Biology subjects

Breznik, B.

Publications and source records attributed to Breznik, B..

3 recordsLinked to original sources

bioRxiv2025

Glioblastoma-natural killer cell crosstalk: insights from dynamic spheroid models reveal the importance of secreted cytokines and the CD155 axis

Glioblastoma (GB) is an aggressive primary brain cancer with poor patient prognosis. Natural killer (NK) cells can recognise and eliminate a range of malignant cells, including GB stem cells, which drive GB recurrence. NK cell-based immunotherapy has emerged as a promising approach for GB treatment, but a better understanding of the complex crosstalk between GB and NK cells is needed, particularly within the immunosuppressive GB tumour microenvironment. In this study, we established a reproducible protocol for the production and dynamic culture of uniformly sized GB spheroids using the Celvivo Clinostar system. Our spheroids recapitulated the heterogeneous structure of GB and expressed ligands for NK cell receptors at levels distinct from those observed in corresponding GB cell lines in standard culture, implicating altered sensitivity of GB cells to NK cells in dynamic 3D cultures. GB-NK cell crosstalk was GB cell type dependent and the ability of NK cells to infiltrate GB did not necessarily correlate with their cytotoxicity against GB cells. Spheroids derived from differentiated GB cells secreted higher levels of immunomodulatory cytokines compared to spheroids from GB stem-like cells, and a prominent increase in the secretion of immune-attracting factors was observed in their co-cultures with NK cells. Finally, the CD155-DNAM1/TIGIT axis was indicated as an important regulator of NK cell cytotoxicity against GB stem-like cells. Collectively, our results highlight important factors in GB-NK cell communication and provide a groundwork for further targeted research as well as therapeutic evaluation of NK cell-based approaches in the established dynamic 3D cultures.

cancer biology↗

bioRxiv2025

Heterogenous mitochondrial ultrastructure and metabolism of human glioblastoma cells: differences between stem-like and differentiated cancer cells in response to chemotherapy

BackgroundGlioblastoma stem-like cells (GSCs) contribute to the resistance of glioblastoma (GBM) tumors to standard therapies. The cellular and molecular background of the resistance of GSCs to the chemotherapeutic agent temozolomide is not yet fully understood, in particular in the context of cellular metabolism and the role of mitochondria. The aim of this study was to perform a detailed ultrastructural characterization of the mitochondria of GSCs prior and post temozolomide exposure and to compare it to differentiated GBM cells. MethodsPatient-derived and established GSC and GBM differentiated cell lines were used for the study. The ultrastructure of the mitochondria of the examined cell lines was assessed by transmission electron microscopy. The microscopic analysis was complemented and compared by an analysis of cell metabolism using cell viability assay and extracellular flux analysis using Seahorse assay. ResultsWe found that the metabolic profile of GSCs is quiescent and aerobic. Their elongated mitochondria with highly organized cristae is indicating increased biogenesis and mitochondrial fusion and corresponds to a more OXPHOS-dependent metabolism. The metabolism of GSCs is dependent on OXPHOS and there are no changes in defective mitochondria fraction after the treatment with temozolomide. In contrast, differentiated GBM cells with fragmented mitochondria, which have less organized cristae, are more energetic and glycolytic. Temozolomide treatment induced significant ultrastructural mitochondrial damage in differentiated GBM cells and had less effect on mitochondria in GSCs, suggesting that mitochondria play an important role in the resistance of GSCs to temozolomide treatment. ConclusionsWe demonstrated differences in mitochondrial ultrastructure and cellular metabolism between GSCs and differentiated GBM cells in response to temozolomide. This study provides a basis for further studies addressing the chemotherapy resistance of GSCs and the effects of different treatment regimens on the mitochondrial structure and function of GSCs. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=136 SRC="FIGDIR/small/638633v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@14674e1org.highwire.dtl.DTLVardef@a38976org.highwire.dtl.DTLVardef@95d7a5org.highwire.dtl.DTLVardef@180b6dd_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2024

Melanoma antigens in pediatric medulloblastoma contribute to tumor heterogeneity and species-specificity of group 3 tumors

BackgroundMedulloblastoma (MB) is the most malignant childhood brain cancer. Group 3 MB subtype accounts for about 25% of MB diagnoses and is associated with the most unfavorable outcomes. Herein, we report that more than half of group 3 MB tumors express melanoma antigens (MAGEs), which are potential prognostic and therapeutic markers. MAGEs are tumor antigens, expressed in several types of adult cancers and associated with poorer prognosis and therapy resistance; however, their expression in pediatric cancers is mostly unknown. The aim of this study was to determine whether MAGEs are activated in pediatric MB. MethodsTo determine MAGE frequency in pediatric MB, we obtained formalin-fixed paraffin-embedded tissue (FFPE) samples of 34 patients, collected between 2008 - 2015, from the Childrens Medical Center Dallas pathology archives and applied our validated reverse transcription quantitative PCR (RT-qPCR) assay to measure the relative expression of 23 MAGE cancer-testis antigen genes. To validate our data, we analyzed several published datasets from pediatric MB patients and patient-derived orthotopic xenografts, totaling 860 patients. We then examined how MAGE expression affects the growth and oncogenic potential of medulloblastoma cells by CRISPR-Cas9- and siRNA-mediated gene depletion. ResultsOur RT-qPCR analysis suggested that MAGEs were expressed in group 3/4 medulloblastoma. Further mining of bulk and single-cell RNA-sequencing datasets confirmed that 50-75% of group 3 tumors activate a subset of MAGE genes. Depletion of MAGEAs, B2, and Cs alter MB cell survival, viability, and clonogenic growth due to decreased proliferation and increased apoptosis. ConclusionsThese results indicate that targeting MAGEs in medulloblastoma may be a potential therapeutic option for group 3 medulloblastomas. Key PointsO_LISeveral Type I MAGE CTAs are expressed in >60% of group 3 MBs. C_LIO_LIType I MAGEs affect MB cell proliferation and apoptosis. C_LIO_LIMAGEs are potential biomarkers and therapeutic targets for group 3 MBs. C_LI Importance of the StudyThis study is the first comprehensive analysis of all Type I MAGE CTAs (MAGEA, -B, and -C subfamily members) in pediatric MBs. Our results show that more than 60% of group 3 MBs express MAGE genes, which are required for the viability and growth of cells in which they are expressed. Collectively, these data provide novel insights into the antigen landscape of pediatric MBs. The activation of MAGE genes in group 3 MBs presents potential stratifying and therapeutic options. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=175 SRC="FIGDIR/small/594201v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@1ac274aorg.highwire.dtl.DTLVardef@1479944org.highwire.dtl.DTLVardef@19f5aaforg.highwire.dtl.DTLVardef@fad069_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