Syngeneic murine models with distinct immune microenvironments represent subsets of human intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: Cholangiocarcinoma (CCA) is a poorly immunogenic malignancy associated with limited survival. Syngeneic immunocompetent mouse models of CCA are an essential tool to elucidate the tumor immune microenvironment (TIME), understand mechanisms of tumor immune evasion, and test novel immunotherapeutic strategies. The scope of this study was to develop and characterize immunocompetent CCA models with distinct genetic drivers, and correlate tumor genomics, immunobiology, and therapeutic response. METHODS: A multifaceted approach including scRNA-seq, CITE-seq, whole exome and bulk RNA sequencing was employed. FDA-approved PD-1/PD-L1 antibodies were tested in humanized PD-1/PD-L1 mice (HuPD-H1). RESULTS: A genetic mouse model of intrahepatic CCA (iCCA) driven by intrabiliary transduction of Fbxw7ΔF/Akt that mimics human iCCA was generated. From the Fbxw7ΔF/Akt tumors, a murine cell line (FAC) and syngeneic model with genetic and phenotypic characteristics of human iCCA were developed. Established SB1 (YAPS127A/Akt) and KPPC (KrasG12Dp53L/L) models were compared to the FAC model. Although the models had transcriptomic similarities, they had substantial differences as well. Mutation patterns of FAC, SB1, and KPPC cells matched different mutational signatures in Western and Japanese CCA patient cohorts. KPPC tumors had a high tumor mutation burden. FAC tumors had a T cell-infiltrated TIME, while SB1 tumors had a preponderance of suppressive myeloid cells. FAC, SB1, and KPPC tumors matched different immune signatures in human iCCA cohorts. Moreover, FAC, SB1, and KPPC tumor-bearing HuPD-H1 mice displayed differential responses to nivolumab or durvalumab. CONCLUSIONS: Syngeneic iCCA models display a correlation between tumor genotype and TIME phenotype, with differential responses to FDA-approved immunotherapies. This study underscores the importance of leveraging multiple preclinical models to understand responses to immunotherapy in different genetic subsets of human CCA. IMPACT AND IMPLICATIONS: Understanding the relationship between tumor genotype and the phenotype of the immune microenvironment is an unmet need in cholangiocarcinoma (CCA). Herein, we use syngeneic murine models of intrahepatic CCA with different genetic drivers to demonstrate a correlation between tumor genotype and immune microenvironment phenotype in murine models, which is associated with differential responses to FDA-approved immunotherapies. This information will help guide other preclinical studies. Additionally, it emphasizes that immune checkpoint inhibition in patients with CCA is not a "one-size-fits-all" approach. Our observations suggest that, as for targeted therapies, patients should be stratified and selected for treatment according to their tumor genetics.

Characterization of Morreton virus as an oncolytic virotherapy platform for liver cancers.

BACKGROUND: Morreton virus (MORV) is an oncolytic Vesiculovirus , genetically distinct from vesicular stomatitis virus (VSV). AIM: To report that MORV induced potent cytopathic effects (CPEs) in cholangiocarcinoma (CCA) and hepatocellular carcinoma (HCC) in vitro models. APPROACH AND RESULTS: In preliminary safety analyses, high intranasal doses (up to 10 10 50% tissue culture infectious dose [TCID 50 ]) of MORV were not associated with significant adverse effects in immune competent, non-tumor-bearing mice. MORV was shown to be efficacious in a Hep3B hepatocellular cancer xenograft model but not in a CCA xenograft HuCCT1 model. In an immune competent, syngeneic murine CCA model, single intratumoral treatments with MORV (1 × 10 7 TCID 50 ) triggered a robust antitumor immune response leading to substantial tumor regression and disease control at a dose 10-fold lower than VSV (1 × 10 8 TCID 50 ). MORV led to increased CD8 + cytotoxic T cells without compensatory increases in tumor-associated macrophages and granulocytic or monocytic myeloid-derived suppressor cells. CONCLUSIONS: Our findings indicate that wild-type MORV is safe and can induce potent tumor regression via immune-mediated and immune-independent mechanisms in HCC and CCA animal models without dose limiting adverse events. These data warrant further development and clinical translation of MORV as an oncolytic virotherapy platform.

Rethinking Immune Check Point Inhibitors Use in Liver Transplantation: Implications and Resistance.

