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This study presents a comprehensive genetic characterization of the H9c2 cell line, a widely used model for cardiac myoblast research. We established a short tandem repeat (STR) profile for H9c2 that is useful to confirm the identity and stability of the cell line. Additionally, we prepared H9c2 metaphase chromosomes and performed karyotyping and molecular cytogenetics to further investigate chromosomal characteristics. The genetic analysis showed that H9c2 cells exhibit chromosomal instability, which may impact experimental reproducibility and data interpretation. Next-generation sequencing (NGS) was performed to analyze the transcriptome, revealing gene expression patterns relevant to cardiac biology. Western blot analysis further validated the expression levels of selected cardiac genes identified through NGS. Additionally, Phalloidin staining was used to visualize cytoskeletal organization, highlighting the morphological features of these cardiac myoblasts. Our findings collectively support that H9c2 cells are a reliable model for studying cardiac myoblast biology, despite some genetic alterations identified resembling sarcoma cells. The list of genes identified through NGS analysis, coupled with our comprehensive genetic analysis, will serve as a valuable resource for future studies utilizing this cell line in cardiovascular medicine.

Immortalized cell lines play a crucial role in biomedical research by enabling reproducible experiments and enhancing our understanding of complex diseases. HuH-7, originally derived from a human hepatocellular carcinoma, is particularly valuable for studying liver cancer dynamics, viral hepatitis, and drug metabolism. However, concerns about cell line misidentification and genetic drift in cell lines highlight the importance of rigorous authentication to maintain the reliability of research outcomes, despite their widespread use. In this study, we present a detailed (cyto)genetic and molecular analysis of HuH-7 cells, focusing on their hepatocellular characteristics and potential applications in translational research. Through thorough genomic profiling and next-generation mRNA expression analyses, we aimed to confirm the authenticity of the cell line and identify key genetic signatures associated with tumorigenic pathways. Our results emphasize the importance of regular identity verification, such as short tandem repeat (STR) profiling, and demonstrate how subtle genetic variations can affect phenotypic traits relevant to modeling liver disease. By providing insights into the genetic and transcriptomic features of HuH-7 cells, this study establishes a robust basis for future research and therapeutic investigations using this widely accepted liver cell model. It also emphasizes the importance for maintaining high-quality standards and robust authentication practices to ensure that cell-based studies produce reliable and reproducible results.

The murine Alpha Mouse Liver 12 (AML12) cell line, established over four decades ago, is one of the most commonly used non-transformed hepatocyte models in basic and pre-clinical liver research. Despite its widespread use, a comprehensive and current molecular characterization has been lacking. In this study, we combined cytogenetics with high-resolution genomic technologies to establish a detailed genetic reference profile of AML12. Inverted DAPI banding and multicolor fluorescence in situ hybridization (m-FISH) revealed a complex yet stable, near-tetraploid karyotype featuring double X-chromosome deletions [del(X)(A3)×2], a recurrent derivative chromosome der(3)t(2;3)(A2;H4), biallelic deletions of 17D1, two dicentric chromosomes dic(X;17), and multiple whole-chromosome gains (e.g., +1, +6, +15, +19×4) and losses (e.g., −4, −12, −16, −18). Multicolor banding (mcb) further pinpointed cryptic inversions on chromosomes 7 and 11. Copy number imbalances were visualized as in silico array comparative genomic hybridization (aCGH)-style profiles inferred from these metaphase-based assays, and no independent array- or sequencing-based copy number variation (CNV) experiment was performed in this study. Short tandem repeat (STR) profiling created a unique 16-locus authentication barcode that unambiguously distinguishes AML12 from other murine cell lines in public databases. Bulk RNA sequencing (RNA-seq) further demonstrated a transcriptional profile in AML12 cells that is indicative of hepatocyte origin while also revealing partial de-differentiation and reduced expression of selected urea cycle, gluconeogenic, and xenobiotic-metabolizing transcripts, consistent with limited mature hepatocyte functions. These functional inferences are likely based on gene expression patterns rather than on direct physiological assays. In summary, our study provides (i) the first integrated cytogenetic, STR, and next-generation sequencing dataset for AML12, (ii) a practical authentication panel for routine laboratory use, and (iii) reference information that will enhance the interpretation, reproducibility, and translational relevance of future studies using this versatile hepatocyte model.

