Van Gogh-like 2 is essential for the architectural patterning of the mammalian biliary tree.

BACKGROUND & AIMS: In the developing liver, bipotent epithelial progenitor cells undergo lineage segregation to form hepatocytes, which constitute the bulk of the liver parenchyma, and biliary epithelial cells (cholangiocytes), which comprise the bile duct (a complex tubular network that is critical for normal liver function). Notch and TGFß signalling promote the formation of a sheet of biliary epithelial cells, the ductal plate, that organises into discontinuous tubular structures. How these structures elongate and connect to form a continuous duct remains undefined. We aimed to define the mechanisms by which the ductal plate transitions from a simple sheet of epithelial cells into a complex and connected bile duct. METHODS: By combining single-cell RNA sequencing of embryonic mouse livers with genetic tools and organoid models we functionally dissected the role of planar cell polarity in duct patterning. RESULTS: We show that the planar cell polarity protein VANGL2 is expressed late in intrahepatic bile duct development and patterns the formation of cell-cell contacts between biliary cells. The patterning of these cell contacts regulates the normal polarisation of the actin cytoskeleton within biliary cells and loss of Vangl2 function results in the abnormal distribution of cortical actin remodelling, leading to the failure of bile duct formation. CONCLUSIONS: Planar cell polarity is a critical step in the post-specification sculpture of the bile duct and is essential for establishing normal tissue architecture. IMPACT AND IMPLICATIONS: Like other branched tissues, such as the lung and kidney, the bile ducts use planar cell polarity signalling to coordinate cell movements; however, how these biochemical signals are linked to ductular patterning remains unclear. Here we show that the core planar cell polarity protein VANGL2 patterns how cell-cell contacts form in the mammalian bile duct and how ductular cells transmit confluent mechanical changes along the length of a duct. This work sheds light on how biological tubes are patterned across mammalian tissues (including within the liver) and will be important in how we promote ductular growth in patients where the duct is mis-patterned or poorly formed.

Molecular portraits of patients with intrahepatic cholangiocarcinoma who diverge as rapid progressors or long survivors on chemotherapy.

OBJECTIVE: Cytotoxic agents are the cornerstone of treatment for patients with advanced intrahepatic cholangiocarcinoma (iCCA), despite heterogeneous benefit. We hypothesised that the pretreatment molecular profiles of diagnostic biopsies can predict patient benefit from chemotherapy and define molecular bases of innate chemoresistance. DESIGN: We identified a cohort of advanced iCCA patients with comparable baseline characteristics who diverged as extreme outliers on chemotherapy (survival <6 m in rapid progressors, RP; survival >23 m in long survivors, LS). Diagnostic biopsies were characterised by digital pathology, then subjected to whole-transcriptome profiling of bulk and geospatially macrodissected tissue regions. Spatial transcriptomics of tumour-infiltrating myeloid cells was performed using targeted digital spatial profiling (GeoMx). Transcriptome signatures were evaluated in multiple cohorts of resected cancers. Signatures were also characterised using in vitro cell lines, in vivo mouse models and single cell RNA-sequencing data. RESULTS: Pretreatment transcriptome profiles differentiated patients who would become RPs or LSs on chemotherapy. Biologically, this signature originated from altered tumour-myeloid dynamics, implicating tumour-induced immune tolerogenicity with poor response to chemotherapy. The central role of the liver microenviroment was confrmed by the association of the RPLS transcriptome signature with clinical outcome in iCCA but not extrahepatic CCA, and in liver metastasis from colorectal cancer, but not in the matched primary bowel tumours. CONCLUSIONS: The RPLS signature could be a novel metric of chemotherapy outcome in iCCA. Further development and validation of this transcriptomic signature is warranted to develop precision chemotherapy strategies in these settings.

Patient-derived precision cut tissue slices from primary liver cancer as a potential platform for preclinical drug testing.

