Diagnostic Accuracy of Transient Elastography in Hepatosteatosis in Youth With Obesity.

Context: Steatotic liver disease is common but overlooked in childhood obesity; diagnostic methods are invasive or expensive. Objective: We sought to determine the diagnostic accuracy of vibration-controlled transient elastography (VCTE) compared with magnetic resonance imaging (MRI) in adolescents with obesity and high risk for hepatosteatosis. Methods: Baseline data in 3 clinical trials enrolling adolescents with obesity were included (NCT03919929, NCT03717935, NCT04342390). Liver fat was assessed using MRI fat fraction and VCTE-based controlled attenuation parameter (CAP). Hepatosteatosis was defined as MRI fat fraction ≥5.0%. The area under the receiver-operating characteristic curves (AUROCs) for CAP against MRI was calculated, and optimal CAP using the Youden index for hepatosteatosis diagnosis was determined. Results: Data from 82 adolescents (age 15.6 ± 1.4 years, body mass index 36.5 ± 5.9 kg/m2, 81% female) were included. Fifty youth had hepatosteatosis by MRI (fat fraction 9.3% ; 95% CI 6.7, 14.0), and 32 participants did not have hepatosteatosis (fat fraction 3.1%; 95% CI 2.2, 3.9; P < .001). The hepatosteatosis group had higher mean CAP compared with no hepatosteatosis (293 dB/m; 95% CI 267, 325 vs 267 dB/m; 95% CI 248, 282; P = .0120). A CAP of 281 dB/m had the highest sensitivity (60%) and specificity (74%) with AUROC of 0.649 (95% CI 0.51-0.79; P = .04) in the entire cohort. In a subset of participants with polycystic ovary syndrome (PCOS), a CAP of 306 dB/m had the highest sensitivity (78%) and specificity (52%) and AUROC of 0.678 (95% CI 0.45-0.90; P = .108). Conclusion: CAP of 281 dB/m has modest diagnostic performance for hepatosteatosis compared with MRI in youth with significant obesity. A higher CAP in youth with PCOS suggests that comorbidities might affect optimal CAP in hepatosteatosis diagnosis.

Accuracy of blood-based biomarkers for staging liver fibrosis in chronic liver disease: A systematic review supporting the AASLD Practice Guideline.

BACKGROUND AND AIMS: Blood-based biomarkers have been proposed as an alternative to liver biopsy for non-invasive liver disease assessment (NILDA) in chronic liver disease (CLD). Our aims for this systematic review were to evaluate the diagnostic utility of selected blood-based tests either alone, or in combination, for identifying significant fibrosis (F2-4), advanced fibrosis (F3-4) and cirrhosis (F4), as compared to biopsy in CLD. APPROACH AND RESULTS: We included a comprehensive search of databases including Ovid MEDLINE(R), EMBASE, Cochrane Database, and Scopus through to April 2022. Two independent reviewers selected 286 studies with 103,162 patients. The most frequently identified studies included the simple aminotransferase-to-platelet ratio index (APRI) and fibrosis (FIB)-4 markers (with low-to-moderate risk of bias) in hepatitis B virus (HBV) and C virus (HCV), HIV-HCV/HBV co-infection, and nonalcoholic fatty liver disease (NAFLD). Positive (LR+) and negative (LR) likelihood ratios across direct and indirect biomarker tests for HCV and HBV for F2-4, F3-4, or F4 were 1.66-6.25 and 0.23-0.80, 1.89-5.24 and 0.12-0.64, and 1.32-7.15 and 0.15-0.86 respectively; LR+ and LR for NAFLD F2-4, F3-4 and F4 were 2-65-3.37 and 0.37-0.39, 2.25-6.76 and 0.07-0.87, and 3.90 and 0.15 respectively. Overall, proportional odds ratio indicated FIB-4 <1.45 was better than APRI <0.5 for F2-4. FIB-4 >3.25 was also better than APRI >1.5 for F3-4 and F4. There was limited data for combined tests. CONCLUSIONS: Blood-based biomarkers are associated with small-to-moderate change in pre-test probability for diagnosing F2-4, F3-4, and F4 in viral hepatitis, HIV-HCV co-infection, and NAFLD, with limited comparative or combination studies for other CLD.

Coffee, adenosine, and the liver.

