An altered liver microenvironment characterized by a dysregulated extracellular matrix (ECM) supports the development and progression of hepatocellular carcinoma (HCC). The development of experimental platforms able to reproduce these physio-pathological conditions is essential in order to identify and validate new therapeutic targets for HCC. The aim of this work was to validate a new in vitro model based on engineering three-dimensional (3D) healthy and cirrhotic human liver scaffolds with HCC cells recreating the micro-environmental features favoring HCC. Healthy and cirrhotic human livers ECM scaffolds were developed using a high shear stress oscillation-decellularization procedure. The scaffolds bio-physical/bio-chemical properties were analyzed by qualitative and quantitative approaches. Cirrhotic 3D scaffolds were characterized by biomechanical properties and microarchitecture typical of the native cirrhotic tissue. Proteomic analysis was employed on decellularized 3D scaffolds and showed specific enriched proteins in cirrhotic ECM in comparison to healthy ECM proteins. Cell repopulation of cirrhotic scaffolds highlighted a unique up-regulation in genes related to epithelial to mesenchymal transition (EMT) and TGFβ signaling. This was also supported by the presence and release of higher concentration of endogenous TGFβ1 in cirrhotic scaffolds in comparison to healthy scaffolds. Fibronectin secretion was significantly upregulated in cells grown in cirrhotic scaffolds in comparison to cells engrafted in healthy scaffolds. TGFβ1 induced the phosphorylation of canonical proteins Smad2/3, which was ECM scaffold-dependent. Important, TGFβ1-induced phosphorylation of Smad2/3 was significantly reduced and ECM scaffold-independent when pre/simultaneously treated with the TGFβ-R1 kinase inhibitor Galunisertib. In conclusion, the inherent features of cirrhotic human liver ECM micro-environment were dissected and characterized for the first time as key pro-carcinogenic components in HCC development.

Coronavirus disease 2019 (COVID-19), caused by infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), comprises mild courses of disease as well as progression to severe disease, characterised by lung and other organ failure. The immune system is considered to play a crucial role for the pathogenesis of COVID-19, although especially the contribution of innate-like T cells remains poorly understood. Here, we analysed the phenotype and function of mucosal-associated invariant T (MAIT) cells, innate-like T cells with potent antimicrobial effector function, in patients with mild and severe COVID-19 by multicolour flow cytometry. Our data indicate that MAIT cells are highly activated in patients with COVID-19, irrespective of the course of disease, and express high levels of proinflammatory cytokines such as IL-17A and TNFα ex vivo. Of note, expression of the activation marker HLA-DR positively correlated with SAPS II score, a measure of disease severity. Upon MAIT cell-specific in vitro stimulation, MAIT cells however failed to upregulate expression of the cytokines IL-17A and TNFα, as well as cytolytic proteins, that is, granzyme B and perforin. Thus, our data point towards an altered cytokine expression profile alongside an impaired antibacterial and antiviral function of MAIT cells in COVID-19 and thereby contribute to the understanding of COVID-19 immunopathogenesis.

