Eugenio Gaudio

Background: Cholangiocarcinoma (CCA) is a highly heterogeneous malignancy with limited therapeutic options and poor prognosis. The increasing complexity of molecular stratification and treatment selection has stimulated interest in computational and biological modeling approaches for precision oncology. Objective. This narrative review aims to provide a comprehensive overview of digital twins (DTs), DT-enabling computational models, and biological twins (BTs) in CCA, discussing their applications, limitations, and potential integration within hybrid precision medicine frameworks. Methods: A narrative literature review was conducted. To inform the twin-focused sections, a structured PubMed search was performed using predefined keywords related to CCA and twin-related technologies, including organoids, xenografts, organ-on-chip systems. Particular attention was devoted to recent studies addressing computational modeling, patient-derived experimental systems, and translational applications. Results: DT development in CCA is supported by an ecosystem of DT-enabling technologies, including radiomics, artificial intelligence, multi-omics integration, and simulation-based models. However, fully realized medical DTs remain unavailable. BTs, including patient-derived organoids, xenografts, and microfluidic platforms, enable functional validation of therapeutic hypotheses but face challenges related to scalability, standardization, and clinical feasibility. Emerging hybrid DT-BT frameworks seek to combine computational prediction with biological validation through iterative feedback loops, potentially improving patient stratification and treatment personalization. Conclusions: DTs and BTs represent complementary components of an evolving precision oncology ecosystem in CCA. Although technical, biological, regulatory, and implementation challenges remain, the convergence of computational models, longitudinal molecular monitoring, and patient-derived systems may facilitate clinically actionable hybrid twin frameworks. Successful translation will require both technological innovation and healthcare-system improvements to precision medicine access.

Cholangiocarcinoma (CCA) is a highly heterogeneous malignancy with limited thera-peutic options and poor prognosis, underscoring the need for more effective precision medicine strategies. In this context, digital twins (DTs) and biological twins (BTs) have emerged as complementary approaches to model disease complexity and personalize treatment. DTs integrate multi-modal patient data, including imaging, clinical, and multi-omics information, into computational frameworks capable of predicting disease trajectories and therapeutic responses. BTs, such as patient-derived organoids and xenografts, enable functional validation of these predictions in patient-specific ex-perimental systems. This review synthesizes current advances in DT- and BT-based approaches in CCA, highlighting their respective strengths and limitations, and dis-cusses emerging hybrid frameworks that iteratively connect computational modeling with biological experimentation. Despite promising developments, significant chal-lenges remain, including data integration, model validation, regulatory alignment, and clinical adoption. Addressing these barriers will be critical to translating twin-based strategies into clinically actionable tools for personalized oncology.

The journal retracts the article titled “The Effects of Taurocholic Acid on Biliary Damage and Liver Fibrosis Are Mediated by Calcitonin-Gene-Related Peptide Signaling”

Background and Aims: Cholangiocarcinoma (CCA) displays remarkable anatomical and histological heterogeneity. Besides diagnosis confirmation, histology currently does not have a major role in the management of CCA. We aimed to study the clinical relevance of histological heterogeneity of CCA and putative tissue biomarkers by creating a multicentric digitalized European CCA Histology Registry. Approach and Results: Nine referral centers, participating in the International Cholangiocarcinoma Clinical Registry, shared samples and data from 293 patients. Histological and immunohistochemistry stains (n=10) were performed. Computed tomography (CT) scans (n=112 cases) were analyzed by morphological and radiomics techniques. A selection of cases (n=18) was processed for spatial transcriptomics analysis. No significant differences in 5-year overall survival (OS) were found in perihilar CCA versus intrahepatic (i) CCA, and in small bile duct (SBD) versus large bile duct (LBD) iCCA. When cases were classified by Periodic acid of Schiff (PAS) positivity (mucin content), PAS ^HIGH LBD iCCA showed a significantly worse 5-year OS compared to PAS ^LOW iCCA. Multivariate Cox regression identified PAS ^HIGH LBD iCCA phenotype as an independent predictor of worse OS. PAS ^HIGH LBD iCCA subtype showed specific molecular characteristics at spatial transcriptomics and immunohistochemistry; CT scans and serology could distinguish PAS ^HIGH LBD iCCA phenotype with excellent accuracy. Conclusions: Our data underline the importance of identifying morphological subclasses with a significant prevalence in CCA as a tool for risk stratification and prognosis. The European CCA Histology Registry represents a valuable platform for integrating digital pathology with clinical, radiological, and molecular information as a framework for digital twin advancement.