A variety of observational studies have demonstrated that coffee, likely acting through caffeine, improves health outcomes in patients with chronic liver disease. The primary pharmacologic role of caffeine is to act as an inhibitor of adenosine receptors. Because key liver cells express adenosine receptors linked to liver injury, regeneration, and fibrosis, it is plausible that the biological effects of coffee are explained by effects of caffeine on adenosinergic signaling in the liver. This review is designed to help the reader make sense of that hypothesis, highlighting key observations in the literature that support or dispute it.

Effects of short-term supervised exercise training on liver fat in adolescents with obesity: a randomized controlled trial.

OBJECTIVE: The objective of this study was to quantify the effects of a 4-week, supervised, high-intensity interval training (HIIT) on intrahepatic triglyceride content (IHTG, percentage), cardiorespiratory fitness (CRF), and cardiometabolic markers in adolescents with obesity. METHODS: A total of 40 adolescents (age 13-18 y, BMI 36.7 ± 5.8 kg/m2 ) at risk for metabolic dysfunction-associated steatotic liver disease (MASLD) based on obesity and elevated Fibroscan measured controlled attenuation parameter (CAP) scores were randomized to HIIT three times a week for 4 weeks (n = 34) or observation (control; n = 6). Liver magnetic resonance imaging proton-density fat-fraction (MRI-PDFF), CAP, oral glucose tolerance test, serum alanine aminotransferase, dual-energy x-ray absorptiometry, and CRF tests were performed before and after intervention. Within- and between-group differences were compared. RESULTS: A total of 13 (38%) and 4 (66%) children had MASLD by MRI-PDFF (IHTG ≥ 5%) in the HIIT and control groups, respectively. The implemented HIIT protocol had no impact on CRF or IHTG (baseline 5.26%, Δ = -0.31 percentage points, 95% CI: -0.77 to 0.15; p = 0.179), but it decreased the 2-h glucose concentration (baseline 116 mg/dL, Δ = -11 mg/dL; 95% CI: -17.6 to -5.5; p < 0.001). When limiting the analysis to participants with MASLD (n = 17), HIIT decreased IHTG (baseline 8.81%, Δ = -1.05 percentage points, 95% CI: -2.08 to -0.01; p = 0.048). Between-group comparisons were not different. CONCLUSIONS: The implemented exercise protocol did not reduce IHTG, but it led to modest improvement in markers of cardiometabolic health.

Coffee as chemoprotectant in fatty liver disease: caffeine-dependent and caffeine-independent effects.

Coffee consumption is associated with a variety of positive health outcomes in patients with chronic liver disease, including decreased liver-related mortality. The evidence for this has come from a wide variety of epidemiological studies over the past decade and remains consistent. Because coffee contains a large number of constituent molecules, many of which vary based on coffee source, roasting approach, and preparation, it has been difficult to identify the mechanisms by which coffee improves liver-related health. The caffeine hypothesis suggests that the primary active ingredient in coffee in this context is caffeine, which is an antagonist of liver adenosine receptors. However, some lines of data suggest caffeine-independent effects as well. This review examines the biological plausibility for caffeine-independent effects in the context of a recent publication in this journal.

Obesity, but not glycemic control, predicts liver steatosis in children with type 1 diabetes.

OBJECTIVE: Nonalcoholic fatty liver disease (NAFLD), the most common liver disease in children, is strongly associated with obesity and insulin resistance. Although type 1 diabetes (T1D) is characterized by insulin deficiency, increasing obesity rates among children with T1D is a major risk factor for NAFLD in this patient population. Predisposing factors for NAFLD in children with T1D are not known. STUDY DESIGN: This is a cross-sectional study comparing children with T1D across the range of body mass index (BMI) to the BMI-matched obese group without T1D. Hepatic steatosis was semi-quantitatively measured via the vibration-controlled transient elastogram (VCTE) method. Linear regression analysis was performed to assess the relationship between controlled-attenuated parameter (CAP) scores and clinical parameters. Receiver-operator curve (ROC) analysis was used to evaluate the diagnostic performance of several clinical parameters against NAFLD status determined via CAP. RESULTS: Two-thirds of subjects with obesity had CAP scores suggestive of NAFLD, while 16 % (n = 6) of T1D patients without obesity had elevated CAP. Obese subjects were different from non-obese subjects in many laboratory and clinical characteristics, regardless of T1D status. CAP score was significantly associated with BMI, HDL-Cholesterol (HDL-c), and HbA1c in all subjects as well as the T1D-only subgroup. Among subjects with obesity only, age, HDL-cand ALT were the most significant predictors. Diagnostic performance of BMI, HDL-c, and BMI/HDL ratio were in the good to the excellent range for predicting NAFLD among all subjects, while performance varied for T1D-only or obesity-only groups. CONCLUSION: The clinical and imaging findings of children with T1D and obesity are comparable to non-diabetic children with a similar degree of obesity. Obesity is the major risk factor for NAFLD in pediatric T1D. BMI, HDL-c, and BMI/HDL ratio may be helpful markers to determine further workup for NAFLD in children with T1D, particularly those with obesity.