BACKGROUND: The exploitation of anti-tumour immunity, harnessed through immunomodulatory therapies, has fundamentally changed the treatment of primary liver cancer (PLC). However, this has posed significant challenges in preclinical research. Novel immunologically relevant models for PLC are urgently required to improve the translation from bench to bedside and back, explore and predict effective combinatorial therapies, aid novel drug discovery and develop personalised treatment modalities. METHODS: We used human precision-cut tissue slices (PCTS) derived from resected tumours to create a patient-specific immunocompetent disease model that captures the multifaceted and intricate heterogeneity of the tumour and the tumour microenvironment. Tissue architecture, tumour viability and treatment response to single agent and combination therapies were assessed longitudinally over 8 days of ex vivo culture by histological analysis, detection of proliferation/cell death markers, ATP content via HPLC. Immune cell infiltrate was assessed using PCR and immunofluorescence. Checkpoint receptor expression was quantified via Quantigene RNA assay. FINDINGS: After optimising the culture conditions, PCTS maintained the original tissue architecture, including tumour morphology, stroma and tumour-infiltrated leukocytes. Moreover, PCTS retained the tumour-specific immunophenotype over time, suggesting the utility of PCTS to investigate immunotherapeutic drug efficacy and identify non-responsiveness. INTERPRETATION: Here we have characterised the PCTS model and demonstrated its effectiveness as a robust preclinical tool that will significantly support the development of successful (immuno)therapeutic strategies for PLC. FUNDING: Foundation for Liver Research, London.

A TGFß-ECM-integrin signaling axis drives structural reconfiguration of the bile duct to promote polycystic liver disease.

The formation of multiple cysts in the liver occurs in a number of isolated monogenic diseases or multisystemic syndromes, during which bile ducts develop into fluid-filled biliary cysts. For patients with polycystic liver disease (PCLD), nonsurgical treatments are limited, and managing life-long abdominal swelling, pain, and increasing risk of cyst rupture and infection is common. We demonstrate here that loss of the primary cilium on postnatal biliary epithelial cells (via the deletion of the cilia gene Wdr35) drives ongoing pathological remodeling of the biliary tree, resulting in progressive cyst formation and growth. The development of cystic tissue requires the activation of transforming growth factor-ß (TGFß) signaling, which promotes the expression of a procystic, fibronectin-rich extracellular matrix and which itself is perceived by a changing profile of integrin receptors on the cystic epithelium. This signaling axis is conserved in liver cysts from patients with either autosomal dominant polycystic kidney disease or autosomal dominant polycystic liver disease, indicating that there are common cellular mechanisms for liver cyst growth regardless of the underlying genetic cause. Cyst number and size can be reduced by inhibiting TGFß signaling or integrin signaling in vivo. We suggest that our findings represent a therapeutic route for patients with polycystic liver disease, most of whom would not be amenable to surgery.

Thymic epithelial cell fate and potency in early organogenesis assessed by single cell transcriptional and functional analysis.

During development, cortical (c) and medullary (m) thymic epithelial cells (TEC) arise from the third pharyngeal pouch endoderm. Current models suggest that within the thymic primordium most TEC exist in a bipotent/common thymic epithelial progenitor cell (TEPC) state able to generate both cTEC and mTEC, at least until embryonic day 12.5 (E12.5) in the mouse. This view, however, is challenged by recent transcriptomics and genetic evidence. We therefore set out to investigate the fate and potency of TEC in the early thymus. Here using single cell (sc) RNAseq we identify a candidate mTEC progenitor population at E12.5, consistent with recent reports. Via lineage-tracing we demonstrate this population as mTEC fate-restricted, validating our bioinformatics prediction. Using potency analyses we also establish that most E11.5 and E12.5 progenitor TEC are cTEC-fated. Finally we show that overnight culture causes most if not all E12.5 cTEC-fated TEPC to acquire functional bipotency, and provide a likely molecular mechanism for this changed differentiation potential. Collectively, our data overturn the widely held view that a common TEPC predominates in the E12.5 thymus, showing instead that sublineage-primed progenitors are present from the earliest stages of thymus organogenesis but that these early fetal TEPC exhibit cell-fate plasticity in response to extrinsic factors. Our data provide a significant advance in the understanding of fetal thymic epithelial development and thus have implications for thymus-related clinical research, in particular research focussed on generating TEC from pluripotent stem cells.

NOTCH signalling - a core regulator of bile duct disease?

The Notch signalling pathway is an evolutionarily conserved mechanism of cell-cell communication that mediates cellular proliferation, fate determination and maintenance of stem/progenitor cell populations across tissues. Although it was originally identified as a critical regulator of embryonic liver development, NOTCH signalling activation has been associated with the pathogenesis of a number of paediatric and adult liver diseases. It remains unclear, however, what role NOTCH actually plays in these pathophysiological processes and whether NOTCH activity represents the reactivation of a conserved developmental programme that is essential for adult tissue repair. In this Review, we explore the concepts that NOTCH signalling reactivation in the biliary epithelium is a reiterative and essential response to bile duct damage and that, in disease contexts in which biliary epithelial cells need to be regenerated, NOTCH signalling supports ductular regrowth. Furthermore, we evaluate the recent literature on NOTCH signalling as a critical factor in progenitor-mediated hepatocyte regeneration, which indicates that the mitogenic role for NOTCH signalling in biliary epithelial cell proliferation has also been co-opted to support other forms of epithelial regeneration in the adult liver.

