Immunosuppressive treatments are broadly used to prevent or treat graft-versus-host disease (GVHD) after allogeneic hematopoietic stem cell transplantation (alloSCT). The resulting severe treatment-related immunodeficiency is the main contributor to high alloSCT-associated mortality, highlighting the medical need for non-immunosuppressive strategies to prevent GVHD. Angiogenesis represents an attractive target because its therapeutic inhibition leads to reduced inflammation without major negative effects on immunity. The major hurdle for translation is safety: current anti-angiogenic drugs lack specificity and disrupt physiological angiogenesis, which is required for repair and regeneration. In search for more specific targets, we identified carnitine palmitoyltransferase 2 (Cpt2), a key enzyme in mitochondrial long-chain fatty acid oxidation, which is selectively upregulated during acute GVHD (aGVHD)-associated pathological angiogenesis. Genetic and therapeutic inhibition of Cpt2 prevented inflammation-associated pathologic endothelial changes and preserved normal endothelial cell functions in vitro . In preclinical alloSCT models, endothelial-specific CPT2 knockout as well as therapeutic CPT2 inhibition with perhexiline reduced aGVHD-associated angiogenesis, mitigated aGVHD severity and promoted functional restoration of the endothelial phenotype. Importantly, CPT2 inhibition did not inhibit immune reconstitution, preserved the graft-versus-leukemia effect and reduced tumor growth in preclinical models. Our data indicate that Cpt2 contributes to endothelial processes driving pathological angiogenesis in aGVHD. The selective targeting of pathologic angiogenesis is a new non-immunosuppressive strategy to mitigate aGVHD.

OBJECTIVE:To investigate the impact of VEGFA and EGF genetic polymorphisms on postoperative survival, susceptibility, transcriptomics, and systemic proteomic regulation in cholangiocarcinoma (CCA). BACKGROUND:Angiogenesis and growth factor signaling via vascular endothelial growth factor A (VEGFA) and epidermal growth factor (EGF) are key drivers of CCA biology, yet the clinical relevance of their genetic variants remains poorly defined. METHODS:A cohort of 221 patients undergoing curative-intent CCA resection at Charité-Berlin was analyzed. Patients were genotyped for 6 angiogenesis-related SNPs, including VEGFA rs3025039 and EGF rs4444903, and survival associations were assessed using Cox regression. The prognostic relevance of intratumoral VEGFA and EGF mRNA expression and pathway co-expression was evaluated in an independent transcriptomic cohort. Population-level associations with CCA susceptibility and serum proteomics were analyzed in the UK Biobank (n>500,000). RESULTS:The VEGFA rs3025039 T allele was associated with improved cancer-specific survival, particularly in intrahepatic CCA, while the EGF rs4444903 G allele was linked to improved survival in perihilar CCA. High intratumoral VEGFA or EGF expression correlated with poorer survival in external validation, but this association was lost for EGF after multivariable adjustment. VEGFA -high tumors showed increased proliferative and suppressed immune transcriptional signatures, whereas EGF -high tumors displayed limited changes. UK Biobank analysis revealed increased intrahepatic CCA susceptibility among VEGFA T allele carriers, while the EGF G allele was associated with elevated circulating EGF levels. CONCLUSIONS:VEGFA and EGF polymorphisms exert distinct effects on CCA, influencing disease susceptibility, postoperative outcomes, and systemic signaling, supporting their potential as biomarkers for clinical stratification.