COVID-19 and the liver: a narrative review of the present state of knowledge.

Novel corona virus disease (COVID-19) is an ongoing pandemic that has spread across the globe. The virus primarily infects type-2 pneumocytes in alveoli and causes lung disease, with severity ranging from mild pneumonia to acute respiratory distress syndrome. The virus also invades gastrointestinal epithelial cells, hepatocytes, and biliary epithelial cells. Derangement of liver function tests is noted in about one third of patients and appears to correlate with more severe disease. There are multiple mechanisms by which the virus can cause liver injury; immune-mediated inflammation and direct viral cytotoxicity are believed to be the predominant mechanisms. Liver injury appears to be transient, usually recovering with resolution of illness. Limited available studies and experience from prior corona virus pandemics seem to suggest that immunosuppressed patients have similar outcomes compared to non-immunosuppressed patients. Age and comorbid conditions seem to influence outcome, irrespective of immune status. Additionally, patients with preexisting comorbid conditions are more prone to acquire infection and should strictly adhere to travel and social distancing advisories. Telemedicine should be utilized to provide uninterrupted care for patients with liver disease, and clinic or hospital visits should be advised only in sick patients with advanced liver disease. In conclusion, liver dysfunction is not uncommon in COVID-19, it generally improves with resolution of disease, and patients with chronic liver disease (CLD) need continued follow up, uninterrupted by the ongoing pandemic, preferably in virtual clinic settings.

Proteomics Indicates Lactate Dehydrogenase Is Prognostic in Acetaminophen-Induced Acute Liver Failure Patients and Reveals Altered Signaling Pathways.

Better biomarkers to predict death early in acute liver failure (ALF) are needed. To that end, we obtained early (study day 1) and later (day 3) serum samples from transplant-free survivors (n = 28) and nonsurvivors (n = 30) of acetaminophen-induced ALF from the NIH-sponsored Acute Liver Failure Study Group and from control volunteers (n = 10). To identify proteins that increase early in serum during ALF, we selected individuals from this cohort for whom alanine aminotransferase was lower on day 1 than day 3, indicating a time point before peak injury (n = 10/group). We then performed untargeted proteomics on their day 1 samples. Out of 1682 quantifiable proteins, 361 were ≥ 4-fold elevated or decreased in ALF patients versus controls and 16 of those were further elevated or decreased ≥ 4-fold in nonsurvivors versus survivors, indicating potential to predict death. Interestingly, 1 of the biomarkers was lactate dehydrogenase (LDH), which is already measured in most clinical laboratories. To validate our proteomics results and to confirm the prognostic potential of LDH, we measured LDH activity in all day 1 and 3 samples from all 58 ALF patients. LDH was elevated in the nonsurvivors versus survivors on both days. In addition, it had prognostic value similar to the model for end-stage liver disease and outperformed the King's College Criteria, while a combination of model for end-stage liver disease and LDH together outperformed either alone. Finally, bioinformatics analysis of our proteomics data revealed alteration of numerous signaling pathways that may be important in liver regeneration. Overall, we conclude LDH can predict death in APAP-induced ALF.

Short-Term Safety of Repeated Acetaminophen Use in Patients With Compensated Cirrhosis.

