Liver Immune Microenvironment Research Articles

Abstract PO026: Bile acid signaling remodels the iCCA immune microenvironment

Abstract Intrahepatic cholangiocarcinoma (iCCA) primarily arises from the transformation of cholangiocytes (epithelial cells of the bile ducts), is diagnosed at late stage and has a dismal prognosis (average 5-year survival after resection is 32%). An alarming increase in its incidence over the past decades and the scarce treatment options available urges for the development of novel therapeutic strategies to tackle this malignancy. Immunotherapy has had limited success in iCCA. Possible explanations are that iCCA immune microenvironment is poor in CD8+ T cells and rich in immunosuppressive innate immune cells. The immune landscape of iCCA is determined by the aetiology of the liver insult (e.g. viral, cholestatic) and these different iCCA immune landscapes have not yet been characterized. Therefore, in depth studies on the immunobiology of iCCA are essential to develop new immunotherapy strategies where innate immune cells play a prominent role. We hypothesized that bile acids (BA) are a potential, valid therapeutic target for iCCA, as they strongly influence liver immune microenvironment and proliferation. This idea is supported by the fact that BA have an immunomodulatory role through inhibition of NF-KB and reduction of macrophage production of pro-inflammatory cytokines, suppressing cholangiocyte transformation in iCCA. Since BA and derivatives have recently been approved for the treatment of human chronic cholestatic diseases and primary sclerosis cholangitis, but have not yet been tested to treat iCCA, the proposed project might directly offer evidence for a second use of these drugs in patients with iCCA. Using novel preclinical mouse models of iCCA and human iCCA samples, our objective is to develop new therapeutic strategies for iCCA treatment based on BA signalling, that would impair tumour growth and foster the immune response against the tumour. In order to investigate the effects that BA can exert in tumour initiation and development, we adopted a iCCA mouse model based on the hydrodynamic tail-vein delivery of genetic elements specifically in hepatocytes, which differentiate into cholangiocytes and ultimately leads to the development of iCCA, while conserving a competent immune system. We found that by feeding the animals with an endogenous BA-enriched diet (BA diet), we reduced the overall tumor burden and strikingly increased overall survival. The positive response to the BA diet was associated to an inhibition of tumor proliferation and a robust increase in the abundance and activation of inflammatory monocytes and T cells. Our preliminary results indicate that mimicking the effect of BA could represent a novel immunotherapeutic strategy for iCCA. We will present these data as well as ongoing studies including fluorescence-activated cell sorting and single-cell RNA seq sequencing, focused on understanding the cooperation between different immune cell types that foster response against the tumor. Citation Format: María García-Beccaria, Mirian Fernández-Vaquero, Maria Reich, Dominik Pfister, Diran Herebian, Benjamin Goeppert, Darjus Felix Tschaharganeh, Verena Keitel, Mathias Heikenwälder. Bile acid signaling remodels the iCCA immune microenvironment [abstract]. In: Proceedings of the AACR Special Conference: Advances in the Pathogenesis and Molecular Therapies of Liver Cancer; 2022 May 5-8; Boston, MA. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(17_Suppl):Abstract nr PO026.

Neuregulin 4 suppresses NASH-HCC development by restraining tumor-prone liver microenvironment

The mammalian liver comprises heterogeneous cell types within its tissue microenvironment that undergo pathophysiological reprogramming in disease states, such as non-alcoholic steatohepatitis (NASH). Patients with NASH are at an increased risk for the development of hepatocellular carcinoma (HCC). However, the molecular and cellular nature of liver microenvironment remodeling that links NASH to liver carcinogenesis remains obscure. Here, we show that diet-induced NASH is characterized by the induction of tumor-associated macrophage (TAM)-like macrophages and exhaustion of cytotoxic CD8+ Tcells in the liver. The adipocyte-derived endocrine factor Neuregulin 4 (NRG4) serves as a hormonal checkpoint that restrains this pathological reprogramming during NASH. NRG4 deficiency exacerbated the induction of tumor-prone liver immune microenvironment and NASH-related HCC, whereas transgenic NRG4 overexpression elicited protective effects in mice. In a therapeutic setting, recombinant NRG4-Fc fusion protein exhibited remarkable potency in suppressing HCC and prolonged survival in the treated mice. These findings pave the way for therapeutic intervention of liver cancer by targeting the NRG4 hormonal checkpoint.