Indian Hedgehog release from TNF-activated renal epithelia drives local and remote organ fibrosis.

Progressive fibrosis is a feature of aging and chronic tissue injury in multiple organs, including the kidney and heart. Glioma-associated oncogene 1 expressing (Gli1+) cells are a major source of activated fibroblasts in multiple organs, but the links between injury, inflammation, and Gli1+ cell expansion and tissue fibrosis remain incompletely understood. We demonstrated that leukocyte-derived tumor necrosis factor (TNF) promoted Gli1+ cell proliferation and cardiorenal fibrosis through induction and release of Indian Hedgehog (IHH) from renal epithelial cells. Using single-cell-resolution transcriptomic analysis, we identified an "inflammatory" proximal tubular epithelial (iPT) population contributing to TNF- and nuclear factor κB (NF-κB)-induced IHH production in vivo. TNF-induced Ubiquitin D (Ubd) expression was observed in human proximal tubular cells in vitro and during murine and human renal disease and aging. Studies using pharmacological and conditional genetic ablation of TNF-induced IHH signaling revealed that IHH activated canonical Hedgehog signaling in Gli1+ cells, which led to their activation, proliferation, and fibrosis within the injured and aging kidney and heart. These changes were inhibited in mice by Ihh deletion in Pax8-expressing cells or by pharmacological blockade of TNF, NF-κB, or Gli1 signaling. Increased amounts of circulating IHH were associated with loss of renal function and higher rates of cardiovascular disease in patients with chronic kidney disease. Thus, IHH connects leukocyte activation to Gli1+ cell expansion and represents a potential target for therapies to inhibit inflammation-induced fibrosis.

Criteria for preclinical models of cholangiocarcinoma: scientific and medical relevance.

Cholangiocarcinoma (CCA) is a rare malignancy that develops at any point along the biliary tree. CCA has a poor prognosis, its clinical management remains challenging, and effective treatments are lacking. Therefore, preclinical research is of pivotal importance and necessary to acquire a deeper understanding of CCA and improve therapeutic outcomes. Preclinical research involves developing and managing complementary experimental models, from in vitro assays using primary cells or cell lines cultured in 2D or 3D to in vivo models with engrafted material, chemically induced CCA or genetically engineered models. All are valuable tools with well-defined advantages and limitations. The choice of a preclinical model is guided by the question(s) to be addressed; ideally, results should be recapitulated in independent approaches. In this Consensus Statement, a task force of 45 experts in CCA molecular and cellular biology and clinicians, including pathologists, from ten countries provides recommendations on the minimal criteria for preclinical models to provide a uniform approach. These recommendations are based on two rounds of questionnaires completed by 35 (first round) and 45 (second round) experts to reach a consensus with 13 statements. An agreement was defined when at least 90% of the participants voting anonymously agreed with a statement. The ultimate goal was to transfer basic laboratory research to the clinics through increased disease understanding and to develop clinical biomarkers and innovative therapies for patients with CCA.

DKK1 drives immune suppressive phenotypes in intrahepatic cholangiocarcinoma and can be targeted with anti-DKK1 therapeutic DKN-01.