Current guidelines recommend restricting acetaminophen (APAP) use in patients with cirrhosis, but evidence to support that recommendation is lacking. Prior studies focused on pharmacokinetics (PK) of APAP in cirrhosis but did not rigorously examine clinical outcomes, sensitive biomarkers of liver damage, or serum APAP-protein adducts, which are a specific marker of toxic bioactivation. Hence, the goal of this pilot study was to test the effects of regularly scheduled APAP dosing in a well-defined compensated cirrhosis group compared to control subjects without cirrhosis, using the abovementioned outcomes. After a 2-week washout, 12 subjects with and 12 subjects without cirrhosis received 650 mg APAP twice per day (1.3 g/day) for 4 days, followed by 650 mg on the morning of day 5. Patients were assessed in-person at study initiation (day 1) and on days 3 and 5. APAP-protein adducts and both conventional (alanine aminotransferase) and sensitive (glutamate dehydrogenase [GLDH], full-length keratin 18 [K18], and total high-mobility group box 1 protein) biomarkers of liver injury were measured in serum on the mornings of days 1, 3, and 5, with detailed PK analysis of APAP, metabolites, and APAP-protein adducts throughout day 5. No subject experienced adverse clinical outcomes. GLDH and K18 were significantly different at baseline but did not change in either group during APAP administration. In contrast, clearance of APAP-protein adducts was dramatically delayed in the cirrhosis group. Minor differences for other APAP metabolites were also detected. Conclusion: Short-term administration of low-dose APAP (650 mg twice per day, <1 week) is likely safe in patients with compensated cirrhosis. These data provide a foundation for future studies to test higher doses, longer treatment, and subjects who are decompensated, especially in light of the remarkably delayed adduct clearance in subjects with cirrhosis.

The Role of Sirtuin 3 in Radiation-Induced Long-Term Persistent Liver Injury.

In patients with abdominal region cancers, ionizing radiation (IR)-induced long-term liver injury is a major limiting factor in the use of radiotherapy. Previously, the major mitochondrial deacetylase, sirtuin 3 (SIRT3), has been implicated to play an important role in the development of acute liver injury after total body irradiation but no studies to date have examined the role of SIRT3 in liver's chronic response to radiation. In the current study, ten-month-old Sirt3-/- and Sirt3+/+ male mice received 24 Gy radiation targeted to liver. Six months after exposure, irradiated Sirt3-/- mice livers demonstrated histopathological elevations in inflammatory infiltration, the loss of mature bile ducts and higher DNA damage (TUNEL) as well as protein oxidation (3-nitrotyrosine). In addition, increased expression of inflammatory chemokines (IL-6, IL-1ß, TGF-ß) and fibrotic factors (Procollagen 1, α-SMA) were also measured in Sirt3-/- mice following 24 Gy IR. The alterations measured in enzymatic activities of catalase, glutathione peroxidase, and glutathione reductase in the livers of irradiated Sirt3-/- mice also implied that hydrogen peroxide and hydroperoxide sensitive signaling cascades in the absence of SIRT3 might contribute to the IR-induced long-term liver injury.

Reduction in SNAP-23 Alters Microfilament Organization in Myofibrobastic Hepatic Stellate Cells.

Hepatic stellate cells (HSC) are critical effector cells of liver fibrosis. In the injured liver, HSC differentiate into a myofibrobastic phenotype. A critical feature distinguishing myofibroblastic from quiescent HSC is cytoskeletal reorganization. Soluble NSF attachment receptor (SNARE) proteins are important in trafficking of newly synthesized proteins to the plasma membrane for release into the extracellular environment. The goals of this project were to determine the expression of specific SNARE proteins in myofibroblastic HSC and to test whether their alteration changed the HSC phenotype in vitro and progression of liver fibrosis in vivo. We found that HSC lack the t-SNARE protein, SNAP-25, but express a homologous protein, SNAP-23. Downregulation of SNAP-23 in HSC induced reduction in polymerization and disorganization of the actin cytoskeleton associated with loss of cell movement. In contrast, reduction in SNAP-23 in mice by monogenic deletion delayed but did not prevent progression of liver fibrosis to cirrhosis. Taken together, these findings suggest that SNAP-23 is an important regular of actin dynamics in myofibroblastic HSC, but that the role of SNAP-23 in the progression of liver fibrosis in vivo is unclear.

Junctional adhesion molecules JAM-B and JAM-C promote autoimmune-mediated liver fibrosis in mice.

