Figure 1. TAMs are the predominant source of PD-L1 in CCA. (A) Representative images (left and middle panels) of PD-L1 (brown staining, black arrowhead) plus CD68 (red staining, red arrowhead) coimmunostaining (n = 33) and PD-L1 (brown staining) plus CK-19 (red staining) coimmunostaining (n = 18) in human resected CCA specimens. Percentage of patients with positive PD-L1/CD68 costaining and PD-L1/CK19 costaining, respectively (right panel). Scale bars: 40 μm. (B) Histograms show expression of PD-L1 + macrophages in human CCA tumors. (C-F) Flow cytometry analysis of normal WT mouse livers (from WT mice without tumors) as well as adjacent livers and tumors of mice 28 days after orthotopic implantation of 1 × 10 6 SB (murine CCA) cells. (C) Percentage of PD-L1 + macrophages (Mφ) of total macrophages (CD45 + CD11b + F4/80 + ) in WT mouse normal liver, tumor-adjacent liver, or tumor. Fluorescence Minus One (FMO) controls were used for each independent experiment to establish gates (See Supplemental Figure 1A for gating strategy) (n ≥ 8). Representative histograms show expression of PD-L1 + macrophages. (D) Percentage of CD206 + TAMs (left panel) and PD-L1 + CD206 + TAMs (middle panel) of F4/80 int macrophages (CD45 + CD11b + F4/80 int ) in WT mouse liver, tumor-adjacent liver, or tumor. Representative contour plots (right panel) show CD206 and PD-L1 expression of F4/80 int macrophages (n ≥ 7). (E) Percentage of PD-L1 + CD206macrophages or PD-L1 + CD206 + macrophages (CD11b + F4/80 + ) of CD45 + cells from SB tumors (n = 28). (F) Percentage of PD-L1 expression in myeloid cells from SB tumors.
Cholangiocarcinoma (CCA) represents a heterogeneous group of epithelial tumours that are classified according to anatomical location as intrahepatic (iCCA), perihilar (pCCA), or distal (dCCA). Although surgical resection and liver transplantation following neoadjuvant therapy are potentially curative options for a subset of patients with early-stage disease, the currently available medical therapies for CCA have limited efficacy. Immunotherapeutic strategies such as immune checkpoint blockade (ICB) harness the host immune system to unleash an effective and durable antitumour response in a subset of patients with a variety of malignancies. However, response to ICB monotherapy has been relatively disappointing in CCA. CCAs are desmoplastic tumours with an abundant tumour immune microenvironment (TIME) that contains immunosuppressive innate immune cells such as tumour-associated macrophages and myeloid-derived suppressor cells. A subset of CCAs may be classified as immune 'hot' tumours with a high density of CD8 + T cells and enhanced expression of immune checkpoint molecules. Immune 'hot' tumour types are associated with higher response rates to ICB. However, the suboptimal response rates to ICB monotherapy in human clinical trials of CCA imply that the preponderance of CCAs are immune 'cold' tumours with a non-T cell infiltrated TIME. An enhanced comprehension of the immunobiology of CCA, particularly the innate immune response to CCA, is essential in the effort to develop effective combination immunotherapeutic strategies that can target a larger subset of CCAs.
Cholangiocarcinoma (CCA) is an aggressive biliary tract malignancy with a poor overall prognosis. There is a critical need to develop effective targeted therapies for the treatment of this lethal disease. In an effort to address this challenge, preclinical in vivo studies have become paramount in understanding CCA carcinogenesis, progression, and therapy. Various CCA animal models exist including carcinogen-based models in which animals develop CCA after exposure to a carcinogen, genetically engineered mouse models in which genetic changes are induced in mice leading to CCA, murine syngeneic orthotopic models, as well as xenograft tumors derived from xenotransplantation of CCA cells, organoids, and patient-derived tissue. Each type has distinct advantages as well as shortcomings. In the ideal animal model of CCA, the tumor arises from the biliary tract in an immunocompetent host with a species-matched tumor microenvironment. Such a model would also be time-efficient, recapitulate the genetic and histopathological features of human CCA, and predict therapeutic response in humans. Recently developed biliary tract transduction and orthotopic syngeneic transplant mouse models encompass several of these elements. Herein, we review the different animal models of CCA, their advantages and deficiencies, as well as features which mimic human CCA.