Research Progress on the Microenvironment and Immunotherapy of Advanced Non-Small Cell Lung Cancer With Liver Metastases.

Lung cancer is a malignant tumor with the highest morbidity and mortality, and more than 75% of patients are diagnosed at an advanced stage. Liver metastases occur in 20% of non-small cell lung cancer patients, and their prognosis are poor. In recent years, immune checkpoint inhibitor monotherapy and combination therapy have made breakthrough progress in advanced Non-small cell lung cancer (NSCLC) patients. However, compared with the overall population, the liver metastases population was an independent prognostic factor for poor immunotherapy response. Whether and how immunotherapy can work in NSCLC patients with liver metastases is a major and unresolved challenge. Although more and more data have been disclosed, the research progress of NSCLC liver metastasis is still limited. How liver metastasis modulates systemic antitumor immunity and the drug resistance mechanisms of the liver immune microenvironment have not been elucidated. We systematically focused on non-small cell lung cancer patients with liver metastases, reviewed and summarized their pathophysiological mechanisms, immune microenvironment characteristics, and optimization of immunotherapy strategies.

SAT364 - Effect of tenofovir disoproxil fumarate treatment on intrahepatic viral burden and liver immune microenvironment in chronic hepatitis B patients with minimally elevated serum alanine aminotransferase

SAT364 - Effect of tenofovir disoproxil fumarate treatment on intrahepatic viral burden and liver immune microenvironment in chronic hepatitis B patients with minimally elevated serum alanine aminotransferase

Abstract 2531: a

Abstract Hepatocellular carcinoma (HCC) is an emerging health crisis due to rapidly increasing incidence rates and an overall 5-year survival rate of 18%. Consequently, there is an urgent need for new therapies and better preventive strategies. Aging and obesity are two of the biggest risk factors for HCC, and with ever-increasing aging and obese populations, uncovering how aging and obesity drive the increased risk for HCC has become imperative. Aging and obesity are well known to be characterized by immune dysregulation leading to persistent inflammation, and a dysregulated immune microenvironment is also a common theme shared by multiple hallmarks of HCC. In this study, we aim to assess the immune microenvironment of aged and obese mouse livers by performing spatial phenotyping of distinct immune signatures which can potentially predispose hepatocytes to HCC. To address this, we used Akoya’s ultra-high multiplexed CODEX (CO-Detection by indEXing) imaging system to interrogate 31 different biomarkers in liver samples at the single cell level. Specifically, we compared the liver immune microenvironment of mice within 2 different treatment groups (old mice on a high fat diet (HFD) and young mice on a normal diet (ND)) to the immune landscape of liver tumor tissue. Our goal is to identify distinct immune phenotypes present in both, liver tumor tissues and livers of HFD mice, compared to ND livers - to better understand how immune dysregulation can predispose an aged and obese liver to HCC. Citation Format: Adarsh Rajesh, Aaron Havas, Siva Karthik Varanasi, Katrina Evans, Bassem B. Cheikh, Clemens Duerrschmid, Najiba Mammadova, Peter Adams, Susan Kaech. a [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2531.

Characterization of the liver immune microenvironment in liver biopsies from patients with chronic HBV infection

Background & AimsWe aim to describe the liver immune microenvironment by analyzing liver biopsies from patients with chronic HBV infection (CHB). Host immune cell signatures and their corresponding localization were characterized by analyzing the intrahepatic transcriptome in combination with a custom multiplex immunofluorescence panel.MethodMatching FFPE and fresh frozen liver biopsies were collected from immune active patients within the open-label phase IV study GS-US-174-0149. RNA-Seq was conducted on 53 CHB liver biopsies from 46 patients. Twenty-eight of the 53 samples had matched FFPE biopsies and were stained with a 12-plex panel including cell segmentation, immune and viral biomarkers. Corresponding serum samples were screened using the MSD Human V-plex Screen Service to identify peripheral correlates for the immune microenvironment.ResultsUsing unsupervised clustering of the transcriptome, we reveal two unique liver immune signatures classified as immune high and immune low based on the quantification of the liver infiltrate gene signatures. Multiplex immunofluorescence analysis demonstrated large periportal lymphoid aggregates in immune high samples consisting of CD4 and CD8 T cells, B cells and macrophages. Differentiation of the high and low immune microenvironments was independent of HBeAg status and peripheral viral antigen levels. In addition, longitudinal analysis indicates that treatment and normalization of ALT correlates with a decrease in liver immune infiltrate and inflammation. Finally, we screened a panel of peripheral biomarkers and identified ICAM-1 and CXCL10 as biomarkers that strongly correlate with these unique immune microenvironments.ConclusionThese data provide a description of immune phenotypes in patients with CHB and show that immune responses are downregulated in the liver following nucleotide analogue treatment. This may have important implications for both the safety and efficacy of immune modulator programs aimed at HBV cure.Lay summaryLiver biopsies from patients with chronic hepatitis B were submitted to RNA-Seq and multiplex immunofluorescence and identified two different liver immune microenvironments: immune high and immune low. Immune high patients showed elevated immune pathways, including interferon signaling pathways, and increase presence of immune cells. Longitudinal analysis of biopsies from treatment experienced patients showed that treatment correlates with a marked decrease in inflammation and these findings may have important implications for both safety and efficacy of immune modulator programs for HBV cure.