BACKGROUND AND AIMS: Dickkopf-1 (DKK1) is associated with poor prognosis in intrahepatic cholangiocarcinoma (iCCA), but the mechanisms behind this are unclear. Here, we show that DKK1 plays an immune regulatory role in vivo and inhibition reduces tumour growth. METHODS: Various in vivo GEMM mouse models and patient samples were utilized to assess the effects of tumour specific DKK1 overexpression in iCCA. DKK1-driven changes to the tumour immune microenvironment were characterized by immunostaining and gene expression analysis. DKK1 overexpressing and damage-induced models of iCCA were used to demonstrate the therapeutic efficacy of DKK1 inhibition in these contexts using the anti-DKK1 therapeutic, DKN-01. RESULTS: DKK1 overexpression in mouse models of iCCA drives an increase in chemokine and cytokine signalling, the recruitment of regulatory macrophages, and promotes the formation of a tolerogenic niche with higher numbers of regulatory T cells. We show a similar association of DKK1 with FOXP3 and regulatory T cells in patient tissue and gene expression data, demonstrating these effects are relevant to human iCCA. Finally, we demonstrate that inhibition of DKK1 with the monoclonal antibody mDKN-01 is effective at reducing tumour burden in two distinct mouse models of the disease. CONCLUSION: DKK1 promotes tumour immune evasion in iCCA through the recruitment of immune suppressive macrophages. Targeting DKK1 with a neutralizing antibody is effective at reducing tumour growth in vivo. As such, DKK1 targeted and immune modulatory therapies may be an effective strategy in iCCA patients with high DKK1 tumour expression or tolerogenic immune phenotypes.

Senolytic treatment preserves biliary regenerative capacity lost through cellular senescence during cold storage.

Liver transplantation is the only curative option for patients with end-stage liver disease. Despite improvements in surgical techniques, nonanastomotic strictures (characterized by the progressive loss of biliary tract architecture) continue to occur after liver transplantation, negatively affecting liver function and frequently leading to graft loss and retransplantation. To study the biological effects of organ preservation before liver transplantation, we generated murine models that recapitulate liver procurement and static cold storage. In these models, we explored the response of cholangiocytes and hepatocytes to cold storage, focusing on responses that affect liver regeneration, including DNA damage, apoptosis, and cellular senescence. We show that biliary senescence was induced during organ retrieval and exacerbated during static cold storage, resulting in impaired biliary regeneration. We identified decoy receptor 2 (DCR2)-dependent responses in cholangiocytes and hepatocytes, which differentially affected the outcome of those populations during cold storage. Moreover, CRISPR-mediated DCR2 knockdown in vitro increased cholangiocyte proliferation and decreased cellular senescence but had the opposite effect in hepatocytes. Using the p21KO model to inhibit senescence onset, we showed that biliary tract architecture was better preserved during cold storage. Similar results were achieved by administering senolytic ABT737 to mice before procurement. Last, we perfused senolytics into discarded human donor livers and showed that biliary architecture and regenerative capacities were better preserved. Our results indicate that cholangiocytes are susceptible to senescence and identify the use of senolytics and the combination of senotherapies and machine-perfusion preservation to prevent this phenotype and reduce the incidence of biliary injury after transplantation.

Toll-like receptor 2 orchestrates a tumor suppressor response in non-small cell lung cancer.

Targeting early-stage lung cancer is vital to improve survival. However, the mechanisms and components of the early tumor suppressor response in lung cancer are not well understood. In this report, we study the role of Toll-like receptor 2 (TLR2), a regulator of oncogene-induced senescence, which is a key tumor suppressor response in premalignancy. Using human lung cancer samples and genetically engineered mouse models, we show that TLR2 is active early in lung tumorigenesis, where it correlates with improved survival and clinical regression. Mechanistically, TLR2 impairs early lung cancer progression via activation of cell intrinsic cell cycle arrest pathways and the proinflammatory senescence-associated secretory phenotype (SASP). The SASP regulates non-cell autonomous anti-tumor responses, such as immune surveillance of premalignant cells, and we observe impaired myeloid cell recruitment to lung tumors after Tlr2 loss. Last, we show that administration of a TLR2 agonist reduces lung tumor growth, highlighting TLR2 as a possible therapeutic target.

In Vivo Modeling of Patient Genetic Heterogeneity Identifies New Ways to Target Cholangiocarcinoma.

Intrahepatic cholangiocarcinoma (ICC) is an aggressive malignancy of the bile ducts within the liver characterized by high levels of genetic heterogeneity. In the context of such genetic variability, determining which oncogenic mutations drive ICC growth has been difficult, and developing modes of patient stratification and targeted therapies remains challenging. Here we model the interactions between rare mutations with more common driver genes and combine in silico analysis of patient data with highly multiplexed in vivo CRISPR-spCas9 screens to perform a functional in vivo study into the role genetic heterogeneity plays in driving ICC. Novel tumor suppressors were uncovered, which, when lost, cooperate with the RAS oncoprotein to drive ICC growth. Focusing on a set of driver mutations that interact with KRAS to initiate aggressive, sarcomatoid-type ICC revealed that tumor growth relies on Wnt and PI3K signaling. Pharmacologic coinhibition of Wnt and PI3K in vivo impeded ICC growth regardless of mutational profile. Therefore, Wnt and PI3K activity should be considered as a signature by which patients can be stratified for treatment independent of tumor genotype, and inhibitors of these pathways should be levied to treat ICC. SIGNIFICANCE: This work shows that, despite significant genetic heterogeneity, intrahepatic cholangiocarcinoma relies on a limited number of signaling pathways to grow, suggesting common therapeutic vulnerabilities across patients.