BackgroundMagnetic resonance colonography (MRC) has been developed to assess inflammatory bowel diseases. We aimed to assess the feasibility of MRC in rats with TNBS-induced chronic colitis and to confront imaging results with fibrosis and stenosing features of the model.Materials and MethodsChronic colitis was induced in 12 rats by weekly intra-rectal injection of increasing doses of TNBS for 6 weeks, while 8 control rats received the vehicle. At week 7, MRC was performed. Fibrosis scores were assessed and fibrosis mediators measured.ResultsChronic colitis was associated with significant body weight loss (p<0.0001) and higher colon weight/length compared to controls (p = 0.0004). Fibrosis mediators and histological scores were significantly higher in rats with TNBS than in controls: α-SMA expression (0.9 versus 0.61, p = 0.0311) and fibrosis score (p = 0.0308). Colon wall thickness was higher in rats with TNBS than in controls: maximal thickness (2.38 versus 0.74 mm, p<0.0001) and minimal thickness (1.33 versus 0.48 mm, p<0.0001). Wall signal intensity on T2w images was higher in rats with TNBS than in controls (9040 versus 6192, p = 0.0101) and correlated with fibrosis score (r = 0.5214; p = 0.04). Luminal narrowing was higher in rats with TNBS (50.08 versus 10.33%, p<0.0001) and correlated with α-SMA expression (r = 0.5618; p = 0.01). Stenosis was observed in 7/9 rats with TNBS and in no controls (p = 0.0053).ConclusionsMRC is feasible and easily distinguishes rats with colitis from controls. MRC signs correlated with fibrosis parameters. MRC evaluation may be part of a new anti-fibrosis drug assessment in experimental models of chronic colitis.
Rats with TNBS-induced chronic colitis exhibited colon fibrosis associated with higher TGF-β signaling. Proteasome inhibition by bortezomib had no effect on fibrosis in our experimental conditions.
Myofibroblasts are matrix-producing cells with contractile properties, usually characterized by de novo expression of alpha-smooth muscle actin, that arise in fibrotic diseases. Hepatic stellate cells (HSCs), known as perisinusoidal cells containing auto-fluorescent vitamin A, are the major although not exclusive source of myofibroblasts in the injured liver. Portal myofibroblasts (PMFs) have been defined as liver myofibroblasts derived from cells that are distinct from HSCs and located in the portal tract. Here, we describe the protocol we have established to obtain rat PMFs in culture. In this method, the biliary tree is (i) separated from the liver parenchyma by in situ enzymatic perfusion of the liver, (ii) minced and further digested in vitro, until bile duct segments are isolated by sequential filtration. Bile duct isolates free of HSC contaminants, form small cell clusters, which initially comprise a large majority of epithelial cells. In culture conditions (fetal bovine serum) that provide a growth advantage to mesenchymal cells over epithelial cells, the epithelial cells die and detach from the substrate, while spindle-shaped cells outgrow from the periphery of the cell clusters, as shown by video-microscopy. These cells are highly proliferative and after 4–5 days, the culture is composed exclusively of fully differentiated myofibroblasts, which express alpha-smooth muscle actin and collagen 1, and secrete abundant collagen. We found no evidence for epithelial-mesenchymal transition, i.e., no co-expression of alpha-smooth muscle actin and cytokeratin at any stage, while cytokeratin becomes undetectable in the confluent cells. PMFs obtained by this method express the genes that were previously reported to be overexpressed in non-HSC or portal fibroblast-derived liver myofibroblasts as compared to HSC-derived myofibroblasts, including the most discriminant, collagen 15, fibulin 2, and Thy-1. After one passage, PMFs retain the same phenotypic features as in primary culture. In conclusion, this straightforward and reproducible method of PMF culture, can be used to identify new markers of PMFs at different stages of differentiation, to compare their phenotype with those of HSC-MFs and ultimately determine their progenitors and specific functions in liver wound-healing.
Morreton virus (MORV) is a novel oncolytic Vesiculovirus, genetically distinct from vesicular stomatitis virus (VSV). we report that MORV induced potent cytopathic effects in a panel of cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) cell lines. In addition, high intranasal doses of MORV were not associated with significant adverse effects and were well tolerated in mice bearing liver tumor xenografts and syngeneic liver cancers. Furthermore, single intratumoral treatments with MORV (1 x 10 7 TCID 50 ) triggered a robust antitumor immune response leading to substantial tumor regression and disease control in a syngeneic CCA model, using 10-fold lower dose compared to VSV (1 x 10 8 TCID 50 ). In addition, MORV and VSV both induced prominent tumor growth delay in immunodeficient mice bearing Hep3B hepatocellular carcinoma (HCC) but not in mice bearing HuCCT-1 CCA xenografts. Our findings indicate that wild-type MORV is safe and can induce potent tumor regression in HCC and CCA animal models without adverse events via immune-mediated and immune-independent mechanisms. Further development and clinical translation of MORV as virotherapy for liver cancers are warranted.
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