Prolonged oral ingestion of microplastics induced inflammation in the liver tissues of C57BL/6J mice through polarization of macrophages and increased infiltration of natural killer cells

Microplastics (< 5 mm diameter) are one of most important environmental pollutants and contaminants worldwide. However, how microplastics affect liver immune microenvironment in not well understood. Microplastics (0.5 µm) were administered orally to C57BL/6J mice for 4 consecutive weeks at the rate of 0.5 mg/day. Non-parenchymal cells were isolated from of the mice through fractionation of fresh hepatic tissues. The immune landscape for four cell populations of B cells, T cells, NK cells and macrophages in the liver tissues was then evaluated using flow cytometry. The secretion level of inflammatory cytokines and associated signaling pathway were investigated using quantitative real-time polymerase chain reaction and western blot. Oral ingestion of microplastics increases liver weight, general liver index as well as expression of serum, liver function-related indicators. Microplastics also increased the infiltration of natural killer cells and macrophages to non-parenchymal liver cells, but reduced that of B cells to the same tissues. However, microplastics had no effect on the infiltration of T cell to non-parenchymal liver cells. Ingestion of MPs also up-regulated the expression of IFN-γ, TNF-α, IL-1β, IL-6 and IL-33 mRNA, but down-regulated that of IL-4, IL-5, IL-10, IL-18 and TGF-β1. Overall, the aforementioned processes were regulated via the NF-κB pathway in the hepatic non-parenchymal cells. Microplastics disrupts inflammatory process in liver tissues via the NF-κB signaling pathway. These findings provide a strong foundation on immune processes in hepatic tissues following prolonged ingestion of microplastics.

Molecular and Cellular Mediators of the Gut-Liver Axis in the Progression of Liver Diseases.

The gut-liver axis covers the bidirectional communication between the gut and the liver, and thus includes signals from liver-to-gut (e.g., bile acids, immunoglobulins) and from gut-to-liver (e.g., nutrients, microbiota-derived products, and recirculating bile acids). In a healthy individual, liver homeostasis is tightly controlled by the mostly tolerogenic liver resident macrophages, the Kupffer cells, capturing the gut-derived antigens from the blood circulation. However, disturbances of the gut-liver axis have been associated to the progression of varying chronic liver diseases, such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and primary sclerosing cholangitis. Notably, changes of the gut microbiome, or intestinal dysbiosis, combined with increased intestinal permeability, leads to the translocation of gut-derived bacteria or their metabolites into the portal vein. In the context of concomitant or subsequent liver inflammation, the liver is then infiltrated by responsive immune cells (e.g., monocytes, neutrophils, lymphoid, or dendritic cells), and microbiota-derived products may provoke or exacerbate innate immune responses, hence perpetuating liver inflammation and fibrosis, and potentiating the risks of developing cirrhosis. Similarly, food derived antigens, bile acids, danger-, and pathogen-associated molecular patterns are able to reshape the liver immune microenvironment. Immune cell intracellular signaling components, such as inflammasome activation, toll-like receptor or nucleotide-binding oligomerization domain-like receptors signaling, are potent targets of interest for the modulation of the immune response. This review describes the current understanding of the cellular landscape and molecular pathways involved in the gut-liver axis and implicated in chronic liver disease progression. We also provide an overview of innovative therapeutic approaches and current clinical trials aiming at targeting the gut-liver axis for the treatment of patients with chronic liver and/or intestinal diseases.

