1. Angiogenesis in gynecological cancers and the options for anti-angiogenesis therapyBahar Yetkin-Arik, Arnoud W. Kastelein, Ingeborg Klaassen, Charlotte H. J. R. Jansen, Yani P. Latul, Miloš Vittori, Aydan Biri, Korhan Kahraman, Arjan W. Griffioen, Frederic Amant, Christianne A. R. Lok, Reinier O. Schlingemann, Cornelis J. F. van Noorden, 2021, review article Abstract: Angiogenesis is required in cancer, including gynecological cancers, for the growth of primary tumors and secondary metastases. Development of anti-angiogenesis therapy in gynecological cancers and improvement of its efficacy have been a major focus of fundamental and clinical research. However, survival benefits of current anti-angiogenic agents, such as bevacizumab, in patients with gynecological cancer, are modest. Therefore, a better understanding of angiogenesis and the tumor microenvironment in gynecological cancers is urgently needed to develop more effective anti-angiogenic therapies, either or not in combination with other therapeutic approaches. We describe the molecular aspects of (tumor) blood vessel formation and the tumor microenvironment and provide an extensive clinical overview of current anti-angiogenic therapies for gynecological cancers. We discuss the different phenotypes of angiogenic endothelial cells as potential therapeutic targets, strategies aimed at intervention in their metabolism, and approaches targeting their (inflammatory) tumor microenvironment.
Keywords: angiogenesis, anti-angiogenic therapy, endothelial cells, endothelial cell metabolism, gynecological cancer, non-tip cells, tip cells, tumor microenvironment, vascular disrupting agents
Published in DiRROS: 05.08.2024; Views: 32; Downloads: 55
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2. Transmembrane protein CD9 is glioblastoma biomarker, relevant for maintenance of glioblastoma stem cellsNeža Podergajs, Helena Motaln, Uroš Rajčević, Urška Verbovšek, Marjan Koršič, Nina Obad, Heidi Espedal, Miloš Vittori, Christel Herold-Mende, Hrvoje Miletic, Rolf Bjerkvig, Tamara Lah Turnšek, 2016, original scientific article Abstract: The cancer stem cell model suggests that glioblastomas contain a subpopulation of stem-like tumor cells that reproduce themselves to sustain tumor growth. Targeting these cells thus represents a novel treatment strategy and therefore more specific markers that characterize glioblastoma stem cells need to be identified. In the present study, we performed transcriptomic analysis of glioblastoma tissues compared to normal brain tissues revealing sensible up-regulation of CD9 gene. CD9 encodes the transmembrane protein tetraspanin which is involved in tumor cell invasion, apoptosis and resistance to chemotherapy. Using the public REMBRANDT database for brain tumors, we confirmed the prognostic value of CD9, whereby a more than two fold up-regulation correlates with shorter patient survival. We validated CD9 gene and protein expression showing selective up-regulation in glioblastoma stem cells isolated from primary biopsies and in primary organotypic glioblastoma spheroids as well as in U87-MG and U373 glioblastoma cell lines. In contrast, no or low CD9 gene expression was observed in normal human astrocytes, normal brain tissue and neural stem cells. CD9 silencing in three CD133+ glioblastoma cell lines (NCH644, NCH421k and NCH660h) led to decreased cell proliferation, survival, invasion, and self-renewal ability, and altered expression of the stem-cell markers CD133, nestin and SOX2. Moreover, CD9-silenced glioblastoma stem cells showed altered activation patterns of the Akt, MapK and Stat3 signaling transducers. Orthotopic xenotransplantation of CD9-silenced glioblastoma stem cells into nude rats promoted prolonged survival. Therefore, CD9 should be further evaluated as a target for glioblastoma treatment.
Keywords: biomarker, CD9, glioblastoma stem cells, neural stem cells, tetraspanin
Published in DiRROS: 26.07.2024; Views: 108; Downloads: 83
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3. Imaging of human glioblastoma cells and their interactions with mesenchymal stem cells in the zebrafish (Danio rerio) embryonic brainMiloš Vittori, Barbara Breznik, Tajda Gredar, Katja Hrovat, Lilijana Bizjak-Mali, Tamara Lah Turnšek, 2016, original scientific article Abstract: Background
An attractive approach in the study of human cancers is the use of transparent zebrafish (Danio rerio) embryos, which enable the visualization of cancer progression in a living animal.
Materials and methods
We implanted mixtures of fluorescently labeled glioblastoma (GBM) cells and bonemarrow-derived mesenchymal stem cells (MSCs) into zebrafish embryos to study the cellular pathways of their invasion and the interactions between these cells in vivo.