The 25 kDa glycoprotein lipocalin-2 (LCN2) is widely expressed and has diverse functions, ranging from physiological to pathophysiological processes. In the liver, LCN2 is primarily associated with inflammatory processes and is considered a potential biomarker in metabolic disorders. However, a significant challenge is the absence of a suitable human in vitro model for studying LCN2 and its associated signaling pathways. Therefore, we have successfully generated patient-derived liver organoids of both male and female origin, providing a novel in vitro model for LCN2 research. Our data show that the self-renewing organoids mimic essential architectural features of hepatocytes, as demonstrated by electron microscopy and F-actin staining. Consistent with the expression profile observed in liver tissue, the isolated 3D organoids exhibit minimal endogenous LCN2 levels. Next, the LCN2 expression was studied at the protein and mRNA levels under inflammatory conditions by treating the organoids with various cytokines and lipopolysaccharides (LPS). Our results show that LCN2 expression is significantly upregulated by IL-1β and TNF-α in an NF-κB-dependent manner, but remains unchanged with IL-6 or LPS. In conclusion, we have established human patient-derived liver organoids as a valuable model for investigating LCN2 signaling mechanisms. This study lays the foundation for future research on the role of LCN2 in liver pathologies, aiding in disease progression understanding and facilitating patient-specific treatment predictions.

Lipocalin-2 (LCN2) is a 25 kDa glycoprotein that has been shown to be a multifunctional player in the metastasis of triple-negative breast cancer (TNBC). In physiological contexts, LCN2 exhibits bacteriostatic properties and plays key roles in iron homeostasis and the transport of hydrophobic molecules. However, several studies have shown that aberrant LCN2 expression is associated with poor prognosis in various malignancies, including breast cancer, which is the most common cancer in women worldwide and can be classified into four molecular subtypes. Among these, TNBC represents a disproportionately aggressive subtype characterized by poor prognosis and high metastatic potential. Although LCN2 has been extensively studied in breast cancer overall, its specific role in TNBC progression and metastasis is only beginning to be understood. Recent evidence suggests that LCN2 contributes to several tumor-promoting processes such as angiogenesis, therapy resistance and modulation of the tumor microenvironment. Moreover, LCN2 appears to influence organ-specific metastasis, particularly to the lung and brain, while its role in liver and bone dissemination remains unclear. Collectively, current data identify LCN2 as a critical mediator of TNBC progression and highlight its potential as a prognostic factor and modulator of disease progression. This review aims to summarize insights from both in vitro and in vivo studies, with particular focus on the role of LCN2 in the metastatic cascade, while also addressing existing research gaps and critically evaluating the current findings.

The immortal murine hepatic stellate cell line Col-GFP HSC was comprehensively characterized using genetic and molecular approaches. Short tandem repeat (STR) profiling and karyotyping combined with multiplex fluorescence in situ hybridization (M-FISH) confirmed the identity of the cell line and revealed no contamination. Col-GFP HSCs showed a near tetraploid karyotype. Additionally, next-generation sequencing (NGS) data, quantitative reverse transcription PCR, and Western blot analyses demonstrated robust expression of genes and proteins associated with hepatic stellate cells, including those involved in extracellular matrix remodeling and fibrogenic pathways. Phalloidin staining revealed filamentous actin patterns characteristic of stellate cells, providing additional support for their cytoskeletal organization and functional status. These findings provide strong evidence that the Col-GFP HSC cell line originates from hepatic stellate cells and can serve as a reliable in vitro model to study stellate cell biology and related pathophysiological processes.

While classical Hodgkin lymphoma (HL) is highly susceptible to anti-programmed death protein 1 (PD1) antibodies, the exact modes of action remain controversial. To elucidate the circulating lymphocyte phenotype and systemic effects during anti-PD1 1st-line HL treatment we applied multicolor flow cytometry, FluoroSpot and NanoString to sequential samples of 81 HL patients from the NIVAHL trial (NCT03004833) compared to healthy controls. HL patients showed a decreased CD4 T-cell fraction, a higher percentage of effector-memory T cells and higher expression of activation markers at baseline. Strikingly, and in contrast to solid cancers, expression for 10 out of 16 analyzed co-inhibitory molecules on T cells (e.g., PD1, LAG3, Tim3) was higher in HL. Overall, we observed a sustained decrease of the exhausted T-cell phenotype during anti-PD1 treatment. FluoroSpot of 42.3% of patients revealed T-cell responses against ≥1 of five analyzed tumor-associated antigens. Importantly, these responses were more frequently observed in samples from patients with early excellent response to anti-PD1 therapy. In summary, an initially exhausted lymphocyte phenotype rapidly reverted during anti-PD1 1st-line treatment. The frequently observed IFN-y responses against shared tumor-associated antigens indicate T-cell-mediated cytotoxicity and could represent an important resource for immune monitoring and cellular therapy of HL.