The Clinical Significance and Mechanism of the Effect for Hepatitis B Virus Protein on Host Immune

The cytotoxic effect targeting hepatitis B virus (HBV) infected hepatocytes from virus-specific cytotoxic T cells and the neutralizing antibodies secreted by virus-specific B cells play an important role in the immune control and elimination of HBV. In patients with chronic hepatitis B, the liver immune microenvironment usually presents a suppression state, and virus-specific immune cells are mostly exhausted. Studies on the interaction between HBV and host immunity during infection, especially the influence of various viral proteins on immune cell function, will contribute to understanding the mechanism of the chronicity of HBV infection, disease progression, and optimization of immunotherapy against HBV. The review summarized the suppressive effects of HBV viral proteins on the host innate immunity and adaptive immune system, to help us understanding the mechanism(s) relevant to the observation that a CHB patient with HBeAg loss and lower HBsAg level is more likley achieving functionall cure. and expect to provide new sights for accelerate virus clearance and achieve functional cure of chronic hepatitis B, by removing the HBV viral proteins and consequently, liberting host immune from suppression state.

Liver Immune Microenvironment and Metastasis from Colorectal Cancer-Pathogenesis and Therapeutic Perspectives.

Simple SummaryLiver metastasis remains the major contributor in colorectal cancer-related death. It has become clear that the unique immune features of liver microenvironment take part in many steps of metastatic cascade, from pre-metastatic niche formation, tumor cell colonization to metastatic tumor establishment. Therefore, better understanding of mechanisms orchestrating the formation of a hospitable hepatic metastatic niche is necessary for the development of effective therapies. This review summarizes the current understandings of the critical role of liver immune microenvironment in metastasis development and provides therapeutic perspective on targeting the metastasis-prone microenvironment. A drastic difference exists between the 5-year survival rates of colorectal cancer patients with localized cancer and distal organ metastasis. The liver is the most favorable organ for cancer metastases from the colorectum. Beyond the liver-colon anatomic relationship, emerging evidence highlights the impact of liver immune microenvironment on colorectal liver metastasis. Prior to cancer cell dissemination, hepatocytes secrete multiple factors to recruit or activate immune cells and stromal cells in the liver to form a favorable premetastatic niche. The liver-resident cells including Kupffer cells, hepatic stellate cells, and liver-sinusoidal endothelial cells are co-opted by the recruited cells, such as myeloid-derived suppressor cells and tumor-associated macrophages, to establish an immunosuppressive liver microenvironment suitable for tumor cell colonization and outgrowth. Current treatments including radical surgery, systemic therapy, and localized therapy have only achieved good clinical outcomes in a minority of colorectal cancer patients with liver metastasis, which is further hampered by high recurrence rate. Better understanding of the mechanisms governing the metastasis-prone liver immune microenvironment should open new immuno-oncology avenues for liver metastasis intervention.

Modulation of the liver immune microenvironment by the adeno-associated virus serotype 8 gene therapy vector

Adeno-associated viruses (AAVs) are emerging as one of the vehicles of choice for gene therapy. However, the potential immunogenicity of these vectors is a major limitation of their use, leading to the necessity of a better understanding of how viral vectors engage the innate immune system. In this study, we demonstrate the immune response mediated by an AAV vector in a mouse model. Mice were infected intravenously with 4 × 1012 copies (cp)/kg of AAV8, and the ensuing immune response was analyzed using intravital microscopy during a period of weeks. Administration of AAV8 resulted in the infection of hepatocytes, and this infection led to a moderate, but significant, activation of the immune system in the liver. This host immune response involved platelet aggregation, neutrophil extracellular trap (NET) formation, and the recruitment of monocytes, B cells, and T cells. The resident liver macrophage population, Kupffer cells, was necessary to initiate this immune response, as its depletion abrogated platelet aggregation and NET formation and delayed the recruitment of immune cells. Moreover, the death of liver cells produced by this AAV was moderate and failed to result in a robust, sustained inflammatory response. Altogether, these data suggest that AAV8 is a suitable vector for gene therapy approaches.

Cell cycle-related kinase reprograms the liver immune microenvironment to promote cancer metastasis.