Immune checkpoint inhibitors (ICIs) have revolutionized cancer therapy, including the two most common liver tumors, hepatocellular carcinoma and cholangiocarcinoma, but their use in the peri-transplantation period is controversial. ICI therapy aims to heighten cytotoxic T lymphocytes response against tumors. However, tumor recurrence is common owing to tumor immune response escape involving ablation of CTL response by interfering with antigen presentation, triggering CLT apoptosis and inducing epigenetic changes that promote ICI therapy resistance. ICI can also affect tissue resident memory T cell population, impact tolerance in the post-transplant period, and induce acute inflammation risking graft survival post-transplant. Their interaction with immunosuppression may be key in reducing tumor burden and may thus, require multimodal therapy to treat these tumors. This review summarizes ICI use in the liver transplantation period, their impact on tolerance and resistance, and new potential therapies for combination or sequential treatments for liver tumors.

Macrophage RAGE activation is proinflammatory in NASH.

Intrahepatic macrophages in nonalcoholic steatohepatitis (NASH) are heterogenous and include proinflammatory recruited monocyte-derived macrophages. The receptor for advanced glycation endproducts (RAGE) is expressed on macrophages and can be activated by damage associated molecular patterns (DAMPs) upregulated in NASH, yet the role of macrophage-specific RAGE signaling in NASH is unclear. Therefore, we hypothesized that RAGE-expressing macrophages are proinflammatory and mediate liver inflammation in NASH. Compared with healthy controls, RAGE expression was increased in liver biopsies from patients with NASH. In a high-fat, -fructose, and -cholesterol-induced (FFC)-induced murine model of NASH, RAGE expression was increased, specifically on recruited macrophages. FFC mice that received a pharmacological inhibitor of RAGE (TTP488), and myeloid-specific RAGE KO mice (RAGE-MKO) had attenuated liver injury associated with a reduced accumulation of RAGE+ recruited macrophages. Transcriptomics analysis suggested that pathways of macrophage and T cell activation were upregulated by FFC diet, inhibited by TTP488 treatment, and reduced in RAGE-MKO mice. Correspondingly, the secretome of ligand-stimulated BM-derived macrophages from RAGE-MKO mice had an attenuated capacity to activate CD8+ T cells. Our data implicate RAGE as what we propose to be a novel and potentially targetable mediator of the proinflammatory signaling of recruited macrophages in NASH.

Noncanonical TRAIL Signaling Promotes Myeloid-Derived Suppressor Cell Abundance and Tumor Growth in Cholangiocarcinoma.

BACKGROUND & AIMS: Proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling as a cause of cancer cell death is a well-established mechanism. However, TRAIL-receptor (TRAIL-R) agonists have had very limited anticancer activity in human beings, challenging the concept of TRAIL as a potent anticancer agent. Herein, we aimed to define mechanisms by which TRAIL+ cancer cells can leverage noncanonical TRAIL signaling in myeloid-derived suppressor cells (MDSCs) promoting their abundance in murine cholangiocarcinoma (CCA). METHODS: Multiple immunocompetent syngeneic, orthotopic models of CCA were used. Single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing of CD45+ cells in murine tumors from the different CCA models was conducted. RESULTS: In multiple immunocompetent murine models of CCA, implantation of TRAIL+ murine cancer cells into Trail-r-/- mice resulted in a significant reduction in tumor volumes compared with wild-type mice. Tumor-bearing Trail-r-/- mice had a significant decrease in the abundance of MDSCs owing to attenuation of MDSC proliferation. Noncanonical TRAIL signaling with consequent nuclear factor-κB activation in MDSCs facilitated enhanced MDSC proliferation. Single-cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing of immune cells from murine tumors showed enrichment of a nuclear factor-κB activation signature in MDSCs. Moreover, MDSCs were resistant to TRAIL-mediated apoptosis owing to enhanced expression of cellular FLICE inhibitory protein, an inhibitor of proapoptotic TRAIL signaling. Accordingly, cellular FLICE inhibitory protein knockdown sensitized murine MDSCs to TRAIL-mediated apoptosis. Finally, cancer cell-restricted deletion of Trail significantly reduced MDSC abundance and murine tumor burden. CONCLUSIONS: Our findings highlight the therapeutic potential of targeting TRAIL+ cancer cells for treatment of a poorly immunogenic cancer.

Noncanonical TRAIL Signaling Promotes Myeloid-Derived Suppressor Cell Abundance and Tumor Progression in Cholangiocarcinoma.