The developmental origins of Notch-driven intrahepatic bile duct disorders.

The Notch signaling pathway is an evolutionarily conserved mechanism of cell-cell communication that mediates cellular proliferation, cell fate specification, and maintenance of stem and progenitor cell populations. In the vertebrate liver, an absence of Notch signaling results in failure to form bile ducts, a complex tubular network that radiates throughout the liver, which, in healthy individuals, transports bile from the liver into the bowel. Loss of a functional biliary network through congenital malformations during development results in cholestasis and necessitates liver transplantation. Here, we examine to what extent Notch signaling is necessary throughout embryonic life to initiate the proliferation and specification of biliary cells and concentrate on the animal and human models that have been used to define how perturbations in this signaling pathway result in developmental liver disorders.

Notch-IGF1 signaling during liver regeneration drives biliary epithelial cell expansion and inhibits hepatocyte differentiation.

In the adult liver, a population of facultative progenitor cells called biliary epithelial cells (BECs) proliferate and differentiate into cholangiocytes and hepatocytes after injury, thereby restoring liver function. In mammalian models of chronic liver injury, Notch signaling is essential for bile duct formation from these cells. However, the continual proliferation of BECs and differentiation of hepatocytes in these models have limited their use for determining whether Notch signaling is required for BECs to replenish hepatocytes after injury in the mammalian liver. Here, we used a temporally restricted model of hepatic repair in which large-scale hepatocyte injury and regeneration are initiated through the acute loss of Mdm2 in hepatocytes, resulting in the rapid, coordinated proliferation of BECs. We found that transient, early activation of Notch1- and Notch3-mediated signaling and entrance into the cell cycle preceded the phenotypic expansion of BECs into hepatocytes. Notch inhibition reduced BEC proliferation, which resulted in failure of BECs to differentiate into hepatocytes, indicating that Notch-dependent expansion of BECs is essential for hepatocyte regeneration. Notch signaling increased the abundance of the insulin-like growth factor 1 receptor (IGF1R) in BECs, and activating IGFR signaling increased BEC numbers but suppressed BEC differentiation into hepatocytes. These results suggest that different signaling mechanisms control BEC expansion and hepatocyte differentiation.

Inhibition of nuclear factor (erythroid-derived 2)-like 2 promotes hepatic progenitor cell activation and differentiation.

The stem cell ability to self-renew and lead regeneration relies on the balance of complex signals in their microenvironment. The identification of modulators of hepatic progenitor cell (HPC) activation is determinant for liver regeneration and may improve cell transplantation for end-stage liver disease. This investigation used different models to point out the Nuclear factor (erythroid-derived 2)-like 2 (NRF2) as a key regulator of the HPC fate. We initially proved that in vivo models of biliary epithelial cells (BECs)/HPC activation show hepatic oxidative stress, which activates primary BECs/HPCs in vitro. NRF2 downregulation and silencing were associated with morphological, phenotypic, and functional modifications distinctive of differentiated cells. Furthermore, NRF2 activation in the biliary tract repressed the ductular reaction in injured liver. To definitely assess the importance of NRF2 in HPC biology, we applied a xenograft model by inhibiting NRF2 in the human derived HepaRG cell line and transplanting into SCID/beige mice administered with anti-Fas antibody to induce hepatocellular apoptosis; this resulted in effective human hepatocyte repopulation with reduced liver injury. To conclude, NRF2 inhibition leads to the activation and differentiation of liver progenitors. This redox-dependent transcription factor represents a potential target to regulate the commitment of undifferentiated hepatic progenitors into specific lineages.

Developing models of cholangiocarcinoma to close the translational gap in cancer research.

