21. Characterization of In vitro 3D cell model developed from human hepatocellular carcinoma (HepG2) cell lineMartina Štampar, Barbara Breznik, Metka Filipič, Bojana Žegura, 2020, izvirni znanstveni članek Povzetek: In genetic toxicology, there is a trend against the increased use of in vivo models as highlighted by the 3R strategy, thus encouraging the development and implementation of alternative models. Two-dimensional (2D) hepatic cell models, which are generally used for studying the adverse effects of chemicals and consumer products, are prone to giving misleading results. On the other hand, newly developed hepatic three-dimensional (3D) cell models provide an attractive alternative, which, due to improved cell interactions and a higher level of liver-specific functions, including metabolic enzymes, reflect in vivo conditions more accurately. We developed an in vitro 3D cell model from the human hepatocellular carcinoma (HepG2) cell line. The spheroids were cultured under static conditions and characterised by monitoring their growth, morphology, and cell viability during the time of cultivation. A time-dependent suppression of cell division was observed. Cell cycle analysis showed time-dependent accumulation of cells in the G0/G1 phase. Moreover, time-dependent downregulation of proliferation markers was shown at the mRNA level. Genes encoding hepatic markers, metabolic phase I/II enzymes, were time-dependently deregulated compared to monolayers. New knowledge on the characteristics of the 3D cell model is of great importance for its further development and application in the safety assessment of chemicals, food products, and complex mixtures. Objavljeno v DiRROS: 22.07.2024; Ogledov: 169; Prenosov: 147
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22. CCR5-mediated signaling is involved in invasion of glioblastoma cells in its microenvironmentMetka Novak, Miha Koprivnikar Krajnc, Barbara Hrastar, Barbara Breznik, Bernarda Majc, Mateja Mlinar, Ana Rotter, Andrej Porčnik, Jernej Mlakar, Katja Stare, Richard G. Pestell, Tamara Lah Turnšek, 2020, izvirni znanstveni članek Povzetek: Abstract
The chemokine CCL5/RANTES is a versatile inflammatory mediator, which interacts with the receptor CCR5, promoting cancer cell interactions within the tumor microenvironment. Glioblastoma is a highly invasive tumor, in which CCL5 expression correlates with shorter patient survival. Using immunohistochemistry, we identified CCL5 and CCR5 in a series of glioblastoma samples and cells, including glioblastoma stem cells. CCL5 and CCR5 gene expression were significantly higher in a cohort of 38 glioblastoma samples, compared to low-grade glioma and non-cancerous tissues. The in vitro invasion of patients-derived primary glioblastoma cells and glioblastoma stem cells was dependent on CCL5-induced CCR5 signaling and is strongly inhibited by the small molecule CCR5 antagonist maraviroc. Invasion of these cells, which was enhanced when co-cultured with mesenchymal stem cells (MSCs), was inhibited by maraviroc, suggesting that MSCs release CCR5 ligands. In support of this model, we detected CCL5 and CCR5 in MSC monocultures and glioblastoma-associated MSC in tissue sections. We also found CCR5 expressing macrophages were in close proximity to glioblastoma cells. In conclusion, autocrine and paracrine cross-talk in glioblastoma and, in particular, glioblastoma stem cells with its stromal microenvironment, involves CCR5 and CCL5, contributing to glioblastoma invasion, suggesting the CCL5/CCR5 axis as a potential therapeutic target that can be targeted with repositioned drug maraviroc. Ključne besede: CCL5, CCR5, chemokines, glioblastoma, invasion, maraviroc, mesenchymal stem cells Objavljeno v DiRROS: 22.07.2024; Ogledov: 178; Prenosov: 39