Hepatocellular carcinoma (HCC) is one of the most frequent causes of cancer-related death worldwide. Mirroring the complexity of human HCC with its underlying liver disease requires multilocular orthotopic tumor models in mice, where objective tumor quantification in vivo is challenging, especially longitudinally. We investigated magnetic resonance imaging (MRI) to noninvasively quantify orthotopic HCC in an immunotherapy setting. Orthotopic HCCs were induced in diethylnitrosamine (DEN)-injected mice with either CCl4-induced hepatic fibrosis or dietary metabolic dysfunction-associated steatotic liver disease (MASLD) to mirror the most frequent etiologies. Growth kinetics over the course of immune checkpoint inhibitor treatment with anti-Programmed Death Ligand 1 (αPD-L1) were modeled by measuring the overall tumor burden (OTB) using MRI and compared to untreated animals. Tumor parameters, such as tumor volume, liver weights at sacrifice and largest tumor diameter were analyzed. We demonstrate that MRI is a reliable imaging tool for both pretreatment tumor confirmation as well as the longitudinal quantification of overall tumor burden under investigational treatment. While measuring only the largest tumor diameter yielded significant differences between αPD-L1-treated animals and untreated controls in the long-term setting, these trends in tumor response were not confirmed by MRI-based OTB measurement. In MASLD-HCC, tumors did not respond to PD-L1 blockade, thus confirming the OTB data and reflecting the immunotherapy resistance observed in the human setting and highlighting the translational relevance of the model. Orthotopic fibrosis-HCC and MASLD-HCC mouse models can be enhanced through the longitudinal use of MRI while reducing endpoints and animal numbers. The DEN-CCl4 and DEN-Western Diet models mirror the αPD-L1 response from the human setting and require OTB measurement, because largest diameters may underestimate the total tumor mass in orthotopic HCC.

Handling-induced stress represents a major burden on laboratory mice and is heavily influenced by the handling technique. As such, tail-handling induces much higher stress than cup- or tunnel-handling, which is further aggravated by interventions, e.g., injections, which require even harsher fixation methods. Previous studies demonstrated that habituation protocols can improve animal welfare during handling and interventions. Here, we developed a medical training program and an alternative fixation method using an application tunnel in the context of a liver cancer model, where frequent intraperitoneal injections in male C57Bl/6 mice over many weeks are needed to induce tumor development. The training regimen consisted of 5 sessions over 2 weeks, which gradually introduced the animals to being touched by handlers and restrained for procedures. The training program was completed once before the start of any interventions, additional training sessions were performed biweekly during the entire course of the experiments. The animals were randomized to receive injections either in the novel application tunnel or using conventional fixation. Training effect was continuously monitored by measuring the latency to interact with the experimenter, the surface body temperature and by activity tracking. The latency to interact rapidly decreased during initial training sessions in both groups, and this effect was sustained throughout the course of treatment. Body temperature did not change over the course of medical training and seemed to be more influenced by environmental influences. Activity tracking demonstrated that mice injected inside the tunnel were more active and returned to their normal activity levels after injections faster than conventionally restrained mice. Those mice also showed less signs of defecation and urination during interventions. Furthermore, the tunnel had a positive influence on the well-being of mice during blood draws demonstrated by reduced signs of pain and faster willingness to interact with the handler after the procedure. In conclusion, habituation of the mice to the interventions with medical training and the improved handling procedure during ip injections and blood draws durably reduces stress levels and improves welfare of mice.

Portal vein tumor thrombus (PVTT) is among the most lethal complications of hepatocellular carcinoma (HCC), yet its molecular mechanisms and immune features remain poorly characterized. To address this gap, we performed a comprehensive multi-omics analysis of 99 specimens from 47 patients, integrating nCounter profiling, single-cell RNA sequencing, digital spatial profiling, and proteomics to construct the first spatial map of the PVTT microenvironment. These analyses revealed marked intratumoral heterogeneity and enrichment of myofibroblast-like cancer-associated fibroblasts (myCAFs) arising through a macrophage-to-myofibroblast transition. Nidogen-1 (NID1) was identified as a stromal driver of immune barriers, highly expressed in PVTT cores and associated with impaired antitumor immunity. Guided by these mechanistic insights, we repurposed acarbose, a Food and Drug Administration-approved drug, to inhibit the NID1 axis. Functional assays demonstrated that acarbose disrupted myCAF-mediated immune barriers, suppressed PVTT progression, and synergized with anti-programmed death-1 (anti-PD-1) therapy in preclinical models. Furthermore, analysis of an independent clinical cohort of 810 HCC patients revealed a substantially lower incidence of PVTT among those receiving acarbose, underscoring its translational potential. Collectively, these findings establish the immune-stromal landscape of PVTT, uncover NID1-driven stromal remodeling as a mechanism of immune evasion, and highlight drug repurposing as an immediately actionable strategy to improve outcomes in HCC with PVTT.