Mucosal-associated invariant T (MAIT) cells constitute a highly abundant innate-like T-cell population in the human liver that is critical for immune surveillance of hepatic cancers. However, MAIT cells are often dysfunctional in human hepatocellular carcinoma (HCC), for reasons that remain unclear. Here, we aimed to identify the mechanisms driving MAIT cell dysfunction in metabolic dysfunction-associated steatotic liver disease (MASLD), a chronic liver condition that predisposes patients to HCC. We studied MAIT cell functionality, metabolism and anti-cancer activity directly ex vivo in patients with MASLD, as well as in co-culture models mimicking MASLD. (Single-cell) RNA-sequencing was used to translate findings into clinical cohorts of patients with MASLD and MASLD-associated HCC. We found that MAIT cells lose their effector functions in patients with MASLD and uncover that this dysfunction is driven by MASLD-associated polyunsaturated fatty acids (PUFAs), which selectively accumulate in MAIT cells but not in conventional CD8⁺ T cells or NK cells. Mechanistically, PUFAs drive MAIT cell dysfunction through intracellular formation of lipid peroxides that promote a state of 'metabolic exhaustion' characterised by compromised mitochondrial respiration and glycolysis in MAIT cells. Excessive signalling through this MASLD-PUFA-lipid peroxide axis results in MAIT cell death by ferroptosis. Interference with PUFA-induced lipid peroxide formation in MAIT cells reversed their metabolic exhaustion and prevented ferroptotic MAIT cell death, thereby restoring MAIT cell effector function and anti-cancer activity. In patients with HCC, high expression of the MAIT cell-PUFA gene signature that was linked to MAIT cell dysfunction was associated with poor survival. Our findings identify a novel immunometabolic axis that impairs MAIT cell-mediated anti-cancer immunity in MASLD and may represent a targetable pathway to enhance the effectiveness of immunotherapy. In this study, we identify a novel immunometabolic axis in which polyunsaturated fatty acids, accumulating in liver tissue in MASLD (metabolic dysfunction-associated steatotic liver disease), drive MAIT (mucosal-associated invariant T) cell dysfunction through lipid peroxide-induced metabolic exhaustion and ferroptosis, ultimately impairing their anti-tumour activity. These findings reveal how MASLD creates an immune-permissive environment that may facilitate hepatocellular carcinoma development and progression and prevent effective immunotherapy. Targeting the polyunsaturated fatty acid-lipid peroxide axis could restore MAIT cell function and enhance current immunotherapeutic anti-cancer strategies.

Atezolizumab/bevacizumab (atezo/bev) and lenvatinib have demonstrated efficacy as first-line therapies for hepatocellular carcinoma (HCC). However, vascular endothelial growth factor (VEGF) inhibition with these therapies may be associated with the risk of bleeding and thromboembolic events. In this study, we evaluated the efficacy and safety with focus on the bleeding and thromboembolic events of atezo/bev vs. lenvatinib in a large, multicenter real-world population. This study is based on HCC cohorts from seven centers in Germany and Austria. Incidences of bleeding or thromboembolic events and efficacy outcomes were assessed and compared. In total, 464 patients treated with atezo/bev (n = 325) or lenvatinib (n = 139) were analyzed. Both groups were balanced with respect to demographics, presence of liver cirrhosis, and variceal status. Duration of therapy did not differ between groups. Within 3 months of therapy, bleeding episodes were described in 57 (18%) patients receiving atezo/bev compared with 15 (11%) patients receiving lenvatinib (p = 0.07). Variceal hemorrhage occurred in 11 (3%) patients treated with atezo/bev compared with 4 (3%) patients treated with lenvatinib (p = 0.99). Thromboembolic events were reported in 19 (6%) of patients in the atezo/bev cohort compared with 5 (4%) patients in the lenvatinib cohort (p = 0.37). In addition, incidence of overall bleeding, variceal hemorrhage, and thromboembolic events did not differ significantly in patients who received either atezo/bev or lenvantinib for 6 months. Safety considerations related to bleeding and thromboembolic events may not be helpful in guiding clinical decision-making when choosing between atezo/bev and lenvatinib. The inhibition of VEGF by current first-line therapies for HCC, such as atezolizumab/bevacizumab or lenvatinib, may be associated with the risk of bleeding and thromboembolic events. Studies comparing the incidence of these side effects between atezolizumab/bevacizumab and lenvatinib, which are preferred treatments over sorafenib for HCC, are needed. Differences in this side effect profile may influence the choice of first-line therapy by treating physicians. Because no significant differences were observed regarding bleeding or thromboembolic events between both therapies in the present study, we conclude that safety considerations related to these events may not be helpful in guiding clinical decision-making when choosing between atezolizumab/bevacizumab and lenvatinib.

