Extravasation of circulating tumor cells (CTCs) is critical for metastasis and is initiated by adhesive interactions between glycoligands on CTCs and E-selectin on endothelia. Here, we show that the clinically approved proteasome inhibitor bortezomib (BZM; Velcade) counteracts the cytokine-dependent induction of E-selectin in the lung mediated by the primary tumor, thereby impairing endothelial adhesion and thus spontaneous lung metastasis in vivo. However, the efficacy of BZM crucially depends on the tumor cells' E-selectin ligands, which determine distinct adhesion patterns. The canonical ligands sialyl-Lewis A (sLeA) and sLeX mediate particularly high-affinity E-selectin binding so that the incomplete E-selectin-reducing effect of BZM is not sufficient to disrupt adhesion or metastasis. In contrast, tumor cells lacking sLeA/X nevertheless bind E-selectin, but with low affinity, so that adhesion and lung metastasis are significantly diminished. Such low-affinity E-selectin ligands apparently consist of sialylated MGAT5 products on CD44. BZM no longer has anti-metastatic activity after CD44 knockdown in sLeA/X-negative tumor cells or E-selectin knockout in mice. sLeA/X can be determined by immunohistochemistry in cancer samples, which might aid patient stratification. These data suggest that BZM might act as a drug for inhibiting extravasation and thus distant metastasis formation in malignancies expressing low-affinity E-selectin ligands.

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: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality. HCC is characterized by high heterogeneity and, subsequently, adaptation by developing resistance to current treatments. Applying individualized models is crucial to understanding the potential of approved therapies. Therefore, we classify a primary-like cell line derived from the core region of an HCC with underlying HIV-HCV co-infection employing deep analysis on the pathway regulation level. Methods: We employed DEG analysis, followed by pathway analysis, to characterize the preservation level of the LC4 cells and the level of adoption. Next, we classify the new model for HCC research by employing healthy donor samples, commonly used HCC cell lines, and global RNAseq datasets. Results: LC4 cells reflect the characteristics of the parental cancer region, including immunosuppression and metabolic reprogramming, characterized by the downregulation of drug-metabolizing enzymes compared to healthy individuals, indicating a transition to alternate metabolic pathways. Moreover, we identified specific biomarkers equally regulated in the parental tissue, in global datasets of the same entities as well as in LC4 cells. Conclusions: We classified LC4 cells as an individual immunosuppressive and highly progressive primary-like HCC cell line. LC4 cells are applicable as a model for preclinical drug testing, minimizing the lack of preclinical models in HCV-HIV-induced HCC research.

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.

Background Hepatocellular carcinoma represents a significant global health challenge, affecting over a million patients annually, arising mainly from chronic liver diseases, with a majority being related to viral infections. However, despite the groundbreaking clinical results of immune checkpoint blockades and adoptive cell therapies, we still face non-responders accompanied by high rebound rates after resection. Considering that the main concern is to overcome a highly specialized immunosuppressive tumor microenvironment of the individual patient, the characterization of the particular tumor microenvironment and the source of the immune cells used in ACT is of immense importance. Approved ACT therapies mainly use modified peripheral blood cells from individuals. At the same time, tumor-infiltrating lymphocytes are underrepresented even if they have garnered interest due to their potential to target tumor-specific antigens more effectively. Methods In this study, we employed allogenic and autologous immune cell sources for expansion and stimulation, resulting in adoptive T-cell transfer experiments determining the effector cell differentiation and the related anti-tumor effects by the possible implementation of re-stimulation. Results We determined a high success rate in expanding and stimulating tumor-infiltrating lymphocytes with consistent CD8 T-cell fractions from HCC patients. To showcase the effectiveness of stimulated T-cells from different sources, we generated cell lines derived from the margin and center of an HBV-induced HCC with a highly immune-suppressive TME. We found effective immune responses supported by cell death induction, ferroptosis, proptosis and apoptosis triggered by all sources of T-cells depending on the area of derived tumor cells. Conclusion Effector T-cell fractions derived from tumor-infiltrating lymphocytes present a viable source for cell-based immune therapy combined with immune checkpoint inhibitors in HCC patients, especially after resection, to suppress rebound strategies of the parental tumor on an individual level.