT25D1 was overexpressed in activated versus quiescence LX-2 cells and regulated in vitro LX-2 cell activation, including proliferation, contraction, and migration. GLT25D1 also significantly increased liver fibrogenic gene and protein expression. GLT25D1 upregulation promoted HSC activation and enhanced collagen expression through the TGF-beta 1/SMAD signaling pathway. Mass spectrometry showed that GLT25D1 regulated the glycosylation of collagen in HSCs, affecting the diameter of collagen fibers. Conclusions: Collectively, the upregulation of GLT25D1 in HSCs promoted the progression of liver fibrosis by affecting HSCs activation and collagen stability.

ABSTRACTOrganoids are novelin vitromodels to study intercellular crosstalk between the different types of cells in the pathophysiology of disease. To better understand the underlying mechanisms driving the progression of primary sclerosing cholangitis (PSC), we developed scaffold-free multi-cellular 3D cholangiocyte organoids (3D-CHO) using ‘primary’ liver cell lines derived from normal and PSC patients. Human liver samples from healthy donors and late-stage PSC patients were used to isolate ‘primary’ cholangiocytes (EPCAM+/CK-19+), liver endothelial cells (LECs, CD31+), and hepatic stellate cells (HSCs, CD31−/CD68−/Desmin+/Vitamin A+). 3D-CHOs were formed using cholangiocytes:HSCs:LECs and kept viable for up to 1 month. Isolated primary cell lines and 3D-CHOs were further characterized by immunofluorescence (IF), qRT-PCR, and transmission electron microscopy. Gene expressions for cholangiocytes (SOX9, CFTR, EpCAM, AE, SCT, SCTR), fibrosis (ACTA2, COL1A1, DESMIN, TGFβ1), angiogenesis (PECAM, VEGF, CDH5, vWF), and inflammation (IL-6, TNF-α) confirmed PSC phenotypes of 3D-CHOs. Since cholangiocytes develop a neuroendocrine phenotype and express neuromodulators, confocal-IF demonstrated that neurokinin-1 receptor (NK-1R, expressed by cholangiocytes and upregulated in PSC), was localized within CK-19+cholangiocytes. Moreover, 3D-CHOs from PSC patients confirmed PSC phenotypes with upregulated NK-1R, tachykinin precursor 1, and downregulated membrane metalloendopeptidase. Our viable scaffold-free multiple-cell 3D-CHOs showed superiority as anin vitromodel in mimicking PSCin vivophenotypes compared to 2D cell culture, which can be used in PSC disease-related research.