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23. TRIM28 selective nanobody reduces glioblastoma stem cell invasionAndrej Porčnik, Metka Novak, Barbara Breznik, Bernarda Majc, Barbara Hrastar, Neja Šamec, Alja Zottel, Ivana Jovchevska, Miloš Vittori, Ana Rotter, Radovan Komel, Tamara Lah Turnšek, 2021, izvirni znanstveni članek Povzetek: Glioblastoma (GB), is the most common and aggressive malignant primary brain tumour in adults. Intra- and inter-tumour heterogeneity, infiltrative GB cell invasion and presence of therapy-resistant GB stem cells (GSCs) represent major obstacles to favourable prognosis and poor therapy response. Identifying the biomarkers of the most aggressive tumour cells and their more efficient targeting strategies are; therefore, crucial. Recently, transcription factor TRIM28 has been identified as a GB biomarker and, in this study, we have shown high expression of TRIM28 in GB and in low grade gliomas as well as higher expression in GSCs vs. differentiated GB cells, although in both cases not significant. We demonstrated significant in vitro inhibition of GB cells and GSCs invasiveness and spread in zebrafish brains in vivo by anti-TRIM28 selective nanobody NB237. TRIM28 was also enriched in GB (tumour) core and associated with the expression of stem cell genes, but was not prognostic for overall survival. However, based on the above results, we conclude that TRIM28 nanobody NB237 offers a new opportunity as a GB therapeutic tool. Objavljeno v DiRROS: 19.07.2024; Ogledov: 148; Prenosov: 148
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24. The pathogens spillover and incidence correlation in bumblebees and honeybees in SloveniaMetka Pislak, Ivan Toplak, Urška Zajc, Danilo Bevk, 2021, izvirni znanstveni članek Povzetek: Slovenia has a long tradition of beekeeping and a high density of honeybee colonies, but less is known about bumblebees and their pathogens. Therefore, a study was conducted to define the incidence and prevalence of pathogens in bumblebees and to determine whether there are links between infections in bumblebees and honeybees. In 2017 and 2018, clinically healthy workers of bumblebees (Bombus spp.) and honeybees (Apis mellifera) were collected on flowers at four different locations in Slovenia. In addition, bumblebee queens were also collected in 2018. Several pathogens were detected in the bumblebee workers using PCR and RT-PCR methods: 8.8% on acute bee paralysis virus (ABPV), 58.5% on black queen cell virus (BQCV), 6.8% on deformed wing virus (DWV), 24.5% on sacbrood bee virus (SBV), 15.6% on Lake Sinai virus (LSV), 16.3% on Nosema bombi, 8.2% on Nosema ceranae, 15.0% on Apicystis bombi and 17.0% on Crithidia bombi. In bumblebee queens, only the presence of BQCV, A. bombi and C. bombi was detected with 73.3, 26.3 and 33.3% positive samples, respectively. This study confirmed that several pathogens are regularly detected in both bumblebees and honeybees. Further studies on the pathogen transmission routes are required. Objavljeno v DiRROS: 19.07.2024; Ogledov: 143; Prenosov: 104
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25. Hepatocellular carcinoma (HepG2/C3A) cell-based 3D model for genotoxicity testing of chemicalsMartina Štampar, Helle Frandsen, Adelina Rogowska-Wrzesinska, Krzysztof Wrzesinski, Metka Filipič, Bojana Žegura, 2021, izvirni znanstveni članek Povzetek: The major weakness of the current in vitro genotoxicity test systems is the inability of the indicator cells to express metabolic enzymes needed for the activation and detoxification of genotoxic compounds, which consequently can lead to misleading results. Thus, there is a significant emphasis on developing hepatic cell models, including advanced in vitro three-dimensional (3D) cell-based systems, which better imitate in vivo cell behaviour and offer more accurate and predictive data for human exposures. In this study, we developed an approach for genotoxicity testing with 21-day old spheroids formed from human hepatocellular carcinoma cells (HepG2/C3A) using the dynamic clinostat bioreactor system (CelVivo BAM/bioreactor) under controlled conditions. The spheroids were exposed to indirect-acting genotoxic compounds, polycyclic aromatic hydrocarbon [PAH; benzo(a) pyrene B(a)P], and heterocyclic aromatic amine [PhIP]) at non-cytotoxic concentrations for 24 and 96 h. The results showed that both environmental pollutants B(a)P and PhIP significantly increased the level of DNA strand breaks assessed by the comet assay. Further, the mRNA level of selected genes encoding metabolic enzymes from phase I and II, and DNA damage responsive genes was determined (qPCR). The 21-day old spheroids showed higher basal expression of genes encoding metabolic enzymes compared to monolayer culture. In spheroids, B(a)P or PhIP induced compound-specific up-regulation of genes implicated in their metabolism, and deregulation of genes implicated in DNA damage and immediate-early response. The study demonstrated that this model utilizing HepG2/C3A spheroids grown under dynamic clinostat conditions represents a very sensitive and promising in vitro model for genotoxicity and environmental studies and can thus significantly contribute to a more reliable assessment of genotoxic activities of pure chemicals, and complex environmental samples even at very low for environmental exposure relevant concentrations. Ključne besede: in vitro 3D cell model, 21-day old spheroids, cytotoxic, genotoxic, gene expression Objavljeno v DiRROS: 19.07.2024; Ogledov: 144; Prenosov: 168