Resolution of inflammation, including of colitis, requires carefully coordinated communication between different tissues. While the role of immune cells in colitis is well established, less is known about epithelial communication to its microenvironment. Most secreted factors that comprise cellular communication are directly regulated by the transcription factor NF-κB. However, which transcriptomes NF-κB regulates in epithelium during colitis progression and whether these transcriptomes exacerbate or protect from colitis remains controversial. Here we use colonic biopsies from patients with ulcerative colitis (UC) and novel mouse models that permit specific visualisation of NF-κB in all tissues. Single-cell RNA sequencing and secretome analysis were combined to define epithelial communication at different stages of murine colitis and UC. We demonstrated that paradoxically, the NF-κB-directed pro-inflammatory secretome from the intestinal epithelial cells (IEC) is essential for recovery and for deterrence of unrestrained inflammation. Mechanistically, IEC NF-κB recruits and reprograms tolerogenic regulatory T cells that mediate protection from colitis. In ulcerative colitis patients, IEC NF-κB is not uniformly activated, as previously believed, but rather switches on transcription of select pro-inflammatory genes with concomitant repression of targets regulating T cell recruitment. Our findings map the dynamic changes of epithelial communication to mucosal leukocytes and identify a crucial protective mechanism initiated by IEC essential for resolution and recovery.

Background & Aims: The combination of locoregional therapies (LRT) with immune checkpoint inhibitors (ICIs) in unresectable hepatocellular carcinoma (HCC) is expected to enhance immune-mediated anti-tumor effects. Although clinical trials are underway, an unmet need exists to understand the immunological effects of LRT and how they evolve. This study aimed to longitudinally assess immune cell subpopulations and checkpoint expression after LRT. Methods: This prospective, single-center study (DRKS00026994) enrolled 128 consecutive patients with unresectable HCC, who underwent conventional transarterial chemoembolization (cTACE), interstitial high-dose-rate brachytherapy (iBT), or a combination of cTACE and iBT (from July 2020 to September 2021). Peripheral blood samples were collected at baseline, 1 day after LRT, and 2 months after LRT. Immune cells were quantified using spectral flow cytometry. Immune cell subpopulations and checkpoint molecule expression were compared longitudinally and among treatment groups. Cluster analyses were used to explore immune profiles and their relationship with treatment response. Results: Changes in absolute immune cell counts were detected 1 day after LRT, which largely diminished by 2 months. Myeloid populations increased significantly, whereas most lymphoid cells decreased after LRT. However, relative proportions of anti-tumoral CD56diminished NK cells (Cohen’s D = 0.40, 95% CI 0.19–0.61, p <0.01), CD8+ T cells (Cohen’s D = 0.15, 95% CI -0.06 to 0.35, p = 0.01), and CTLA-4 expression on T cells (CD4+: Cohen’s D = 0.54, 95% CI 0.33–0.75, p <0.01; CD8+: Cohen’s D = 0.15, 95% CI 0.36–0.78, p <0.01) were upregulated at 1 day, particularly after cTACE. Cluster analysis distinguished responders from non-responders based on distinct immune profiles. Conclusions: LRT induce an early pro-inflammatory immune response with increased myeloid, CTLA-4+ T cells, and cytotoxic lymphocytes, particularly after cTACE. These findings support the potential of immune profiling to guide personalized combination strategies with LRT and systemic immunotherapies. Impact and implications: Combining locoregional therapies (LRT) with immune checkpoint inhibitors (ICI) in unresectable hepatocellular carcinoma (HCC) aims to enhance immune-mediated anti-tumor effects. However, potential immunological targets remain unknown. Immune profiling could be facilitated as a tool to predict tumor response to LRT and may inform personalized treatment planning, selecting patients who may benefit from an additional ICI therapy. The study’s design may guide future investigations to identify the temporal dynamics of immune cell alterations following LRT to identify the appropriate time point to co-administer the ICI application. Clinical trial number: DRKS00026994 (https://drks.de/search/de/trial/DRKS00026994).

