Hepatocellular carcinoma (HCC) results in high mortality due to ineffective systemic therapy. Human immortalized cell lines are commonly used to study anti-tumor effects in the context of new anti-tumor therapies and tumor biology. As immortalized cell lines have limited biological relevance and heterogeneity compared to primary cells, patient-derived tumor tissues, and corresponding immune cells are the gold standards for studying the complexity of individual tumor entities. However, culturing primary HCC cells has a low success rate. Here, we aimed to establish a reproducible approach to preserve the patient-derived liver cancer cells for in vitro and in vivo studies. The underlying study aimed to establish an in vitro pre-screening platform to test treatment options’ effectivity and dosage, e.g., for new substances, autologous modified immune cells, or combined therapies in HCC. We initially employed 15 surgical resection specimens from patients with different HCC entities for isolation and preservation. The isolated liver cancer cells from four HCC-diagnosed patients were used for orthotopic transplantation into the healthy liver of immunodeficient mice, allowing them to grow for six months before human liver cancer cells were isolated and cultured. As a result, we generated and characterized four new primary-like liver cancer cell lines. Compared to immortalized HCC cell lines, freshly generated liver cancer cells displayed individual morphologies and heterogeneous protein-level characteristics. We assessed their ability to proliferate, migrate, form spheroids, and react to common medications compared to immortalized HCC cell lines. All four liver cancer cell lines exhibit strong migration and colony-forming characteristics in vitro, comparable to extensively investigated immortalized HCC cell lines. Moreover, the four etiological different liver cancer cell lines displayed differences in the response to 5-FU, Sorafenib, Axitinib, and interferon-alpha treatment, ranking from non-responders to responders depending on the applicated medication. In sum, we generated individual patient-derived liver cancer cell lines suitable for predictive in vitro drug screenings and for xenograft transplantations to realize the in vivo investigation of drug candidates. We overcame the low cultivation success rate of liver cancer cells derived from patients and analyzed their potential to serve a pre-clinical model.

Hepatocellular carcinoma (HCC) results in high mortality due to ineffective systemic therapy. Human immortalized cell lines are commonly used to study anti-tumour effects in the context of new anti-tumour therapies and tumour biology. As immortalized cell lines have limited biological relevance and heterogeneity compared to primary cells, patient-derived tumour tissues and corresponding immune cells are the gold standards for studying the complexity of individual tumour entities. However, culturing primary HCC cells has a low success rate. Here, we aimed to establish a reproducible approach to preserve the patient-derived liver cancer cells for in vitro and in vivo studies. The underlying study aimed to establish an in vitro pre-screening platform to test treatment options' effectivity and dosage, e.g., for new substances, autologous modified immune cells, or combined therapies in HCC. We initially employed 15 surgical resection specimens from patients with different HCC entities for isolation and preservation. The isolated liver cancer cells from four HCC-diagnosed patients were used for orthotopic transplantation into the healthy liver of immunodeficient mice, allowing them to grow for six months before human liver cancer cells were isolated and cultured. As a result, we generated and characterized four new primary-like liver cancer cell lines. Compared to immortalized HCC cell lines, freshly generated liver cancer cells displayed individual morphologies and heterogenous protein-level characteristics. We assessed their ability to proliferate, migrate, form spheroids, and react to common medications compared to immortalized HCC cell lines. All four liver cancer cell lines exhibit strong migration and colony-forming characteristics in vitro, comparable to extensively investigated immortalized HCC cell lines. Moreover, the four etiological different liver cancer cell lines displayed differences in the response to 5-FU, Sorafenib, Axitinib and Interferon-alpha treatment, ranking from non-responders to responders depending on the applicated medication. In sum, we generated individual patient-derived liver cancer cell lines suitable for predictive in vitro drug screenings and moreover for xenograft transplantations to realize the in vivo investigation of drug candidates. We overcame the low cultivation success rate of liver cancer cells derived from patients and analyzed their potential to serve a pre-clinical model.

Background: Steatotic liver disease (SLD) is characterised by profound metabolic reprogramming, yet no single biomarker reliably distinguishes disease entities, stages or sex-specific risk profiles. By integrating serum metabolomic signatures as a liquid biopsy with tumour-associated CSC marker profiles in a sex-stratified analytical framework, we aimed to identify biologically meaningful differences and improve strategies for early, presymptomatic detection of SLD progression and HCC. Methods: The present study focuses on a targeted panel of strongly dysregulated serum metabolites as candidate biomarkers of disease progression, quantified by NMR-based metabolomics and ELISA and complemented by CSC marker staining. We combined these NMR-based metabolomic ‘liquid biopsy’ data with circulating tumour-associated biomarkers, MELD-based risk assessment and tissue-level CSC marker expression across MetALD, MASLD, immune-mediated and cancerogenic liver disease, HCC and healthy controls. Results: Female MetALD patients showed the second highest mortality after HCC, with lower survival than male cancer patients, despite MELD 3.0 assigning ~50% higher scores in women. MetALD mortality clustered with GP73, CD44, metabolomics and AA/3HB ratio, indicating a distinct, high-risk female phenotype. Conclusion: Integrating liquid-based metabolomic profiling, AA/3HB redox assessment, CSC markers and MELD 3.0 into sex-sensitive diagnostic pathways may improve early detection and risk stratification of alcohol-associated CLD, especially in women.