Fibrosis remains a serious health concern in patients with chronic liver disease. We recently reported that chemically induced chronic murine liver injury triggers increased expression of junctional adhesion molecules (JAMs) JAM-B and JAM-C by endothelial cells and de novo synthesis of JAM-C by hepatic stellate cells (HSCs). Here, we demonstrate that biopsies of patients suffering from primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) or autoimmune hepatitis (AIH) display elevated levels of JAM-C on portal fibroblasts (PFs), HSCs, endothelial cells and cholangiocytes, whereas smooth muscle cells expressed JAM-C constitutively. Therefore, localization and function of JAM-B and JAM-C were investigated in three mouse models of autoimmune-driven liver inflammation. A PBC-like disease was induced by immunization with 2-octynoic acid-BSA conjugate, which resulted in the upregulation of both JAMs in fibrotic portal triads. Analysis of a murine model of PSC revealed a role of JAM-C in PF cell-cell adhesion and contractility. In mice suffering from AIH, endothelial cells increased JAM-B level and HSCs and capsular fibroblasts became JAM-C-positive. Most importantly, AIH-mediated liver fibrosis was reduced in JAM-B-/- mice or when JAM-C was blocked by soluble recombinant JAM-C. Interestingly, loss of JAM-B/JAM-C function had no effect on leukocyte infiltration, suggesting that the well-documented function of JAMs in leukocyte recruitment to inflamed tissue was not effective in the tested chronic models. This might be different in patients and may even be complicated by the fact that human leukocytes express JAM-C. Our findings delineate JAM-C as a mediator of myofibroblast-operated contraction of the liver capsule, intrahepatic vasoconstriction and bile duct stricture. Due to its potential to interact heterophilically with endothelial JAM-B, JAM-C supports also HSC/PF mural cell function. Together, these properties allow JAM-B and JAM-C to actively participate in vascular remodeling associated with liver/biliary fibrosis and suggest them as valuable targets for anti-fibrosis therapies.

Coffee Consumption and Prevention of Cirrhosis: In Support of the Caffeine Hypothesis.

Coffee is acknowledged as the most widely used drug worldwide. Coffee is also a foodstuff, so its use is often used to satisfy dietary urges. When used as a drug, coffee is normally consumed as a stimulant rather than to treat or prevent particular diseases. Recently, coffee consumption has been inversely related to progression of liver fibrosis to cirrhosis and even hepatocellular carcinoma. Experiments in cellular and animal models have provided biological plausibility for coffee as an antifibrotic agent in the liver. A recent article examined one of the key questions regarding the antifibrotic role of coffee-specifically what is the primary antifibrotic agent in coffee? This article briefly reviews the relevant issues with regard to coffee as an antifibrotic agent for patients with chronic liver disease.

Liver myofibroblasts of murine origins express mesothelin: Identification of novel rat mesothelin splice variants.

Liver myofibroblasts are specialized effector cells that drive hepatic fibrosis, a hallmark process of chronic liver diseases, leading to progressive scar formation and organ failure. Liver myofibroblasts are increasingly recognized as heterogeneous with regards to their origin, phenotype, and functions. For instance, liver myofibroblasts express cell markers that are universally represented such as, ItgαV and Pdgfrß, or restricted to a given subpopulation such as, Lrat exclusively expressed in hepatic stellate cells, and Gpm6a in mesothelial cells. To study liver myofibroblasts in vitro, we have previously generated and characterized a SV40-immortalized polyclonal rat activated portal fibroblast cell line called RGF-N2 expressing multiple mesothelin mRNA transcripts. Mesothelin, a cell-surface molecule expressed in normal mesothelial cells and overexpressed in several cancers such as, mesothelioma and cholangiocarcinoma, was recently identified as a key regulator of portal myofibroblast proliferation, and fibrosis progression in the setting of chronic cholestatic liver disease. Here, we identify novel mesothelin splice variants expressed in rat activated portal fibroblasts. RGF-N2 portal fibroblast cDNA was used as template for insertion of hemagglutinin tag consensus sequence into the complete open reading frame of rat mesothelin variant coding sequences by extension PCR. Purified amplicons were subsequently cloned into an expression vector for in vitro translation and transfection in monkey COS7 fibroblasts, before characterization of fusion proteins by immunoblot and immunofluorescence. We show that rat activated portal fibroblasts, hepatic stellate cells, and cholangiocarcinoma cells express wild-type mesothelin and additional splice variants, while mouse activated hepatic stellate cells appear to only express wild-type mesothelin. Notably, rat mesothelin splice variants differ from the wild-type isoform by their protein properties and cellular distribution in transfected COS7 fibroblasts. We conclude that mesothelin is a marker of activated murine liver myofibroblasts. Mesothelin gene expression and regulation may be critical in liver myofibroblasts functions and fibrosis progression.

The Cholangiocyte Adenosine-IL-6 Axis Regulates Survival During Biliary Cirrhosis.