The gut-liver axis includes the bidirectional communication between the gut and the liver, and thus covers signals from liver-to-gut and from gut-to-liver. Disruptions of the gut-liver axis have been associated with the progression of chronic liver diseases, including alcohol-related and metabolic dysfunction-associated steatotic liver disease and cholangiopathies. Immune cells and their expression of pattern recognition receptors, activation markers or immune checkpoints might play an active role in the communication between gut and liver. Here, we present a 26-color full spectrum flow cytometry panel for human cells to decipher the role of circulating immune cells in gut-liver communication during the progression of chronic liver diseases in a non-invasive manner, which has been optimized to be used on patient-derived whole blood samples, the most abundantly available clinical material. Our panel focuses on changes in pattern recognition receptors, including toll-like receptors (TLRs) or Dectin-1, and also includes other immunomodulatory molecules such as bile acid receptors and checkpoint molecules. Moreover, this panel can be utilized to follow the progression of chronic liver diseases and could be used as a tool to evaluate the efficiency of therapeutic targets directed against microbial mediators or modulating immune cell activation.

Hepatocellular carcinoma (HCC) is the most common form of primary liver cancer and one of the leading causes of cancer-related deaths worldwide due to limited treatment options. The tumor microenvironment (TME), which is usually immunosuppressive in HCC, appears to be a decisive factor for response to immunotherapy and strategies aimed at inducing a more inflamed TME hold promise to overcome resistance to immunotherapy. Within the TME, the interplay of various cell types determines whether immunotherapy is successful. Liver macrophages, in particular tumor associated macrophages (TAMs), are known to play a crucial role in tumor progression and represent potential future therapeutic targets. The presence of C-C motif chemokine receptor 2 (CCR2) expressing macrophages is known to be associated with pathogenic angiogenesis and bad prognosis for HCC patients. A recent study published in Cancer Research describes how immunosuppressive macrophages in the TME can be repolarized through targeting Signaling Lymphocyte Activation Molecule Family member 7 (SLAMF7)-regulated CCchemokine ligand 2 (CCL2) signaling, which sensitizes HCC tumors to immunotherapy in a mouse model. This mini-review gives a brief overview about the current knowledge on SLAMF7 in the context of anticancer immunity and how the recent findings could be integrated into new therapeutic strategies for HCC.

Ferroptosis is a pervasive non-apoptotic form of cell death highly relevant in various degenerative diseases and malignancies. The hallmark of ferroptosis is uncontrolled and overwhelming peroxidation of polyunsaturated fatty acids contained in membrane phospholipids, which eventually leads to rupture of the plasma membrane. Ferroptosis is unique in that it is essentially a spontaneous, uncatalyzed chemical process based on perturbed iron and redox homeostasis contributing to the cell death process, but that it is nonetheless modulated by many metabolic nodes that impinge on the cells' susceptibility to ferroptosis. Among the various nodes affecting ferroptosis sensitivity, several have emerged as promising candidates for pharmacological intervention, rendering ferroptosis-related proteins attractive targets for the treatment of numerous currently incurable diseases. Herein, the current members of a Germany-wide research consortium focusing on ferroptosis research, as well as key external experts in ferroptosis who have made seminal contributions to this rapidly growing and exciting field of research, have gathered to provide a comprehensive, state-of-the-art review on ferroptosis. Specific topics include: basic mechanisms, in vivo relevance, specialized methodologies, chemical and pharmacological tools, and the potential contribution of ferroptosis to disease etiopathology and progression. We hope that this article will not only provide established scientists and newcomers to the field with an overview of the multiple facets of ferroptosis, but also encourage additional efforts to characterize further molecular pathways modulating ferroptosis, with the ultimate goal to develop novel pharmacotherapies to tackle the various diseases associated with - or caused by - ferroptosis.