The present review provides a comprehensive examination of the intricate dynamics between α-synuclein, a protein crucially involved in the pathogenesis of several neurodegenerative diseases, including Parkinson’s disease and multiple system atrophy, and endogenously-produced bioactive lipids, which play a pivotal role in neuroinflammation and neurodegeneration. The interaction of α-synuclein with bioactive lipids is emerging as a critical factor in the development and progression of neurodegenerative and neuroinflammatory diseases, offering new insights into disease mechanisms and novel perspectives in the identification of potential biomarkers and therapeutic targets. We delve into the molecular pathways through which α-synuclein interacts with biological membranes and bioactive lipids, influencing the aggregation of α-synuclein and triggering neuroinflammatory responses, highlighting the potential of bioactive lipids as biomarkers for early disease detection and progression monitoring. Moreover, we explore innovative therapeutic strategies aimed at modulating the interaction between α-synuclein and bioactive lipids, including the development of small molecules and nutritional interventions. Finally, the review addresses the significance of the gut-to-brain axis in mediating the effects of bioactive lipids on α-synuclein pathology and discusses the role of altered gut lipid metabolism and microbiota composition in neuroinflammation and neurodegeneration. The present review aims to underscore the potential of targeting α-synuclein-lipid interactions as a multifaceted approach for the detection and treatment of neurodegenerative and neuroinflammatory diseases.

Gut-dysbiosis-induced lipopolysaccharides (LPS) translocation into systemic circulation has been suggested to be implicated in nonalcoholic fatty liver disease (NAFLD) pathogenesis. This study aimed to assess if oleuropein (OLE), a component of extra virgin olive oil, lowers high-fat-diet (HFD)-induced endotoxemia and, eventually, liver steatosis. An immunohistochemistry analysis of the intestine and liver was performed in (i) control mice (CTR; n = 15), (ii) high-fat-diet fed (HFD) mice (HFD; n = 16), and (iii) HFD mice treated with 6 µg/day of OLE for 30 days (HFD + OLE, n = 13). The HFD mice developed significant liver steatosis compared to the controls, an effect that was significantly reduced in the HFD + OLE-treated mice. The amount of hepatocyte LPS localization and the number of TLR4+ macrophages were higher in the HFD mice in the than controls and were lowered in the HFD + OLE-treated mice. The number of CD42b+ platelets was increased in the liver sinusoids of the HFD mice compared to the controls and decreased in the HFD + OLE-treated mice. Compared to the controls, the HFD-treated mice showed a high percentage of intestine PAS+ goblet cells, an increased length of intestinal crypts, LPS localization and TLR4+ expression, and occludin downregulation, an effect counteracted in the HFD + OLE-treated mice. The HFD-fed animals displayed increased systemic levels of LPS and zonulin, but they were reduced in the HFD + OLE-treated animals. It can be seen that OLE administration improves liver steatosis and inflammation in association with decreased LPS translocation into the systemic circulation, hepatocyte localization of LPS and TLR4 downregulation in HFD-induced mouse model of NAFLD.

The term cholangiocarcinoma (CCA) defines a class of epithelial malignancies originating from bile ducts. Although it has been demonstrated that CCA patients with perineural invasion (PNI) have a worse prognosis, the biological features of this phenomenon are yet unclear. Our data show that in human intrahepatic CCA specimens with documented PNI, nerve-infiltrating CCA cells display positivity of the epithelial marker cytokeratin 7, lower with respect to the rest of the tumor mass. In an in vitro 3D model, CCA cells move towards a peripheral nerve explant allowing contact with Schwann cells (SCs) emerging from the nerve. Here, we show that SCs produce soluble factors that favor the migration, invasion, survival and proliferation of CCA cells in vitro. This effect is accompanied by a cadherin switch, suggestive of an epithelial–mesenchymal transition. The influence of SCs in promoting the ability of CCA cells to migrate and invade the extracellular matrix is hampered by a specific TGFβ receptor 1 (TGFBR1) antagonist. Differential proteomic data indicate that the exposure of CCA cells to SC secreted factors induces the upregulation of key oncogenes and the concomitant downregulation of some tumor suppressors. Taken together, these data concur in identifying SCs as possible promoters of a more aggressive CCA phenotype, ascribing a central role to TGFβ signaling in regulating this process.