Epithelial response to injury is critical to the pathogenesis of biliary cirrhosis, and IL-6 has been suggested as a mediator of this phenomenon. Several liver cell types can secrete IL-6 following activation by various signaling molecules including circulating adenosine. The aims of this study were to assess whether adenosine can induce IL-6 secretion by cholangiocytes via the A2b adenosine receptor (A2bAR) and to determine the effect of A2bAR-sensitive IL-6 release on injury response in biliary cirrhosis. Human normal cholangiocyte H69 cells were used for in vitro studies to determine the mechanism by which adenosine and the A2bAR induce release of IL-6. In vivo, control and A2bAR-deficient mice were used to determine the roles of A2bAR-sensitive IL-6 release in biliary cirrhosis induced by common bile duct ligation (BDL). Additionally, the response to exogenous IL-6 was assessed in C57BL/6 and A2bAR-deficient mice. Adenosine induced IL-6 mRNA expression and protein secretion via A2bAR activation. Although activation of A2bAR induced cAMP and intracellular Ca2+ signals, only the Ca2+ signals were linked to IL-6 upregulation. After BDL, A2bAR-deficient mice have impaired survival, which is further impaired by exogenous IL-6; however, decreased survival is not due to changes in fibrosis and no changes in inflammatory cells. Exogenous IL-6 is associated with the increased presence of bile infarcts. Extracellular adenosine induces cholangiocyte IL-6 release via the A2bAR. This signaling pathway is important in the pathogenesis of injury response in biliary cirrhosis but does not alter fibrosis. Adenosine upregulates IL-6 release by cholangiocytes via the A2bAR in a calcium-sensitive fashion. Mice deficient in A2bAR experience impaired survival after biliary cirrhosis induced by common bile duct ligation independent of changes in fibrosis.

An Elf2-like transcription factor acts as repressor of the mouse ecto-5'-nucleotidase gene expression in hepatic myofibroblasts.

Hepatic fibrosis represents a pathological wound healing and tissue repair process triggered in response to chronic liver injury. A heterogeneous population of activated non-parenchymal liver cells, known as liver myofibroblasts, functions as the effector cells in hepatic fibrosis. Upon activation, liver myofibroblasts become fibrogenic, acquiring contractile properties and increasing collagen production capacity, while developing enhanced sensitivity to endogenous molecules and factors released in the local microenvironment. Hepatic extracellular adenosine is a bioactive small molecule, increasingly recognized as an important regulator of liver myofibroblast functions, and an important mediator in the pathogenesis of liver fibrosis overall. Remarkably, ecto-5'-nucleotidase/Nt5e/Cd73 enzyme, which accounts for the dominant adenosine-generating activity in the extracellular medium, is expressed by activated liver myofibroblasts. However, the molecular signals regulating Nt5e gene expression in liver myofibroblasts remain poorly understood. Here, we show that activated mouse liver myofibroblasts express Nt5e gene products and characterize the putative Nt5e minimal promoter in the mouse species. We describe the existence of an enhancer sequence upstream of the mouse Nt5e minimal promoter and establish that the mouse Nt5e minimal promoter transcriptional activity is negatively regulated by an Elf2-like Ets-related transcription factor in activated mouse liver myofibroblasts.

Transforming Growth Factors α and ß Are Essential for Modeling Cholangiocarcinoma Desmoplasia and Progression in a Three-Dimensional Organotypic Culture Model.

To gain insight into the cellular and molecular interactions mediating the desmoplastic reaction and aggressive malignancy of mass-forming intrahepatic cholangiocarcinoma (ICC), we modeled ICC desmoplasia and progression in vitro. A unique three-dimensional (3D) organotypic culture model was established; within a dilute collagen-type I hydrogel, a novel clonal strain of rat cancer-associated myofibroblasts (TDFSM) was co-cultured with a pure rat cholangiocarcinoma cell strain (TDECC) derived from the same ICC type as TDFSM. This 3D organotypic culture model reproduced key features of desmoplastic reaction that closely mimicked those of the in situ tumor, as well as promoted cholangiocarcinoma cell growth and progression. Our results supported a resident liver mesenchymal cell origin of the TDFSM cells, which were not neoplastically transformed. Notably, 3D co-culturing of TDECC cells with TDFSM cells provoked the formation of a dense fibrocollagenous stroma in vitro that was associated with significant increases in both proliferative TDFSM myofibroblastic cells and TDECC cholangiocarcinoma cells accumulating within the gel matrix. This dramatic desmoplastic ICC-like phenotype, which was not observed in the TDECC or TDFSM controls, was highly dependent on transforming growth factor (TGF)-ß, but not promoted by TGF-α. However, TGF-α was determined to be a key factor for promoting cholangiocarcinoma cell anaplasia, hyperproliferation, and higher malignant grading in this 3D culture model of desmoplastic ICC.