Background/Objectives: Pneumocystis jirovecii pneumonia (PJP) is the most frequently diagnosed AIDS-defining illness in Europe, with especially high mortality in HIV-negative patients caused by delayed diagnosis and low awareness. This study aims to evaluate cytokine release assays (CRA) to facilitate a less invasive and resource-efficient PJP specific diagnostic test. We focus on the P. jirovecii antigens Kexin 1 (KEX1), MSG1, and MSG2, which were identified in prior studies as immunologically relevant. Methods: Whole blood samples from 50 participants—22 healthy individuals and 28 immunocompromised individuals, including 8 with proven PJP—were stimulated in vitro with full-length and partial KEX1, MSG1, MSG2, and a combination of all three antigens (PJ-MIX). Following 24 h incubation at 37 °C, cytokine levels of IL-2, IFN-γ, IL-17A, and IL-17F were measured. Results: Stimulation with full-length KEX1, MSG1, MSG2, and PJ-MIX antigens induced higher IL-2 concentrations in the healthy control group compared to the groups IL-2 baseline levels and to the group of proven PJP cases. Similarly, stimulation with full-length KEX1, MSG1, and PJ-MIX elevated IFN-γ levels in the healthy control group compared to baseline IFN-γ levels. Conclusions: Our findings highlight the potential of IL-2 and IFN-γ release following stimulation with PJ antigens, with PJ-MIX eliciting the strongest and most significant responses, suggesting a cumulative antigen effect. This pilot study establishes a foundation for a PJP-specific CRA, deepening our knowledge of T-cell immunity against PJP. Clinically, such a test could, among other applications, evaluate at-risk patients who should receive prophylaxis and may consequently reduce PJP-related morbidity and mortality.

Steatotic liver disease (SLD) is characterised by profound metabolic reprogramming, yet no single biomarker reliably distinguishes disease entities, stages or sex-specific risk profiles. By integrating serum metabolomic signatures as a liquid biopsy with tumour-associated CSC marker profiles in a sex-stratified analytical framework, we aimed to identify biologically meaningful differences and improve strategies for early, presymptomatic detection of SLD progression and HCC. The present study focuses on a targeted panel of 12 strongly dysregulated serum metabolites as candidate biomarkers of disease progression, quantified by NMR-based metabolomics and ELISA and complemented by CSC marker staining. We combined these NMR-based metabolomic ‘liquid biopsy’ data with circulating tumour-associated biomarkers, MELD-based risk assessment and tissue-level CSC marker expression across MetALD, MASLD, immune-mediated and cancerogenic liver disease, HCC and healthy controls. Female MetALD patients showed the second highest mortality after HCC, with lower survival than male cancer patients, despite MELD 3.0 assigning ~50% higher scores in women. MetALD mortality clustered with GP73, CD44, metabolomics and AA/3HB ratio, indicating a distinct, high-risk female phenotype. Integrating liquid-based metabolomic profiling, AA/3HB redox assessment, CSC markers and MELD 3.0 into sex-sensitive diagnostic pathways may improve early detection and risk stratification of alcohol-associated SLD, especially in women.

Background: T-cell-based therapies achieved milestones in targeting solid tumors, such as hepatocellular carcinoma, by leveraging the cytotoxic potential of effector memory T-cells. However, a key challenge lies in the ex-vivo expansion of functional memory T-cells while simultaneously preventing over-differentiation into senescent TEMRA cells. Methods: PBMC derived from 10 HCC patients and two healthy donors were used for expansion in small bioreactors equipped with a permeable membrane for 2 weeks. During expansion, surface marker composition, cytokine and chemokine production were observed. Enriched T-cell subsets from three patients with chronic HBV infection were analysed in detail. We extracted the enriched complement of one chronically infected patient to elucidate the therapeutic potential. Results: We successfully expanded the effector T-cell subsets for all investigated samples and consistently enriched the cell amount over time. Subsequently, we showed that the expanded patient-derived T-cells showed functionality against autologous liver cancer-derived cells by inducing receptor and protein-mediated cell death. Conclusion: We showed the consistent ex-vivo expansion of T-cell subsets of initial patient-derived PBMCs. The enriched subsets exhibit cytotoxic functionality and shift to cytolytic TEMRA cells in an immune evasion setting in the context of chronic HBV-infected patient-derived liver cancer cells.