Immunotherapy with atezolizumab and bevacizumab (a + b) has improved the prognosis of patients with unresectable hepatocellular carcinoma (HCC). However, the outcome for individual patients is highly variable. This study aimed to (i) develop and validate a prognostic prediction model to estimate individual prognosis and (ii) compare it with established models. In this multicenter retrospective study, patients with HCC undergoing first-line immunotherapy with a + b from 24 centers (Europe, USA) were included. Statistical analysis and reporting followed the TRIPOD guidelines. The primary objective was overall survival (OS). A Cox model was developed and externally validated. In total, 683 patients were included (training: 526, validation: 157). The C-reactive protein, albumin, bilirubin, lymphocytes, ECOG performance status, and extrahepatic spread (CABLE score) remained significantly associated with OS in Cox regression analysis. In the training set, the CABLE score had a higher discriminatory accuracy relative to ALBI, EZ-ALBI, mALBI, CRAFITY, PNI, NLR, PLR, and GPS (time-dependent AUC 0.79 and C-index 0.75 (95% CI 0.71–0.78) at 12 months). In the external validation set, the discriminatory performance of the CABLE score was comparable to ALBI, EZ-ALBI, and mALBI, but on average higher than PNI, CRAFITY, NLR, PLR, and GPS. In patients with Child-Pugh A, the CABLE score outperformed ALBI, EZ-ALBI, and mALBI in the first 9 months. We provide a web-based calculator for the CABLE score to allow estimation of individual prognosis for these patients (http://shiny.imbei.uni-mainz.de:3838/CABLE_Score/). The CABLE score shows good discriminatory performance in assessing the individual prognosis of patients undergoing first-line immunotherapy with a + b. Further validation studies are needed to investigate its performance compared with the ALBI score, in particular in subgroup analysis. The CABLE score allows estimation of the prognosis of patients with unresectable hepatocellular carcinoma undergoing first-line immunotherapy with atezolizumab and bevacizumab at an individual level using our web-based calculator. This feature, as well as the evaluation of the score’s added benefit through an extensive comparison with other established scores, can inform clinicians on their significance and may guide clinical decision-making in the context of a malignant disease where the prognosis has become highly variable. Further large validation studies are needed to investigate the incremental value of the CABLE score compared with the ALBI score, in particular in subgroups such as patients designated as Child-Pugh A.

Atezolizumab/bevacizumab (atezo/bev) has revolutionized the standard of care for patients with unresectable hepatocellular carcinoma (HCC). However, only a subgroup of patients responds to atezo/bev and derives durable clinical benefit. This study aims to analyze the frequency and risk factors of early mortality (EM) in patients with HCC treated with atezo/bev. This study uses data from a large, European real-world cohort and flow cytometry-based immunophenotyping of patient’s baseline PBMC. EM was defined as death from any cause within 90 days of treatment initiation. Logistic regression analysis was used to identify parameters associated with EM. A total of 317 patients with unresectable HCC treated with first-line atezo/bev were included. EM rate in the cohort was 15.8%, with a median survival of 12.6 months. The proportion of patients with preserved liver function and BCLC stage B was significantly lower in the EM cohort. The strongest predictor of EM was advanced liver disease in univariate analysis, as reflected by surrogates of impaired liver function such as Child–Pugh score (CPS) B (p <0.0001), albumin–bilirubin grade 2/3 (p = 0.026, p <0.0001) or high model for end-stage liver disease score (p <0.0001). CPS B remained a significant risk factor after adjusting for other variables. Biomarker analysis and immunophenotyping revealed high C-reactive protein, reduced lymphocyte frequencies, increased CD44 expression on regulatory T cells and elevated PD-L1 levels on CD8⁺ T cells to be associated with EM. EM rate was 15.8% in patients with HCC treated with atezo/bev. Significant risk factors for early death involved impaired liver function, elevated biomarkers of inflammation and alterations of systemic immunity. Although rare in clinical trial populations, early mortality (EM) is a significant concern following the initiation of immune checkpoint inhibitors in patients with hepatocellular carcinoma (HCC) in real-world cohorts, highlighting the need for adequate risk stratification and patient selection. Using data of a large, European real-world cohort as well as FACS-based immunophenotyping of baseline peripheral blood mononuclear cells, we comprehensively characterized the frequency, risk factors, and biomarkers associated with EM in atezo/bev-treated patients with HCC. We identified EM to represent a common event in patients with HCC after treatment initiation and demonstrated impaired liver function, elevated biomarkers of inflammation, and reduced lymphocyte frequencies and increased regulatory T cell activity to be associated with increased EM risk. These findings can help clinicians identify patients with HCC at high risk of EM, enabling adequate and informed decision-making regarding end-of-life care and palliative treatment.