Results
By developing and applying a carbocyanine-dye-compatible clearing protocol for observation of cells in deep tissues, we showed that U87 and U373 GBM cells rapidly aggregated into tumor masses in the ventricles and midbrain hemispheres of the zebrafish embryo brain, and invaded the central nervous system, often using the ventricular system and the central canal of the spinal cord. However, the GBM cells did not leave the central nervous system. With co-injection of differentially labeled cultured GBM cells and MSCs, the implanted cells formed mixed tumor masses in the brain. We observed tight associations between GBM cells and MSCs, and possible cell-fusion events. GBM cells and MSCs used similar invasion routes in the central nervous system.
Conclusions
This simple model can be used to study the molecular pathways of cellular processes in GBM cell invasion, and their interactions with various types of stromal cells in double or triple cell co-cultures, to design anti-GBM cell therapies that use MSCs as vectors.
Keywords: brain tumors, tumor microenvironment, animal models, xenotransplantation
Published in DiRROS: 25.07.2024; Views: 108; Downloads: 130
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4. RECQ1 helicase silencing decreases the tumour growth rate of U87 glioblastoma cell xenografts in zebrafish embryosMiloš Vittori, Barbara Breznik, Katja Hrovat, Saša Kenig, Tamara Lah Turnšek, 2017, original scientific article Abstract: RECQ1 helicase has multiple roles in DNA replication, including restoration of the replication fork and DNA repair, and plays an important role in tumour progression. Its expression is highly elevated in glioblastoma as compared to healthy brain tissue. We studied the effects of small hairpin RNA (shRNA)-induced silencing of RECQ1 helicase on the increase in cell number and the invasion of U87 glioblastoma cells. RECQ1 silencing reduced the rate of increase in the number of U87 cells by 30%. This corresponded with a 40% reduction of the percentage of cells in the G2 phase of the cell cycle, and an accumulation of cells in the G1 phase. These effects were confirmed in vivo, in the brain of zebrafish (Danio rerio) embryos, by implanting DsRed-labelled RECQ1 helicase-silenced and control U87 cells. The growth of resulting tumours was quantified by monitoring the increase in xenograft fluorescence intensity during a three-day period with fluorescence microscopy. The reduced rate of tumour growth, by approximately 30% in RECQ1 helicase-silenced cells, was in line with in vitro measurements of the increase in cell number upon RECQ1 helicase silencing. However, RECQ1 helicase silencing did not affect invasive behaviour of U87 cells in the zebrafish brain. This is the first in vivo confirmation that RECQ1 helicase is a promising molecular target in the treatment of glioblastoma.
Keywords: cancer, cell cycle, DNA damage, intravital imaging, RNA interference, theranostics
Published in DiRROS: 25.07.2024; Views: 99; Downloads: 105
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5. Mesenchymal stem cells differentially affect the invasion of distinct glioblastoma cell linesBarbara Breznik, Helena Motaln, Miloš Vittori, Ana Rotter, Tamara Lah Turnšek, 2017, original scientific article Abstract: Glioblastoma multiforme are an aggressive form of brain tumors that are characterized by distinct invasion of single glioblastoma cells, which infiltrate the brain parenchyma. This appears to be stimulated by the communication between cancer and stromal cells. Mesenchymal stem cells (MSCs) are part of the glioblastoma microenvironment, and their ‘cross-talk’ with glioblastoma cells is still poorly understood. Here, we examined the effects of bone marrow-derived MSCs on two different established glioblastoma cell lines U87 and U373. We focused on mutual effects of direct MSC/glioblastoma contact on cellular invasion in three-dimensional invasion assays in vitro and in a zebrafish embryo model in vivo. This is the first demonstration of glioblastoma cell-type-specific responses to MSCs in direct glioblastoma co-cultures, where MSCs inhibited the invasion of U87 cells and enhanced the invasion of U373. Inversely, direct cross-talk between MSCs and both of glioblastoma cell lines enhanced MSC motility. MSC-enhanced invasion of U373 cells was assisted by overexpression of proteases cathepsin B, calpain1, uPA/uPAR, MMP-2, -9 and -14, and increased activities of some of these proteases, as determined by the effects of their selective inhibitors on invasion. In contrast, these proteases had no effect on U87 cell invasion under MSC co-culturing. Finally, we identified differentially expressed genes, in U87 and U373 cells that could explain different response of these cell lines to MSCs. In conclusion, we demonstrated that MSC/glioblastoma cross-talk is different in the two glioblastoma cell phenotypes, which contributes to tumor heterogeneity.
Keywords: glioblastoma multiforme, proteases, mesenchymal stem cells, tumor heterogeneity, zebrafish model
Published in DiRROS: 24.07.2024; Views: 114; Downloads: 83
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6. 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, original scientific article Abstract: 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.