Introduction: Cholangiocarcinoma (CCA) is an aggressive primary liver malignancy with abysmal prognosis and increasing global incidence. Individuals afflicted with CCA often remain asymptomatic until late stages of disease, resulting in very limited possibilities for therapeutic intervention. The emergence of numerous preclinical models in vitro and in vivo has expanded the tool kit for CCA researchers; nonetheless, how these tools can be best applied to understand CCA biology and accelerate drug development requires further scrutiny.Areas covered: The paper reviews the literature on animal and organoid models of CCA (available through PubMed between September 2020 and January 2021) and examines their investigational role in CCA therapeutics. Finally, the potential of these systems for screening therapeutics to improve CCA patient outcomes is illuminated.Expert Opinion: The expansion of CCA models has yielded a diverse and interesting tool kit for preclinical research. However, investigators should consider which tools are best suited to answer key preclinical questions for real progress. A combination of advanced in vitro cell systems and in vivo testing will be necessary to accelerate translational medicine in cholangiocarcinoma.

The fibrotic and immune microenvironments as targetable drivers of metastasis.

Although substantial progress has been made over the past 40 years in treating patients with cancer, effective therapies for those who are diagnosed with advanced metastatic disease are still few and far between. Cancer cells do not exist in isolation: rather, they exist within a complex microenvironment composed of stromal cells and extracellular matrix. Within this tumour microenvironment exists an interplay between the two main stromal cell subtypes, cancer-associated fibroblasts (CAFs) and immune cells, that are important in controlling metastasis. A complex network of paracrine signalling pathways between CAFs, immune cells and tumour cells are involved at multiple stages of the metastatic process, from invasion and intravasation at the primary tumour site to extravasation and colonisation in the metastatic site. Heterogeneity and plasticity within stromal cell populations also contribute to the complexity. Although many of these processes are likely to be common to a number of metastatic sites, we will describe in detail the interplay within the liver, a preferred site of metastasis for many tumours. A greater understanding of these networks provides opportunities for the design of new therapeutic approaches for targeting the metastatic disease.

TWEAK/Fn14 signalling promotes cholangiocarcinoma niche formation and progression.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a cancer of the hepatic bile ducts that is rarely resectable and is associated with poor prognosis. Tumour necrosis factor-like weak inducer of apoptosis (TWEAK) is known to signal via its receptor fibroblast growth factor-inducible 14 (Fn14) and induce cholangiocyte and myofibroblast proliferation in liver injury. We aimed to characterise its role in CCA. METHODS: The expression of the TWEAK ligand and Fn14 receptor was assessed immunohistochemically and by bulk RNA and single cell transcriptomics of human liver tissue. Spatiotemporal dynamics of pathway regulation were comprehensively analysed in rat and mouse models of thioacetamide (TAA)-mediated CCA. Flow cytometry, qPCR and proteomic analyses of CCA cell lines and conditioned medium experiments with primary macrophages were performed to evaluate the downstream functions of TWEAK/Fn14. In vivo pathway manipulation was assessed via TWEAK overexpression in NICD/AKT-induced CCA or genetic Fn14 knockout during TAA-mediated carcinogenesis. RESULTS: Our data reveal TWEAK and Fn14 overexpression in multiple human CCA cohorts, and Fn14 upregulation in early TAA-induced carcinogenesis. TWEAK regulated the secretion of factors from CC-SW-1 and SNU-1079 CCA cells, inducing polarisation of proinflammatory CD206+ macrophages. Pharmacological blocking of the TWEAK downstream target chemokine monocyte chemoattractant protein 1 (MCP-1 or CCL2) significantly reduced CCA xenograft growth, while TWEAK overexpression drove cancer-associated fibroblast proliferation and collagen deposition in the tumour niche. Genetic Fn14 ablation significantly reduced inflammatory, fibrogenic and ductular responses during carcinogenic TAA-mediated injury. CONCLUSION: These novel data provide evidence for the action of TWEAK/Fn14 on macrophage recruitment and phenotype, and cancer-associated fibroblast proliferation in CCA. Targeting TWEAK/Fn14 and its downstream signals may provide a means to inhibit CCA niche development and tumour growth. LAY SUMMARY: Cholangiocarcinoma is an aggressive, chemotherapy-resistant liver cancer. Interactions between tumour cells and cells that form a supportive environment for the tumour to grow are a source of this aggressiveness and resistance to chemotherapy. Herein, we describe interactions between tumour cells and their supportive environment via a chemical messenger, TWEAK and its receptor Fn14. TWEAK/Fn14 alters the recruitment and type of immune cells in tumours, increases the growth of cancer-associated fibroblasts in the tumour environment, and is a potential target to reduce tumour formation.