Full spectrum flow cytometry is a powerful tool for immune monitoring on a single-cell level and with currently available machines, panels of 40 or more markers per sample are possible. However, with an increased panel size, spectral unmixing issues arise, and appropriate single stain reference controls are required for accurate experimental results and to avoid unmixing errors. In contrast to conventional flow cytometry, full spectrum flow cytometry takes into account even minor differences in spectral signatures and requires the full spectrum of each fluorochrome to be identical in the reference control and the fully stained sample to ensure accurate and reliable results. In general, using the cells of interest is considered optimal, but certain markers may not be expressed at sufficient levels to generate a reliable positive control. In this case, compensation beads show some significant advantages as they bind a consistent amount of antibody independent of its specificity. In this study, we evaluated two types of manufactured compensation beads for use as reference controls for 30 of the most commonly used and commercially available fluorochromes in full spectrum cytometry and compared them to human and murine primary leukocytes. While most fluorochromes show the same spectral profile on beads and cells, we demonstrate that specific fluorochromes show a significantly different spectral profile depending on which type of compensation beads is used, and some fluorochromes should be used on cells exclusively. Here, we provide a list of important considerations when selecting optimal reference controls for full spectrum flow cytometry.

Primary liver cancer, represented mainly by hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (CCA), is one of the most common and deadliest tumors worldwide. While surgical resection or liver transplantation are the best option in early disease stages, these tumors often present in advanced stages and systemic treatment is required to improve survival time. The emergence of immune checkpoint inhibitor (ICI) therapy has had a positive impact especially on the treatment of advanced cancers, thereby establishing immunotherapy as part of first-line treatment in HCC and CCA. Nevertheless, low response rates reflect on the usually cold or immunosuppressed tumor microenvironment of primary liver cancer. In this review, we aim to summarize mechanisms of resistance leading to tumor immune escape with a special focus on the composition of tumor microenvironment in both HCC and CCA, also reflecting on recent important developments in ICI combination therapy. Furthermore, we discuss how combination of ICIs with established primary liver cancer treatments (e.g. multikinase inhibitors and chemotherapy) as well as more complex combinations with state-of-the-art therapeutic concepts may reshape the tumor microenvironment, leading to higher response rates and long-lasting antitumor immunity for primary liver cancer patients.

Chronic liver diseases such as nonalcoholic fatty liver disease (NAFLD) or viral hepatitis are characterized by persistent inflammation and subsequent liver fibrosis. Liver fibrosis critically determines long-term morbidity (for example, cirrhosis or liver cancer) and mortality in NAFLD and nonalcoholic steatohepatitis (NASH). Inflammation represents the concerted response of various hepatic cell types to hepatocellular death and inflammatory signals, which are related to intrahepatic injury pathways or extrahepatic mediators from the gut–liver axis and the circulation. Single-cell technologies have revealed the heterogeneity of immune cell activation concerning disease states and the spatial organization within the liver, including resident and recruited macrophages, neutrophils as mediators of tissue repair, auto-aggressive features of T cells as well as various innate lymphoid cell and unconventional T cell populations. Inflammatory responses drive the activation of hepatic stellate cells (HSCs), and HSC subsets, in turn, modulate immune mechanisms via chemokines and cytokines or transdifferentiate into matrix-producing myofibroblasts. Current advances in understanding the pathogenesis of inflammation and fibrosis in the liver, mainly focused on NAFLD or NASH owing to the high unmet medical need, have led to the identification of several therapeutic targets. In this Review, we summarize the inflammatory mediators and cells in the diseased liver, fibrogenic pathways and their therapeutic implications.