Published in DiRROS: 19.07.2024; Views: 98; Downloads: 110
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7. Anti-vimentin nanobody decreases glioblastoma cell invasion in vitro and in vivoAlja Zottel, Metka Novak, Neja Šamec, Bernarda Majc, Sara Colja, Mojca Katrašnik, Miloš Vittori, Barbara Hrastar, Ana Rotter, Andrej Porčnik, Tamara Lah Turnšek, Radovan Komel, Barbara Breznik, Ivana Jovchevska, 2023, original scientific article Abstract: Purpose: Glioblastoma (GBM) is the most common primary brain tumour and one of the deadliest cancers. In addition to late diagnosis and inadequate treatment, the extremely low survival rate is also due to the lack of appropriate therapeutic biomarkers and corresponding therapeutic agents. One of the potential therapeutic biomarkers is the intermediate filament vimentin, which is associated with epithelial-mesenchymal transition (EMT). The purpose of this study was to analyse the effect of the anti-vimentin nanobody Nb79 on cell invasion in vitro and in vivo. To further our understanding of the mechanism of action, we investigated the association between Nb79 and EMT in GBM and GBM stem cells by analysing the expression levels of key EMT-related proteins. Methods: The expression of vimentin in glioma tissues and cells was determined by RT-qPCR. An invasion assay was performed on differentiated glioblastoma cell line U-87 MG and stem cell line NCH421k in vitro as well as in vivo in zebrafish embryos. The effect of Nb79 on expression of EMT biomarkers beta-catenin, vimentin, ZEB-1 and ZO1 was determined by Western blot and immunocytochemistry. Results: Our study shows that vimentin is upregulated in glioblastoma tissue compared to lower grade glioma and non-tumour brain tissue. We demonstrated that treatment with Nb79 reduced glioblastoma cell invasion by up to 64% in vitro and up to 21% in vivo. In addition, we found that the tight junction protein ZO-1 had higher expression on the cell membrane, when treated with inhibitory anti-vimentin Nb79 compared to control. Conclusion: In conclusion, our results suggest that anti-vimentin nanobody Nb79 is a promising tool to target glioblastoma cell invasion.
Keywords: glioblastoma, vimentin, nanobody
Published in DiRROS: 12.07.2024; Views: 112; Downloads: 101
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8. New insights in ATP synthesis as therapeutic target in cancer and angiogenic ocular diseasesCornelis J. F. van Noorden, Bahar Yetkin-Arik, Paola Serrano Martinez, Noëlle Bakker, Mathilda E. van Breest Smallenburg, Reinier O. Schlingemann, Ingeborg Klaassen, Bernarda Majc, Anamarija Habič, Urban Bogataj, Katrin S. Galun, Miloš Vittori, Mateja Erdani-Kreft, Metka Novak, Barbara Breznik, Vashendriya V. V. Hira, 2024, review article Abstract: Lactate and ATP formation by aerobic glycolysis, the Warburg effect, is considered a hallmark of cancer. During angiogenesis in non-cancerous tissue, proliferating stalk endothelial cells (ECs) also produce lactate and ATP by aerobic glycolysis. In fact, all proliferating cells, both non-cancer and cancer cells, need lactate for the biosynthesis of building blocks for cell growth and tissue expansion. Moreover, both non-proliferating cancer stem cells in tumors and leader tip ECs during angiogenesis rely on glycolysis for pyruvate production, which is used for ATP synthesis in mitochondria through oxidative phosphorylation (OXPHOS). Therefore, aerobic glycolysis is not a specific hallmark of cancer but rather a hallmark of proliferating cells and limits its utility in cancer therapy. However, local treatment of angiogenic eye conditions with inhibitors of glycolysis may be a safe therapeutic option that warrants experimental investigation. Most types of cells in the eye such as photoreceptors and pericytes use OXPHOS for ATP production, whereas proliferating angiogenic stalk ECs rely on glycolysis for lactate and ATP production.
Keywords: aerobic glycolysis, anaerobic glycolysis, angiogenesis, ATP synthesis, cancer cells, cancer stem cells, endothelial cells, energy metabolism, eye diseases, oxidative phosphorylation, pericytes, retina, Warburg effect
Published in DiRROS: 18.06.2024; Views: 182; Downloads: 113
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9. Imaging of human glioblastoma cells and their interactions with mesenchymal stem cells in the zebrafish (Danio rerio) embryonic brainMiloš Vittori, Barbara Breznik, Tajda Gredar, Katja Hrovat, Lilijana Bizjak-Mali, Tamara Lah Turnšek, 2016, original scientific article Keywords: brain tumors, tumor microenvironment, animal models, xenotransplantation
Published in DiRROS: 09.05.2024; Views: 220; Downloads: 466
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