Proapoptotic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling as a cause of cancer cell death is a well-established mechanism. However, TRAIL-receptor (TRAIL-R) agonists have had very limited anticancer activity in humans, challenging the concept of TRAIL as a potent anticancer agent. Herein, we demonstrate that TRAIL + cancer cells can leverage noncanonical TRAIL signaling in myeloid-derived suppressor cells (MDSCs) promoting their abundance in murine cholangiocarcinoma (CCA). In multiple immunocompetent syngeneic, orthotopic murine models of CCA, implantation of TRAIL + murine cancer cells into Trail-r -/- mice resulted in a significant reduction in tumor volumes compared to wild type mice. Tumor bearing Trail-r -/- mice had a significant decrease in the abundance of MDSCs due to attenuation of MDSC proliferation. Noncanonical TRAIL signaling with consequent NF-κB activation in MDSCs facilitated enhanced MDSC proliferation. Single cell RNA sequencing and cellular indexing of transcriptomes and epitopes by sequencing (CITE-Seq) of CD45 + cells in murine tumors from three distinct immunocompetent CCA models demonstrated a significant enrichment of an NF-κB activation signature in MDSCs. Moreover, MDSCs were resistant to TRAIL-mediated apoptosis due to enhanced expression of cellular FLICE inhibitory protein (cFLIP), an inhibitor of proapoptotic TRAIL signaling. Accordingly, cFLIP knockdown sensitized murine MDSCs to TRAIL-mediated apoptosis. Finally, cancer cell-restricted deletion of Trail significantly reduced MDSC abundance and murine tumor burden. In summary, our findings define a noncanonical TRAIL signal in MDSCs and highlight the therapeutic potential of targeting TRAIL + cancer cells for the treatment of a poorly immunogenic cancer.

Cholangiocarcinoma: what are the most valuable therapeutic targets - cancer-associated fibroblasts, immune cells, or beyond T cells?

INTRODUCTION: CCAs are dense and desmoplastic tumors with an abundant tumor microenviroment (TME). The evolving TME is characterized by reciprocal interactions between cancer cells and their environment and is essential in facilitating tumor progression. The TME has nonimmune and immune components. Nonimmune cell types include cancer-associated fibroblasts (CAFs) and endothelial cells accompanying tumor angiogenesis. Immune cell types include elements of the innate and adaptive immune response, and can have pro-tumor or antitumor roles. The TME can shape treatment response and resistance. Therefore, elements of the TME are attractive therapeutic targets. TME targeting therapies have been evaluated in preclinical and clinical studies but only a small subset of patients has a meaningful response. AREAS COVERED: We discuss the TME components and potential TME targeting strategies. Literature search was performed on PubMed and ClinicalTrials.gov until October 2021. EXPERT OPINION: Elucidating the CCA TME is essential for developing effective treatment strategies. Preclinical models that recapitulate the disease (such as organoids) are important tools in uncovering the intricate cross talk in the CCA TME. Characterization of patient-derived specimens using multi-omic and single-omic technologies can dissect the cellular interplay in the CCA TME, which can guide development of effective treatment strategies and identify biomarkers for patient stratification.

Targeting tumor-associated macrophages and granulocytic myeloid-derived suppressor cells augments PD-1 blockade in cholangiocarcinoma.

Immune checkpoint blockade (ICB) has revolutionized cancer therapeutics. Desmoplastic malignancies, such as cholangiocarcinoma (CCA), have an abundant tumor immune microenvironment (TIME). However, to date, ICB monotherapy in such malignancies has been ineffective. Herein, we identify tumor-associated macrophages (TAMs) as the primary source of programmed death-ligand 1 (PD-L1) in human and murine CCA. In a murine model of CCA, recruited PD-L1+ TAMs facilitated CCA progression. However, TAM blockade failed to decrease tumor progression due to a compensatory emergence of granulocytic myeloid-derived suppressor cells (G-MDSCs) that mediated immune escape by impairing T cell response. Single-cell RNA sequencing (scRNA-Seq) of murine tumor G-MDSCs highlighted a unique ApoE G-MDSC subset enriched with TAM blockade; further analysis of a human scRNA-Seq data set demonstrated the presence of a similar G-MDSC subset in human CCA. Finally, dual inhibition of TAMs and G-MDSCs potentiated ICB. In summary, our findings highlight the therapeutic potential of coupling ICB with immunotherapies targeting immunosuppressive myeloid cells in CCA.

Animal models of cholangiocarcinoma.

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.