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26. The cytotoxic effects of cannabidiol and cannabigerol on glioblastoma stem cells may mostly involve GPR55 and TRPV1 signallingTamara Lah Turnšek, Bernarda Majc, Metka Novak, Ajda Sušnik, Barbara Breznik, Andrej Porčnik, Roman Bošnjak, Aleksander Sadikov, Marta Malavolta, Selma Halilčević, Jernej Mlakar, Roby Zomer, 2022, izvirni znanstveni članek Povzetek: Glioblastoma (GBM) is one of the most aggressive cancers, comprising 60–70% of all gliomas. The large G-protein-coupled receptor family includes cannabinoid receptors CB1, CB2, GPR55, and non-specific ion receptor protein transporters TRPs. First, we found up-regulated CNR1, GPR55, and TRPV1 expression in glioma patient-derived tissue samples and cell lines compared with non-malignant brain samples. CNR1 and GPR55 did not correlate with glioma grade, whereas TRPV1 negatively correlated with grade and positively correlated with longer overall survival. This suggests a tumour-suppressor role of TRPV1. With respect to markers of GBM stem cells, preferred targets of therapy, TRPV1 and GPR55, but not CNR1, strongly correlated with different sets of stemness gene markers: NOTCH, OLIG2, CD9, TRIM28, and TUFM and CD15, SOX2, OCT4, and ID1, respectively. This is in line with the higher expression of TRPV1 and GPR55 genes in GSCs compared with differentiated GBM cells. Second, in a panel of patient-derived GSCs, we found that CBG and CBD exhibited the highest cytotoxicity at a molar ratio of 3:1. We suggest that this mixture should be tested in experimental animals and clinical studies, in which currently used Δ9-tetrahydrocannabinol (THC) is replaced with efficient and non-psychoactive CBG in adjuvant standard-of-care therapy. Ključne besede: glioblastoma, glioma, cannabigerol, cannabidiol, cannabinoid receptors, stem cells Objavljeno v DiRROS: 18.07.2024; Ogledov: 193; Prenosov: 147
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27. Infiltrating natural killer cells bind, lyse and increase chemotherapy efficacy in glioblastoma stem-like tumorospheresBarbara Breznik, Meng-Wei Ko, Christopher Tse, Po-Chun Chen, Emanuela Senjor, Bernarda Majc, Anamarija Habič, Nicolas Angelillis, Metka Novak, Vera Župunski, Jernej Mlakar, David Nathanson, Anahid Jewett, 2022, izvirni znanstveni članek Povzetek: Glioblastomas remain the most lethal primary brain tumors. Natural killer (NK) cell-based therapy is a promising immunotherapeutic strategy in the treatment of glioblastomas, since these cells can select and lyse therapy-resistant glioblastoma stem-like cells (GSLCs). Immunotherapy with super-charged NK cells has a potential as antitumor approach since we found their efficiency to kill patient-derived GSLCs in 2D and 3D models, potentially reversing the immunosuppression also seen in the patients. In addition to their potent cytotoxicity, NK cells secrete IFN-γ, upregulate GSLC surface expression of CD54 and MHC class I and increase sensitivity of GSLCs to chemotherapeutic drugs. Moreover, NK cell localization in peri-vascular regions in glioblastoma tissues and their close contact with GSLCs in tumorospheres suggests their ability to infiltrate glioblastoma tumors and target GSLCs. Due to GSLC heterogeneity and plasticity in regards to their stage of differentiation personalized immunotherapeutic strategies should be designed to effectively target glioblastomas. Ključne besede: glioblastoma, natural killer cells, translational oncology Objavljeno v DiRROS: 16.07.2024; Ogledov: 224; Prenosov: 203
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28. Upregulation of cathepsin X in glioblastoma : interplay with γ-enolase and the effects of selective cathepsin X inhibitorsBernarda Majc, Anamarija Habič, Metka Novak, Ana Rotter, Andrej Porčnik, Jernej Mlakar, Vera Župunski, Urša Pečar Fonović, Damijan Knez, Nace Zidar, Stanislav Gobec, Janko Kos, Tamara Lah Turnšek, Anja Pišlar, Barbara Breznik, 2022, izvirni znanstveni članek Ključne besede: glioblastoma, cathepsin X, γ-enolase, tumor microenvironment, glioblastoma stem cells, cathepsin X inhibitors Objavljeno v DiRROS: 16.07.2024; Ogledov: 166; Prenosov: 149