The liver is an immunologically tolerant organ and a common metastatic site of multiple cancer types. Although a role for cancer cell invasion programs has been well characterized, whether and how liver-intrinsic factors drive metastatic spread is incompletely understood. Here, we show that aberrantly activated hepatocyte-intrinsic cell cycle-related kinase (CCRK) signaling in chronic liver diseases is critical for cancer metastasis by reprogramming an immunosuppressive microenvironment. Using an inducible liver-specific transgenic model, we found that CCRK overexpression dramatically increased both B16F10 melanoma and MC38 colorectal cancer (CRC) metastasis to the liver, which was highly infiltrated by polymorphonuclear-myeloid-derived suppressor cells (PMN-MDSCs) and lacking natural killer T (NKT) cells. Depletion of PMN-MDSCs in CCRK transgenic mice restored NKT cell levels and their interferon gamma production and reduced liver metastasis to 2.7% and 0.7% (metastatic tumor weights) in the melanoma and CRC models, respectively. Mechanistically, CCRK activated nuclear factor-kappa B (NF-κB) signaling to increase the PMN-MDSC-trafficking chemokine C-X-C motif ligand 1 (CXCL1), which was positively correlated with liver-infiltrating PMN-MDSC levels in CCRK transgenic mice. Accordingly, CRC liver metastasis patients exhibited hyperactivation of hepatic CCRK/NF-κB/CXCL1 signaling, which was associated with accumulation of PMN-MDSCs and paucity of NKT cells compared to healthy liver transplantation donors. In summary, this study demonstrates that immunosuppressive reprogramming by hepatic CCRK signaling undermines antimetastatic immunosurveillance. Our findings offer new mechanistic insights and therapeutic targets for liver metastasis intervention.

Intrahepatic immune changes after hepatitis c virus eradication by direct-acting antiviral therapy.

The recent approval of direct acting anti-virals (DAA) has dramatically changed the landscape of hepatitis C virus (HCV) therapy. Whether viral clearance could promote liver carcinogenesis is debated. It has been hypothesized that changes in intrahepatic immune surveillance following viral cure could favour tumour growth. This study aimed at characterizing the intrahepatic immune changes induced by HCV cure following DAA therapy. Patients with compensated cirrhosis who underwent surgical resection for hepatocellular carcinoma (HCC) after sustained virological response (SVR) to DAA therapy were included. A control group of untreated HCV-infected patients with compensated cirrhosis was selected. RNA was extracted from tumoral and non-tumoral tissues and analysed using the Nanostring Immuno-Oncology-360 panel. Immune cells were quantified by immunohistochemistry. Twenty patients were included: 10 patients with a DAA-induced SVR and 10 untreated controls. All of them had a de novo BCLC 0/A HCC. Non-tumoral tissue profiling showed down-regulation of interferon-related genes (including MX1, ISG15 and IFIT1) after DAA therapy. No other differences in immune profiles/immune cell densities were identified between the two groups. The intra-tumoral immune profiles of HCCs that occurred after DAA therapy were not qualitatively or quantitatively different from those of tumours occurring in untreated patients. In conclusion, removal of HCV infection after DAA-based therapy results only in a down-regulation of interferon-stimulated genes in non-tumoral tissues from patients with cirrhosis who develop HCC. These minor changes in the liver immune microenvironment are unlikely to favour HCC occurrence or recurrence after DAA-induced SVR.