Immunobiology of cholangiocarcinoma.

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.

Endoplasmic reticulum stress induces inverse regulations of major functions in portal myofibroblasts during liver fibrosis progression.

Portal myofibroblasts (PMF) form a sub-population of highly proliferative and proangiogenic liver myofibroblasts that derive from portal mesenchymal progenitors. Endoplasmic reticulum (ER) stress was previously shown to modulate fibrogenesis, notably in the liver. Our aim was to determine if ER stress occurred in PMF and affected their functions. PMF were obtained after their expansion in vivo from bile duct-ligated (BDL) rats and referred to as BDL PMF. Compared to standard PMF obtained from normal rats, BDL PMF were more myofibroblastic, as assessed by higher alpha-smooth muscle actin expression and collagen 1 production. Their proangiogenic properties were also higher, whereas their proliferative and migratory capacities were lower. CHOP expression was detected in the liver of BDL rats, at the leading edge of portal fibrosis where PMF accumulate. BDL PMF displayed ER dilatation and an overexpression of the PERK pathway downstream targets, Chop, Gadd34 and Trb3, in comparison with standard PMF. In vitro, the induction of ER stress by tunicamycin in standard PMF, caused a decrease in their proliferative and migratory activity, and an increase in their proangiogenic activity, without affecting their myofibroblastic differentiation. Conversely, the treatment of BDL PMF with the PERK inhibitor GSK2656157 reduced ER stress, which caused a decrease in their angiogenic properties, and restored their proliferative and migratory capacity. In conclusion, PMF develop ER stress as they expand with the progression of fibrosis, which further increases their proangiogenic activity, but also inhibits their proliferation and migration. This phenotypic switch may restrict PMF expansion while they support angiogenesis.

Culture Model of Rat Portal Myofibroblasts.

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.

2,4,6-trinitrobenzene sulfonic acid-induced chronic colitis with fibrosis and modulation of TGF-ß1 signaling.

AIM: To investigate whether targeting proteasome might reverse intestinal fibrosis in rats. METHODS: Chronic colitis was induced in rats by repeated administration of increasing dose of 2,4,6-trinitrobenzene sulfonic acid (TNBS, 15, 30, 45, 60, 60, 60 mg) by rectal injection for 6 wk (from day 0 to day 35), while control rats received the vehicle. TNBS + bortezomib (BTZ) rats received intraperitoneal injections of BTZ twice weekly (from day 37 to day 44) at a dose of 25 mg/kg, whereas the control and TNBS groups received the same amount of the vehicle. Histologic scoring of inflammation and fibrosis was performed. Colonic production of transforming growth factor (TGF)-ß was measured by ELISA. Colon fibrosis-related proteins such as phospho-p38, phospho-SMAD2/3, Akt and peroxisome proliferator activated receptor γ (PPARγ) were studied by western blot. Expression of the tight junction proteins, occludin and claudin-1, were assessed by Western blot. Colon proteasome activities (chymotrypsin-like and trypsin-like activities) were assessed. RESULTS: TNBS-treated rats had a higher colon weight/length ratio compared to control rats (P < 0.01). Furthermore, fibrosis and inflammation scores were higher in TNBS-treated rats compared to control rats (P < 0.01 for both). Colonic production of TGF-ß production tended to be higher in TNBS-treated rats (P < 0.06). Fibrosis-related proteins such as phospho-p38, phospho-SMAD2/3, and PPARγ were significantly higher in TNBS-treated rats compared to control rats (all P < 0.05). TNBS rats had a higher expression of Akt compared to control rats (P < 0.01). Tight junction proteins were modified by repeated TNBS challenge: colon occludin expression rose significantly (P < 0.01), whereas claudin-1 expression fell (P < 0.01). Bortezomib inhibition significantly decreased chymotrypsin-like activity (P < 0.05), but had no significant effect on trypsin-like activity (P > 0.05). In contrast, bortezomib had no effect on other studied parameters such as fibrosis score, TGF-ß signaling, or tight junction expression (P > 0.05 for all). CONCLUSION: Rats with TNBS-induced chronic colitis exhibited colon fibrosis associated with higher TGF-ß signaling. Proteasome inhibition by bortezomib had no effect on fibrosis in our experimental conditions.

Magnetic resonance colonography for fibrosis assessment in rats with chronic colitis.

BACKGROUND: Magnetic 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 METHODS: Chronic 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. RESULTS: Chronic 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). CONCLUSIONS: MRC 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.