The protease ADAM17 plays an important role in inflammation and cancer and is regulated by iRhom2. Mutations in the cytosolic N-terminus of human iRhom2 cause tylosis with oesophageal cancer (TOC). In mice, partial deletion of the N-terminus results in a curly hair phenotype (cub). These pathological consequences are consistent with our findings that iRhom2 is highly expressed in keratinocytes and in oesophageal cancer. Cub and TOC are associated with hyperactivation of ADAM17-dependent EGFR signalling. However, the underlying molecular mechanisms are not understood. We have identified a non-canonical, phosphorylation-independent 14-3-3 interaction site that encompasses all known TOC mutations. Disruption of this site dysregulates ADAM17 activity. The larger cub deletion also includes the TOC site and thus also dysregulated ADAM17 activity. The cub deletion, but not the TOC mutation, also causes severe reductions in stimulated shedding, binding, and stability of ADAM17, demonstrating the presence of additional regulatory sites in the N-terminus of iRhom2. Overall, this study contrasts the TOC and cub mutations, illustrates their different molecular consequences, and reveals important key functions of the iRhom2 N-terminus in regulating ADAM17.

BackgroundThe individual HLA-I genotype is associated with cancer, autoimmune diseases and infections. This study elucidates the role of germline homozygosity or allelic imbalance of HLA-I loci in esophago-gastric adenocarcinoma (EGA) and determines the resulting repertoires of potentially immunogenic peptides.MethodsHLA genotypes and sequences of either (1) 10 relevant tumor-associated antigens (TAAs) or (2) patient-specific mutation-associated neoantigens (MANAs) were used to predict good-affinity binders using an in silico approach for MHC-binding (www.iedb.org). Imbalanced or lost expression of HLA-I-A/B/C alleles was analyzed by transcriptome sequencing. FluoroSpot assays and TCR sequencing were used to determine peptide-specific T-cell responses.ResultsWe show that germline homozygosity of HLA-I genes is significantly enriched in EGA patients (n=80) compared with an HLA-matched reference cohort (n=7605). Whereas the overall mutational burden is similar, the repertoire of potentially immunogenic peptides derived from TAAs and MANAs was lower in homozygous patients. Promiscuity of peptides binding to different HLA-I molecules was low for most TAAs and MANAs and in silico modeling of the homozygous to a heterozygous HLA genotype revealed normalized peptide repertoires. Transcriptome sequencing showed imbalanced expression of HLA-I alleles in 75% of heterozygous patients. Out of these, 33% showed complete loss of heterozygosity, whereas 66% had altered expression of only one or two HLA-I molecules. In a FluoroSpot assay, we determined that peptide-specific T-cell responses against NY-ESO-1 are derived from multiple peptides, which often exclusively bind only one HLA-I allele.ConclusionThe high frequency of germline homozygosity in EGA patients suggests reduced cancer immunosurveillance leading to an increased cancer risk. Therapeutic targeting of allelic imbalance of HLA-I molecules should be considered in EGA.

BackgroundSpecific immune response is a hallmark of cancer immunotherapy and shared tumor-associated antigens (TAAs) are important targets. Recent advances using combined cellular therapy against multiple TAAs renewed the interest in this class of antigens. Our study aims to determine the role of TAAs in esophago-gastric adenocarcinoma (EGA).MethodsRNA expression was assessed by NanoString in tumor samples of 41 treatment-naïve EGA patients. Endogenous T cell and antibody responses against the 10 most relevant TAAs were determined by FluoroSpot and protein-bound bead assays. Digital image analysis was used to evaluate the correlation of TAAs and T-cell abundance. T-cell receptor sequencing, in vitro expansion with autologous CD40-activated B cells (CD40Bs) and in vitro cytotoxicity assays were applied to determine specific expansion, clonality and cytotoxic activity of expanded T cells.Results68.3% of patients expressed ≥5 TAAs simultaneously with coregulated clusters, which were similar to data from The Cancer Genome Atlas (n=505). Endogenous cellular or humoral responses against ≥1 TAA were detectable in 75.0% and 53.7% of patients, respectively. We found a correlation of T-cell abundance and the expression of TAAs and genes related to antigen presentation. TAA-specific T-cell responses were polyclonal, could be induced or enhanced using autologous CD40Bs and were cytotoxic in vitro. Despite the frequent expression of TAAs co-occurrence with immune responses was rare.ConclusionsWe identified the most relevant TAAs in EGA for monitoring of clinical trials and as therapeutic targets. Antigen-escape rather than missing immune response should be considered as mechanism underlying immunotherapy resistance of EGA.