Abstract A02: Gut microbiome controls growth of liver tumors

Abstract Aim: The gut microbiome can modify tumor immunity and has been suggested to be involved in the development and growth of liver cancer as well as metastasis in the liver. However, it remains unknown how the gut microbiome controls hepatic immune responses. This study was designed to exam the effect of the gut microbiome on liver antitumor immunity, and to study potential mechanism. Experimental Procedure: An antibiotic cocktail containing 0.5g/L vancomycin, 0.5 g/L neomycin and 0.6 g/L primaxin in drinking water was given to reduce mouse gut microbiota. Control mice were kept on regular water. EL4 thymoma cells were injected s.c. to induces spontaneous liver metastasis. B16 melanoma and CT26 colon cancer cells were injected intrasplenically to form liver metastasis. Lung metastasis was induced by tail injection of tumor cells. Spontaneous hepatocellular carcinomas were studied in TRE-MYC mice. Gut bacteria and metabolic studies were performed. Results: Antibiotic cocktail efficiently depleted gut bacteria. Removing gut commensal bacteria did not affect the growth of primary s.c. EL4 tumors, but impaired formation of liver metastasis in different models. The inhibitory effect on liver metastasis by removing gut microbiome was found after intrasplenic injection of tumor cells to form liver metastasis using both B16 melanoma and CT26 colon cancer tumor cells as well as in TRE-MYC mice. Interestingly, formation of lung metastasis caused by tail vein injection of B16 cells was not impaired by antibiotics treatment, suggesting a liver specific effect. The inhibition of liver metastasis by antibiotic treatment was absent in Rag1 knockout mice, suggesting that the observed mechanism is mediated by the adaptive immune system. A detailed mechanism how the gut microbiome causes metabolic liver changes and thereby growth of liver tumors will be presented. Conclusion: Our results suggest that the gut microbiome affects the liver immune microenvironment and modulates antitumor immunity Citation Format: Chi Ma, Miaojun Han, Bernd Heinrich, Qiong Fu, Qianfei Zhang, Xin W. Wang, Giorgio Trinchieri, Tim Greten. Gut microbiome controls growth of liver tumors [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr A02.

Hepatic Dendritic Cells, the Tolerogenic Liver Environment, and Liver Disease.

The unique liver immune microenvironment favors resistance to inflammation that promotes normal physiological function. At the same time, it endows the liver with tolerogenic properties that may promote pathological processes. Hepatic dendritic cells (HDCs) initiate and orchestrate immune responses depending on signals they receive from the local environment and are thought to contribute to liver tolerance. Thus, HDCs facilitate impaired T cell responses that are observed in persistent hepatitis C virus (HCV) infection, hepatocellular carcinoma progression, and liver allograft transplantation. HDCs also participate in anti-inflammatory responses in liver ischemia-reperfusion injury (IRI). Moreover, they promote the regression of fibrosis from various fibrogenic liver injuries. These findings suggest that HDCs regulate intrahepatic immune responses, allowing the liver to maintain homeostasis and integrity even under pathological conditions. This review focuses on the tolerogenic properties of HDCs based on recent research and in relation to liver disease pathogenesis and its therapy.

Myeloid Cells and Chronic Liver Disease: a Comprehensive Review.

Myeloid cells play a major role in the sensitization to liver injury, particularly in chronic inflammatory liver diseases with a biliary or hepatocellular origin, and the interplay between myeloid cells and the liver may explain the increased incidence of hepatic osteodystrophy. The myeloid cell-liver axis involves several mature myeloid cells as well as immature or progenitor cells with the complexity of the liver immune microenvironment aggravating the mist of cell differentiation. The unique positioning of the liver at the junction of the peripheral and portal circulation systems underlines the interaction of myeloid cells and hepatic cells and leads to immune tolerance breakdown. We herein discuss the scenarios of different chronic liver diseases closely modulated by myeloid cells and illustrate the numerous potential targets, the understanding of which will ultimately steer the development of solid immunotherapeutic regimens. Ultimately, we are convinced that an adequate modulation of the liver microenvironment to modify the functional and quantitative characteristics of myeloid cells will be a successful approach to treating chronic liver diseases of different etiologies.

Immune Gene Expression Profile in Hepatocellular Carcinoma and Surrounding Tissue Predicts Time to Tumor Recurrence

Background: The antitumor immune response may play a major role in the clinical outcome of hepatocellular carcinoma (HCC). We characterized the liver immune microenvironment by direct hybridization of RNA extracted from HCC and nontumorous tissues. Methods: RNA was extracted from frozen liver tissue samples of HCC (T; n = 30) and nontumorous tissues (NT; n = 33) obtained from 38 patients. Matched samples were available for 25 patients. The immune gene expression profile was analyzed with the nCounter GX Human Immunology v2 system (NanoString Technologies), which detects the expression levels of 579 immune response-related genes simultaneously. Results: Since the immune gene expression profile of T and NT tissues was significantly different, the prognostic relevance of the liver immune microenvironment was evaluated in the T and NT samples separately. Unsupervised clustering detected two main clusters of immune gene expression both in T and in NT liver samples. In both cases, the expression clusters identified groups of patients with a significantly different median time to HCC recurrence (TTR) but similar overall survival. Based on T tissue, two groups with median TTR of 19 and 127 months, respectively, were detected (p < 0.005). Expression of genes related to T-cell activation was associated with longer TTR. The analysis of NT tissue discriminated subsets of patients with median TTR of 22 and 68 months (p < 0.05). In contrast to T tissue, a predominant inflammatory immune environment was associated with shorter TTR. Conclusions: Immune gene expression profiles predictive of TTR could be identified both in HCC and in adjacent cirrhotic tissues. Longer TTR was associated with overexpression in T tissue and downregulation in NT tissue of the immune response and of inflammation-related genes.