Cholangiocytes, the epithelial cells that line the biliary tree, can proliferate under the stimulation of several factors through both autocrine and paracrine pathways. The cocaine-amphetamine-regulated-transcript (CART) peptide has several physiological functions, and it is widely expressed in several organs. CART increases the survival of hippocampal neurons by upregulating brain-derived neurotrophic factor (BDNF), whose expression has been correlated to the proliferation rate of cholangiocytes. In the present study, we aimed to evaluate the expression of CART and its role in modulating cholangiocyte proliferation in healthy and bile duct ligated (BDL) rats in vivo, as well as in cultured normal rat cholangiocytes (NRC) in vitro. Liver samples from both healthy and BDL (1 week) rats, were analyzed by immunohistochemistry and immunofluorescence for CART, CK19, TrkB and p75NTR BDNF receptors. PCNA staining was used to evaluate the proliferation of the cholangiocytes, whereas TUNEL assay was used to evaluate biliary apoptosis. NRC treated or not with CART were used to confirm the role of CART on cholangiocytes proliferation and the secretion of BDNF. Cholangiocytes proliferation, apoptosis, CART and TrkB expression were increased in BDL rats, compared to control rats. We found a higher expression of TrkB and p75NTR, which could be correlated with the proliferation rate of biliary tree during BDL. The in vitro study demonstrated increased BDNF secretion by NRC after treatment with CART compared with control cells. As previously reported, proliferating cholangiocytes acquire a neuroendocrine phenotype, modulated by several factors, including neurotrophins. Accordingly, CART may play a key role in the remodeling of biliary epithelium during cholestasis by modulating the secretion of BDNF.

Background and Aims: NAFLD is characterized by steatosis, hepatic inflammation, and fibrosis, which can develop into NASH. Patients with NAFLD/NASH have increased ductular reaction (DR) and biliary senescence. High fat/high cholesterol diet feeding increases biliary senescence, DR, and biliary insulin-like growth factor-1 (IGF-1) expression in mice. p16/IGF-1 converges with fork-head box transcription factor O1 (FOXO1) through E2F1. We evaluated p16 inhibition on NAFLD phenotypes and biliary E2F1/FOXO1/IGF-1 signaling. Approach and Results: 4-week wild-type (C57BL/6J) male mice were fed a control diet (CD) or high fat/high cholesterol diet and received either p16 or control Vivo Morpholino (VM) by tail vein injection 2× during the 16th week of feeding. We confirmed p16 knockdown and examined: (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling. Human normal, NAFLD, and NASH liver samples and isolated cholangiocytes treated with control or p16 VM were evaluated for p16/E2F1/FOXO1/IGF-1 signaling. p16 VM treatment reduced cholangiocyte and hepatocyte p16. In wild-type high fat/high cholesterol diet mice with control VM, there were increased (i) NAFLD phenotypes; (ii) DR and biliary senescence; (iii) serum metabolites; and (iv) biliary E2F1/FOXO1/IGF-1 signaling; however, p16 VM treatment reduced these parameters. Biliary E2F1/FOX-O1/IGF-1 signaling increased in human NAFLD/NASH but was blocked by p16 VM. In vitro, p16 VM reduced biliary E2f1 and Foxo1 transcription by inhibiting RNA pol II binding and E2F1 binding at the Foxo1 locus, respectively. Inhibition of E2F1 reduced biliary FOXO1 in vitro. Conclusion: Attenuating hepatic p16 expression may be a therapeutic approach for improving NAFLD/NASH phenotypes.

Background and Aims: Secretin (SCT) and secretin receptor (SR, only expressed on cholangiocytes within the liver) play key roles in modulating liver phenotypes. Forkhead box A2 (FoxA2) is required for normal bile duct homeostasis by preventing the excess of cholangiocyte proliferation. Short-term administration of the SR antagonist (SCT 5–27) decreased ductular reaction and liver fibrosis in bile duct ligated and Mdr2^−/− [primary sclerosing cholangitis (PSC), model] mice. We aimed to evaluate the effectiveness and risks of long-term SCT 5–27 treatment in Mdr2^−/− mice. Approach and Results: In vivo studies were performed in male wild-type and Mdr2^−/− mice treated with saline or SCT 5–27 for 3 months and human samples from late-stage PSC patients and healthy controls. Compared with controls, biliary SCT/SR expression and SCT serum levels increased in Mdr2^−/− mice and late-stage PSC patients. There was a significant increase in ductular reaction, biliary senescence, liver inflammation, angiogenesis, fibrosis, biliary expression of TGF-β1/VEGF-A axis, and biliary phosphorylation of protein kinase A and ERK1/2 in Mdr2^−/− mice. The biliary expression of miR-125b and FoxA2 decreased in Mdr2^−/− compared with wild-type mice, which was reversed by long-term SCT 5–27 treatment. In vitro, SCT 5–27 treatment of a human biliary PSC cell line decreased proliferation and senescence and SR/TGF-β1/VEGF-A axis but increased the expression of miR-125b and FoxA2. Downregulation of FoxA2 prevented SCT 5–27–induced reduction in biliary damage, whereas overexpression of FoxA2 reduced proliferation and senescence in the human PSC cell line. Conclusions: Modulating the SCT/SR axis may be critical for managing PSC.