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29. Lethal and sub-lethal effects and modulation of gene expression induced by T kinase inhibitors in zebrafish (Danio Rerio) embryosTina Eleršek, Matjaž Novak, Mateja Mlinar, Igor Virant, Nika Bahor, Karin Leben, Bojana Žegura, Metka Filipič, 2022, izvirni znanstveni članek Povzetek: Tyrosine kinase inhibitors (TKIs) are designed for targeted cancer therapy. The consumption of these drugs during the last 20 years has been constantly rising. In the zebrafish (Danio rerio) embryo toxicity test, we assessed the toxicity of six TKIs: imatinib mesylate, erlotinib, nilotinib, dasatinib, sorafenib and regorafenib. Imatinib mesylate and dasatinib induced lethal effects, while regorafenib, sorfenib and dasatinib caused a significant increase of sub-lethal effects, predominantly oedema, no blood circulation and formation of blood aggregates. The analyses of the changes in the expression of selected genes associated with the hormone system after the exposure to imatinib mesylate, dasatinib and regorafenib demonstrated that all three tested TKIs deregulated the expression of oestrogen receptor esr1, cytochrome P450 aromatase (cypa19b) and hydroxysteroid-dehydrogenase (hsd3b), regorafenib, and also thyroglobulin (tg). The expression of genes involved in the DNA damage response (gadd45 and mcm6) and apoptosis (bcl2) was deregulated only by exposure to regorafenib. The data indicate that common mechanisms, namely antiangiogenic activity and interference with steroidogenesis are involved in the TKI induced sub-lethal effects and potential hormone disrupting activity, respectively. The residues of TKIs may represent an environmental hazard; therefore, further ecotoxicological studies focusing also on the effects of their mixtures are warranted. Ključne besede: aquatic toxicity, tyrosine kinase inhibitors, zebrafish embryo toxicity test, gene expression, environmental hazard Objavljeno v DiRROS: 16.07.2024; Ogledov: 183; Prenosov: 122
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30. HepG2 spheroids as a biosensor-like cell-based system for (geno)toxicity assessmentMartina Štampar, Sonja Žabkar, Metka Filipič, Bojana Žegura, 2022, izvirni znanstveni članek Povzetek: 3D spheroids developed from HepG2 cells were used as a biosensor-like system for the detection of (geno)toxic effects induced by chemicals. Benzo(a)pyrene (B(a)P) and amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) with well-known mechanisms of action were used for system validation. HepG2 spheroids grown for 3 days were exposed to BaP and PhIP for 24 and 72 h. The growth and viability of spheroids were monitored by planimetry and Live/Dead staining of cells. Multi-parametric flow cytometric analysis was applied for simultaneous detection of specific end-effects including cell cycle analysis (Hoechst staining), cell proliferation (KI67 marker), and DNA double-strand breaks (ℽH2AX) induced by genotoxic compounds. Depending on the exposure concentration/time, BaP reduced spheroid growth, affected cell proliferation by arresting cells in S and G2 phase and induced DNA double-strand breaks (DSB). Simultaneous staining of ℽH2AX formation and cell cycle analysis revealed that after BaP (10 μM; 24 h) exposure 60% of cells in G0/G1 phase had DNA DSB, while after 72 h only 20% of cells contained DSB indicating efficient repair of DNA lesions. PhIP did not influence the spheroid size whereas accumulation of cells in the G2 phase occurred after both treatment times. The evaluation of DNA damage revealed that at 200 μM PhIP 50% of cells in G0/G1 phase had DNA DSB, which after 72-h exposure dropped to 40%, showing lower repair capacity of PhIP-induced DSB compared to BaP-induced. The developed approach using simultaneous detection of several parameters provides mechanistic data and thus contributes to more reliable genotoxicity assessment of chemicals as a high-content screening tool. Ključne besede: in vitro 3D cell model, HepG2, flow cytometry, cell cycle, proliferation, DNA strand, breaks Objavljeno v DiRROS: 16.07.2024; Ogledov: 192; Prenosov: 124
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