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The ectopic xenograft mouse model of cancer is a commonly employed tool for in vivo investigations, particularly for studying cell tumorigenicity and testing the efficacy and tolerability of systemic or local anti-cancer therapies. The model displays advantageous features with an easy-access to visualize and monitor tumor growth in real-time with a caliper. Although the tumor development occurs in an ectopic location, the histology of the tumor resembles that of human cancer upon pathological examination. This suggests that when human malignant cells are transplanted into immunocompromised mice, they can educate and attract murine cells from the surrounding environment to recapitulate a tumor structure. The experimental protocol for ectopic xenograft models is straightforward, making them reproducible, cost-effective, and conductive to shorter experimental durations. Here, we detail the utilization of ectopic xenograft models in studying biliary tract cancers (BTC), which involves subcutaneously grafting human BTC cell lines originating from different biliary tree locations onto immunocompromised nude mice., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Background & Aims: The class I- phosphatidylinositol-3 kinases (PI3Ks) signalling is dysregulated in almost all human cancers whereas the isoform-specific roles remain poorly investigated. We reported that the isoform δ (PI3Kδ) regulated epithelial cell polarity and plasticity and recent developments have heightened its role in hepatocellular carcinoma (HCC) and solid tumour progression. However, its role in cholangiocarcinoma (CCA) still lacks investigation., Approach & Results: Immunohistochemical analyses of CCA samples reveal a high expression of PI3Kδ in the less differentiated CCA. The RT-qPCR and immunoblot analyses performed on CCA cells stably overexpressing PI3Kδ using lentiviral construction reveal an increase of mesenchymal and stem cell markers and the pluripotency transcription factors. CCA cells stably overexpressing PI3Kδ cultured in 3D culture display a thick layer of ECM at the basement membrane and a wide single lumen compared to control cells. Similar data are observed in vivo, in xenografted tumours established with PI3Kδ-overexpressing CCA cells in immunodeficient mice. The expression of mesenchymal and stemness genes also increases and tumour tissue displays necrosis and fibrosis, along with a prominent angiogenesis and lymphangiogenesis, as in mice liver of AAV8-based-PI3Kδ overexpression. These PI3Kδ-mediated cell morphogenesis and stroma remodelling were dependent on TGFβ/Src/Notch signalling. Whole transcriptome analysis of PI3Kδ using the cancer cell line encyclopedia allows the classification of CCA cells according to cancer progression., Conclusions: Overall, our results support the critical role of PI3Kδ in the progression and aggressiveness of CCA via TGFβ/src/Notch-dependent mechanisms and open new directions for the classification and treatment of CCA patients., (© 2023 The Authors. Liver International published by John Wiley & Sons Ltd.)

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., (© 2023. Springer Nature Limited.)

During the last decade, immunotherapy has radically changed perspectives on anti-tumor treatments. However, solid tumor treatment by immunotherapy has not met expectations. Indeed, poor clinical response to treatment has highlighted the need to understand and avoid immunotherapy resistance. Cholangiocarcinoma (CCA) is the second cause of hepatic cancer-related deaths because of drug inefficacy and chemo-resistance in a majority of patients. Thus, intense research is ongoing to better understand the mechanisms involved in the chemo-resistance processes. The tumor microenvironment (TME) may be involved in tumor therapy resistance by limiting drug access. Indeed, cells such as cancer-associated fibroblasts (CAFs) alter TME by producing in excess an aberrant extracellular matrix (ECM). Interestingly, CAFs are the dominant stromal component in CCA that secrete large amounts of stiff ECM. Stiff ECM could contribute to immune exclusion by limiting anti-tumor T-cells drop-in. Herein, we summarize features, functions, and interactions among CAFs, tumor-associated ECM, and immune cells in TME. Moreover, we discuss the strategies targeting CAFs and the remodeling of the ECM to improve immunotherapy and drug therapies.

Correct actin cytoskeleton organization is vital in the liver organ homeostasis and disease control. Rearrangements of the actin cytoskeleton may play a vital role in the bile duct cells cholangiocytes. An abnormal actin network leads to aberrant cell morphology, deregulated signaling networks and ultimately triggering the development of cholangiocarcinoma (CCA) and paving the route for cancer cell dissemination (metastasis). In this review, we will outline alterations of the actin cytoskeleton and the potential role of this dynamic network in initiating CCA, as well as regulating the course of this malignancy. Actin rearrangements not only occur because of signaling pathways, but also regulate and modify cellular signaling. This emphasizes the importance of the actin cytoskeleton itself as cause for aberrant signaling and in promoting tumorigenic phenotypes. We will highlight the impact of aberrant signaling networks on the actin cytoskeleton and its rearrangement as potential cause for CCA. Often, these exact mechanisms in CCA are limited understood and still must be elucidated. Indeed, focusing future research on how actin affects and regulates other signaling pathways may provide more insights into the mechanisms of CCA development, progression, and metastasis. Moreover, manipulation of the actin cytoskeleton organization highlights the potential for a novel therapeutic area., Competing Interests: Declaration of Competing Interest JBA declares consultancy roles for Flagship Pioneering, SEALD and QED therapeutics., (Copyright © 2022. Published by Elsevier Inc.)