Abstract 3057: Gut microbiome controls liver metastasis

Abstract Aim: The gut microbiome can modify tumor immunity and has been suggested to be involved in the development of liver cancer. However, it remains unknown how the gut microbiome controls hepatic immune responses. This study was designed to exam the effect of the gut microbiome on liver antitumor immunity, and to study potential mechanism. Experimental procedure: Antibiotic cocktail in drinking water was given to remove mouse gut microbiota. Control mice were kept on regular water. EL4 thymoma cells were injected s.c. to induce spontaneous liver metastasis. B16 melanoma and CT26 colon cancer cells were injected intrasplenically to form liver metastasis. Lung metastasis was induced by tail injection of tumor cells. Results: Antibiotic cocktail efficiently depleted gut bacteria. Removing gut commensal bacteria did not affect the growth of primary s.c. tumors, but impaired formation of liver metastasis in different models. The inhibitory effect on liver metastasis by removing gut microbiome was found after intrasplenic injection of tumor cells to form liver metastasis using both B16 melanoma and CT26 colon cancer tumor cells. Interestingly, formation of lung metastasis caused by tail vein injection of B16 cells was not impaired by antibiotics treatment, suggesting a liver specific effect. The inhibition of liver metastasis by antibiotic treatment was absent in Rag1 knockout mice, suggesting that the mechanism is mediated by the adaptive immune system. Conclusion: Our results suggest that the gut microbiome affects the liver immune microenvironment and modulates antitumor immunity. Citation Format: Chi Ma, Qiong Fu, Tim Greten. Gut microbiome controls liver metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3057. doi:10.1158/1538-7445.AM2017-3057

Abstract A04: Systemic agonistic anti-CD40 treatment of tumor bearing mice modulates hepatic myeloid suppressive cells and causes immune-mediated liver damage

Abstract Immune stimulatory monoclonal antibodies are currently evaluated as antitumor agents in clinical trials. Although overall toxicity appears to be moderate, liver toxicities have been reported and are not completely understood. Therefore, we decided to study the effect of systemic CD40 antibody treatment on the liver immune microenvironment. Agonistic CD40 antibody or isotype control was injected intraperitoneally into naïve and tumor-bearing mice. Liver enzymes, histologies and immune infiltrates were analyzed after antibody administration. The effect of agonistic CD40 administration was evaluated using bone marrow chimeras and genetically modified mouse strains Cd40-/- and Nox2-/-. Mouse and human tumor-induced myeloid suppressive cells were cultured to address changes in their immune function upon CD40 ligation. Agonistic CD40 antibody caused liver damage within 24 hours after injection in two unrelated tumor models and mice strains. Using bone marrow chimeras we could demonstrate that CD40 antibody-induced hepatitis in tumor-bearing mice was dependent on the presence of CD40+ immune cells. Agonistic CD40 ligation-dependent liver damage was induced by the generation of reactive oxygen species. Furthermore, agonistic CD40 antibody resulted in increased CD80 and CD40 positive liver CD11b+Gr-1+ immature myeloid cells. CD40 ligation on tumor-induced hepatic myeloid cells or CD14+HLA-DRlow PBMC from cancer patients reduced their immune suppressor function. Collectively, agonistic CD40 antibody activates the tumor-induced liver myeloid suppressive compartment, enhancing acute inflammation and thus liver damage. Our data in murine and human myeloid suppressive cells point towards a pivotal role for CD40 as a key molecule in modulating myeloid suppressive cell plasticity. Citation Format: Jose Medina-Echeverz, Chi Ma, Tobias Eggert, Tim F. Greten. Systemic agonistic anti-CD40 treatment of tumor bearing mice modulates hepatic myeloid suppressive cells and causes immune-mediated liver damage. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A04.