The Epidermal Growth Factor Receptor (EGFR) has been targeted through the development of selective tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAb). These molecules have shown effectiveness in a subset of patients with specific genetic alterations (i.e. gain-of-function EGFR mutations or EGFR gene amplification) and have been approved for their use in non-small-cell lung cancer (NSCLC), colorectal cancer (CRC), pancreatic cancer and head and neck cancer. In addition, extensive research is being performed in many other tumour types hoping for a future approval. However, the majority of the patients show no benefit from these molecules due to primary mechanisms of resistance, already present before treatment or show disease progression upon the acquisition of drug resistance mechanisms during the treatment. At present, the majority of patients display resistance due to alterations in genes related to the EGFR signalling pathway that eventually circumvent EGFR inhibition and allow cancer progression. Thus, in this review article we focus on the molecular mechanisms underlying drug resistance via genetic alterations leading to resistance to all anti-EGFR drugs approved by the FDA and/or EMA. We also discuss novel approaches to surmount these chemoresistance modalities., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background and Aims: Zinc finger E-box binding homeobox 1 (ZEB1) is a transcription factor that promotes metastatic and stem cell features, which has been associated with poor prognosis in cholangiocarcinoma (CCA), a desmoplastic cancer enriched in cancer-associated fibroblasts (CAFs). We aimed to define ZEB1 regulatory functions in malignant and stromal compartments of CCA., Approach and Results: Bioinformatic and immunohistochemical analyses were performed to determine correlations between ZEB1 and markers of progressiveness in human intrahepatic CCA (iCCA). Gain-of-function and loss-of-function models were generated in CCA cells and liver myofibroblasts as a model of CAFs. Conditioned media (CM) was used to unravel tumor-stroma interplay. In vivo experiments were performed using a xenograft CCA model. ZEB1 expression in tumor cells of human iCCA was associated with undifferentiated tumor and vascular invasion. In vitro, ZEB1 promoted epithelial-mesenchymal transition and stemness in tumor cells, leading to cell migration and spheroid formation. In vivo, ZEB1-overexpressing CCA cells formed larger tumors with more abundant stroma. Expression of cellular communication network factor 2 (CCN2, encoding connective tissue growth factor [CTGF]) was increased in tumor cells from ZEB1-overexpressing xenografts and correlated with ZEB1 expression in human tumors. In vitro, CM from ZEB1-overexpressing tumor cells or recombinant CTGF induced myofibroblast proliferation. ZEB1 was also expressed by CAFs in human CCA, and its expression correlated with CCN2 in myofibroblasts and CCA stroma. In mice, cotransplantation of CCA cells with ZEB1-depleted myofibroblasts reduced CCA progressiveness compared to CCA cells/ZEB1-expressing myofibroblasts. Furthermore, ZEB1 controls the expression of paracrine signals (i.e., HGF and IL6) in tumor cells and myofibroblasts., Conclusions: ZEB1 plays a key role in CCA progression by regulating tumor cell-CAF crosstalk, leading to tumor dedifferentiation and CAF activation., (© 2021 by the American Association for the Study of Liver Diseases.)

Cholangiocarcinoma (CCA) results from the malignant transformation of cholangiocytes. Primary sclerosing cholangitis (PSC) and primary biliary cholangitis (PBC) are chronic diseases in which cholangiocytes are primarily damaged. Although PSC is an inflammatory condition predisposing to CCA, CCA is almost never found in the autoimmune context of PBC. Here, we hypothesized that PBC might favor CCA immunosurveillance. In preclinical murine models of cholangitis challenged with syngeneic CCA, PBC (but not PSC) reduced the frequency of CCA development and delayed tumor growth kinetics. This PBC-related effect appeared specific to CCA as it was not observed against other cancers, including hepatocellular carcinoma. The protective effect of PBC was relying on type 1 and type 2 T cell responses and, to a lesser extent, on B cells. Single-cell TCR/RNA sequencing revealed the existence of TCR clonotypes shared between the liver and CCA tumor of a PBC host. Altogether, these results evidence a mechanistic overlapping between autoimmunity and cancer immunosurveillance in the biliary tract., Competing Interests: Disclosures: J. Tosello Boari reported personal fees from Egle Therapeutics and personal fees from Institut Curie outside the submitted work. L. Zitvogel reported "other" from everImmune, grants from Transgene, grants from Daichi Sankyo, and grants from Kaleido outside the submitted work. E. Piaggio reported personal fees from Egle Therapeutics outside the submitted work. G. Kroemer reported grants from Daiichi Sankyo, Eleor, Kaleido, Lytix Pharma, PharmaMar, Sotio, Vasculox/Tioma, Samsara Therapeutics, and Sanofi outside the submitted work; in addition, G. Kroemer is on the board of directors of the Bristol Myers Squibb Foundation France and is a scientific co-founder of everImmune, Samsara Therapeutics, and Therafast Bio. No other disclosures were reported., (© 2021 Paillet et al.)

Only a fraction of cancer patients benefits from immune checkpoint inhibitors. This may be partly due to the dense extracellular matrix (ECM) that forms a barrier for T cells. Comparing five preclinical mouse tumor models with heterogeneous tumor microenvironments, we aimed to relate the rate of tumor stiffening with the remodeling of ECM architecture and to determine how these features affect intratumoral T cell migration. An ECM-targeted strategy, based on the inhibition of lysyl oxidase, was used. In vivo stiffness measurements were found to be strongly correlated with tumor growth and ECM crosslinking but negatively correlated with T cell migration. Interfering with collagen stabilization reduces ECM content and tumor stiffness leading to improved T cell migration and increased efficacy of anti-PD-1 blockade. This study highlights the rationale of mechanical characterizations in solid tumors to understand resistance to immunotherapy and of combining treatment strategies targeting the ECM with anti-PD-1 therapy., Competing Interests: AN, JV, LV, TG, SB, CK, MP, GR, PD, KP, RB, IC, JC, LF, FG, ED No competing interests declared, (© 2021, Nicolas-Boluda et al.)